Activation of the AMP-activated protein kinase enhances glucose-stimulated insulin secretion in mouse β-cells

The AMP-activated protein kinase (AMPK) is one of the key players in cellular energy regulation adapting cellular demands to nutritional and metabolic variations. Oral antidiabetic drugs like metformin and glitazones (thiazolidinediones) are known to stimulate this enzyme. Besides their established action on peripheral organs including liver and muscles, it has been claimed that these drugs may affect β-cell function. However, it is still a matter of debate whether pharmacological AMPK stimulation increases or decreases insulin secretion. To study this point and to reveal mechanisms underlying changes in insulin secretion we used the specific AMPK activator AICAR and investigated its effects on stimulus-secretion coupling. Membrane potential and currents were measured by the patch-clamp technique, [Ca²⁺]_c, mitochondrial membrane potential, and NAD(P)H by fluorescence techniques and insulin secretion by a radioimmunoassay. AICAR enhanced glucose-stimulated insulin release, an effect that can be attributed to the augmentation of electrical activity and [Ca²⁺]_c resulting from an AICAR-evoked inhibition of the KATP current. This latter effect was not due to a direct interaction of AICAR with the KATP channels but was dependent on cell metabolism. AICAR did not affect mitochondrial membrane potential or NAD(P)H autofluorescence. Metformin mimicked the action of AICAR on electrical activity, [Ca²⁺]_c, and KATP current. However, compared to AICAR the effects were less pronounced and not sufficient to stimulate insulin secretion. In conclusion, activation of AMPK augments nutrient-induced insulin secretion. Thus, targeting AMPK of β-cells may     

胰岛素和β肾上腺素受体激动剂对大鼠脂联素分泌和表达的影响

目的 在体探讨大鼠脂联素表达和分泌的调节机制.方法 大鼠在空腹后重新进食、高胰岛素钳夹试验、应用β肾上腺素受体激动剂情况下,放射免疫法测定血浆中脂联素水平,实时定量PCR方法检测脂肪组织脂联素基因表达的变化.结果 (1)大鼠空腹18 h后,进食可显著增加血浆脂联素水平(约2倍)和脂肪组织脂联素的表达(约3倍),且此作用可被β肾上腺素受体激动剂所拮抗.(2)大鼠空腹18 h后,高胰岛素钳夹可显著增加血浆脂联素水平和脂肪组织脂联素的表达.结论 在体内,脂联素的合成和分泌可迅速被营养状态所调节.胰岛素和β肾上腺素受体激动剂可调节脂肪组织脂联素的分泌和表达.

Abstract：

Objective To explore the potential mechanisms of regulating adiponectin secretion and expression in vivo in rats.Methods To observe the regulation of adiponectin by fasing-refeeding and β-adrenergic agonists, male Wistar rats were fasted for 18 h and allowed to refeed or a β3-adrenergic receptor agonist was infused into refeeding rats.The effects of insulin clamp on adiponectin secretion and expression, including euglycemichyperinsulinemic clamp and hyperglycemic-hyperinsulinemic clamp, were also investigated.Plasma adiponectin level was determined by radioimmunoassay.Adiponectin mRNA expression in adipose tissue of rats was detected by realtime PCR.Results (1) Refeeding 18 h fasted rats increased plasma adiponectin concentration (about 2-fold) and adipose tissue adiponectin expression (about 3-fold), which were completely blocked by administration of β-adrenergic agonist.(2) Hyperinsulinemic clamp increased plasma adiponectin concentration and adiponectin gene expression in adipose tissue.Conclusions Adiponectin secretion and expression are acutely regulated in vivo by nutritional status.Insulin and β-adrenergic agonists regulate adiponectin secretion and expression in adipose tissue.     

Serine-385 phosphorylation of inwardly rectifying K+ channel subunit (Kir6.2) by AMP-dependent protein kinase plays a key role in rosiglitazone-induced closure of the KATP channel and insulin secretion in rats

