Myriam Nenquin

Mechanisms of the stimulation of insulin release by saturated fatty acids. A study of palmitate effects in mouse beta-cells

The mechanisms by which fatty acids increase insulin release are not known. In this study, mouse islets were used as a model and palmitate as a reference compound to study in vitro how saturated fatty acids influence pancreatic β-cells. Palmitate (625 μM) was bound to albumin. It did not affect basal insulin release (3 mM glucose) but increased the release induced by 10–15 mM glucose. This effect was dependent on the concentration of free rather than total palmitate. It was reversible and abolished by epinephrine, diazoxide, nimodipine, or omission of extracellular Ca. Bromopalmitate and methyl palmoxirate, two inhibitors of fatty acid oxidation, were ineffective alone, and only bromopalmitate partially inhibited the effects of palmitate on insulin release. The increase in insulin release produced by palmitate could not be ascribed to a blockade of ATP-sensitive K+-channels because the fatty acid only barely decreased 86Rb efflux and did not depolarize β-cells in 3 mM glucose. The small effect on 86Rb efflux might be attributed to a slight rise in the ATP/ADP ratio. No such rise occurred when palmitate was tested in 15 mM glucose, and the fatty acid consistently accelerated 86Rb efflux under these conditions. Measurements of β-cell membrane potential (intracellular microelectrodes) and of free cytoplasmic calcium (Cai 2+) in β-cells (Fura 2 technique) showed that palmitate increases Ca2+ influx; it also caused a very small mobilization of intracellular Ca. The persistence of this stimulation of Ca2+ influx in the presence of diazoxide and high K+ suggests that palmitate might act on Ca2+ channels. The rise in Ca12+ produced by palmitate was accompanied by an increase in insulin release only if the concentration of glucose was sufficiently high. The β-cell response to palmitate thus differs from the responses to glucose and other metabolized nutrients in several respects. Saturated fatty acids appear to potentiate insulin release through an increase in Ca12+ and another, yet unidentified, fuel-dependent mechanism.

Shortcomings of current models of glucose-induced insulin secretion.

Glucose‐induced insulin secretion by pancreatic β‐cells is generally schematized by a ‘consensus model’ that involves the following sequence of events: acceleration of glucose metabolism, closure of ATP‐sensitive potassium channels (KATP channels) in the plasma membrane, depolarization, influx of Ca2+ through voltage‐dependent calcium channels and a rise in cytosolic‐free Ca2+ concentration that induces exocytosis of insulin‐containing granules. This model adequately depicts the essential triggering pathway but is incomplete. In this article, we first make a case for a model of dual regulation in which a metabolic amplifying pathway is also activated by glucose and augments the secretory response to the triggering Ca2+ signal under physiological conditions. We next discuss experimental evidence, largely but not exclusively obtained from β‐cells lacking KATP channels, which indicates that these channels are not the only possible transducers of glucose effects on the triggering Ca2+signal. We finally address the identity of the widely neglected background inward current (Cl− efflux vs. Na+ or Ca2+ influx through voltage‐independent channels) that is necessary to cause β‐cell depolarization when glucose closes KATP channels. More attention should be paid to the possibility that some components of this background current are influenced by glucose metabolism and have their place in a model of glucose‐induced insulin secretion.

Quinine-induced modifications of insulin release and glucose metabolism by isolated pancreatic islets

The striking analogies between the processes regulating secretion and contraction led Douglas and Rubin [l] to introduce the term ‘stimulus-secretion coupling’ to parallel the concept of ‘excitation-contraction coupling’ proposed earlier by Sandow [2] . The effects of several alkaloids on muscle contraction have long been recognized and their site and mechanism of action extensively studied [3]. Among these alkaloids, methylxanthines are well-known potentiators of contraction [4] and, more recently, have been shown to increase secretion in various glands, in particular insulin release [5-71. The effects of quinine and of its optical isomer quinidine on contraction are, in some respects, similar to those of methylxanthines, but are more complex, depending on the concentration used and on the type of muscle. As no attention has been paid on possible modifications of secretion by these latter alkaloids, we investigated the action of quinine on isolated islets of Langerhans. In this report, we show that the drug stimulates, potentiates or inhibits insulin release according to the experimental conditions and also alters glucose metabolism by islet cells.

