Amino acid-stimulated insulin secretion: a path forward in type 2 diabetes.

Qualitative and quantitatively appropriate insulin secretion is essential for optimal control of blood glucose. Beta-cells of the pancreas produce and secrete insulin in response to glucose and non-glucose stimuli including amino acids. In this manuscript, we review the literature on amino acid-stimulated insulin secretion in oral and intravenous in vivo studies, in addition to the in vitro literature, and describe areas of consensus and gaps in understanding. We find promising evidence that the synergism of amino acid-stimulated insulin secretion could be exploited to develop novel therapeutics, but that a systematic approach to investigating these lines of evidence is lacking. We highlight evidence that supports the relative preservation of amino acid-stimulated insulin secretion compared to glucose-stimulated insulin secretion in type 2 diabetes, and make the case for the therapeutic potential of amino acids. Finally, we make recommendations for research and describe the potential clinical utility of nutrient-based treatments for type 2 diabetes including remission services.

Insulin secretion induced by glucose-dependent insulinotropic polypeptide requires phosphatidylinositol 3-kinase γ in rodent and human ß-cells.

PI3Kγ, a G-protein-coupled type 1B phosphoinositol 3-kinase, exhibits a basal glucose-independent activity in ß-cells and can be activated by the glucose-dependent insulinotropic polypeptide (GIP). We therefore investigated the role of the PI3Kγ catalytic subunit (p110γ) in insulin secretion and ß-cell exocytosis stimulated by GIP. We inhibited p110γ with AS604850 (1 µmol/liter) or knocked it down using an shRNA adenovirus or siRNA duplex in mouse and human islets and ß-cells. Inhibition of PI3Kγ blunted the exocytotic and insulinotropic response to GIP receptor activation, whereas responses to the glucagon-like peptide-1 or the glucagon-like peptide-1 receptor agonist exendin-4 were unchanged. Downstream, we find that GIP, much like glucose stimulation, activates the small GTPase protein Rac1 to induce actin remodeling. Inhibition of PI3Kγ blocked these effects of GIP. Although exendin-4 could also stimulate actin remodeling, this was not prevented by p110γ inhibition. Finally, forced actin depolymerization with latrunculin B restored the exocytotic and secretory responses to GIP during PI3Kγ inhibition, demonstrating that the loss of GIP-induced actin depolymerization was indeed limiting insulin exocytosis.

Web-based multi-omics integration using the Analyst software suite.

The growing number of multi-omics studies demands clear conceptual workflows coupled with easy-to-use software tools to facilitate data analysis and interpretation. This protocol covers three key components involved in multi-omics analysis, including single-omics data analysis, knowledge-driven integration using biological networks and data-driven integration through joint dimensionality reduction. Using the dataset from a recent multi-omics study of human pancreatic islet tissue and plasma samples, the first section introduces how to perform transcriptomics/proteomics data analysis using ExpressAnalyst and lipidomics data analysis using MetaboAnalyst. On the basis of significant features detected in these workflows, the second section demonstrates how to perform knowledge-driven integration using OmicsNet. The last section illustrates how to perform data-driven integration from the normalized omics data and metadata using OmicsAnalyst. The complete protocol can be executed in ~2 h. Compared with other available options for multi-omics integration, the Analyst software suite described in this protocol enables researchers to perform a wide range of omics data analysis tasks via a user-friendly web interface.

Proteomic predictors of individualized nutrient-specific insulin secretion in health and disease.

Population level variation and molecular mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized despite ramifications for personalized nutrition. Here, we define prototypical insulin secretion dynamics in response to the three macronutrients in islets from 140 cadaveric donors, including those diagnosed with type 2 diabetes. While islets from the majority of donors exhibited the expected relative response magnitudes, with glucose being highest, amino acid moderate, and fatty acid small, 9% of islets stimulated with amino acid and 8% of islets stimulated with fatty acids had larger responses compared with high glucose. We leveraged this insulin response heterogeneity and used transcriptomics and proteomics to identify molecular correlates of specific nutrient responsiveness, as well as those proteins and mRNAs altered in type 2 diabetes. We also examine nutrient-responsiveness in stem cell-derived islet clusters and observe that they have dysregulated fuel sensitivity, which is a hallmark of functionally immature cells. Our study now represents the first comparison of dynamic responses to nutrients and multi-omics analysis in human insulin secreting cells. Responses of different people's islets to carbohydrate, protein, and fat lay the groundwork for personalized nutrition. ONE-SENTENCE SUMMARY: Deep phenotyping and multi-omics reveal individualized nutrient-specific insulin secretion propensity.

