**2. Incretin effect, incretin hormones, secretion and functions**

### **2.1. The incretin effect**

Pancreas secrete insulin in response to the food content in the gastrointestinal lumen. Endo‐ crine pancreas senses food ingestion via incretin hormones, nerve inputs and substrates to secrete insulin. This chain of secretion which starts with food ingestion and result with insulin secretion by endocrine pancreas is called enteroinsulinar axis [1, 2]. The first definition of incretin effect depend on the fact that, much more insulin secretion is induced by oral glucose than with iv glucose administration. So two-to three fold augmented insulin response to oral glucose compared with iv glucose is known as the incretin effect [3].

A duodenal exctract has been found to reduce glucosuria first in early 20th century before the discovery of this phenomenon. The elements of the incretin effect were recognised much more

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before their insulinotropic effects. Glucagon like insulinotropic peptide (GIP) is the first, which was discovered in 1973 by its inhibitory effect on gastric acid secretion and insulinotropic effect was defined later. This discovery was followed by the definiton of another intestinal peptide called glucagon like peptide 1 (GLP-1) ten years later. Discovery of incretin system, and its pathogenetic role in T2 DM caused an important evolution in pathogenesis and management of diabetes. This discovery pointed the role of gastrointestinal system and derived peptides on insulin secretion and glucose metabolism, which has not been taken into account for a very long time.

Postprandial rate of insulin secretion is assumed to be solely affected by the stimulatory effect of incretin hormones, and the role of gastrointestinal motility seems not to be accounted. Passage rate of the ingested foods through the gastrointestinal tract directly affects the secretion rate, secretion amount and the type of incretin hormone [4-6]. These mentioned gastrointestinal motility dependent events play an important role on postprandial glucose homeostasis. Effect of gastrointestinal motor function on glucose metabolism, pathogenesis of diabetes and glycemic regulation still need to be further evaluated.

#### **2.2. Incretin hormones**

There are two incretin hormones known to function on postprandial insulin secretion, which called GIP and GLP-1. Their malfunction and malsecretion have been shown to have role in the pathogenesis of T2 DM.

GIP is a large peptide hormone which is processed from a larger prohormone. Expression of GIP is widely distributed in the body, but the functions are not well understood at these locations. GIP is secreted from enteroendocrine, so called K cells, which predominantly located in the proximal duodenal mucosa but may be seen anywhere in the entire intestinal mucosa [7].

The other incretine hormone GLP-1 is derived from proglucagon peptide. Proglucagon gene is dominantly expressed in pancreatic alpha cells, brain stem and distal intestinal mucosal endocrine, so called L cells [8]. Posttranslational processing of proglucagon peptide differ between pancreatic alpha, brain and intestinal cells, resulting with different endproducts [9-10]. Proglucagon peptide contains two proglucagon peptides named glicentin and major proglucagon fragment. Pancreas contains these two proglucagon peptides in one molecule and secrete glucagon along with major proglukagon fragment. Pancreas processes glicentin to glicentin related pancreatic polypeptide (GRPP), glucagon, and intervening peptide 1 (IP-1), while major proglucagon fragment is not further processed in pancreas. Intestinal L cells secrete these two glucagon like peptide seperately. Unlike alpha cells, intestinal cells have the ability to process major proglucagon fragment to GLP-1, GLP-2 and IP-2. Glicentin is not cleaved or partly cleaved into GRPP and oxyntomodulin in the intestinal cells [Figure 1].

