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]. 

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... metabolism of glucose by glycolysis, and further metab‐ olism of pyruvate via the downstream tricarboxylic acid (TCA) cycle, leads to elevated NADH, FADH 2 and ultimately ATP levels [4]. The increased intracellular ATP:ADP ratio closes membrane-bound ATP-sensitive K + channels, resulting in plasma membrane depolarisation and a subsequent opening of membrane-bound voltage activated Ca 2+ channels. A rapid influx of calcium ions is promoted, causing the exocytosis of insulin through fusion of the insulin containing vesicles with the plasma membrane via VAMP (vesicle-associated membrane protein) and SNARE (soluble NH2-ethylmaleimide-sensitive fusion protein attachment protein receptor) association [5]. This specific process of insulin secretion is known as K ATP - dependent GSIS, and since ATP generation is critical, the metabolic control points of glycolysis, the TCA cycle and oxidative phosphorylation ( i.e. activity of metabolic enzymes such as hexokinase, phosphofructokinase, pyruvate kinase, pyruvate dehydrogenase, pyruvate carboxylase, glutamate dehydrogenase and mitochondrial redox-shuttles) have a significant impact on regulation of insulin release. However, there also remains the possibility that K ATP -independent GSIS can occur in the β- cell, although the exact methodology is still not fully understood. K ATP -independent GSIS has been illustrated in studies utilising diazoxide to maintain K + channels in the open position [6] and in mice with disrupted/deleted K + channels [7, 8]. GSIS was subsequently shown to be possible in a K ATP -independent manner and it is believed that these two co-ordinate mecha‐ nisms of insulin secretion ( i.e. K ATP -dependent & K ATP -independent GSIS), are responsible for the bi-phasic insulin response in animals. It is thought that the initial rise in insulin secretion is K ATP -dependent, while the second phase is mediated through K ATP -independent interactions dependent on mitochondrial activity [4, 9]. 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, 13, 14]. Conversely, chronic exposure to monounsa‐ turated oleic acid and polyunsaturated arachidonic acid can increase insulin production in β- cells [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 acids will be discussed below. 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, 18, 19, 20, 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. 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 FADH 2 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 Ca 2+ 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 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-cell glucose uptake at low levels, but at high concentration may be toxic [26]. Interaction of NO 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 with its conjugate peroxynitrous acid (ONOOH, pK a ≈ 6.8) [28], is a highly reactive oxidant species produced by the combination of the oxygen free radical O 2- and NO [29] and has been demon‐ strated to be a more potent oxidant and cytotoxic mediator than NO or O 2- 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 (H 2 O 2 )-induced cell death and prevented glucotoxicity in mouse β-cells [39, 40]. However, at elevated concentrations, it impaired GSIS through excessive hydrogen sulphide (H 2 S) 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 ...