**The Role of the Pituitary-Growth Hormone-IGF Axis in Glucose Homeostasis**

## Stephen F. Kemp

*University of Arkansas for Medical Sciences Arkansas Children's Hospital U. S. A.* 

### **1. Introduction**

126 Diabetes – Damages and Treatments

[51] Kabadi UM ,Kabadi MU Early Postprandial Insulin Secretion:Its Relation to Insulin

[52] American Diabetes association. Standard of Medical Care in Diabetes Diabetes Care :

Sensitivity J Diabetes Mellitus1(1),1-5,2011.

34,S11-S61,2011

Hypoglycemia results when either carbohydrate intake is low, tissue use is high (glycolysis or glucagons synthesis), or endogenous production of glucose is low (glycogenolysis and glyuconeogenesis)(Berry, Nathan et al. 2009). Glucose levels are controlled by the hormone insulin, and also by the counterregulatory hormones glucagons, cortisol, growth hormone (GH), epinephrine, and norepinephrine. The counterregulatory hormones stimulate production and release of glucose. Hypoglycemia is the most common metabolic problem in neonates, and is also seen in children and adults.

## **2. The pituitary-growth hormone-IGF axis**

### **2.1 Embryology of the pituitary gland**

The pituitary gland develops from invagination of the oral ectoderm (Rathke's pouch)(Frohnert and Miller 2009). Nearby neuroectoderm becomes the posterior pituitary, which secretes the hormones oxytocin and vasopressin. Signalling factors involved in the initial differentiation of the anterior pituitary (thickening of the oral ectoderm) include the transcription factors HESX1, PITX1, LHX3, and LHX4. Under the influence of the transcription factor TPIT some of the cells develop into corticotrophs which secrete ACTH. When influenced by transcription factors PROP1, PIT1 (now called POU1F1), PITX1 and PITX2 the remaining cells differentiate into gonadotrophs (which secrete FSH and LH), thyrotrophs (which secrete TSH), somatotrophs (which secrete GH), and lactotrophs (which secrete PRL). During this process the oral ectoderm and the neuroectoderm remain in contact with each other, and both migrate together to form the pituitary with distinct anterior and posterior lobes. All of the hormones of the anterior pituitary are influenced by secretions from the hypothalamus and are regulated through specific feedback loops. Two hormones of the anterior pituitary protect against hypoglycemia— GH and ACTH. ACTH stimulates secretion of cortisol by the adrenal gland. Both GH and cortisol protect against hypoglycemia by countering the effects of insulin.

#### **2.2 The GH-IGF system**

The GH/IGF axis is shown in Figure 1. It is regulated by three peptides, two from the hypothalamus (that part of the brain closest to the pituitary gland) (Growth Hormone

The Role of the Pituitary-Growth Hormone-IGF Axis in Glucose Homeostasis 129

Releasing Hormone (GHRH) and somatostatin), and one from the gastrointestinal tract (ghrelin). Growth hormone releasing hormone (GHRH), signals the pituitary to secrete GH into the general circulation. The other hypothalamic signal is somatostatin, which inhibits GH secretion. These two signals alternate in their expression, so that when GHRH is high, somatostatin is low, and vice versa. The third factor, ghrelin, also stimulates GH secretion. GH is secreted in discrete bursts, and, once secreted, remains in circulation for about 20

GH circulates bound to GH binding protein (GHBP), which in humans is identical to the extracellular portion of the GH receptor. GHBP is produced as a result of cleavage of the extracellular portion of the GH receptor. In order for GH to act two GH molecules bind to adjacent GH receptors, resulting in a conformational shift in the GH receptor, which activates the JAK-STAT pathway in the cell. Activation of the JAK-STAT pathway initiates a cascade of intracellular events, ultimately increasing production of Insulin-like Growth Factor I (IGF-I). GH stimulates statural growth by action directly at the growth plate and indirectly through the production of IGF-I. The name growth hormone is somewhat unfortunate, in that it suggests that its only function is to stimulate growth. In addition to its involvement in the growth process, GH has many metabolic functions, such as increasing

There are two GH genes, located on chromosome 17 in the human, GH-1 and GH-2. The GH-1 (or GH-N) gene encodes for GH. It consists of five exons separated by four introns (Parks, Nielson et al. 1985). The most abundant hormone of the pituitary gland, GH is a single chain α-helical non-glycosylated polypeptide consisting of 191 amino acids with two intramolecular disulphide bonds between amino acid 52-165 and 282-189. Different forms of GH exist with the most common form of GH being the one with a molecular weight of 22 kD, which accounts for about 75% of the GH produced in the pituitary gland. Alternative splicing of the second codon results in a 20-kD form that make up about 5-10% of the total GH. There is structural homology between the GH molecule and prolactin and human placental lactogen (human chorionic somatotropin), suggesting that they may all have

GH acts by binding to its receptor primarily at two sites, the liver and the growth plate. In the liver activation of the GH receptor stimulates production of IGF-I, its binding protein IGFBP-3, and the acid labile subunit (ALS). Growth hormone circulates at least 50% bound to its binding protein, GHBP (Rosenfeld 2005). It binds specifically with high affinity and low capacity. In humans the circulating GHBP is actually the extracellular domain of the GH receptor; it is thought that GHBP is shed or cleaved from intact receptors. The physiological significance of GH binding by GHBP is not completely understood; it may act to prolong the half-life of GH

Insulin-like growth factors (IGF-I and IGF-II) are small peptide hormones (~7.5 kDa) which share a high degree of homology with proinsulin (Rinderknecht and Humbel 1978; Rinderknecht and Humbel 1978). Almost ubiquitously produced, they circulate at high concentrations in serum. Beyond their insulin-like effects, these growth-promoting peptides influence cellular proliferation and differentiation in numerous tissues, including at the growth plate (Nilsson, Marino et al. 2005). For IGFs to exert their effects at the cell surface, they must first bind specific, high affinity cell-surface receptors, principally the type I IGF receptor. The interaction of IGFs with cell-surface receptors, however, is tightly regulated by

at least six distinct high affinity carrier proteins, the IGF- binding proteins (IGFBPs).

muscle mass and bone mineral density. Generally, the effects of GH are anabolic.

descended from a common ancestral gene.

in the serum or it may compete with the GH receptor for binding.

mintues.


Table 1. Transcription factors involved in the differentiation of the anterior pituitary.

Fig. 1. The Hypothalamic-GH-IGF system 1. The hypothalamus secretes GHRH and somatostatin. 2. GH is secreted into the general circulation. 3. GH circulates bound to its binding protein, GHBP. 4. GH binds to the GH receptor. 5. ALS, 6. IGFBP-3, and 7. IGF-I circulate together in a 140 kDa complex. 8. IGF-I acts at the growth plate.

PITX2 Formation of pituitary. Differentiation of pituitary cell into a somatotroph (GH) or a lactotroph (PRL)

Formation of pituitary. Involved in differentiation of pituitary cell into a corticotroph (secreting ACTH) or a gonadotroph

Formation of pituitary. Differentiation of pituitary cell into precursor for gonadotrophs, thyrotrophs, samatotrophs, and

Formation of pituitary. Differentiation of pituitary cell into precursor for gonadotrophs, thyrotrophs, samatotrophs, and

HESX1 Involved in formation of Rathke's pouch

(LH/FSH)

lactotrophs.

lactotrophs. SF1 Differentiation of gonadotrophs. TPIT Differentiation of corticotrophs. NEUROD1 Differentiation of corticotrophs

POU1F1 (PIT1) Differentiation of cells into precursors of thyrotrophs, somatotrophs, and lactotrophs. PROP1 Differentiation of cells into precursors of gonadotrophs,

Table 1. Transcription factors involved in the differentiation of the anterior pituitary.

Fig. 1. The Hypothalamic-GH-IGF system 1. The hypothalamus secretes GHRH and somatostatin. 2. GH is secreted into the general circulation. 3. GH circulates bound to its binding protein, GHBP. 4. GH binds to the GH receptor. 5. ALS, 6. IGFBP-3, and 7. IGF-I

circulate together in a 140 kDa complex. 8. IGF-I acts at the growth plate.

thyrotrophs, somatotrophs, and lactotrophs.

**Transcription Factor Function** 

PITX1

LHX3

LHX4

Releasing Hormone (GHRH) and somatostatin), and one from the gastrointestinal tract (ghrelin). Growth hormone releasing hormone (GHRH), signals the pituitary to secrete GH into the general circulation. The other hypothalamic signal is somatostatin, which inhibits GH secretion. These two signals alternate in their expression, so that when GHRH is high, somatostatin is low, and vice versa. The third factor, ghrelin, also stimulates GH secretion. GH is secreted in discrete bursts, and, once secreted, remains in circulation for about 20 mintues.

GH circulates bound to GH binding protein (GHBP), which in humans is identical to the extracellular portion of the GH receptor. GHBP is produced as a result of cleavage of the extracellular portion of the GH receptor. In order for GH to act two GH molecules bind to adjacent GH receptors, resulting in a conformational shift in the GH receptor, which activates the JAK-STAT pathway in the cell. Activation of the JAK-STAT pathway initiates a cascade of intracellular events, ultimately increasing production of Insulin-like Growth Factor I (IGF-I). GH stimulates statural growth by action directly at the growth plate and indirectly through the production of IGF-I. The name growth hormone is somewhat unfortunate, in that it suggests that its only function is to stimulate growth. In addition to its involvement in the growth process, GH has many metabolic functions, such as increasing muscle mass and bone mineral density. Generally, the effects of GH are anabolic.

There are two GH genes, located on chromosome 17 in the human, GH-1 and GH-2. The GH-1 (or GH-N) gene encodes for GH. It consists of five exons separated by four introns (Parks, Nielson et al. 1985). The most abundant hormone of the pituitary gland, GH is a single chain α-helical non-glycosylated polypeptide consisting of 191 amino acids with two intramolecular disulphide bonds between amino acid 52-165 and 282-189. Different forms of GH exist with the most common form of GH being the one with a molecular weight of 22 kD, which accounts for about 75% of the GH produced in the pituitary gland. Alternative splicing of the second codon results in a 20-kD form that make up about 5-10% of the total GH. There is structural homology between the GH molecule and prolactin and human placental lactogen (human chorionic somatotropin), suggesting that they may all have descended from a common ancestral gene.

GH acts by binding to its receptor primarily at two sites, the liver and the growth plate. In the liver activation of the GH receptor stimulates production of IGF-I, its binding protein IGFBP-3, and the acid labile subunit (ALS). Growth hormone circulates at least 50% bound to its binding protein, GHBP (Rosenfeld 2005). It binds specifically with high affinity and low capacity. In humans the circulating GHBP is actually the extracellular domain of the GH receptor; it is thought that GHBP is shed or cleaved from intact receptors. The physiological significance of GH binding by GHBP is not completely understood; it may act to prolong the half-life of GH in the serum or it may compete with the GH receptor for binding.

Insulin-like growth factors (IGF-I and IGF-II) are small peptide hormones (~7.5 kDa) which share a high degree of homology with proinsulin (Rinderknecht and Humbel 1978; Rinderknecht and Humbel 1978). Almost ubiquitously produced, they circulate at high concentrations in serum. Beyond their insulin-like effects, these growth-promoting peptides influence cellular proliferation and differentiation in numerous tissues, including at the growth plate (Nilsson, Marino et al. 2005). For IGFs to exert their effects at the cell surface, they must first bind specific, high affinity cell-surface receptors, principally the type I IGF receptor. The interaction of IGFs with cell-surface receptors, however, is tightly regulated by at least six distinct high affinity carrier proteins, the IGF- binding proteins (IGFBPs).

The Role of the Pituitary-Growth Hormone-IGF Axis in Glucose Homeostasis 131

**SHP2**

SH

**RAS**

**IRS-1**

**PI3K AKT**

**Metabolic actions**

**Raf**

**ERK**

**TF TF P <sup>P</sup> Accessory**

Fig. 2. The JAK-STAT Signalling System: The GH Receptor

**MEK**

**ERK**

**ERK**

**Extracellular**

**Cytoplasm**

**Nucleus**

**3. GH action** 

**3.2 IGF proteins** 

**3.1 IGF-I** 

R**AC**

**GRB2**

**TF Motif ISRE/GAS**

In 1957 Salmon and Daughaday (Salmon and Daughaday 1957) described a "sulfation factor" to explain the observation that while normal serum stimulated sulfate incorporation into cartilage tissue (a marker for synthesis of glycosaminoglycan, a component of cartilage), this effect was reduced using serum from growth hormone deficient patients, and could not be restored by treating cartilage directly with GH. Sulfation factor was re-named somatomedin, and became the basis of the classical somatomedin hypothesis (Rosenfeld 2005); namely, that most of the actions of growth hormone are carried out by factors originally named somatomedins. At the same time others were studying a compound in the serum which they called "non-suppressible insulin-like activity" (NSILA), whose insulinlike action persisted even after removing insulin by the addition of anti-insulin antibodies. Rinderknecht and Humbel (Rinderknecht and Humbel 1978; Rinderknecht and Humbel 1978) identified and sequenced two proteins, NSILA-I and NSILA-II, which were structurally similar to proinsulin. In the early 1980's it became apparent that NSILA-I and somatomedin C were identical, which led to the renaming of NSILA-I and NSILA-II to the insulin-like growth factors IGF-I and IGF-II. Of these two proteins IGF-I is the most growth hormone dependent. It is also now apparent that there are distinct cell membrane receptors for insulin, IGF-I and IGF-II. Even though each receptor binds most strongly to its own ligand, there is cross-reactivity among these ligands for all the receptors (see 3.3, below) .

There are three peptide hormones in the IGF family—insulin, IGF-I and IGF-II (Rosenfeld 2005). Insulin-like growth factors (IGF-I and IGF-II) are small peptide hormones (~7.5 kDa) which share a high degree of homology with proinsulin. As with insulin, the IGFs have A and

**JAK JAK P P P STAT STAT**

> **STAT STAT <sup>P</sup> <sup>P</sup>**

**STAT STATP P P P**

**STAT**

**PIAS**

**PTPases**

**SHP1**

**CIS**

**SOCS**

**CIS, SOCS, TFs, IGFBP-3, IGFs, etc** 

**Feedback inhibition**

IGF-I is the IGF most directly under GH control. It circulates in serum as part of a 140-kDa complex consisting of IGF-I, IGFBP3, and a third 85 KDa factor named acid-labile subunit (ALS) (Baxter 1994). It is probably binding of free (unbound) IGF-I to receptors on chondrocytes in the epiphyseal growth plates that stimulates linear growth. Although the primary site of synthesis of IGF binding proteins is the liver, it has been shown that most tissues produce IGFBPs locally. They may act as part of paracrine and autocrine functions of the IGFs. Functions that the IGFBPs may perform include; 1) increasing the half-life of IGF-I in serum; 2) decreasing binding of IGF-I with the insulin receptor reducing the risk of IGFinduced hypoglycemia; 3) being involved in the transport of IGF-I between the intravascular and the extravascular space ; 4) blocking the local effects of IGF-I; 5) enhancing IGF-I action by keeping the IGF-I in a slowly-releasing pool and 6) modulating cellular proliferation and apoptosis through interaction with receptors other than the IGF-I receptor. Disruption of the IGF:IGFBP:ALS complex is probably a prerequisite for IGFs to exert mitogenic and metabolic effects through the type I IGF receptor.

IGF-I, IGFBP3 and ALS all appear to be regulated by GH, since they are all low in GH deficiency and are restored with GH treatment (Jorgensen, Blum et al. 1991). About 80% of circulating IGF-I is produced the in the liver (IGF-I and ALS are produced by hepatocytes, while ALS is produced by Kupffer cells and sinusoidal endothelial cells), although locally produced IGF-I may be important for skeletal growth (Sjogren, Liu et al. 1999; Yakar, J. et al. 2002). It is not clear whether GH regulates all components of the 140-kDa complex directly, or whether one of the components may be regulated by GH, which, in turn, regulates synthesis of the others (Binoux 1997). Transcription of the rat ALS gene and ALS promoter activity has been shown to be stimulated by GH (Ooi, Cohen et al. 1997). In Growth Hormone Insensitivity Syndrome (GHIS) the patient is unresponsive to growth hormone; that is, the GHIS patient has high circulating levels of growth hormone, but low circulating levels of IGF-I and IGFBP3. In the case of a patient described with the IGF-I gene deletion (Woods, Camacho-Hubner et al. 1996), there are high circulating levels of growth hormone and low circulating levels of IGF-I, but normal circulating levels of IGFBP3.

#### **2.3 GH receptor/signaling**

The GH receptor is a member of the cytokine family of receptors. The gene for the human growth hormone receptor is located on chromosome 5p13.1-p12, and spans a region that is greater than 87 kb. The receptor consists of 620 amino acids (molecular weight 70 kD before glycosylation). It is highly homologous with the prolactin receptor, as well as receptors for interleukins 2,3,4,6, and 7, erythropoietin, granulocyte-macrophage colony-stimulating factor, and interferon. The GH receptor has extracellular, transmembrane, and intracellular domains, but it lacks intrinsic tyrosine kinase activity. Similar to other members of the cytokine family of receptors, it uses the JAK-STAT pathway for signal transduction (see Figure 2). Initially GH binds one GH receptor and then recruits a second GH receptor. This dimerization is followed by a conformational shift, which initiates the JAK-STAT cascade. Janus kinase (JAK2) is activated (it is a receptor-associated kinase which both autophosphorylates and phosphorylates the GH receptor). Once phosphorylated, these sites act as docking sites for molecules which undergo phosphorylation by JAK2, resulting in activation of STAT1, STAT3, and STAT5 (STAT stands for Signal Transducers and Activators of Transcription proteins). Once phosphorylated, cytoplasmic proteins form homodimers and heterodimers, travel to the nucleus, and bind specific DNA sequences, which activate gene transcription.

Fig. 2. The JAK-STAT Signalling System: The GH Receptor

### **3. GH action**

### **3.1 IGF-I**

130 Diabetes – Damages and Treatments

IGF-I is the IGF most directly under GH control. It circulates in serum as part of a 140-kDa complex consisting of IGF-I, IGFBP3, and a third 85 KDa factor named acid-labile subunit (ALS) (Baxter 1994). It is probably binding of free (unbound) IGF-I to receptors on chondrocytes in the epiphyseal growth plates that stimulates linear growth. Although the primary site of synthesis of IGF binding proteins is the liver, it has been shown that most tissues produce IGFBPs locally. They may act as part of paracrine and autocrine functions of the IGFs. Functions that the IGFBPs may perform include; 1) increasing the half-life of IGF-I in serum; 2) decreasing binding of IGF-I with the insulin receptor reducing the risk of IGFinduced hypoglycemia; 3) being involved in the transport of IGF-I between the intravascular and the extravascular space ; 4) blocking the local effects of IGF-I; 5) enhancing IGF-I action by keeping the IGF-I in a slowly-releasing pool and 6) modulating cellular proliferation and apoptosis through interaction with receptors other than the IGF-I receptor. Disruption of the IGF:IGFBP:ALS complex is probably a prerequisite for IGFs to exert mitogenic and

IGF-I, IGFBP3 and ALS all appear to be regulated by GH, since they are all low in GH deficiency and are restored with GH treatment (Jorgensen, Blum et al. 1991). About 80% of circulating IGF-I is produced the in the liver (IGF-I and ALS are produced by hepatocytes, while ALS is produced by Kupffer cells and sinusoidal endothelial cells), although locally produced IGF-I may be important for skeletal growth (Sjogren, Liu et al. 1999; Yakar, J. et al. 2002). It is not clear whether GH regulates all components of the 140-kDa complex directly, or whether one of the components may be regulated by GH, which, in turn, regulates synthesis of the others (Binoux 1997). Transcription of the rat ALS gene and ALS promoter activity has been shown to be stimulated by GH (Ooi, Cohen et al. 1997). In Growth Hormone Insensitivity Syndrome (GHIS) the patient is unresponsive to growth hormone; that is, the GHIS patient has high circulating levels of growth hormone, but low circulating levels of IGF-I and IGFBP3. In the case of a patient described with the IGF-I gene deletion (Woods, Camacho-Hubner et al. 1996), there are high circulating levels of growth hormone

The GH receptor is a member of the cytokine family of receptors. The gene for the human growth hormone receptor is located on chromosome 5p13.1-p12, and spans a region that is greater than 87 kb. The receptor consists of 620 amino acids (molecular weight 70 kD before glycosylation). It is highly homologous with the prolactin receptor, as well as receptors for interleukins 2,3,4,6, and 7, erythropoietin, granulocyte-macrophage colony-stimulating factor, and interferon. The GH receptor has extracellular, transmembrane, and intracellular domains, but it lacks intrinsic tyrosine kinase activity. Similar to other members of the cytokine family of receptors, it uses the JAK-STAT pathway for signal transduction (see Figure 2). Initially GH binds one GH receptor and then recruits a second GH receptor. This dimerization is followed by a conformational shift, which initiates the JAK-STAT cascade. Janus kinase (JAK2) is activated (it is a receptor-associated kinase which both autophosphorylates and phosphorylates the GH receptor). Once phosphorylated, these sites act as docking sites for molecules which undergo phosphorylation by JAK2, resulting in activation of STAT1, STAT3, and STAT5 (STAT stands for Signal Transducers and Activators of Transcription proteins). Once phosphorylated, cytoplasmic proteins form homodimers and heterodimers, travel to the nucleus, and bind specific DNA sequences,

and low circulating levels of IGF-I, but normal circulating levels of IGFBP3.

metabolic effects through the type I IGF receptor.

**2.3 GH receptor/signaling** 

which activate gene transcription.

In 1957 Salmon and Daughaday (Salmon and Daughaday 1957) described a "sulfation factor" to explain the observation that while normal serum stimulated sulfate incorporation into cartilage tissue (a marker for synthesis of glycosaminoglycan, a component of cartilage), this effect was reduced using serum from growth hormone deficient patients, and could not be restored by treating cartilage directly with GH. Sulfation factor was re-named somatomedin, and became the basis of the classical somatomedin hypothesis (Rosenfeld 2005); namely, that most of the actions of growth hormone are carried out by factors originally named somatomedins. At the same time others were studying a compound in the serum which they called "non-suppressible insulin-like activity" (NSILA), whose insulinlike action persisted even after removing insulin by the addition of anti-insulin antibodies. Rinderknecht and Humbel (Rinderknecht and Humbel 1978; Rinderknecht and Humbel 1978) identified and sequenced two proteins, NSILA-I and NSILA-II, which were structurally similar to proinsulin. In the early 1980's it became apparent that NSILA-I and somatomedin C were identical, which led to the renaming of NSILA-I and NSILA-II to the insulin-like growth factors IGF-I and IGF-II. Of these two proteins IGF-I is the most growth hormone dependent. It is also now apparent that there are distinct cell membrane receptors for insulin, IGF-I and IGF-II. Even though each receptor binds most strongly to its own ligand, there is cross-reactivity among these ligands for all the receptors (see 3.3, below) .

#### **3.2 IGF proteins**

There are three peptide hormones in the IGF family—insulin, IGF-I and IGF-II (Rosenfeld 2005). Insulin-like growth factors (IGF-I and IGF-II) are small peptide hormones (~7.5 kDa) which share a high degree of homology with proinsulin. As with insulin, the IGFs have A and

The Role of the Pituitary-Growth Hormone-IGF Axis in Glucose Homeostasis 133

were identified who presented with hypoglycemia. Of these 148 had hypopituitarism. There were 12 patients with isolated GH deficiency (GHD), and 9 were without hypothalamic or pituitary pathology. Structural central nervous system (CNS) lesions and/or midline facial defects were present in 39%. Of the males 55% had micropenis. Eighty-nine percent of the

A number of developmental occurrences may cause hypopituitarism. These include failure of LHX3 or LHX4. In mice the absence of LHX3 results in Rathke's pouch not growing or differentiating. Three patients were studied from a family with a mutation in LHX3. In humans LHX3 deletion has presented with severe growth failure. Other clinical features have included elevated and anteverted shoulders and a severe restriction of neck rotation, although vertebrae were not abnormal on MRI. The neck conformation appeared to be muscular in origin. One patient with an LHX3 deletion had severe pituitary hypoplasia, one had an enlarged anterior pituitary on MRI, and the third had a pituitary adenoma (Netchine, Sobrier et al. 2000). Individuals with LHX4 deficiency have been shown to be deficient in GH, TSH, and ACTH, and therefore, had short stature at the time of investigation. MRI findings include a small sella turcica with a hypoplastic anterior pituitary, a persistant craniopharyngeal canal, and a Chiari I malformation, as well as an ectopic posterior pituitary ("ectopic bright spot") on MRI. Deletions of LHX4 appear to be transmitted in an autosomal dominant fashion (Machinis, Pantel et al. 2001). Deficiencies in PROP1 ("Prophet of PIT1") may have a small pituitary on MRI, they can present with a large pituitary. They usually have deficiencies in GH, TSH, prolactin, FSH, and LH (Deladoëy, Flück et al. 1999). Mutations in the POU1F1 (formerly PIT1) gene result in deficiencies in GH, prolactin, and the β-subunit of TSH, but ACTH, FSH, and LH are not affected (Hendriks-Stegeman, Augustijn et al. 2001). At least 14 distinct mutations (some dominant and some recessive) in *POU1F1* have been described (Dattani and C. 2000; Hendriks-Stegeman, Augustijn et al. 2001; Malvagia, Poggi et al. 2003; Salemi, Besson et al. 2003) . Both dominant and recessive mutations have been reported. A mutation in the gene HESX1 is associated with hypopituitarism as part of a De Morsier syndrome, also known as Septo-optic Dysplasia (Dattani, Martinez-Barbera et al. 1999). Septo-optic Dysplasia includes optic nerve hypoplasia, midline anomolies (especially absence of the septum pellucidum and occasionally the corpus collosum), and varying degrees of hypopituitarism (usually of the anterior pituitary, but may

infants required multiple hormone replacement therapy (Bell, August et al. 2004).

also include the posterior pituitary) (Brickman, Clements et al. 2001).

with GHD with 100% sensitivity and 98% specificity.