Glucose Stimulates Ca2+ Influx and Insulin Secretion in 2-Week-old β-Cells Lacking ATP-sensitive K+ Channels

In adult β-cells glucose-induced insulin secretion involves two mechanisms (a) a KATP channel-dependent Ca2+ influx and rise of cytosolic [Ca2+]c and (b) a KATP channel-independent amplification of secretion without further increase of [Ca2+]c. Mice lacking the high affinity sulfonylurea receptor (Sur1KO), and thus KATP channels, have been developed as a model of congenital hyperinsulinism. Here, we compared [Ca2+]c and insulin secretion in overnight cultured islets from 2-week-old normal and Sur1KO mice. Control islets proved functionally mature: the magnitude and biphasic kinetics of [Ca2+]c and insulin secretion changes induced by glucose, and operation of the amplifying pathway, were similar to adult islets. Sur1KO islets perifused with 1 mm glucose showed elevation of both basal [Ca2+]c and insulin secretion. Stimulation with 15 mm glucose produced a transient drop of [Ca2+]c followed by an overshoot and a sustained elevation, accompanied by a monophasic, 6-fold increase in insulin secretion. Glucose also increased insulin secretion when [Ca2+]c was clamped by KCl. When Sur1KO islets were cultured in 5 instead of 10 mm glucose, [Ca2+]c and insulin secretion were unexpectedly low in 1 mm glucose and increased following a biphasic time course upon stimulation by 15 mm glucose. This KATP channel-independent first phase [Ca2+]c rise was attributed to a Na+-, Cl--, and Na+-pump-independent depolarization of β-cells, leading to Ca2+ influx through voltage-dependent calcium channels. Glucose indeed depolarized Sur1KO islets under these conditions. It is suggested that unidentified potassium channels are sensitive to glucose and subserve the acute and long-term metabolic control of [Ca2+]c in β-cells without functional KATP channels.

Glucose Controls Cytosolic Ca2+ and Insulin Secretion in Mouse Islets Lacking Adenosine Triphosphate-Sensitive K+ Channels Owing to a Knockout of the Pore-Forming Subunit Kir6.2

Glucose-induced insulin secretion is classically attributed to the cooperation of an ATP-sensitive potassium (K ATP) channel-dependent Ca2+ influx with a subsequent increase of the cytosolic free Ca2+ concentration ([Ca2+]c) (triggering pathway) and a K ATP channel-independent augmentation of secretion without further increase of [Ca2+]c (amplifying pathway). Here, we characterized the effects of glucose in beta-cells lacking K ATP channels because of a knockout (KO) of the pore-forming subunit Kir6.2. Islets from 1-yr and 2-wk-old Kir6.2KO mice were used freshly after isolation and after 18 h culture to measure glucose effects on [Ca2+]c and insulin secretion. Kir6.2KO islets were insensitive to diazoxide and tolbutamide. In fresh adult Kir6.2KO islets, basal [Ca2+]c and insulin secretion were marginally elevated, and high glucose increased [Ca2+]c only transiently, so that the secretory response was minimal (10% of controls) despite a functioning amplifying pathway (evidenced in 30 mm KCl). Culture in 10 mm glucose increased basal secretion and considerably improved glucose-induced insulin secretion (200% of controls), unexpectedly because of an increase in [Ca2+]c with modulation of [Ca2+]c oscillations. Similar results were obtained in 2-wk-old Kir6.2KO islets. Under selected conditions, high glucose evoked biphasic increases in [Ca2+]c and insulin secretion, by inducing K ATP channel-independent depolarization and Ca2+ influx via voltage-dependent Ca2+ channels. In conclusion, Kir6.2KO beta-cells down-regulate insulin secretion by maintaining low [Ca2+]c, but culture reveals a glucose-responsive phenotype mainly by increasing [Ca2+]c. The results support models implicating a K ATP channel-independent amplifying pathway in glucose-induced insulin secretion, and show that K ATP channels are not the only possible transducers of metabolic effects on the triggering Ca2+ signal.

Unbound rather than total concentration and saturation rather than unsaturation determine the potency of fatty acids on insulin secretion.

