Initiation Of Insulin Secretion Research Articles

Glucose Regulation of β-Cell KATP Channels: It Is Time for a New Model!

An agreed-upon consensus model of glucose-stimulated insulin secretion from healthy β-cells is essential for understanding diabetes pathophysiology. Since the discovery of the KATP channel in 1984, an oxidative phosphorylation (OxPhos)-driven rise in ATP has been assumed to close KATP channels to initiate insulin secretion. This model lacks any evidence, genetic or otherwise, that mitochondria possess the bioenergetics to raise the ATP/ADP ratio to the triggering threshold, and conflicts with genetic evidence demonstrating that OxPhos is dispensable for insulin secretion. It also conflates the stoichiometric yield of OxPhos with thermodynamics, and overestimates OxPhos by failing to account for established features of β-cell metabolism, such as leak, anaplerosis, cataplerosis, and NADPH production that subtract from the efficiency of mitochondrial ATP production. We have proposed an alternative model, based on the spatial and bioenergetic specializations of β-cell metabolism, in which glycolysis initiates insulin secretion. The evidence for this model includes that 1) glycolysis has high control strength over insulin secretion; 2) glycolysis is active at the correct time to explain KATP channel closure; 3) plasma membrane-associated glycolytic enzymes control KATP channels; 4) pyruvate kinase has favorable bioenergetics, relative to OxPhos, for raising ATP/ADP; and 5) OxPhos stalls before membrane depolarization and increases after. Although several key experiments remain to evaluate this model, the 1984 model is based purely on circumstantial evidence and must be rescued by causal, mechanistic experiments if it is to endure.

1465-P: Identification of GCK Gene Variants and Clinical Characterization in Japanese Pediatric MODY2 Cases

Maturity-onset diabetes of the young (MODY) is traditionally characterized by early onset and a heterogeneous autosomal dominant form of inheritance. MODY2 may be common in the Japanese, but the difference in clinical characteristics compared to Europeans and Americans remains unclear. Here, we examined the characteristics in Japanese pediatric patients diagnosed with MODY2. We retrospectively reviewed the diagnostic data of Japanese MODY2 patients diagnosed at our hospital between 2009 and 2022. Genetic screening for MODY2 was performed by direct sequencing followed by multiplex ligation amplification assays. We identified 20 missense mutations in 25 patients and 2 nonsense mutations in 2 patients. We detected 5 deletions in 6 patients. The mean age at diagnosis was 10 years (range 2-14 years), the mean BMI percentile was 46.2%, with none being obese. Mean HbA1c was 6.54%. A glucose tolerance test was performed on 29 patients, 15 of whom were diabetic (7 were diabetic at fasting, 5 were diabetic at 120 min, 3 were diabetic at both) and 14 of borderline diabetes (IFG + IGT: 11, IFG: 2, and IGT: 1). The average glucose tolerance test values were 121.6 mg/dL at 0 min and 182.2 mg/dL at 120 min. No correlation was found between age at onset and plasma glucose value at 0 and 120 min. Reportedly, MODY2 causes a slight increase in post-load blood glucose levels, however the difference between 120 min and 0 min was 56.0 mg/dL (−32 to 140). A negative correlation was observed between 0 min and 120 min blood glucose levels. HOMAIR average was 1.85, 27% of which patients were 2.5 or higher; HOMA-β average was 37.0%, 39% of which showed <30%; and insulinogenic index averaged 0.63, 25% of which showed <0.4, indicating decreased initial insulin secretion. In this study, it is suggested that in Japanese MODY2 cases, glucose sensor dysfunction due to GCK gene mutation causes decreased initial insulin secretion. Disclosure R.Katashima: None. S.Yoshida: None. Y.Watanabe: None. M.Moritani: None. I.Yokota: None.

Pyruvate kinase and GLP-1 receptor signaling converge on cAMP production to enhance insulin secretion

Pyruvate kinase (PK), by converting ADP and phosphoenolpyruvate (PEP) to ATP and pyruvate, initiates insulin secretion via KATP closure and enhances insulin secretion via mitochondrial PEP production. The physiological drivers of mitochondrial PEP production include amino acids, which also work on neighboring α-cells to stimulate insulin secretion through activation of β-cell glucagon and GLP-1 receptors. We therefore hypothesized that PK would augment α-cell paracrine signaling upon amino acid (AA) stimulation.