Aims/hypothesis  Rosiglitazone, an insulin sensitiser, not only improves insulin sensitivity but also enhances insulin secretory capacity by ameliorating gluco- and lipotoxicity in beta cells. Rosiglitazone can stimulate insulin secretion at basal and high glucose levels via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. We hypothesised that regulation of phosphorylation of the ATP-sensitive potassium (K_ATP) channel might serve as a key step in the regulation of insulin secretion. Methods  Insulin secretory responses were studied in an isolated pancreas perfusion system, cultured rat islets and MIN6 and RINm5F beta cells. Signal transduction pathways downstream of PI3K were explored to link rosiglitazone to K_ATP channel conductance with patch clamp techniques and insulin secretion measured by ELISA. Results  Rosiglitazone stimulated AMP-activated protein kinase (AMPK) activity and induced inhibition of the K_ATP channel conductance in islet beta cells; both effects were blocked by the PI3K inhibitor LY294002. Following stimulation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a pharmacological activator, both AICAR-stimulated insulin secretion and inhibition of K_ATP channel conductance were unaffected by LY294002, indicating that AMPK activation occurs at a site downstream of PI3K activity. The serine residue at amino acid position 385 of Kir6.2 was found to be the substrate phosphorylation site of AMPK when activated by rosiglitazone or AICAR. Conclusions/interpretation  Our data indicate that PI3K-dependent activation of AMPK is required for rosiglitazone-stimulated insulin secretion in pancreatic beta cells. Phosphorylation of the Ser³⁸⁵ residue of the Kir6.2 subunit of the K_ATP channel by AMPK may play a role in insulin secretion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. T.-J. Chang and W.-P. Chen contributed equally to this study.     

Ablation of AMP-activated protein kinase α1 and α2 from mouse pancreatic beta cells and RIP2.Cre neurons suppresses insulin release in vivo

Aims/hypothesis

AMP-activated protein kinase (AMPK) is an evolutionarily conserved enzyme and a target of glucose-lowering agents, including metformin. However, the precise role or roles of the enzyme in controlling insulin secretion remain uncertain.

Methods

The catalytic α1 and α2 subunits of AMPK were ablated selectively in mouse pancreatic beta cells and hypothalamic neurons by breeding Ampkα1 [also known as Prkaa1]-knockout mice, bearing floxed Ampkα2 [also known as Prkaa2] alleles (Ampkα1 ^?/? ,α2 ^fl/fl ,), with mice expressing Cre recombinase under the rat insulin promoter (RIP2). RIP2 was used to express constitutively activated AMPK selectively in beta cells in transgenic mice. Food intake, body weight and urinary catecholamines were measured using metabolic cages. Glucose and insulin tolerance were determined after intraperitoneal injection. Beta cell mass and morphology were analysed by optical projection tomography and confocal immunofluorescence microscopy, respectively. Granule docking, insulin secretion, membrane potential and intracellular free Ca²⁺ were measured with standard techniques.

Results

Trigenic Ampkα1 ^?/? ,α2 ^fl/fl expressing Cre recombinase and lacking both AMPKα subunits in the beta cell, displayed normal body weight and increased insulin sensitivity, but were profoundly insulin-deficient. Secreted catecholamine levels were unchanged. Total beta cell mass was unaltered, while mean islet and beta cell volume were reduced. AMPK-deficient beta cells displayed normal glucose-induced changes in membrane potential and intracellular free Ca²⁺, while granule docking and insulin secretion were enhanced. Conversely, βAMPK transgenic mice were glucose-intolerant and displayed defective insulin secretion.

Conclusions/interpretation

Inhibition of AMPK activity within the beta cell is necessary, but not sufficient for stimulation of insulin secretion by glucose to occur. AMPK activation in extrapancreatic RIP2.Cre-expressing cells might also influence insulin secretion in vivo.     

Fuel-induced amplification of insulin secretion in mouse pancreatic islets exposed to a high sulfonylurea concentration: role of the NADPH/NADP+ ratio

Aims/hypothesis The aim of this study was to examine whether the cytosolic NADPH/NADP⁺ ratio of beta cells serves as an amplifying signal in fuel-induced insulin secretion and whether such a function is mediated by cytosolic α-ketoglutarate. Methods Pancreatic islets and islet cells were isolated from albino mice by collagenase digestion. Insulin secretion of incubated or perifused islets was measured by ELISA. The NADPH and NADP⁺ content of incubated islets was determined by enzymatic cycling. The cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in islets was measured by microfluorimetry and the activity of ATP-sensitive K⁺ channels in islet cells by patch-clamping. Results Both 30 mmol/l glucose and 10 mmol/l α-ketoisocaproate stimulated insulin secretion and elevated the NADPH/NADP⁺ ratio of islets preincubated in the absence of fuel. The increase in the NADPH/NADP⁺ ratio was abolished in the presence of 2.7 μmol/l glipizide (closing all ATP-sensitive K⁺ channels). However, α-ketoisocaproate, but not glucose, still stimulated insulin secretion. That glipizide did not inhibit α-ketoisocaproate-induced insulin secretion was not the result of elevated [Ca²⁺]_c, as glucose caused a more marked [Ca²⁺]_c increase. Insulin release triggered by glipizide alone was moderately amplified by dimethyl α-ketoglutarate (which is cleaved to produce cytosolic α-ketoglutarate), but there was no indication of a signal function of cytosolic α-ketoglutarate. Conclusions/interpretation The results strongly suggest that the NADPH/NADP⁺ ratio in the beta cell cytosol does not serve as an amplifying signal in fuel-induced insulin release. The study supports the view that amplification results from the intramitochondrial production of citrate by citrate synthase and from the associated export of citrate into the cytosol.     