Mechanism of organ spesific posttranslational progulcagon processing is not fully determined. Several factors have been defined to have role in organ spesific processing. Transcription factor named pax6 and the other novel regulator is β-catenin, which is the major effector in Wnt signalisation system are among these regulators. T cell factor 4 (TCF-4 or known as TCF7L2) has been discovered to mediate the Wnt pathway, and shown to induce proglucagon gene

Incretin System in the Pathogenesis of Type 2 Diabetes and the Role of Incretin Based Therapies… http://dx.doi.org/10.5772/59241 273

before their insulinotropic effects. Glucagon like insulinotropic peptide (GIP) is the first, which was discovered in 1973 by its inhibitory effect on gastric acid secretion and insulinotropic effect was defined later. This discovery was followed by the definiton of another intestinal peptide called glucagon like peptide 1 (GLP-1) ten years later. Discovery of incretin system, and its pathogenetic role in T2 DM caused an important evolution in pathogenesis and management of diabetes. This discovery pointed the role of gastrointestinal system and derived peptides on insulin secretion and glucose metabolism, which has not been taken into account for a very

Postprandial rate of insulin secretion is assumed to be solely affected by the stimulatory effect of incretin hormones, and the role of gastrointestinal motility seems not to be accounted. Passage rate of the ingested foods through the gastrointestinal tract directly affects the secretion rate, secretion amount and the type of incretin hormone [4-6]. These mentioned gastrointestinal motility dependent events play an important role on postprandial glucose homeostasis. Effect of gastrointestinal motor function on glucose metabolism, pathogenesis of

There are two incretin hormones known to function on postprandial insulin secretion, which called GIP and GLP-1. Their malfunction and malsecretion have been shown to have role in

GIP is a large peptide hormone which is processed from a larger prohormone. Expression of GIP is widely distributed in the body, but the functions are not well understood at these locations. GIP is secreted from enteroendocrine, so called K cells, which predominantly located in the proximal duodenal mucosa but may be seen anywhere in the entire intestinal mucosa [7].

The other incretine hormone GLP-1 is derived from proglucagon peptide. Proglucagon gene is dominantly expressed in pancreatic alpha cells, brain stem and distal intestinal mucosal endocrine, so called L cells [8]. Posttranslational processing of proglucagon peptide differ between pancreatic alpha, brain and intestinal cells, resulting with different endproducts [9-10]. Proglucagon peptide contains two proglucagon peptides named glicentin and major proglucagon fragment. Pancreas contains these two proglucagon peptides in one molecule and secrete glucagon along with major proglukagon fragment. Pancreas processes glicentin to glicentin related pancreatic polypeptide (GRPP), glucagon, and intervening peptide 1 (IP-1), while major proglucagon fragment is not further processed in pancreas. Intestinal L cells secrete these two glucagon like peptide seperately. Unlike alpha cells, intestinal cells have the ability to process major proglucagon fragment to GLP-1, GLP-2 and IP-2. Glicentin is not cleaved or partly cleaved into GRPP and oxyntomodulin in the intestinal cells [Figure 1].

Mechanism of organ spesific posttranslational progulcagon processing is not fully determined. Several factors have been defined to have role in organ spesific processing. Transcription factor named pax6 and the other novel regulator is β-catenin, which is the major effector in Wnt signalisation system are among these regulators. T cell factor 4 (TCF-4 or known as TCF7L2) has been discovered to mediate the Wnt pathway, and shown to induce proglucagon gene

diabetes and glycemic regulation still need to be further evaluated.

long time.

272 Treatment of Type 2 Diabetes

**2.2. Incretin hormones**

the pathogenesis of T2 DM.

**Figure 1.** Post-translational processing of proglucagon peptide in pancreas, intestine and brain (GRPP: Glicentin relat‐ ed polypeptide, IP-1: intervening peptide 1).

expression to produce GLP-1 in the intestinal endocrine cells, but not in alpha cells [11, 12]. Later a TCF-4 gene polymorphism has been found to be involved in susceptibity to T2 DM. This is an important evidence which proved a link between disrupted incretin effect and development of T2 DM.

