**3. Pancreatic β-cell metabolism and influencing factors**

Pancreatic β-cells are unique and can be distinguished from other cell types by their metabolic profile. Several key characteristics of β-cells include the ability to utilise glucose in the physiological range of 2-20mmol/L, express low levels of lactate dehydrogenase (LDH) and plasma membrane monocarboxylate pyruvate/lactate transporter, have a corresponding high activity of glycerol-3-phosphate and malate/aspartate redox shuttles, and finally possess an elevated level of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) activity, ensuring that both oxidative and anaplerotic metabolism of glucose and pyruvate can occur preferentially in the near absence of lactate generation (Fig. 2) (further details can be found in [4,10,11,17-21]). These adaptions are designed to specifically accelerate oxidative phosphor‐ ylation and TCA activity as a means to increase ATP output and consequently insulin exocytosis.

and in mice with disrupted/deleted K+

130 Type 1 Diabetes

dependent on mitochondrial activity [4,9].

acids will be discussed below.

exocytosis.

**3. Pancreatic β-cell metabolism and influencing factors**

Pancreatic β-cells are unique and can be distinguished from other cell types by their metabolic profile. Several key characteristics of β-cells include the ability to utilise glucose in the physiological range of 2-20mmol/L, express low levels of lactate dehydrogenase (LDH) and plasma membrane monocarboxylate pyruvate/lactate transporter, have a corresponding high activity of glycerol-3-phosphate and malate/aspartate redox shuttles, and finally possess an elevated level of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) activity, ensuring that both oxidative and anaplerotic metabolism of glucose and pyruvate can occur preferentially in the near absence of lactate generation (Fig. 2) (further details can be found in [4,10,11,17-21]). These adaptions are designed to specifically accelerate oxidative phosphor‐ ylation and TCA activity as a means to increase ATP output and consequently insulin

channels [7, 8]. GSIS was subsequently shown to be

possible in a KATP-independent manner and it is believed that these two co-ordinate mecha‐ nisms of insulin secretion (*i.e.* KATP-dependent & KATP-independent GSIS), are responsible for the bi-phasic insulin response in animals. It is thought that the initial rise in insulin secretion is KATP-dependent, while the second phase is mediated through KATP-independent interactions

Mitochondrial, lipid and amino acid metabolism plays a significant role in regulation of insulin secretion and GSIS. Lipid and amino acid metabolites can generate, or can directly become MCFs that enhance or inhibit GSIS. While individual amino acids alone at physiological concentrations do not enhance GSIS, some specific amino acids at higher concentrations, or in combination with others, can cause increments in GSIS [10]. Arginine, alanine, leucine and glutamine can increase GSIS, while homocysteine and cysteine at elevated concentration can inhibit GSIS [10]. The effect of amino acids is also dependent on whether β-cells are exposed acutely or chronically, as chronic exposure may influence the expression of genes involved in the control of insulin secretion [10,11]. In addition, another nutrient source, fatty acids, can also regulate GSIS in both a positive or negative manner depending on the level of saturation, carbon chain length, and whether exposure is under acute or chronic conditions. Saturated fatty acids like palmitic and stearic acid are known to chronically decrease GSIS *in vitro*, but palmitic acid can acutely enhance GSIS [12-14]. Conversely, chronic exposure to monounsa‐ turated oleic acid and polyunsaturated arachidonic acid can increase insulin production in βcells [13,15]. Fatty acids can amplify β-cell GSIS, and it is likely that they elevate insulin levels by causing changes in calcium influx and proteins associated with ion channel activity [16]. Mitochondrial metabolism of amino and fatty acid is at the hub of the reported effects on insulin secretion and GSIS, mainly because TCA-mediated metabolism of both leads to increased ATP production and protein biosynthesis, which is a prerequisite for insulin secretion (Fig. 1). The intricacies of mitochondrial-mediated metabolism of amino and fatty

**Figure 2.** Schematic diagram representing the metabolism of selected amino acids, highlighting related metabolic stimulus-secretion coupling factors involved in insulin release. The pathway of glutamine metabolism via glutaminase, GDH, and entry into the TCA cycle (glutaminolysis) is shown along with key points of amino acid interaction with glu‐ tamine and glucose metabolism. KG, -ketoglutarate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AT, aminotransferase; BCKDH, branched-chain-keto-acid dehydrogenase; PC, pyruvate carboxylase; PDH, pyruvate de‐ hydrogenase; KIC, ketoisocaproic acid. Adapted from [21].

Pancreatic β-cells regenerate NAD+ for glycolysis primarily through high expression of mitochondrial NADH shuttles like glycerol-3-phosphate and the malate/aspartate shuttle (Fig. 3), for specific details refer to [11,22]. Briefly, the glycerol-3-phosphate shuttle consists of cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase that operate in unison to convert dihydroxyacetone phosphate to glycerol-3-phosphate and NAD+ , with a subsequent generation of FADH2 from NAD+ [4]. In contrast, the malate/aspartate shuttle is the main shuttle responsible for transferring glycolytic reducing equivalents to the mitochondria in the β-cell [11]. Here, cytosolic malate dehydrogenase reduces oxaloacetate to malate and NAD+ , with a subsequent generation of NADH inside the mitochondria. Using an amino group provided by glutamate, mitochondrial oxaloacetate can be converted back to aspartate maintaining this cyclic event. The malate/aspartate shuttle is dominantly expressed in β-cells, eloquently linking glycolysis to mitochondrial & amino acid metabolism.

