Glucose-stimulated Insulin Release Research Articles

Transgenic expression of the positive selected human GLUD2 gene improves in vivo glucose homeostasis by regulating basic insulin secretion

Glutamate dehydrogenase 1 (GDH1) contributes to glucose-stimulated insulin secretion in murine β-cells, but not to basic insulin release. The implications of these findings for human biology are unclear as humans have two GDH-specific enzymes: hGDH1 (GLUD1-encoded) and hGDH2 (GLUD2-encoded), a novel enzyme that is highly activated by ADP and L-leucine. Here we studied in vivo glucose homeostasis in transgenic (Tg) mice generated by inserting the GLUD2 gene and its putative regulatory elements into their genome. Using specific antibodies, we observed that hGDH2 was co-expressed with the endogenous murine GDH1 in pancreatic β-cells of Tg mice. Fasting blood glucose (FBG) levels were lower and of a narrower range in Tg (95% CI: 90.6–96.8 mg/dl; N = 26) than in Wt mice (95% CI: 136.2–151.4 mg/dl; N = 23; p < 0.0001), closely resembling those of healthy humans. GLUD2 also protected the host mouse from developing diabetes with advancing age. Tg animals maintained 2.6-fold higher fasting serum insulin levels (mean ± SD: 1.63 ± 0.15 ng/ml; N = 12) than Wt mice (0.63 ± 0.05 ng/ml; N = 12; p < 0.0001). Glucose loading (1 mg/g, given i.p.) induced comparable serum insulin increases in Tg and Wt mice, suggesting no significant GLUD2 effect on glucose-stimulated insulin release. L-leucine (0.25 mg/g given orally) induced a 2-fold increase in the serum insulin of the Wt mice, implying significant activation of the endogenous GDH1. However, L-leucine had little effect on the high insulin levels of the Tg mice, suggesting that, under the high ADP levels that prevail in β-cells in the fasting state, glutamate flux through hGDH2 is close to maximal. Hence, the present data, showing that GLUD2 expression in Tg mice improves in vivo glucose homeostasis by boosting fasting serum insulin levels, suggest that evolutionary adaptation of hGDH2 has enabled humans to achieve narrow-range euglycemia by regulating glutamate-mediated basal insulin secretion.

Ghrelin in rat pancreatic islets decreases islet blood flow.

The peptide ghrelin is mainly produced in some of the epithelial cells in the stomach, but also, during starvation, by the ε-cells in the endocrine pancreas. Ghrelin, as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R1α), exerts a variety of metabolic functions including stimulation of appetite and weight gain. Its complete role is not yet fully understood, including whether it has any vascular functions. The present study evaluated if ghrelin affects pancreatic and islet blood flow. Ghrelin and the GHS-R1α receptor antagonist GHRP-6 were injected intravenously in rats followed by blood flow measurements using a microsphere technique. Ghrelin decreased, while GHRP-6 in fasted, but not fed, rats selectively increased islet blood flow fourfold. GHS-R1α was identified not only on glucagon-producing cells but also seemed to be present in the islet arterioles. GHRP-6 in fasted rats, only, also improved the peak insulin response to glucose in vivo, thereby substantially blunting the hyperglycemia. GHRP-6 doubled glucose-stimulated insulin release in vitro of both islets obtained from fed and fasted rats. Our results indicate a novel role for endogenous ghrelin acting directly or indirectly as a local vasoconstrictor in the islets during fasting, thereby restricting the insulin response to hyperglycemia. This is to the best of our knowledge the first report that shows this physiological mechanism to restrict insulin delivery from the islets by acting on the vasculature; a mode of action that can be envisaged to complement the previously well-described mechanisms of ghrelin acting directly on the islet endocrine cells.

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human isletsamples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

Improved cryopreservation yield of pancreatic islets using combination of lower dose permeable cryoprotective agents