Even children born with severe isolated GH deficiency also frequently have hypoglycemia, which usually resolves with GH therapy. A part of the work-up of hypoglycemia is to measure insulin, ketones, free fatty acids, GH, and cortisol levels on a specimen when the blood sugar is hypoglycemic (in children and adults, usually a specimen with a glucose < 50 mg/dl) (Berry, Nathan et al. 2009). A normal GH response should be a GH level >10 ng/ml, and a normal cortisol response should be >15 mg/dl. Elevated free fatty acids and ketones, along with a normal GH and cortisol response suggests hyperinsulinism, which can be further suspected if the insulin level is high. Either low GH or low cortisol alone suggests isolated GH deficiency or cortisol (or ACTH) deficiency. If GH and cortisol are both low, hypopituitarism is suspected. Binder et al.(G., Weidenkeller et al. 2010) have recently suggested that it is possible to diagnose severe congenital GH deficiency in neonates using a random blood sample in which the GH level is less than 7 mg/L. This test identified infants

B chains connected by disulfide bonds, and a C-peptide region that bears no homology with that of proinsulin. IGF-I and IGF-II have a carboxy-terminal extension of variable amino acid lengths. Almost ubiquitously produced, compared with insulin which circulates at picomolar concentrations and has a half-life of minutes, IGF-I and IGF-II circulate at much higher (i. e., nanomolar) concentrations in serum and have much longer half-lives, primarily because they are part of a complex with IGF binding proteins. Beyond their insulin-like effects, these growth-promoting peptides influence cellular proliferation and differentiation in numerous tissues. For IGFs to exert their effects at the cell surface, they first must bind specific, high affinity cell-surface receptors, principally the type I IGF receptor. The interaction of IGFs with cell-surface receptors, however, is tightly regulated by at least six distinct high affinity carrier proteins, the IGF- binding proteins (IGFBPs), and possibly by several low-affinity IGFBP-like molecules. IGFBPs 1-6, which are present in serum and many biologic fluids, have similar or higher affinities for IGF-I and IGF-II than does the type I IGF receptor. Therefore, the interaction of IGFs with IGFBPs can prevent untoward IGF effects, such as uncontrolled cellular proliferation or hypoglycemia. Conversely, disruption of the IGF;IGFBP complex is probably a prerequisite for IGFs to exert their mitogenic and metabolic effects through the type I IGF receptor. It is probably binding of unbound IGF-I to receptors on chondrocytes in the epiphyseal growth plate that stimulates linear growth.

#### **3.3 IGF receptors**

The IGF-I receptor is similar in molecular structure to the insulin receptor (Rosenfeld 2005); in fact, it has approximately 60% homology in amino acid composition. There are two membrane-spanning alpha subunits connected by disulfide bonds, which form a pocket that mediates binding of IGF-I. There are two intracellular beta subunits which contain a transmembrane domain, an ATP binding site, and a tyrosine kinase domain that accounts for the presumed signal transduction mechanism for the receptor. The type I IGF receptor binds IGF-I, IGF-II, and insulin with high affinity and mediates the actions of IGF on all tissue specific cell types. Likewise, the insulin receptor can also be bound by IGF-I and IGF-II, which means that if either of these growth factors is in abundance in the serum without being part of a larger complex, it can bind the insulin receptor and cause hypoglycemia. IGF-II also binds to a second receptor that has neither an intrinsic tyrosine kinase domain nor a known signal transduction mechanism. This receptor was first called the mannose-6 phosphate receptor that binds lysomal enzymes at binding sites distinct from that of IGF-II. Given that this receptor binds mannose-6-phosphate-containing enzymes and to transport them between intracellular compartments, it may serve as a biological sink which would remove IGF-II as well as enzymes such as cathepsin and urokinase from the cellular environment.

#### **4. Disorders of the pituitary-growth hormone-IGF axis**

#### **4.1 Congenital hypopituitarism**

Children who are born with lack of pituitary function are at risk for hypoglycemia because they lack ACTH (and, thus, do not produce adequate cortisol) and GH. Male infants with hypopituitarism often present in the newborn period with hypoglycemia and micropenis (because of the inability to secrete the gonadotropins LH and FSH; LH is necessary for testosterone production, which is required for penile growth). In an analysis of a large GH registry (all patients in the registry were being treated with growth hormone) 169 infants

B chains connected by disulfide bonds, and a C-peptide region that bears no homology with that of proinsulin. IGF-I and IGF-II have a carboxy-terminal extension of variable amino acid lengths. Almost ubiquitously produced, compared with insulin which circulates at picomolar concentrations and has a half-life of minutes, IGF-I and IGF-II circulate at much higher (i. e., nanomolar) concentrations in serum and have much longer half-lives, primarily because they are part of a complex with IGF binding proteins. Beyond their insulin-like effects, these growth-promoting peptides influence cellular proliferation and differentiation in numerous tissues. For IGFs to exert their effects at the cell surface, they first must bind specific, high affinity cell-surface receptors, principally the type I IGF receptor. The interaction of IGFs with cell-surface receptors, however, is tightly regulated by at least six distinct high affinity carrier proteins, the IGF- binding proteins (IGFBPs), and possibly by several low-affinity IGFBP-like molecules. IGFBPs 1-6, which are present in serum and many biologic fluids, have similar or higher affinities for IGF-I and IGF-II than does the type I IGF receptor. Therefore, the interaction of IGFs with IGFBPs can prevent untoward IGF effects, such as uncontrolled cellular proliferation or hypoglycemia. Conversely, disruption of the IGF;IGFBP complex is probably a prerequisite for IGFs to exert their mitogenic and metabolic effects through the type I IGF receptor. It is probably binding of unbound IGF-I to receptors on chondrocytes in the

The IGF-I receptor is similar in molecular structure to the insulin receptor (Rosenfeld 2005); in fact, it has approximately 60% homology in amino acid composition. There are two membrane-spanning alpha subunits connected by disulfide bonds, which form a pocket that mediates binding of IGF-I. There are two intracellular beta subunits which contain a transmembrane domain, an ATP binding site, and a tyrosine kinase domain that accounts for the presumed signal transduction mechanism for the receptor. The type I IGF receptor binds IGF-I, IGF-II, and insulin with high affinity and mediates the actions of IGF on all tissue specific cell types. Likewise, the insulin receptor can also be bound by IGF-I and IGF-II, which means that if either of these growth factors is in abundance in the serum without being part of a larger complex, it can bind the insulin receptor and cause hypoglycemia. IGF-II also binds to a second receptor that has neither an intrinsic tyrosine kinase domain nor a known signal transduction mechanism. This receptor was first called the mannose-6 phosphate receptor that binds lysomal enzymes at binding sites distinct from that of IGF-II. Given that this receptor binds mannose-6-phosphate-containing enzymes and to transport them between intracellular compartments, it may serve as a biological sink which would remove IGF-II as well as enzymes such as cathepsin and urokinase from the cellular

Children who are born with lack of pituitary function are at risk for hypoglycemia because they lack ACTH (and, thus, do not produce adequate cortisol) and GH. Male infants with hypopituitarism often present in the newborn period with hypoglycemia and micropenis (because of the inability to secrete the gonadotropins LH and FSH; LH is necessary for testosterone production, which is required for penile growth). In an analysis of a large GH registry (all patients in the registry were being treated with growth hormone) 169 infants

epiphyseal growth plate that stimulates linear growth.

**4. Disorders of the pituitary-growth hormone-IGF axis** 

**3.3 IGF receptors** 

environment.

**4.1 Congenital hypopituitarism** 

were identified who presented with hypoglycemia. Of these 148 had hypopituitarism. There were 12 patients with isolated GH deficiency (GHD), and 9 were without hypothalamic or pituitary pathology. Structural central nervous system (CNS) lesions and/or midline facial defects were present in 39%. Of the males 55% had micropenis. Eighty-nine percent of the infants required multiple hormone replacement therapy (Bell, August et al. 2004).

A number of developmental occurrences may cause hypopituitarism. These include failure of LHX3 or LHX4. In mice the absence of LHX3 results in Rathke's pouch not growing or differentiating. Three patients were studied from a family with a mutation in LHX3. In humans LHX3 deletion has presented with severe growth failure. Other clinical features have included elevated and anteverted shoulders and a severe restriction of neck rotation, although vertebrae were not abnormal on MRI. The neck conformation appeared to be muscular in origin. One patient with an LHX3 deletion had severe pituitary hypoplasia, one had an enlarged anterior pituitary on MRI, and the third had a pituitary adenoma (Netchine, Sobrier et al. 2000). Individuals with LHX4 deficiency have been shown to be deficient in GH, TSH, and ACTH, and therefore, had short stature at the time of investigation. MRI findings include a small sella turcica with a hypoplastic anterior pituitary, a persistant craniopharyngeal canal, and a Chiari I malformation, as well as an ectopic posterior pituitary ("ectopic bright spot") on MRI. Deletions of LHX4 appear to be transmitted in an autosomal dominant fashion (Machinis, Pantel et al. 2001). Deficiencies in PROP1 ("Prophet of PIT1") may have a small pituitary on MRI, they can present with a large pituitary. They usually have deficiencies in GH, TSH, prolactin, FSH, and LH (Deladoëy, Flück et al. 1999). Mutations in the POU1F1 (formerly PIT1) gene result in deficiencies in GH, prolactin, and the β-subunit of TSH, but ACTH, FSH, and LH are not affected (Hendriks-Stegeman, Augustijn et al. 2001). At least 14 distinct mutations (some dominant and some recessive) in *POU1F1* have been described (Dattani and C. 2000; Hendriks-Stegeman, Augustijn et al. 2001; Malvagia, Poggi et al. 2003; Salemi, Besson et al. 2003) . Both dominant and recessive mutations have been reported. A mutation in the gene HESX1 is associated with hypopituitarism as part of a De Morsier syndrome, also known as Septo-optic Dysplasia (Dattani, Martinez-Barbera et al. 1999). Septo-optic Dysplasia includes optic nerve hypoplasia, midline anomolies (especially absence of the septum pellucidum and occasionally the corpus collosum), and varying degrees of hypopituitarism (usually of the anterior pituitary, but may also include the posterior pituitary) (Brickman, Clements et al. 2001).

Even children born with severe isolated GH deficiency also frequently have hypoglycemia, which usually resolves with GH therapy. A part of the work-up of hypoglycemia is to measure insulin, ketones, free fatty acids, GH, and cortisol levels on a specimen when the blood sugar is hypoglycemic (in children and adults, usually a specimen with a glucose < 50 mg/dl) (Berry, Nathan et al. 2009). A normal GH response should be a GH level >10 ng/ml, and a normal cortisol response should be >15 mg/dl. Elevated free fatty acids and ketones, along with a normal GH and cortisol response suggests hyperinsulinism, which can be further suspected if the insulin level is high. Either low GH or low cortisol alone suggests isolated GH deficiency or cortisol (or ACTH) deficiency. If GH and cortisol are both low, hypopituitarism is suspected. Binder et al.(G., Weidenkeller et al. 2010) have recently suggested that it is possible to diagnose severe congenital GH deficiency in neonates using a random blood sample in which the GH level is less than 7 mg/L. This test identified infants with GHD with 100% sensitivity and 98% specificity.

The Role of the Pituitary-Growth Hormone-IGF Axis in Glucose Homeostasis 135

treating growth hormone deficiency has expanded and at the same time the number of approved indications for growth hormone therapy has also increased. When GH therapy is used in a patient with GH deficiency who has hypoglycemia, the hypoglycemia usually resolves as soon as treatment is started. The etiology of this effect is somewhat complicated; GH administration reduces insulin sensitivity, which corrects the hypoglycemia. GH also increases insulin secretion. With GH excess these effects can lead to carbohydrate intolerance, and with GH deficiency they may result in hypoglycemia(Allen, Johanson et al.

For populations where treatment with growth hormone is not possible, such as in the case of GH Insenstivity Syndrome, STAT5b deficiency, or IGF-I Deficiency (Woods, Camacho-Hübner et al. 2000), treatment with IGF-I is now possible. Patents with GHIS or (Laron Syndrome) also frequently report problems with hypoglycemia. Because GH cannot activate the receptor, these individuals have very low concentrations of GH, GHBP-3, and ALS. It is not clear why this situation leads to hypoglycemia. There have been two compounds which have IGF-I as the major component. One, mecasermin, which is rhIGF-I alone (IncrelexTM, Tercica, Inc., Brisbane, CA), received approval from the FDA for treatment of severe growth hormone resistance in August, 2005 and approval from the European Agency for the Evaluation of Medical Products (EMEA) in 2007 (Collett-Solberg and Misra 2008). The second compound is mecasermin rinfabate (iPlexTM, Insmed, Richmond, VA). It is a complex of equimolar amounts of rhIGF-I and its most abundant binding protein Insulin-like Growth Factor I binding protein 3 (rhIGFBP-3). The combination of IGF-I and IGFBP-3 was postulated to have the advantage and an increased serum half-life and protection against hypoglycemia, although there was never a head-to-head comparison of the two preparations which compared the propensity for hypoglycemia. Mecaserin rinfabate received approval from the FDA in December 2005, but is no longer available for the

treatment of short stature due to a legal agreement (Collett-Solberg and Misra 2008).

an intercurrent illness resulting in loss of appetite.

**6. Summary** 

Since hypoglycemia risk appears to be dose dependent, hypoglycemia risk has been reduced by dividing the IGF-I dose into two daily injections, twelve hours apart, and it is recommended to give the injection of IGF-I along with a meal. In a recent report, hypoglycemia was reported by 49% of subjects treated with recombinant IGF-I (Chernausek, Backeljauw et al. 2007). Most hypoglycemic events occurred during the first month of treatment. Seven of the events were reported as severe, and four resulted in seizures. Of the subjects reporting hypoglycemia, 32% had a history of hypoglycemia before starting treatment with IGF-I. It seemed to occur in younger, shorter subjects who had already had problems with hypoglycemia. This observation seems to be consistent with an earlier report, in which hypoglycemia occurred in some of the patients receiving IGF-I, but at the same rate as in those receiving placebo injections (Guevara-Aguirre, Vasconez et al. 1995), only rarely resulting in seizures (Backeljauw, Underwood et al. 2001). Hypoglycemia was lessened by giving the IGF-I dose with meals, and hypoglycemia was usually a problem when there was

The GH-IGF axis plays an import role in glucose homeostasis, which is somewhat complicated. Growth hormone decreases insulin sensitivity and stimulates insulin secretion.

1996).

**5.2 Administration of IGF-I** 

### **4.2 Idiopathic isolated GH deficiency**

Idiopathic isolated GH deficiency is the most common form of GH deficiency, accounting for as many as 44% of patients treated with growth hormone (Root, Kemp et al. 1998). Because GH is secreted episodically and GH is present in the serum for only about 20 minutes after it is secreted, random assessment of serum GH levels is rarely helpful. In addition to measuring serum IGF-I and IGFBP3 levels, provocative testing of the GH axis is a means of evaluating a patient suspected of having GH deficiency. Provocative agents which stimulate GH secretion include dopaminergic agents (L-dOPA and clonidine), glucagon, the amino acid arginine, and insulin-induced hypoglycemia. When the pituitary gland was visualized using MRI, as many as 15% of patients diagnosed as having idiopathic isolated GH deficiency had abnormal findings (Frindik 2001). These abnormalities included small pituitary glands, empty sella tursicas and ectopic posterior pituitaries (i.e., the posterior pituitary had never descended into the sella tursica, but remained near its origin in the brain).

### **4.3 Growth hormone insensitivity syndrome**

Growth hormone insensitivity syndrome (GHIS) is a rare autosomal recessive condition characterized by a failure to synthesize insulin-like growth factor-I (IGF-I) in spite of elevated levels of growth hormone. It was first described in 1966 (Laron, Pertzelan et al. 1966), and there have been a variety of different mutations described which account for this condition (Rosenfeld, Rosenbloom et al. 1994), most involving mutations in the growth hormone receptor. GHIS is characterized by growth failure starting in infancy which is unresponsive to GH administration, associated with elevated levels of GH and decreased levels of IGF-I and IGFBP3 (Laron, Lillos et al. 1993; Rosenbloom 2000; Savage, Burren et al. 2001). There has been a patient described who had a normal GH receptor, but a mutation in STAT5 (Kofoed, Hwa et al. 2003). A patient has also been described with a similar presentation who has a deletion of the gene for IGF-I (Woods, Camacho-Hubner et al. 1996). Patients with GHIS have frequently reported hypoglycemia without being treated with IGF-I. Because there is a GH receptor which is not fully functional, it may be that the hypoglycemia in this condition results from an inability of GH to function as a glucose counterregulatory hormone, independent of its function in stimulating production of IGF-I.

## **5. Treatment of disorders of the pituitary-growth hormone-IGF axis**

### **5.1 Treatment of GH deficiency**

Therapy for GH deficiency dates to 1958 when Rabin (Raben 1958) reported using growth hormone isolated from human pituitary glands to treat a patient who was growth hormone deficient. Between 1960 and 1985 human-derived growth hormone was available to treat this population. Because of limited supply, treatment was limited to the most GH deficient patients; in fact, as the supply became more plentiful the criteria for growth hormone deficiency gradually rose from peak GH responses to provocative stimuli of 5 ng/ml to allow treatment of patients who had peak GH responses to provocative stimuli of 10 ng/ml in the early 1980's. In 1985 the distribution of human-derived growth hormone was abruptly stopped with the discovery of Creutzfeld-Jacob disease in recipients of these preparations (Brown 1988; Hintz 1995), with the exception of GH deficient patients who experienced hypoglycemia in the absence of GH therapy. At about the same time a recombinant source of growth hormone was approved for use by the FDA, which allowed an almost limitless supply, albeit at a rather expensive cost. Since that time the use of growth hormone in treating growth hormone deficiency has expanded and at the same time the number of approved indications for growth hormone therapy has also increased. When GH therapy is used in a patient with GH deficiency who has hypoglycemia, the hypoglycemia usually resolves as soon as treatment is started. The etiology of this effect is somewhat complicated; GH administration reduces insulin sensitivity, which corrects the hypoglycemia. GH also increases insulin secretion. With GH excess these effects can lead to carbohydrate intolerance, and with GH deficiency they may result in hypoglycemia(Allen, Johanson et al. 1996).

### **5.2 Administration of IGF-I**

134 Diabetes – Damages and Treatments

Idiopathic isolated GH deficiency is the most common form of GH deficiency, accounting for as many as 44% of patients treated with growth hormone (Root, Kemp et al. 1998). Because GH is secreted episodically and GH is present in the serum for only about 20 minutes after it is secreted, random assessment of serum GH levels is rarely helpful. In addition to measuring serum IGF-I and IGFBP3 levels, provocative testing of the GH axis is a means of evaluating a patient suspected of having GH deficiency. Provocative agents which stimulate GH secretion include dopaminergic agents (L-dOPA and clonidine), glucagon, the amino acid arginine, and insulin-induced hypoglycemia. When the pituitary gland was visualized using MRI, as many as 15% of patients diagnosed as having idiopathic isolated GH deficiency had abnormal findings (Frindik 2001). These abnormalities included small pituitary glands, empty sella tursicas and ectopic posterior pituitaries (i.e., the posterior pituitary had never descended into

Growth hormone insensitivity syndrome (GHIS) is a rare autosomal recessive condition characterized by a failure to synthesize insulin-like growth factor-I (IGF-I) in spite of elevated levels of growth hormone. It was first described in 1966 (Laron, Pertzelan et al. 1966), and there have been a variety of different mutations described which account for this condition (Rosenfeld, Rosenbloom et al. 1994), most involving mutations in the growth hormone receptor. GHIS is characterized by growth failure starting in infancy which is unresponsive to GH administration, associated with elevated levels of GH and decreased levels of IGF-I and IGFBP3 (Laron, Lillos et al. 1993; Rosenbloom 2000; Savage, Burren et al. 2001). There has been a patient described who had a normal GH receptor, but a mutation in STAT5 (Kofoed, Hwa et al. 2003). A patient has also been described with a similar presentation who has a deletion of the gene for IGF-I (Woods, Camacho-Hubner et al. 1996). Patients with GHIS have frequently reported hypoglycemia without being treated with IGF-I. Because there is a GH receptor which is not fully functional, it may be that the hypoglycemia in this condition results from an inability of GH to function as a glucose counterregulatory hormone, independent of its function in stimulating production of IGF-I.

**5. Treatment of disorders of the pituitary-growth hormone-IGF axis** 

Therapy for GH deficiency dates to 1958 when Rabin (Raben 1958) reported using growth hormone isolated from human pituitary glands to treat a patient who was growth hormone deficient. Between 1960 and 1985 human-derived growth hormone was available to treat this population. Because of limited supply, treatment was limited to the most GH deficient patients; in fact, as the supply became more plentiful the criteria for growth hormone deficiency gradually rose from peak GH responses to provocative stimuli of 5 ng/ml to allow treatment of patients who had peak GH responses to provocative stimuli of 10 ng/ml in the early 1980's. In 1985 the distribution of human-derived growth hormone was abruptly stopped with the discovery of Creutzfeld-Jacob disease in recipients of these preparations (Brown 1988; Hintz 1995), with the exception of GH deficient patients who experienced hypoglycemia in the absence of GH therapy. At about the same time a recombinant source of growth hormone was approved for use by the FDA, which allowed an almost limitless supply, albeit at a rather expensive cost. Since that time the use of growth hormone in

**4.2 Idiopathic isolated GH deficiency** 

the sella tursica, but remained near its origin in the brain).

**4.3 Growth hormone insensitivity syndrome** 

**5.1 Treatment of GH deficiency** 

For populations where treatment with growth hormone is not possible, such as in the case of GH Insenstivity Syndrome, STAT5b deficiency, or IGF-I Deficiency (Woods, Camacho-Hübner et al. 2000), treatment with IGF-I is now possible. Patents with GHIS or (Laron Syndrome) also frequently report problems with hypoglycemia. Because GH cannot activate the receptor, these individuals have very low concentrations of GH, GHBP-3, and ALS. It is not clear why this situation leads to hypoglycemia. There have been two compounds which have IGF-I as the major component. One, mecasermin, which is rhIGF-I alone (IncrelexTM, Tercica, Inc., Brisbane, CA), received approval from the FDA for treatment of severe growth hormone resistance in August, 2005 and approval from the European Agency for the Evaluation of Medical Products (EMEA) in 2007 (Collett-Solberg and Misra 2008). The second compound is mecasermin rinfabate (iPlexTM, Insmed, Richmond, VA). It is a complex of equimolar amounts of rhIGF-I and its most abundant binding protein Insulin-like Growth Factor I binding protein 3 (rhIGFBP-3). The combination of IGF-I and IGFBP-3 was postulated to have the advantage and an increased serum half-life and protection against hypoglycemia, although there was never a head-to-head comparison of the two preparations which compared the propensity for hypoglycemia. Mecaserin rinfabate received approval from the FDA in December 2005, but is no longer available for the treatment of short stature due to a legal agreement (Collett-Solberg and Misra 2008).

Since hypoglycemia risk appears to be dose dependent, hypoglycemia risk has been reduced by dividing the IGF-I dose into two daily injections, twelve hours apart, and it is recommended to give the injection of IGF-I along with a meal. In a recent report, hypoglycemia was reported by 49% of subjects treated with recombinant IGF-I (Chernausek, Backeljauw et al. 2007). Most hypoglycemic events occurred during the first month of treatment. Seven of the events were reported as severe, and four resulted in seizures. Of the subjects reporting hypoglycemia, 32% had a history of hypoglycemia before starting treatment with IGF-I. It seemed to occur in younger, shorter subjects who had already had problems with hypoglycemia. This observation seems to be consistent with an earlier report, in which hypoglycemia occurred in some of the patients receiving IGF-I, but at the same rate as in those receiving placebo injections (Guevara-Aguirre, Vasconez et al. 1995), only rarely resulting in seizures (Backeljauw, Underwood et al. 2001). Hypoglycemia was lessened by giving the IGF-I dose with meals, and hypoglycemia was usually a problem when there was an intercurrent illness resulting in loss of appetite.

### **6. Summary**

The GH-IGF axis plays an import role in glucose homeostasis, which is somewhat complicated. Growth hormone decreases insulin sensitivity and stimulates insulin secretion.

The Role of the Pituitary-Growth Hormone-IGF Axis in Glucose Homeostasis 137

Deladoëy J, Flück C, Büyükgebiz A et al. (1999). Hot spot" in the PROP1 gene responsible for

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Hintz RL. (1995). The prismatic case of Creutzfeldt-Jacob disease associated with pituitary

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Most of the actions of growth hormone take place through Insulin-like Growth Factor-I. IGF-I can bind with the insulin receptor, but under normal circumstances is protected from direct contact insulin receptors, since this 6000 Kda protein circulates bound to Binding Protein 3 and acid labile subunit in a 140,000 Kda complex. With GH deficiency many patients experience hypoglycemia, which is corrected with GH treatment. Growth hormone resistant states also may present with hypoglycemia, but are not sensitive to growth hormone. Treatment of GH resistant states with IGF-I does cause an increase in the linear growth velocity, but IGF-I treatment carries a risk for hypoglycemia, presumably because its similarity to proinsulin allows it to bind the insulin receptor, and it is not protected by its binding protein or ALS.