Kisspeptin stimulation of insulin secretion: mechanisms of action in mouse islets and rats

Aims/hypothesis  Kisspeptin is a novel peptide identified as an endogenous ligand of the G-protein-coupled receptor 54 (GPR-54), which plays a crucial role in puberty and reproductive function. High levels of GPR-54 and kisspeptin have been reported in the pancreas and we have previously shown that kisspeptin potentiates glucose-induced insulin release from isolated islets, although the mechanisms underlying this effect were unclear. Methods  Insulin secretion from isolated mouse islets was measured to characterise the effects of kisspeptin. The effects of kisspeptin on both p42/44 mitogen-activated protein kinase (MAPK) phosphorylation and intracellular Ca²⁺([Ca²⁺]_i) in mouse islets were also investigated. Furthermore, kisspeptin was administered to rats in vivo and effects on plasma insulin levels measured. Results  In the current study, kisspeptin induced a concentration-dependent potentiation of glucose-induced (20 mmol/l) insulin secretion from mouse islets, with maximal effects at 1 μmol/l, but had no effect on insulin secretion at a substimulatory concentration of glucose (2 mmol/l). Activation of GPR-54 by kisspeptin also caused reversible increases in [Ca²⁺]_i in Fura-2 loaded dispersed islet cells. The kisspeptin-induced potentiation of glucose-induced insulin secretion was completely abolished by inhibitors of phospholipase C and p42/44 MAPK, but not by inhibitors of protein kinase C or p38 MAPK. Intravenous administration of kisspeptin into conscious, unrestrained rats caused an increase in circulating insulin levels, whilst central administration of kisspeptin had no effect, indicating a peripheral site of action. Conclusions/interpretation  These observations suggest that neither typical protein kinase C isoforms nor p38 MAPK are involved in the potentiation of glucose-induced insulin release by kisspeptin, but intracellular signalling pathways involving phospholipase C, p42/44 MAPK and increased [Ca²⁺]_i are required for the stimulatory effects on insulin secretion. The observation that kisspeptin is also capable of stimulating insulin release in vivo supports the conclusion that kisspeptin is a regulator of beta cell function.     

Somatostatin release, electrical activity, membrane currents and exocytosis in human pancreatic delta cells

Aims/hypothesis  The aim of this study was to characterise electrical activity, ion channels, exocytosis and somatostatin release in human delta cells/pancreatic islets. Methods  Glucose-stimulated somatostatin release was measured from intact human islets. Membrane potential, currents and changes in membrane capacitance (reflecting exocytosis) were recorded from individual human delta cells identified by immunocytochemistry. Results  Somatostatin secretion from human islets was stimulated by glucose and tolbutamide and inhibited by diazoxide. Human delta cells generated bursting or sporadic electrical activity, which was enhanced by tolbutamide but unaffected by glucose. Delta cells contained a tolbutamide-insensitive, Ba²⁺-sensitive inwardly rectifying K⁺ current and two types of voltage-gated K⁺ currents, sensitive to tetraethylammonium/stromatoxin (delayed rectifying, Kv2.1/2.2) and 4-aminopyridine (A current). Voltage-gated tetrodotoxin (TTX)-sensitive Na⁺ currents contributed to the action potential upstroke but TTX had no effect on somatostatin release. Delta cells are equipped with Ca²⁺ channels blocked by isradipine (L), ω-agatoxin (P/Q) and NNC 55-0396 (T). Blockade of any of these channels interferes with delta cell electrical activity and abolishes glucose-stimulated somatostatin release. Capacitance measurements revealed a slow component of depolarisation-evoked exocytosis sensitive to ω-agatoxin. Conclusions/interpretation  Action potential firing in delta cells is modulated by ATP-sensitive K⁺-channel activity. The membrane potential is stabilised by Ba²⁺-sensitive inwardly rectifying K⁺ channels. Voltage-gated L- and T-type Ca²⁺ channels are required for electrical activity, whereas Na⁺ currents and P/Q-type Ca²⁺ channels contribute to (but are not necessary for) the upstroke of the action potential. Action potential repolarisation is mediated by A-type and Kv2.1/2.2 K⁺ channels. Exocytosis is tightly linked to Ca²⁺-influx via P/Q-type Ca²⁺ channels. Glucose stimulation of somatostatin secretion involves both K_ATP channel-dependent and -independent processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. M. Braun and R. Ramracheya contributed equally to this study     

