Second-phase Insulin Release Research Articles

Management of Diabetes Mellitus, Risk Factors and Complications

Chronic progressive metabolic problem and chronic hyperglycemia caused by a dysregulation of protein, lipid, and carbohydrate metabolism are two symptoms of diabetes mellitus, a complicated condition. Verapamil belongs to a group of calcium channel blockers that are not dihydropyridines. It works by preventing calcium from entering beta cells' cytoplasm, preventing the second phase of insulin release driven by glucose, as well as sulfonylurea and glucagon. Nephropathy, neuropathy, and retinopathy are examples of microvascular consequences of diabetes mellitus (cardiovascular and cerebrovascular disease). In both kinds of diabetes mellitus, higher urine albumin excretion (proteinuria) or decreased kidney glomerular filtration rate are symptoms of diabetic nephropathy, a microvascular consequence. By lowering patients' blood sugar levels and reducing their risk of cardiovascular disease, diabetes mellitus therapy aims to reduce mortality, delay the onset of disease complications, and slow the disease's progression. Metformin increases peripheral glucose utilization, liver, muscle, and adipose tissue sensitivity to insulin, inhibits gluconeogenesis, and reduces glucose absorption from the gastrointestinal system.

Less sedentary time is associated with a more favourable glucose-insulin axis in obese pregnant women\u2014a secondary analysis of the DALI study

Background/objectivesObese pregnant women are at high risk of developing gestational diabetes mellitus (GDM), which might be reduced by sufficient physical activity (PA) and reduced sedentary time (ST). We assessed whether PA and ST are longitudinally associated with the glucose-insulin axis in obese pregnant women.Subjects/methodsIn this secondary analysis of the DALI (vitamin D And Lifestyle Intervention for gestational diabetes mellitus prevention) study, pregnant women, <20 weeks gestation, with a pre-pregnancy body mass index (BMI) ≥ 29 kg/m2, without GDM on entry were included. Time spent in moderate-to-vigorous PA (MVPA) and ST were measured objectively with accelerometers at <20 weeks, 24–28 weeks and 35–37 weeks of gestation. Fasting glucose (mmol/l) and insulin (mU/l), insulin resistance (HOMA-IR) and first-phase and second-phase insulin release (Stumvoll first and second phase) were assessed at the same time. Linear mixed regression models were used to calculate between-participant differences and within-participant changes over time. Analyses were adjusted for gestational age, randomisation, pre-pregnancy BMI, education and age. MVPA, Insulin, HOMA-IR and Stumvoll first and second phase were log-transformed for analyses due to skewness.Results232 women were included in the analysis. Concerning differences between participants, more ST was associated with higher fasting glucose (Estimate: 0.008; 95% CI: 0.002, 0.014), fasting insulin (0.011; 0.002, 0.019), HOMA-IR (0.012; 0.004, 0.021) and Stumvoll first and second phase (0.008; 0.001, 0.014 and 0.007; 0.001, 0.014). Participants with more MVPA had lower Stumvoll first and second phase (−0.137; −0.210, −0.064 and −0.133; −0.202, −0.063). Concerning changes over time, an increase in ST during gestation was associated with elevated Stumvoll first and second phase (0.006; 0.000, 0.011).ConclusionsAs the glucose-insulin axis is more strongly associated with ST than MVPA in our obese population, pregnant women could be advised to reduce ST in addition to increasing MVPA. Moreover, our findings suggest that behaviour change interventions aiming at GDM risk reduction should start in early or pre-pregnancy.

In Vitro Platform for Studying Human Insulin Release Dynamics of Single Pancreatic Islet Microtissues at High Resolution.

Insulin is released from pancreatic islets in a biphasic and pulsatile manner in response to elevated glucose levels. This highly dynamic insulin release can be studied in vitro with islet perifusion assays. Herein, a novel platform to perform glucose-stimulated insulin secretion (GSIS) assays with single islets is presented for studying the dynamics of insulin release at high temporal resolution. A standardized human islet model is developed and a microfluidic hanging-drop-based perifusion system is engineered, which facilitates rapid glucose switching, minimal sample dilution, low analyte dispersion, and short sampling intervals. Human islet microtissues feature robust and long-term glucose responsiveness and demonstrate reproducible dynamic GSIS with a prominent first phase and a sustained, pulsatile second phase. Perifusion of single islet microtissues produces a higher peak secretion rate, higher secretion during the first and second phases of insulin release, as well as more defined pulsations during the second phase in comparison to perifusion of pooled islets. The developed platform enables to study compound effects on both phases of insulin secretion as shown with two classes of insulin secretagogs. It provides a new tool for studying physiologically relevant dynamic insulin secretion at comparably low sample-to-sample variation and high temporal resolution.