### **7. References**


Most of the actions of growth hormone take place through Insulin-like Growth Factor-I. IGF-I can bind with the insulin receptor, but under normal circumstances is protected from direct contact insulin receptors, since this 6000 Kda protein circulates bound to Binding Protein 3 and acid labile subunit in a 140,000 Kda complex. With GH deficiency many patients experience hypoglycemia, which is corrected with GH treatment. Growth hormone resistant states also may present with hypoglycemia, but are not sensitive to growth hormone. Treatment of GH resistant states with IGF-I does cause an increase in the linear growth velocity, but IGF-I treatment carries a risk for hypoglycemia, presumably because its similarity to proinsulin allows it to bind the insulin receptor, and it is not protected by its

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**7. References** 


**8** 

*1U.S.A. 2Japan* 

**Molecular Mechanism Underlying the** 

*Department of Medicine, Harvard Medical School, Boston, MA,* 

Dan Kawamori1,2 and Rohit N. Kulkarni1

*Graduate School of Medicine, Osaka,* 

**Intra-Islet Regulation of Glucagon Secretion** 

*1Section of Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center and* 

*2Medical Education Center and Department of Metabolic Medicine, Osaka University* 

Glucagon secreted from pancreatic α-cells plays central roles for counteracting hypoglycemia by modulating hepatic glucose metabolism (Gromada et al., 2007). In addition, glucagon also contributes to the maintenance of glucose homeostasis together with insulin from β-cells. During hyperglycemia such as post-prandial state, insulin secretion from β-cells is stimulated while glucagon secretion from α-cells is suppressed, leading to a lowering of blood glucose levels due to enhanced hepatic- and adipo- glucose uptake and suppressed hepatic glucose output. In contrast, in hypoglycemia such as starvation, glucagon secretion is promoted while insulin secretion is reduced, causing elevated blood glucose levels via the effects of glucagon, including enhanced hepatic glucose output and breakdown of lipids and proteins to provide glucose that is critical to the central nervous system. Thus, both glucagon and insulin are pivotal in systemic energy homeostasis, and the balance between these two hormones determines the metabolic state of various organs in

In both type 1 and type 2 diabetes, both of which exhibit a global increase in incidence, an imbalance between the two hormones appears to significantly impact glucose homeostasis (Unger, 1978). Insufficient insulin secretion and systemic insulin resistance both contribute to hyperglycemia due to quantitative and qualitative insulin shortage. In addition, abnormal elevations in circulating glucagon, due to lack of normal suppression mechanisms, worsens the hyperglycemia via enhanced hepatic glucose output. On the other hand, in patients undergoing treatment for diabetes, an increased incidence of hypoglycemia likely occurs due to a poor glucagon response. Whether this poor glucagon response is a consequence of impaired effects of insulin due to repeated treatment with exogenous insulin or other factors is not fully understood (Gerich et al., 1973). Therefore, diabetes can be recognized as "state where adequate hormones cannot work appropriately" when intra-islet hormone balance is focused on. These observations have prompted consideration of glucagon in the overall therapeutic approach to treat patients with both type 1 and type 2 diabetes. Furthermore, novel therapeutic approaches targeting Glucagon-like peptide (GLP)-1 action in α-cells

**1. Introduction** 

response to changes in energy status.


## **Molecular Mechanism Underlying the Intra-Islet Regulation of Glucagon Secretion**

Dan Kawamori1,2 and Rohit N. Kulkarni1

*1Section of Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, MA, 2Medical Education Center and Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, 1U.S.A. 2Japan* 

### **1. Introduction**

138 Diabetes – Damages and Treatments

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Rosenbloom AL. (2000). IGF-I treatment of growth hormone insensitivity. In: *IGF in Health and* 

Rosenfeld RG (2005). The IGF system: new developments relevant to pediatric practice.

Rosenfeld RG, Rosenbloom RL, Guevara-Aguirre J. (1994). Growth hormone (GH) insensitivity due to pituitary GH receptor deficiency. *Endocr Rev,* 15, 3, (June, 1994), pp. 369-390 Salemi S, Besson A, Eblé A et al. (2003). New N-terminal located mutation (Q4ter) within the

variable phenotype. *Growth Horm IGF Res,* 13, 5, (October, 2003), pp. 264-8 Salmon WD, Daughaday WH. (1957). A hormonally controlled serum factor which

Savage MO, Burren CP, Blair JC et al. (2001). Growth hormone insensitivity:

Sjogren K, Liu JL, Blad K et al. (1999). Liver-derived insulin-like growth factor-I (IGF-I) is the

Woods KA, Camacho-Hübner C, Bergman RN et al. (2000). Effects of insulin-like growth

Woods KA, Camacho-Hubner C, Savage MO et al. (1996). Intrauterine growth retardation

Yakar S, Rosen CJ, Beamer WG et al. (2002). Circulating levels of IGF-I directly regulate bone growth and density. *J Clin Invest,* 110, 6, (September, 2002), pp. 771-781

*Proc Natl Acad Sci, USA*, 96, 12, (June, 1999), pp. 7088-7092

deletion, *J Clin Endocrinol Metab*, 85, 4. (April, 2000), pp. 1407-11

factor I gene. *N. Engl. J. Med.,* 335, 18 (October, 1996), pp. 1363-1367

*Endocrinol* 11, 7. (June, 1997), pp. 997-1007

II. *FEBS Lett*, 89, 2, (May, 1978), pp. 283-286

*Endocr Dev, 9,1*, (January, 2005), pp. 1-10

1978), pp. 2769-2776

1957), pp. 825-836

*Research* , 55, Suppl 2, pp. 32-5

*Endocrinol Metab,* 18, 8, (August, 1958), pp. 901-903

*J Pediatr Endocrinol Metab*, 11, 3, (March, 1998), pp. 403-412

gene encoding the acid-labile subunit (ALS) of the circulating insulin-like growth factor-binding protein complex and ALS promoter activity in rat liver. *Mol* 

factor I and its strucutral homology with proinsulin. *J. Biol. Chem,* 253, 8, (April,

hormone therapy in children National Cooperative Growth Study, USA, 1985-1994.

*Disease*, R. G. Rosenfeld and C. T. Roberts. pp. 739-770, Humana Press, Inc., Totowa, NJ

POU1F1-gene (PIT-1) causes recessive combined pituitary hormone deficiency and

stimulates sulfate incorporation by cartilage in vitro. *J. Lab. Clin. Med,* 49, 6, (June,

Pathophysiology, diagnosis, clincal variation and future perspective. *Hormone* 

principle source of IGF-I in blood but is not required for postnatal growth in mice.

factor I (IGF-I) therapy on body composition and insulin resistance in IGF-I gene

and postnatal growth failure associated with deletion of the insulin-like growth

Glucagon secreted from pancreatic α-cells plays central roles for counteracting hypoglycemia by modulating hepatic glucose metabolism (Gromada et al., 2007). In addition, glucagon also contributes to the maintenance of glucose homeostasis together with insulin from β-cells. During hyperglycemia such as post-prandial state, insulin secretion from β-cells is stimulated while glucagon secretion from α-cells is suppressed, leading to a lowering of blood glucose levels due to enhanced hepatic- and adipo- glucose uptake and suppressed hepatic glucose output. In contrast, in hypoglycemia such as starvation, glucagon secretion is promoted while insulin secretion is reduced, causing elevated blood glucose levels via the effects of glucagon, including enhanced hepatic glucose output and breakdown of lipids and proteins to provide glucose that is critical to the central nervous system. Thus, both glucagon and insulin are pivotal in systemic energy homeostasis, and the balance between these two hormones determines the metabolic state of various organs in response to changes in energy status.

In both type 1 and type 2 diabetes, both of which exhibit a global increase in incidence, an imbalance between the two hormones appears to significantly impact glucose homeostasis (Unger, 1978). Insufficient insulin secretion and systemic insulin resistance both contribute to hyperglycemia due to quantitative and qualitative insulin shortage. In addition, abnormal elevations in circulating glucagon, due to lack of normal suppression mechanisms, worsens the hyperglycemia via enhanced hepatic glucose output. On the other hand, in patients undergoing treatment for diabetes, an increased incidence of hypoglycemia likely occurs due to a poor glucagon response. Whether this poor glucagon response is a consequence of impaired effects of insulin due to repeated treatment with exogenous insulin or other factors is not fully understood (Gerich et al., 1973). Therefore, diabetes can be recognized as "state where adequate hormones cannot work appropriately" when intra-islet hormone balance is focused on. These observations have prompted consideration of glucagon in the overall therapeutic approach to treat patients with both type 1 and type 2 diabetes. Furthermore, novel therapeutic approaches targeting Glucagon-like peptide (GLP)-1 action in α-cells

Molecular Mechanism Underlying the Intra-Islet Regulation of Glucagon Secretion 141

targets leading to the suppression of glycolysis and glycogenesis, and the enhancement of gluconeogenesis and glycogenolysis (Jiang and Zhang, 2003). In islet cells, the elevation of cAMP by glucagon has been reported to stimulate insulin and glucagon secretion from βand α-cells respectively (Huypens et al., 2000; Ma et al., 2005) by PKA dependent and independent mechanisms. Upregulation of cAMP activates cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs / Epac), which modulates intracellular Ca2+-ion

Pancreatic islets possess unique anatomical characteristics and are composed of five different endocrine cell types distributed as islands randomly within the exocrine pancreas. Among these five endocrine cells in islets, the α-cells account for approximately 20% of islet

In adult rodents, β-cells are restricted mostly to the islet core, while α-cells, somatostatinsecreting δ-cells, pancreatic polypeptide-secreting PP-cells, and ghrelin-secreting ε-cells, are scattered along the periphery of the islet and surrounding β-cells. It is likely that this distribution and arrangement of different islet cell types is teleologically important for physiological regulation between the cells since the blood flows from the center of the islets toward periphery; i.e.β-cells to non-β-cells in the islet microcirculation system (Bonner-Weir and Orci, 1982; Stagner and Samols, 1986), suggesting that secreted insulin regulates hormone secretion from other islet cell types. This architecture is typically preserved in rodent islets, while in humans, non-β-cells are often observed both at the periphery and also seemingly in clusters within the center of islets (Cabrera et al., 2006). This implies several possibilities; 1) rodent cellular hierarchy in the islets does not apply to human islets, or 2) human islets consist of several clover-leaf like 'rosettes', with each rosette resembling the basic islet architecture observed in rodent islets (Bonner-Weir and O'Brien, 2008) suggesting

**islets** 

**-cells intra-islet**

**-cells -cells -cells PP-cells** 

Schematic image for the structure of mouse and human islets adapted from the recent

publication of (Bosco, 2010) (10).

mobilization, enhancing exocytosis (Holz et al., 2006; Ma et al., 2005).

**2.3 Anatomical characteristics of pancreatic islets and α-cells** 

**mouse islets human**

**vessels** 

cells.

(GLP-1 analogues and DPP-4 inhibitors) are also being considered given the potential for GLP-1 to have direct suppressive effects on α-cells, thus these enabled comprehensive control of islet hormone balance including improvement of both insulin and glucagon secretion.

Therefore, it becomes more important to understand the underlying molecular mechanisms for the regulation of glucagon secretion to apply new therapeutic approaches to diabetes targeting α-cell dysfunction.

## **2. Functions of glucagon**

### **2.1 Functions of glucagon**

Glucagon is a 29 amino acid peptide hormone, secreted by pancreatic α-cells mainly in hypoglycemic state, and exerts multiple biological effects on a wide range of organs (Kawamori et al., 2010). Glucagon has important functions *in vivo* for sustaining appropriate blood glucose level. In physiological states, glucagon is released into the bloodstream in response to hypoglycemia to oppose the action of insulin in peripheral tissues, and works as a counter-regulatory hormone to restore normoglycemia. Secreted glucagon works predominantly on the liver, and promotes hepatic gluconeogenesis, glycogenolysis, and simultaneously inhibits glycolysis and glycogenesis (Exton et al., 1966; Unger and Orci, 1977), thus contributing to restoring glucose homeostasis by counteracting the action of insulin. In contrast, insulin suppresses hepatic glucose output while enhancing hepatic glucose uptake and glycogenesis, indicating that a balance between these two hormones ath the hepatocyte determines hepatic glucose metabolism, thus systemic glycemic homeostasis. In addition to countering hypoglycemia and opposing the effects of insulin in the liver, glucagon has impacts the function of several metabolic organs together favoring the maintenance of glucose homeostasis. For example, in the adipose tissue, glucagon enhances lipid decomposition, while, in contrast, the lack of detectable glucagon receptors in skeletal muscle indicates glucagon has little effect in regulating systemic glucose metabolism by acting on skeletal muscle (Christophe, 1996). Glucagon can also stimulate insulin secretion from pancreatic β-cells (Scheen et al., 1996) and indirectly impact hepatic glucose output. Taken together, these actions indicate an important role for glucagon in maintaining glucose homeostasis.

### **2.2 Molecular mechanism underlying glucagon action**

The glucagon receptor is a G-protein (Gs/Gq) coupled type receptor (Jelinek et al., 1993) and is widely expressed in insulin target organs, such as liver, adipose, β-cells and brain, with the exception of skeletal muscle (Burcelin et al., 1995). Following binding and conformational changes of the receptor the activation of Gs leads to recruitment of adenylate cyclase to the cellular membrane, causing an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and subsequent activation of protein kinase A (PKA) (Weinstein et al., 2001). On the other hand, activation of Gq induces activation of phospholipase C, upregulation of inositol 1,4,5-triphosphate, and the subsequent release of intracellular calcium (Ca2+) (Wakelam et al., 1986). The action of glucagon is relatively complex and involves the coordinate regulation of transcription factors and signal transduction networks which converge to regulate amino acid, lipid and carbohydrate metabolism. For example, in the liver, elevated PKA activity activates various downstream

(GLP-1 analogues and DPP-4 inhibitors) are also being considered given the potential for GLP-1 to have direct suppressive effects on α-cells, thus these enabled comprehensive control of islet hormone balance including improvement of both insulin and glucagon

Therefore, it becomes more important to understand the underlying molecular mechanisms for the regulation of glucagon secretion to apply new therapeutic approaches to diabetes

Glucagon is a 29 amino acid peptide hormone, secreted by pancreatic α-cells mainly in hypoglycemic state, and exerts multiple biological effects on a wide range of organs (Kawamori et al., 2010). Glucagon has important functions *in vivo* for sustaining appropriate blood glucose level. In physiological states, glucagon is released into the bloodstream in response to hypoglycemia to oppose the action of insulin in peripheral tissues, and works as a counter-regulatory hormone to restore normoglycemia. Secreted glucagon works predominantly on the liver, and promotes hepatic gluconeogenesis, glycogenolysis, and simultaneously inhibits glycolysis and glycogenesis (Exton et al., 1966; Unger and Orci, 1977), thus contributing to restoring glucose homeostasis by counteracting the action of insulin. In contrast, insulin suppresses hepatic glucose output while enhancing hepatic glucose uptake and glycogenesis, indicating that a balance between these two hormones ath the hepatocyte determines hepatic glucose metabolism, thus systemic glycemic homeostasis. In addition to countering hypoglycemia and opposing the effects of insulin in the liver, glucagon has impacts the function of several metabolic organs together favoring the maintenance of glucose homeostasis. For example, in the adipose tissue, glucagon enhances lipid decomposition, while, in contrast, the lack of detectable glucagon receptors in skeletal muscle indicates glucagon has little effect in regulating systemic glucose metabolism by acting on skeletal muscle (Christophe, 1996). Glucagon can also stimulate insulin secretion from pancreatic β-cells (Scheen et al., 1996) and indirectly impact hepatic glucose output. Taken together, these actions indicate an important role for glucagon in maintaining glucose

The glucagon receptor is a G-protein (Gs/Gq) coupled type receptor (Jelinek et al., 1993) and is widely expressed in insulin target organs, such as liver, adipose, β-cells and brain, with the exception of skeletal muscle (Burcelin et al., 1995). Following binding and conformational changes of the receptor the activation of Gs leads to recruitment of adenylate cyclase to the cellular membrane, causing an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and subsequent activation of protein kinase A (PKA) (Weinstein et al., 2001). On the other hand, activation of Gq induces activation of phospholipase C, upregulation of inositol 1,4,5-triphosphate, and the subsequent release of intracellular calcium (Ca2+) (Wakelam et al., 1986). The action of glucagon is relatively complex and involves the coordinate regulation of transcription factors and signal transduction networks which converge to regulate amino acid, lipid and carbohydrate metabolism. For example, in the liver, elevated PKA activity activates various downstream

secretion.

homeostasis.

**2.2 Molecular mechanism underlying glucagon action** 

targeting α-cell dysfunction.

**2. Functions of glucagon 2.1 Functions of glucagon** 

targets leading to the suppression of glycolysis and glycogenesis, and the enhancement of gluconeogenesis and glycogenolysis (Jiang and Zhang, 2003). In islet cells, the elevation of cAMP by glucagon has been reported to stimulate insulin and glucagon secretion from βand α-cells respectively (Huypens et al., 2000; Ma et al., 2005) by PKA dependent and independent mechanisms. Upregulation of cAMP activates cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs / Epac), which modulates intracellular Ca2+-ion mobilization, enhancing exocytosis (Holz et al., 2006; Ma et al., 2005).

### **2.3 Anatomical characteristics of pancreatic islets and α-cells**

Pancreatic islets possess unique anatomical characteristics and are composed of five different endocrine cell types distributed as islands randomly within the exocrine pancreas. Among these five endocrine cells in islets, the α-cells account for approximately 20% of islet cells.

In adult rodents, β-cells are restricted mostly to the islet core, while α-cells, somatostatinsecreting δ-cells, pancreatic polypeptide-secreting PP-cells, and ghrelin-secreting ε-cells, are scattered along the periphery of the islet and surrounding β-cells. It is likely that this distribution and arrangement of different islet cell types is teleologically important for physiological regulation between the cells since the blood flows from the center of the islets toward periphery; i.e.β-cells to non-β-cells in the islet microcirculation system (Bonner-Weir and Orci, 1982; Stagner and Samols, 1986), suggesting that secreted insulin regulates hormone secretion from other islet cell types. This architecture is typically preserved in rodent islets, while in humans, non-β-cells are often observed both at the periphery and also seemingly in clusters within the center of islets (Cabrera et al., 2006). This implies several possibilities; 1) rodent cellular hierarchy in the islets does not apply to human islets, or 2) human islets consist of several clover-leaf like 'rosettes', with each rosette resembling the basic islet architecture observed in rodent islets (Bonner-Weir and O'Brien, 2008) suggesting

Schematic image for the structure of mouse and human islets adapted from the recent publication of (Bosco, 2010) (10).

Molecular Mechanism Underlying the Intra-Islet Regulation of Glucagon Secretion 143

The secretion of glucagon from α-cells is stimulated in response to hypoglycemia, and suppressed by hyperglycemia *in vivo*. However, the regulation of glucagon secretion is not simply determined only by glucose concentration, but is complex and finely controlled by additional contribution of neural, hormonal, and intra-islet interactions (Gromada et al., 2007). While it is still not conclusive whether α-cells can directly sense glucose concentration outside the cells and subsequently respond in glucagon secretion (section 3.2.), additional mechanisms which contribute to the secretion of glucagon have recently been revealed. For example, the central nervous system is reported to sense glucose concentration largely through the hypothalamus, and to modulate secretion of islet hormones via the autonomic nervous system (section 3.3.). In addition, circulating autonomic neurotransmitters such as γ-amino-butyric acid (GABA), epinephrine and norepinephrine can stimulate glucagon secretion from α-cells. As described above, various regulatory mechanisms for the glucagon secretion than glucose were uncovered. Among them, it is recently revealed that intra-islet regulation by neighboring β-cells plays critical roles in the physiology of glucagon secretion

The secretion of glucagon from α-cells is elevated in response to hypoglycemia and suppressed by hyperglycemia *in vivo*. While some studies suggest a direct suppressive effect of glucose on α-cell secretory function (Ravier and Rutter, 2005; Vieira et al., 2007), the paradoxical stimulation of glucagon secretion by high glucose in isolated islets and α-cell lines (Franklin et al., 2005; Olsen et al., 2005; Salehi et al., 2006) suggests that additional mechanisms contribute to the secretion of glucagon in response to glucose. Also, it is still not conclusive whether α-cells can directly sense glucose concentration outside the cells then

Amino acids such as L-arginine are potent stimulators of glucagon secretion (Gerich et al., 1974). This is physiologically relevant to prevent hypoglycemia after protein intake since amino acids also stimulate insulin secretion. L-glutamate is produced, secreted by various cell types including neural cells, and acts as a neurotransmitter. In islet α-cells, glutamate is contained in glucagon secretory vesicles (Yamada et al., 2001). Interestingly, a recent study shows that glutamate secreted by α-cells functions as an autocrine positive feedback signal for glucagon secretion (Cabrera et al., 2008), as α-cells express glutamate transporters and receptors (Hayashi et al., 2001). Low glucose stimulates glutamate release from α-cells, which in turn acts on α-cells in an autocrine manner leading to membrane depolarization

While glycemia might modulate glucagon secretion directly, several reports indicate the involvement of the central and/or autonomic nervous systems in the regulation of glucagon secretion (Ahren, 2000; Bloom et al., 1978; Evans et al., 2004; Marty et al., 2005). Hypoglycemia is a critical condition for body especially since glucose is an essential fuel for the central nervous system. Thus in response to hypoglycemia, the nervous response immediately triggers various counterregulatory mechanisms to protect the brain from

**3.2 Regulation of glucagon secretion by glucose and other nutrients** 

**3. Regulation of glucagon secretion 3.1 Factors involved in glucagon secretion** 

from α-cells (see section 4).

respond in glucagon secretion or not.

and glucagon secretion (Cabrera et al., 2008).

**3.3 Involvement of nervous system and neurotransmitters** 

energy deprivation, including the stimulation of glucagon secretion.

the arrangement and interaction of the different cell types in human islets is similar to that in rodents. Recent studies report that in large human islets blood vessels penetrate and branch inside islets, and α-cells located within the core of islets are placed along these vessels and surrounded by β-cells (Bosco et al.). Thus, according to this report, in human islets, α-cells which appear to be placed in the islet core are still 'peripheral' in the islets since blood vessels are usually considered to be placed outside the islets. Given the direction of intraislet microcirculation described above, intraislet auto-/paracrine effects between islet cells especially from β- to non-β-cells can be applied to human islets.

#### **2.4 Excessive glucagon secretion in diabetes**

Glucagon plays critical roles in glucose homeostasis largely by regulating hepatic glucose metabolism. However, circulating glucagon levels are often elevated in both type 1 and type 2 diabetes, thus are suggested to contribute to the development of insulin resistance (e.g. hepatic insulin resistance) and exacerbation of diabetes (Ahren and Larsson, 2001; Dinneen et al., 1995; Larsson and Ahren, 2000; Unger, 1978). In addition, the absence of postprandial glucagon suppression in diabetes patients also contributes to postprandial hyperglycemia (Mitrakou et al., 1992; Raskin and Unger, 1978; Sherwin et al., 1976). Another potential contributor to the excess glucagon levels is a relative increase in α-cells compared to -cells in pancreatic islets in both type 1 (Orci et al., 1976) and type 2 diabetes (Rahier et al., 1983; Yoon et al., 2003). Moreover, in type 1 diabetic islets, an increase in α-cell area and number, and dysregulated cell-type distribution in islets is due to specific -cell destruction. Although the precise mechanism(s) of relative hyperglucagonemia in the diabetic state is still obscure, -cell dysfunction is a possible candidate since -cell secretory products, including insulin, are known to suppress glucagon secretion (see section 4.1.). Thus altered (impaired) -cell function in diabetes can potentially induce inappropriately elevated glucagon in hyperglycemic states by impairing the intraislet influence of -cells on glucagon regulation (Meier et al., 2006a).

#### **2.5 Defective glucagon response to hypoglycemia in diabetes**

Diabetes patients (both type 1 and type 2) frequently develop defective counter-regulatory responses to hypoglycemia that is associated with reduced or absent glucagon secretory responses. A defective glucagon secretory response to hypoglycemia in hyperinsulinemic states frequently exacerbates a hypoglycemic attack, and limits intensive glucose control by insulin therapy (Amiel et al., 1988; Gerich et al., 1973). Moreover, hypoglycemia associated autonomic failure is induced especially in patients with frequent exposure to hypoglycemia leading to a worsening phenotype (Cryer, 1994). This defective response to hypoglycemia includes sympathoadrenal and neurohormonal responses against hypoglycemia such as epinephrine, cortisol and growth hormone that act to decrease blood glucose further, finally leading to sudden states of hypoglycemia and hypoglycemia unawareness (Amiel et al., 1988; Gerich et al., 1973). How diabetes induces these defective responses to hypoglycemia is still under investigation and suggested theories include alteration in brain glucose transport and metabolism by frequent exposure to hypoglycemia (Criego et al., 2005) and/or defective intraislet -cell effects on α-cell function, such as the "switch-off" of insulin (Hope et al., 2004; Zhou et al., 2004) or Zinc iron (Zhou et al., 2007) (see section 4.).