HumanIslets: An integrated platform for human islet data access and analysis.

Comprehensive molecular and cellular phenotyping of human islets can enable deep mechanistic insights for diabetes research. We established the Human Islet Data Analysis and Sharing (HI-DAS) consortium to advance goals in accessibility, usability, and integration of data from human islets isolated from donors with and without diabetes at the Alberta Diabetes Institute (ADI) IsletCore. Here we introduce HumanIslets.com , an open resource for the research community. This platform, which presently includes data on 547 human islet donors, allows users to access linked datasets describing molecular profiles, islet function and donor phenotypes, and to perform various statistical and functional analyses at the donor, islet and single-cell levels. As an example of the analytic capacity of this resource we show a dissociation between cell culture effects on transcript and protein expression, and an approach to correct for exocrine contamination found in hand-picked islets. Finally, we provide an example workflow and visualization that highlights links between type 2 diabetes status, SERCA3b Ca 2+ -ATPase levels at the transcript and protein level, insulin secretion and islet cell phenotypes. HumanIslets.com provides a growing and adaptable set of resources and tools to support the metabolism and diabetes research community.

Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance.

Insulin receptor (Insr) protein is present at higher levels in pancreatic ß-cells than in most other tissues, but the consequences of ß-cell insulin resistance remain enigmatic. Here, we use an Ins1cre knock-in allele to delete Insr specifically in ß-cells of both female and male mice. We compare experimental mice to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined ß-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout ß-cells from female, but not male mice, whereas only male ßInsrKO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female ßInsrKO and ßInsrHET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter ß-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include ß-cell insulin resistance, which predicts that ß-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female ßInsrKO and ßInsrHET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of ß-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that ß-cell insulin resistance in the form of reduced ß-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.

Differential Effects of Voclosporin and Tacrolimus on Insulin Secretion From Human Islets.

The incidence of new onset diabetes after transplant (NODAT) has increased over the past decade, likely due to calcineurin inhibitor-based immunosuppressants, including tacrolimus (TAC) and cyclosporin. Voclosporin (VCS), a next-generation calcineurin inhibitor, is reported to cause fewer incidences of NODAT but the reason is unclear. While calcineurin signaling plays important roles in pancreatic ß-cell survival, proliferation, and function, its effects on human ß-cells remain understudied. In particular, we do not understand why some calcineurin inhibitors have more profound effects on the incidence of NODAT. We compared the effects of TAC and VCS on the dynamics of insulin secretory function, programmed cell death rate, and the transcriptomic profile of human islets. We studied 2 clinically relevant doses of TAC (10 ng/mL, 30 ng/mL) and VCS (20 ng/mL, 60 ng/mL), meant to approximate the clinical trough and peak concentrations. TAC, but not VCS, caused a significant impairment of 15 mM glucose-stimulated and 30 mM KCl-stimulated insulin secretion. This points to molecular defects in the distal stages of exocytosis after voltage-gated Ca2+ entry. No significant effects on islet cell survival or total insulin content were identified. RNA sequencing showed that TAC significantly decreased the expression of 17 genes, including direct and indirect regulators of exocytosis (SYT16, TBC1D30, PCK1, SMOC1, SYT5, PDK4, and CREM), whereas VCS has less broad, and milder, effects on gene expression. Clinically relevant doses of TAC, but not VCS, directly inhibit insulin secretion from human islets, likely via transcriptional control of exocytosis machinery.

Specialized Hub Beta Cells Trade Maximal Insulin Production for Perfect Timing.

The pulsatility of insulin release is disturbed early in type 2 diabetes, but it is not clear whether specialized pacemaker cells drive islet oscillations. In this issue of Cell Metabolism, Johnston et al. (2016) show that specialized hubs, identified as 1%-10% of beta cells with more active mitochondria and less insulin, synchronize beta cell oscillations.

PI3 kinases p110α and PI3K-C2ß negatively regulate cAMP via PDE3/8 to control insulin secretion in mouse and human islets.