A Mouse Model of Beta-Cell Dysfunction as Seen in Human Type 2 Diabetes.

Loss of first-phase insulin release is an early pathogenic feature of type 2 diabetes (T2D). Various mouse models exist to study T2D; however, few recapitulate the early β-cell defects seen in humans. We sought to develop a nongenetic mouse model of T2D that exhibits reduced first-phase insulin secretion without a significant deficit in pancreatic insulin content. C57BL/6J mice were fed 10% or 60% fat diet for three weeks, followed by three consecutive, once-daily intraperitoneal injections of the β-cell toxin streptozotocin (STZ; 30, 50, or 75 mg/kg) or vehicle. Four weeks after injections, the first-phase insulin response to glucose was reduced in mice when high-fat diet was combined with 30, 50, or 75 mg/kg STZ. This was accompanied by diminished second-phase insulin release and elevated fed glucose levels. Further, body weight gain, pancreatic insulin content, and β-cell area were decreased in high fat-fed mice treated with 50 and 75 mg/kg STZ, but not 30 mg/kg STZ. Low fat-fed mice were relatively resistant to STZ, with the exception of reduced pancreatic insulin content and β-cell area. Together, these data demonstrate that in high fat-fed mice, three once-daily injections of 30 mg/kg STZ produces a model of β-cell failure without insulin deficiency that may be useful in studies investigating the etiology and progression of human T2D.

Early deficits in insulin secretion, beta cell mass and islet blood perfusion precede onset of autoimmune type 1 diabetes in BioBreeding rats

Aims/hypothesisGenetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available.MethodsWe used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells.ResultsDRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3 ± 121.3 vs 633.3 ± 148.7; p < 0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5–7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9 ± 1593.7 vs 4416.8 ± 1230.5 pg islet−1 h−1; p < 0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1 × 109 ± 9.5 × 107 vs 3.8 × 109 ± 5.8 × 107 μm3; p = 0.044) and small sized islets (1.6 × 109 ± 5.1 × 107 vs 1.4 × 109 ± 4.5 × 107 μm3; p = 0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1 ± 16.8 vs 76.9 ± 11.8 μl min−1 [g pancreas]−1; p = 0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats.Conclusions/interpretationThe present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.

Plasma kisspeptin levels are associated with insulin secretion in nondiabetic individuals.

To evaluate if plasma kisspeptin concentrations are associated with insulin secretion, as suggested by recent in vitro studies, independently of confounders. 261 nondiabetic subjects were stratified into tertiles according to kisspeptin values. Insulin secretion was assessed using indexes derived from oral glucose tolerance test (OGTT). After adjusting for age, gender, and BMI, subjects in the highest (tertile 3) kisspeptin group exhibited significantly lower values of insulinogenic index, corrected insulin response (CIR30), and Stumvoll indexes for first-phase and second-phase insulin release as compared with low (tertile 1) or intermediate (tertile 2) kisspeptin groups. Univariate correlations between kisspeptin concentration and metabolic variables showed that kisspeptin concentration was significantly and positively correlated with age, blood pressure, and 2-h post-load glucose, and inversely correlated with BMI, and waist circumference. There was an inverse relationship between kisspeptin levels and OGTT-derived indexes of glucose-stimulated insulin secretion. A multivariable regression analysis in a model including all the variables significantly correlated with kisspeptin concentration showed thar age (β = -0.338, P<0.0001), BMI (β = 0.272, P<0.0001), 2-h post-load glucose (β = -0.229, P<0.0001), and kisspeptin (β = -0.105, P = 0.03) remained associated with insulinogenic index. These factors explained 34.6% of the variance of the insulinogenic index. In conclusion, kisspeptin concentrations are associated with insulin secretion independently of important determinants of glucose homeostasis such as gender, age, adiposity, 2-h post-load glucose, and insulin sensitivity.