Active forms of GLP-1 are GLP-1 (7-36) and GLP-1 (7-37). Lower than %25 of total amount of active form secretion leaves intestine, then 40-50% of this degraded in the liver. In conclusion a very low amount of active GLP-1 reaches into the systemic circulation [13, 14]. GLP-1 (7-36) is cleaved by dipeptidylpeptidase 4 (DPP-4) to GLP-1 (9-36). This enzyme is highly expressed in the brush border of the enterocytes and also in the endothelial cells of the enteric vasculature [15]. Inactive GLP-1 (9-36) and active GLP-1 (7-36) are also degraded by neutral endopeptidase 24.11 (NEP 24,11) to form another inactive form named GLP-1 (28-36) [16]. Although GLP-1 (9-36) and GLP-1 (28-36) are known as inactive forms, it has been shown that they may be as beneficial as their active counterparts on glucose metabolism [17, 18]. Active GLP-1 and its metabolites are cleared from kidneys [19].

Both incretin hormones has been shown to be important in food induced insulin secretion but their potency and molar secretion amounts differ. GIP is secreted into the circulation 10-fold higher amount than GLP-1, but the potency of GLP-1 exceeds GIP [20].

### **2.3. Incretin hormone secretion and regulation of secretion**

Both incretin hormones are secreted from gastrointestinal endocrine cells response to food ingestion. Although it is very low in amount, incretins are also has been shown to secreted during fasting [21]. Proximal intestinal cells secrete GIP, while GLP-1 is secreted from distal ileal and colonic L cells. It is the amount of ingested foods and the gastric emptying rate which effect the type of incretin hormone secreted [22, 23]. For example small amounts of food and a rapid gastric emptying induce GIP secretion, while slow gastric emptying and large complex food portions induce GLP-1 secretion. The exact mechanism of how food components induce the selective secretion of incretin hormones are still not clear. Elements of glucose transport system, such as sodium glucose transporter 1 (SGLT-1) and G protein coupled long chain fatty acid receptors on L cells has been shown to mediate the pathways which induce enteric endocrine cells to secrete incretin hormones in a selective manner [24, 25]. In conclusion incretin hormone levels are very low during fasting state, they are secreted in response to ingested glucose and lipids. Food ingestion is the trigger which starts enteroinsulinar axis result with insulin secretion from pancreatic β-cells. Although neuronal pathways modulate insulin secretion, neuronal pathways do not have role in the induction of enteroinsulinar axis, since GLP-1 does not increase during cephalic phase of insulin secretion [26].

#### **2.4. Functions of incretin hormones**

The incretin GIP shows its actions via a G protein coupled membrane receptor which belongs to the secretin-glucagon receptor family [27, 28]. The other incretin GLP-1 also shows its effects on target cells via a G protein coupled GLP-1 receptor (GLP-1R), which is widely expressed in the body unlike limited secretion sites of GLP-1 [29]. Only one type GLP-1R has been defined in the body, and the organ spesific effects of GLP-1 is believed to be determined by the difference in the glycosilation of the receptor. Wide distribution of the receptor such as endocrine pancreas, brain, heart, gastrointestinal system and kidney, is responsible for the extrapancreatic and extraintestinal effects of the peptide.

Because GIP has been reported to be nearly not affected in diabetic patients, and there is a clear evidence of diminished GLP-1 secretion in T2 DM, this chapter will mention GLP-1 as a representative of incretine hormones [30, 31].

#### **1. Effects of GLP-1 on β-cells:**

It has been shown that GLP-1 has insulinomimetic, insulinotropic and insulinotrophic effects, which mean insulin-like, insulin secretory and regenerative and proliferative effects respectively.

Insulin-like effect of GLP-1 has been shown in several studies, in which GLP-1 inhbibited hepatic glucose output [32, 33]. The mechanisms of inhibition of hepatic glucose output and involving receptors need to be clarified, since hepatocytes do not express GLP-1R.

