Glutamate biosynthesis

1.8.4 Glutamate biosynthesis

Several enzymes are implicated in the generation of glutamate. The amino acid can be produced from glutamine either through the action of phosphate-activated glutaminase (PAG) or during γ-glutamyl cycle activity via the generation of 5-oxoproline. Furthermore, glutamate can be generated from the TCA cycle intermediate α-ketoglutarate during transamination reactions. Transamination reactions are either mediated by the mitochondrial enzyme glutamate dehydrogenase (GDH) or cytoplasmic aspartate aminotransferase (cAAT). Another possible pathway

Figure 1.13: Glutamate generation pathways and function in pancreatic β-cells.

Glutamate is synthesized either from glutamine in the cytoplasm through the action of phosphate-activated glutaminase (PAG) or from the TCA cycle intermediate α -ketoglutarate (αKG) by glutamate dehydrogenase (GDH) or by cytoplasmic aspartate aminotransferase (cAAT). Glutamate is implicated in several cellular processes, including neurotransmitter signalling (transfer into secretory granules), redox metabolism (via glutathione generation), protein synthesis, GABA shunt activity and glutamine re-synthesis (modified from Frigerio et al 2008 [249]).

1.8.4.1 Glutamine as glutamate precursor

Glutamate can be produced from glutamine in the cytoplasm by the action of phosphate-activated glutaminase (PAG), an enzyme found in high concentration and associated with the outer mitochondrial membrane in cells. Endocrine pancreas expresses two isoforms of glutaminase, denoted as kidney-type (KGA) and liver-type (LGA) [250]. Whereas KGA was mainly present in α-cells, LGA was very abundant in β-cells. Interestingly, all glutaminase activity detected in pancreatic islets was attributed to KGA and was confined to the α-cell rich islet mantle [251].

1.8.4.2 GABA shunt and glutamate generation

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain [252, 253] but it is also present in the endocrine pancreas [253]. GABA is synthesized from

been detected in pancreatic islets. The 65kDa-isoform of GAD (GAD65) was predominantly found in pancreatic β-cells whereas GAD67 is mainly localized to the α- and δ-cells rich islet mantle [253-256]. However, the GAD67 isoform was only detected in rat but not mouse and human islets. Glutamate serves as GABA precursor and is either generated from glutamine by glutaminase or from the TCA cycle intermediate α-ketoglutarate (αKG) through cytoplasmic aspartate aminotransferase activity (see chapter 1.8.4.4). The amount of GABA released and produced from pancreatic β-cells depends not only on GAD-mediated GABA formation but also on GABA catabolism, also referred to as GABA shunt [256]. Glucose stimulation was shown to reduce GABA net production from pancreatic β-cells. This reduction results from increased GABA metabolism to succinate semialdehyde (SSA), a reaction catalysed by GABA-transaminase [257]. Of note, it is not caused by an inhibition of GAD expression or activity [257].

The following model was suggested: Glucose stimulates GABA catabolism to generate succinate semialdehyde by GABA-transaminase which is converted to succinate by succinate semialdehyde dehydrogenase (SSADH) to provide an intermediate to the TCA cycle (GABA shunt). Hence, GABA level decrease in response to glucose. Glucose metabolism/oxidation also causes a stimulation of TCA cycle activity resulting in αKG formation. αKG serves as precursor to generate glutamate either by GABA-transaminase or by cytoplasmic aspartate aminotransferase reaction.

Figure 1.14: Regulation of GABA net production in pancreatic β-cells. cAAT, cytoplasmic aspartate aminotransferase; α-KG, α-ketoglutarate; GAD, glutamate decarboxylase;

GABA-T, GABA-transaminase (modified from Wang et al 2006 [257]).

1.8.4.3 GDH & glutamate generation

GDH catalyses the reversible reaction α-ketoglutarate + NH₄⁺ + NAD(P)H ↔ glutamate + NAD(P)⁺ [258]. The enzyme is allosterically activated by leucine and ADP while inhibited by GTP or the ADP-ribosylating enzyme SIRT4 [259-264].