Islet transplantation has been shown to restore normoglycemia in animal models and for type 1 diabetic patients in clinical trials. One method of storing islets intended for transplantation is via cryobanking at very low temperatures (−196 °C). Cryobanking islets without the use of cryoprotecting agents (CPAs) contributes to cellular shear stress and cell death. Although current CPA protocols vary, high concentrations of these agents are toxic to islets cells. This study tested the effects of the permeating CPA dimethyl sulfoxide (Me2SO) with the addition of ethylene glycol (EG), both at reduced concentrations, on rat and human islet cell yield, viability, and glucose stimulated insulin release (GSIR). To test this, islets were treated using three combinations of CPAs (2M ME2SO, 1M ME2SO + 1M EG, and 1M ME2SO + 0.5M EG). Next, fresh islets, 2M ME2SO islets, and 1M ME2SO + 0.5M EG isolated rat islets were transplanted into streptozotocin-induced (STZ) diabetic mice. Our data showed that cryopreservation with a reduced concentration of ME2SO (1M ME2SO + multimolar EG) achieved a higher percent yield and viability when compared to the current standard 2M ME2SO treatment for both rat and human islets. Furthermore, STZ-induced diabetic mice achieved normoglycemia after transplantation with 1000 islet equivalents (IE), an average 12 days sooner, with islets cryopreserved with reduced-concentration (ME2SO + 0.5M EG), compared to islets preserved with 2M ME2SO. In conclusion, reduced concentration of penetrating CPAs during islet cryopreservation increases islet yield and viability in vitro and reduces delay before normoglycemia in diabetic mice.

Effect of broth from meat of linseed-fed cattle on glucose-stimulated insulin release in healthy male volunteers.

Polyunsaturated fatty acid consumption has been shown to improve insulin sensitivity. We studied if administration of broth with beef meat enriched with polyunsaturated fatty acids influenced glucose-stimulated insulin release in healthy male volunteers. Broth was made either from cattles undergone dietary supplementation with lightly bruised whole linseed in addition to feeding ad libitum on grass silage (test meal) or from those fed grass silage alone (control meal). Oral glucose tolerance tests (OGTT) were performed in patients after a 6-day period of eating 300ml broth containing 100g meat once a day in addition to their otherwise normal mixed nourishment. During OGTT, blood samples were taken for blood glucose level and plasma insulin immunoreactivity before and 15, 30, 60, 90, 120, and 180min after the glucose load. Glucose-stimulated maximum increase in plasma insulin immunoreactivity was 42±6.6 and 81±7.4mU/ml (p<0.05) after the test and the control meals, respectively. However, both fasting and postload blood glucose levels were the same after either meal period. The results suggest an insulin-sensitizing effect of food produced from beef cattle maintained on linseed diet in healthy human volunteers.

Low Cost, Enriched Collagenase-Purified Protease Enzyme Mixtures Successfully Used for Human Islet Isolation

Background: Purified bacterial collagenase and protease enzymes are commonly used to recover human islets from clinical grade pancreata for subsequent clinical islet transplantation. The high cost of purified enzymes can be prohibitive for recovering human islets from research pancreata used for translational research or pre-clinical studies. In this study, we successfully isolated islets from human research pancreata using enriched collagenase products supplemented with BP Protease (DE Collagenase 800 or Collagenase Gold-BP Protease mixtures) that cost significantly less than purified collagenase-protease enzymes used for clinical islet isolation. Materials & methods: Defined, enriched collagenase products (DE Collagenase 800 and Collagenase Gold) with a purity comparable to purified clinical collagenase blends (> 85% purity) were prepared from Clostridium histolyticum culture supernatants. These products contained primarily intact class I (C1) and class II (C2) at about a 75:25 C1:C2 ratio as determined by analytical anion exchange chromotography Enriched collagenase-BP Protease mixtures were used at either 19,000-48,000 or 32,500-78,800 neutral protease U (NP U) per g pancreas, and hereafter referred to as low or high protease enzyme mixtures. Each enzyme mixture was diluted up to 350 mL of HBSS and used in a modified Ricordi procedure with with isolations performed at three different islet processing centers. Results: Comparison of the biochemical characteristics of enriched collagenase to the purified collagenase products showed the enriched product is comparable and in some cases superior to those higher priced purified collagenase products. The mean post-purification islet yields from three different centers were 4444 and 4261 IEQ/g pancreas when using the high and low protease enzyme mixtures, respectively. The mean stimulation indices calculated after analysis of glucose-stimulated insulin release were 2.68 and 2.11 when using the high and low protease enzyme mixtures, respectively. Conclusions: The use of a low cost enriched collagenase products with biochemical characteristics comparable to purified collagenase enzymes were shown to be as effective in human islet isolation as higher priced products. These data indicate that using enzyme mixtures with high Wunsch activity may not be required for successful human islet isolation. The amount of Wunsch activity/g tissue can be reduced to 25-50% of the amount currently used by some islet transplant centers for isolating human islets. Further studies are needed to validate enzyme compositions required for successful human islet isolation.

