Short-Term Inhibition of Translation by Cycloheximide Concurrently Affects Mitochondrial Function and Insulin Secretion in Islets from Female Mice.

Since glucose stimulates protein biosynthesis in beta cells concomitantly with the stimulation of insulin release, the possible interaction of both processes was explored. The protein biosynthesis was inhibited by 10 μM cycloheximide (CHX) 60 min prior to the stimulation of perifused, freshly isolated or 22 h-cultured NMRI mouse islets. CHX reduced the insulinotropic effect of 25 mM glucose or 500 μM tolbutamide in fresh but not in cultured islets.

Regulation of insulin secretion in mouse islets: metabolic amplification by alpha-ketoisocaproate coincides with rapid and sustained increase in acetyl-CoA content.

Glucose and alpha-ketoisocaproate, the keto acid analogue of leucine, stimulate insulin secretion in the absence of other exogenous fuels. Their mitochondrial metabolism in the beta-cell raises the cytosolic ATP/ADP ratio, thereby providing the triggering signal for the exocytosis of the insulin granules. However, additional amplifying signals are required for the full extent of insulin secretion stimulated by these fuels.

What Is the Metabolic Amplification of Insulin Secretion and Is It (Still) Relevant?

The pancreatic beta-cell transduces the availability of nutrients into the secretion of insulin. While this process is extensively modified by hormones and neurotransmitters, it is the availability of nutrients, above all glucose, which sets the process of insulin synthesis and secretion in motion. The central role of the mitochondria in this process was identified decades ago, but how changes in mitochondrial activity are coupled to the exocytosis of insulin granules is still incompletely understood.

Fresh and cultured mouse islets differ in their response to nutrient stimulation.

Observing different kinetics of nutrient-induced insulin secretion in fresh and cultured islets under the same condition we compared parameters of stimulus secretion coupling in freshly isolated and 22-h-cultured NMRI mouse islets. Stimulation of fresh islets with 30 mM glucose after perifusion without nutrient gave a continuously ascending secretion rate. In 22-h-cultured islets the same protocol produced a brisk first phase followed by a moderately elevated plateau, a pattern regarded to be typical for mouse islets.

Metabolic amplification of insulin secretion is differentially desensitized by depolarization in the absence of exogenous fuels.

Objective: The metabolic amplification of insulin secretion is the sequence of events which enables the secretory response to a fuel secretagogue to exceed the secretory response to a purely depolarizing stimulus. The signals in this pathway are incompletely understood. Here, we have characterized an experimental procedure by which the amplifying response to glucose is reversibly desensitized, while the response to α-ketoisocaproic acid (KIC) is unchanged.

Acute metabolic amplification of insulin secretion in mouse islets: Role of cytosolic acetyl-CoA.

Objective: Stimulation of the ß-cell metabolism by glucose and other fuels triggers insulin release by enhancing the mitochondrial ATP production and acutely amplifies the secretory response by increase in mitochondrial export of metabolites. We aimed to narrow down the uniform final reaction steps mediating fuel-induced acute amplification of insulin secretion.

Material/methods: Insulin secretion and metabolic parameters were measured in isolated mouse islets exposed to the sulfonylurea glipizide in high concentration (closing all ATP-sensitive K(+) channels) during the entire experiment.

Acute metabolic amplification of insulin secretion in mouse islets is mediated by mitochondrial export of metabolites, but not by mitochondrial energy generation.

Objective: The β-cell metabolism of glucose and of some other fuels (e.g. α-ketoisocaproate) generates signals triggering and acutely amplifying insulin secretion.

Studies of first phase insulin secretion using imposed plasma membrane depolarization.

The first phase of glucose-induced insulin secretion is generally regarded to represent the release of a finite pool of secretion-ready granules, triggered by the depolarization-induced influx of Ca2+ through L-type Ca2+ channels. However, the experimental induction of insulin secretion by imposed plasma membrane depolarization may be more complicated than currently appreciated. A comparison of the effects of high K+ concentrations with those of KATP channel closure, which initiates the electrical activity of the beta cell, suggests that 40 mM K+, which is a popular tool to produce a first phase-like secretion, is of supraphysiological strength, whereas the 20 mV depolarization by 15 mM K+ is nearly inefficient.

Triggering and amplification of insulin secretion by dimethyl alpha-ketoglutarate, a membrane permeable alpha-ketoglutarate analogue.

Cytosolic alpha-ketoglutarate is a potential signalling compound at late steps of stimulus-secretion-coupling in the course of insulin secretion induced by glucose and other fuels. This hypothesis is mainly based on the insulin-releasing effect of the membrane permeable ester dimethyl alpha-ketoglutarate which enters the beta-cell and is cleaved to produce cytosolic monomethyl alpha-ketoglutarate and eventually alpha-ketoglutarate. The present study tested this hypothesis.

Mechanism of the insulin-releasing action of alpha-ketoisocaproate and related alpha-keto acid anions.

Alpha-ketoisocaproate directly inhibits the ATP-sensitive K(+) channel (K(ATP) channel) in pancreatic beta-cells, but it is unknown whether direct K(ATP) channel inhibition contributes to insulin release by alpha-ketoisocaproate and related alpha-keto acid anions, which are generally believed to act via beta-cell metabolism. In membranes from HIT-T15 beta-cells and COS-1 cells expressing sulfonylurea receptor 1, alpha-keto acid anions bound to the sulfonylurea receptor site of the K(ATP) channel with affinities increasing in the order alpha-ketoisovalerate < alpha-ketovalerate < alpha-ketoisocaproate < alpha-ketocaproate < beta-phenylpyruvate. Patch-clamp experiments revealed a similar order for the K(ATP) channel-inhibitory potencies of the compounds (applied at the cytoplasmic side of inside-out patches from mouse beta-cells).

Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic beta-cell ATP-sensitive K+ currents.

Hypoglycemic sulfonylureas (e.g. tolbutamide, glibenclamide) exert their stimulatory effects on pancreatic beta-cells by closure of ATP-sensitive K(+) (K(ATP)) channels.

Inhibition of ATP-sensitive K(+)-channels by a sulfonylurea analogue with a phosphate group.

Hypoglycaemic sulfonylureas initiate insulin secretion by direct inhibition of ATP-sensitive K(+)-channels in the pancreatic beta-cells. These channels are composed of two proteins, a pore-forming subunit (K(IR)6.2 in the case of beta-cells) and a regulatory subunit, the sulfonylurea receptor (SUR).