Metabolomics Study of the Effects of Inflammation, Hypoxia, and High Glucose on Isolated Human Pancreatic Islets.

The transplantation of human pancreatic islets is a therapeutic possibility for a subset of type 1 diabetic patients who experience severe hypoglycemia. Pre- and post-transplantation loss in islet viability and function, however, is a major efficacy-limiting impediment. To investigate the effects of inflammation and hypoxia, the main obstacles hampering the survival and function of isolated, cultured, and transplanted islets, we conducted a comprehensive metabolomics evaluation of human islets in parallel with dynamic glucose-stimulated insulin release (GSIR) perifusion studies for functional evaluation. Metabolomics profiling of media and cell samples identified a total of 241 and 361 biochemicals, respectively. Metabolites that were altered in highly significant manner in both included, for example, kynurenine, kynurenate, citrulline, and mannitol/sorbitol under inflammation (all elevated) plus lactate (elevated) and N-formylmethionine (depressed) for hypoxia. Dynamic GSIR experiments, which capture both first- and second-phase insulin release, found severely depressed insulin-secretion under hypoxia, whereas elevated baseline and stimulated insulin-secretion was measured for islet exposed to the inflammatory cytokine cocktail (IL-1β, IFN-γ, and TNF-α). Because of the uniquely large changes observed in kynurenine and kynurenate, they might serve as potential biomarkers of islet inflammation, and indoleamine-2,3-dioxygenase on the corresponding pathway could be a worthwhile therapeutic target to dampen inflammatory effects.

Dynamics of Insulin Secretion from EndoC-βH1 β-Cell Pseudoislets in Response to Glucose and Other Nutrient and Nonnutrient Secretagogues.

The dynamics of insulin secretion were characterized in response to a variety of physiological and pharmacological stimulators and other compounds in perifused pseudoislets generated from cells of the EndoC-βH1 β-cell line. Perifusion of EndoC-βH1 pseudoislets with the physiological stimulus glucose (16.7 mM) induced sustained insulin secretion, which was inhibited by mannoheptulose. The adenylate cyclase activators IBMX and forskolin strongly potentiated this secretion. Glibenclamide, a Kir 6.2 potassium channel blocker, and Bay K 8644, an opener of the voltage-sensitive Ca2+ channel, also potentiated glucose-induced insulin secretion. The dynamics of insulin secretion from EndoC-βH1 pseudoislets were characterized by an insulin secretory response to glucose starting within 1-2 min and passing over without interruption into a sustained phase of insulin release for the whole stimulation period. This lack of a transient decline between the first and the second phases of insulin release is an indication for a quick supply of insulin secretory granules from the reserve pool to the docking sites below the plasma membrane. Thereby, new secretory granules are directly made available for sustained exocytosis of insulin in EndoC-βH1 β-cells. The study shows that EndoC-βH1 β-cell pseudoislets are well suited for kinetic analyses of insulin secretion.

Estimation of second-phase insulin secretion in the Zucker fatty rat.

The purpose of the present study was to test the efficacy of the empiric index SPIR (Second-phase Insulin Release) in the quantification of second-phase insulin secretion in the Zucker Fatty Rat. SPIR index is defined as the area under the curve of insulin between 8 and 90 min after an Intravenous Glucose Tolerance Test (IVGTT). The validation of such index was performed against the second-phase β-cell responsiveness index (Φ2) provided by C-peptide minimal model. To this aim, Φ2 and SPIR were simultaneously computed from IVGTT data, measured in six Zucker fatty rats (ZFR), 7-to-9week-old, and seven age-matched Zucker lean rats (ZLR). SPIR index showed a significant linear correlation with Φ2 (Pearson's correlation coefficient, r = 0.91, R-square = 0.82, P<;0.001). Moreover, both Φ2 (P<;0.001) and SPIR (P<;0.001) showed a significant increase, in the ZFRs, compared to control group (ZLR). These findings suggest that the SPIR index is able to provide similar information of Φ2, in the evaluation of the second-phase insulin secretion and of its alteration in Zucker Fatty Rats. Thus, the study proposes the SPIR, as a suitable index for a simple, reliable and low-cost quantification of the second-phase insulin secretion in ZFR.

