Glucotoxicity Inhibits Late Steps of Insulin Exocytosis

Abstract

Prolonged exposure of beta-cells to high glucose (glucotoxicity) diminishes insulin secretion in response to glucose and has been linked to altered generation of metabolism-secretion coupling factors. We have investigated whether glucotoxicity may also alter calcium handling and late steps in secretion such as exocytosis. Clonal INS-1E beta-cells cultured at high glucose (20 or 30 mM vs. 5.5 mM) for 72 h exhibited elevated basal intracellular calcium ([Ca2+]i), which was KATP-channel dependent and due to long-term activation of protein kinase A. An increased amplitude and shortened duration of depolarization-evoked rises in [Ca2+]i were apparent. These changes were probably linked to the observed increased filling of intracellular stores and to short-term activation of protein kinase A. Insulin secretion was reduced not only by acute stimulation with either glucose or KCl but more importantly by direct calcium stimulation of permeabilized cells. These findings indicate a defect in the final steps of exocytosis. To confirm this, we measured expression levels of some 30 proteins implicated in trafficking/exocytosis of post-Golgi vesicles. Several proteins required for calcium-induced exocytosis of secretory granules were down-regulated, such as the soluble N-ethylmaleimide-sensitive factor-sensitive factor attachment receptor (SNARE) proteins VAMP-2 [vesicle (v)-SNARE, vesicle-associated membrane protein 2] and syntaxin 1 as well as complexin. VAMP-2 was also reduced in human islets. In contrast, cell immunostaining and expression levels of several fluorescent proteins suggested that other post-trans-Golgi trafficking steps and compartments are preserved and that cells were not degranulated. Thus, these studies indicate that, in addition to known metabolic changes, glucotoxicity impedes generation of signals for secretion and diminishes the efficiency of late steps in exocytosis.

Quantitative proteomic analysis of single pancreatic islets

“…4). In the case of vesicle-associated membrane protein (VAMP)-2, synaptotagmin-like protein 4 (Sytl4), and Rab3b, who interact upon Ca 2ϩ increase, this has been observed before (26,27 expression of Sytl4 reduces the number of docked vesicles to the plasma membrane, which is suggested to effect insulin secretion (19,28). Decreased mRNA and protein expression of VAMP2 and various other secretory proteins have also been associated with impaired protein secretion in diabetic patients (29) and animal models for diabetes (30).…”

Cellular responses of novel human pancreatic β-cell line, 1.1B4 to hyperglycemia

“…[16][17][18][19][20][21][22] Basal insulin release at 5.6 mM glucose was not affected by hyperglycemia but the secretory response to 16.7 mM glucose was impaired by 48 and 72 h exposure to 25 mM glucose. We also observed that insulin release from 1.1B4 cells chronically exposed to hyperglycemia for 48 and 72 h was significantly reduced in response to alanine, GLP-1, KCl, elevated Ca 2+ , or forskolin.…”

Adenylyl cyclase 8 is central to glucagon-like peptide 1 signalling and effects of chronically elevated glucose in rat and human pancreatic beta cells

“…Plasmids encoding ADCY8, ADCY8-eGFP and myc-GLP1R were generously donated by D. Cooper (University of Cambridge, UK) and A. Bisello (University of Pittsburgh, PA, USA). Plasmids encoding control short hairpin RNA (shRNA) or shRNA directed against ADCY8 were from SABiosciences (Frederick, MD, USA), the plasmid encoding human growth hormone (hGH) has been described [16,17]. CD8-eGFP was obtained by replacing the cytosolic domain of CD8 with eGFP.…”

“…Prolonged exposure to elevated levels of glucose alters calcium handling in a cAMP-dependent manner [15,16]. As the incretin GLP-1 exerts most of its effects through these second messengers and its efficacy is partially lost in type 2 diabetes, we used this hormone to investigate the interplay between cAMP and calcium under exposure to normal and chronically elevated glucose levels to identify underlying changes in signal transduction and gene expression.…”