Impact of Nutrient Type and Sequence on Glucose Tolerance: Physiological Insights and Therapeutic Implications.

Pharmacological and dietary interventions targeting postprandial glycemia have proved effective in reducing the risk for type 2 diabetes and its cardiovascular complications. Besides meal composition and size, the timing of macronutrient consumption during a meal has been recently recognized as a key regulator of postprandial glycemia. Emerging evidence suggests that premeal consumption of non-carbohydrate macronutrients (i.e., protein and fat “preloads”) can markedly reduce postprandial glycemia by delaying gastric emptying, enhancing glucose-stimulated insulin release, and decreasing insulin clearance. The same improvement in glucose tolerance is achievable by optimal timing of carbohydrate ingestion during a meal (i.e., carbohydrate-last meal patterns), which minimizes the risk of body weight gain when compared with nutrient preloads. The magnitude of the glucose-lowering effect of preload-based nutritional strategies is greater in type 2 diabetes than healthy subjects, being comparable and additive to current glucose-lowering drugs, and appears sustained over time. This dietary approach has also shown promising results in pathological conditions characterized by postprandial hyperglycemia in which available pharmacological options are limited or not cost-effective, such as type 1 diabetes, gestational diabetes, and impaired glucose tolerance. Therefore, preload-based nutritional strategies, either alone or in combination with pharmacological treatments, may offer a simple, effective, safe, and inexpensive tool for the prevention and management of postprandial hyperglycemia. Here, we survey these novel physiological insights and their therapeutic implications for patients with diabetes mellitus and altered glucose tolerance.

Enhancing pseudoislet biofunctionality using gelatin bead technology

Central necrosis hampers the formation of highly biofunctional Pseudoislets (PIs), which consist of aggregates of insulin-secreting pancreatic β-cells. Necrosis arises because of a shortage of nutrient and oxygen diffusion to the core of the PIs during culture, especially when PIs exceed >200 µm. This study aimed to generate ‘vents’ by incorporating gelatin beads (GBs) into the center of PIs and to examine if this promotes nutrient and oxygen diffusion by blocking the center for cell residence. In addition, we examined the impact of delivering GBs loaded with anti-necrosis or anti-apoptosis drugs to the center of PIs. The BRIN-BD11 rat pancreatic β-cell line was used to generate PIs by suspension culture. PIs were generated at a seeding density of 32,000 cells/PI and cultured for up to 7 days. GBs of 40 µm diameter were produced from Gelatin A and crosslinked with 5% glutaraldehyde for 6 h. The neat GBs or GBs loaded with 100 ng/mL IL-10, or 5 µg/mL anti-IL-1β were incorporated into PIs. The cell viability of the PIs was assessed using cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) assays. Glucose-stimulated insulin release (GSIS) from PIs was evaluated after stimulation with 16.7 mM glucose for 20 min. Incorporating IL-10, or anti-IL-1β -loaded GBs to PIs synergistically enhanced cell proliferation and reduced cell death. Importantly, PIs cultured for 1 week following incorporation of cytokine-loaded GBs displayed enhanced biofunctionality in terms of higher GSIS.

Advances in Human Islet Processing: Manufacturing Steps to Achieve Predictable Islet Outcomes from Research Pancreases

Background This presentation of a six-year study processing human islets for research and transplantation includes a review of multi-center transplant studies identifying key variables critical for successful islet processing and defines standardized processing procedures required to provide highly purified, functional Human Islets. Methods Human islet processing methods are defined in detail with pancreas retrieval, shipping, trimming for processing, collagenase distension, controlled digestion by digestion/filtration method, islet purification and islet culture. Islet processing results are summarized from 27 published reports (2003-2017) from 21 international clinical islet transplant centers with 13 single islet centers and 8 from multi-center clinical trials that averaged islet yields of 5,680 IEQ/Gm (Pre-Purification), 4,101 IEQ/Gm (Post-Purification), and 3,599 IEQ/Gm (Post-Culture) with 59.2% purity at time of islet transplant into the liver. Results Their results were compared to this study of 226 Non-DM (Non-Diabetes Mellitus) donor processing islet yields that averaged 6,942.7±201 IEQ/Gm (Pre-Purification), 5,484.5±199 IEQ/Gm (Post-Purification), and 4,351.0±167 IEQ/Gm (Post-Culture) per pancreas with 90.0% purity at time of islet distribution. Islet processing from 29 Type 2 Diabetes donors resulted in reduced islet yields of 5,797±734 IEQ/Gm (Pre-Purification), 4,371±866 IEQ/Gm (Post-Purification), and 3,323±423 IEQ/Gm (Post-Culture) with similar purity but reduced insulin content. Glucose Stimulated Insulin Release testing in T2D islets showed significantly reduced insulin release at 20mM and 20mM+IBMX versus Non-DM islets and showed significantly reduced Total Stimulated Insulin Release of 0.722±0.142 (T2D) versus 1.069±0.067 ng insulin/ng DNA (Non-DM). Conclusions Of the significant process variables, short Switch Times were predominant in greatly increasing islet yields, GSIR results, and insulin content with any time

