Deletion of Glutamate Dehydrogenase in ß-Cells Abolishes Part of the Insulin Secretory Response Not Required for Glucose Homeostasis

Abstract

Insulin exocytosis is regulated in pancreatic ß-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been tested yet in animal models. Here, we generated transgenic mice, named ßGlud1 ؊/؊ , with ß-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin secretion was reduced by 37% in ßGlud1 ؊/؊ . Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in ßGlud1 ؊/؊ islets fully restored glucose-induced insulin release. Thus, GDH appears to account for about 40% of glucose-stimulated insulin secretion and to lack redundant mechanisms. In ßGlud1 ؊/؊ mice, the reduced secretory capacity resulted in lower plasma insulin levels in response to both feeding and glucose load, while body weight gain was preserved. The results demonstrate that GDH is essential for the full development of the secretory response in ß-cells. However, maximal secretory capacity is not required for maintenance of glucose homeostasis in normo-caloric conditions.Pancreatic ß-cells produce the hormone insulin that is essential for glucose homeostasis. Upon nutrient stimulation, elevation of cytosolic calcium in the ß-cell is the primary and necessary signal for insulin exocytosis (1). Then, increasing the magnitude of the secretory response requires amplification of the calcium signal supported by metabolism-derived additive factors (2). The enzyme glutamate dehydrogenase (GDH, 2 EC 1.4.1.3) has been proposed to participate to the development of the secretory response. GDH is a homohexamer located in the mitochondrial matrix that catalyzes the reversible reaction: ␣-ketoglutarate ϩ NH 3 ϩ NAD(P)H % glutamate ϩ NAD(P) ϩ ; inhibited by GTP and activated by ADP (3, 4). Regarding ß-cell, allosteric activation of GDH by L-leucine or its non-metabolized analogue BCH has triggered most of the attention over the last three decades (5).To date, the role of GDH in ß-cell function remains unclear and debated. Specifically, GDH might play a role in glucoseinduced amplifying pathway through generation of glutamate (6, 7). GDH is also an amino acid sensor triggering insulin release upon glutamine stimulation in conditions of GDH allosteric activation (8 -10). Recently, the importance of GDH has been further highlighted by studies showing that SIRT4, a mitochondrial ADP-ribosyltransferase, down-regulates GDH activity and thereby modulates insulin secretion (11,12).GDH is encoded by a well-conserved 45-kb gene named GLUD1, which is organized into 13 exons (13). A decade ago, clinical data and associated genetic studies revealed GDH as a key enzyme for the control ...

Epigallocatechin-3-gallate (EGCG) activates AMPK through the inhibition of glutamate dehydrogenase in muscle and pancreatic ß-cells: A potential beneficial effect in the pre-diabetic state?

“…EGCG inhibited glucose-stimulated insulin secretion by 70% and that induced by glutamine + BCH by 76%. As reported previously (Vetterli et al, 2012;Carobbio et al, 2009), insulin secretion stimulated by either glucose or glutamine + BCH was reduced respectively by 63% and 56% in islets lacking ß-cell GDH (ßGlud1 −/− ). Of note, EGCG did not alter insulin secretion in ß-cell GDH knockout islets (Fig.…”

“…Mouse pancreatic islets were obtained from either C57BL/6J wild type mice or ß-cell-specific GDH knockout mice (ßGlud1 −/− ) described previously (Carobbio et al, 2009). Islets were isolated by collagenase digestion (collagenase P, Roche) as detailed elsewhere (Carobbio et al, 2004) and cultured overnight free floating in RPMI-1640 medium (Gibco) supplemented with 10% (v/v) heatinactivated fetal calf serum (FCS), 2 mM glutamine, 10 mM Hepes, 100 U/ml penicillin, 100 g/ml streptomycin, 1 mM sodium pyruvate and 50 M 2-mercaptoethanol.…”

“…To study whether GDH is the main target of EGCG in ß-cells we first compared the insulin secretory responses of wild type islets with those of ß-cell-specific GDH knockout islets isolated from ßGlud1 −/− mice (Carobbio et al, 2009). In wild type control mouse islets, 22.8 mM glucose and the combination of 5 mM glutamine plus 10 mM BCH stimulated insulin secretion 3.3-fold and 3.1-fold over basal release, respectively (Fig.…”

The Zinc Finger Protein ZBTB20 Regulates Transcription of Fructose-1,6-Bisphosphatase 1 and β Cell Function in Mice

“…We found that ZBTB20 protein expression was not significantly affected in hypothalamus, liver, or skeletal muscle from ␤-ZB20KO mice compared with their control counterparts ( Figure 2D and Supplementary Figure 1), which is consistent with previous reports about this RIP-Cre line. 19,21 Born at the expected frequencies, ␤-ZB20KO mice were fertile, grew normally, and did not differ in size or weight (Supplementary Figure 2) from their littermate controls. In addition, there was no significant difference of food intake, oxygen consumption, CO 2 production, or activity between ␤-ZB20KO and control adult mice (Supplementary Figure 3), indicating that ZBTB20 ablation mediated by this RIP-Cre line did not affect energy homeostasis.…”

“…These were consistent with previous reports that this RIP-Cre line has normal glucose tolerance. 19,21 Collectively, these results suggested that the ␤ cell dysfunction in ␤-ZB20KO mice was caused by ␤ cell-specific deletion of ZBTB20.…”