As alluded to previously, amino acid metabolism is essential for nutrient- and glucose-stimulat‐ ed insulin secretion, and the effects of several amino acids have been reviewed extensively [3,10, 11]. To summarise these findings briefly, both arginine and alanine have been shown to promote insulin release through changes in electrogenic transport, progressing to activation of Ca2+ ion channels [10,23,24]. It has also been demonstrated that they enhance glutamate production and consequently may play a role in malate/aspartate shuttle-mediated generation of NADH, and/or in glutathione synthesis and antioxidant defence [25]. Therefore, both arginine and alanine may

such as diabetic nephropathy [11,33]. It has been suggested that homocysteine can decrease GSIS in rat pancreatic β-cells [34], although the inhibitory mechanism is still not fully under‐ stood. It may decrease insulin secretion by altering enzyme and/or protein activity, or by causing oxidative stress [35,36]. In addition, homocysteine can be converted to asymmetric dimethylarginine, which is inhibitor of neuronal NOS and can also inhibit iNOS to a lesser extent and therefore may reduce NO production, which is important for β-cell insulin secretion and function [10,37]. In contrast, cysteine has been shown to increase β-cell GSIS at low concentrations [38] and is essential for antioxidant defence and glutathione synthesis, along with glycine and glutamate. Cysteine supplementation was found to protect β-cells from hydrogen peroxide (H2O2)-induced cell death and prevented glucotoxicity in mouse β-cells [39,40]. However, at elevated concentrations, it impaired GSIS through excessive hydrogen

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Glutamine is required for β-cell metabolism and function, and is consumed at rapid rates [10]. Glutamine supplementation does not induce insulin release [10], but co-treatment with leucine significantly enhances GSIS via activation of glutamate dehydrogenase (GDH), allowing entry of glutamine into the TCA cycle (Fig. 2) [42]. It has been suggested that glutamine alone does not induce insulin secretion because it is not oxidised during its metabolism. Instead, metab‐ olism of glutamine may yield aspartate and GABA (γ-aminobutyric acid), a potent inhibitor of glucagon secretion (Fig. 2) [3]. However, using NMR studies, we found that the major products of glutamine metabolism were aspartate and glutamate. Here, glutamate entered the γ-glutamyl cycle and increased the synthesis of the antioxidant, glutathione [43]. Formation of glutamate from glutamine also has important implications in activation of the aspartate/ glutamate shuttles and in ATP production from the TCA cycle, via glutamate metabolism to α-ketoglutarate. Consequently, glutamine may function to enhance ATP production and insulin release by changes in down-stream metabolism, most notably via glutamine-derived glutamate. Alternatively, glutamate can be transported externally from the cell and into the surrounding matrix, which may cause glutamate receptor activation and desensitisation if the rate of release is over extended periods [44]. Since glucagon secretion from pancreatic α-cells is sensitive to glutamate exposure, its release may represent a novel paracrine control mech‐ anism for modulation of blood carbohydrate levels [44]. Some groups have reported that total intracellular glutamate levels increased in response to glucose, while others reported no significant change [25,45,46]. Recently, it has been suggested that glutamate is transported into insulin-containing vesicles, thereby promoting Ca2+-dependent insulin secretion [47]. How‐

ever, the role of glutamate in mediating insulin secretion remains hotly debated.

acid metabolism like acetyl CoA carboxylase (ACC) and fatty acid synthase.

Taken together, this evidence suggests that a variety of amino acids may contribute signifi‐ cantly to regulation of pancreatic β-cell insulin secretion. However, other β-cell metabolic processes are important to insulin secretion and must be considered. These include four key metabolic shunts that divert glucose from being utilised by TCA cycle (*i.e.,* aldose reductase, pentose-phosphate, glycogen synthesis and hexosamine pathways; please, see Fig. 4) as well as down-stream glycolytic enzymes such as PC and PDH, and also enzymes involved in fatty

sulphide (H2S) formation [41].

**Figure 3.** The malate–aspartate shuttle is the principal mechanism for the movement of reducing equivalents in the form of NADH from the cytoplasm to the mitochondrion in β-cells. Cytoplasmic malate dehydrogenase (MDH) reduces oxaloacetate (OAA) to malate while oxidizing NADH to NAD+. Malate then enters the mitochondrion where the re‐ verse reaction is performed by mitochondrial malate dehydrogenase. Movement of mitochondrial oxaloacetate to the cytoplasm to maintain this cycle is achieved by transamination to aspartate with the amino group being donated by glutamate. The 2-oxoglutarate (α-ketoglutarate) generated leaves the mitochondrion for the cytoplasm. Adapted from [11].