The atypical antipsychotic clozapine impairs insulin secretion by inhibiting glucose metabolism and distal steps in rat pancreatic islets

Aims/hypothesis Diabetogenic effects of some atypical antipsychotic drugs have been reported, although the mechanisms are not fully understood. We investigated the long-term effects of culturing isolated rat pancreatic islets with atypical antipsychotic clozapine.Methods Glucose- and non-glucose-stimulated insulin secretion, glucose metabolism and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were measured in islets cultured with or without clozapine.Results Although acute incubation or 3-day culture with clozapine did not affect glucose-stimulated insulin secretion, clozapine suppressed glucose-stimulated insulin secretion by 53.2% at 1.0 μmol/l (therapeutic concentration) after 7 days of culture. Islet glucose oxidation and [Ca²⁺]_i elevation by high glucose were not affected after 3 days of culture, but clozapine significantly inhibited islet glucose oxidation, ATP production, and [Ca²⁺]_i elevation by high glucose after 7 days of culture. Moreover, 7 days of culture with clozapine inhibited insulin secretion stimulated by: (1) membrane depolarisation induced by high K⁺; (2) protein kinase C activation; and (3) mastoparan at 16.7 mmol/l glucose under stringent Ca²⁺-free conditions. Elevation of [Ca²⁺]_i by high K⁺-induced membrane depolarisation was similar in control and clozapine-treated islets. Clozapine, a muscarinic blocker, acutely inhibited carbachol-induced insulin secretion, as did atropine, whereas after 7 days of culture atropine did not have the inhibitory effect shown by clozapine after 7 days. The impairment of glucose-stimulated insulin secretion recovered 3 days after the removal of clozapine treatment.Conclusions/interpretation The present study demonstrated that the atypical antipsychotic drug clozapine directly impaired insulin secretion via multiple sites including glucose metabolism and the distal step in insulin exocytosis in a long-term culture condition. These mechanisms may be involved in the form of diabetes mellitus associated with atypical antipsychotic drugs.     

A beta cell-specific knockout of hormone-sensitive lipase in mice results in hyperglycaemia and disruption of exocytosis

Aims/hypothesis  The enzyme hormone-sensitive lipase (HSL) is produced and is active in pancreatic beta cells. Because lipids are known to play a crucial role in normal control of insulin release and in the deterioration of beta cell function, as observed in type 2 diabetes, actions of HSL in beta cells may be critical. This notion has been addressed in different lines of HSL knockout mice with contradictory results. Methods  To resolve this, we created a transgenic mouse lacking HSL specifically in beta cells, and characterised this model with regard to glucose metabolism and insulin secretion, using both in vivo and in vitro methods. Results  We found that fasting basal plasma glucose levels were significantly elevated in mice lacking HSL in beta cells. An IVGTT at 12 weeks revealed a blunting of the initial insulin response to glucose with delayed elimination of the sugar. Additionally, arginine-stimulated insulin secretion was markedly diminished in vivo. Investigation of the exocytotic response in single HSL-deficient beta cells showed an impaired response to depolarisation of the plasma membrane. Beta cell mass and islet insulin content were increased, suggesting a compensatory mechanism, by which beta cells lacking HSL strive to maintain normoglycaemia. Conclusions/interpretation  Based on these results, we suggest that HSL, which is located in close proximity of the secretory granules, may serve as provider of a lipid-derived signal essential for normal insulin secretion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Crosstalk between membrane potential and cytosolic Ca2+ concentration in beta cells from Sur1 −/− mice