### **3. Regulation of glucagon secretion**

142 Diabetes – Damages and Treatments

the arrangement and interaction of the different cell types in human islets is similar to that in rodents. Recent studies report that in large human islets blood vessels penetrate and branch inside islets, and α-cells located within the core of islets are placed along these vessels and surrounded by β-cells (Bosco et al.). Thus, according to this report, in human islets, α-cells which appear to be placed in the islet core are still 'peripheral' in the islets since blood vessels are usually considered to be placed outside the islets. Given the direction of intraislet microcirculation described above, intraislet auto-/paracrine effects between islet

Glucagon plays critical roles in glucose homeostasis largely by regulating hepatic glucose metabolism. However, circulating glucagon levels are often elevated in both type 1 and type 2 diabetes, thus are suggested to contribute to the development of insulin resistance (e.g. hepatic insulin resistance) and exacerbation of diabetes (Ahren and Larsson, 2001; Dinneen et al., 1995; Larsson and Ahren, 2000; Unger, 1978). In addition, the absence of postprandial glucagon suppression in diabetes patients also contributes to postprandial hyperglycemia (Mitrakou et al., 1992; Raskin and Unger, 1978; Sherwin et al., 1976). Another potential contributor to the excess glucagon levels is a relative increase in α-cells compared to -cells in pancreatic islets in both type 1 (Orci et al., 1976) and type 2 diabetes (Rahier et al., 1983; Yoon et al., 2003). Moreover, in type 1 diabetic islets, an increase in α-cell area and number, and dysregulated cell-type distribution in islets is due to specific -cell destruction. Although the precise mechanism(s) of relative hyperglucagonemia in the diabetic state is still obscure, -cell dysfunction is a possible candidate since -cell secretory products, including insulin, are known to suppress glucagon secretion (see section 4.1.). Thus altered (impaired) -cell function in diabetes can potentially induce inappropriately elevated glucagon in hyperglycemic states by impairing the intraislet influence of -cells on glucagon

Diabetes patients (both type 1 and type 2) frequently develop defective counter-regulatory responses to hypoglycemia that is associated with reduced or absent glucagon secretory responses. A defective glucagon secretory response to hypoglycemia in hyperinsulinemic states frequently exacerbates a hypoglycemic attack, and limits intensive glucose control by insulin therapy (Amiel et al., 1988; Gerich et al., 1973). Moreover, hypoglycemia associated autonomic failure is induced especially in patients with frequent exposure to hypoglycemia leading to a worsening phenotype (Cryer, 1994). This defective response to hypoglycemia includes sympathoadrenal and neurohormonal responses against hypoglycemia such as epinephrine, cortisol and growth hormone that act to decrease blood glucose further, finally leading to sudden states of hypoglycemia and hypoglycemia unawareness (Amiel et al., 1988; Gerich et al., 1973). How diabetes induces these defective responses to hypoglycemia is still under investigation and suggested theories include alteration in brain glucose transport and metabolism by frequent exposure to hypoglycemia (Criego et al., 2005) and/or defective intraislet -cell effects on α-cell function, such as the "switch-off" of insulin (Hope et al., 2004; Zhou et al., 2004) or Zinc

cells especially from β- to non-β-cells can be applied to human islets.

**2.5 Defective glucagon response to hypoglycemia in diabetes** 

**2.4 Excessive glucagon secretion in diabetes** 

regulation (Meier et al., 2006a).

iron (Zhou et al., 2007) (see section 4.).

### **3.1 Factors involved in glucagon secretion**

The secretion of glucagon from α-cells is stimulated in response to hypoglycemia, and suppressed by hyperglycemia *in vivo*. However, the regulation of glucagon secretion is not simply determined only by glucose concentration, but is complex and finely controlled by additional contribution of neural, hormonal, and intra-islet interactions (Gromada et al., 2007). While it is still not conclusive whether α-cells can directly sense glucose concentration outside the cells and subsequently respond in glucagon secretion (section 3.2.), additional mechanisms which contribute to the secretion of glucagon have recently been revealed. For example, the central nervous system is reported to sense glucose concentration largely through the hypothalamus, and to modulate secretion of islet hormones via the autonomic nervous system (section 3.3.). In addition, circulating autonomic neurotransmitters such as γ-amino-butyric acid (GABA), epinephrine and norepinephrine can stimulate glucagon secretion from α-cells. As described above, various regulatory mechanisms for the glucagon secretion than glucose were uncovered. Among them, it is recently revealed that intra-islet regulation by neighboring β-cells plays critical roles in the physiology of glucagon secretion from α-cells (see section 4).

### **3.2 Regulation of glucagon secretion by glucose and other nutrients**

The secretion of glucagon from α-cells is elevated in response to hypoglycemia and suppressed by hyperglycemia *in vivo*. While some studies suggest a direct suppressive effect of glucose on α-cell secretory function (Ravier and Rutter, 2005; Vieira et al., 2007), the paradoxical stimulation of glucagon secretion by high glucose in isolated islets and α-cell lines (Franklin et al., 2005; Olsen et al., 2005; Salehi et al., 2006) suggests that additional mechanisms contribute to the secretion of glucagon in response to glucose. Also, it is still not conclusive whether α-cells can directly sense glucose concentration outside the cells then respond in glucagon secretion or not.

Amino acids such as L-arginine are potent stimulators of glucagon secretion (Gerich et al., 1974). This is physiologically relevant to prevent hypoglycemia after protein intake since amino acids also stimulate insulin secretion. L-glutamate is produced, secreted by various cell types including neural cells, and acts as a neurotransmitter. In islet α-cells, glutamate is contained in glucagon secretory vesicles (Yamada et al., 2001). Interestingly, a recent study shows that glutamate secreted by α-cells functions as an autocrine positive feedback signal for glucagon secretion (Cabrera et al., 2008), as α-cells express glutamate transporters and receptors (Hayashi et al., 2001). Low glucose stimulates glutamate release from α-cells, which in turn acts on α-cells in an autocrine manner leading to membrane depolarization and glucagon secretion (Cabrera et al., 2008).

#### **3.3 Involvement of nervous system and neurotransmitters**

While glycemia might modulate glucagon secretion directly, several reports indicate the involvement of the central and/or autonomic nervous systems in the regulation of glucagon secretion (Ahren, 2000; Bloom et al., 1978; Evans et al., 2004; Marty et al., 2005). Hypoglycemia is a critical condition for body especially since glucose is an essential fuel for the central nervous system. Thus in response to hypoglycemia, the nervous response immediately triggers various counterregulatory mechanisms to protect the brain from energy deprivation, including the stimulation of glucagon secretion.

Molecular Mechanism Underlying the Intra-Islet Regulation of Glucagon Secretion 145

Schematic image for the β-cell-mediated suppression of glucagon secretion from α-cells via a paracrine mechanism. The β-cell secretes insulin, γ-amino-butyric acid (GABA), and zinc irons (Zn) which suppress glucagon secretion. High glucose/hyperglycaemia suppresses glucagon secretion through the nervous system and by stimulation of β-cell secretion. Somatostatin also suppresses glucagon secretion. GLP-1 suppresses glucagon secretion

that insulin suppresses glucagon secretion *in vivo*. In insulinopenic animal models, exogenous insulin suppressed glucagon secretion (Greenbaum et al., 1991; Stagner and Samols, 1986; Weir et al., 1976). Conversely, suppression of insulin action by infusion of an anti-insulin antibody increased glucagon release (Maruyama et al., 1984). These studies clearly indicate the suppressive effect of insulin on glucagon secretion. Thus, it is conceivable that chronic and post-prandial hyperglucagonemia seen in diabetes patients (see section 2.4) is due to a lack of the direct suppression of insulin on glucagon secretion induced either by an absolute lack of insulin and/or α-cell insulin resistance(Meier et al.,

In addition, insulin is reported to stimulate glucagon secretion through a "switch-off" mechanism (Hope et al., 2004; Zhou et al., 2004). During hypoglycemia, a decrease in intraislet insulin may act as a trigger for glucagon secretion as α-cells can sense the decrease in ambient insulin. This concept is proposed by studies wherein cessation of insulin administration in *in vivo* pancreas perfusion experiments in insulinopenic diabetic rats induces glucagon secretion in response to hypoglycemia (Hope et al., 2004; Zhou et al., 2004). It is also possible that the defective secretory response of glucagon to hypoglycemia in diabetes patients occurs secondary to a defect in insulin sensing in -cells (see section 2.5). Thus, insulin is a center player not only in the suppression of glucagon secretion but also the

through β-cell mediated and direct pathways.

2006a; Raju and Cryer, 2005).

stimulation of glucagon secretion.

The dense innervations of the islets suggests that both α- and β-cells are regulated by the nervous system (Ahren, 2000). The autonomic nervous system (ANS) transmits stimuli to promote glucagon secretion especially under hypoglycemia when blood glucose must be increased to supply fuel for the body. The ANS can modulate all islet cells and regulate glucagon secretion directly via the parasympathetic pathway or indirectly by pathways that can modulate islet paracrine factors (see section 4.) (Ahren, 2000). In addition, circulating autonomic neurotransmitters epinephrine and norepinephrine have been reported to stimulate glucagon secretion from α-cells through adrenergic receptors (Schuit and Pipeleers, 1986; Vieira et al., 2004). Glucagon secretion is also modulated by other neurotransmitters including GABA (see section 4.2.) and glutamate (see section 3.2.).

The precise mechanism by which the central nervous system (CNS) senses blood glucose and affects glucagon secretion is not fully understood, although several possibilities have been suggested. Glucose sensing in the CNS is suggested to be an interaction between neurons and glial cells. For example, neurons in the ventro-medial hypothalamus (VMH) have been reported to play a role in sensing hypoglycemia in the brain and triggering the responses of counter-regulatory hormones to impact hypoglycemia (Borg et al., 1995), through AMPK (McCrimmon et al., 2004), K+ATP channels (Evans et al., 2004), and corticoptrophin releasing factor receptors (Cheng et al., 2007) in rat models. Moreover, it has also been reported that GLUT2 in cerebral astrocytes acts as a central glucose sensor in the modulation of glucagon secretion in mice (Marty et al., 2005).

### **4. Intra-islet regulation of glucagon secretion**

In addition to glucose, various regulatory mechanisms for glucagon secretion have been detected. Among these mechanisms is the emerging concept that intra-islet regulation by secretory products from neighboring β-cells plays a critical role in determining α-cell function. This concept is supported, at least in the rodent, by the direction of the intraislet microcirculation which occurs from the core to the periphery and implicates α-cells as potential direct targets of β-cell secretory products such as insulin, (Asplin et al., 1981; Kawamori et al., 2009; Maruyama et al., 1984; Weir et al., 1976), GABA (Rorsman et al., 1989; Xu et al., 2006) and Zinc ions (Ishihara et al., 2003). In addition, another islet hormone somatostatin is reported to modulate glucagon secretion. Interestingly, glucagon itself is reported to regulate glucagon secretion. GLP-1 can suppress glucagon secretion directly and possibly indirectly by enhancing insulin secretion.

#### **4.1 Insulin**

Insulin, the major secretory product of β-cells, has been proposed as one of the intra-islet paracrine factors that can modulate the secretion of glucagon from neighboring α-cells (Asplin et al., 1981; Kawamori et al., 2009; Maruyama et al., 1984; Weir et al., 1976). Furthermore, proteins in the insulin signaling pathway are abundantly expressed in α-cells supporting an important role for insulin signaling in α-cells(Bhathena et al., 1982; Franklin et al., 2005; Patel et al., 1982).

#### **4.1.1 Modulation of glucagon secretion by insulin**

In clinical studies in human type 1 diabetes patients whose β-cell function is considered to be extinct (Asplin et al., 1981; Gerich et al., 1975), along with basic studies in insulinopenic animal models (Maruyama et al., 1984; Stagner and Samols, 1986; Weir et al., 1976), indicate

The dense innervations of the islets suggests that both α- and β-cells are regulated by the nervous system (Ahren, 2000). The autonomic nervous system (ANS) transmits stimuli to promote glucagon secretion especially under hypoglycemia when blood glucose must be increased to supply fuel for the body. The ANS can modulate all islet cells and regulate glucagon secretion directly via the parasympathetic pathway or indirectly by pathways that can modulate islet paracrine factors (see section 4.) (Ahren, 2000). In addition, circulating autonomic neurotransmitters epinephrine and norepinephrine have been reported to stimulate glucagon secretion from α-cells through adrenergic receptors (Schuit and Pipeleers, 1986; Vieira et al., 2004). Glucagon secretion is also modulated by other

neurotransmitters including GABA (see section 4.2.) and glutamate (see section 3.2.).

modulation of glucagon secretion in mice (Marty et al., 2005).

**4. Intra-islet regulation of glucagon secretion** 

possibly indirectly by enhancing insulin secretion.

**4.1.1 Modulation of glucagon secretion by insulin** 

**4.1 Insulin** 

et al., 2005; Patel et al., 1982).

The precise mechanism by which the central nervous system (CNS) senses blood glucose and affects glucagon secretion is not fully understood, although several possibilities have been suggested. Glucose sensing in the CNS is suggested to be an interaction between neurons and glial cells. For example, neurons in the ventro-medial hypothalamus (VMH) have been reported to play a role in sensing hypoglycemia in the brain and triggering the responses of counter-regulatory hormones to impact hypoglycemia (Borg et al., 1995), through AMPK (McCrimmon et al., 2004), K+ATP channels (Evans et al., 2004), and corticoptrophin releasing factor receptors (Cheng et al., 2007) in rat models. Moreover, it has also been reported that GLUT2 in cerebral astrocytes acts as a central glucose sensor in the

In addition to glucose, various regulatory mechanisms for glucagon secretion have been detected. Among these mechanisms is the emerging concept that intra-islet regulation by secretory products from neighboring β-cells plays a critical role in determining α-cell function. This concept is supported, at least in the rodent, by the direction of the intraislet microcirculation which occurs from the core to the periphery and implicates α-cells as potential direct targets of β-cell secretory products such as insulin, (Asplin et al., 1981; Kawamori et al., 2009; Maruyama et al., 1984; Weir et al., 1976), GABA (Rorsman et al., 1989; Xu et al., 2006) and Zinc ions (Ishihara et al., 2003). In addition, another islet hormone somatostatin is reported to modulate glucagon secretion. Interestingly, glucagon itself is reported to regulate glucagon secretion. GLP-1 can suppress glucagon secretion directly and

Insulin, the major secretory product of β-cells, has been proposed as one of the intra-islet paracrine factors that can modulate the secretion of glucagon from neighboring α-cells (Asplin et al., 1981; Kawamori et al., 2009; Maruyama et al., 1984; Weir et al., 1976). Furthermore, proteins in the insulin signaling pathway are abundantly expressed in α-cells supporting an important role for insulin signaling in α-cells(Bhathena et al., 1982; Franklin

In clinical studies in human type 1 diabetes patients whose β-cell function is considered to be extinct (Asplin et al., 1981; Gerich et al., 1975), along with basic studies in insulinopenic animal models (Maruyama et al., 1984; Stagner and Samols, 1986; Weir et al., 1976), indicate

Schematic image for the β-cell-mediated suppression of glucagon secretion from α-cells via a paracrine mechanism. The β-cell secretes insulin, γ-amino-butyric acid (GABA), and zinc irons (Zn) which suppress glucagon secretion. High glucose/hyperglycaemia suppresses glucagon secretion through the nervous system and by stimulation of β-cell secretion. Somatostatin also suppresses glucagon secretion. GLP-1 suppresses glucagon secretion through β-cell mediated and direct pathways.

that insulin suppresses glucagon secretion *in vivo*. In insulinopenic animal models, exogenous insulin suppressed glucagon secretion (Greenbaum et al., 1991; Stagner and Samols, 1986; Weir et al., 1976). Conversely, suppression of insulin action by infusion of an anti-insulin antibody increased glucagon release (Maruyama et al., 1984). These studies clearly indicate the suppressive effect of insulin on glucagon secretion. Thus, it is conceivable that chronic and post-prandial hyperglucagonemia seen in diabetes patients (see section 2.4) is due to a lack of the direct suppression of insulin on glucagon secretion induced either by an absolute lack of insulin and/or α-cell insulin resistance(Meier et al., 2006a; Raju and Cryer, 2005).

In addition, insulin is reported to stimulate glucagon secretion through a "switch-off" mechanism (Hope et al., 2004; Zhou et al., 2004). During hypoglycemia, a decrease in intraislet insulin may act as a trigger for glucagon secretion as α-cells can sense the decrease in ambient insulin. This concept is proposed by studies wherein cessation of insulin administration in *in vivo* pancreas perfusion experiments in insulinopenic diabetic rats induces glucagon secretion in response to hypoglycemia (Hope et al., 2004; Zhou et al., 2004). It is also possible that the defective secretory response of glucagon to hypoglycemia in diabetes patients occurs secondary to a defect in insulin sensing in -cells (see section 2.5). Thus, insulin is a center player not only in the suppression of glucagon secretion but also the stimulation of glucagon secretion.

Molecular Mechanism Underlying the Intra-Islet Regulation of Glucagon Secretion 147

analogue glulisine were evaluated. Continuous glulisine infusion suppressed glucagon secretion both under normo- and hypoglycemic states, while discontinuation of glulisine infusion stimulated glucagon secretion in hypoglycemic state. From these studies, it is proposed that insulin overrides the effects of glucose and suppresses glucagon secretion in the hyperglycemic state, and decreasing insulin levels triggers glucagon response to

**-cell** 

**insulin secretion** 

**stimulation** 

**-cell IR**

**glucagon**

**low glucose**

In high glucose state, stimulated insulin secretion from β-cells acts on insulin receptor on the surface of α-cells then suppresses glucagon secretion by paracrine manner. In low glucose state, decreased insulin secretion from β-cells is recognized by α-cells as a reduction of insulin signaling in α-cells through insulin receptor, then α-cells increase glucagon secretion

γ-amino-butyric acid (GABA) is produced from the excitatory amino acid glutamate by glutamic acid decarboxylase (GAD) and works as an important inhibitory neurotransmitter in neural synapses, mainly in the central nervous system (Kittler and Moss, 2003). In neurons, GABA is released by the presynaptic terminal into synaptic junctions and binds to GABA receptors on the postsynaptic membrane, inhibiting cellular electrical firing through modulation of ion channels and consequent membrane hyperpolarization (Kittler and Moss, 2003). Islets are also innerved by GABA-ergic neurons (Sorenson et al., 1991), suggesting

In addition, GABA has also been reported to be secreted from β-cells and suppress glucagon secretion from α-cells in an intraislet paracrine manner (Rorsman et al., 1989; Wendt et al., 2004; Xu et al., 2006). High glucose or glutamate levels stimulate secretion of GABA from βcells and the secreted GABA then binds to its receptor expressed on α-cells, inhibiting glucagon secretion through cellular membrane hyperpoloarization. Importantly, the GABA-A receptor is recruited to the cellular membrane by insulin-Akt signaling (Xu et al., 2006), and its activation suppresses glucagon secretion through desensitization of K+ATP channels. These observations suggest a cooperative role between insulin and GABA in the inhibition

hypoglycemia and precedes the direct effect of low glucose.

**-cell**

**insulin secretion**

**-cell IR**

**suppression**

**glucagon**

**high glucose**

that GABA is a potential inhibitor of -cell function.

in response.

**4.2 GABA** 

of glucagon secretion.

#### **4.1.2 Molecular mechanisms underlying the modulation of glucagon secretion by insulin signaling**

These *in vivo* reports suggest a direct effect of insulin in modulating glucagon secretion. On the other hand, recent *in vitro* studies in α-cell lines using gene knock-down techniques indicate a role for the insulin receptor and its signaling pathway in suppressing glucagon secretion by high glucose (Ravier and Rutter, 2005), as well as in stimulating glucagon secretion by low glucose concentration (Diao et al., 2005).

The direct inhibitory effects of insulin to suppress glucagon secretion has been reported to occur either by 1) reducing the sensitivity of K+ATP channels (Franklin et al., 2005) which regulate glucagon secretion machinery via phosphatidyl inositol 3-kinase (PI3K) (Leung et al., 2006), or by 2) modulating Akt, a critical downstream effector of PI3K, leading to recruitment of the GABA-A receptor to the cellular membrane to allow its ligand, GABA, to inhibit glucagon secretion (see section 4.2) (Rorsman et al., 1989; Xu et al., 2006).

#### **4.1.3 The α-cell specific insulin receptor knockout mouse model**

While numerous reports indicate a pivotal role for insulin in the regulation of glucagon secretion, direct molecular evidence for the importance of insulin signaling in α-cells *in vivo* has been lacking until recently. The significance of systemic insulin signaling in glucose homeostasis is well known as insulin resistance is induced in insulin target organs including the liver, the skeletal muscle and the adipose tissues under diabetic state, and impacts on glycemic metabolism in these organs. Eventually, the genetic evidence of the *in vivo* significance of insulin signaling in α-cells in the regulation of glucagon secretion was provided by investigation of the α-cell specific insulin receptor knockout (αIRKO) mice (Kawamori et al., 2009).

The αIRKO mice exhibited glucose intolerance, hyperglycemia and hyperglucagonemia in the fed state together with enhanced glucagon secretion in response to L-arginine. These results indicate that disruption of insulin receptor in α-cells enhanced glucagon secretion by diminishing the glucagonostatic effect of insulin, and provided direct *in vivo* evidence for the suppression of glucagon secretion by insulin from β-cells through intra-islet paracrine manner. Interestingly, the mutant mice also displayed blunted glucagon response to hypoglycemia indicating a defective glucagon response through insulin "switch-off" mechanism (Hope et al., 2004; Zhou et al., 2004) by disruption of insulin signaling in α-cells. The results using αIRKO mice clearly demonstrate a critical role for insulin in the regulation of α-cell function in both normo- and hypoglycemic states *in vivo*.

#### **4.1.4 Model for the intraislet regulation of glucagon secretion from α-cells by insulin**

From these findings, a possible model for the intraislet regulation of glucagon secretion by insulin can be proposed. In states of hyperglycemia, the greater insulin secretion from βcells is stimulated and would activate insulin signaling in α-cells via paracrine manner, and represses glucagon secretion. On the other hand, in hypoglycemic state, the consequent levels of low insulin would allow the α-cells to sense the reduction in ambient insulin leading to a lack of activation of insulin signaling that in turn leads to the stimulation of glucagon secretion. This would occur in addition to possible direct stimulation by low glucose itself. Indeed, a recent clinical study reported that this proposed mechanism is actually feasible in humans (Cooperberg and Cryer, 2010). In this report, patients with type 1 diabetes were subjected to normo- and hypoglycemic clamps and the effects of insulin analogue glulisine were evaluated. Continuous glulisine infusion suppressed glucagon secretion both under normo- and hypoglycemic states, while discontinuation of glulisine infusion stimulated glucagon secretion in hypoglycemic state. From these studies, it is proposed that insulin overrides the effects of glucose and suppresses glucagon secretion in the hyperglycemic state, and decreasing insulin levels triggers glucagon response to hypoglycemia and precedes the direct effect of low glucose.

In high glucose state, stimulated insulin secretion from β-cells acts on insulin receptor on the surface of α-cells then suppresses glucagon secretion by paracrine manner. In low glucose state, decreased insulin secretion from β-cells is recognized by α-cells as a reduction of insulin signaling in α-cells through insulin receptor, then α-cells increase glucagon secretion in response.

### **4.2 GABA**

146 Diabetes – Damages and Treatments

These *in vivo* reports suggest a direct effect of insulin in modulating glucagon secretion. On the other hand, recent *in vitro* studies in α-cell lines using gene knock-down techniques indicate a role for the insulin receptor and its signaling pathway in suppressing glucagon secretion by high glucose (Ravier and Rutter, 2005), as well as in stimulating glucagon

The direct inhibitory effects of insulin to suppress glucagon secretion has been reported to occur either by 1) reducing the sensitivity of K+ATP channels (Franklin et al., 2005) which regulate glucagon secretion machinery via phosphatidyl inositol 3-kinase (PI3K) (Leung et al., 2006), or by 2) modulating Akt, a critical downstream effector of PI3K, leading to recruitment of the GABA-A receptor to the cellular membrane to allow its ligand, GABA, to

While numerous reports indicate a pivotal role for insulin in the regulation of glucagon secretion, direct molecular evidence for the importance of insulin signaling in α-cells *in vivo* has been lacking until recently. The significance of systemic insulin signaling in glucose homeostasis is well known as insulin resistance is induced in insulin target organs including the liver, the skeletal muscle and the adipose tissues under diabetic state, and impacts on glycemic metabolism in these organs. Eventually, the genetic evidence of the *in vivo* significance of insulin signaling in α-cells in the regulation of glucagon secretion was provided by investigation of the α-cell specific insulin receptor knockout (αIRKO) mice

The αIRKO mice exhibited glucose intolerance, hyperglycemia and hyperglucagonemia in the fed state together with enhanced glucagon secretion in response to L-arginine. These results indicate that disruption of insulin receptor in α-cells enhanced glucagon secretion by diminishing the glucagonostatic effect of insulin, and provided direct *in vivo* evidence for the suppression of glucagon secretion by insulin from β-cells through intra-islet paracrine manner. Interestingly, the mutant mice also displayed blunted glucagon response to hypoglycemia indicating a defective glucagon response through insulin "switch-off" mechanism (Hope et al., 2004; Zhou et al., 2004) by disruption of insulin signaling in α-cells. The results using αIRKO mice clearly demonstrate a critical role for insulin in the regulation

**4.1.4 Model for the intraislet regulation of glucagon secretion from α-cells by insulin**  From these findings, a possible model for the intraislet regulation of glucagon secretion by insulin can be proposed. In states of hyperglycemia, the greater insulin secretion from βcells is stimulated and would activate insulin signaling in α-cells via paracrine manner, and represses glucagon secretion. On the other hand, in hypoglycemic state, the consequent levels of low insulin would allow the α-cells to sense the reduction in ambient insulin leading to a lack of activation of insulin signaling that in turn leads to the stimulation of glucagon secretion. This would occur in addition to possible direct stimulation by low glucose itself. Indeed, a recent clinical study reported that this proposed mechanism is actually feasible in humans (Cooperberg and Cryer, 2010). In this report, patients with type 1 diabetes were subjected to normo- and hypoglycemic clamps and the effects of insulin

inhibit glucagon secretion (see section 4.2) (Rorsman et al., 1989; Xu et al., 2006).