Study of phases of insulin secretion in pre-diabetes and newly diagnosed type 2 diabetes mellitus

Background: Insulin is released from the pancreas in a biphasic manner in response to arterial glucose concentration. The assumption has been generally made that the 30-minute response reflected first-phase insulin release, whereas the 120-minute response reflected second-phase insulin release.Objectives: The aim of this study was to identify the defect in first and second phases of insulin secretion in pre-diabetes and newly diagnosed T2DM.Methods: This case-control study was conducted in the department of Biochemistry, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka from March 2013 to June 2014. All the study subjects (n = 94) were collected from the one point centre, BSMMU as newly diagnosed T2DM, pre-diabetes and healthy normal glucose tolerant subjects according to fasting plasma glucose and 2 hour plasma glucose status. A total of 32 newly diagnosed T2DM and 32 pre-diabetes were included on the basis of inclusion criteria as cases. Another 30 healthy normal glucose tolerant subjects were emolled as control. Fasting blood samples were collected from study subjects to estimate the plasma glucose and insulin level. Again blood samples were taken for measurement of plasma glucose and insulin level at 30 minute and 120 minute on OGTT.Results: Fasting plasma insulin was significantly higher in pre-diabetes than control and T2DM (p = 0.011). Plasma insulin at 30 minute and 120 minute of OGTT were significantly lower in T2DM than control and pre- diabetes (p = 0.001 &amp; 0.016). The insulin secretion in first and second phases were significantly lower in T2DM patients than controls and pre-diabetes (p = 0.000). Beta-cell function was also significantly lower in T2DM than controls and pre-diabetes (p = 0.000). Median values of HOMA-IR were higher in pre-diabetes (1.68) and T2DM (1.53) than control (1.37), but not statistically significant (p = 0.153). There was significant positive correlation of both phases of insulin secretion with FPI, beta-cell function and insulin resistance in T2DM, pre-diabetes and controls.Conclusions: The study reveals that 1st and 2nd phase insulin secretory defect was detected in T2DM, but in pre-diabetes, we have failed to identify insulin secretory defects in both phases.

KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS–1 cells. Taking advantage of hemicannels’opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.

YES, a Src Family Kinase, Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.

Calcium Channel α2δ-1 Subunit Knockout Causes Diabetes Due to Impaired Insulin Release

Pancreatic β-cells, the major cellular component of the island of Langerhans, are responsible for the synthesis of insulin and its secretion in response to elevated blood glucose. High voltage-gated calcium channels (HVCC) are intimately involved in excitation-secretion coupling in pancreatic islet cells and calcium entering through HVCC is an important regulator of insulin synthesis. HVCC are multi-subunit protein complexes comprised of the main pore-forming α1 subunit and auxiliary extracellular α2δ and intracellular β subunits. Here we show that genetic ablation of the α2δ-1 subunit (the main pancreatic α2δ isoform) results in the postnatal development of diabetes. Homozygous α2δ-1 KO mice show highly elevated urine production and develop ∼9-fold higher blood glucose levels compared to WT littermates. Morphological analysis of the pancreas shows a reduction in the number of islets and their size due to a dramatic decrease in β-cell mass in an age-dependant manner. The reduced β-cell mass is not caused by an islet-specific autoimmune reaction, but might result from prolonged hyperglycaemia toxicity. Voltage-clamp recording in dissociated pancreatic β-cells shows a more than two-fold decrease in calcium current amplitude. Glucose stimulated calcium oscillations in whole isolated pancreatic islets shows a strong decrease in amplitude of both the first and second phase of insulin release, and an increased oscillation frequency of the second phase. On-going pharmacological experiments will identify which pore-forming α1 subunits are primarily affected by α2δ-1 deletion and how this effects insulin secretion. These findings indicate that α2δ-1 is an important determinant of normal β-cell physiology, critical for insulin release. Support: FWF W1101, P23479, LFU-P7400-027-011.