Aims/hypothesis Islets or beta cells from Sur1^–/– mice were used to determine whether changes in plasma membrane potential (V_m) remain coupled to changes in cytosolic Ca²⁺ ([Ca²⁺]_i) in the absence of K_ATP channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca²⁺]_i influences oscillations in V_m in sur1^–/– beta cells.Methods Plasma membrane potential and ion currents were measured with microelectrodes and the patch–clamp technique. [Ca²⁺]_i was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations.Results Membrane depolarisation of Sur1^–/– islets by arginine or increased extracellular K⁺, elevated [Ca²⁺]_i and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1^–/– islets. Oscillations in V_m were influenced by [Ca²⁺]_i as follows: (1) elevation of extracellular Ca²⁺ lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca²⁺-dependent outward current that was augmented by increased Ca²⁺ influx through L-type Ca²⁺ channels; (3) Ca²⁺ depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1^–/– islets, elevating [Ca²⁺]_i and insulin secretion; (4) store depletion activated a Ca²⁺ influx that was not inhibitable by the L-type Ca²⁺ channel blocker D600.Conclusions/interpretation Although V_m is largely uncoupled from glucose metabolism in the absence of K_ATP channels, increased electrical activity leads to elevations of [Ca²⁺]_i that are sufficient to stimulate insulin secretion. In Sur1^–/– beta cells, [Ca²⁺]_i exerts feedback mechanisms on V_m by activating a hyperpolarising outward current and by depolarising V_m via store-operated ion channels.     

Glutamate is an essential mediator in glutamine-amplified insulin secretion

Aims/IntroductionGlutamine is the most abundant amino acid in the circulation. In this study, we investigated cell signaling in the amplification of insulin secretion by glutamine.Materials and MethodsClonal pancreatic β‐cells MIN6‐K8, wild‐type B6 mouse islets, glutamate dehydrogenase (GDH) knockout clonal β‐cells (Glud1KOβCL), and glutamate‐oxaloacetate transaminase 1 (GOT1) knockout clonal β‐cells (Got1KOβCL) were studied. Insulin secretion from these cells and islets was examined under various conditions, and intracellular glutamine metabolism was assessed by metabolic flux analysis. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also measured.ResultsGlutamine dose‐dependently amplified insulin secretion in the presence of high glucose in both MIN6‐K8 cells and Glud1KOβCL. Inhibition of glutaminases, the enzymes that convert glutamine to glutamate, dramatically reduced the glutamine‐amplifying effect on insulin secretion. A substantial amount of glutamate was produced from glutamine through direct conversion by glutaminases. Glutamine also increased [Ca²⁺]_i at high glucose, which was abolished by inhibition of glutaminases. Glutamic acid dimethylester (dm‐Glu), a membrane permeable glutamate precursor that is converted to glutamate in cells, increased [Ca²⁺]_i as well as induced insulin secretion at high glucose. These effects of glutamine and dm‐Glu were dependent on calcium influx. Glutamine also induced insulin secretion in clonal β‐cells MIN6‐m14, which otherwise exhibit no insulin secretory response to glucose.ConclusionsGlutamate converted from glutamine is an essential mediator that enhances calcium signaling in the glutamine‐amplifying effect on insulin secretion. Our data also suggest that glutamine exerts a permissive effect on glucose‐induced insulin secretion.     

Globular adiponectin augments insulin secretion from pancreatic islet beta cells at high glucose concentrations

Adiponectin plays an important role in improving insulin resistance and preventing atherosclerosis. However it has been rarely reported that adiponectin influences insulin secretion because its receptor was identified in human islet β cells. In order to investigate the direct effect of adiponectin on pancreatic islet β cells, we performed an insulin secretion test in purified rat islets, which were incubated with adiponectin (100 ng/mL) at low (3.3 mM) and high (16.7 mM) glucose concentrations. Furthermore, cell lysates were extracted from the adiponectin-treated islets for p-AMPKα assay. RTPCR and immunohistochemical examination showed both adiponectin receptor 1 (AdipoR1) and receptor 2 (A dipoR2) were expressed in islet cells and AdipoR1 was predominantly expressed. Insulin secretion was significantly increased in the presence of adiponectin for 6 h at high glucose concentration. Meanwhile, the levels of phosphorylated AMPK increased with adiponectin treatment at high glucose concentrations. It is concluded that adiponectin augments insulin secretion from pancreatic islet β cells at high glucose concentration through AMPK activation.     