protect β-cells from oxidative insult in addition to promoting insulin secretion. However, prolonged exposure of β-cells to alanine results in decreased alanine-induced insulin secre‐ tion, while reaction of arginine with inducible nitric oxide synthase (iNOS) can promote nitric oxide (NO) production [10,19]. NO is an important signalling molecule, which is essential for β-cellglucoseuptake atlowlevels,but athighconcentrationmaybe toxic [26].InteractionofNO with superoxide (O -) can also lead to the formation of peroxynitrite (ONOO- ), a damaging free radical that can disrupt mitochondrial function [27]. In fact, ONOO- , which is in equilibrium withitsconjugateperoxynitrousacid(ONOOH, pK<sup>a</sup> ≈6.8)[28],isahighlyreactiveoxidantspecies produced by the combination of the oxygen free radical O2 and NO [29] and has been demon‐ strated to be a more potent oxidant and cytotoxic mediator than NO or O2 individually, in a variety of inflammatory conditions [30]. ONOO is extremely cytotoxic to rat and human islet cells *in vitro* [31] and its *in vivo* formation has been reported in pancreatic islets where it has been associated with β-cell destruction and development of T1DM in NOD mice [32].

High levels of homocysteine and cysteine have also been shown to elicit a negative effect on β-cell function. In obese hyperinsulinaemic T2DM patients, homocysteine levels are increased, while they are increased in T1DM patients, but only following disease-related complications such as diabetic nephropathy [11,33]. It has been suggested that homocysteine can decrease GSIS in rat pancreatic β-cells [34], although the inhibitory mechanism is still not fully under‐ stood. It may decrease insulin secretion by altering enzyme and/or protein activity, or by causing oxidative stress [35,36]. In addition, homocysteine can be converted to asymmetric dimethylarginine, which is inhibitor of neuronal NOS and can also inhibit iNOS to a lesser extent and therefore may reduce NO production, which is important for β-cell insulin secretion and function [10,37]. In contrast, cysteine has been shown to increase β-cell GSIS at low concentrations [38] and is essential for antioxidant defence and glutathione synthesis, along with glycine and glutamate. Cysteine supplementation was found to protect β-cells from hydrogen peroxide (H2O2)-induced cell death and prevented glucotoxicity in mouse β-cells [39,40]. However, at elevated concentrations, it impaired GSIS through excessive hydrogen sulphide (H2S) formation [41].

Glutamine is required for β-cell metabolism and function, and is consumed at rapid rates [10]. Glutamine supplementation does not induce insulin release [10], but co-treatment with leucine significantly enhances GSIS via activation of glutamate dehydrogenase (GDH), allowing entry of glutamine into the TCA cycle (Fig. 2) [42]. It has been suggested that glutamine alone does not induce insulin secretion because it is not oxidised during its metabolism. Instead, metab‐ olism of glutamine may yield aspartate and GABA (γ-aminobutyric acid), a potent inhibitor of glucagon secretion (Fig. 2) [3]. However, using NMR studies, we found that the major products of glutamine metabolism were aspartate and glutamate. Here, glutamate entered the γ-glutamyl cycle and increased the synthesis of the antioxidant, glutathione [43]. Formation of glutamate from glutamine also has important implications in activation of the aspartate/ glutamate shuttles and in ATP production from the TCA cycle, via glutamate metabolism to α-ketoglutarate. Consequently, glutamine may function to enhance ATP production and insulin release by changes in down-stream metabolism, most notably via glutamine-derived glutamate. Alternatively, glutamate can be transported externally from the cell and into the surrounding matrix, which may cause glutamate receptor activation and desensitisation if the rate of release is over extended periods [44]. Since glucagon secretion from pancreatic α-cells is sensitive to glutamate exposure, its release may represent a novel paracrine control mech‐ anism for modulation of blood carbohydrate levels [44]. Some groups have reported that total intracellular glutamate levels increased in response to glucose, while others reported no significant change [25,45,46]. Recently, it has been suggested that glutamate is transported into insulin-containing vesicles, thereby promoting Ca2+-dependent insulin secretion [47]. How‐ ever, the role of glutamate in mediating insulin secretion remains hotly debated.

protect β-cells from oxidative insult in addition to promoting insulin secretion. However, prolonged exposure of β-cells to alanine results in decreased alanine-induced insulin secre‐ tion, while reaction of arginine with inducible nitric oxide synthase (iNOS) can promote nitric oxide (NO) production [10,19]. NO is an important signalling molecule, which is essential for β-cellglucoseuptake atlowlevels,but athighconcentrationmaybe toxic [26].InteractionofNO

**Figure 3.** The malate–aspartate shuttle is the principal mechanism for the movement of reducing equivalents in the form of NADH from the cytoplasm to the mitochondrion in β-cells. Cytoplasmic malate dehydrogenase (MDH) reduces oxaloacetate (OAA) to malate while oxidizing NADH to NAD+. Malate then enters the mitochondrion where the re‐ verse reaction is performed by mitochondrial malate dehydrogenase. Movement of mitochondrial oxaloacetate to the cytoplasm to maintain this cycle is achieved by transamination to aspartate with the amino group being donated by glutamate. The 2-oxoglutarate (α-ketoglutarate) generated leaves the mitochondrion for the cytoplasm. Adapted

