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. In cultured islets the second phase of glucose stimulation was even enhanced. In fresh and in cultured islets CHX strongly reduced the content of proinsulin, but not of insulin, and moderately diminished the [Ca2+]i increase during stimulation. The oxygen consumption rate (OCR) of fresh islets was about 50% higher than that of cultured islets at basal glucose and was significantly increased by glucose but not tolbutamide. In fresh, but not in cultured, islets CHX diminished the glucose-induced OCR increase and changes in the NAD(P)H- and FAD-autofluorescence. It is concluded that short-term CHX exposure interferes with the signal function of the mitochondria, which have different working conditions in fresh and in cultured islets. The interference may not be an off-target effect but may result from the inhibited cytosolic synthesis of mitochondrial proteins.

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. While it is generally recognized that the amplifying signals are also derived from the mitochondrial metabolism, their exact nature is still unclear. The current study tests the hypothesis that the supply of cytosolic acetyl-CoA is a signal in the amplifying pathway. The contents of acetyl-CoA and acetyl-CoA plus CoA-SH were measured in isolated mouse islets. Insulin secretion was recorded in isolated perifused islets. In islets, the ATP-sensitive K+ channels of which were pharmacologically closed and which were preincubated without exogenous fuel, 10 mmol/L alpha-ketoisocaproate enhanced the acetyl-CoA content after 5 and 20 min incubations and decreased the acetyl-CoA plus CoA-SH within 5 min, but not after 20 min. In islets not exposed to drugs, the preincubation with 3 mmol/L glucose, a non-triggering concentration, elevated the acetyl-CoA content. This content was further increased after 5 min and 20 min incubations with 30 mmol/L glucose, concurrent with a strong increase in insulin secretion. Alpha-ketoisocaproate and glucose increase the supply of acetyl-CoA in the beta-cell cytosol during both phases of insulin secretion. Most likely, this increase provides a signal for the metabolic amplification.

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. The identification of ATP-sensitive K+-channels provided the link between the level of adenine nucleotides and the electrical activity of the beta cell, but the depolarization-induced Ca2+-influx into the beta cells, although necessary for stimulated secretion, is not sufficient to generate the secretion pattern as produced by glucose and other nutrient secretagogues. The metabolic amplification of insulin secretion is thus the sequence of events that enables the secretory response to a nutrient secretagogue to exceed the secretory response to a purely depolarizing stimulus and is thus of prime importance. Since the cataplerotic export of mitochondrial metabolites is involved in this signaling, an orienting overview on the topic of nutrient secretagogues beyond glucose is included. Their judicious use may help to define better the nature of the signals and their mechanism of action.

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. This was also the response of cultured islets to the nutrient secretagogue alpha-ketoisocaproic acid, whereas the secretion of fresh islets increased similarly fast but remained strongly elevated. The responses of fresh and cultured islets to purely depolarizing stimuli (tolbutamide or KCl), however, were closely similar. Signs of apoptosis and necrosis were rare in both preparations. In cultured islets, the glucose-induced rise of the cytosolic Ca2+ concentration started from a lower value and was larger as was the increase of the ATP/ADP ratio. The prestimulatory level of mitochondrial reducing equivalents, expressed as the NAD(P)H/FAD fluorescence ratio, was lower in cultured islets, but increased more strongly than in fresh islets. When culture conditions were modified by replacing RPMI with Krebs-Ringer medium and FCS with BSA, the amount of released insulin varied widely, but the kinetics always showed a predominant first phase. In conclusion, the secretion kinetics of fresh mouse islets is more responsive to variations of nutrient stimulation than cultured islets. The more uniform kinetics of the latter may be caused by a different use of endogenous metabolites.

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. MATERIALS/METHODS: Insulin secretion, NAD(P)H- and FAD-autofluorescence, Fura-2 fluorescence and oxygen consumption were measured in perifused NMRI mouse islets. The ATP- and ADP-contents were measured in statically incubated mouse islets. All islets were freshly isolated. RESULTS: While the original observation on the dissociation between glucose- and KIC-amplification was obtained with islets that had been exposed to a high concentration of the sulfonylurea glipizide in the absence of glucose, we now show that in the absence of exogenous fuel a moderate depolarization, irrespective of its mechanism, progressively decreased the amplification in response to both glucose and KIC. However, the amplification in response to glucose declined faster, so a time window exists where glucose was already inefficient, whereas KIC was of unimpaired efficiency. Measurements of adenine nucleotides, NAD(P)H- and FAD-autofluorescence, and oxygen consumption point to a central role of the mitochondrial metabolism in this process. The desensitization could be quickly reversed by increasing oxidative deamination of glutamate and consequently anaplerosis of the citrate cycle. CONCLUSION: Depolarization in the absence of exogenous fuel may be a useful model to identify those signals which are indispensable for the generation of metabolic amplification.