Possible Insulinotropic Action of Apolipoprotein A-I Through the ABCA1/Cdc42/cAMP/PKA Pathway in MIN6 Cells.

Aims/Introduction: We studied the mechanisms for the possible insulinotropic action of apolipoprotein (Apo) A–I in mouse insulinoma (MIN6) cells.Materials and Methods: The effects of ApoA-I on cAMP production and glucose-stimulated insulin secretion (GSIS), and the dose dependency (ApoA-I at 5, 10, 25, and 50 μg/ml) were determined using MIN6 cells. The effects of the small-interference ribonucleic acid (siRNA) of ATP-binding cassette transporter A1(ABCA1) and Cell division control protein 42 homolog (Cdc42) on the insulinotropic action of ApoA-I was studied, as well as mRNA and protein levels of ABCA1 and Cdc42. Then, the influence of cAMP inhibitor SQ22536, and the cAMP-dependent protein kinase inhibitor Rp-cAMPS on ApoA-I action were studied.Results: Addition of ApoA-I produced cAMP and increased insulin secretion, dose-dependently in high glucose concentration (25 mmmol/l). and ABCA1 protein and Cdc42 mRNA and protein were also enhanced. Specific ABCA1 and Cdc42 siRNA significantly decreased the effects of ApoA-I on insulin secretion compared with negative controls. Manifestations of ABCA1 and Cdc42 mRNA and protein were less than that of the negative control group. Both cAMP inhibiror (SQ22536) and protein kinases inhibitor (Rp-cAMPS) strongly inhibited the effects of ApoA-I on insulin secretion.Conclusions: We demonstrated that ApoA-I enhances glucose-stimulated insulin release in high glucose at least partially through the ABCA1/Cdc42/cAMP/ Protein kinase A (PKA) pathway.

Characterization of glucose-stimulated insulin release protocols in african green monkeys (Chlorocebus aethiops).

BackgroundManagement of diabetes remains a major health and economic challenge, demanding test systems in which to develop new therapies. These studies assessed different methodologies for determining glucose tolerance in green monkeys.MethodsTwenty‐eight African green monkeys between 4 and 24 years old underwent single or repeat intravenous glucose tolerance testing (IVGTT), oral glucose tolerance testing (OGTT), and/or graded glucose infusion testing.ResultsGeriatric monkeys exhibited glucose intolerance with impaired glucose‐stimulated insulin secretion following IVGTT. Repeat IVGTT and OGTT assessments were inconsistent. Monkeys with low glucose‐stimulated insulin secretion after graded glucose infusion exhibited elevated blood glucose levels.Conclusion IVGTT and graded glucose infusion protocols revealed differences in glucose tolerance among green monkeys at single time points, including age‐dependent differences suggestive of shifts in pancreatic beta‐cell functional capacity, but care should be applied to study design and the interpretation of data in the setting of longitudinal studies.

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix.

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17%±7.5%) and higher postpurification islet recovery (83.8%±5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

On the effects of NLRP3 inflammasome on mice pancreatic β-cell damage induced by advanced glycation end products