**4.1.3 The α-cell specific insulin receptor knockout mouse model** 

of α-cell function in both normo- and hypoglycemic states *in vivo*.

**4.1.2 Molecular mechanisms underlying the modulation of glucagon secretion by** 

secretion by low glucose concentration (Diao et al., 2005).

**insulin signaling** 

(Kawamori et al., 2009).

γ-amino-butyric acid (GABA) is produced from the excitatory amino acid glutamate by glutamic acid decarboxylase (GAD) and works as an important inhibitory neurotransmitter in neural synapses, mainly in the central nervous system (Kittler and Moss, 2003). In neurons, GABA is released by the presynaptic terminal into synaptic junctions and binds to GABA receptors on the postsynaptic membrane, inhibiting cellular electrical firing through modulation of ion channels and consequent membrane hyperpolarization (Kittler and Moss, 2003). Islets are also innerved by GABA-ergic neurons (Sorenson et al., 1991), suggesting that GABA is a potential inhibitor of -cell function.

In addition, GABA has also been reported to be secreted from β-cells and suppress glucagon secretion from α-cells in an intraislet paracrine manner (Rorsman et al., 1989; Wendt et al., 2004; Xu et al., 2006). High glucose or glutamate levels stimulate secretion of GABA from βcells and the secreted GABA then binds to its receptor expressed on α-cells, inhibiting glucagon secretion through cellular membrane hyperpoloarization. Importantly, the GABA-A receptor is recruited to the cellular membrane by insulin-Akt signaling (Xu et al., 2006), and its activation suppresses glucagon secretion through desensitization of K+ATP channels. These observations suggest a cooperative role between insulin and GABA in the inhibition of glucagon secretion.

Molecular Mechanism Underlying the Intra-Islet Regulation of Glucagon Secretion 149

reported to suppress glucagon secretion by directly acting on α-cells or indirectly by stimulating insulin secretion or modulating other non-β-cell hormones (e.g. somatostatin) which can in turn suppress glucagon secretion. However, the defects in GLP-1 secretion and action in type 2 diabetes likely impact the pathophysiology of the disease via abnormal

Paradoxically, another incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), can stimulate glucagon secretion despite stimulating insulin secretion from β-cells in a manner similar to GLP-1 (de Heer et al., 2008; Meier et al., 2003; Pederson and Brown, 1978). On the other hand, GLP-2, although derived from the same proglucagon gene as GLP-1, in intestinal L-cells, has not been reported to affect the secretory properties of -cells but stimulates glucagon secretion in human subjects (Meier et al., 2006b), by activation of

GLP-1 is reported to suppress glucagon secretion directly and/or indirectly through other cell-types; β- and δ-cells. In this point, many studies were conducted and displayed pros and cons to both theories. However, considering these reports comprehensively, it is less possible that only one mechanism is working in the suppressive effect of GLP-1 on glucagon, and it is conceivable that these direct and indirect manners are both regulating

There are conflicting reports concerning the expression of GLP-1 receptors in α-cells (Heller et al., 1997; Moens et al., 1996). Previous studies investigating GLP-1 receptor expression in α-cells by RNA expression and immunohistochemical analyses indicate that GLP-1 receptors are not expressed in α-cells or if present are expressed at low levels (Tornehave et al., 2008), or by only a few α-cells (Heller et al., 1997). A recent study using *in situ* hybridization and immunofluorescence microscopy in mouse, rat, and human pancreas identified the islet cell types that express GLP-1 receptors (Tornehave et al., 2008) and concluded that GLP-1 receptors are not expressed in α-cells. Thus, it is unlikely that GLP-1 can exert its direct effects on α-cells to impact glucagon secretion. On the other hand, GLP-1 is a strong secretagogue for insulin from β-cells, and considering the central role for insulin in the regulation of glucagon secretion, it is reasonable to suggest that GLP-1 suppresses glucagon secretion by secreted insulin. GLP-1 is also reported to stimulate somatostatin secretion from δ-cells in response to high glucose (Orskov et al., 1988), and it is possible that the secreted somatostatin suppresses glucagon secretion (de Heer and Holst, 2007; Hauge-Evans et al., 2009). This suggestion is supported by the observation that expression of a highly specific somatostatin receptor subtype 2 (SSTR2) antagonist completely abolished the GLP-1 effect on glucagon secretion in isolated perfused rat pancreas (de Heer et al., 2008). However, considering that the direction of intra-islet microcirculation occurs from the core of islets to the mantle; from β-α-δ at least in rodents (Stagner and Samols, 1986), additional

In contrast, reports that GLP-1 (Creutzfeldt et al., 1996) and DPP-4 inhibitor (Foley et al., 2008) treatment suppressed excessive glucagon secretion in type 1 diabetes patients even in the absence of secretory products from β-cells, suggest a potential direct effect of GLP-1 on glucagon suppression. A recent study by De Marinis et al reported that the expression of

regulation of both insulin and glucagon secretion (Holst et al., 2009).

**4.6.1 Indirect suppression of glucagon secretion by GLP-1** 

GLP-2 receptors on α-cells (de Heer et al., 2007).

glucagon secretion with interacting each other.

studies are necessary to explore these possibilities.

**4.6.2 Direct suppression of glucagon secretion by GLP-1** 

### **4.3 Zinc**

Zinc ions (Zn2+), co-released with insulin by β-cells in response to high glucose levels, have been reported to activate K+ATP channels on α-cells, desensitize the channels and suppress glucagon secretion (Ishihara et al., 2003). Zn2+ is also reported to stimulate glucagon secretion from α-cells when its concentration falls as part of a "switch-off" mechanism (Zhou et al., 2007). However, another study reports a lack of inhibitory effect of exogenous Zn2+ on glucagon secretion (Ravier and Rutter, 2005), indicating that the effects of Zn2+ on glucagon secretion are complex and require further investigation.

### **4.4 Somatostatin**

Somatostatin, an inhibitory hormone, secreted by neuronal and pancreatic δ-cells in islets inhibits both insulin and glucagon in a paracrine manner in the islet (Barden et al., 1977; Gerich et al., 1974; Starke et al., 1987). Somatostatin is considered to exert its suppressive effect on glucagon secretion largely through interstitial communication between α- and δcells (Stagner and Samols, 1986). Following binding to its receptors on α-cells somatostatin inhibits glucagon secretion by inducing plasma membrane hyperpolarization (Yoshimoto et al., 1999), suppression of cAMP elevation (Schuit et al., 1989) and direct inhibition of the exocytotic machinery via a G-protein-dependent mechanism (Gromada et al., 2001).

Somatostatin secretion from islet -cells is stimulated by glucose (Gerber et al., 1981; Honey et al., 1980), consistent with the report that the suppressive effect of high glucose on glucagon secretion may be mediated by glucose-induced secretion of somatostatin (Hauge-Evans et al., 2009). Interestingly, global somatostatin knockout mice exhibit enhanced insulin and glucagon secretion *in vivo* and *ex vivo*. In addition the ability of exogenous glucose to suppress glucagon secretion is lost in islets isolated from somatostatin knockout mice (Hauge-Evans et al., 2009) and highlights the intra-islet interactions between somatostatin, glucagon, and insulin. These observations from a global knockout of somatostatin should be interpreted with caution since extra-pancreatic neuronal effects cannot be ruled out. It should also be noted that somatostatin involvement in glucagon suppression during hyperglycemia might be less important than the effects of β-cell secretion *in vivo* according to the direction of intraislet microcirculation, β-α-δ (Gerich, 1990; Stagner and Samols, 1986). Interestingly, somatostatin is also reported to be involved in GLP-1 mediated suppression of glucagon secretion (see section 4.6). Further investigation is thus necessary to clarify the intra-islet relationship of islet hormones.

### **4.5 Glucagon**

Interestingly, glucagon which is secreted by α-cells is reported to stimulate glucagon secretion (Ma et al., 2005). Upregulation of cAMP by glucagon signaling is suggested to stimulate glucagon exocytosis via a mechanism that is similar to the stimulatory effects of glucagon on insulin and somatostatin secretion (Huypens et al., 2000; Stagner et al., 1989).

### **4.6 Glucagon like-peptide-1 (GLP-1)**

The incretin hormone, glucagon-like peptide-1 (GLP-1), is secreted by intestinal L-cells in response to food intake and is a strong stimulator of insulin secretion and also regulates βcell mass through modulation of cellular proliferation and death (Drucker, 2006). Therefore, GLP-1 contributes to glucose homeostasis acutely by enhancing β-cell secretory function and chronically by maintaining β-cell mass. In addition to these effects on β-cells, GLP-1 is

Zinc ions (Zn2+), co-released with insulin by β-cells in response to high glucose levels, have been reported to activate K+ATP channels on α-cells, desensitize the channels and suppress glucagon secretion (Ishihara et al., 2003). Zn2+ is also reported to stimulate glucagon secretion from α-cells when its concentration falls as part of a "switch-off" mechanism (Zhou et al., 2007). However, another study reports a lack of inhibitory effect of exogenous Zn2+ on glucagon secretion (Ravier and Rutter, 2005), indicating that the effects of Zn2+ on

Somatostatin, an inhibitory hormone, secreted by neuronal and pancreatic δ-cells in islets inhibits both insulin and glucagon in a paracrine manner in the islet (Barden et al., 1977; Gerich et al., 1974; Starke et al., 1987). Somatostatin is considered to exert its suppressive effect on glucagon secretion largely through interstitial communication between α- and δcells (Stagner and Samols, 1986). Following binding to its receptors on α-cells somatostatin inhibits glucagon secretion by inducing plasma membrane hyperpolarization (Yoshimoto et al., 1999), suppression of cAMP elevation (Schuit et al., 1989) and direct inhibition of the

Somatostatin secretion from islet -cells is stimulated by glucose (Gerber et al., 1981; Honey et al., 1980), consistent with the report that the suppressive effect of high glucose on glucagon secretion may be mediated by glucose-induced secretion of somatostatin (Hauge-Evans et al., 2009). Interestingly, global somatostatin knockout mice exhibit enhanced insulin and glucagon secretion *in vivo* and *ex vivo*. In addition the ability of exogenous glucose to suppress glucagon secretion is lost in islets isolated from somatostatin knockout mice (Hauge-Evans et al., 2009) and highlights the intra-islet interactions between somatostatin, glucagon, and insulin. These observations from a global knockout of somatostatin should be interpreted with caution since extra-pancreatic neuronal effects cannot be ruled out. It should also be noted that somatostatin involvement in glucagon suppression during hyperglycemia might be less important than the effects of β-cell secretion *in vivo* according to the direction of intraislet microcirculation, β-α-δ (Gerich, 1990; Stagner and Samols, 1986). Interestingly, somatostatin is also reported to be involved in GLP-1 mediated suppression of glucagon secretion (see section 4.6). Further investigation is

Interestingly, glucagon which is secreted by α-cells is reported to stimulate glucagon secretion (Ma et al., 2005). Upregulation of cAMP by glucagon signaling is suggested to stimulate glucagon exocytosis via a mechanism that is similar to the stimulatory effects of glucagon on insulin and somatostatin secretion (Huypens et al., 2000; Stagner et al., 1989).

The incretin hormone, glucagon-like peptide-1 (GLP-1), is secreted by intestinal L-cells in response to food intake and is a strong stimulator of insulin secretion and also regulates βcell mass through modulation of cellular proliferation and death (Drucker, 2006). Therefore, GLP-1 contributes to glucose homeostasis acutely by enhancing β-cell secretory function and chronically by maintaining β-cell mass. In addition to these effects on β-cells, GLP-1 is

exocytotic machinery via a G-protein-dependent mechanism (Gromada et al., 2001).

thus necessary to clarify the intra-islet relationship of islet hormones.

glucagon secretion are complex and require further investigation.

**4.3 Zinc** 

**4.4 Somatostatin** 

**4.5 Glucagon** 

**4.6 Glucagon like-peptide-1 (GLP-1)** 

reported to suppress glucagon secretion by directly acting on α-cells or indirectly by stimulating insulin secretion or modulating other non-β-cell hormones (e.g. somatostatin) which can in turn suppress glucagon secretion. However, the defects in GLP-1 secretion and action in type 2 diabetes likely impact the pathophysiology of the disease via abnormal regulation of both insulin and glucagon secretion (Holst et al., 2009).

Paradoxically, another incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), can stimulate glucagon secretion despite stimulating insulin secretion from β-cells in a manner similar to GLP-1 (de Heer et al., 2008; Meier et al., 2003; Pederson and Brown, 1978). On the other hand, GLP-2, although derived from the same proglucagon gene as GLP-1, in intestinal L-cells, has not been reported to affect the secretory properties of -cells but stimulates glucagon secretion in human subjects (Meier et al., 2006b), by activation of GLP-2 receptors on α-cells (de Heer et al., 2007).

#### **4.6.1 Indirect suppression of glucagon secretion by GLP-1**

GLP-1 is reported to suppress glucagon secretion directly and/or indirectly through other cell-types; β- and δ-cells. In this point, many studies were conducted and displayed pros and cons to both theories. However, considering these reports comprehensively, it is less possible that only one mechanism is working in the suppressive effect of GLP-1 on glucagon, and it is conceivable that these direct and indirect manners are both regulating glucagon secretion with interacting each other.

There are conflicting reports concerning the expression of GLP-1 receptors in α-cells (Heller et al., 1997; Moens et al., 1996). Previous studies investigating GLP-1 receptor expression in α-cells by RNA expression and immunohistochemical analyses indicate that GLP-1 receptors are not expressed in α-cells or if present are expressed at low levels (Tornehave et al., 2008), or by only a few α-cells (Heller et al., 1997). A recent study using *in situ* hybridization and immunofluorescence microscopy in mouse, rat, and human pancreas identified the islet cell types that express GLP-1 receptors (Tornehave et al., 2008) and concluded that GLP-1 receptors are not expressed in α-cells. Thus, it is unlikely that GLP-1 can exert its direct effects on α-cells to impact glucagon secretion. On the other hand, GLP-1 is a strong secretagogue for insulin from β-cells, and considering the central role for insulin in the regulation of glucagon secretion, it is reasonable to suggest that GLP-1 suppresses glucagon secretion by secreted insulin. GLP-1 is also reported to stimulate somatostatin secretion from δ-cells in response to high glucose (Orskov et al., 1988), and it is possible that the secreted somatostatin suppresses glucagon secretion (de Heer and Holst, 2007; Hauge-Evans et al., 2009). This suggestion is supported by the observation that expression of a highly specific somatostatin receptor subtype 2 (SSTR2) antagonist completely abolished the GLP-1 effect on glucagon secretion in isolated perfused rat pancreas (de Heer et al., 2008). However, considering that the direction of intra-islet microcirculation occurs from the core of islets to the mantle; from β-α-δ at least in rodents (Stagner and Samols, 1986), additional studies are necessary to explore these possibilities.

#### **4.6.2 Direct suppression of glucagon secretion by GLP-1**

In contrast, reports that GLP-1 (Creutzfeldt et al., 1996) and DPP-4 inhibitor (Foley et al., 2008) treatment suppressed excessive glucagon secretion in type 1 diabetes patients even in the absence of secretory products from β-cells, suggest a potential direct effect of GLP-1 on glucagon suppression. A recent study by De Marinis et al reported that the expression of

Molecular Mechanism Underlying the Intra-Islet Regulation of Glucagon Secretion 151

While glucagon was believed to elevate or decline simply in response to blood glucose levels, emerging work reveals a complex but sophisticated regulatory mechanism for the modulation of glucagon output from the α-cells with effects from pancreatic and endocrine hormones including insulin, somatostatin, epinephrine and incretins, nutrients and central and autonomic nervous pathways. The concept of intra-islet regulation of glucagon secretion that is mediated by insulin in a paracrine manner is now recognized as an important pathway that determines α-cell functions. Thus, disorder in intra-islet regulation of glucagon secretion is deeply involved in pathophysiology of diabetes. Considering that the diabetic state is characterized by systemic insulin resistance, that includes non-classical targets such as β-cells (Gunton et al., 2005; Kulkarni et al., 1999), it would be important to explore whether insulin resistance at the level of the α-cell underlies some of the early defects that lead to enhanced glucagon output and a consequent defect in glucose

Recently, new therapeutic approaches targeting excessive glucagon by suppression of glucagon secretion or inhibition of glucagon receptors and their function were tried in the treatment of diabetes, but simple inhibition of glucagon effect does not result in improvement of glucose homeostasis because of hypoglycemia by lack of glucagon effect. In future therapy in diabetes, we need to aim glucagon to work appropriately and rest properly, then improve its effects on other organs and hormonal balance between glucagon and insulin. Further studies are necessary to explore whether cells in the central and/or autonomic nervous systems can be targeted to modulate glucagon secretion for therapeutic

D.K. is the recipient of a Research Fellowship (Manpei Suzuki Diabetes Foundation, Japan) and a JDRF Postdoctoral Fellowship. The authors acknowledge support from the American Diabetes Association Research Grant (R.N.K.) and National Institutes of Health (R.N.K.).

Ahren, B. (2000). Autonomic regulation of islet hormone secretion--implications for health

Ahren, B., and Larsson, H. (2001). Impaired glucose tolerance (IGT) is associated with

Amiel, S. A., Sherwin, R. S., Simonson, D. C., and Tamborlane, W. V. (1988). Effect of

Asplin, C. M., Paquette, T. L., and Palmer, J. P. (1981). In vivo inhibition of glucagon secretion by paracrine beta cell activity in man. J Clin Invest *68*, 314-318. Barden, N., Lavoie, M., Dupont, A., Cote, J., and Cote, J. P. (1977). Stimulation of glucagon

reduced insulin-induced suppression of glucagon concentrations. Diabetologia *44*,

intensive insulin therapy on glycemic thresholds for counterregulatory hormone

release by addition of anti-stomatostatin serum to islets of Langerhans in vitro.

and disease. Diabetologia *43*, 393-410.

release. Diabetes *37*, 901-907.

Endocrinology *101*, 635-638.

**5. Conclusion and future perspectives** 

homeostasis.

purposes.

**6. Acknowledgments** 

1998-2003.

**7. References** 

GLP-1 receptors in α-cells is less than 0.2 % of its expression in β-cells, and consequently GLP-1 can induce a small elevation in cAMP activating PKA followed by selective inhibition of N-type Ca2+ ion channels, thus suppressing glucagon exocytosis (De Marinis et al.). In contrast, receptors for epinephrine or GIP are expressed abundantly in α-cells, and these molecules stimulate electrical activity significantly leading to an increase in Ca2+ in α-cells, causing glucagon exocytosis to accelerate through activation of L-type Ca2+ ion channels (De Marinis et al.). Studies using isolated islets indicated that GLP-1 effect on glucagon suppression is independent of insulin and intra-islet paracrine effect.

### **4.6.3 Model for the GLP-1 mediated suppression of glucagon secretion**

Considering these reports together, it is possible that GLP-1 suppresses glucagon secretion directly, but in postprandial state, GLP-1 enhances insulin secretion from β-cells together with another incretin GIP, and subsequently exerts suppressive effects on glucagon secretion. Further urgent investigations are necessary to understand the effects of GLP-1 on α-cell function. However, reports of GLP-1 induced suppression of glucagon secretion, in addition to its beneficial role on β-cells including augmentation of glucose-stimulated insulin secretion, promotion of β-cell proliferation, and protection of β-cells from various cytotoxicities, emphasizes the potential of GLP-1 therapy for the treatment of diabetes.

GLP-1 directly suppresses glucagon secretion from α-cells through slight increase of cAMP followed by inhibition of N-type Ca2+ channels (De Marinis, 2010) (57). GLP-1 also potentiates insulin secretion from β-cells then suppresses glucagon secretion through insulin effects on αcells. Glucose stimulates insulin secretion from β-cells and suppresses glucagon from α-cells through insulin effects, while glucose can stimulate glucagon secretion from α-cells.

### **5. Conclusion and future perspectives**

150 Diabetes – Damages and Treatments

GLP-1 receptors in α-cells is less than 0.2 % of its expression in β-cells, and consequently GLP-1 can induce a small elevation in cAMP activating PKA followed by selective inhibition of N-type Ca2+ ion channels, thus suppressing glucagon exocytosis (De Marinis et al.). In contrast, receptors for epinephrine or GIP are expressed abundantly in α-cells, and these molecules stimulate electrical activity significantly leading to an increase in Ca2+ in α-cells, causing glucagon exocytosis to accelerate through activation of L-type Ca2+ ion channels (De Marinis et al.). Studies using isolated islets indicated that GLP-1 effect on glucagon

Considering these reports together, it is possible that GLP-1 suppresses glucagon secretion directly, but in postprandial state, GLP-1 enhances insulin secretion from β-cells together with another incretin GIP, and subsequently exerts suppressive effects on glucagon secretion. Further urgent investigations are necessary to understand the effects of GLP-1 on α-cell function. However, reports of GLP-1 induced suppression of glucagon secretion, in addition to its beneficial role on β-cells including augmentation of glucose-stimulated insulin secretion, promotion of β-cell proliferation, and protection of β-cells from various cytotoxicities, emphasizes the potential of GLP-1 therapy for the treatment of diabetes.

GLP-1 directly suppresses glucagon secretion from α-cells through slight increase of cAMP followed by inhibition of N-type Ca2+ channels (De Marinis, 2010) (57). GLP-1 also potentiates insulin secretion from β-cells then suppresses glucagon secretion through insulin effects on αcells. Glucose stimulates insulin secretion from β-cells and suppresses glucagon from α-cells

through insulin effects, while glucose can stimulate glucagon secretion from α-cells.

suppression is independent of insulin and intra-islet paracrine effect.

**4.6.3 Model for the GLP-1 mediated suppression of glucagon secretion** 

While glucagon was believed to elevate or decline simply in response to blood glucose levels, emerging work reveals a complex but sophisticated regulatory mechanism for the modulation of glucagon output from the α-cells with effects from pancreatic and endocrine hormones including insulin, somatostatin, epinephrine and incretins, nutrients and central and autonomic nervous pathways. The concept of intra-islet regulation of glucagon secretion that is mediated by insulin in a paracrine manner is now recognized as an important pathway that determines α-cell functions. Thus, disorder in intra-islet regulation of glucagon secretion is deeply involved in pathophysiology of diabetes. Considering that the diabetic state is characterized by systemic insulin resistance, that includes non-classical targets such as β-cells (Gunton et al., 2005; Kulkarni et al., 1999), it would be important to explore whether insulin resistance at the level of the α-cell underlies some of the early defects that lead to enhanced glucagon output and a consequent defect in glucose homeostasis.

Recently, new therapeutic approaches targeting excessive glucagon by suppression of glucagon secretion or inhibition of glucagon receptors and their function were tried in the treatment of diabetes, but simple inhibition of glucagon effect does not result in improvement of glucose homeostasis because of hypoglycemia by lack of glucagon effect. In future therapy in diabetes, we need to aim glucagon to work appropriately and rest properly, then improve its effects on other organs and hormonal balance between glucagon and insulin. Further studies are necessary to explore whether cells in the central and/or autonomic nervous systems can be targeted to modulate glucagon secretion for therapeutic purposes.

### **6. Acknowledgments**

D.K. is the recipient of a Research Fellowship (Manpei Suzuki Diabetes Foundation, Japan) and a JDRF Postdoctoral Fellowship. The authors acknowledge support from the American Diabetes Association Research Grant (R.N.K.) and National Institutes of Health (R.N.K.).

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**Part 4** 

**Section D** 


**Part 4** 

**Section D** 

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a G protein-gated K+ channel. FEBS Lett *444*, 265-269.

rats: the "switch-off" hypothesis. Diabetes *53*, 1482-1487.

1112.

Somatostatin induces hyperpolarization in pancreatic islet alpha cells by activating

Regulation of alpha-cell function by the beta-cell during hypoglycemia in Wistar

regulates the rat alpha-cell response to hypoglycemia in vivo. Diabetes *56*, 1107-

**9** 

*1Romania 2,3Israel* 

**Insulin Therapy and** 

*2InsuLine Medical Ltd., Petach-Tikva, 3Diabetes Center, Hadassah-Hebrew University Medical School, Jerusalem,* 

**Hypoglycemia - Present and Future** 

*1Diabetes, Nutrition and Metabolic Diseases Outpatient Unit,* 

*Emergency County Clinical Hospital, Târgu Mureş,* 

Simona Cernea1, Ron Nagar2, Gabriel Bitton2 and Itamar Raz3

Over the last few decades the prevalence of diabetes has dramatically grown in most regions of the world. In 2010, 285 million people had diabetes and it is estimated that the number

Both types of diabetes are characterized by a progressive decline of pancreatic beta cell function and mass. In type 1 diabetes, the chronic autoimmune process causes the selective destruction of insulin-producing beta cells by the auto-reactive T cells in genetically predisposed individuals. There is a continuous loss of functional C-peptide responses and at the time of clinical presentation the beta cell mass is reduced by 70–90 %, as suggested by anatomic studies (2, 3). This results in an inability to secrete sufficient amounts of insulin and loss of metabolic control. As a consequence, exogenous insulin replacement in the form of multiple subcutaneous injections or continuous subcutaneous insulin infusions (CSII) is essential for patients with type 1 diabetes. It prevents death from acute metabolic complications and assures normal growth and development, maintenance of

Type 2 diabetes results from an entirely different pathophysiological process*.* It is characterized by an increased resistance to insulin action in the peripheral tissues with decreased glucose uptake and enhanced hepatic glucose output associated with impaired insulin-secretory capacity caused by a progressive decline of beta cell function over time. Studies indicate a substantial loss of beta cell mass (of about 25-60 %) by the time of diagnosis, mainly secondary to increased apoptosis and impaired augmentation of cell mass through neogenesis (4, 5). The clinical onset is due to the reduction of beta cell mass per se and to a concomitant dysfunction of residual beta cells (6, 7). The beta cell failure, which seems to occur much earlier during the natural history of the disease than previously thought, results in significant insulin deficiency and by then, insulin administration is

will increase to 438 million in 2030 (1). About 5-10% of them have type 1 diabetes.

normoglycemia and prevention of end-organ complications.

required in order to achieve glycemic control (8, 9).

