Regulation of Leucine-stimulated Insulin Secretion and Glutamine Metabolism in Isolated Rat Islets

Abstract

Glutamate dehydrogenase (GDH) is regulated by both positive (leucine and ADP) and negative (GTP and ATP) allosteric factors. We hypothesized that the phosphate potential of ␤-cells regulates the sensitivity of leucine stimulation. These predictions were tested by measuring leucine-stimulated insulin secretion in perifused rat islets following glucose depletion and by tracing the nitrogen flux of [2-15 N]glutamine using stable isotope techniques. The sensitivity of leucine stimulation was enhanced by long time (120-min) energy depletion and inhibited by glucose pretreatment. After limited 50-min glucose depletion, leucine, not ␣-ketoisocaproate, failed to stimulate insulin release. ␤-Cells sensitivity to leucine is therefore proposed to be a function of GDH activation. Leucine increased the flux through GDH 3-fold compared with controls while causing insulin release. High glucose inhibited flux through both glutaminase and GDH, and leucine was unable to override this inhibition. These results clearly show that leucine induced the secretion of insulin by augmenting glutaminolysis through activating glutaminase and GDH. Glucose regulates ␤-cell sensitivity to leucine by elevating the ratio of ATP and GTP to ADP and P i and thereby decreasing the flux through GDH and glutaminase. These mechanisms provide an explanation for hypoglycemia caused by mutations of GDH in children.In addition to glucose, amino acids and other metabolic fuels are important stimulants of insulin secretion from pancreatic ␤-cells. Leucine, which has been studied intensively, may stimulate insulin release through two different mechanisms. The first involves transamination of leucine to ␣-ketoisocaproate (KIC) 1 and subsequent mitochondrial oxidation. The second promotes insulin release via allosteric activation of glutamate dehydrogenase (GDH) causing oxidation of glutamate to the Krebs cycle intermediate, ␣-ketoglutarate, plus ammonia. The importance of the latter mechanism has been highlighted recently by the discovery of a dominant form of congenital hyperinsulinism associated with mutations of GDH leading to a gain of enzyme activity, because sensitivity to inhibition by GTP and ATP is impaired (1-3). Affected children have increased ␤-cell responsiveness to leucine and are susceptible to acute hypoglycemia following a high protein meal (4). The involvement of GDH may explain the observation that, in contrast to other amino acids, leucine-stimulated insulin secretion (LSIS) is suppressed by high glucose. For example, Gao et al. (5) reported that glucose inhibits leucine stimulation of glutaminolysis and insulin secretion in isolated mouse islets, presumably by increasing intracellular ATP and GTP while decreasing ADP and thus inhibiting GDH activity.GDH has also been proposed by Maechler and Wollheim (6) to play an essential role in glucose-mediated insulin secretion by acting in the reverse direction to catalyze production of glutamate, which is hypothesized to work as a cofactor in the process leading to exocytosis of insulin granules. T...

Anaphase-promoting complex/cyclosome-Cdh1 coordinates glycolysis and glutaminolysis with transition to S phase in human T lymphocytes

“…Moreover, it has been shown that the rate of utilization of glutamine in proliferating T lymphocytes is even higher than that of glucose (19). Glutaminase is a critical enzyme in glutaminolysis (13,20), and its activity is known to be increased in proliferating T lymphocytes (13) and in cancer cells (21,22). The fact that this enzyme contains a KEN box and, as our experiments show, is a substrate for APC/ C-Cdh1 indicates that its activity is regulated in a similar manner to that of PFKFB3.…”

Insulin secretion profiles are modified by overexpression of glutamate dehydrogenase in pancreatic islets

“…Principally, GDH is also considered important for its anaplerotic function, mediating deamination of glutamate to α-ketoglutarate [51]. Therefore, in pancreatic beta cells, the preferred directional flux of the enzyme is still under discussion [16,29,30,31,52], depending both on stimulation conditions and allosteric regulation.…”

“…Therefore, islet glutamate levels can be markedly increased by exposure to extracellular glutamine and, because of scarce conversion of glutamate to α-ketoglutarate [24], without stimulation of insulin secretion [24,55]. Activation of GDH by L-leucine or its non-metabolisable analogue BCH increases glutamine oxidation and insulin secretion, essentially by enhancing the oxidative deamination of glutamate [25,27,28,29,32,33,56]. GDH activity is under the tight control of allosteric effectors, although the flux direction depends chiefly on the relative supply of substrates and cofactors, i.e.…”

“…Indeed, glutamine oxidation can be prompted by allosteric activation of GDH, an effect correlating with stimulation of insulin secretion [25,27]. However, glucose exposure inhibits glutamine oxidation [28] and glutamate formation from glutamine [29]. Therefore, GDH could play a dual role in glutamate handling [30].…”

Mitochondrial regulation of insulin production in rat pancreatic islets

“…The fact that mitochondrial activation via glutamate dehydrogenase does not increase specific proinsulin biosynthesis contrasts with the strong specific effect of succinate, emphasising its unique role in signalling proinsulin biosynthesis. Allosteric activation of glutamate dehydrogenase by L-leucine or its non-metabolisable analogue BCH increases glutamine oxidation and insulin secretion [21,30]. The conversion of glutamate to alphaketoglutarate eventually increases mitochondrial succinate levels.…”