Acute Metabolic Response in Adults to Toddler Milk Formulas with Alternating Higher and Lower Protein and Fat Contents, a Randomized Cross-Over Trial.

Protein intake in early life influences metabolism, weight gain, and later obesity risk. As such, a better understanding of the effects of protein intake on the postprandial metabolism and its dynamics over time may elucidate underlying mechanisms. In a randomized crossover study, we observed fasted adults who consumed two isocaloric toddler milk formulas concentrated as meals of 480 kcal with 67 g of carbohydrates 30 g (HP) or 7 g (LP) protein, and 10 g or 20 g fat, respectively. Anthropometry and body plethysmography were assessed, and blood samples collected at baseline and over five hours. Time-specific concentrations, areas under concentration curves (AUC), and maximum values of metabolites were compared by paired t-tests to examine the effects of protein content of toddler milks on postprandial plasma concentrations of insulin, glucose, branched-chain amino acids (BCAA), urea and triglycerides. Twenty-seven men and women aged 26.7 ± 5.0 years (BMI: 22.2 ± 2.5 kg/m2) (mean ± SD) participated. BCAA AUC, and Cmax values were significantly higher with HP than LP (144,765 ± 21,221 vs. 97,089 ± 14,650 µmol·min/L, p < 0.001; 656 ± 120 vs. 407 ± 66 µmol/L, p < 0.001), as were insulin AUC and Cmax values (6674 ± 3013 vs. 5600 ± 2423 µmol·min/L, p = 0.005; 71 ± 37 vs. 55 ± 28 µmol/L, p = 0.001). Higher glucose, urea, and triglyceride concentrations occurred in the late postprandial phase (≥180 min) with HP. In conclusion, we noted that higher milk protein intake induces increased postprandial BCAA concentrations for at least 5 h and led to higher initial insulin secretion. Gluconeogenesis due to an influx of amino acids and their degradation after HP meal might explain the late effects of protein intake on glucose and insulin.

38-OR: Deletion of the Mitofusins 1 and 2 (Mfn1 and Mfn2) from the Pancreatic Beta Cell Disrupts Mitochondrial Structure and Impairs Glucose-, but Not Incretin-, Stimulated Insulin Secretion

Background and Aims: Mitochondrial metabolism of glucose is vital for the initiation of insulin secretion from pancreatic β-cells. Whether mitochondrial ultra-structure, and the proteins controlling mitochondrial fission and fusion, are essential for glucose recognition is less clear. Here, β-cell-restricted deletion of Mfn1 and Mfn2 in adult mice was used to investigate the role of mitochondrial dynamics in insulin secretion and glucose homeostasis in vivo and in vitro. Methods: C57BL6 mice bearing Mfn1 and Mfn2 alleles with FloxP sites, were crossed to animals carrying an inducible Cre recombinase expressed under Pdx1 promoter control (PdxCreERT). Following tamoxifen induction, Mfn1 and Mfn2 knockdown was confirmed in KO islets. Live β-cell fluorescence imaging of Ca2+ accumulation and membrane potential were performed on isolated islets and in the living mouse. Mitochondrial ultra-structure was measured using super resolution fluorescence imaging and EM. Results: Mitochondrial length was strongly and selectively diminished in β-cells from βMfn1/2 KO mice. KO animals displayed higher fasting and fed glycaemia than control animals at 14 weeks and a considerable (&amp;gt;5-fold) decrease in plasma insulin post-IP glucose injection. Additionally, mitochondrial length, glucose-induced hyperpolarization, ATP synthesis, Ca2+ accumulation, O2 consumption and β-cell connectivity were all significantly reduced in βMfn1/2-KO mouse islets. Ca2+ dynamics and mitochondrial membrane potential changes were also suppressed in vivo. Remarkably, defective GSIS in islets isolated from βMfn1/2-KO mice was normalised by the addition of GLP-1 receptor agonists. Conclusions: Mitochondrial fusion and fission cycles are indispensable in the β-cell for normal glucose sensing in vitro and in vivo. GLP-1 receptor agonism may mitigate the effects of mitochondrial abnormalities in some forms of diabetes. Disclosure E. Georgiadou: None. M. Ibberson: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. A. K. Linnemann: None. Y. Ali: None. T. Rodriguez: None. G. A. Rutter: Advisory Panel; Self; Sun Pharmaceutical Industries Ltd. C. Muralidharan: None. P. L. Chabosseau: None. A. Tomas: Other Relationship; Self; Sun Pharmaceutical Industries Ltd. T. Stylianides: None. C. Legido-quigley: None. N. Alsabeeh: None. C. Cruciani-guglielmacci: Consultant; Self; The Healthy Aging Company. C. Magnan: None. Funding UK Wellcome Trust (WT098424AIA, 212625/Z/18/Z); Medical Research Council (MR/R022259/1, MR/J0003042/1, MR/L020149/1, MR/N00275X/1); Experimental Challenge Grant (DIVA, MR/L02036X/1); Diabetes UK (BDA/11/0004210, BDA/15/0005275, BDA16/0005485)