Insulin degradation in human erythrocyte: Effects of cations

Summary Insulin degradation by human erythrocyte fractions was studied using the TCA-precipitation method. Hemolysate exhibited an insulin degrading activity higher than membranes. Triton X-100 treatment of membranes led to the appraisal of Triton-soluble degrading activity and of a more efficient Triton-not-soluble degrading activity. Monovalent cations (Na⁺, K⁺, Li⁺) did not modify the insulin degradation by any of the erythrocyte fractions. Divalent cations, Ca⁺⁺ and Zn⁺⁺ selectively enhanced insulin degradation by the membranous fractions, and Cu⁺⁺ and Zn⁺⁺ strongly inhibited insulin degradation by all the erythrocyte fractions. The results supported the hypothesis of the existence of at least two different degrading systems in human erythrocytes: soluble (cytosolic) Ca⁺⁺ and Mg⁺⁺ insensitive system(s) and membrane associated Ca⁺⁺ and Mg⁺⁺ sensitive system(s).     

Insulin signalling downstream of protein kinase B is potentiated by 5′AMP-activated protein kinase in rat hearts in vivo

Aims/hypothesis 5′AMP-activated protein kinase (AMPK) and insulin stimulate glucose transport in heart and muscle. AMPK acts in an additive manner with insulin to increase glucose uptake, thereby suggesting that AMPK activation may be a useful strategy for ameliorating glucose uptake, especially in cases of insulin resistance. In order to characterise interactions between the insulin- and AMPK-signalling pathways, we investigated the effects of AMPK activation on insulin signalling in the rat heart in vivo. Methods Male rats (350–400 g) were injected with 1 g/kg 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or 250 mg/kg metformin in order to activate AMPK. Rats were administered insulin 30 min later and after another 30 min their hearts were removed. The activities and phosphorylation levels of components of the insulin-signalling pathway were subsequently analysed in individual rat hearts. Results AICAR and metformin administration activated AMPK and enhanced insulin signalling downstream of protein kinase B in rat hearts in vivo. Insulin-induced phosphorylation of glycogen synthase kinase 3 (GSK3) β, p70 S6 kinase (p70^S6K)(Thr389) and IRS1(Ser636/639) were significantly increased following AMPK activation. To the best of our knowledge, this is the first report of heightened insulin responses of GSK3β and p70^S6K following AMPK activation. In addition, we found that AMPK inhibits insulin stimulation of IRS1-associated phosphatidylinositol 3-kinase activity, and that AMPK activates atypical protein kinase C and extracellular signal-regulated kinase in the heart. Conclusions/interpretations Our data are indicative of differential effects of AMPK on the activation of components in the cardiac insulin-signalling pathway. These intriguing observations are critical for characterisation of the crosstalk between AMPK and insulin signalling.     

Pioglitazone stimulates AMP-activated protein kinase signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation in human skeletal muscle in vivo: a randomised trial

Aims/hypothesis  The molecular mechanisms by which thiazolidinediones improve insulin sensitivity in type 2 diabetes are not fully understood. We hypothesised that pioglitazone would activate the adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway and increase the expression of genes involved in adiponectin signalling, NEFA oxidation and mitochondrial function in human skeletal muscle. Methods  A randomised, double-blind, parallel study was performed in 26 drug-naive type 2 diabetes patients treated with: (1) pioglitazone (n = 14) or (2) aggressive nutritional therapy (n = 12) to reduce HbA_1c to levels observed in the pioglitazone-treated group. Participants were assigned randomly to treatment using a table of random numbers. Before and after 6 months, patients reported to the Clinical Research Center of the Texas Diabetes Institute for a vastus lateralis muscle biopsy followed by a 180 min euglycaemic–hyperinsulinaemic (80 mU m⁻² min⁻¹) clamp. Results  All patients in the pioglitazone (n = 14) or nutritional therapy (n = 12) group were included in the analysis. Pioglitazone significantly increased plasma adiponectin concentration by 79% and reduced fasting plasma NEFA by 35% (both p < 0.01). Following pioglitazone, insulin-stimulated glucose disposal increased by 30% (p < 0.01), and muscle AMPK and acetyl-CoA carboxylase (ACC) phosphorylation increased by 38% and 53%, respectively (p < 0.05). Pioglitazone increased mRNA levels for adiponectin receptor 1 and 2 genes (ADIPOR1, ADIPOR2), peroxisome proliferator-activated receptor gamma, coactivator 1 gene (PPARGC1) and multiple genes involved in mitochondrial function and fat oxidation. Despite a similar reduction in HbA_1c and similar improvement in insulin sensitivity with nutritional therapy, there were no significant changes in muscle AMPK and ACC phosphorylation, or the expression of ADIPOR1, ADIPOR2, PPARGC1 and genes involved in mitochondrial function and fat oxidation. No adverse (or unexpected) effects or side effects were reported from the study. Conclusions/interpretations  Pioglitazone increases plasma adiponectin levels, stimulates muscle AMPK signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation. These changes may represent an important cellular mechanism by which thiazolidinediones improve skeletal muscle insulin sensitivity. Trial registration: NCT 00816218 Funding: This trial was funded by National Institutes of Health Grant DK24092, VA Merit Award, GCRC Grant RR01346, Executive Research Committee Research Award from the University of Texas Health Science Center at San Antonio, American Diabetes Association Junior Faculty Award, American Heart Association National Scientist Development Grant, Takeda Pharmaceuticals North America Grant and Canadian Institute of Health Research Grant. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Subplasmalemmal Ca2+ measurements in mouse pancreatic beta cells support the existence of an amplifying effect of glucose on insulin secretion