**1. Introduction** 

## **Insulin Therapy and Hypoglycemia - Present and Future**

Simona Cernea1, Ron Nagar2, Gabriel Bitton2 and Itamar Raz3

*1Diabetes, Nutrition and Metabolic Diseases Outpatient Unit, Emergency County Clinical Hospital, Târgu Mureş, 2InsuLine Medical Ltd., Petach-Tikva, 3Diabetes Center, Hadassah-Hebrew University Medical School, Jerusalem, 1Romania 2,3Israel* 

### **1. Introduction**

Over the last few decades the prevalence of diabetes has dramatically grown in most regions of the world. In 2010, 285 million people had diabetes and it is estimated that the number will increase to 438 million in 2030 (1). About 5-10% of them have type 1 diabetes.

Both types of diabetes are characterized by a progressive decline of pancreatic beta cell function and mass. In type 1 diabetes, the chronic autoimmune process causes the selective destruction of insulin-producing beta cells by the auto-reactive T cells in genetically predisposed individuals. There is a continuous loss of functional C-peptide responses and at the time of clinical presentation the beta cell mass is reduced by 70–90 %, as suggested by anatomic studies (2, 3). This results in an inability to secrete sufficient amounts of insulin and loss of metabolic control. As a consequence, exogenous insulin replacement in the form of multiple subcutaneous injections or continuous subcutaneous insulin infusions (CSII) is essential for patients with type 1 diabetes. It prevents death from acute metabolic complications and assures normal growth and development, maintenance of normoglycemia and prevention of end-organ complications.

Type 2 diabetes results from an entirely different pathophysiological process*.* It is characterized by an increased resistance to insulin action in the peripheral tissues with decreased glucose uptake and enhanced hepatic glucose output associated with impaired insulin-secretory capacity caused by a progressive decline of beta cell function over time. Studies indicate a substantial loss of beta cell mass (of about 25-60 %) by the time of diagnosis, mainly secondary to increased apoptosis and impaired augmentation of cell mass through neogenesis (4, 5). The clinical onset is due to the reduction of beta cell mass per se and to a concomitant dysfunction of residual beta cells (6, 7). The beta cell failure, which seems to occur much earlier during the natural history of the disease than previously thought, results in significant insulin deficiency and by then, insulin administration is required in order to achieve glycemic control (8, 9).

Insulin Therapy and Hypoglycemia - Present and Future 163

It is well established that poorly controlled diabetes is associated with development of chronic micro- and macrovascular complications. Experimental studies demonstrated the atherogenic role of postprandial glycemic peaks and the link between the post-meal or postchallenge hyperglycemia (2hPG) and cardiovascular morbidity and mortality. Two metaanalyses have shown an exponential relationship between incidence of cardiovascular events and fasting glucose or 2hPG (31, 32). The relationship was stronger and highly significant for 2hPG and there seemed to be no threshold for 2hPG. Several populationbased studies have basically confirmed this finding indicating an increased relative risk (in the range of 1.18 to 3.3) of cardiovascular or coronary heart disease mortality in patients with increased 2hPG (33). It has been reported that in individuals with type 2 diabetes, especially women, postprandial plasma glucose is a stronger predictor of cardiovascular events than fasting glucose levels (34). Another study indicated that both fasting and postmeal glycemia were predictive for cardiovascular events after adjusting for other risk factors

A growing body of evidence shows that there is a relationship between postprandial hyperglycemia and markers of cardiovascular disease such as oxidative stress, carotid IMT and endothelial dysfunction. Oxidative stress has been implicated as a cause of both macroand microvascular complications of diabetes. The proposed mechanism is that hyperglycemia, insulin resistance and free fatty acids feed into oxidative stress, activation of RAGE and PKC, which leads to vascular inflammation, thrombosis and vasoconstriction (36). Furthermore, increased risk of retinopathy, certain cancers and cognitive dysfunction in elderly was shown

The Kumamoto study demonstrated that postprandial glycemia was strongly associated with onset of retinopathy and nephropathy (as were fasting blood glucose and HbA1c) and that control of both fasting glucose levels < 110 mg/dl and post-meal glucose levels < 180 mg/dl prevented the onset and progression of diabetic microvascular complications (19, 20). On the other hand, the cost of strict glycemic control and intensive therapy is an increased risk of hypoglycemia, which per-se is a limiting factor in achieving long-term near-normal glucose control in patients with diabetes (40). Depending on its degree, hypoglycemia can affect physical and cognitive functions and can induce negative psychological and social consequences (41). Studies have consistently indicated a higher rate of hypoglycemia in patients with type 1 diabetes treated to lower HbA1c targets (40, 42). In the DCCT, the frequency of severe hypoglycemia was three times higher in subjects treated with intensive insulin therapy compared with those on conventional therapy, while in the Stockholm Diabetes Intervention Study - severe hypoglycemia occurred 2.5 times more frequently in the intensively treated group (43, 44). Insulin-treated subjects with type 2 diabetes experience severe hypoglycemia less frequently than patients with type 1 diabetes. This fact is explainable in part by the maintenance of some beta cell function (which allows a decrease of insulin secretion when blood glucose falls) and by insulin resistance (41). However, data from UKPDS provide evidence that the risk of hypoglycemia increases with longer duration of insulin treatment. Another study reported similar frequencies of severe hypoglycemia in patients with type 2 and type 1 diabetes after matching for duration of insulin therapy (45, 46). It is plausible that in real life patients on intensive insulin regimens experience higher rates of hypoglycemia, but since there is relatively limited data on the actual frequency of asymptomatic and mild hypoglycemia, episodes of mild hypoglycemia may be

to be associated with postprandial hyperglycemia in type 2 diabetic patients (37-39).

in type 2 diabetic subjects (35).

underestimated and/or underreported (41).

### **2. Intensive insulin regimens: Evidence for benefit**

It is well established that in patients with both types of diabetes obtaining a good metabolic control is of paramount importance because the risk of developing chronic micro- and macrovascular complications is dependent on the degree of glycemic control (10). Current guidelines from professional organizations recommend achieving glycated hemoglobin (HbA1c) levels lower than 7% (and closer to normal values in selected individuals, if this could be achieved without significant increase in hypoglycemic events or other side effects) (11). Several landmark studies emphasize the importance of more physiologic insulin profiles in reaching these goals.

The Diabetes Control and Complications Trial (DCCT) proved that tighter glycemic control after onset obtained with intensive insulin regimens can prevent / delay microvascular complications in patients with type 1 diabetes compared with conventional insulin regimens (12). The follow-up of the DCCT, the Epidemiology of Diabetes Interventions and Complications (EDIC) study provided evidence for the sustained benefit in subjects with prior intensive treatment, even if during the follow-up period the glycemic control was similar to that of subjects previously receiving conventional therapy (13-16). These studies demonstrated that the risk of developing long-term complications is determined both by the degree and the total duration of glycemic exposure. In addition, the DCCT established the relationship between glucose control and residual beta cell function as subjects with stimulated C-peptide concentrations > 0.2 pmol/ml had better outcomes (17, 18). The maintenance of endogenous beta cell function was associated with diminished disease progression, improved long term metabolic control and reduced chronic complications. These studies highlighted the role of insulin therapy over long-term.

In patients with type 2 diabetes similar benefits of intensive insulin regimens have been shown. In the Kumamoto study, which included a smaller patient population, intensive glycemic control obtained by multiple insulin injection therapy delayed the onset and progression of the early stages of diabetic microvascular complications (19, 20). Likewise, the United Kingdom Prospective Diabetes Study (UKPDS) emphasized the role of glycemic control in reducing the incidence of chronic complications in patients with type 2 diabetes, although in this study the intensive treatments were not limited to insulin regimens (21-23). Similar to EDIC, the follow-up of the UKPDS cohort showed the persistence of microvascular benefits in patients formerly treated with intensive regimens (24). A more recent study in subjects with newly diagnosed type 2 diabetes demonstrated that transient intensive insulin therapy (with continuous subcutaneous insulin infusion or multiple daily insulin injections) resulted in favorable outcomes on glycemic control and beta cell function compared to oral hypoglycemic agents (25). Trials in patients with type 2 diabetes of longer duration have also supported the benefits (even if more modest) on the onset / progression of chronic complications (26-28).

### **3. The importance of controlling postprandial hyperglycemia and hypoglycemic events**

To date, the therapeutic interventions have mainly been focused on lowering HbA1c with emphasis on fasting blood glucose levels. However, in order to obtain optimal glycemic control with HbA1c levels < 7%, controlling both fasting and post-meal glycemia is necessary (29, 30).

It is well established that in patients with both types of diabetes obtaining a good metabolic control is of paramount importance because the risk of developing chronic micro- and macrovascular complications is dependent on the degree of glycemic control (10). Current guidelines from professional organizations recommend achieving glycated hemoglobin (HbA1c) levels lower than 7% (and closer to normal values in selected individuals, if this could be achieved without significant increase in hypoglycemic events or other side effects) (11). Several landmark studies emphasize the importance of more physiologic insulin

The Diabetes Control and Complications Trial (DCCT) proved that tighter glycemic control after onset obtained with intensive insulin regimens can prevent / delay microvascular complications in patients with type 1 diabetes compared with conventional insulin regimens (12). The follow-up of the DCCT, the Epidemiology of Diabetes Interventions and Complications (EDIC) study provided evidence for the sustained benefit in subjects with prior intensive treatment, even if during the follow-up period the glycemic control was similar to that of subjects previously receiving conventional therapy (13-16). These studies demonstrated that the risk of developing long-term complications is determined both by the degree and the total duration of glycemic exposure. In addition, the DCCT established the relationship between glucose control and residual beta cell function as subjects with stimulated C-peptide concentrations > 0.2 pmol/ml had better outcomes (17, 18). The maintenance of endogenous beta cell function was associated with diminished disease progression, improved long term metabolic control and reduced chronic complications.

In patients with type 2 diabetes similar benefits of intensive insulin regimens have been shown. In the Kumamoto study, which included a smaller patient population, intensive glycemic control obtained by multiple insulin injection therapy delayed the onset and progression of the early stages of diabetic microvascular complications (19, 20). Likewise, the United Kingdom Prospective Diabetes Study (UKPDS) emphasized the role of glycemic control in reducing the incidence of chronic complications in patients with type 2 diabetes, although in this study the intensive treatments were not limited to insulin regimens (21-23). Similar to EDIC, the follow-up of the UKPDS cohort showed the persistence of microvascular benefits in patients formerly treated with intensive regimens (24). A more recent study in subjects with newly diagnosed type 2 diabetes demonstrated that transient intensive insulin therapy (with continuous subcutaneous insulin infusion or multiple daily insulin injections) resulted in favorable outcomes on glycemic control and beta cell function compared to oral hypoglycemic agents (25). Trials in patients with type 2 diabetes of longer duration have also supported the benefits (even if more modest) on the onset / progression

**2. Intensive insulin regimens: Evidence for benefit** 

These studies highlighted the role of insulin therapy over long-term.

**3. The importance of controlling postprandial hyperglycemia and** 

To date, the therapeutic interventions have mainly been focused on lowering HbA1c with emphasis on fasting blood glucose levels. However, in order to obtain optimal glycemic control with HbA1c levels < 7%, controlling both fasting and post-meal glycemia is

profiles in reaching these goals.

of chronic complications (26-28).

**hypoglycemic events** 

necessary (29, 30).

It is well established that poorly controlled diabetes is associated with development of chronic micro- and macrovascular complications. Experimental studies demonstrated the atherogenic role of postprandial glycemic peaks and the link between the post-meal or postchallenge hyperglycemia (2hPG) and cardiovascular morbidity and mortality. Two metaanalyses have shown an exponential relationship between incidence of cardiovascular events and fasting glucose or 2hPG (31, 32). The relationship was stronger and highly significant for 2hPG and there seemed to be no threshold for 2hPG. Several populationbased studies have basically confirmed this finding indicating an increased relative risk (in the range of 1.18 to 3.3) of cardiovascular or coronary heart disease mortality in patients with increased 2hPG (33). It has been reported that in individuals with type 2 diabetes, especially women, postprandial plasma glucose is a stronger predictor of cardiovascular events than fasting glucose levels (34). Another study indicated that both fasting and postmeal glycemia were predictive for cardiovascular events after adjusting for other risk factors in type 2 diabetic subjects (35).

A growing body of evidence shows that there is a relationship between postprandial hyperglycemia and markers of cardiovascular disease such as oxidative stress, carotid IMT and endothelial dysfunction. Oxidative stress has been implicated as a cause of both macroand microvascular complications of diabetes. The proposed mechanism is that hyperglycemia, insulin resistance and free fatty acids feed into oxidative stress, activation of RAGE and PKC, which leads to vascular inflammation, thrombosis and vasoconstriction (36). Furthermore, increased risk of retinopathy, certain cancers and cognitive dysfunction in elderly was shown to be associated with postprandial hyperglycemia in type 2 diabetic patients (37-39).

The Kumamoto study demonstrated that postprandial glycemia was strongly associated with onset of retinopathy and nephropathy (as were fasting blood glucose and HbA1c) and that control of both fasting glucose levels < 110 mg/dl and post-meal glucose levels < 180 mg/dl prevented the onset and progression of diabetic microvascular complications (19, 20). On the other hand, the cost of strict glycemic control and intensive therapy is an increased risk of hypoglycemia, which per-se is a limiting factor in achieving long-term near-normal glucose control in patients with diabetes (40). Depending on its degree, hypoglycemia can affect physical and cognitive functions and can induce negative psychological and social consequences (41). Studies have consistently indicated a higher rate of hypoglycemia in patients with type 1 diabetes treated to lower HbA1c targets (40, 42). In the DCCT, the frequency of severe hypoglycemia was three times higher in subjects treated with intensive insulin therapy compared with those on conventional therapy, while in the Stockholm Diabetes Intervention Study - severe hypoglycemia occurred 2.5 times more frequently in the intensively treated group (43, 44). Insulin-treated subjects with type 2 diabetes experience severe hypoglycemia less frequently than patients with type 1 diabetes. This fact is explainable in part by the maintenance of some beta cell function (which allows a decrease of insulin secretion when blood glucose falls) and by insulin resistance (41). However, data from UKPDS provide evidence that the risk of hypoglycemia increases with longer duration of insulin treatment. Another study reported similar frequencies of severe hypoglycemia in patients with type 2 and type 1 diabetes after matching for duration of insulin therapy (45, 46). It is plausible that in real life patients on intensive insulin regimens experience higher rates of hypoglycemia, but since there is relatively limited data on the actual frequency of asymptomatic and mild hypoglycemia, episodes of mild hypoglycemia may be underestimated and/or underreported (41).

Insulin Therapy and Hypoglycemia - Present and Future 165

attempt to replicate the normal insulin secretion by combining basal and meal-time insulin

The "gold-standard" of insulin replacement is CSII by means of a pump, which delivers shortacting insulin in a continuous manner at determined rates and assures a peakless insulin profile between meals and insulin surges at meal-time (56, 58). The short-acting insulin analogues are better suited for CSII because of their faster absorption from subcutaneous tissue (59). The basal insulin rates can be adjusted on an hourly basis according to blood glucose oscillations to meet the 24-hour requirements of each individual and should provide about half of the total daily dose. The prandial doses are calculated by the patients and delivered according to blood glucose monitoring results, target glucose levels, carbohydrate content of the meal, physical activity, insulin sensitivity and other factors (58). Several studies have shown that CSII offers more flexibility and provides better glycemic control with improved HbA1c levels and fewer hypoglycemic events due to lower variability and better reproducibility of insulin absorption (probably resulting from the fact that the subcutaneous insulin depot is smaller) (60, 61). However, the cost of such therapy is too high to be widely available and it also requires significant patient involvement, education and motivation. Alternatively the basal/bolus replacement can be supplied in the form of multiple daily injections. Traditionally, the regimens consisted of two injections of NPH (in the morning and at bedtime) plus 2-3 injections of regular insulin with meals. The problem with the intermediate-acting insulin preparations like NPH is that their pharmacokinetic profile does not provide a physiologic basal replacement: they have a peak at about 4-6 hours post subcutaneous injection and the action wanes rapidly at about 5-6 hours after the peak (56, 58). This profile increases the risk of nocturnal hypoglycemia (even with a bedtime snack intake), because at the time of their highest concentration (which usually occurs between midnight and 2-3 a.m.) the insulin sensitivity is higher and patients would require less basal insulin (56). Nocturnal hypoglycemia is a serious concern because it causes morning hyperglycemia through the release of counter-regulatory hormones (glucagon, epinephrine, growth hormone, cortisol) and prolonged insulin resistance and influences different physical and psychological functions during the following day. In addition, undetected nocturnal hypoglycemic episodes contribute to hypoglycemia counter-regulatory failure and unawareness, which in turn predisposes to severe hypoglycemia and profoundly impacts on patients' quality of life (62). On the other hand, the time-action profile of the intermediate-acting insulin poses another problem: during the morning hours (after 4 a.m.) the requirement for basal insulin is greater due to increased release of counter-regulatory hormones and by then the insulin action is waning, which results in morning hyperglycemia. An attempt to correct this by increasing the bedtime insulin dose may result

A different approach of multiple daily injections which attempts to alleviate these problems uses short-acting insulin analogues at meal-time with one or two injections of long-acting insulin analogues (glargine or detemir) and it is the preferred regimen in recent years (58). The long-acting analogues afford less glycemic fluctuations, less variability, reduced risk of hypoglycemic events and a significantly prolonged duration of action (17-24 hours) due to a steady absorption into the circulation and more stable serum concentrations (63, 64). Studies have indicated fewer overall, nocturnal and severe hypoglycemic episodes in both types of diabetes (especially in type 1), while providing similar or slightly improved metabolic

replacements (58).

in higher risk of nocturnal hypoglycemia.

control compared with NPH insulin (65-67).

Hypoglycemia, even mild (especially if it occurs recurrently), can be associated with negative effects, such as impaired autonomic counter-regulation, compromised behavioral defenses against subsequent decreasing glucose concentrations and hypoglycemia unawareness, which causes a vicious cycle of recurrent hypoglycemia (41, 47). Severe hypoglycemia may exert even more serious side effects, such as seizures, unconsciousness (which may be particularly debilitating in the elderly), coma and even death (48). In older patients with type 2 diabetes and a history of severe hypoglycemia, an increased risk of dementia has been reported, particularly for patients who have a history of multiple episodes (49). In the UKPDS, recurrent hypoglycemia was associated with decreased quality of life in patients treated with insulin (50). Moreover, the unpleasant symptoms and negative consequences of hypoglycemia may result in fear and anxiety, lower treatment satisfaction, which in turn may negatively impact the diabetes management and adherence to therapy, precluding a full attainment of the benefits offered by improved glycemic control (48).

Evidence exist that hypoglycemic episodes, especially severe ones, are associated with adverse cardiovascular events (such as prolongation of the QT interval, cardiac arrhythmias, sudden cardiac arrest, and acute myocardial infarction), which are triggered by the stimulation of the sympathetic nervous system and the catecholamine surge (51, 52). Hypoglycemia also has proinflammatory consequences that may augment the risk of plaque inflammation and rupture, causing subsequent cardiovascular events (51). Hypoglycemia, mainly the recurrent and severe episodes, and the presumed ensuing cardiovascular toxicity may increase the susceptibility to poor cardiovascular outcomes, especially in subjects with significant atherosclerosis and functional / structural heart abnormalities. The cause of excess mortality during intensive therapy seen in the ACCORD study is not entirely clear, but it is thought that the most plausible cause is iatrogenic hypoglycemia (51).

Thus, it is equally important to avoid both hyperglycemic surges and hypoglycemic events while striving to obtain a tight metabolic control.

### **4. Restoring physiological insulin secretory profiles**

In the normal, physiologic conditions there is a low basal insulin output that suppresses endogenous hepatic glucose production (overnight and between meals) as well as incremental responses of insulin secretion following food ingestion.

After a meal, blood glucose concentrations start rising within 15 minutes, reach a peak at about 30-45 minutes and within 1-2 hours return to basal levels and remain stable until the next food ingestion (53, 54). The maximal amplitude of glucose excursion depends on the amount and type of carbohydrates ingested (53). These dynamics are mirrored by the prandial insulin secretion profile: there is an initial (first) phase, which peaks in 2-3 minutes and lasts about 10 minutes, then there is a second phase of insulin release that becomes apparent after 10 minutes and continues as long as the glucose concentrations remain elevated and is concordant with the amount of carbohydrates absorbed (54-56). Once the blood glucose levels decrease, insulin secretion returns to baseline values, in order to prevent hypoglycemia in the post-absorptive phase (56).

It is believed that insulin regimens that best mimic the physiological pattern of insulin production are most likely to reach near-normal glycemic control by regulating both fasting and postprandial blood glucose levels (56, 57). These regimens require a sharp increase of insulin levels after meals and flat, nearly constant plasma insulin concentrations in the postabsorbtive / interprandial periods. They are known as basal-bolus therapy because they

Hypoglycemia, even mild (especially if it occurs recurrently), can be associated with negative effects, such as impaired autonomic counter-regulation, compromised behavioral defenses against subsequent decreasing glucose concentrations and hypoglycemia unawareness, which causes a vicious cycle of recurrent hypoglycemia (41, 47). Severe hypoglycemia may exert even more serious side effects, such as seizures, unconsciousness (which may be particularly debilitating in the elderly), coma and even death (48). In older patients with type 2 diabetes and a history of severe hypoglycemia, an increased risk of dementia has been reported, particularly for patients who have a history of multiple episodes (49). In the UKPDS, recurrent hypoglycemia was associated with decreased quality of life in patients treated with insulin (50). Moreover, the unpleasant symptoms and negative consequences of hypoglycemia may result in fear and anxiety, lower treatment satisfaction, which in turn may negatively impact the diabetes management and adherence to therapy, precluding a full attainment of the

Evidence exist that hypoglycemic episodes, especially severe ones, are associated with adverse cardiovascular events (such as prolongation of the QT interval, cardiac arrhythmias, sudden cardiac arrest, and acute myocardial infarction), which are triggered by the stimulation of the sympathetic nervous system and the catecholamine surge (51, 52). Hypoglycemia also has proinflammatory consequences that may augment the risk of plaque inflammation and rupture, causing subsequent cardiovascular events (51). Hypoglycemia, mainly the recurrent and severe episodes, and the presumed ensuing cardiovascular toxicity may increase the susceptibility to poor cardiovascular outcomes, especially in subjects with significant atherosclerosis and functional / structural heart abnormalities. The cause of excess mortality during intensive therapy seen in the ACCORD study is not entirely clear,

Thus, it is equally important to avoid both hyperglycemic surges and hypoglycemic events

In the normal, physiologic conditions there is a low basal insulin output that suppresses endogenous hepatic glucose production (overnight and between meals) as well as

After a meal, blood glucose concentrations start rising within 15 minutes, reach a peak at about 30-45 minutes and within 1-2 hours return to basal levels and remain stable until the next food ingestion (53, 54). The maximal amplitude of glucose excursion depends on the amount and type of carbohydrates ingested (53). These dynamics are mirrored by the prandial insulin secretion profile: there is an initial (first) phase, which peaks in 2-3 minutes and lasts about 10 minutes, then there is a second phase of insulin release that becomes apparent after 10 minutes and continues as long as the glucose concentrations remain elevated and is concordant with the amount of carbohydrates absorbed (54-56). Once the blood glucose levels decrease, insulin secretion returns to baseline values, in order to

It is believed that insulin regimens that best mimic the physiological pattern of insulin production are most likely to reach near-normal glycemic control by regulating both fasting and postprandial blood glucose levels (56, 57). These regimens require a sharp increase of insulin levels after meals and flat, nearly constant plasma insulin concentrations in the postabsorbtive / interprandial periods. They are known as basal-bolus therapy because they

but it is thought that the most plausible cause is iatrogenic hypoglycemia (51).

benefits offered by improved glycemic control (48).

while striving to obtain a tight metabolic control.

**4. Restoring physiological insulin secretory profiles** 

prevent hypoglycemia in the post-absorptive phase (56).

incremental responses of insulin secretion following food ingestion.

attempt to replicate the normal insulin secretion by combining basal and meal-time insulin replacements (58).

The "gold-standard" of insulin replacement is CSII by means of a pump, which delivers shortacting insulin in a continuous manner at determined rates and assures a peakless insulin profile between meals and insulin surges at meal-time (56, 58). The short-acting insulin analogues are better suited for CSII because of their faster absorption from subcutaneous tissue (59). The basal insulin rates can be adjusted on an hourly basis according to blood glucose oscillations to meet the 24-hour requirements of each individual and should provide about half of the total daily dose. The prandial doses are calculated by the patients and delivered according to blood glucose monitoring results, target glucose levels, carbohydrate content of the meal, physical activity, insulin sensitivity and other factors (58). Several studies have shown that CSII offers more flexibility and provides better glycemic control with improved HbA1c levels and fewer hypoglycemic events due to lower variability and better reproducibility of insulin absorption (probably resulting from the fact that the subcutaneous insulin depot is smaller) (60, 61). However, the cost of such therapy is too high to be widely available and it also requires significant patient involvement, education and motivation.