2072-P: Deletion of the Mitofusins 1 and 2 (Mfn1 and Mfn2) in the Pancreatic Beta Cell Disrupts Mitochondrial Structure and Function In Vitro and Strongly Impairs Glucose-Stimulated Insulin Secretion In Vivo

Background and Aims: Mitochondrial metabolism of glucose is vital for the initiation of insulin secretion in pancreatic beta cells. Whether mitochondrial ultra-structure, and the proteins controlling their fission and fusion, are essential for glucose recognition is less clear. In the present study, beta cell-selective adult mouse restricted deletion of Mfn1 and Mfn2 was used to investigate the role of mitochondrial fusion in insulin secretion and glucose homeostasis in vivo and in vitro. Methods: C57BL6 mice bearing Mfn1 and Mfn2 alleles with FloxP sites, were crossed to animals carrying an inducible Cre recombinase at the Pdx1 locus (PdxCreERT). After induction of recombination with tamoxifen, live beta cell fluorescence imaging of mitochondrial [Ca2+] and membrane potential were performed on isolated islets. Mitochondrial ultra-structure was measured using super resolution fluorescence and transmission electron microscopy. Intraperitoneal glucose tolerance, and insulin secretion in vivo and in vitro, were measured using standard techniques. Results: Mitochondrial length was strongly (to 77±0.9% of controls, p&amp;lt;0.0001) and selectively diminished in beta cells from Mfn1/2 KO mice. KO animals displayed higher fasting glycaemia than control animals at 11-12 weeks (8.6 vs. 6.4 mmol/L, p&amp;gt;0.05). An increase in circulating glucose levels was also observed (p&amp;lt;0.05 at 30 min and p&amp;lt;0.01 at 60 min) and was linked to a considerable (&amp;gt;five-fold) decrease in plasma insulin (5-15 min, p&amp;lt;0.0001) post-intraperitoneal glucose injection. Mitochondrial Ca2+ accumulation and membrane potential were significantly reduced (p&amp;lt;0.01) in response to high glucose in KO animals. Conclusions: Mitochondrial fusion and fission cycles are indispensable in the beta cell in order to preserve normal mitochondrial bioenergetics and glucose sensing, and may be disrupted in some forms of diabetes. Disclosure E. Georgiadou: None. P.L. Chabosseau: None. A. Tomas: None. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. Research Support; Spouse/Partner; Servier. G.A. Rutter: Consultant; Self; Sun Pharmaceutical Industries Ltd. Research Support; Self; Servier, Sun Pharmaceutical Industries Ltd. Funding UK Wellcome Trust (WT098424AIA, 212625/Z/18/Z); UK Medical Researh Council (MR/R022259/1, MR/J0003042/1, MR/L020149/1, MR/L02036X/1, MR/N00275X/1); Diabetes UK (BDA/11/0004210, BDA/15/0005275, BDA16/0005485)

Presenilin-1 Established ER-Ca2+ Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and β-Cells upon Elevated Glucose.

In our recent work, the importance of GSK3β-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in β-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3β-phosphorylated presenilin-1 for responsiveness of pancreatic islets and β-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. Freshly isolated pancreatic islets and the two pancreatic β-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in β-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3β in the pancreatic β-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3β in the pancreatic β-cells and isolated pancreatic islets, respectively. These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and β-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3β, a phenomenon that might be involved in the development of type 2 diabetes.