Objective To explore the role of the pyrin domain-containing 3 (NLRP3) inflammasome in advanced glycation end products (AGEs)-induced mice pancreatic β-cell damage. Methods AGEs were administered intraperitoneally for 6 weeks in NLRP3 knockout mice or C57BL/6J mice. Intraperitoneal glucose tolerance test and insulin releasing test were performed. Pancreatic sections were stained with haematoxylin and eosin, or with F4/80 and NLRP3 antibodies. Insulin and pancreatic tissue monocyte chemotactic protein 1 (MCP-1) as well as interleukin-1β (IL-1β) levels were measured with ELISA kits. Expression of MCP-1 protein was determined by western blot. MIN6 cells and mouse peritoneal macrophages cells were treated with AGEs and different interventions (antioxidant NAC, adenovirus NLRP3 shRNA or NLRP3 knockout). Reactive oxygen species production, NLRP3 mRNA expression, IL-1β secretion, caspase 1 activity, apoptosis and glucose stimulated insulin release were determined. Results Injection of AGEs induced an abnormal response to glucose, enhanced the insulitis score, and increased the levels of pancreatic tissue MCP-1 and IL-1β, as well as raised the expression of NLRP3 and F4/80 in pancreatic islet. Remarkably, co-localization of NLRP3 and macrophage marker F4/80 was observed in islet. The damages were improved in NLRP3 knockout mice. After incubation with AGEs, reactive oxygen species production and cell apoptosis was enhanced, NLRP3 inflammasome activated, with glucose-stimulated insulin release impaired in MIN6 cells. NAC treatment alliviated the above damages, but NLRP3 gene silencing had no effect on ROS level, apoptosis, and insulin secretion. Finally NAC treatment and NLRP3 gene knockout inhibited activation of NLRP3 inflammasome induced by AGEs in mouse peritoneal macrophages cells. Conclusion NLRP3 knockout ameliorates the islet β-cell damage induced by AGEs. These effects were associated with AGEs-induced islets macrophage infiltrating by up-regulation of MCP-1 expression, and AGEs-induced activation of NLRP3 inflammasome in macrophage through ROS pathway, which results in the release of active IL-1β and leads to the lesions of β-cell. (Chin J Endocrinol Metab, 2018, 34: 690-695) Key words: Advanced glycation end products; NLRP3 inflammasome; β-cell damage

A Positive Allosteric Modulator Enhances the Ability of Beta Cell M3 Muscarinic Receptors to Simulate Insulin Secretion In Vitro and In Vivo

Several studies have shown that activation of M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic beta-cells leads to striking increases in glucose-stimulated insulin release. As a result, we hypothesized that agents that can promote signaling through beta-cell M3Rs might become useful in the treatment of type 2 diabetes. The M3R is a prototypic class A GPCR that is selectively coupled to G proteins of the Gq family. At present, selective M3R agonists that bind to the orthosteric ACh binding site are not available. However, the lab of Craig Lindsley (Bridges et al., 2010) recently described a compound (VU0119498) that binds to an allosteric site on the M3R, thus enhancing ACh-induced signaling through M3Rs. In this study, we tested the ability of this positive allosteric modulator (PAM) to stimulate insulin release in vitro and in vivo. The use of PAMs has the great advantage that it respects the spatio-temporal control of receptor activation, i.e., the PAM is only active upon receptor occupation by an endogenous orthosteric agonist. Studies with cultured MIN6 cells showed that VU0119498 enhanced ACh-induced insulin secretion in an M3R-dependent fashion. In vivo studies demonstrated that PAM treatment of WT mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These VU0119498 effects were mediated by beta-cell M3Rs since they were absent in mutant mice selectively lacking M3Rs in beta-cells. In future studies, we will use mouse models of diabetes to examine whether this M3R-PAM is endowed with antidiabetic activity. This approach may lead to the development of a novel generation of antidiabetic drugs. Disclosure L. Zhu: None. J. Wess: None.

Predicting Insulin Secretion Profiles for Immunoisolating Devices with Transplanted Islets

Avascular islets or stem cells transplanted under immunoisolating conditions offer considerable hope in overcoming the challenges of islet transplantation; however, because of diffusional limitations, implants face long-term viability and efficiency-of-response problems. To quantitatively characterize the glucose-stimulated insulin release of pancreatic islets, we conducted parallel dynamic perifusion experiments with free and hydrogel encapsulated islets. Perifused human islets secreted less insulin than murine islets per unit mass (IEQ) and had a less pronounced first-phase peak. The data was used to develop a computational model of insulin secretion. Our model couples hormone secretion and nutrient consumption kinetics with diffusive and convective transport, fits well the experimental data, and can be used to predict insulin release profiles for arbitrary geometries and glucose challenges. We found that larger diffusion distances (larger capsules) unavoidably dampen the first-phase insulin response, resulting in a sustained-release type insulin secretion that sluggishly responds to changes in glucose concentration. Hence, bioartificial pancreas devices can provide long-term and physiologically desirable solutions only if immunoisolation and biocompatibility considerations are considered together with optimized nutrient diffusion and insulin release characteristics. Disclosure P. Buchwald: None. A.A. Tomei: Consultant; Self; EyePharma. Research Support; Self; Semma Therapeutics, Inc.. Stock/Shareholder; Self; Converge Biotech Pvt. Ltd.. C.L. Stabler: None.