) can also lead to the formation of peroxynitrite (ONOO-

withitsconjugateperoxynitrousacid(ONOOH, pK<sup>a</sup> ≈6.8)[28],isahighlyreactiveoxidantspecies

cells *in vitro* [31] and its *in vivo* formation has been reported in pancreatic islets where it has been

High levels of homocysteine and cysteine have also been shown to elicit a negative effect on β-cell function. In obese hyperinsulinaemic T2DM patients, homocysteine levels are increased, while they are increased in T1DM patients, but only following disease-related complications


radical that can disrupt mitochondrial function [27]. In fact, ONOO-

strated to be a more potent oxidant and cytotoxic mediator than NO or O2

associated with β-cell destruction and development of T1DM in NOD mice [32].

produced by the combination of the oxygen free radical O2

variety of inflammatory conditions [30]. ONOO-

), a damaging free

individually, in a

, which is in equilibrium

and NO [29] and has been demon‐


is extremely cytotoxic to rat and human islet

with superoxide (O -

from [11].

132 Type 1 Diabetes

Taken together, this evidence suggests that a variety of amino acids may contribute signifi‐ cantly to regulation of pancreatic β-cell insulin secretion. However, other β-cell metabolic processes are important to insulin secretion and must be considered. These include four key metabolic shunts that divert glucose from being utilised by TCA cycle (*i.e.,* aldose reductase, pentose-phosphate, glycogen synthesis and hexosamine pathways; please, see Fig. 4) as well as down-stream glycolytic enzymes such as PC and PDH, and also enzymes involved in fatty acid metabolism like acetyl CoA carboxylase (ACC) and fatty acid synthase.

accumulation of pyruvate, succinate, fumarate, malate, α-ketoglutarate, dihydroxyacetone phosphate (DHAP), (iso)citrate, palmitate, glucose-6-phosphate and 6-phosphogluconate whereas aspartate was consumed in response to glucose [48]. Here, the authors have clearly demonstrated that under glucose stimulus, β-cells strongly enhance metabolic flux towards glycolysis and TCA cycle. Indeed, there is a very delicate poise to coordinately regulate the flux of glucose towards the formation of NADPH (through the pentose-phosphate shunt) avoiding excessive formation of sorbitol (via the polyol-aldose reductase shunt) which would empty glycolytic flux (Fig. 4). It has long been recognised, for instance, that overexpression of the aldose reductase gene is able to induce apoptosis in pancreatic β-cells by causing a redox imbalance [49] while, on the contrary, pharmacological blockage of aldose reductase may impair GSIS, thus suggesting that the conversion of free intracellular glucose to sorbitol in the

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β-cell is an essential step in the glucose-induced release mechanism (Fig.4) [50].

resistance [55].

Although the physiological significance is still under debate, glucose 6-phosphate may also be targeted towards glycogen synthesis in pancreatic islets, which is enhanced during GSIS and impaired in STZ-diabetic rats (Fig. 4) [51,52]. Finally, glucose may be deviated from ultimate metabolism through further glycolytic steps via the reaction of fructose-6-phosphate with glutamine through the hexosamine biochemical pathway (HBP) (Fig. 4). Increased fluxes through HBP may, on the one hand, block glycogen synthase kinase-3β (GSK-3β), thus liberating glycogen synthesis by glycogen synthase and, on the other, may reduce heat shock factor-1 (HSF-1) degradation thus allowing enhanced expression of the 70-kDa heat shock protein (HSP70), which is cytoprotective to β-cells (Fig. 4) [53,54]. Over-enhanced flux through HBP is an inducer of endoplasmic reticulum (ER) stress, while being associated with insulin-

PC and PDH are both highly expressed in β-cells and allow conversion of pyruvate to oxaloacetate (PC) and acetyl-CoA (PDH), with subsequent entry into the TCA cycle [4]. Interestingly, siRNA inhibition of PC reduces cell proliferation and GSIS in insulinoma cells and rat islets, while overexpression in rat islets could enhance GSIS and cell proliferation [56, 57]. The role of PDH is less understood and it is thought to support PC activity by providing acetyl-CoA for citrate production. Both enzymes are important regulators of the pyruvate/ malate and pyruvate/citrate shuttles. Common to each pathway is the conversion of glycolyticderived pyruvate to oxaloacetate by PC, as described above. In the case of the pyruvate/malate shuttle, oxaloacetate is then converted to malate and translocated to the cytosol, where malic enzyme1 (ME1) converts malate back to pyruvate along with generation of NADPH. Pyruvate can re-enter the mitochondria to repeat the cycle with further generation of NADPH [4]. However, for the pyruvate/citrate shuttle, PC-mediated oxaloacetate leads to condensation with acetyl CoA (possibly generated by PDH), and the subsequent formation of citrate. Translocation of citrate to the cytosol results in oxaloacetate and acetyl CoA regeneration from citrate by ATP-citrate lyase (ACL). Oxaloacetate re-enters the pyruvate/malate cycle with generation of NADPH as outlined previously, while acetyl CoA is carboxylated to malonyl CoA by acetyl CoA carboxylase (ACC). Malonyl CoA is then converted to long chain acyl CoA by fatty acid synthase leading to fatty acid production. Additionally, malonyl CoA can also inhibit carnitine palmitoyl transferase 1 (CPT-1), which in a low glucose state, transports fatty