2179-P: Overexpression Screening Identifies Mitochondrial Carbonic Anhydrase 5b as an Important Regulator of Glucose-Stimulated Insulin Secretion in Beta Cells

Despite extensive research efforts, a precise picture of how glucose itself and its metabolites initiates insulin secretion in a concentration-dependent manner has unveiled. To identify candidate genes important for glucose-stimulated insulin secretion (GSIS), we have conducted screening of differentially expressed genes identified by microarray analyses between highly glucose-responsive and less responsive MIN6 subclones. For this purpose, a platform containing zeocin resistant gene flanked with a wild type and mutated flippase recognition target sites was integrated in the genome of MIN6 cells expressing Tet3G. The modified MIN6 cells have been used to generate stable transfectants expressing mRNA or shRNA of genes of interest by means of the recombinease-mediated cassette exchange method. Using this system, more than ten stable MIN6 cell transformants can be gnenerated at a time. By creating approximately 100 overexpressing transfectants, we have identified mitochondrial carbonic anhydrase 5b (Car5b) as an important regulator of GSIS. Car5b is a one of 15 members of carbonic anhydrases, which catalyze the interconversion of carbon dioxide and water to bicarbonate and protons, and modulate various intracellular processes, such as urea formation, HCO3- supply for carboxylation and regulation of intracellular acid-base balance. Car5b overexpression in MIN6 cells increased 14CO2 formation from H14CO3- (37 ± 14% increases, p &amp;lt; 0.05) in isolated mitochondria and resulted in 32 ± 12% increases (P &amp;lt; 0.05) in GSIS at 20 mM, while shCar5b expression reduced Car5b protein levels to 31.4 ± 4.4% (p &amp;lt; 0.05), 14CO2 formation to 68.7 ± 8.1% (p &amp;lt; 0.05), and GSIS at 20 mM to 77 ± 5% (p &amp;lt; 0.05). In addition, we found that Car5b expressions in ob/ob mice were 3.7 ± 0.4-fold higher (p &amp;lt; 0.01) and tended to be higher (p = 0.073) in HFD-mice. These data indicated that mitochondrial carbonic anhydrase Car5b plays a role in GSIS in pancreatic β-cells. Disclosure M. Kosuda: None. S. Yamaguchi: None. A. Nagasawa: None. M. Yamana: None. M. Koike: None. H. Nishioka: None. H. Ishihara: Research Support; Self; Daiichi Sankyo Company, Limited, Lilly Diabetes. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

Mitochondrial Dynamin-Related Protein 1 (DRP1) translocation in response to cerebral glucose is impaired in a rat model of early alteration in hypothalamic glucose sensing

ObjectiveHypothalamic glucose sensing (HGS) initiates insulin secretion (IS) via a vagal control, participating in energy homeostasis. This requires mitochondrial reactive oxygen species (mROS) signaling, dependent on mitochondrial fission, as shown by invalidation of the hypothalamic DRP1 protein. Here, our objectives were to determine whether a model with a HGS defect induced by a short, high fat-high sucrose (HFHS) diet in rats affected the fission machinery and mROS signaling within the mediobasal hypothalamus (MBH). MethodsRats fed a HFHS diet for 3 weeks were compared with animals fed a normal chow. Both in vitro (calcium imaging) and in vivo (vagal nerve activity recordings) experiments to measure the electrical activity of isolated MBH gluco-sensitive neurons in response to increased glucose level were performed. In parallel, insulin secretion to a direct glucose stimulus in isolated islets vs. insulin secretion resulting from brain glucose stimulation was evaluated. Intra-carotid glucose load-induced hypothalamic DRP1 translocation to mitochondria and mROS (H2O2) production were assessed in both groups. Finally, compound C was intracerebroventricularly injected to block the proposed AMPK-inhibited DRP1 translocation in the MBH to reverse the phenotype of HFHS fed animals. ResultsRats fed a HFHS diet displayed a decreased HGS-induced IS. Responses of MBH neurons to glucose exhibited an alteration of their electrical activity, whereas glucose-induced insulin secretion in isolated islets was not affected. These MBH defects correlated with a decreased ROS signaling and glucose-induced translocation of the fission protein DRP1, as the vagal activity was altered. AMPK-induced inhibition of DRP1 translocation increased in this model, but its reversal through the injection of the compound C, an AMPK inhibitor, failed to restore HGS-induced IS. ConclusionsA hypothalamic alteration of DRP1-induced fission and mROS signaling in response to glucose was observed in HGS-induced IS of rats exposed to a 3 week HFHS diet. Early hypothalamic modifications of the neuronal activity could participate in a primary defect of the control of IS and ultimately, the development of diabetes.

