Glucose, adrenaline and palmitate antagonistically regulate insulin and glucagon secretion in human pseudoislets.
ABSTRACT: Isolated human islets do not always meet the quality standards required for transplant survival and reliable functional in vitro studies. The formation of pseudoislets, i.e. the reaggregation of a defined number of islet cells after dissociation, improves insulin secretion. We present a simple method of pseudoislet formation from human islet cells and assess the transcriptome and function of isolated human islets and pseudoislets from the same organ donors. Following pseudoislet formation, insulin content/DNA and mRNA/RPS13 resembled that of islets. In pseudoislets, glucose-stimulated insulin secretion (GSIS) was significantly higher (8-13-fold) than in islets (2-4-fold). GSIS of pseudoislets was partly inhibited by the glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin-9. The stimulatory effects of palmitate and forskolin at 12?mM glucose were also significantly higher in pseudoislets than in islets. Further analysis of pseudoislets revealed that regulation of secretion and insulin and glucagon content was maintained over a longer culture period (6-14 d). While adrenaline inhibited GSIS, adrenaline together with palmitate stimulated glucagon secretion 2-fold at low glucose, an effect suppressed by high glucose. Transcriptome analysis revealed that, unlike islets, pseudoislets were deprived of exocrine and endothelial cells. In conclusion, pseudoislet formation restores functional integrity of human islet cells and allows long-term in vitro testing.
Project description:We report that intra-islet glucagon secreted from α-cells signals through β-cell glucagon and GLP-1 receptors (GcgR and GLP-1R), thereby conferring to rat islets their competence to exhibit first-phase glucose-stimulated insulin secretion (GSIS). Thus, in islets not treated with exogenous glucagon or GLP-1, first-phase GSIS is abolished by a GcgR antagonist (LY2786890) or a GLP-1R antagonist (Ex[9-39]). Mechanistically, glucose competence in response to intra-islet glucagon is conditional on β-cell cAMP signaling because it is blocked by the cAMP antagonist prodrug Rp-8-Br-cAMPS-pAB. In its role as a paracrine hormone, intra-islet glucagon binds with high affinity to the GcgR, while also exerting a "spillover" effect to bind with low affinity to the GLP-1R. This produces a right shift of the concentration-response relationship for the potentiation of GSIS by exogenous glucagon. Thus, 0.3 nM glucagon fails to potentiate GSIS, as expected if similar concentrations of intra-islet glucagon already occupy the GcgR. However, 10 to 30 nM glucagon effectively engages the β-cell GLP-1R to potentiate GSIS, an action blocked by Ex[9-39] but not LY2786890. Finally, we report that the action of intra-islet glucagon to support insulin secretion requires a step-wise increase of glucose concentration to trigger first-phase GSIS. It is not measurable when GSIS is stimulated by a gradient of increasing glucose concentrations, as occurs during an oral glucose tolerance test in vivo. Collectively, such findings are understandable if defective intra-islet glucagon action contributes to the characteristic loss of first-phase GSIS in an intravenous glucose tolerance test, that is, diagnostic of type 2 diabetes in the clinical setting.
Project description:Misregulated hormone secretion from the islet of Langerhans is central to the pathophysiology of diabetes. Although insulin plays a key role in glucose regulation, the importance of glucagon is increasingly acknowledged. However, the mechanisms that regulate glucagon secretion from ?-cells are still unclear. We used pseudoislets reconstituted from dispersed islet cells to study ?-cells with and without various indirect effects from other islet cells. Dispersed islet cells secrete aberrant levels of glucagon and insulin at basal and elevated glucose levels. When cultured, murine islet cells reassociate to form pseudoislets, which recover normal glucose-regulated hormone secretion, and human islet cells follow a similar pattern. We created small (?40-µm) pseudoislets using all of the islet cells or only some of the cell types, which allowed us to characterize novel aspects of regulated hormone secretion. The recovery of regulated glucagon secretion from ?-cells in small pseudoislets depends upon the combined action of paracrine factors, such as insulin and somatostatin, and juxtacrine signals between EphA4/7 on ?-cells and ephrins on ?-cells. Although these signals modulate different pathways, both appear to be required for proper inhibition of glucagon secretion in response to glucose. This improved understanding of the modulation of glucagon secretion can provide novel therapeutic routes for the treatment of some individuals with diabetes.
Project description:We have recently shown that overnight exposure of INS-1E insulinoma cells to palmitate in the presence of high glucose causes defects in both mitochondrial energy metabolism and glucose-stimulated insulin secretion (GSIS). Here we report experiments designed to test the involvement of mitochondrial uncoupling protein-2 (UCP2) in these glucolipotoxic effects. Measuring real-time oxygen consumption in siRNA-transfected INS-1E cells, we show that deleterious effects of palmitate on the glucose sensitivity of mitochondrial respiration and on the coupling efficiency of oxidative phosphorylation are independent of UCP2. Consistently, palmitate impairs GSIS to the same extent in cells with and without UCP2. Furthermore, we knocked down UCP2 in spheroid INS-1E cell clusters (pseudoislets) to test whether or not UCP2 regulates insulin secretion during prolonged glucose exposure. We demonstrate that there are no differences in temporal GSIS kinetics between perifused pseudoislets with and without UCP2. We conclude that UCP2 is not involved in palmitate-induced impairment of GSIS in INS-1E insulinoma cells and is not needed for the amplification of insulin release. These conclusions inform ongoing debate on the disputed biochemical and physiological functions of the beta cell UCP2.