**Figure 4.** Flux balance analysis of glucose utilisation in β-cells. The fluxes through the biochemical pathways shown here were calculated by using Michaelis-Menten function, intracellular metabolite concentrations estimated from dif‐ ferent works. Percentages in parentheses refer to the proportional amount of the metabolite consumed through that step. AR, aldose reductase; ARE, antioxidant response (ARE) elements in the promoter regions of target genes; F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; G6PI, glucose-6 phosphate isomerase (a.k. as phosphoglucoisomerase); γ-GCS, glutamate cysteine ligase, a.k. as γ-glutamylcysteine synthetase; GFAT, glutamine:fructose-6-phosphate amidotransferase a.k. as GFPT, for glutamine-fructose-6-phos‐ phate transaminase; Gluc, glucose; GS, glycogen synthase; GSIS, glucose-stimulated insulin secretion; GSK-3β, glyco‐ gen synthase kinase-3β; HSF-1, heat shock transcription factor-1; HSP70, the 70-kDa family of heat shock proteins (includes both hsp72, encoded by the HSPA1A gene, and hsp73, a.k. as hsc70, encoded by the HSPA8 gene); Keap1, Kelch-like ECH-associated protein 1; Nrf2, Nuclear factor erythroid 2-related transcription factor 2; OGT, *O*-*N*-acetylglu‐ cosamine transferase, a.k. as UDP-*N*-acetyl-D-glucosamine:protein-*O*-β-*N*-acetyl-D-glucosaminyl transferase and uri‐ dine diphospho-*N*-acetylglucosamine:polypeptide β-*N*-acetylglucosaminyl transferase; PFK, phosphofructokinase; PGmutase, phosphoglucomutase; UGP, UDP-glucose pyrophosphorylase.

Highlighting the peculiarities of β-cell metabolism in a coordinated effort to increase the activity of a number of metabolic pathways in response to glucose, Huang & Joseph (2012) have shown, by using metabolomic analysis, during GSIS in clonal β-cells, a conspicuous accumulation of pyruvate, succinate, fumarate, malate, α-ketoglutarate, dihydroxyacetone phosphate (DHAP), (iso)citrate, palmitate, glucose-6-phosphate and 6-phosphogluconate whereas aspartate was consumed in response to glucose [48]. Here, the authors have clearly demonstrated that under glucose stimulus, β-cells strongly enhance metabolic flux towards glycolysis and TCA cycle. Indeed, there is a very delicate poise to coordinately regulate the flux of glucose towards the formation of NADPH (through the pentose-phosphate shunt) avoiding excessive formation of sorbitol (via the polyol-aldose reductase shunt) which would empty glycolytic flux (Fig. 4). It has long been recognised, for instance, that overexpression of the aldose reductase gene is able to induce apoptosis in pancreatic β-cells by causing a redox imbalance [49] while, on the contrary, pharmacological blockage of aldose reductase may impair GSIS, thus suggesting that the conversion of free intracellular glucose to sorbitol in the β-cell is an essential step in the glucose-induced release mechanism (Fig.4) [50].

Although the physiological significance is still under debate, glucose 6-phosphate may also be targeted towards glycogen synthesis in pancreatic islets, which is enhanced during GSIS and impaired in STZ-diabetic rats (Fig. 4) [51,52]. Finally, glucose may be deviated from ultimate metabolism through further glycolytic steps via the reaction of fructose-6-phosphate with glutamine through the hexosamine biochemical pathway (HBP) (Fig. 4). Increased fluxes through HBP may, on the one hand, block glycogen synthase kinase-3β (GSK-3β), thus liberating glycogen synthesis by glycogen synthase and, on the other, may reduce heat shock factor-1 (HSF-1) degradation thus allowing enhanced expression of the 70-kDa heat shock protein (HSP70), which is cytoprotective to β-cells (Fig. 4) [53,54]. Over-enhanced flux through HBP is an inducer of endoplasmic reticulum (ER) stress, while being associated with insulinresistance [55].

PC and PDH are both highly expressed in β-cells and allow conversion of pyruvate to oxaloacetate (PC) and acetyl-CoA (PDH), with subsequent entry into the TCA cycle [4]. Interestingly, siRNA inhibition of PC reduces cell proliferation and GSIS in insulinoma cells and rat islets, while overexpression in rat islets could enhance GSIS and cell proliferation [56, 57]. The role of PDH is less understood and it is thought to support PC activity by providing acetyl-CoA for citrate production. Both enzymes are important regulators of the pyruvate/ malate and pyruvate/citrate shuttles. Common to each pathway is the conversion of glycolyticderived pyruvate to oxaloacetate by PC, as described above. In the case of the pyruvate/malate shuttle, oxaloacetate is then converted to malate and translocated to the cytosol, where malic enzyme1 (ME1) converts malate back to pyruvate along with generation of NADPH. Pyruvate can re-enter the mitochondria to repeat the cycle with further generation of NADPH [4]. However, for the pyruvate/citrate shuttle, PC-mediated oxaloacetate leads to condensation with acetyl CoA (possibly generated by PDH), and the subsequent formation of citrate. Translocation of citrate to the cytosol results in oxaloacetate and acetyl CoA regeneration from citrate by ATP-citrate lyase (ACL). Oxaloacetate re-enters the pyruvate/malate cycle with generation of NADPH as outlined previously, while acetyl CoA is carboxylated to malonyl CoA by acetyl CoA carboxylase (ACC). Malonyl CoA is then converted to long chain acyl CoA by fatty acid synthase leading to fatty acid production. Additionally, malonyl CoA can also inhibit carnitine palmitoyl transferase 1 (CPT-1), which in a low glucose state, transports fatty