Alternatively the basal/bolus replacement can be supplied in the form of multiple daily injections. Traditionally, the regimens consisted of two injections of NPH (in the morning and at bedtime) plus 2-3 injections of regular insulin with meals. The problem with the intermediate-acting insulin preparations like NPH is that their pharmacokinetic profile does not provide a physiologic basal replacement: they have a peak at about 4-6 hours post subcutaneous injection and the action wanes rapidly at about 5-6 hours after the peak (56, 58). This profile increases the risk of nocturnal hypoglycemia (even with a bedtime snack intake), because at the time of their highest concentration (which usually occurs between midnight and 2-3 a.m.) the insulin sensitivity is higher and patients would require less basal insulin (56). Nocturnal hypoglycemia is a serious concern because it causes morning hyperglycemia through the release of counter-regulatory hormones (glucagon, epinephrine, growth hormone, cortisol) and prolonged insulin resistance and influences different physical and psychological functions during the following day. In addition, undetected nocturnal hypoglycemic episodes contribute to hypoglycemia counter-regulatory failure and unawareness, which in turn predisposes to severe hypoglycemia and profoundly impacts on patients' quality of life (62). On the other hand, the time-action profile of the intermediate-acting insulin poses another problem: during the morning hours (after 4 a.m.) the requirement for basal insulin is greater due to increased release of counter-regulatory hormones and by then the insulin action is waning, which results in morning hyperglycemia. An attempt to correct this by increasing the bedtime insulin dose may result in higher risk of nocturnal hypoglycemia.

A different approach of multiple daily injections which attempts to alleviate these problems uses short-acting insulin analogues at meal-time with one or two injections of long-acting insulin analogues (glargine or detemir) and it is the preferred regimen in recent years (58). The long-acting analogues afford less glycemic fluctuations, less variability, reduced risk of hypoglycemic events and a significantly prolonged duration of action (17-24 hours) due to a steady absorption into the circulation and more stable serum concentrations (63, 64). Studies have indicated fewer overall, nocturnal and severe hypoglycemic episodes in both types of diabetes (especially in type 1), while providing similar or slightly improved metabolic control compared with NPH insulin (65-67).

Insulin Therapy and Hypoglycemia - Present and Future 167

the insulin analogues have a smaller intra- and inter-individual variability compared to regular insulin which could provide a somewhat improved glycemic control and potentially

Insulin Onset of action Peak action Total duration of action

5-15 min 1-2 h 4 h

Table 1. The pharmacodynamic profiles of currently available prandial insulin formulations

However, even with the insulin analogues the synchronization between insulin action and glucose absorption from a meal is still less than ideal, as they do not replicate normal physiology, and many patients still have suboptimal glucose control. Several meta-analyses have suggested that insulin analogues offer rather modest or inconsistent clinical advantages over conventional insulin in terms of lowering HbA1c and reducing hypoglycemia, in children and adults with type 1 diabetes (72-76). Data on the influence on hypoglycemia is particularly inconsistent. Some studies have shown that in fact the overall frequency of hypoglycemic episodes were similar with analogue insulin and regular insulin use in adults with type 1 diabetes and were modestly decreased in children (72-77). Moreover, some reports indicated that the frequency of severe and nocturnal hypoglycemia seemed to be reduced with analogues in adults, but not in prepubertal children, while others found no difference in the frequency of severe or nocturnal hypoglycemia and no evidence for reduction in patient awareness for hypoglycemia with insulin analogues (72-78). It should be noted that hypoglycemia occurrence is not fully attributable to the pharmacokinetic profile of the insulin preparations, but may also result from a mismatch between insulin dose and the carbohydrate

On the other hand, postprandial hyperglycemia still occurs with the new insulin analogues (80, 81). Hyperglycemic postprandial glucose excursions were found to reach levels over 300 mg/dl in about 50% and over 180 mg/dl in almost 90% of children with type 1 diabetes with good overall metabolic control (82). The findings were confirmed by other studies that indicated postprandial glucose levels higher than 300 mg/dl in subjects with type 1 diabetes receiving multiple insulin injection therapy (83). Targeting postprandial hyperglycemia is important in order to improve HbA1c levels and this has also been recently highlighted by

In everyday life the control of postprandial hyperglycemia poses even more challenges due to variations in dietary intake and physical exercise or insulin dosage and timing changes (patients may modify the timing of insulin administration in the sense of dosing immediately before or even after meals in order to fit their lifestyle / daily activity requirements) (86). In addition, lack of predictable insulin response may occur with insulin analogues because their absorption can be affected by various factors such as: mechanics of injection, the injection site, and metabolic factors, similar to regular human insulin (87).

Regular 30-45 min 2-3 h 5-8 h

content of the meal, delayed food intake or other factors (79).

the International Diabetes Federation guidelines (84, 85).

reduced risk of hypoglycemia (69).

**Short-acting**

**Rapid-acting** 

Lispro Aspart Glulisine

(68)

### **5. The limitations of current prandial insulin treatment for type 1 and type 2 diabetes**

Multiple daily insulin injections are the mainstay of insulin delivery for many patients with type 1 diabetes and patients with type 2 diabetes that cannot be controlled with other regimens, especially those with longer duration of the disease and severe insulin deficiency. Despite the evidence and increased awareness of the necessity to achieve strict glycemic control, current insulin therapy has some limitations that preclude reaching the goal of maintaining near-normal glycemia in the long-term, even in compliant patients. The major challenges are related to avoiding postprandial hyperglycemia and late hypoglycemia, which are mainly caused by the mismatch between the time-action profile of the administered insulin and postprandial glucose excursions.

The "conventional" prandial insulin therapy with regular human insulin has its shortcomings in terms of the pharmacokinetic properties which limit their clinical efficacy: the onset of action is slow, the peak is reached in about 2-3 hours and the total duration of action lasts 5-8 hours (68). This is caused by the fact that the dissociation rate of human insulin from hexamers into monomers in the subcutaneous tissues is slow and the absorption into the bloodstream is gradual. Thus, the maximal insulin concentrations do not occur at the time when glucose levels are the highest, and so the short-acting insulin has to be administered 30-45 minutes before meals in order to minimize postprandial hyperglycemia. This is quite inconvenient for patients (and poses a risk of pre-meal hypoglycemia if the food intake is inadvertently delayed) and even so, the time-action profile is not optimal. Glycemic excursions are not properly covered and 4-5 hours postinjection, after the food absorption is completed, there is still some insulin absorption from subcutaneous depot (58). This results in relative hyperinsulinemia, which increases the risk of late postprandial hypoglycemia and would require a snack intake to prevent it. Moreover, the regular human insulin preparations have important intra- and interindividual variations that result in unpredictable effects and makes it even more difficult to avoid hyper- and hypoglycemia (69).

In order to overcome the problems of non-physiologic pharmacokinetics, the regular human insulins have been largely substituted with the newer insulin analogues that were developed by means of protein engineering and recombinant DNA technology to enable better glycemic control by faster action (70). The insulin analogues have been obtained by substitution or minimal alterations in the amino acid sequence in regions of the molecule not essential for binding to the insulin receptor but pivotal for dimer formation in order to diminish the tendency of self-association between insulin molecules and allow a faster absorption from injection site (70). There are three rapid-acting insulin analogues available at the moment: insulin lispro (based on amino-acid substitution of proline at position B28 and lysine at position B29), insulin aspart (with aspartic acid substituted for proline at position B28) and insulin glulisine (that has an asparagine to lysine substitution at position B3, and a lysine to glutamine acid substitution at position B29) (71). Despite the differences in structure, the three analogues have similar pharmacokinetic and pharmacodynamic properties (70). Their onset of action is more rapid, which permits an administration within 10-15 minutes before meals, the peak is greater and occurs at about 1-2 hours and the total duration of action is shorter (4-5 hours) compared to regular human insulin (Table 1) (58, 68). This allows an improved replacement of mealtime insulin needs with regards to postprandial plasma glucose control and more flexibility than regular insulin. In addition,

**5. The limitations of current prandial insulin treatment for type 1 and type 2** 

Multiple daily insulin injections are the mainstay of insulin delivery for many patients with type 1 diabetes and patients with type 2 diabetes that cannot be controlled with other regimens, especially those with longer duration of the disease and severe insulin deficiency. Despite the evidence and increased awareness of the necessity to achieve strict glycemic control, current insulin therapy has some limitations that preclude reaching the goal of maintaining near-normal glycemia in the long-term, even in compliant patients. The major challenges are related to avoiding postprandial hyperglycemia and late hypoglycemia, which are mainly caused by the mismatch between the time-action profile of the

The "conventional" prandial insulin therapy with regular human insulin has its shortcomings in terms of the pharmacokinetic properties which limit their clinical efficacy: the onset of action is slow, the peak is reached in about 2-3 hours and the total duration of action lasts 5-8 hours (68). This is caused by the fact that the dissociation rate of human insulin from hexamers into monomers in the subcutaneous tissues is slow and the absorption into the bloodstream is gradual. Thus, the maximal insulin concentrations do not occur at the time when glucose levels are the highest, and so the short-acting insulin has to be administered 30-45 minutes before meals in order to minimize postprandial hyperglycemia. This is quite inconvenient for patients (and poses a risk of pre-meal hypoglycemia if the food intake is inadvertently delayed) and even so, the time-action profile is not optimal. Glycemic excursions are not properly covered and 4-5 hours postinjection, after the food absorption is completed, there is still some insulin absorption from subcutaneous depot (58). This results in relative hyperinsulinemia, which increases the risk of late postprandial hypoglycemia and would require a snack intake to prevent it. Moreover, the regular human insulin preparations have important intra- and interindividual variations that result in unpredictable effects and makes it even more difficult to

In order to overcome the problems of non-physiologic pharmacokinetics, the regular human insulins have been largely substituted with the newer insulin analogues that were developed by means of protein engineering and recombinant DNA technology to enable better glycemic control by faster action (70). The insulin analogues have been obtained by substitution or minimal alterations in the amino acid sequence in regions of the molecule not essential for binding to the insulin receptor but pivotal for dimer formation in order to diminish the tendency of self-association between insulin molecules and allow a faster absorption from injection site (70). There are three rapid-acting insulin analogues available at the moment: insulin lispro (based on amino-acid substitution of proline at position B28 and lysine at position B29), insulin aspart (with aspartic acid substituted for proline at position B28) and insulin glulisine (that has an asparagine to lysine substitution at position B3, and a lysine to glutamine acid substitution at position B29) (71). Despite the differences in structure, the three analogues have similar pharmacokinetic and pharmacodynamic properties (70). Their onset of action is more rapid, which permits an administration within 10-15 minutes before meals, the peak is greater and occurs at about 1-2 hours and the total duration of action is shorter (4-5 hours) compared to regular human insulin (Table 1) (58, 68). This allows an improved replacement of mealtime insulin needs with regards to postprandial plasma glucose control and more flexibility than regular insulin. In addition,

administered insulin and postprandial glucose excursions.

avoid hyper- and hypoglycemia (69).

**diabetes** 

the insulin analogues have a smaller intra- and inter-individual variability compared to regular insulin which could provide a somewhat improved glycemic control and potentially reduced risk of hypoglycemia (69).


Table 1. The pharmacodynamic profiles of currently available prandial insulin formulations (68)

However, even with the insulin analogues the synchronization between insulin action and glucose absorption from a meal is still less than ideal, as they do not replicate normal physiology, and many patients still have suboptimal glucose control. Several meta-analyses have suggested that insulin analogues offer rather modest or inconsistent clinical advantages over conventional insulin in terms of lowering HbA1c and reducing hypoglycemia, in children and adults with type 1 diabetes (72-76). Data on the influence on hypoglycemia is particularly inconsistent. Some studies have shown that in fact the overall frequency of hypoglycemic episodes were similar with analogue insulin and regular insulin use in adults with type 1 diabetes and were modestly decreased in children (72-77). Moreover, some reports indicated that the frequency of severe and nocturnal hypoglycemia seemed to be reduced with analogues in adults, but not in prepubertal children, while others found no difference in the frequency of severe or nocturnal hypoglycemia and no evidence for reduction in patient awareness for hypoglycemia with insulin analogues (72-78). It should be noted that hypoglycemia occurrence is not fully attributable to the pharmacokinetic profile of the insulin preparations, but may also result from a mismatch between insulin dose and the carbohydrate content of the meal, delayed food intake or other factors (79).

On the other hand, postprandial hyperglycemia still occurs with the new insulin analogues (80, 81). Hyperglycemic postprandial glucose excursions were found to reach levels over 300 mg/dl in about 50% and over 180 mg/dl in almost 90% of children with type 1 diabetes with good overall metabolic control (82). The findings were confirmed by other studies that indicated postprandial glucose levels higher than 300 mg/dl in subjects with type 1 diabetes receiving multiple insulin injection therapy (83). Targeting postprandial hyperglycemia is important in order to improve HbA1c levels and this has also been recently highlighted by the International Diabetes Federation guidelines (84, 85).

In everyday life the control of postprandial hyperglycemia poses even more challenges due to variations in dietary intake and physical exercise or insulin dosage and timing changes (patients may modify the timing of insulin administration in the sense of dosing immediately before or even after meals in order to fit their lifestyle / daily activity requirements) (86). In addition, lack of predictable insulin response may occur with insulin analogues because their absorption can be affected by various factors such as: mechanics of injection, the injection site, and metabolic factors, similar to regular human insulin (87).

Insulin Therapy and Hypoglycemia - Present and Future 169

A clinical study in healthy volunteers that evaluated the pharmacokinetics and pharmacodynamics of intradermal administration of insulin lispro compared to subcutaneous injections under euglycemic clamp conditions, has basically confirmed these findings (90). Delivery via microneedles resulted in faster insulin uptake with decreased time to maximal insulin concentration (by approximately 24 minutes), higher relative bioavailability at early post-injection times and a more physiologic metabolic effect, with faster onset of action (shorter times to maximal and early half-maximal glucose infusion rates) and more rapid offset of action (shorter time to late half-maximal glucose infusion rates) (90). Another clinical study was conducted in patients with type 1 diabetes in order to determine if the more rapid absorption of insulin resulting from microneedle administration translates into a significant reduction in postprandial glucose levels under standardized meal conditions (91). The results indicated that postprandial glucose levels were improved when regular human insulin was delivered intradermaly vs. subcutaneously, but were similar for analogue insulin. In clinical studies the intradermal delivery was generally well tolerated (although some transient, localized wheal formation and redness were noticed at injection sites), but the potential effects of high level or repetitive exposure of protein drugs such as insulin on the lymphatics and immune system need full investigation (90, 91). Another area of research focuses on the combination of available insulin products with a human recombinant hyaluronidase, which facilitates the local dispersion and absorption of co-administered molecules (92, 93). The human recombinant hyaluronidase is a highly purified neutral pH-active enzyme that depolymerizes hyaluronan in the hypodermis under physiologic conditions. Thus, it decreases the resistance to fluid flow and further contributes to the drug dispersion for better exposure to a larger capillary network (92). Following this, concomitant injection with proteins / drugs such as insulin, is expected to lead to an enhanced absorption and improved bioavailability. Recombinant human hyaluronidase (rHuPH20) is a genetically engineered soluble hyaluronidase approved by the Food and Drug Administration as an adjuvant to enhance permeation of other injected drugs (94). Since rHuPH20 is rapidly metabolized locally, without systemic exposure and because

hyaluronan has a fast turn-over, the permeation effects are transient (94).

A phase 1 glucose clamp study in healthy volunteers evaluated the insulin timeconcentration curve and pharmacodynamic profiles of insulin analogue (lispro) and of regular human insulin combined with rHuPH20 and reported significantly faster systemic absorption, enhanced insulin plasma concentrations and faster metabolic effects compared with either insulin formulation alone (95). A rise in insulin concentration was observed within 3 minutes following the injection and the enhanced pharmacokinetic and glucodynamic effects early after injection were accompanied by reduced late effects. A second study in healthy subjects also reported a lower intra-subject variability with rHuPH20 coadministration (94). A phase 2 standardized meal-test study in patients with type 1 diabetes examined whether the accelerated insulin absorption has favorable consequences on the control of postprandial glycemic excursions (94). As in the phase 1 studies, the coadministration of rHuPH20 with regular insulin or lispro yielded an accelerated insulin concentration profile that was accompanied by a significant reduction in both mean peak and total post-meal glucose concentrations compared to either insulin alone. Post-meal hypoglycemia was reported to be generally mild and the overall hypoglycemic risk comparable for lispro with or without rHuPH20 and reduced for regular insulin with rHuPH20 compared with regular insulin alone (94). Clinical studies reported a

Two meta-analyses indicated that regular human insulin and rapid-acting analogues have comparable frequencies and types of adverse events (other than hypoglycemia), i.e. local site reactions, ketoacidosis and the discontinuation rates during the clinical studies were similar for the two types of insulin preparations (75, 77).

## **6. Current ultrafast insulin formulations**

Thus, the limitations of current insulin formulations and the need for proper postprandial glycemic control have led to research of novel, ultrafast insulin formulations /delivery systems that could eventually better match post-prandial glucose excursions (by speeding the onset of insulin absorption and action coupled with a faster offset of action) and that would offer improved flexibility in terms of injection time relative to a meal (Table 2). By a closer approximation of the normal insulin release, several outcomes could be obtained, i.e. improvement of HbA1c through a better control of postprandial blood glucose, reduced incidence of late-phase hypoglycemia, lower intra-subject variability, and less weight gain.

Recently, stainless steel microneedle syringe devices have been under investigation for intradermal delivery of insulin and their potential to improve postprandial glycemia has been evaluated. The microneedles (34-gauge; an external diameter of approximately 260 μm, 1.25-1.75-mm long) penetrate the stratum corneum and epidermis to reach the dense beds of capillaries and lymphatic vessels of the dermis (88). The dermis layer can facilitate a faster insulin absorption compared to injection into the subcutaneous layer by an increased lymphatic absorption and reach blood circulation.


Table 2. The pharmacodynamic profiles of ultrafast insulin formulations / delivery systems under development

In animal models the intradermal delivery of insulin by microneedles provided a unique pharmacokinetic profile more closely resembling the intravenous rather than the subcutaneous administration (89). The profile is characterized by an extremely rapid uptake and systemic distribution from the injection site: the time to maximum concentration was significantly reduced (with 64%) for insulin lispro administered intradermaly vs. subcutaneously. In addition, the maximum circulating peak concentrations were elevated several fold (349% for insulin lispro) compared to subcutaneous delivery. Moreover, both regular and analogue insulins, despite their differences in molecular weight, when delivered by microneedles showed a more rapid onset of action than subcutaneous delivery of insulin analogue (lispro) (89).

Two meta-analyses indicated that regular human insulin and rapid-acting analogues have comparable frequencies and types of adverse events (other than hypoglycemia), i.e. local site reactions, ketoacidosis and the discontinuation rates during the clinical studies were similar

Thus, the limitations of current insulin formulations and the need for proper postprandial glycemic control have led to research of novel, ultrafast insulin formulations /delivery systems that could eventually better match post-prandial glucose excursions (by speeding the onset of insulin absorption and action coupled with a faster offset of action) and that would offer improved flexibility in terms of injection time relative to a meal (Table 2). By a closer approximation of the normal insulin release, several outcomes could be obtained, i.e. improvement of HbA1c through a better control of postprandial blood glucose, reduced incidence of late-phase hypoglycemia, lower intra-subject variability, and less weight gain. Recently, stainless steel microneedle syringe devices have been under investigation for intradermal delivery of insulin and their potential to improve postprandial glycemia has been evaluated. The microneedles (34-gauge; an external diameter of approximately 260 μm, 1.25-1.75-mm long) penetrate the stratum corneum and epidermis to reach the dense beds of capillaries and lymphatic vessels of the dermis (88). The dermis layer can facilitate a faster insulin absorption compared to injection into the subcutaneous layer by an increased

for the two types of insulin preparations (75, 77).

**6. Current ultrafast insulin formulations** 

lymphatic absorption and reach blood circulation.

(early T50%)

InsuPatch101 NA 95 min NA Technosphere105,106 NA 42-79 min NA

Intradermal90 28-35 min 105-110 min 271-287 min rHUPH20+insulin93 43-44 min 72-114 min 119-275 min VIAject95 31-35 min 111-136 min 270-297 min

Oral-lyn119-121 23-35 min 40-50 min 56-101 min

Table 2. The pharmacodynamic profiles of ultrafast insulin formulations / delivery systems

In animal models the intradermal delivery of insulin by microneedles provided a unique pharmacokinetic profile more closely resembling the intravenous rather than the subcutaneous administration (89). The profile is characterized by an extremely rapid uptake and systemic distribution from the injection site: the time to maximum concentration was significantly reduced (with 64%) for insulin lispro administered intradermaly vs. subcutaneously. In addition, the maximum circulating peak concentrations were elevated several fold (349% for insulin lispro) compared to subcutaneous delivery. Moreover, both regular and analogue insulins, despite their differences in molecular weight, when delivered by microneedles showed a more rapid onset of action than subcutaneous delivery of insulin

Peak (T GIRmax) Offset (late T50%)

Insulin Onset

under development

analogue (lispro) (89).

A clinical study in healthy volunteers that evaluated the pharmacokinetics and pharmacodynamics of intradermal administration of insulin lispro compared to subcutaneous injections under euglycemic clamp conditions, has basically confirmed these findings (90). Delivery via microneedles resulted in faster insulin uptake with decreased time to maximal insulin concentration (by approximately 24 minutes), higher relative bioavailability at early post-injection times and a more physiologic metabolic effect, with faster onset of action (shorter times to maximal and early half-maximal glucose infusion rates) and more rapid offset of action (shorter time to late half-maximal glucose infusion rates) (90). Another clinical study was conducted in patients with type 1 diabetes in order to determine if the more rapid absorption of insulin resulting from microneedle administration translates into a significant reduction in postprandial glucose levels under standardized meal conditions (91). The results indicated that postprandial glucose levels were improved when regular human insulin was delivered intradermaly vs. subcutaneously, but were similar for analogue insulin. In clinical studies the intradermal delivery was generally well tolerated (although some transient, localized wheal formation and redness were noticed at injection sites), but the potential effects of high level or repetitive exposure of protein drugs such as insulin on the lymphatics and immune system need full investigation (90, 91).

Another area of research focuses on the combination of available insulin products with a human recombinant hyaluronidase, which facilitates the local dispersion and absorption of co-administered molecules (92, 93). The human recombinant hyaluronidase is a highly purified neutral pH-active enzyme that depolymerizes hyaluronan in the hypodermis under physiologic conditions. Thus, it decreases the resistance to fluid flow and further contributes to the drug dispersion for better exposure to a larger capillary network (92). Following this, concomitant injection with proteins / drugs such as insulin, is expected to lead to an enhanced absorption and improved bioavailability. Recombinant human hyaluronidase (rHuPH20) is a genetically engineered soluble hyaluronidase approved by the Food and Drug Administration as an adjuvant to enhance permeation of other injected drugs (94). Since rHuPH20 is rapidly metabolized locally, without systemic exposure and because hyaluronan has a fast turn-over, the permeation effects are transient (94).

A phase 1 glucose clamp study in healthy volunteers evaluated the insulin timeconcentration curve and pharmacodynamic profiles of insulin analogue (lispro) and of regular human insulin combined with rHuPH20 and reported significantly faster systemic absorption, enhanced insulin plasma concentrations and faster metabolic effects compared with either insulin formulation alone (95). A rise in insulin concentration was observed within 3 minutes following the injection and the enhanced pharmacokinetic and glucodynamic effects early after injection were accompanied by reduced late effects. A second study in healthy subjects also reported a lower intra-subject variability with rHuPH20 coadministration (94). A phase 2 standardized meal-test study in patients with type 1 diabetes examined whether the accelerated insulin absorption has favorable consequences on the control of postprandial glycemic excursions (94). As in the phase 1 studies, the coadministration of rHuPH20 with regular insulin or lispro yielded an accelerated insulin concentration profile that was accompanied by a significant reduction in both mean peak and total post-meal glucose concentrations compared to either insulin alone. Post-meal hypoglycemia was reported to be generally mild and the overall hypoglycemic risk comparable for lispro with or without rHuPH20 and reduced for regular insulin with rHuPH20 compared with regular insulin alone (94). Clinical studies reported a

Insulin Therapy and Hypoglycemia - Present and Future 171

minutes of glucose concentrations were lowered" before by 39%) (99). The InsuPatchTM was also tested in youth with type 1 diabetes using a euglycemic clamp procedure. The use of the InsuPatchTM was found to decrease time to peak action by more than 40 minutes, whereas the bioavailability and peak responses remained unchanged (101, 102). Such improvements in time-action responses may provide a better control of post-meal glucose excursions (101). Another study that evaluated the effect of the InsuPatchTM heating device on postprandial blood glucose levels after different standardized meals in patients with type 1 diabetes on CSII has confirmed that local heating of the skin around the infusion site significantly increases early post-delivery insulin levels (AUC 0-60 minutes for insulin concentrations above baseline) as well as significantly reduces post-prandial blood glucose (blood glucose at 90 minutes and AUC 0-120 minutes of blood glucose levels) without causing more hypoglycemia (103). Current efforts are being employed in order to optimize the effect of the device on the pharmacokinetic and pharmacodynamic parameters by improving the heating process. The InsuPatchTM device was well tolerated and no serious

A different strategy that attempts to overcome the barriers and limitations of subcutaneous insulin administration is engaging a diverse route of delivery (i.e. pulmonary). After the discontinuation of the first inhaled insulin product (Exubera), the development of most of the pulmonary administration systems has ceased. One of them though, TechnosphereTM insulin, is still being developed and it appears to overcome some of the barriers that contributed to the withdrawal of Exubera (104, 105). Technosphere*™* insulin is an ordered lattice array containing recombinant human insulin, formulated as a crystalline dry powder. The TechnosphereTM carrier is created with microcrystallized plates of fumaryl diketopiperazine that undergo self-assembly into microparticles with a very large surface area and a high internal porosity which are then lyophilized into a dry powder (104). Insulin is absorbed onto the surface of the particles and is delivered by a high-impedance, low-flow, breath-powered inhaler with a powder de-agglomeration mechanism that allows for a high percentage of the administered insulin to be absorbed. At the neutral pH environment of the lungs, the microparticles dissolve rapidly and insulin is absorbed across the pulmonary epithelium into

The pharmacokinetic clamp studies performed in healthy volunteers and patients with type 2 diabetes revealed a very rapid systemic insulin uptake (time to maximal insulin concentration around 15 minutes) with a subsequent fast onset of action (time to maximal metabolic effect of about 40-80 minutes) and a short duration of action (106-109). These characteristics had basically been confirmed by a meal-test study in patients with type 2 diabetes, which demonstrated a more rapid absorption and higher peak insulin levels as well as markedly improved postprandial glycemic control with the inhaled insulin compared with subcutaneous regular human insulin (110). A linear systemic insulin uptake profile was noted in studies employing healthy volunteers inhaling three doses of insulin (106-108). In addition, the within-subject variability of insulin exposure following inhalation of Technosphere*™* insulin was lower compared to regular insulin (109). The relative bioavailability was reported to be 26-50% in the first 3 hours after administration (111). Given that other inhaled insulin preparations have been associated with reduced absorption in patients with chronic obstructive pulmonary disease, a study assessing the pharmacokinetic profile and safety of Technosphere*™* insulin in nondiabetic patients with chronic obstructive pulmonary disease has shown that insulin absorption was not

the systemic circulation, while the carrier is cleared unmetabolized (104, 106).

adverse effects were reported with its use to date (99).

good tolerability profile without severe adverse effects, but there is no safety data so far regarding the repeated or long term exposure to recombinant hyaluronidase.