β-Cell Dysfunction Is Associated with Metabolic Syndrome Severity in Adults

Metabolic syndrome is prevalent in adults characterized by increased visceral adiposity and insulin resistance (IR). However, the link between pancreatic β-cell function and metabolic syndrome severity in adults across the glucose spectrum is unknown. We hypothesized that poor β-cell function would independently predict a higher metabolic syndrome Z-score (i.e., severity). Seventy (12 normal glucose tolerant, 37 prediabetic, 21 type 2 diabetic) obese adults [62.4±1.1 year; 34.6±0.6 kg/m(2); data are mean±standard error of the mean (SEM)] participated in this cross-sectional study. A 2-hr 75-gram oral glucose tolerance test (OGTT) was administered, and insulin and glucose area under the curve was determined for calculations of insulin action. Fasting and glucose-stimulated insulin secretion was calculated using homeostasis model assessment of insulin secretion (HOMA-B) and the insulinogenic index (i.e., I(0-30)/Glc(0-30) or I(60-120)/Glc(60-120)), respectively. Fasting and postprandial insulin sensitivity was assessed by HOMA-IR and the Matsuda Index, respectively. β-cell function was estimated using the disposition index via HOMA-B/HOMA-IR, I(0-30)/Glc(0-30) or I(60-120)/Glc(60-120) × Matsuda Index, which represents basal, first-, and second-phase insulin release, respectively. Body composition (via computerized tomography and dual X-ray absorptiometry) and sex-specific metabolic syndrome Z-scores were calculated from waist circumference, blood pressure, fasting glucose, triglycerides, and high-density lipoproteins. Compared to those with normal glucose tolerance, visceral fat and IR were higher and β-cell function was lower in adults with glucose intolerance and type 2 diabetes mellitus. Elevated visceral fat and IR (HOMA-IR and Matsuda Index) correlated with elevated Z-scores (r=0.51, r=0.54, r=-0.49; all P<0.002, respectively). Basal, first-, and second-phase β-cell function correlated with low Z-scores (r=-0.59, r=-0.51, and r=-0.43, all P<0.001). Insulin secretion significantly predicted the Z-score independent of sex, body fat, blood lipids, blood pressure, IR, and glucose metabolism (P<0.005). β-cell dysfunction is highly correlated with the severity of metabolic syndrome in adults. Future work is warranted to elucidate the mechanism by which cardiometabolic disturbances influence insulin secretion.

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB1 cannabinoid receptors (CB1Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB1R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB1Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB1Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.

Endogenous Somatostatin Is Critical in Regulating the Acute Effects of l-Arginine on Growth Hormone and Insulin Release in Mice

l-arginine (l-Arg) rapidly stimulates GH and insulin release in vivo. It has been hypothesized that l-Arg stimulates GH release by lowering hypothalamic somatostatin (SST) tone. l-Arg may also act directly at the pituitary to stimulate GH release. Moreover, l-Arg has a direct stimulatory effect on β-cells, which is thought to be blunted by the release of SST from pancreatic δ-cells. To confirm the role of endogenous SST on l-Arg-induced GH and insulin release, wild-type (WT) and SST-knockout (SST-KO) mice were injected with l-Arg (ip; 0.8 g/kg), and pre-/post-injection GH, insulin, and glucose levels were measured. In WT mice, l-Arg evoked a 6-fold increase in circulating GH. However, there was only a modest increase in GH levels in WT pituitary cell cultures treated with l-Arg. In contrast, l-Arg failed to increase GH in SST-KO beyond their already elevated levels. These results further support the hypothesis that the primary mechanism by which l-Arg acutely increases GH in vivo is by lowering hypothalamic SST input to the pituitary and not via direct pituitary effects. Additionally, l-Arg induced a clear first-phase insulin secretion in WT mice, but not in SST-KO. However, SST-KO, but not WT mice, displayed a robust and sustained second-phase insulin release. These results further support a role for endogenous SST in regulating l-Arg-mediated insulin release.

Cool-1/βPIX functions as a guanine nucleotide exchange factor in the cycling of Cdc42 to regulate insulin secretion.

