Amino acid-dependent stimulation of insulin secretion

Several amino acids have been proposed to participate in the amplifying pathway of glucose-stimulated insulin secretion. Among them, L-glutamine, L-glutamate, L-alanine, L-arginine and L-leucine have been suggested [57, 118-122]. Their physiological levels in the plasma vary depending on the amino acid (glutamine: 390-650 µmol/L; glutamate:

18-98 µmol/L; alanine: 230-510 µmol/L; arginine: 13-64 µmol/L; leucine: 66-170 µmol/L; www.nlm.nih.gov/medlineplus/ency/article/003361.html). Mitochondrial metabolism is crucial for the coupling of amino acid and glucose recognition to insulin exocytosis. Consequently, key enzymes and transporters (e.g. aspartate aminotransferase, malate-aspartate shuttle, and glutamate dehydrogenase) play an important role in the control of insulin secretion.

Figure 1.11: Amino acids implicated in the amplifying pathway of glucose stimulated insulin secretion (modified from Newsholme et al 2007 [123]).

L-glutamine

Glutamine is the most abundant free amino acid in the body and it plays an important role in promoting and maintaining function of various organs (e.g. liver, neurons). It is implicated in several cell specific processes such as metabolism (e.g., oxidative fuel), cell integrity (apoptosis, cell proliferation), protein synthesis, and degradation, insulin resistance and insulin secretion [124]. Furthermore, glutamine has been demonstrated to regulate the expression of many genes related to metabolism, signal transduction, cell defence and repair, and to activate intracellular signalling pathways. Thus, glutamine is involved in a broad spectrum of cellular processes.

Previous studies have shown that glutamine is consumed at high rates by both islets and clonal β-cells [120]. The amino acid is rapidly taken up and metabolized by islets but glutamine alone fails to stimulate insulin secretion both in the absence and presence of glucose. However, a marked stimulation of insulin release by L-glutamine occurs in the presence of leucine [121]. This action was attributed to activation of the mitochondrial enzyme glutamate dehydrogenase (GDH) by leucine. GDH catalyses the reversible generation of α-ketoglutarate from glutamate. The proposed model for the role of

cycle, hence increasing the ATP/ADP ratio and stimulation of insulin release. In this model, glutamate is generated from glutamine through the action of phosphate-activated glutaminase whereas leucine acts as allosteric activator of GDH resulting in α -ketoglutarate generation [125]. However, at stimulatory glucose concentrations glutaminolysis is inhibited, presumably via GTP-mediated allosteric inhibition of GDH [126].

Paradoxically glutamine alone fails to stimulate insulin secretion. It has been suggested that glutamine is metabolized to GABA and aspartate which accumulates in islets [127, 128]. In the absence of leucine, there was no oxidation of glutamine via the TCA cycle.

These results were taken to explain the poor ability of glutamine to stimulate insulin secretion. In contrast, a NMR study performed in clonal β-cells demonstrated that the main products of glutamine metabolism are glutamate and aspartate [129]. There was no production of GABA. However, glutamine-derived glutamate increased the production of glutathione. Addition of glucose stimulated glutamate generation but did not affect glutamine consumption [126, 129]. Furthermore, glutamine increased the flux through GDH without affecting insulin secretion [102]. Interestingly, SUR1-/- mice mice revealed an increased glucose-stimulated glutamate production compared to control [130].

However, GSIS was dramatically decreased compared to control whereas basal insulin secretion rates were increased 6-fold [95]. The transgenic mice showed a suppression of glutamate decarboxylase (GAD) expression which catalyzes the conversion of glutamate to GABA. Concomitantly, GABA levels were decreased 10-fold. The reduction of GAD level was caused by an increase in intracellular Ca²⁺-concentration, explaining also elevated basal insulin secretion rates of SUR1-/- mice and the reduced GSIS compared to controls [130, 131].

Leucine

Leucine can stimulate insulin secretion through two different mechanisms, both leading to increased mitochondrial metabolism: (1) Allosteric activation of GDH and (2) Deamination of leucine to α-ketoisocaproate (αKIC) followed by entry into the TCA cycle via acetyl-CoA [121, 132, 133]. However, leucine-induced insulin secretion is inhibited in the presence of high glucose concentrations due to allosteric inhibitory effects of GTP and ATP on the activity of GDH. Thus, GDH flux towards oxidative deamination of glutamate to aKG is inhibited at elevated glucose concentrations [134, 135] (see also glutamine paragraph). Interestingly, patients with hyperinsulinism (increased insulin secretion rates) were shown to have mutations in the inhibitory allosteric GTP-binding site of GDH.

Affected patients are more responsive to leucine and develop hypoglycaemia [135-137].

L-glutamate

The role of glutamate in insulin secretion is debated. Extracellular and intracellular messenger functions have been proposed for this amino acid. The participation of glutamate in metabolism secretion coupling will be discussed in chapter 1.8.3.

L-alanine

Evidence suggests that L-alanine promotes insulin secretion in clonal β-cells and islets at basal glucose concentrations by a mechanism requiring oxidative metabolism [120]. This stimulatory effect was caused by electrogenic Na⁺ co-transport, followed by membrane depolarization, generation of Ca²⁺ spike potentials and an increase in [Ca²⁺]i [138, 139].

Furthermore, metabolism and oxidation of alanine is important for its insulinotropic effects [140, 141]. It has been demonstrated that alanine is metabolised to glutamate, aspartate and lactate in clonal β-cells [118]. Additonally, alanine stimulates glucose oxidation and thereby insulin secretion. In contrast to clonal cells, alanine is a weak secretagogue in native β-cells.

L-Arginine

The stimulation of insulin secretion in response to this cationic amino acid is caused by membrane depolarization [122, 142-144]. Arginine is transported into the β-cells via the electrogenic transporter mCAT2A. Membrane depolarization, activation of voltage-gated Ca²⁺-channels and Ca²⁺-influx result in stimulated insulin release.

Homocystein

In contrast to the other amino acids, homocystein dose-dependently inhibited insulin secretion at moderate and stimulatory glucose concentrations from clonal β-cells [145].

In the presence of homocystein TCA cycle-dependent glucose metabolism was reduced [146]. Similar effects were observed when insulin release was stimulated with alanine, arginine or α-KIC. Homocystein might play a role in Type 2 diabetes as elevated homocystein levels have been reported in these patients [147].