Long‐term culture and in vitro maturation of macroencapsulated adult and neonatal porcine islets

Encapsulated porcine islets could be used to treat type I diabetes without necessitating severe immunosuppression. Islet survival and secretory function in the encapsulation device need to be preserved to ensure efficient insulin output in response to surrounding stimuli. In the present study, we evaluated stimulated insulin secretion from adult and neonatal pig islets seeded on an acellular collagen matrix and encapsulated in alginate during long-term culture. Pig islets survived longer and secreted more insulin when cultured on acellular porcine dermis compared to human fascia. Islets from neonatal pigs could survive up to 33weeks in vitro, and their insulin secretion increased during the first 5weeks of culture in a beta-cell maturation medium. In fact, by the 4th week of culture, insulin secretion from neonatal islets attained the same level as adult islets and even surpassed it by the 18th week. Our results show that in vitro maturation of encapsulated neonatal porcine islets is possible and can actually compensate the initial low insulin secretion from these islets while allowing enough time to perform complete functional and biosafety characterization of islets before transplantation.

Duodenal long noncoding RNAs are associated with glycemic control after bariatric surgery in high-fat diet-induced diabetic mice

BackgroundThe duodenum plays a role in the mechanism of type 2 diabetes remission after bariatric surgery. Roux-en-Y gastric bypass (RYGB) may change gene expression in the duodenum and metabolism. Long noncoding RNAs (lncRNAs) constitute a novel class of RNAs that regulate gene expression. Little is known about how duodenal lncRNAs respond to RYGB. Logically, studies on the changes of duodenal lncRNAs potentially can lead to an understanding of the mechanisms of bariatric surgery, as well as discovery of antidiabetic drug targets and biomarkers predicting postoperative outcome. ObjectivesTo investigate the expression signature of duodenal lncRNAs associated with glycemic improvement by duodenal-jejunal bypass (DJB), a component of RYGB, on a genome-wide scale in high-fat diet–induced diabetic mice. SettingUniversity medical center. MethodsHigh fat diet–induced diabetic mice were randomized into 2 groups receiving either the DJB or a sham procedure. Microarray was applied to screen the differentially expressed lncRNAs and messenger RNAs (mRNAs) in the duodenum between the DJB and sham groups, and the result was validated by quantitative real-time polymerase chain reaction in another cohort of animals. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to predict the potential lncRNA functions. Based on Pearson correlation analysis, the lncRNA-mRNA and lncRNA-transcription factor (TF) interaction networks were constructed to identify and rank core regulatory lncRNAs and transcription factors. ResultsA total of 301 lncRNAs, including 232 that were upregulated and 69 downregulated (fold change≥2.0), were differentially expressed in the duodenum between the DJB and sham groups. GO enrichment indicated that these lncRNA-coexpressed mRNAs were correlated with biological processes including cell proliferation, digestion, and catabolic and biosynthetic processes. KEGG pathway analysis revealed that in addition to the digestion and absorption signaling pathways, pancreatic secretion– and inflammatory process–related signaling pathways were mostly enriched in the DJB group. In addition, the lncRNA-mRNA interaction network combined with GO and KEGG pathway analysis suggested that as a top-ranked gene, NONMMUG021726 may play an important role in the mechanism of type 2 diabetes remission after DJB. ConclusionDJB leads to drastic changes in lncRNA and mRNA expressions in the duodenum. The majority of top-ranked lncRNAs and mRNAs have roles in pancreatic secretion and inflammatory processes, implying that bypass of the duodenum may initiate insulin secretion and attenuate inflammation. In addition, modulators of such lncRNAs, most likely NONMMUG021726, have potential to become therapeutic targets or biomarkers for prediction of the outcomes of bariatric surgery.

Involvement of cAMP/EPAC/TRPM2 activation in glucose- and incretin-induced insulin secretion.

