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

Stefania Carobbio,Francesca Frigerio,Blanca Rubi,Lauréne Vetterli,Maria Bloksgaard,Asllan Gjinovci,Shirin Pournourmohammadi,Pedro L Herrera,Walter Reith,Susanne Mandrup,Pierre Maechler

doi:10.1074/jbc.m806295200

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 betaGlud1(-/-), 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 betaGlud1(-/-). 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 betaGlud1(-/-) 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 betaGlud1(-/-) 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 beta-cells. However, maximal secretory capacity is not required for maintenance of glucose homeostasis in normo-caloric conditions.

Highlights

The calcium signal supported by metabolism-derived additive factors [2]
Our results show that glutamate dehydrogenase (GDH) plays an essential role in the full development of the insulin secretory response
The results demonstrate that GDH is essential for the full development of the secretory response in ß-cells

Summary

EXPERIMENTAL PROCEDURES

Transgene Cloning—By screening a RPCI-22 mouse 129 BAC library For acute in vitro GDH knock-out, islets isolated from control and transgenic Glud1lox/lox mice were subjected to adenoviral treatment for 90 min with 0.4 ␮l/ml of the recombinant adenovirus rAdInsPNCre, thereby enabling expression of nuclear-localized Cre recombinase in ß-cells [20]. Pancreatic insulin content was determined in mouse pancreata that were first frozen in liquid nitrogen, homogenized, resuspended in cold acid ethanol, and left at 4 °C for 48 h with sonication after 24 h Insulin concentration in these different collected samples was measured by radioimmunoassay (Linco Research Inc., St. Charles, MO). A bioluminescent assay kit (Roche Applied Science) was used to measure ATP levels in batches of 10 isolated islets following a 30-min incubation period at 37 °C in the presence of 2.8 and 22.8 mM glucose. A p value lower than 0.05 was considered statistically significant

RESULTS

Secretory Responses of Isolated

Insulin secretion stimulated with

DISCUSSION