Insulin secretion is potentiated by GLP-1 only in the presence of glucose. This effect starts with the interaction between the GLP-1 and its G protein coupled membrane receptor. GLP-1 induces insulin secretion only in the presence of glucose in the β-cell [34]. GLP-1

and glucose both increase intracellular cAMP levels sinergistically, then cAMP induces protein kinase A (PKA) and cAMP regulated guanine nucleotide exchange factor II (cAMP-GEF II), also known as Epac2. These two system induce β-cells to secrete insulin by several mechanisms. Closure of ATP sensitive potassium channel and activation of calcium channels both cause depolarisation of β-cell, and then insulin secretion occurs. Both calcium derived from intracellular stores and extracellular space contribute in increase in intracellular calcium levels. Increase in intracellular calcium further stimulate insulin secretion via granule exocytosis. The latter calcium dependent insulin secretion may contribute to >70 % of overall GLP-1 induced insulin secretion. Induction of insulin secretion is not the only effect of GLP-1 on β-cells. Insulin gene promoter region which is mediated with PKA and possibly mitogen activated protein (MAP) kinase pathway are also modulated by GLP-1 [35]. Pancreatic duodenal homeobox-1 (PDX-1), which is a key regulator of developing pancreas, and essential for β-cell growth and insulin gene transcription in adulthood, has been shown to be regulated by GLP-1 [36]. Intracellular glucose concentration depends on the function of glucose transporter system, predomi‐ nantly GLUT-2, and β-cells sense the presence of high glucose levels by the action of the enzyme glucokinase. These two effectors modulate insulin secretion and GLP-1 upregu‐ lates the transcription of glucose transporter and glucokinase genes [37].

**2.3. Incretin hormone secretion and regulation of secretion**

Both incretin hormones are secreted from gastrointestinal endocrine cells response to food ingestion. Although it is very low in amount, incretins are also has been shown to secreted during fasting [21]. Proximal intestinal cells secrete GIP, while GLP-1 is secreted from distal ileal and colonic L cells. It is the amount of ingested foods and the gastric emptying rate which effect the type of incretin hormone secreted [22, 23]. For example small amounts of food and a rapid gastric emptying induce GIP secretion, while slow gastric emptying and large complex food portions induce GLP-1 secretion. The exact mechanism of how food components induce the selective secretion of incretin hormones are still not clear. Elements of glucose transport system, such as sodium glucose transporter 1 (SGLT-1) and G protein coupled long chain fatty acid receptors on L cells has been shown to mediate the pathways which induce enteric endocrine cells to secrete incretin hormones in a selective manner [24, 25]. In conclusion incretin hormone levels are very low during fasting state, they are secreted in response to ingested glucose and lipids. Food ingestion is the trigger which starts enteroinsulinar axis result with insulin secretion from pancreatic β-cells. Although neuronal pathways modulate insulin secretion, neuronal pathways do not have role in the induction of enteroinsulinar axis,

since GLP-1 does not increase during cephalic phase of insulin secretion [26].

The incretin GIP shows its actions via a G protein coupled membrane receptor which belongs to the secretin-glucagon receptor family [27, 28]. The other incretin GLP-1 also shows its effects on target cells via a G protein coupled GLP-1 receptor (GLP-1R), which is widely expressed in the body unlike limited secretion sites of GLP-1 [29]. Only one type GLP-1R has been defined in the body, and the organ spesific effects of GLP-1 is believed to be determined by the difference in the glycosilation of the receptor. Wide distribution of the receptor such as endocrine pancreas, brain, heart, gastrointestinal system and kidney, is responsible for the

Because GIP has been reported to be nearly not affected in diabetic patients, and there is a clear evidence of diminished GLP-1 secretion in T2 DM, this chapter will mention GLP-1 as a

It has been shown that GLP-1 has insulinomimetic, insulinotropic and insulinotrophic effects, which mean insulin-like, insulin secretory and regenerative and proliferative

Insulin-like effect of GLP-1 has been shown in several studies, in which GLP-1 inhbibited hepatic glucose output [32, 33]. The mechanisms of inhibition of hepatic glucose output and involving receptors need to be clarified, since hepatocytes do not express GLP-1R.

Insulin secretion is potentiated by GLP-1 only in the presence of glucose. This effect starts with the interaction between the GLP-1 and its G protein coupled membrane receptor. GLP-1 induces insulin secretion only in the presence of glucose in the β-cell [34]. GLP-1

**2.4. Functions of incretin hormones**

274 Treatment of Type 2 Diabetes

extrapancreatic and extraintestinal effects of the peptide.

representative of incretine hormones [30, 31].