In pancreatic β-cells, closure of the ATP-sensitive K(+) (K(ATP)) channel is an initial process triggering glucose-stimulated insulin secretion. In addition, constitutive opening of background nonselective cation channels (NSCCs) is essentially required to effectively evoke depolarization as a consequence of K(ATP) channel closure. Thus, it is hypothesized that further opening of NSCC facilitates membrane excitability. We identified a class of NSCC that was activated by exendin (ex)-4, GLP-1, and its analog liraglutide at picomolar levels. This NSCC was also activated by increasing the glucose concentration. NSCC activation by glucose and GLP-1 was a consequence of the activated cAMP/EPAC-mediated pathway and was attenuated in TRPM2-deficient mice. The NSCC was not activated by protein kinase A (PKA) activators and was activated by ex-4 in the presence of PKA inhibitors. These results suggest that glucose- and incretin-activated NSCC (TRPM2) works in concert with closure of the KATP channel to effectively induce membrane depolarization to initiate insulin secretion. The current study reveals a new mechanism for regulating electrical excitability in β-cells and for mediating the action of glucose and incretin to evoke insulin secretion, thereby providing an innovative target for the treatment of type 2 diabetes.

Glucose Control of Glucagon Secretion: There Is More to It Than KATP Channels

Diabetes is generally attributed to insulin deficiency, but it has become increasingly evident that glucagon excess accounts for many diabetic manifestations (1). In contrast to the consensus mechanisms by which hyperglycemia initiates insulin secretion, there are fundamentally different ideas regarding how glucagon release is stimulated by hypoglycemia. Current hypotheses fall into three categories. First, it is possible that glucose concentration is measured by extrapancreatic sensors that feed back information to glucagon-releasing α-cells via neurons (2,3). Second, since glucagon release is also regulated independently of neural coupling, glucose sensing may be accomplished within the pancreatic islets by β- and δ-cells that control the α-cells via paracrine release of inhibitory insulin (4), Zn2+ (5), γ-aminobutyric acid (6), or somatostatin (7). Third, there is evidence supporting intrinsic glucose sensing by α-cells. In this category, two hypotheses imply that glucose deficiency causes a depolarization with influx of Ca2+ that initiates glucagon secretion by providing less energy to the electrogenic Na+/K+ pump (8) or by activation of a depolarizing store-operated influx of Ca2+ when energy is insufficient to maintain Ca2+ in the endoplasmic reticulum (9,10). However, the most frequently cited hypothesis proposes that glucose deficit hyperpolarizes the α-cells, relieving a depolarization-induced inactivation of ion channels involved in action potential firing and glucagon release (11). AMP-activated protein kinase may also contribute to stimulation of glucagon secretion downstream of Ca2+ (12). Clarification of the mechanisms by which glucose regulates glucagon release should facilitate development of α-cell–centered therapies to improve glucose homeostasis in diabetes. The …

Population analysis of ethnicity and first-phase insulin release

Objective A mechanistic, physiologically-based pharmacokinetic (PK/PD) model was developed to describe the biphasic insulin release and evaluate the racial effects on the glucose–insulin kinetics in response to intravenous glucose. Methods Fifteen African-American and 18 Caucasian children and adolescents between 8 and 18 years of age were enrolled in the study. Intravenous bolus of glucose (250 mg/kg) was administered and blood samples collected at frequent intervals for three hours following the glucose injection. A nonlinear mixed-effect population kinetic analysis with covariate structure was performed using Monolix. Results A significantly higher initial insulin secretion from a readily releasable pool, which is responsible for the first-phase insulin secretion, was detected in African-Americans compared to Caucasians ( p < 0.05). Conclusions The proposed kinetic model is able to describe the glucose-stimulated insulin response, account for the first-phase insulin release and identify a racially-based pharmacokinetic difference in insulin's biphasic secretion behavior. It is hypothesized that the first-phase insulin component may play an important role in the development of type 2 diabetes. The proposed mechanistic model provides a quantitative analysis of the biphasic insulin release that may be useful in the early detection of diabetes.