OBJECTIVES: Phosphatidylinositol-3-OH kinase (PI3K) signalling in the endocrine pancreas contributes to glycaemic control. However, the mechanism by which PI3K modulates insulin secretion from the pancreatic beta cell is poorly understood. Thus, our objective was two-fold; to determine the signalling pathway by which acute PI3K inhibition enhances glucose-stimulated insulin secretion (GSIS) and to examine the role of this pathway in islets from type-2 diabetic (T2D) donors. METHODS: Isolated islets from mice and non-diabetic or T2D human donors, or INS 832/13 cells, were treated with inhibitors of PI3K and/or phosphodiesterases (PDEs). The expression of PI3K-C2ß was knocked down using siRNA. We measured insulin release, single-cell exocytosis, intracellular Ca(2+) responses ([Ca(2+)]i) and Ca(2+) channel currents, intracellular cAMP concentrations ([cAMP]i), and activation of cAMP-dependent protein kinase A (PKA) and protein kinase B (PKB/AKT). RESULTS: The non-specific PI3K inhibitor wortmannin amplifies GSIS, raises [cAMP]i and activates PKA, but is without effect in T2D islets. Direct inhibition of specific PDE isoforms demonstrates a role for PDE3 (in humans and mice) and PDE8 (in mice) downstream of PI3K, and restores glucose-responsiveness of T2D islets. We implicate a role for the Class II PI3K catalytic isoform PI3K-C2ß in this effect by limiting beta cell exocytosis. CONCLUSIONS: PI3K limits GSIS via PDE3 in human islets. While inhibition of p110α or PIK-C2ß signalling per se, may promote nutrient-stimulated insulin release, we now suggest that this signalling pathway is perturbed in islets from T2D donors.

TCF1 links GIPR signaling to the control of beta cell function and survival.

The glucagon-like peptide-1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor transduce nutrient-stimulated signals to control beta cell function. Although the GLP-1 receptor (GLP-1R) is a validated drug target for diabetes, the importance of the GIP receptor (GIPR) for the function of beta cells remains uncertain. We demonstrate that mice with selective ablation of GIPR in beta cells (MIP-Cre:Gipr(Flox/Flox); Gipr(-/-ßCell)) exhibit lower levels of meal-stimulated insulin secretion, decreased expansion of adipose tissue mass and preservation of insulin sensitivity when compared to MIP-Cre controls. Beta cells from Gipr(-/-ßCell) mice display greater sensitivity to apoptosis and markedly lower islet expression of T cell-specific transcription factor-1 (TCF1, encoded by Tcf7), a protein not previously characterized in beta cells. GIP, but not GLP-1, promotes beta cell Tcf7 expression via a cyclic adenosine monophosphate (cAMP)-independent and extracellular signal-regulated kinase (ERK)-dependent pathway. Tcf7 (in mice) or TCF7 (in humans) levels are lower in islets taken from diabetic mice and in humans with type 2 diabetes; knockdown of TCF7 in human and mouse islets impairs the cytoprotective responsiveness to GIP and enhances the magnitude of apoptotic injury, whereas restoring TCF1 levels in beta cells from Gipr(-/-ßCell) mice lowers the number of apoptotic cells compared to that seen in MIP-Cre controls. Tcf7(-/-) mice show impaired insulin secretion, deterioration of glucose tolerance with either aging and/or high-fat feeding and increased sensitivity to beta cell injury relative to wild-type (WT) controls. Hence the GIPR-TCF1 axis represents a potential therapeutic target for preserving both the function and survival of vulnerable, diabetic beta cells.

cAMP-independent effects of GLP-1 on ß cells.

The ability of glucose to stimulate insulin secretion from the pancreatic islets of Langerhans is enhanced by the intestinal hormone glucagon-like peptide 1 (GLP-1), which is secreted from the gut in response to nutrient ingestion. This action, called the incretin effect, accounts for as much as half of the postprandial insulin response and is exploited therapeutically for diabetes treatment through the use of incretin mimetic drugs and inhibitors of dipeptidyl peptidase 4, which degrades GLP-1. Despite a prominent role for incretin mimetics in diabetes treatment, several key questions remain about GLP-1-induced insulin secretion. Most studies have examined the effects of GLP-1 at concentrations several orders of magnitude higher than those found in vivo; therefore, one might question the physiological (and perhaps even pharmacological) relevance of pathways identified in these studies and whether other important mechanisms might have been obscured. In this issue of the JCI, Shigeto and colleagues demonstrate that physiological GLP-1 does indeed amplify the insulin secretory response. Intriguingly, while much of this response is PKA dependent, as might be expected, the use of picomolar GLP-1 reveals a new and important mechanism that contributes to GLP-1-induced insulin secretion.

Functional plasticity of the human infant ß-cell exocytotic phenotype.