Effects of acute NEFA manipulation on incretin-induced insulin secretion in participants with and without type 2 diabetes.

Incretin effect-the potentiation of glucose-stimulated insulin release induced by the oral vs the i.v. route-is impaired in dysglycaemic states. Despite evidence from human islet studies that NEFA interfere with incretin function, little information is available about the effect in humans. We tested the impact of acute bidirectional NEFA manipulation on the incretin effect in humans. Thirteen individuals with type 2 diabetes and ten non-diabetic volunteers had a 3h OGTT, and, a week later, an i.v. isoglycaemic glucose infusion (ISO; OGTT matched). Both pairs of studies were repeated during an exogenous lipid infusion in the non-diabetic volunteers, and following acipimox administration (to inhibit lipolysis) in people with diabetes. Mathematical modelling of insulin secretion dynamics assessed total insulin secretion (TIS), beta cell glucose sensitivity (β-GS), glucose-induced potentiation (PGLU) and incretin-induced potentiation (PINCR); the oral glucose sensitivity index was used to estimate insulin sensitivity. Lipid infusion increased TIS (from 61 [interquartile range 26] to 78 [31] nmol/m2 on OGTT and from 29nmol/m2 [26] to 57nmol/m2 [30] on ISO) and induced insulin resistance. PINCR decreased from 1.6 [1.1] to 1.3 [0.1] (p < 0.05). β-GS, PGLU and glucagon, glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) responses were unaffected. Acipimox (lowering NEFA by ~55%) reduced plasma glucose and TIS and enhanced insulin sensitivity, but did not change β-GS, PINCR, PGLU or glucagon, GLP-1 or GIP responses. As the per cent difference, incretin effect was decreased in non-diabetic participants and unchanged in those with diabetes. Raising NEFA selectively impairs incretin effect and insulin sensitivity in non-diabetic individuals, while acute NEFA reduction lowers plasma glucose and enhances insulin sensitivity in people with diabetes but does not correct the impaired incretin-induced potentiation.

Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction.

Beta cell dysfunction accompanies and drives the progression of type 2 diabetes mellitus (T2D), but there are few clinical biomarkers available to assess islet cell stress in humans. Secretagogin, a protein enriched in pancreatic islets, demonstrates protective effects on beta cell function in animals. However, its potential as a circulating biomarker released from human beta cells and islets has not been studied. In this study primary human islets, beta cells and plasma samples were used to explore secretion and expression of secretagogin in relation to the T2D pathology. Secretagogin was abundantly and specifically expressed and secreted from human islets. Furthermore, T2D patients had an elevated plasma level of secretagogin compared with matched healthy controls, which was confirmed in plasma of diabetic mice transplanted with human islets. Additionally, the plasma secretagogin level of the human cohort had an inverse correlation to clinical assessments of beta cell function. To explore the mechanism of secretagogin release in vitro, human beta cells (EndoC-βH1) were exposed to elevated glucose or cellular stress-inducing agents. Secretagogin was not released in parallel with glucose stimulated insulin release, but was markedly elevated in response to endoplasmic reticulum stressors and cytokines. These findings indicate that secretagogin is a potential novel biomarker, reflecting stress and islet cell dysfunction in T2D patients.

An engineered cell sheet composed of human islets and human fibroblast, bone marrow–derived mesenchymal stem cells, or adipose–derived mesenchymal stem cells: An in vitro comparison study