Isolated mouse islets were used to compare the effects of three saturated (myristate, palmitate and stearate) and three unsaturated (oleate, linoleate and linolenate) long-chain fatty acids on insulin secretion. By varying the concentrations of fatty acid (250-1250 micromol/l) and albumin simultaneously or independently, we also investigated whether the insulinotropic effect is determined by the unbound or total concentration of the fatty acids. Only palmitate and stearate slightly increased basal insulin secretion (3 mmol/l glucose). All tested fatty acids potentiated glucose-induced insulin secretion (10-15 mmol/l), and the following rank order of potency was obtained when they were compared at the same total concentrations: palmitate approximately = stearate > myristate > or = oleate > or = linoleate approximately = linolenate. The effect of a given fatty acid varied with the fatty acid to albumin molar ratio, in a way which indicated that the unbound fraction is the important one for the stimulation of beta cells. When the potentiation of insulin secretion was expressed as a function of the unbound concentrations, the following rank order emerged: palmitate > myristate > stearate approximately = oleate > linoleate approximately = linolenate. In conclusion, the acute and direct effects of long-chain fatty acids on insulin secretion are due to their unbound fraction. They are observed only at fatty acid/albumin ratios higher than those normally occurring in plasma. Saturated fatty acids are stronger insulin secretagogues than unsaturated fatty acids. Unbound palmitate is by far the most potent of the six common long-chain fatty acids.

Mechanism of the stimulation of insulin release in vitro by HB 699, a benzoic acid derivative similar to the non-sulphonylurea moiety of glibenclamide

SummaryHB 699 is a benzoic acid derivative similar to the non-sulphonylurea moiety of glibenclamide. The mechanisms whereby it affects B-cell function have been studied in vitro with mouse islets. In the presence of 3 mmol/l glucose, HB 699 decreased 86Rb+ efflux and accelerated 45Ca2+ efflux from islet cells, depolarized the B-cell membrane and induced an electrical activity similar to that triggered by stimulatory concentrations of glucose, and increased insulin release. The changes in 45Ca2+ efflux and insulin release, but not the inhibition of 86Rb+ efflux, were abolished in the absence of Ca2+. In the presence of 10 mmol/l glucose, HB 699 increased 86Rb+ and 45Ca2+ efflux from the islets, caused a persistent depolarization of the B-cell membrane with continuous electrical activity, and markedly potentiated insulin release. All these changes were suppressed by omission of extracellular Ca2+. In the presence of 15 mmol/l glucose, diazoxide increased 86Rb+ efflux, hyperpolarized the B-cell membrane, suppressed electrical activity and inhibited insulin release. HB 699 reversed these effects of diazoxide. It is suggested that HB 699 decreases K+ permeability of the B-cell membrane, thereby causing a depolarization which leads to activation of voltage-dependent Ca channnels and Ca2+ influx, and eventually increases insulin release. A sulphonylurea group is thus not a prerequisite to trigger the sequence of events that is also thought to underlie the releasing effects of tolbutamide and glibenclamide.

Metabolic amplifying pathway increases both phases of insulin secretion independently of β-cell actin microfilaments

Two pathways control glucose-induced insulin secretion (IS) by beta-cells. The triggering pathway involves ATP-sensitive potassium (K(ATP)) channel-dependent depolarization, Ca(2+) influx, and a rise in the cytosolic Ca(2+) concentration ([Ca(2+)](c)), which triggers exocytosis of insulin granules. The metabolic amplifying pathway augments IS without further increasing [Ca(2+)](c). The underlying mechanisms are unknown. Here, we tested the hypothesis that amplification implicates actin microfilaments. Mouse islets were treated with latrunculin B and cytochalasin B to depolymerize actin or jasplakinolide to polymerize actin. They were then perifused to measure [Ca(2+)](c) and IS. Metabolic amplification was studied during imposed steady elevation of [Ca(2+)](c) by tolbutamide or KCl or by comparing the magnitude of [Ca(2+)](c) and IS changes produced by glucose and tolbutamide. Both actin polymerization and depolymerization augmented IS triggered by all stimuli without increasing (sometimes decreasing) [Ca(2+)](c), which indicates a predominantly inhibitory function of microfilaments in exocytosis at a step distal to [Ca(2+)](c) increase. When [Ca(2+)](c) was elevated and controlled by KCl or tolbutamide, the amplifying action of glucose was facilitated by actin depolymerization and unaffected by polymerization. Both phases of IS were larger in response to high-glucose than to tolbutamide in low-glucose, although triggering [Ca(2+)](c) was lower. This difference in IS, due to amplification, persisted when the IS rate was doubled by actin depolymerization or polymerization. In conclusion, metabolic amplification is rapid and influences the first as well as the second phase of IS. It is a late step of stimulus-secretion coupling, which does not require functional actin microfilaments and could correspond to acceleration of the priming process conferring release competence to insulin granules.