**Figure 4.** Flux balance analysis of glucose utilisation in β-cells. The fluxes through the biochemical pathways shown here were calculated by using Michaelis-Menten function, intracellular metabolite concentrations estimated from dif‐ ferent works. Percentages in parentheses refer to the proportional amount of the metabolite consumed through that step. AR, aldose reductase; ARE, antioxidant response (ARE) elements in the promoter regions of target genes; F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; G6PI, glucose-6 phosphate isomerase (a.k. as phosphoglucoisomerase); γ-GCS, glutamate cysteine ligase, a.k. as γ-glutamylcysteine synthetase; GFAT, glutamine:fructose-6-phosphate amidotransferase a.k. as GFPT, for glutamine-fructose-6-phos‐ phate transaminase; Gluc, glucose; GS, glycogen synthase; GSIS, glucose-stimulated insulin secretion; GSK-3β, glyco‐ gen synthase kinase-3β; HSF-1, heat shock transcription factor-1; HSP70, the 70-kDa family of heat shock proteins (includes both hsp72, encoded by the HSPA1A gene, and hsp73, a.k. as hsc70, encoded by the HSPA8 gene); Keap1, Kelch-like ECH-associated protein 1; Nrf2, Nuclear factor erythroid 2-related transcription factor 2; OGT, *O*-*N*-acetylglu‐ cosamine transferase, a.k. as UDP-*N*-acetyl-D-glucosamine:protein-*O*-β-*N*-acetyl-D-glucosaminyl transferase and uri‐ dine diphospho-*N*-acetylglucosamine:polypeptide β-*N*-acetylglucosaminyl transferase; PFK, phosphofructokinase;

Highlighting the peculiarities of β-cell metabolism in a coordinated effort to increase the activity of a number of metabolic pathways in response to glucose, Huang & Joseph (2012) have shown, by using metabolomic analysis, during GSIS in clonal β-cells, a conspicuous

PGmutase, phosphoglucomutase; UGP, UDP-glucose pyrophosphorylase.

134 Type 1 Diabetes

acids into the mitochondria to generate ATP by oxidation [4,10]. However, in high glucose situations, inhibition of CPT-1 leads to fatty acid accumulation in the cytosol and this accu‐ mulation may increase insulin exocytosis by augmenting calcium influx and ion channel proteins [10,16]. Interestingly, formation of malonyl CoA from acetyl CoA by ACC is positively regulated by the glutamine-sensitive protein phosphatase type 2A (PP2A), while it is nega‐ tively regulated by the amino acid-sensitive AMP-activated kinase (AMPK) [11,58,59]. These concepts again fully illustrate the inherent relationship between β-cell metabolism of glucose, amino acids and lipids with insulin exocytosis [11,58,59].

cellular glutathione molecules generating S-nitrosoglutathione (SNOG) that induces HSP70,

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HSP70 confers protection against sepsis-related circulatory fatality via inhibition of iNOS (NOS-2) gene expression in the rostral ventrolateral medulla through the prevention of NFκB activation, inhibition of IκB kinase activation and consequent inhibition of IκB degradation [69]. This is corroborated by the finding that HSP70 assembles with liver NF-κB/IκB complex in the cytosol thus impeding further transcription of NF-κB-dependent TNF-α and NOS-2 genes that worsen sepsis [70]. This may also be unequivocally demonstrated by treating cells or tissues with HSP70 antisense oligonucleotides that completely reverse the beneficial NFκB-inhibiting effect of HSP70 and inducible HSP70 expression (see [68,69]). Hence, HSP70 is anti-inflammatory per se, when intracellularly located, which also explains why cyclopente‐ none prostaglandins (cp-PGs), which are the most powerful physiological inducers of HSP70 by activating HSF-1, are at the same time powerful anti-inflammatory autacoids [71-73].