The insulinotropic effect of fluoroquinolones

Antimicrobial fluoroquinolones induce, with strongly varying frequency, life-threatening hypoglycemias, which is explained by their ability to block K ATP channels in pancreatic B-cells and thus to initiate insulin secretion. In apparent contradiction to this, we observed that none of the fluoroquinolones in this study (gatifloxacin, moxifloxacin, ciprofloxacin, and a number of fluorophenyl-substituted compounds) initiated insulin secretion of perifused mouse islets when the glucose concentration was basal (5 mM). Only when the glucose concentration was stimulatory by itself (10 mM), the fluoroquinolones enhanced secretion. The fluoroquinolones were ineffective on SUR1 Ko islets, which do not have functional K ATP channels. All of these fluoroquinolones depolarized the membrane potential of mouse B-cells (patch-clamping in the whole-cell mode). Using metabolically intact B-cells (perforated-patch mode) however, 100 μM of gatifloxacin, ciprofloxacin or moxifloxacin were unable to depolarize when the glucose concentration was 5 mM, whereas other K ATP channel blockers (tolbutamide and efaroxan) remained effective. Only at a very high concentration (500 μM) gatifloxacin and moxifloxacin, but not ciprofloxacin induced repetitive depolarizations which could be antagonized by diazoxide. In the presence of 10 mM glucose all fluoroquinolones which enhanced secretion markedly elevated cytosolic calcium concentration ([Ca 2+] i). In the presence of 5 mM glucose gatifloxacin and moxifloxacin at 500 μM but not at 100 μM elevated [Ca 2+] i. It is concluded that fluoroquinolones in the clinically relevant concentration range are not initiators, but rather enhancers of glucose-induced insulin secretion. The block of K ATP channels appears necessary but not sufficient to explain the hypoglycemic effect of fluoroquinolones.

Activation of the extracellular calcium-sensing receptor initiates insulin secretion from human islets of Langerhans: involvement of protein kinases

The extracellular calcium-sensing receptor (CaR) is usually associated with systemic Ca(2+) homeostasis, but the CaR is also expressed in many other tissues, including pancreatic islets of Langerhans. In the present study, we have used human islets and an insulin-secreting cell line (MIN6) to investigate the effects of CaR activation using the calcimimetic R-568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca(2+). CaR activation initiated a marked but transient insulin secretory response from both human islets and MIN6 cells at a sub-stimulatory concentration of glucose, and further enhanced glucose-induced insulin secretion. CaR-induced insulin secretion was reduced by inhibitors of phospholipase C or calcium-calmodulin-dependent kinases, but not by a protein kinase C inhibitor. CaR activation was also associated with an activation of p42/44 mitogen-activated protein kinases (MAPK), and CaR-induced insulin secretion was reduced by an inhibitor of p42/44 MAPK activation. We suggest that the beta-cell CaR is activated by divalent cations co-released with insulin, and that this may be an important mechanism of intra-islet communication between beta-cells.

Selective loss of glucose-induced amplification of insulin secretion in mouse pancreatic islets pretreated with sulfonylurea in the absence of fuels

The beta cell metabolism of glucose, and some other fuels, initiates insulin secretion by closure of ATP-sensitive K+ channels and amplifies the secretory response via unknown metabolic intermediates. The aim of this study was to further characterise the mechanism responsible for the metabolic amplification of insulin secretion. Pancreatic islets were isolated from albino mice by collagenase digestion. Insulin secretion in perifused islets was determined by ELISA. Bioluminometry was used to determine the ATP and ADP content of the incubated islets. After perifusing islets for 60 min with 2.7 micromol/l glipizide (closing all ATP-sensitive K+ channels) in the absence of any fuel, perifusion with a test medium containing 2.7 micromol/l glipizide plus 30 mmol/l glucose did not enhance insulin secretion. However, test media supplemented with 2.7 micromol/l glipizide plus either 10 mmol/l alpha-ketoisocaproate or 10 mmol/l 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid amplified the glipizide-induced insulin secretion. In pancreatic islets preincubated for 60 min with 2.7 micromol/l glipizide in the absence of any fuel, 40 min incubations in the presence of 2.7 micromol/l glipizide plus 30 mmol/l glucose or plus 10 mmol/l alpha-ketoisocaproate produced an increase in the ATP content, no change in the ADP content and a rather small increase in the ATP:ADP ratio. The corresponding effects of glucose and alpha-ketoisocaproate were similar. These results suggest that metabolic amplification of fuel-induced insulin secretion is not mediated by changes in the beta cell content of ATP and ADP, but might be due to export of citrate cycle intermediates to the beta cell cytosol.