A third novel ultrafast insulin formulation, VIAjectTM, is currently under clinical development. The main concept of the approach is that instead of altering the structure of insulin molecule, the zinc ions are pulled away from human insulin hexamers and simultaneously charges on the surface of the insulin molecule are masked by the addition of ethylene diamine tetraacetic acid and citric acid (96). This results in destabilization and dissociation of the insulin hexamer and prevents re-association to the hexameric state after subcutaneous injection.

A glucose clamp study in healthy volunteers evaluated the pharmacodynamic, pharmacokinetic and the dose-response properties of the VIAject in comparison with regular human insulin and insulin lispro (96). The results indicated a more rapid increase and decline in serum insulin concentrations after VIAject injection compared to regular human insulin and insulin lispro, but the difference between the later and VIAject failed to reach statistical significance (96). The three dose of ultrafast insulin used in the study showed a linear dose-response relationship. The time-action profile induced by VIAject was faster than either subcutaneously injected human insulin or lispro, with a more rapid onset of action and maximal metabolic activity, while the activity in the first 2 hours after injection was higher. A second glucose clamp study in patients with type 1 diabetes confirmed the faster absorption kinetics and the more rapid onset of insulin action compared to regular human insulin and showed that upon repeated administration, the within-subject variability is lower than that of human insulin (97). Moreover, a more recent meal-test study conducted in patients with type 2 diabetes indicated that treatment with VIAject determined a significant decrease of postprandial oxidative stress and improved endothelial function compared with regular insulin or insulin lispro, while all insulin formulations resulted in comparable improvements in central arterial elasticity (98).

Another innovative approach developed in order to accelerate insulin absorption into the bloodstream is using a technology (InsuPatchTM) that heats the tissue locally around the injection site (99). Changes in temperature at injection site are partially responsible for variability in insulin absorption (87). Increased skin temperature results in vasodilatation and improved local perfusion, which enables accelerated and enhanced insulin absorption (100). The InsuPatchTM device is an add-on to the insulin pump and is comprised of a heating pad attached to an insulin infusion set and a controller that monitors the temperature of the pad (99). The heating pad warms in a controlled manner the tissue surrounding the injection site for 30 minutes after insulin delivery, without heating the insulin itself.

A study using a meal tolerance test in subjects with type 1 diabetes treated by CSII tested the effect of InsuPatchTM on rapid-acting insulin absorption and post-challenge glucose excursions. The study found a significant effect of the heating device on the pharmacokinetic parameters: the maximum insulin concentrations increased (by 38%), as well as the total insulin absorption during the first 30, 60 and 90 minutes, (by 57%, 45% and 27%, respectively) as measured by area under the curve (AUC). The time to maximal concentration and time to half maximal concentration significantly decreased, indicating an accelerated insulin absorption. The changes were accompanied by significant reductions in post-challenge glucose levels (both 90 minutes post-meal glucose excursion and AUC 0-120

good tolerability profile without severe adverse effects, but there is no safety data so far

A third novel ultrafast insulin formulation, VIAjectTM, is currently under clinical development. The main concept of the approach is that instead of altering the structure of insulin molecule, the zinc ions are pulled away from human insulin hexamers and simultaneously charges on the surface of the insulin molecule are masked by the addition of ethylene diamine tetraacetic acid and citric acid (96). This results in destabilization and dissociation of the insulin hexamer and prevents re-association to the hexameric state after

A glucose clamp study in healthy volunteers evaluated the pharmacodynamic, pharmacokinetic and the dose-response properties of the VIAject in comparison with regular human insulin and insulin lispro (96). The results indicated a more rapid increase and decline in serum insulin concentrations after VIAject injection compared to regular human insulin and insulin lispro, but the difference between the later and VIAject failed to reach statistical significance (96). The three dose of ultrafast insulin used in the study showed a linear dose-response relationship. The time-action profile induced by VIAject was faster than either subcutaneously injected human insulin or lispro, with a more rapid onset of action and maximal metabolic activity, while the activity in the first 2 hours after injection was higher. A second glucose clamp study in patients with type 1 diabetes confirmed the faster absorption kinetics and the more rapid onset of insulin action compared to regular human insulin and showed that upon repeated administration, the within-subject variability is lower than that of human insulin (97). Moreover, a more recent meal-test study conducted in patients with type 2 diabetes indicated that treatment with VIAject determined a significant decrease of postprandial oxidative stress and improved endothelial function compared with regular insulin or insulin lispro, while all insulin formulations resulted in

Another innovative approach developed in order to accelerate insulin absorption into the bloodstream is using a technology (InsuPatchTM) that heats the tissue locally around the injection site (99). Changes in temperature at injection site are partially responsible for variability in insulin absorption (87). Increased skin temperature results in vasodilatation and improved local perfusion, which enables accelerated and enhanced insulin absorption (100). The InsuPatchTM device is an add-on to the insulin pump and is comprised of a heating pad attached to an insulin infusion set and a controller that monitors the temperature of the pad (99). The heating pad warms in a controlled manner the tissue surrounding the injection site for 30 minutes after insulin delivery, without heating the

A study using a meal tolerance test in subjects with type 1 diabetes treated by CSII tested the effect of InsuPatchTM on rapid-acting insulin absorption and post-challenge glucose excursions. The study found a significant effect of the heating device on the pharmacokinetic parameters: the maximum insulin concentrations increased (by 38%), as well as the total insulin absorption during the first 30, 60 and 90 minutes, (by 57%, 45% and 27%, respectively) as measured by area under the curve (AUC). The time to maximal concentration and time to half maximal concentration significantly decreased, indicating an accelerated insulin absorption. The changes were accompanied by significant reductions in post-challenge glucose levels (both 90 minutes post-meal glucose excursion and AUC 0-120

regarding the repeated or long term exposure to recombinant hyaluronidase.

comparable improvements in central arterial elasticity (98).

subcutaneous injection.

insulin itself.

minutes of glucose concentrations were lowered" before by 39%) (99). The InsuPatchTM was also tested in youth with type 1 diabetes using a euglycemic clamp procedure. The use of the InsuPatchTM was found to decrease time to peak action by more than 40 minutes, whereas the bioavailability and peak responses remained unchanged (101, 102). Such improvements in time-action responses may provide a better control of post-meal glucose excursions (101). Another study that evaluated the effect of the InsuPatchTM heating device on postprandial blood glucose levels after different standardized meals in patients with type 1 diabetes on CSII has confirmed that local heating of the skin around the infusion site significantly increases early post-delivery insulin levels (AUC 0-60 minutes for insulin concentrations above baseline) as well as significantly reduces post-prandial blood glucose (blood glucose at 90 minutes and AUC 0-120 minutes of blood glucose levels) without causing more hypoglycemia (103). Current efforts are being employed in order to optimize the effect of the device on the pharmacokinetic and pharmacodynamic parameters by improving the heating process. The InsuPatchTM device was well tolerated and no serious adverse effects were reported with its use to date (99).

A different strategy that attempts to overcome the barriers and limitations of subcutaneous insulin administration is engaging a diverse route of delivery (i.e. pulmonary). After the discontinuation of the first inhaled insulin product (Exubera), the development of most of the pulmonary administration systems has ceased. One of them though, TechnosphereTM insulin, is still being developed and it appears to overcome some of the barriers that contributed to the withdrawal of Exubera (104, 105). Technosphere*™* insulin is an ordered lattice array containing recombinant human insulin, formulated as a crystalline dry powder. The TechnosphereTM carrier is created with microcrystallized plates of fumaryl diketopiperazine that undergo self-assembly into microparticles with a very large surface area and a high internal porosity which are then lyophilized into a dry powder (104). Insulin is absorbed onto the surface of the particles and is delivered by a high-impedance, low-flow, breath-powered inhaler with a powder de-agglomeration mechanism that allows for a high percentage of the administered insulin to be absorbed. At the neutral pH environment of the lungs, the microparticles dissolve rapidly and insulin is absorbed across the pulmonary epithelium into the systemic circulation, while the carrier is cleared unmetabolized (104, 106).

The pharmacokinetic clamp studies performed in healthy volunteers and patients with type 2 diabetes revealed a very rapid systemic insulin uptake (time to maximal insulin concentration around 15 minutes) with a subsequent fast onset of action (time to maximal metabolic effect of about 40-80 minutes) and a short duration of action (106-109). These characteristics had basically been confirmed by a meal-test study in patients with type 2 diabetes, which demonstrated a more rapid absorption and higher peak insulin levels as well as markedly improved postprandial glycemic control with the inhaled insulin compared with subcutaneous regular human insulin (110). A linear systemic insulin uptake profile was noted in studies employing healthy volunteers inhaling three doses of insulin (106-108). In addition, the within-subject variability of insulin exposure following inhalation of Technosphere*™* insulin was lower compared to regular insulin (109). The relative bioavailability was reported to be 26-50% in the first 3 hours after administration (111). Given that other inhaled insulin preparations have been associated with reduced absorption in patients with chronic obstructive pulmonary disease, a study assessing the pharmacokinetic profile and safety of Technosphere*™* insulin in nondiabetic patients with chronic obstructive pulmonary disease has shown that insulin absorption was not

Insulin Therapy and Hypoglycemia - Present and Future 173

levels was similar across doses (118-121). Additional meal-test studies indicated that the 30 and 60-min glucose levels were significantly lower with oral insulin spray treatment (122, 123). The metabolic effects of Oral-lyn were evaluated in subjects with type 2 diabetes suboptimally controlled with oral hypoglycemic agents and showed that oral insulin spray significantly decreased the 2-hour postprandial glucose increments in comparison with the oral agents alone and that the difference was more pronounced at the end of the 4-h period, due to the rapid onset and wane of action of oral insulin spray (124). In all of the studies Oral-lyn was generally well tolerated, although some individuals experienced transient (1–2 min), mild and self-limited dizziness during dosing with both the oral insulin and placebo spray (122-124). No other significant side effects (including severe hypoglycemia) were

It should be mentioned that although some of the ultrafast insulin formulations / insulin delivery systems are in early phases of development and/or have not specifically reported for hypoglycemic events, based on their pharmacokinetic properties it can be reasonably expected that they may benefit patients with diabetes by reducing post-meal hyperglycemia

The main goal of insulin therapy is to obtain a near-normal glycemic control by mimicking the time-action profile of physiologic insulin secretion as close as possible and with minimal side effects. Management of both types of diabetes is continually evolving as new therapies, including new insulins / insulin delivery systems are still emerging. In real life, with all progress of the recent years, all the above mentioned objectives are difficult to be reached and successful implementation of intensive diabetes management poses true challenges. Ideally, an insulin-replacement therapeutic approach would keep in check both the fasting and the postprandial glucose concentrations while attaining target HbA1c values, without high glycemic variations and without causing hypoglycemia. Current rapid-acting insulin analogues have a faster pharmacokinetics and action compared with regular human insulin following subcutaneous administration (Table 1). This allows improved control of the postmeal early hyperglycemic surge and late relative hyperinsulinemia, the cause of postprandial hypoglycemia. However, recent meta-analyses showed that in fact the use of insulin analogues had only a modest impact on overall glycemic control and on the rates of side effects, mainly hypoglycemia, compared to conventional insulins (72-76). This is because although improved, the time-action profile still does not exactly replicate normal insulin secretion and therefore there is a mismatch with the blood glucose concentrations curve. Both postprandial hyperglycemia and hypoglycemia have important health consequences as well as on quality of life and failure to address them both may compromise

The extent to which these goals can be met depends on many factors, including the type of diabetes, the stage in the progression of the disease and the pharmacokinetic profile of insulin formulation. If some of these factors are unmodifiable, others are, and efforts are being employed to develop new, improved ultrafast insulin products / delivery systems. They provide even more rapid pharmacokinetic and pharmacodynamic properties compared with current prandial insulin products, which may offer some advantages. The short interval between insulin administration and the appearance of the maximal serum

with decreasing (or at least without increasing) the risk of hypoglycemic events.

noted in studies involving subjects with type 2 diabetes (122).

the success of treatment in the short- and long-term.

**7. Conclusions** 

significantly altered in this group (112). Similarly, the absorption of inhaled insulin appeared not to be altered in a clinically significant manner in smokers (105).

The clinical efficacy of Technosphere*™* insulin was assessed in studies of 11 or 12 weekduration in patients with type 2 diabetes (either insulin-naive or treated with basal insulin glargine), which demonstrated significant reductions in postprandial glucose excursions as well as clinically meaningful improvement of glycemic control as evaluated by HbA1c (113, 114). Moreover, a study of longer duration (52 weeks) in subjects with type 2 diabetes compared the inhaled insulin plus insulin glargine with twice daily biaspart insulin and indicated that changes in HbA1c determined by the treatment with inhaled insulin were similar and non-inferior to that with biaspart insulin (115). In addition, the weight gain and the incidence of both mild-to-moderate and severe hypoglycemic events were lower with inhaled insulin therapy.

Considering the issues associated with Exubera in the past, patient satisfaction and acceptance has been evaluated with the new inhaled insulin product. Overall, significant improvements in attitudes toward insulin therapy, treatment satisfaction, and treatment preference were reported with Technosphere*™* insulin (105, 116). The therapeutical approach using the new inhaled insulin was implemented without a negative impact on health-related quality of life (116).

To date, Technosphere*™* insulin has demonstrated a favorable safety and tolerability profile in clinical studies that collected data in healthy volunteers and patients with diabetes (105). The most frequent treatment-emergent adverse events associated with inhaled insulin in clinical studies were cough and hypoglycemia. Weight gain is commonly associated with insulin therapy. However, data so far indicated that with Technosphere*™* insulin the weight gain was actually less compared with subcutaneous prandial insulins (105). While there are no reports of lung cancer or other serious side effects associated with Technosphere*™* insulin to date, longer-term safety follow-up and evaluation should be done in subjects treated with this inhaled insulin formulation, especially in smokers and in subjects with respiratory disorders.

Finally, another alternative approach of insulin delivery is through the oral (buccal) route, which offers some advantages: good accessibility, high level of vascularization, relatively large surface for absorption (100–200 cm²), avoidance of presystemic metabolism in the liver, robustness, direct contact of the drug with the mucosa, weak variations of pH (117, 118). Orallyn is a liquid formulation of human regular insulin with very small amounts of generally regarded as safe (GRAS) ingredients, which is delivered to the buccal mucosa with a metereddose, slightly modified asthma-like spray and used for prandial insulin therapy (117). The device spray the uniform-sized insulin droplets with high speed (100 mph) into the mouth, which then penetrate the superficial layers of the mucosa and get absorbed into the bloodstream.

The pharmacokinetic and pharmacodynamic properties of Oral-lyn have been evaluated in a number of glucose clamp studies, which have demonstrated a significantly more rapid absorption (about 25 minutes) to higher levels than subcutaneous injection of regular human insulin and a rapid return to baseline values (90 minutes after application) (118-121). The profile was paralleled by the glucose infusion rates that reached maximal levels significantly earlier (at about 45 minutes) and then returned back to baseline concentrations after approximately 120 minutes. Increasing doses of Oral-lyn determined a linear dose-response relationship with respect to maximal insulin concentrations, while time to maximal insulin levels was similar across doses (118-121). Additional meal-test studies indicated that the 30 and 60-min glucose levels were significantly lower with oral insulin spray treatment (122, 123). The metabolic effects of Oral-lyn were evaluated in subjects with type 2 diabetes suboptimally controlled with oral hypoglycemic agents and showed that oral insulin spray significantly decreased the 2-hour postprandial glucose increments in comparison with the oral agents alone and that the difference was more pronounced at the end of the 4-h period, due to the rapid onset and wane of action of oral insulin spray (124). In all of the studies Oral-lyn was generally well tolerated, although some individuals experienced transient (1–2 min), mild and self-limited dizziness during dosing with both the oral insulin and placebo spray (122-124). No other significant side effects (including severe hypoglycemia) were noted in studies involving subjects with type 2 diabetes (122).

It should be mentioned that although some of the ultrafast insulin formulations / insulin delivery systems are in early phases of development and/or have not specifically reported for hypoglycemic events, based on their pharmacokinetic properties it can be reasonably expected that they may benefit patients with diabetes by reducing post-meal hyperglycemia with decreasing (or at least without increasing) the risk of hypoglycemic events.

#### **7. Conclusions**

172 Diabetes – Damages and Treatments

significantly altered in this group (112). Similarly, the absorption of inhaled insulin

The clinical efficacy of Technosphere*™* insulin was assessed in studies of 11 or 12 weekduration in patients with type 2 diabetes (either insulin-naive or treated with basal insulin glargine), which demonstrated significant reductions in postprandial glucose excursions as well as clinically meaningful improvement of glycemic control as evaluated by HbA1c (113, 114). Moreover, a study of longer duration (52 weeks) in subjects with type 2 diabetes compared the inhaled insulin plus insulin glargine with twice daily biaspart insulin and indicated that changes in HbA1c determined by the treatment with inhaled insulin were similar and non-inferior to that with biaspart insulin (115). In addition, the weight gain and the incidence of both mild-to-moderate and severe hypoglycemic events were lower with

Considering the issues associated with Exubera in the past, patient satisfaction and acceptance has been evaluated with the new inhaled insulin product. Overall, significant improvements in attitudes toward insulin therapy, treatment satisfaction, and treatment preference were reported with Technosphere*™* insulin (105, 116). The therapeutical approach using the new inhaled insulin was implemented without a negative impact on

To date, Technosphere*™* insulin has demonstrated a favorable safety and tolerability profile in clinical studies that collected data in healthy volunteers and patients with diabetes (105). The most frequent treatment-emergent adverse events associated with inhaled insulin in clinical studies were cough and hypoglycemia. Weight gain is commonly associated with insulin therapy. However, data so far indicated that with Technosphere*™* insulin the weight gain was actually less compared with subcutaneous prandial insulins (105). While there are no reports of lung cancer or other serious side effects associated with Technosphere*™* insulin to date, longer-term safety follow-up and evaluation should be done in subjects treated with this inhaled insulin formulation, especially in smokers and in subjects with

Finally, another alternative approach of insulin delivery is through the oral (buccal) route, which offers some advantages: good accessibility, high level of vascularization, relatively large surface for absorption (100–200 cm²), avoidance of presystemic metabolism in the liver, robustness, direct contact of the drug with the mucosa, weak variations of pH (117, 118). Orallyn is a liquid formulation of human regular insulin with very small amounts of generally regarded as safe (GRAS) ingredients, which is delivered to the buccal mucosa with a metereddose, slightly modified asthma-like spray and used for prandial insulin therapy (117). The device spray the uniform-sized insulin droplets with high speed (100 mph) into the mouth, which then penetrate the superficial layers of the mucosa and get absorbed into the

The pharmacokinetic and pharmacodynamic properties of Oral-lyn have been evaluated in a number of glucose clamp studies, which have demonstrated a significantly more rapid absorption (about 25 minutes) to higher levels than subcutaneous injection of regular human insulin and a rapid return to baseline values (90 minutes after application) (118-121). The profile was paralleled by the glucose infusion rates that reached maximal levels significantly earlier (at about 45 minutes) and then returned back to baseline concentrations after approximately 120 minutes. Increasing doses of Oral-lyn determined a linear dose-response relationship with respect to maximal insulin concentrations, while time to maximal insulin

appeared not to be altered in a clinically significant manner in smokers (105).

inhaled insulin therapy.

respiratory disorders.

bloodstream.

health-related quality of life (116).

The main goal of insulin therapy is to obtain a near-normal glycemic control by mimicking the time-action profile of physiologic insulin secretion as close as possible and with minimal side effects. Management of both types of diabetes is continually evolving as new therapies, including new insulins / insulin delivery systems are still emerging. In real life, with all progress of the recent years, all the above mentioned objectives are difficult to be reached and successful implementation of intensive diabetes management poses true challenges.

Ideally, an insulin-replacement therapeutic approach would keep in check both the fasting and the postprandial glucose concentrations while attaining target HbA1c values, without high glycemic variations and without causing hypoglycemia. Current rapid-acting insulin analogues have a faster pharmacokinetics and action compared with regular human insulin following subcutaneous administration (Table 1). This allows improved control of the postmeal early hyperglycemic surge and late relative hyperinsulinemia, the cause of postprandial hypoglycemia. However, recent meta-analyses showed that in fact the use of insulin analogues had only a modest impact on overall glycemic control and on the rates of side effects, mainly hypoglycemia, compared to conventional insulins (72-76). This is because although improved, the time-action profile still does not exactly replicate normal insulin secretion and therefore there is a mismatch with the blood glucose concentrations curve. Both postprandial hyperglycemia and hypoglycemia have important health consequences as well as on quality of life and failure to address them both may compromise the success of treatment in the short- and long-term.

The extent to which these goals can be met depends on many factors, including the type of diabetes, the stage in the progression of the disease and the pharmacokinetic profile of insulin formulation. If some of these factors are unmodifiable, others are, and efforts are being employed to develop new, improved ultrafast insulin products / delivery systems. They provide even more rapid pharmacokinetic and pharmacodynamic properties compared with current prandial insulin products, which may offer some advantages. The short interval between insulin administration and the appearance of the maximal serum

Insulin Therapy and Hypoglycemia - Present and Future 175

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#### **8. References**


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**10** 

**Prevention of Hospital Hypoglycemia** 

**by Algorithm Design: A Programming** 

Susan S. Braithwaite1, Lisa Clark1, Lydia Dacenko-Grawe1,

Radha Devi1, Josefina Diaz2, Mehran Javadi1 and Harley Salinas1

Caregivers treating hospitalized patients are confronted with the necessity both to control hyperglycemia and also to avoid iatrogenic hypoglycemia. Despite controversy about optimal glycemic targets, a large body of evidence associates uncontrolled hyperglycemia with adverse outcomes, both in the intensive care unit and also on general hospital wards (American Diabetes Association, 2011; Moghissi et al., 2009). On general wards, glycemic control during use of scheduled subcutaneous insulin is superior to that seen during use of sliding scale regimens (Baldwin et al., 2005; Umpierrez et al., 2007). When scheduled insulin was compared to sliding scale treatment among general surgical patients, glycemic control was improved (mean blood glucose 145 ± 32 mg/dL vs. 172 ± 47 mg/dL, p < 0.01), and a composite outcome of complications was reduced from 24.3 to 8.6%with odds ratio 3.39 (95% CI 1.50-7.65), p = 0.003 (Umpierrez et al., 2011). Nevertheless, the problem of hypoglycemia is a barrier to successful control of hospital hyperglycemia. Among 1718 adult patients admitted at academic medical centers and having hyperglycemia or receiving insulin therapy, hypoglycemia occurred on 2.8% of all hospital days (Boord et al., 2009). Predisposing factors and adverse outcomes associated with hypoglycemia have been examined in observational studies and in clinical trials studying the effect of glycemic control upon nonglycemic outcomes (Bagshaw et al., 2009; Fischer et al., 1986; Finfer et al., 2009; Kagansky et al., 2003; Krinsley et al., 2007; Maynard et al., 2008; Smith et al., 2005; Stagnaro-Green et al., 1995; Turchin et al., 2009; Van den Berghe et al., 2006; Varghese et al., 2007; Vriesendorp et al., 2006; Wexler et al., 2007). Mortality of patients having myocardial infarction is higher at the lowest as well as the highest ranges glucose, such that the relationship between mortality and glucose is described by a J-shaped curve (Kosiborod et al., 2008). Outcomes of hospitalized patients that have been linked to hypoglycemia include increased ICU mortality or hospital mortality rates, adverse events such as seizures, and increased length of stay. In the intensive care unit and on general wards, associated factors

**1. Introduction** 

**Pathway for Electronic Order Entry** 

*1University of Illinois-Chicago; Saint Francis Hospital,* 

*2University of Illinois-Chicago; Saint Joseph Hospital,* 

*Resurrection Health Care* 

 *Resurrection Health Care United States of America* 