Our understanding of adult human ß-cells is advancing, but we know little about the function and plasticity of ß-cells from infants. We therefore characterized islets and single islet cells from human infants after isolation and culture. Although islet morphology in pancreas biopsies was similar to that in adults, infant islets after isolation and 24-48 hours of culture had less insulin staining, content, and secretion. The cultured infant islets expressed pancreatic and duodenal homeobox 1 and several (Glut1, Cav1.3, Kir6.2) but not all (syntaxin 1A and synaptosomal-associated protein 25) markers of functional islets, suggesting a loss of secretory phenotype in culture. The activity of key ion channels was maintained in isolated infant ß-cells, whereas exocytosis was much lower than in adults. We examined whether a functional exocytotic phenotype could be reestablished under conditions thought to promote ß-cell differentiation. After a 24- to 28-day expansion and maturation protocol, we found preservation of endocrine markers and hormone expression, an increased proportion of insulin-positive cells, elevated expression of syntaxin 1A and synaptosomal-associated protein 25, and restoration of exocytosis to levels comparable with that in adult ß-cells. Thus, human infant islets are prone to loss of their exocytotic phenotype in culture but amenable to experimental approaches aimed at promoting expansion and functional maturation. Control of exocytotic protein expression may be an important mechanism underlying the plasticity of the secretory machinery, an increased understanding of which may lead to improved regenerative approaches to treat diabetes.

SUMOylation regulates Kv2.1 and modulates pancreatic beta-cell excitability.

The covalent attachment of small ubiquitin-like modifier (SUMO) proteins regulates protein localization and function. SUMOylation has recently been shown to modulate ion-channel function; however, the extent to which this affects native currents and cellular excitability remains to be determined. The voltage-dependent K(+) (Kv) channel Kv2.1 regulates pancreatic beta-cell excitability and insulin secretion. We found that YFP-tagged SUMO1 (SUMO1-YFP) can be immunoprecipitated with Kv2.1 when these two proteins are coexpressed in HEK 293 cells. Furthermore, direct infusion of recombinant SUMO1 peptide or coexpression of SUMO1-YFP inhibited current through cloned Kv2.1 by 80% and 48%, respectively. Insulin-secreting cells express SUMO variants 1 and 3, and expression of the SUMO1-YFP in human beta-cells or insulinoma cells inhibited native Kv currents (by 49% and 33%, respectively). Inhibition of the channel resulted from an acceleration of channel inactivation and an inhibition of recovery from inactivation, resulting in the widening of beta-cell action potentials and a decreased firing frequency. Finally, these effects on channel function and excitability were augmented by the conjugating enzyme Ubc9 and rescued by the SUMO protease SENP1. Thus, protein SUMOylation can exert a strong inhibitory action on the voltage-dependent K(+) channel Kv2.1 and can regulate cellular excitability in native beta-cells.

Insulin granule recruitment and exocytosis is dependent on p110gamma in insulinoma and human beta-cells.

OBJECTIVE: Phosphatidylinositol 3-OH kinase (PI3K) has a long-recognized role in beta-cell mass regulation and gene transcription and is implicated in the modulation of insulin secretion. The role of nontyrosine kinase receptor-activated PI3K isoforms is largely unexplored. We therefore investigated the role of the G-protein-coupled PI3Kgamma and its catalytic subunit p110gamma in the regulation of insulin granule recruitment and exocytosis. RESEARCH DESIGN AND METHODS: The expression of p110gamma was knocked down by small-interfering RNA, and p110gamma activity was selectively inhibited with AS605240 (40 nmol/l). Exocytosis and granule recruitment was monitored by islet perifusion, whole-cell capacitance, total internal reflection fluorescence microscopy, and electron microscopy in INS-1 and human beta-cells. Cortical F-actin was examined in INS-1 cells and human islets and in mouse beta-cells lacking the phosphatase and tensin homolog (PTEN). RESULTS: Knockdown or inhibition of p110gamma markedly blunted depolarization-induced insulin secretion and exocytosis and ablated the exocytotic response to direct Ca(2+) infusion. This resulted from reduced granule localization to the plasma membrane and was associated with increased cortical F-actin. Inhibition of p110gamma had no effect on F-actin in beta-cells lacking PTEN. Finally, the effect of p110gamma inhibition on granule localization and exocytosis could be rapidly reversed by agents that promote actin depolymerization. CONCLUSIONS: The G-protein-coupled PI3Kgamma is an important determinant of secretory granule trafficking to the plasma membrane, at least in part through the negative regulation of cortical F-actin. Thus, p110gamma activity plays an important role in maintaining a membrane-docked, readily releasable pool of secretory granules in insulinoma and human beta-cells.