Project description:Pancreatic islets secrete insulin from ? cells and glucagon from ? cells, and dysregulated secretion of these hormones is a central component of diabetes. Thus, an improved understanding of the pathways governing coordinated ? and ? cell hormone secretion will provide insight into islet dysfunction in diabetes. However, the 3D multicellular islet architecture, essential for coordinated islet function, presents experimental challenges for mechanistic studies of intracellular signaling pathways in primary islet cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles. We demonstrate the utility of this experimental approach by studying the effects of Gi and Gq GPCR pathways on insulin and glucagon secretion by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion, which occur through changes in intracellular Ca2+. The experimental approach of combining pseudoislets with a microfluidic system allowed the coregistration of intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human ? and ? cells.
Project description:Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous beta-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL(-/-) mice indicated the presence of other TG lipase(s) in the beta-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K(ATP)-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL(-/-) mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL(-/-) mice. Accordingly, isolated islets from ATGL(-/-) mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL(-/-) islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.
Project description:<h4>Aims/hypothesis</h4>Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets.<h4>Methods</h4>MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, α-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca(2+) concentration ([Ca(2+)]c; microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured.<h4>Results</h4>Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca(2+)]c response.<h4>Conclusions/interpretation</h4>We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca(2+)]c.
Project description:Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse β-cells, LDs are prominent in human β-cells. However, the regulation of LD mobilization (lipolysis) in human β-cells remains unclear. We found that glucose increases lipolysis in nondiabetic human islets but not in islets in patients with type 2 diabetes (T2D), indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets with shRNA targeting ATGL (shATGL) increased triglycerides (TGs) and the number and size of LDs, indicating that ATGL is the principal lipase in human β-cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS), and insulin secretion to 3-isobutyl-1-methylxanthine and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose-responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL-deficient INS1 cells and human pseudoislets showed reduced SNARE protein syntaxin 1a (STX1A), a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL-deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human β-cells and supports insulin secretion by stabilizing STX1A. The dysregulated lipolysis may contribute to LD accumulation and β-cell dysfunction in T2D islets.
Project description:OBJECTIVE: We have previously shown the existence of a muscle-pancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine ?-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated. METHODS: Gene expression was determined using RNA sequencing in human islets, sorted ?- and non-?-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1-50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted ?-cells exposed to CX3CL1 for 48 h alone or in the presence of TNF? (20 ng/ml). Rat and human ?-cell apoptosis (TUNEL) and rat ?-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1. RESULTS: Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-?-cells than in ?-cells while its receptor is more expressed in ?-cells. CX3CL1 decreased human (but not rat) ?-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted ?-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNF? on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes. CONCLUSIONS: We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human ?-cells. We further demonstrate that CX3CL1 protects ?-cells from the adverse effects of TNF? on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.
Project description:The expression of short chain fatty acid receptors FFA2 and FFA3 in pancreatic islets raised interest in using them as drug targets for treating hyperglycemia in humans. This study aims to examine the efficacy of synthetic FFA2- and FFA3-ligands to modulate glucose-stimulated insulin secretion (GSIS) in human pseudoislets which display intact glucose responsiveness. The FFA2-agonists 4-CMTB and TUG-1375 inhibited GSIS, an effect reversed by the FFA2-antagonist CATPB. GSIS itself was not augmented by CATPB. The FFA3-agonists FHQC and 1-MCPC did not affect GSIS in human pseudoislets. For further drug evaluation we used mouse islets. The CATPB-sensitive inhibitory effect of 100 µM 4-CMTB on GSIS was recapitulated. The inhibition was partially sensitive to the Gi/o-protein inhibitor pertussis toxin. A previously described FFA2-dependent increase of GSIS was observed with lower concentrations of 4-CMTB (10 and 30 µM). The stimulatory effect of 4-CMTB on secretion was prevented by the Gq-protein inhibitor FR900359. As in human pseudoislets, in mouse islets relative mRNA levels were FFAR2?>?FFAR3 and FFA3-agonists did not affect GSIS. The FFA3-agonists, however, inhibited GSIS in a pertussis toxin-sensitive manner in INS-1E cells and this correlated with relative mRNA levels of Ffar3?>?>?Ffar2. Thus, in humans, when FFA2-activation impedes GSIS, FFA2-antagonism may reduce glycemia.
Project description:Long-term exposure to NEFAs leads to inhibition of glucose-induced insulin secretion. We tested whether the release of somatostatin and glucagon, the two other major islet hormones, is also affected.Mouse pancreatic islets were cultured for 72 h at 4.5 or 15 mmol/l glucose with or without 0.5 mmol/l oleate or palmitate. The release of glucagon and somatostatin during subsequent 1 h incubations at 1 or 20 mmol/l glucose as well as the islet content of the two hormones were determined. Lipid-induced changes in islet cell ultrastructure were assessed by electron microscopy.Culture at 15 mmol/l glucose increased islet glucagon content by approximately 50% relative to that observed following culture at 4.5 mmol/l glucose. Inclusion of oleate or palmitate reduced islet glucagon content by 25% (at 4.5 mmol/l glucose) to 50% (at 15 mmol/l glucose). Long-term exposure to the NEFA increased glucagon secretion at 1 mmol/l glucose by 50% (when islets had been cultured at 15 mmol/l glucose) to 100% (with 4.5 mmol/l glucose in the culture medium) and abolished the inhibitory effect of 20 mmol/l glucose on glucagon secretion. Somatostatin content was unaffected by glucose and lipids, but glucose-induced somatostatin secretion was reduced by approximately 50% following long-term exposure to either of the NEFA, regardless of whether the culture medium contained 4.5 or 15 mmol/l glucose. Ultrastructural evidence of lipid deposition was seen in <10% of non-beta cells but in >80% of the beta cells.Long-term exposure to high glucose and/or NEFA affects the release of somatostatin and glucagon. The effects on glucagon secretion are very pronounced and in type 2 diabetes in vivo may aggravate the hyperglycaemic effects due to lack of insulin.