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. Fuel-induced effects were examined after treating the islets for one hour with medium devoid of fuels. This experimental design prevented acute amplification, but only when glucose was the sole fuel. RESULTS: Strong amplification of insulin secretion by α-ketoisocaproate or glucose combined with α-ketoisovalerate (supplying mitochondrial oxaloacetate) was abolished within 14min after transition to medium devoid of fuels. After transition from medium containing glucose plus α-ketoisovalerate to medium containing solely glucose or α-ketoisovalerate, amplification (strong or weak, respectively) occurred until the end of the experiment. Glucose (alone or combined with α-ketoisovalerate) increased the total acetyl-CoA content as intensely as α-ketoisocaproate. Low concentrations of α-ketoisovalerate or α-ketoisocaproate were sufficient for saturation of acetyl-CoA increase, but caused no or only weak amplification, respectively. No acetyl-CoA increases occurred in the absence of glipizide. CONCLUSIONS: Glucose and other fuels regulate acute amplification of insulin secretion by controlling the supply of acetyl-CoA to the ß-cell cytosol. Cytosolic acetyl-CoA does not amplify by serving as substrate for syntheses of metabolic intermediates, but amplifies by acting as substrate for cytosolic protein acetylation.

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

OBJECTIVE: The ß-cell metabolism of glucose and of some other fuels (e.g. α-ketoisocaproate) generates signals triggering and acutely amplifying insulin secretion. As the pathway coupling metabolism with amplification is largely unknown, we aimed to narrow down the putative amplifying signals. MATERIALS/METHODS: An experimental design was used which previously prevented glucose-induced, but not α-ketoisocaproate-induced insulin secretion. Isolated mouse islets were pretreated for one hour with medium devoid of fuels and containing the sulfonylurea glipizide in high concentration which closed all ATP-sensitive K(+) channels. This concentration was also applied during the subsequent examination of fuel-induced effects. In perifused or incubated islets, insulin secretion and metabolic parameters were measured. RESULTS: The pretreatment decreased the islet ATP/ADP ratio. Whereas glucose and α-ketoisovalerate were ineffective or weakly effective, respectively, when tested separately, their combination strongly enhanced the insulin secretion. Compared with glucose, the strong amplifier α-ketoisocaproate caused less increase in NAD(P)H-fluorescence and less mitochondrial hyperpolarization. Compared with α-ketoisovalerate, α-ketoisocaproate caused greater increase in NAD(P)H-fluorescence and greater mitochondrial hyperpolarization. Neither α-ketoacid anion enhanced the islet ATP/ADP ratio during onset of the insulin secretion. α-Ketoisocaproate induced a higher pyruvate content than glucose, slowly elevated the citrate content which was not changed by glucose and generated a much higher acetoacetate content than other fuels. α-Ketoisovalerate alone or in combination with glucose did not increase the citrate content. CONCLUSIONS: In ß-cells, mitochondrial energy generation does not mediate acute metabolic amplification, but mitochondrial production of acetyl-CoA and supplemental acetoacetate supplies cytosolic metabolites which induce the generation of specific amplifying signals.

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. A major conceptual problem consists in the occurrence of action potentials during KATP channel closure, but not during K+ depolarization, which leaves the K+ channel conductance unchanged. Recent observations suggest that the signal function of the endogenously generated depolarization is not homogeneous, but may rather differ between the component mainly determined by KATP channel closure (slow waves) and that mainly determined by Ca2+ influx (action potentials).

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. Insulin release, K(ATP) channel currents, membrane potential, ATP/ADP ratio and fluorescence of NAD(P)H (reduced pyridine nucleotides) were measured in mouse pancreatic islets and beta-cells. At a substimulatory glucose concentration (5 mM), dimethyl alpha-ketoglutarate (15 mM) produced a sustained insulin release, but no change of the islet ATP/ADP ratio and NAD(P)H fluorescence. In the absence of glucose, however, dimethyl alpha-ketoglutarate (15 mM) did not stimulate insulin release although it increased the ATP/ADP ratio and NAD(P)H fluorescence. Insulin secretion induced by a maximally effective concentration of the K(ATP) channel-blocking sulfonylurea glipizide was strongly amplified by dimethyl alpha-ketoglutarate in the presence of 5 mM glucose, but only moderately in the absence of glucose. Dimethyl alpha-ketoglutarate directly inhibited K(ATP) channels in inside-out membrane patches, depolarized the plasma membrane of intact beta-cells and generated action potentials. In conclusion, the stimulation of insulin secretion by extracellularly applied dimethyl alpha-ketoglutarate depends on inhibition of beta-cell K(ATP) channels by direct action of dimethyl alpha-ketoglutarate. The metabolism of alpha-ketoglutarate generated intracellularly by ester cleavage contributes to stimulation of insulin secretion both by indirect K(ATP) channel inhibition (via activation of ATP production) and by an amplifying effect.