Second-phase insulin release requires the sustained mobilization of insulin granules from internal storage pools to the cell surface for fusion with the plasma membrane. However, the detailed mechanisms underlying this process remain largely unknown. GTP-loading of the small GTPase Cdc42 is the first glucose-specific activation step in the process, although how glucose triggers Cdc42 activation is entirely unknown. In a directed candidate screen for guanine nucleotide exchange factors (GEFs), which directly activate small GTPases, Cool-1/βPix was identified in pancreatic islet beta cells. In support of its role as the beta cell Cdc42 GEF, βPix coimmunoprecipitated with Cdc42 in human islets and MIN6 beta cells in a glucose-dependent manner, peaking just prior to Cdc42 activation. Furthermore, RNAi-mediated βPix reduction by 50% corresponded to full ablation of glucose-induced Cdc42 activation and significant attenuation of basal and glucose-stimulated insulin secretion. Of the two Cdc42 guanine nucleotide dissociation inhibitor (GDI) proteins identified in beta cells, βPix competed selectively with caveolin-1 (Cav-1) but not RhoGDI in coimmunoprecipitation and GST-Cdc42-GDP interaction assays. However, a phospho-deficient Cav-1-Y14F mutant failed to compete with βPix; Cav-1(Tyr14) is an established phosphorylation site for Src kinase. Taken together, these data support a new model, wherein glucose stimulates Cav-1 and induces its dissociation from Cdc42, possibly via Src kinase activation to phosphorylate Cav-1(Tyr14), to promote Cdc42-βPix binding and Cdc42 activation, and to trigger downstream signaling and ultimately sustain insulin release.

The dynamic insulin sensitivity and secretion test—a novel measure of insulin sensitivity

The objective was to validate the methodology for the dynamic insulin sensitivity and secretion test (DISST) and to demonstrate its potential in clinical and research settings. One hundred twenty-three men and women had routine clinical and biochemical measurements, an oral glucose tolerance test, and a DISST. For the DISST, participants were cannulated for blood sampling and bolus administration. Blood samples were drawn at t = 0, 10, 15, 25, and 35 minutes for measurement of glucose, insulin, and C-peptide. A 10-g bolus of intravenous glucose at t = 5 minutes and 1 U of intravenous insulin immediately after the t = 15 minute sample were given. Fifty participants also had a hyperinsulinemic-euglycemic clamp. Relationships between DISST insulin sensitivity (SI) and the clamp, and both DISST SI and secretion and other metabolic variables were measured. A Bland-Altman plot showed little bias in the comparison of DISST with the clamp, with DISST underestimating the glucose clamp by 0.1·10−2·mg·L·kg−1·min−1·pmol−1 (90% confidence interval, −0.2 to 0). The correlation between SI as measured by DISST and the clamp was 0.82; the c unit for the receiver operating characteristic curve analysis for the 2 tests was 0.96. Metabolic variables showed significant correlations with DISST SI and the second phase of insulin release. The DISST also appears able to distinguish different insulin secretion patterns in individuals with identical SI values. The DISST is a simple, dynamic test that compares favorably with the clamp in assessing SI and allows simultaneous assessment of insulin secretion. The DISST has the potential to provide even more information about the pathophysiology of diabetes than more complicated tests.

Effects of increasing doses of glucagon-like peptide-1 on insulin-releasing phases during intravenous glucose administration in mice

The increase in insulin secretion caused by glucagon-like peptide-1 (GLP-1) and GLP-1 mimetics observed during an intravenous glucose test (IVGTT) has been reported in both normal and disease animal models, as well as in humans. In this study, a hierarchical population modeling approach is used, together with a previously reported model relating glucose to insulin appearance, to determine quantitative in vivo dose-response relationships between GLP-1 dose level and both first- and second-phase insulin release. Parameters of the insulin kinetic model were estimated from the complete set of glucose and insulin data collected in 219 anesthetized nonfasted NMR-imaged mice after intravenous injection of glucose (1 g/kg) alone or with GLP-1 (0.03-100 nmol/kg). The resulting dose-response curves indicate a difference in GLP-1 effect on the two release phases, as is also evident from the different ED(50) parameter values (0.107 vs. 6.65 nmol/kg for phase 1 vs. phase 2 insulin release parameters). The first phase of insulin release is gradually augmented with increasing GLP-1 dose, reaching saturation at a dose of ~1 nmol/kg, while the second-phase release changes more abruptly at GLP-1 doses between 3 and 10 nmol/kg and shows a more pronounced 100-fold increase between control and the high GLP-1 dose of 100 nmol/kg Moreover, separate disposition indices calculated for phase 1 and 2 insulin release, show a different pattern of increase with increasing GLP-1 dose.

Insulin Release: Shedding Light on a Complex Matter

SNAP25 and syntaxin-1 are core proteins of the exocytosis machinery for insulin granules. Takahashi et al. (2010) show that the distribution and kinetics of the SNAP25/syntaxin-1 complex assembly play a key role in regulating the first and second phase of insulin release.