ABSTRACTBackground: We previously reported the utility of engineered cell sheets composed of human islets and supporting cells in vitro and in vivo. It is unclear which type of supporting cell is most suitable for constructing cell sheets with human islets. The present study aimed to compare human fibroblasts, bone marrow–derived mesenchymal stem cells (BM–MSCs), and adipose–derived mesenchymal stem cells (ADSCs) as a supporting source for cell sheets. Methods: Engineered cell sheets were fabricated with human islets using human fibroblasts, BM–MSCs, or ADSCs as supporting cells. The islet viability, recovery rate, glucose–stimulated insulin release (determined by the stimulation index), and cytokine secretion (TGF–β1, IL–6, and VEGF) of groups—including an islet–alone group as a control-were compared. Results: All three sheet groups consistently exhibited higher viability, recovery rate, and stimulation index values than the islet-alone group. The ADSC group showed the highest viability and recovery rate among the three sheet groups. There were no discernible differences in the stimulation index values of the groups. The fibroblast group exhibited significantly higher TGF–β1 values in comparison to the other groups. The IL–6 level of the ADSC group was more than five times higher than that of the other groups. The ADSC group showed the VEGF level; however, it did not differ from that of the BM–MSC group to a statistically significant extent. Conclusion: Engineered cell sheets composed of islets and supporting cells had a cytoprotective effect on islets. These results suggest that individual cell types could be a more attractive source for crafting engineered cell sheets in comparison to islets alone.

View Insulin Resistance from an Interaction Between Pancreatic Islets and Peripheral Tissues

Curl rent hypotheses of insulin resistance mostly emphasize cellular mechanisms in peripheral tissues. Although received broad recognition, these opinions may have overlooked the interaction between pancreatic endocrine cells and the peripheral tissues in the process of establishment and maintenance of insulin resistance in the whole body. It has been suggested that basal hyperinsulinemia is the root cause of insulin resistance. Basal insulin release does not share the same intracellular mechanism of high glucose stimulated insulin release; instead, it is regulated by local Ca2+ fluctuation and activation of the cAMP-Epac2/Rap1 signaling pathway. Basal insulin release is controlled by the interaction between pancreatic head β-cells and pancreatic tail α-cells, which release insulin and glucagon, respectively. In diabetes, an elevated basal insulin level would mitigate the sensitivity of peripheral tissues to insulin; the decreased insulin sensitivity and elevated plasma glucose concentration could further stimulate more basal insulin release partially by increasing T-type Ca2+ channel expression and activity in β-cells. This interaction forms a positive feedback loop. Therefore, T-type Ca2+ channel antagonists can potentially be employed to break this positive feedback loop, thus reversing insulin resistance.

Activation of ACE2/angiotensin (1–7) attenuates pancreatic β cell dedifferentiation in a high-fat-diet mouse model

ObjectiveAngiotensin-converting enzyme 2 (ACE2) has been identified in pancreatic islets and can preserve β cells. In this study, we aimed to examine the possible role of ACE2 and its end product, angiotensin 1–7 (A1–7), in reducing β cell dedifferentiation during metabolic stress. MethodsFirst, a lineage-tracing experiment was performed to track β cells in mice fed a high-fat diet (HFD). Second, the ACE2/A1–7 axis was evaluated in the HFD mouse model. Intraperitoneal glucose tolerance tests (IPGTTs) and intraperitoneal insulin tolerance tests (IPITTs) were conducted. Phenotypic changes in β cells were detected by immunohistochemistry and quantitative real-time PCR. Pancreatic sections were immunostained for vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Finally, the effects of the ACE2/A1–7 axis were explored in isolated mouse islets exposed to different concentrations of glucose. Glucose-stimulated insulin release and levels of insulin mRNA and OCT4 mRNA were measured. ResultsPancreatic β cell dedifferentiation occurred both in vitro and in vivo in response to metabolic stress and was accompanied by ACE2 reduction. HFD-induced insulin resistance and glucose intolerance were exacerbated in ACE2-knockout (ACE2KO) mice but were alleviated by exogenous A1–7 in C57BL/6J mice. Approximately 20% of β cells were dedifferentiated in ACE2KO mice fed a standard rodent chow diet (SD). A higher percentage of dedifferentiated β cells was detected in ACE2KO mice than in wild-type (WT) mice under HFD conditions. In contrast, the administration of A1–7 alleviated HFD-induced β cell dedifferentiation in C57BL/6J mice. Moreover, the exogenous injection of A1–7 improved microcirculation in islets and decreased the production of iNOS in islets of C57BL/6J mice fed an HFD. Additionally, ACE2 was found to be mainly expressed in α cells of mice, while Mas, the receptor of A1–7, was distributed in β cells. ConclusionsOverall, this study is the first to demonstrate that the ACE2/A1–7/Mas axis may be one of the intra-islet paracrine mechanisms of communication between α and β cells. Enhancing the ACE2/A1–7 axis exerts a protective effect by ameliorating β cell dedifferentiation, and this effect might be partially mediated through improvements in islet microcirculation and suppression of islet iNOS.