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). These findings were compared with the insulin secretion stimulated in isolated mouse islets by alpha-keto acid anions (10 mM). When all K(ATP) channels were closed by the sulfonylurea glipizide, alpha-keto acid anions amplified the insulin release in the order beta-phenylpyruvate < alpha-ketoisovalerate < alpha-ketovalerate approximately alpha-ketocaproate < alpha-ketoisocaproate. The differences in amplification apparently reflected special features of the metabolism of the individual alpha-keto acid anions. In islets with active K(ATP) channels, the first peak of insulin secretion triggered by alpha-keto acid anions was similar for alpha-ketoisocaproate, alpha-ketocaproate, and beta-phenylpyruvate but lower for alpha-ketovalerate and insignificant for alpha-ketoisovalerate. This difference from the above orders indicates that direct K(ATP) channel inhibition is not involved in the secretory responses to alpha-ketoisovalerate and alpha-ketovalerate, moderately contributes to initiation of insulin secretion by alpha-ketoisocaproate and alpha-ketocaproate, and is a major component of the insulin-releasing property of beta-phenylpyruvate.

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. Pancreatic K(ATP) channels are composed of two subunits, a pore-forming inwardly rectifying K(+) channel (Kir6.2) subunit and a regulatory subunit (the sulfonylurea receptor of subtype 1 (SUR1)) in a (SUR1/Kir6.2)(4) stoichiometry. The aim of the present study was to characterize the interaction of green-fluorescent 3-[3-(4,4 difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacen-3-yl)propanamido] glibenclamide (Bodipy-glibenclamide) with pancreatic beta-cell K(ATP) channels using patch-clamp and fluorescence microscopy techniques. Bodipy-glibenclamide inhibited K(ATP) currents from the clonal insulinoma cell line RINm5F half-maximally at a concentration of 0.6nM. Using laser-scanning confocal microscopy Bodipy-glibenclamide was shown to induce a diffuse fluorescence across the RINm5F cell, but only about 17% of total Bodipy-glibenclamide-induced fluorescence intensity in RINm5F cells was due to specific binding to SUR1. Using fluorescence correlation spectroscopy, it could be demonstrated that the fluorescence label contributes to the protein binding and, therefore, possibly also to the non-specific binding of Bodipy-glibenclamide observed in RINm5F cells. Specific binding of Bodipy-glibenclamide to SUR1 in RINm5F cells might be localized to different intracellular structures (nuclear envelope, endoplasmic reticulum, Golgi compartment, insulin secretory granules) as well as to the plasma membrane. In conclusion, Bodipy-glibenclamide is a high-affinity blocker of pancreatic beta-cell K(ATP) currents and can be used for visualizing SUR1 in intact pancreatic beta-cells, although non-specific binding must be taken into account in confocal microscopy experiments on intact beta-cells.

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). In the present study we characterised the interaction with SURs of the new sulfonylurea analogues 5-chloro-N-[2-(4-hydroxyphenyl)ethyl]-2-methoxybenzamide (compound I) and (4-[2-(5-chloro-2-methoxybenzamido)ethyl]phenyl)phosphate (compound II). Compounds I and II differ from the sulfonylurea analogue meglitinide only in so far as the carboxylic group of meglitinide is replaced by a hydroxyl group or a phosphate group, respectively. The binding affinities of compound II for the SUR subtypes SUR1 (identified in beta-cells) and SUR2A (identified in heart and skeletal muscle) were higher by 55 or 21-fold, respectively, than the corresponding affinities for compound I. In inside-out patch-clamp experiments compound II inhibited ATP-sensitive K(+)-channels of the SUR1/K(IR)6.2-type (characteristic of beta-cells) with an IC(50) value of 0.16 microM which is 6-fold lower than the corresponding value for meglitinide. These findings strongly support the conclusion that the interaction of sulfonylureas and acidic analogues with SURs is favoured by the anionic group of these drugs and that a phosphate group allows more efficient ligand interaction with SUR1 than a carboxylic group.