**1. Effects of GLP-1 on β-cells:**

effects respectively.

Research on carcinogenesis and embryogenesis revealed an important intracellular signaling pathway named wnt. Insulinotropic, pancreatic and extrapanceratic insulino‐ mimetic effects of GLP-1 and possibly its metabolites, has shown to be mediated by the activation of wnt pathway. Role of GLP-1 metabolites and GLP-1R in the induc‐ tion of wnt pathway are not clear [38]. The pathway starts with a wnt ligand and LRP5/6-frizzled receptor complex interaction. The cytosolic effector of pathway is βcatenin (β-cat) and is tightly regulated by a phophorylation-destruction complex. This complex is formed by glycogene synthase kinase 3β (GSK-3β), casein kinase 1 (CK-1), axin/conductin, adenomatosis poliposis coli (APC), and phosphorylated extracellular signal-regulated kinase (pERK) [Figure 2]. When the receptor complex is stimulated by a wnt ligand, phosphorylation complex which stimulate the degredation of β-cat by phosphorylation is disrupted. So β-cat escapes from phosphorylation and remains free. Free β-cat then enter to the nucleus and make complex with nuclear coactivator transcription factor named TCF-7. This β-cat-TCF-7 complex induce the target gene expression. Effector β-cat is degraded due to phosphorylation when the receptor is not stimulated. TCF-7 remain free when β-cat does not reach into the nucleus and free TCF-7 regulates the repression of target genes. The most attractive function of GLP-1 is its insulinotrophic effect, which means a regenerative effect on β-cells and progeni‐ tor cells of pancreas [39]. Regeneration of β-cells is maintained by stimulation of βcell proliferation and differentiation of ductal epithelial progenitor cells into β-cells by GLP-1 [40-44]. GLP-1 increases free β-cat in β-cells, which then induce wnt pathway to show its insulinotrophic effects and decreasing glucotoxicity on β-cells. Antiapoptotic effect of GLP-1 on β-cells has been currently defined and may be a promis‐ ing cure for diabetes [45]. Oxidative stress, which play role in β-cell death is another possible target of GLP-1. Thioredoxin (TRX), the thiol oxidoreductase is an important intracellular anti-oxidant. The function of TRX is downregulated by a binding pro‐ tein, called TRX binding protein-2 (TBP-2). This binding protein has been shown to induce β-cell apoptosis, by increasing intracellular oxidative stress. Intracellular levels of TBP-2 closely correlate with blood glucose level [46, 47]. Several studies have found that GLP-1 decreases TBP-2 levels, which in turn increases intracellular TRX and decreases oxidative stress, and further β-cell damage [47, 48].

**Figure 2.** The wnt pathway and the role of GLP-1 on wnt pathway (APC:adenomatosis polyposis coli, CK-1: casein kinase 1, pERK: phosphorylated extracellular signal-regulated kinase, GSK-3β: glycogene synthase kinase 3β, β-cat:be‐ ta catenin, TCF-7: T cell like factor 7) (A and B: Inactive wnt pathway, C: Activation of wnt pathway by a wnt ligand, D: Activation of wnt pathway by GLP-1).

#### **2. Effects of GLP-1 on alpha cells**:

Glucagon plays an important role in pathogenesis of T2 DM. Glucagon hypersecretion has been shown during both fasting and postprandial states in patients with diabetes [49]. GLP-1 decrease glucagon secretion. The exact mechanism of this inhibition is not yet elucidated, but the most possible mechanism is the induction of pancreatic somatostatin secretion, which inhibit the glucagon secretion by paracrine manner [50, 51].