It is still debated if GDH has an anaplerotic role forming α-ketoglutarate from glutamate or a cataplerotic role generating glutamate at the expense of α-ketoglutarate [57].

GDH is expressed in many mammalian tissues, including liver, brain, pancreas etc. In pancreatic β-cells GDH has been proposed to play a central role in the amplifying pathway of glucose-stimulated insulin secretion (GSIS) ([57] (see also chapter 1.8.3). It has been suggested that glutamate is generated from the TCA cycle intermediate α-ketoglutarate through the action of GDH. The suggestion that net flux through GDH is toward synthesis rather than oxidation of glutamate has been contradicted by other reports [135, 242-244, 265]. It has been demonstrated that stimulation of insulin release by the GDH activating mutations or by leucine-mediated GDH activation was associated with increased oxidative deamination of glutamate via GDH [135, 265]. These studies suggest that GDH functions predominantly in the direction of glutamate oxidation rather than glutamate synthesis in mouse islets. Furthermore, glucose stimulation of insulin releasewas associated with a suppression of flux through GDH, consistentwith the well known allosteric inhibitory effect of GTP andATP on activity of the enzyme [266-268]. In addition, mice lacking the ADP-ribosylating enzyme SIRT4, which is known to inhibit GDH activity, showed increased GSIS [262].

In summary, the flux through GDH might depend on the redox state of the mitochondria.

1.8.4.4 The role of aspartate aminotransferases & the malate-aspartate shuttle in glutamate generation

The malate-aspartate shuttle transfers glycolysis-derived reducing equivalents in NADH from the cytosol into the mitochondria where the electrons enter the electron transport chain in order to promote ATP synthesis. It has been proposed that this shuttle activity is implicated in the generation of glutamate, in particular through the action of cytoplasmic aspartate aminotransferase [129, 269, 270]. The malate-aspartate shuttle involves two carrier proteins: The malate/α-ketoglutarate exchanger (dicarboxylate carrier, DC) and the aspartate/glutamate exchanger (Aralar1). Cytoplasmic malate dehydrogenase (cMDH) reduces oxalacetate to malate while oxidizing NADH to NAD⁺. Malate enters the mitochondria in exchange for the TCA cycle intermediate α-ketoglutarate where the

generate oxalacetate and reduce NAD⁺ to NADH. In the cytoplasm, glutamate is generated from its precursor α-ketoglutarate through a transamination reaction at the expense of aspartate which is converted into oxalacetate. This reaction is catalyzed by the cytoplasmic aspartate aminotransferase (cAAT). Glutamate can enter the mitochondria in exchange for aspartate where it is deaminated to α-ketoglutarate through the action of mitochondrial aspartate aminotransferase (mAAT) while aspartate is generated from oxalacetate.

Figure 1.15: Malate-aspartate shuttle system for transferring reducing equivalents from the cytosol to the mitochondria. 1, glutamate-aspartate exchanger (Aralar1 & citrin); 2, malate-αKG exchanger; m/cAAT, aspartate aminotransferase (mitochondrial,

cytoplasmic); αKG, α-ketoglutarate; MDH, malate dehydrogenase; OAA, oxalacetate.

An association between malate-aspartate shuttle activity and glutamate biosynthesis in neuronal tissue as well as in clonal β-cells has been reported [129, 269, 270]. Several studies observed a glucose-stimulated glutamate generation in islets and β-cells, concomitantly with a reduction of aspartate level [130, 135, 269].

In islets lacking glycerol-phosphate shuttle activity (the second NADH shuttle system in β-cells) GSIS was almost abolished when aspartate aminotransferase activity and cytosolic NADH oxidation were suppressed using the inhibitor aminooxyacetate (AOA) [271]. Furthermore, the same inhibitor was reported to lower glutamate production from glucose [269].

Therefore, it was concluded that the malate-aspartate shuttle system is of major importance in glutamate biosynthesis. In particular, the cytoplasmic isoform of aspartate aminotransferases might play a key role in glutamate generation.