In vitro insulin secretion by pancreatic tissue from infants with diazoxide-resistant congenital hyperinsulinism deviates from model predictions.

Congenital hyperinsulinism (CHI) is the major cause of persistent neonatal hypoglycemia. CHI most often occurs due to mutations in the ABCC8 (which encodes sulfonylurea receptor 1) or KCNJ11 (which encodes the potassium channel Kir6.2) gene, which result in a lack of functional KATP channels in pancreatic β cells. Diffuse forms of CHI (DiCHI), in which all β cells are abnormal, often require subtotal pancreatectomy, whereas focal forms (FoCHI), which are characterized by localized hyperplasia of abnormal β cells, can be cured by resection of the lesion. Here, we characterized the in vitro kinetics of insulin secretion by pancreatic fragments from 6 DiCHI patients and by focal lesion and normal adjacent pancreas from 18 FoCHI patients. Responses of normal pancreas were similar to those reported for islets from adult organ donors. Compared with normal pancreas, basal insulin secretion was elevated in both FoCHI and DiCHI tissue. Affected tissues were heterogeneous in their secretory responses, with increased glucose levels often producing a rapid increase in insulin secretion that could be followed by a paradoxical decrease below prestimulatory levels. The KATP channel blocker tolbutamide was consistently ineffective in stimulating insulin secretion; conversely, the KATP channel activator diazoxide often caused an unanticipated increase in insulin secretion. These observed alterations in secretory behavior were similar in focal lesion and DiCHI tissue, and independent of the specific mutation in ABCC8 or KCNJ11. They cannot be explained by classic models of β cell function. Our results provide insight into the excessive and sometimes paradoxical changes in insulin secretion observed in CHI patients with inactivating mutations of KATP channels.

The Oscillatory Behavior of Pancreatic Islets from Mice with Mitochondrial Glycerol-3-phosphate Dehydrogenase Knockout

Glucose stimulation of pancreatic β cells induces oscillations of the membrane potential, cytosolic Ca2+ ([Ca2+] i ), and insulin secretion. Each of these events depends on glucose metabolism. Both intrinsic oscillations of metabolism and repetitive activation of mitochondrial dehydrogenases by Ca2+ have been suggested to be decisive for this oscillatory behavior. Among these dehydrogenases, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate NADH shuttle, is activated by cytosolic [Ca2+] i . In the present study, we compared different types of oscillations in β cells from wild-type and mGPDH−/− mice. In clusters of 5–30 islet cells and in intact islets, 15 mm glucose induced an initial drop of [Ca2+] i , followed by an increase in three phases: a marked initial rise, a partial decrease with rapid oscillations and eventually large and slow oscillations. These changes, in particular the frequency of the oscillations and the magnitude of the [Ca2+] rise, were similar in wild-type and mGPDH−/− mice. Glucose-induced electrical activity (oscillations of the membrane potential with bursts of action potentials) was not altered in mGPDH−/− β cells. In single islets from either type of mouse, insulin secretion strictly followed the changes in [Ca2+] i during imposed oscillations induced by pulses of high K+ or glucose and during the biphasic elevation induced by sustained stimulation with glucose. An imposed and controlled rise of [Ca2+] i in β cells similarly increased NAD(P)H fluorescence in control and mGDPH−/− islets. Inhibition of the malate-aspartate NADH shuttle with aminooxyacetate only had minor effects in control islets but abolished the electrical, [Ca2+] i and secretory responses in mGPDH−/− islets. The results show that the two distinct NADH shuttles play an important but at least partially redundant role in glucose-induced insulin secretion. The oscillatory behavior of β cells does not depend on the functioning of mGPDH and on metabolic oscillations that would be generated by cyclic activation of this enzyme by Ca2+.