#### **3. Effects of GLP-1 on gastrointestinal system**:

intracellular anti-oxidant. The function of TRX is downregulated by a binding pro‐ tein, called TRX binding protein-2 (TBP-2). This binding protein has been shown to induce β-cell apoptosis, by increasing intracellular oxidative stress. Intracellular levels of TBP-2 closely correlate with blood glucose level [46, 47]. Several studies have found that GLP-1 decreases TBP-2 levels, which in turn increases intracellular TRX and

**Figure 2.** The wnt pathway and the role of GLP-1 on wnt pathway (APC:adenomatosis polyposis coli, CK-1: casein kinase 1, pERK: phosphorylated extracellular signal-regulated kinase, GSK-3β: glycogene synthase kinase 3β, β-cat:be‐ ta catenin, TCF-7: T cell like factor 7) (A and B: Inactive wnt pathway, C: Activation of wnt pathway by a wnt ligand,

Glucagon plays an important role in pathogenesis of T2 DM. Glucagon hypersecretion has been shown during both fasting and postprandial states in patients with diabetes [49]. GLP-1 decrease glucagon secretion. The exact mechanism of this inhibition is not yet elucidated, but the most possible mechanism is the induction of pancreatic somatostatin

secretion, which inhibit the glucagon secretion by paracrine manner [50, 51].

D: Activation of wnt pathway by GLP-1).

276 Treatment of Type 2 Diabetes

**2. Effects of GLP-1 on alpha cells**:

decreases oxidative stress, and further β-cell damage [47, 48].

Gastrointestinal system has a central role in nutrient metabolism with its absorption and endocrine functions. GLP-1 inhibits gastrointestinal system motility, gastrin induced gastric acid and exocrine pancreatic secretions, which lead to a physiological malabsorp‐ tion state [52, 53]. This malabsorbtive state contributes to alleviation of postprandial glucose excursions in diabetic patients.

#### **4. Effects of GLP-1 on central nervous system and satiety**:

Low levels of GLP-1 in the systemic circulation may not reach to the central nervous system, but it has been shown that GLP-1 mediated vagal stimulation may play role in decreased gastrointestinal motility. The effect of GLP-1 on vagal afferent sensorial neurons may be a local effect, which in turn these afferent neurons transmit the inputs to solitary tract nucleus, then inhibit the gastrointestinal motility [54, 55]. GLP-1 decreases food intake by inducing satiety. Hypotalamic satiety centers, predominantly arcuate nucleus has been shown to express GLP-1 receptors. But the exact mechanism of how peripheral GLP-1 stimulate these central receptors is not yet elucidated.

#### **5. Pleitropic effects of GLP-1**

Nontraditional (pleitropic) effects of GLP-1 and its metabolites is an evolving area of research. Wide expression of GLP-1R mediate the widespread action of the peptide. Insulin like effects of GLP-1 has been shown in heart and vasculature. Since cardiovascular diseases are the major contributor of mortality and morbidity in patients with T2 DM, scientific concerns about cardiovascular effects of GLP-1 and based therapies are growing. Preclinical and clinical studies revealed several cardioprotective effects of GLP-1. There are two possible mechanism of action of GLP-1 on cardivascular system, one via GLP-1R, and the other one is receptor-independent [56]. Preliminary cinical studies show that GLP-1 decreases post-ischemic left ventricular dysfunction in patients with coronary heart disease [57, 58].

Invitro studies revealed that GLP-1 improves endothelial dysfunction via decreasing TNF-α, PAI-1 and cellular adhesion molecules [59]. But these observations need to be suggested by clinical studies.

A study with an insulin resistant patient population showed that GLP-1 increases renal sodium and fluid excretion, which is oppose to the mechanism of hypertension in T2 DM [60]. This finding raise the possible blood pressure lowering and renoprotective effect of GLP-1 and based thearpies. Improved endothelial function and anti-oxidant effects of GLP-1 may be another contributory effect in their renoprotective action.

Favorable effects on lipids is another important metabolic action of GLP-1. Preliminary studies reveal that GLP-1 decreases triglyceride, apo B-48 and cholesterol levels [61].

GLP-1 is proposed to be a new therapeutic option for neurodegenerative diseases with its neuroprotective effects which has been shown in animal studies [62].