Another striking effect of HSP70 is the inhibition of apoptosis. The intrinsic apoptotic pathway is characterized by the release of mitochondrial pro-apoptotic factors and activation of caspase enzymes, while stimulation of cell surface receptors triggers the extrinsic death-pathway. The inhibitory potential of HSP70 over apoptosis occurs via many intracellular downstream pathways (e.g. JNK, NF-κB and Akt), which are both directly and indirectly blocked by HSP70, or through inhibition of mitochondrial Bcl-2 release. Together, these mechanisms are respon‐

In conclusion, intracellularly activated HSPs of the 70-kDa family are cytoprotective and antiinflammatory by avoiding protein denaturation and excessive NF-κB activation which may be damaging to the cells [75]. These observations link energy sensing (AMPK) to antiinflammation (HSP70) and points out to the complexity of the impact of metabolic regulation for cell survival and function. In addition, expression of cytokines such as interleukin-1β (IL-1β), tumour necrosis factorα (TNFα), and interferon-γ (INF-γ) in pancreatic islets is important in inflammation and progression of both T1 and T2DM, and is associated with βcell dysfunction and death. Therefore, agents or nutrients that promote anti-inflammatory responses may be beneficial as anti-diabetic therapies. Since interaction of the immune system with pancreatic islets is central to T1DM and is becoming increasing linked to T2DM, the precise mechanisms of pancreatic cell death in relation to immunological function will now be

**4. Immune-like characteristics of β-cells and response to cytokines**

The pathophysiology of pancreatic islets in T1 and T2DM is characterised by an inflammatory process that includes immune cell infiltration, presence of apoptotic cells, expression of cytokines or adipokines and even amyloid deposits [76]. Although the aetiology of T1DM differs from T2DM, a common feature of both is an immune system-mediated destruction of pancreatic β-cells, ultimately leading to pancreatic dysfunction and reduced β-cell mass. However, the immunological-mediated attack does not solely originate from invading

sible for HSP70's anti-apoptotic function in stressed-cells [74].

discussed.

and, consequently, HSP70 expression [68].

AMPK is crucial in lipid metabolism control and can chronically regulate β-cell function by altering the expression of vital transcription factors that govern lipogenic and glycolytic enzymes [10]. Chronic exposure of β-cells to high circulatory lipid levels, as occurs in T2DM, can inhibit glucose oxidation and result in a decreased ATP/AMP ratio along with a subsequent activation of AMPK, which inhibits fatty acid synthesis, while enhancing fatty acid oxidation, and impairing GSIS [10]. The exact metabolic mechanisms of how lipids can augment GSIS are still not fully understood but are believed to involve modulation of Ca2+ mobilisation via interaction with G-protein coupled receptors [60]. Recent evidence has shown that these Gprotein coupled receptors are highly expressed in β-cells and correlated with insulinogenic index [10,61]. It has also been demonstrated that interaction of omega-3 fatty acids and the GPR120 receptor, plays an instrumental role in mediating insulin-sensitisation and antiinflammatory effects in obese mice models [62].

AMPK also occupies a central position in metabolic regulation in order to avoid inflammatory dysregulation. Accordingly, in different cell types, AMPK phosphorylates and inhibit gluta‐ mine:fructose-6-phosphate amidotransferase-1 (GFAT-1), the flux-generating step of HBP (Fig. 4), thus allowing for the down-regulation of such a shunt from glycolysis under low glucose situations [63], while chronic hexosamine flux stimulates fatty acid oxidation by activating AMPK [64]. However, regulatory pathways under AMPK control are not solely intended to divert metabolic fluxes. Rather, AMPK regulation of GSK-3β allows the concom‐ itant regulation of inflammatory cytokine production, since the inhibition of GSK-3β elicits the deinhibition of HSF-1, thus triggering the expression of HSP70, which is an intracellular antiinflammatory protein.

It is of note that, besides the now classical molecular chaperone action, the most remarkable intracellular effect of HSP70 is the inhibition of NF- κB activation, which has profound implications for immunity, inflammation, cell survival and apoptosis. HSP70 blocks nuclear factor κB (NF—κB) activation at different levels. For instance, HSP70 inhibits the phosphory‐ lation of inhibitor of κB (IκBs), while heat-induced HSP70 protein molecules are able to directly bind to IκB kinase gamma (IKKγ) thus inhibiting tumor necrosis factor- α (TNFα)-induced apoptosis [65]. In fact, the supposition that HSP70 might act intracellularly as a suppressor of NF- κB pathways has been raised after a number of seminal discoveries in which HSP70 was intentionally induced, such as the inhibition of TNFα-induced activation of phospholipase A2 in murine fibrosarcoma cells [66], the suppression of astroglial iNOS expression paralleled by decreased NF— κB activation [67] and the protection of rat hepatocytes from TNFα-induced apoptosis by treating cells with the nitric oxide (NO)-donor SNAP, which reacts with intra‐ cellular glutathione molecules generating S-nitrosoglutathione (SNOG) that induces HSP70, and, consequently, HSP70 expression [68].

acids into the mitochondria to generate ATP by oxidation [4,10]. However, in high glucose situations, inhibition of CPT-1 leads to fatty acid accumulation in the cytosol and this accu‐ mulation may increase insulin exocytosis by augmenting calcium influx and ion channel proteins [10,16]. Interestingly, formation of malonyl CoA from acetyl CoA by ACC is positively regulated by the glutamine-sensitive protein phosphatase type 2A (PP2A), while it is nega‐ tively regulated by the amino acid-sensitive AMP-activated kinase (AMPK) [11,58,59]. These concepts again fully illustrate the inherent relationship between β-cell metabolism of glucose,