Aims/hypothesis  

Glucose-induced insulin secretion is attributed to a rise of beta cell cytosolic free [Ca²⁺] ([Ca²⁺]_c) (triggering pathway) and amplification of the action of Ca²⁺. This concept of amplification rests on observations that glucose can increase Ca²⁺-induced insulin secretion without further elevating an imposed already high [Ca²⁺]_c. However, it remains possible that this amplification results from an increase in [Ca²⁺] just under the plasma membrane ([Ca²⁺]_SM), which escaped detection by previous measurements of global [Ca²⁺]_c. This was the hypothesis that we tested here by measuring [Ca²⁺]_SM.     

Anaplerosis via pyruvate carboxylase is required for the fuel-induced rise in the ATP:ADP ratio in rat pancreatic islets

Aims/hypothesis The molecular mechanisms of insulin release are only partially known. Among putative factors for coupling glucose metabolism to insulin secretion, anaplerosis has lately received strong support. The anaplerotic enzyme pyruvate carboxylase is highly expressed in beta cells, and anaplerosis influences insulin secretion in beta cells. By inhibiting pyruvate carboxylase in rat islets, we aimed to clarify the hitherto unknown metabolic events underlying anaplerotic regulation of insulin secretion.Methods Phenylacetic acid (5 mmol/l) was used to inhibit pyruvate carboxylase in isolated rat islets, which were then assessed for insulin secretion, fuel oxidation, ATP:ADP ratio, respiration, mitochondrial membrane potential, exocytosis and ATP-sensitive K⁺ channel (K_ATP-channel) conductance.Results We found that the glucose-provoked rise in ATP:ADP ratio was suppressed by inhibition of pyruvate carboxylase. In contrast, fuel oxidation, respiration and mitochondrial membrane potential, as well as Ca²⁺-induced exocytosis and K_ATP-channel conductance in single cells, were unaffected. Insulin secretion induced by α-ketoisocaproic acid was suppressed, whereas methyl-succinate-stimulated secretion remained unchanged. Perifusion of rat islets revealed that inhibition of anaplerosis decreased both the second phase of insulin secretion, during which K_ATP-independent actions of fuel secretagogues are operational, as well as the first and K_ATP-dependent phase.Conclusions/interpretation Our results are consistent with the concept that anaplerosis via pyruvate carboxylase determines pyruvate cycling, which has previously been shown to correlate with glucose responsiveness in clonal beta cells. These processes, controlled by pyruvate carboxylase, seem crucial for generation of an appropriate ATP:ADP ratio, which may regulate both phases of fuel-induced insulin secretion.     