Effect of 17??-Estradiol on Insulin Secretion and Cytosolic Calcium in Min6 Mouse Insulinoma Cells and Human Islets of Langerhans

Female gonadal steroids can exert an insulinotropic effect in vivo. The objective of this study was to investigate the effects in vitro of 17-beta-estradiol (17beta-E2) on changes in cytosolic calcium ([Ca]i) and on insulin secretion from the MIN6 mouse insulinoma cell line and human primary islets of Langerhans. Stimulus-induced changes in [Ca]i were measured in Fura-2-loaded cells by single cell microfluorimetry. The effects of 17beta-E2 on insulin secretion were measured in static incubation experiments, and the rate and pattern of secretory responses were studied in multi-channel perifusion experiments. 17Beta-E2 (1-100 nmol/L) enhanced basal (2 mmol/L glucose) insulin secretion but had no effect on secretory responses to 20 mmol/L glucose or to depolarizing stimuli (100 micromol/L tolbutamide, 20 mmol/L KCl). Approximately 60% of MIN6 cells responded to 17beta-E2 (1-100 nmol/L) with a small but sustained increase in [Ca]i, whereas 98% of MIN6 cells responded to tolbutamide (100 micromol/L). Similar effects were observed in experiments using human primary beta cells. In contrast, 17beta-E2 had no detectable effect on the increases in [Ca]i evoked by tolbutamide (100 micromol/L) or glucose (20 mmol/L). Our observations are consistent with a rapid effect of 17beta-E2 to depolarize beta cells leading to an influx of extracellular Ca and the initiation of insulin secretion by the consequent elevations in [Ca]i. We suggest that this may offer a mechanism through which circulating estradiol can influence beta-cell responsiveness to other signals.

β-Cell Function in Mild Type 2 Diabetic Patients

We studied the effects of the oral insulin secretagogue nateglinide on insulin secretion using a modeling approach to obtain beta-cell function parameters from a meal test and examined the impact of the beta-cell improvement on glucose tolerance. Mild type 2 diabetic men and women (n = 108; fasting glucose 7.0-8.3 mmol/l) on diet treatment alone randomly received 30, 60, or 120 mg nateglinide or placebo for 24 weeks. Beta-cell function parameters were derived by modeling (based on C-peptide deconvolution) from a standardized meal test at baseline and after 24 weeks of treatment. The baseline demographic and metabolic characteristics of the four groups were similar. Nateglinide treatment resulted in dose-dependent reductions in the mean postprandial glucose response and at the 120-mg dose in fasting glucose. Fasting or total insulin secretion during the meal were not different. In contrast, we found differences in the model parameters. Rate sensitivity (expressing early insulin secretion when glucose is rising) was significantly enhanced at 24 weeks with the lowest nateglinide dose, with no further stimulation at higher doses. Early potentiation (expressing an initial insulin secretion enhancement), glucose sensitivity (the slope of the glucose-insulin secretion relationship), and insulin secretion at a fixed- reference 7-mmol/l glucose concentration all showed a trend toward increasing, with increasing nateglinide dose, and were significantly greater than placebo at the 120-mg dose. In multiple regression analyses, changes in rate sensitivity, glucose sensitivity, and potentiation all contributed to the observed glucose changes. The model-derived parameters are sensitive measures of beta-cell function, showing improvements after nateglinide treatment and predicting changes in glucose tolerance.

Glucose Intolerance Is Common in Japanese Patients With Acute Coronary Syndrome Who Were Not Previously Diagnosed With Diabetes

Postprandial hyperglycemia has emerged as a new glycometabolic condition associated with an excessive risk for coronary artery disease. We therefore attempted to evaluate the frequency of postchallenge hyperglycemia in patients with acute coronary syndrome (ACS) who were not previously diagnosed to have diabetes and did not have a fasting glucose concentration of > or =7 mmol/l or an HbA(1c) level >6.0%. We further correlated the presence of postchallenge hyperglycemia with the extent of coronary atherosclerosis. In all, 134 consecutive ACS patients who met the above inclusion criteria were studied. An oral glucose tolerance test was performed before discharge. The mean age, fasting glucose, and HbA(1c) were 60 years, 5.15 mmol/l, and 5.4%, respectively. Among ACS patients, impaired glucose tolerance (IGT) and diabetes were found in 50 (37%) and 13 patients (10%), respectively. The homeostasis model assessment for insulin resistance did not differ substantially among the normal glucose tolerance (NGT), IGT, and diabetic groups. Insulinogenic index, however, was lower and the number of stenosed vessels higher in diabetic patients compared with NGT patients. Postchallenge hyperglycemia, caused primarily by impaired initial insulin secretion, is commonly found in Japanese ACS patients who have not been previously diagnosed with diabetes, and this phenomenon is considered to be associated with advanced coronary atherosclerosis. Therefore, the present study strongly supports the notion that oral glucose tolerance test assessment of postchallenge hyperglycemia is essential to identify any previously undiagnosed diabetes cases among Japanese ACS patients.