AMPK is crucial in lipid metabolism control and can chronically regulate β-cell function by altering the expression of vital transcription factors that govern lipogenic and glycolytic enzymes [10]. Chronic exposure of β-cells to high circulatory lipid levels, as occurs in T2DM, can inhibit glucose oxidation and result in a decreased ATP/AMP ratio along with a subsequent activation of AMPK, which inhibits fatty acid synthesis, while enhancing fatty acid oxidation, and impairing GSIS [10]. The exact metabolic mechanisms of how lipids can augment GSIS are still not fully understood but are believed to involve modulation of Ca2+ mobilisation via interaction with G-protein coupled receptors [60]. Recent evidence has shown that these Gprotein coupled receptors are highly expressed in β-cells and correlated with insulinogenic index [10,61]. It has also been demonstrated that interaction of omega-3 fatty acids and the GPR120 receptor, plays an instrumental role in mediating insulin-sensitisation and anti-

AMPK also occupies a central position in metabolic regulation in order to avoid inflammatory dysregulation. Accordingly, in different cell types, AMPK phosphorylates and inhibit gluta‐ mine:fructose-6-phosphate amidotransferase-1 (GFAT-1), the flux-generating step of HBP (Fig. 4), thus allowing for the down-regulation of such a shunt from glycolysis under low glucose situations [63], while chronic hexosamine flux stimulates fatty acid oxidation by activating AMPK [64]. However, regulatory pathways under AMPK control are not solely intended to divert metabolic fluxes. Rather, AMPK regulation of GSK-3β allows the concom‐ itant regulation of inflammatory cytokine production, since the inhibition of GSK-3β elicits the deinhibition of HSF-1, thus triggering the expression of HSP70, which is an intracellular anti-

It is of note that, besides the now classical molecular chaperone action, the most remarkable intracellular effect of HSP70 is the inhibition of NF- κB activation, which has profound implications for immunity, inflammation, cell survival and apoptosis. HSP70 blocks nuclear factor κB (NF—κB) activation at different levels. For instance, HSP70 inhibits the phosphory‐ lation of inhibitor of κB (IκBs), while heat-induced HSP70 protein molecules are able to directly bind to IκB kinase gamma (IKKγ) thus inhibiting tumor necrosis factor- α (TNFα)-induced apoptosis [65]. In fact, the supposition that HSP70 might act intracellularly as a suppressor of NF- κB pathways has been raised after a number of seminal discoveries in which HSP70 was intentionally induced, such as the inhibition of TNFα-induced activation of phospholipase A2 in murine fibrosarcoma cells [66], the suppression of astroglial iNOS expression paralleled by decreased NF— κB activation [67] and the protection of rat hepatocytes from TNFα-induced apoptosis by treating cells with the nitric oxide (NO)-donor SNAP, which reacts with intra‐

amino acids and lipids with insulin exocytosis [11,58,59].

inflammatory effects in obese mice models [62].

inflammatory protein.

136 Type 1 Diabetes

HSP70 confers protection against sepsis-related circulatory fatality via inhibition of iNOS (NOS-2) gene expression in the rostral ventrolateral medulla through the prevention of NFκB activation, inhibition of IκB kinase activation and consequent inhibition of IκB degradation [69]. This is corroborated by the finding that HSP70 assembles with liver NF-κB/IκB complex in the cytosol thus impeding further transcription of NF-κB-dependent TNF-α and NOS-2 genes that worsen sepsis [70]. This may also be unequivocally demonstrated by treating cells or tissues with HSP70 antisense oligonucleotides that completely reverse the beneficial NFκB-inhibiting effect of HSP70 and inducible HSP70 expression (see [68,69]). Hence, HSP70 is anti-inflammatory per se, when intracellularly located, which also explains why cyclopente‐ none prostaglandins (cp-PGs), which are the most powerful physiological inducers of HSP70 by activating HSF-1, are at the same time powerful anti-inflammatory autacoids [71-73].

Another striking effect of HSP70 is the inhibition of apoptosis. The intrinsic apoptotic pathway is characterized by the release of mitochondrial pro-apoptotic factors and activation of caspase enzymes, while stimulation of cell surface receptors triggers the extrinsic death-pathway. The inhibitory potential of HSP70 over apoptosis occurs via many intracellular downstream pathways (e.g. JNK, NF-κB and Akt), which are both directly and indirectly blocked by HSP70, or through inhibition of mitochondrial Bcl-2 release. Together, these mechanisms are respon‐ sible for HSP70's anti-apoptotic function in stressed-cells [74].

In conclusion, intracellularly activated HSPs of the 70-kDa family are cytoprotective and antiinflammatory by avoiding protein denaturation and excessive NF-κB activation which may be damaging to the cells [75]. These observations link energy sensing (AMPK) to antiinflammation (HSP70) and points out to the complexity of the impact of metabolic regulation for cell survival and function. In addition, expression of cytokines such as interleukin-1β (IL-1β), tumour necrosis factorα (TNFα), and interferon-γ (INF-γ) in pancreatic islets is important in inflammation and progression of both T1 and T2DM, and is associated with βcell dysfunction and death. Therefore, agents or nutrients that promote anti-inflammatory responses may be beneficial as anti-diabetic therapies. Since interaction of the immune system with pancreatic islets is central to T1DM and is becoming increasing linked to T2DM, the precise mechanisms of pancreatic cell death in relation to immunological function will now be discussed.