Effect of age onAgaricus bisporus PHA-B stimulated insulin release and45Ca2+ uptakein vitro by islets of langerhans

Summary Thein vitro basal insulin release and⁴⁵Ca²⁺ uptake by rat islets of Langerhans remain unaffected by the animal’s age. However, whenAgaricus bisporus lectin PHA-B is added to the medium, there is a significant stimulation of basal insulin secretion and⁴⁵Ca²⁺ uptake. The islets isolated from younger rats are more sensitive toA. bisporus PHA-B stimulation and register 1.5-fold and 3-fold more increases of insulin release and⁴⁵Ca²⁺ uptake respectively as compared to islets isolated from 12-month-old rats. Age-related conformational changes in the B-cell membrane are suggested as a possible explanation for the above observation. CDRI Communication No. 3428     

Adiponectin in relation to insulin sensitivity and insulin secretion in the development of type 2 diabetes: a prospective study in 64-year-old women

Abstract. Fagerberg B, Kellis D, Bergström G, Behre CJ (Sahlgrenska Academy at Gothenburg University, Gothenburg, Sweden). Adiponectin in relation to insulin sensitivity and insulin secretion in the development of type 2 diabetes: a prospective study in 64‐year‐old women. J Intern Med 2011; 269 : 636–643. Objectives. To examine how serum adiponectin levels predict the incidence of type 2 diabetes, from different prediabetic states, in relation to insulin sensitivity and β‐cell function during 5.5 years of follow‐up. Methods. In a population‐based cohort of 64‐year‐old Caucasian women, we assessed glucose tolerance, insulin sensitivity as homeostasis model assessment, insulin secretion as acute insulin response, lifestyle factors and serum concentrations of adiponectin and high‐sensitivity C‐reactive protein. After 5.5 years of follow‐up, 167 women with normal glucose tolerance (NGT) and 174 with impaired glucose tolerance (IGT) at baseline were re‐examined and incidence of diabetes was assessed. Results. A total of 69 new cases of diabetes were detected during follow‐up. Diabetes incidence was independently predicted by low levels of serum adiponectin, insulin resistance and insulin secretion, cigarette smoking, impaired fasting glucose (IFG) and IGT at baseline. Serum adiponectin below 11.54 g L^?1 was associated with an odds ratio of 3.6 (95% confidence interval 1.4–8.6) for future type 2 diabetes. At baseline, a high serum adiponectin concentration correlated positively with high levels of insulin sensitivity and insulin secretion. Women with incident diabetes had lower serum adiponectin levels in the NGT, IFG and IGT groups at baseline compared to those who did not develop diabetes during follow‐up. Conclusions. Low adiponectin concentrations were associated with future diabetes independently of insulin secretion and sensitivity, as well as IGT, IFG, smoking and abdominal obesity.     

Essential role of the imidazoline moiety in the insulinotropic effect but not the KATP channel-blocking effect of imidazolines; a comparison of the effects of efaroxan and its imidazole analogue, KU14R

Aims/hypothesis Imidazolines are a class of investigational antidiabetic drugs. It is still unclear whether the imidazoline ring is decisive for insulinotropic characteristics. Materials and methods We studied the imidazoline efaroxan and its imidazole analogue, KU14R, which is currently classified as an imidazoline antagonist. The effects of both on stimulus secretion-coupling in normal mouse islets and beta cells were compared by measuring K_ATP channel activity, plasma membrane potential, cytosolic calcium concentration ([Ca²⁺]_c) and dynamic insulin secretion. Results In the presence of 10 mmol/l but not of 5 mmol/l glucose, efaroxan (100 μmol/l) strongly enhanced insulin secretion by freshly isolated perifused islets, whereas KU14R (30, 100 or 300 μmol/l) was ineffective at both glucose concentrations. Surprisingly, the insulinotropic effect of efaroxan was not antagonised by KU14R. K_ATP channels were blocked by efaroxan (IC₅₀ 8.8 μmol/l, Hill slope −1.1) and by KU14R (IC₅₀ 31.9 μmol/l, Hill slope −1.5). Neither the K_ATP channel-blocking effect nor the depolarising effect of efaroxan was antagonised by KU14R. Rather, both compounds strongly depolarised the beta cell membrane potential and induced action potential spiking. However, KU14R was clearly less efficient than efaroxan in raising [Ca²⁺]_c in single beta cells and whole islets at 5 mmol/l glucose. The increase in [Ca²⁺]_c induced by 10 mmol/l glucose was affected neither by efaroxan nor by KU14R. Again, KU14R did not antagonise the effects of efaroxan. Conclusions/interpretation The presence of an imidazole instead of an imidazoline ring leads to virtually complete loss of the insulinotropic effect in spite of a preserved ability to block K_ATP channels. The imidazole compound is less efficient in raising [Ca²⁺]_c; in particular, it lacks the ability of the imidazoline to potentiate the enhancing effect of energy metabolism on Ca²⁺-induced insulin secretion.