Intra-islet GLP-1, but not CCK, is necessary for β-cell function in mouse and human islets.
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ABSTRACT:
SUBMITTER: de Souza AH
PROVIDER: S-EPMC7028949 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Type 1 and type 2 diabetes result from a deficit in insulin production and beta-cell mass. Methods to expand beta-cell mass are under intensive investigation for the treatment of type 1 and type 2 diabetes. We tested the hypothesis that cholecystokinin (CCK) can promote beta-cell proliferation. We treated isolated mouse and human islets with an adenovirus containing the CCK cDNA (AdCMV-CCK). We measured [(3)H]thymidine and BrdU incorporation into DNA and additionally, performed flow cytometry analysis to determine whether CCK overexpression stimulates beta-cell proliferation. We studied islet function by measuring glucose-stimulated insulin secretion and investigated the cell cycle regulation of proliferating beta-cells by quantitative RT-PCR and Western blot analysis. Overexpression of CCK stimulated [(3)H]thymidine incorporation into DNA 5.0-fold and 15.8-fold in mouse and human islets, respectively. AdCMV-CCK treatment also stimulated BrdU incorporation into DNA 10-fold and 21-fold in mouse and human beta-cells, respectively. Glucose-stimulated insulin secretion was unaffected by CCK expression. Analysis of cyclin and cdk mRNA and protein abundance revealed that CCK overexpression increased cyclin A, cyclin B, cyclin E, cdk1, and cdk2 with no change in cyclin D1, cyclin D2, cyclin D3, cdk4, or cdk6 in mouse and human islets. Additionally, AdCMV-CCK treatment of CCK receptor knockout and wild-type mice resulted in equal [(3)H]thymidine incorporation. CCK is a beta-cell proliferative factor that is effective in both mouse and human islets. CCK triggers beta-cell proliferation without disrupting islet function, up-regulates a distinct set of cell cycle regulators in islets, and signals independently of the CCK receptors.
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Project description:Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that regulates cellular stress responses. While the levels of HIF-1alpha protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1alpha is required for normal beta cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Here we show that HIF-1alpha protein is present at low levels in mouse and human normoxic beta cells and islets. Decreased levels of HIF-1alpha impaired glucose-stimulated ATP generation and beta cell function. C57BL/6 mice with beta cell-specific Hif1a disruption (referred to herein as beta-Hif1a-null mice) exhibited glucose intolerance, beta cell dysfunction, and developed severe glucose intolerance on a high-fat diet. Increasing HIF-1alpha levels by inhibiting its degradation through iron chelation markedly improved insulin secretion and glucose tolerance in control mice fed a high-fat diet but not in beta-Hif1a-null mice. Increasing HIF-1alpha levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. Further analysis indicated that HIF-1alpha was bound to the Arnt promoter in a mouse beta cell line, suggesting direct regulation. Taken together, these findings suggest an important role for HIF-1alpha in beta cell reserve and regulation of ARNT expression and demonstrate that HIF-1alpha is a potential therapeutic target for the beta cell dysfunction of T2D.
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Project description:Glucagon-like peptide-1 (GLP-1) shows robust protective effects on β-cell survival and function and GLP-1 based therapies are successfully applied for type-2 diabetes (T2D) and obesity. Another cleavage product of pro-glucagon, Glucagon-like peptide-2 (GLP-2; both GLP-1 and GLP-2 are inactivated by DPP-4) has received little attention in its action inside pancreatic islets. In this study, we investigated GLP-2 production, GLP-2 receptor (GLP-2R) expression and the effect of GLP-2R activation in human islets. Isolated human islets from non-diabetic donors were exposed to diabetogenic conditions: high glucose, palmitate, cytokine mix (IL-1β/IFN-γ) or Lipopolysaccharide (LPS) in the presence or absence of the DPP4-inhibitor linagliptin, the TLR4 inhibitor TAK-242, the GLP-2R agonist teduglutide and/or its antagonist GLP-2(3-33). Human islets under control conditions secreted active GLP-2 (full-length, non-cleaved by DPP4) into the culture media, which was increased by combined high glucose/palmitate, the cytokine mix and LPS and highly potentiated by linagliptin. Low but reproducible GLP-2R mRNA expression was found in all analyzed human islet isolations from 10 donors, which was reduced by pro-inflammatory stimuli: the cytokine mix and LPS. GLP-2R activation by teduglutide neither affected acute or glucose stimulated insulin secretion nor insulin content. Also, teduglutide had no effect on high glucose/palmitate- or LPS-induced dysfunction in cultured human islets but dampened LPS-induced macrophage-dependent IL1B and IL10 expression, while its antagonist GLP-2(3-33) abolished such reduction. In contrast, the expression of islet macrophage-independent cytokines IL6, IL8 and TNF was not affected by teduglutide. Medium conditioned by teduglutide-exposed human islets attenuated M1-like polarization of human monocyte-derived macrophages, evidenced by a lower mRNA expression of pro-inflammatory cytokines, compared to vehicle treated islets, and a reduced production of itaconate and succinate, marker metabolites of pro-inflammatory macrophages. Our results reveal intra-islet production of GLP-2 and GLP-2R expression in human islets. Despite no impact on β-cell function, local GLP-2R activation reduced islet inflammation which might be mediated by a crosstalk between endocrine cells and macrophages.
| S-EPMC8287580 | biostudies-literature
Project description:Aims/hypothesisOur understanding of the transcription factors that control the development and function of rodent islet beta cells is advancing rapidly, yet less is known of the role they play in similar processes in human islets.MethodsTo characterise the abundance and regulation of key proteins involved in glucose-regulated insulin secretion in human islets, we examined the expression of MAFA, MAFB, GLUT2 (also known as SLC2A2), ?GK (also known as GCK) and PDX1 in isolated, highly purified human islets with an intact insulin secretory pattern. We also assessed these features in islets from two different mouse strains (C57BL/6J and FVB).ResultsCompared with mouse islets, human islets secreted more insulin at baseline glucose (5.6 mmol/l), but less upon stimulation with high glucose (16.7 mmol/l) or high glucose plus 3-isobutyl-1-methyl-xanthine. Human islets had relatively more MAFB than PDX1 mRNA, while mouse islets had relatively more Pdx1 than Mafb mRNA. However, v-maf musculoaponeurotic fibrosarcoma oncogene homologue (MAF) B protein was found in human islet alpha and beta cells. This is unusual as this regulator is only produced in islet alpha cells in adult mice. The expression of insulin, MAFA, ?GK and PDX1 was not glucose-regulated in human islets with an intact insulin secretory pattern.Conclusions/interpretationOur results suggest that human islets have a distinctive distribution and function of key regulators of the glucose-stimulated insulin secretion pathway, emphasising the urgent need to understand the processes that regulate human islet beta cell function.
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Project description:Whereas it is believed that the pancreatic duct contains endocrine precursors, the presence of insulin progenitor cells residing in islets remain controversial. We tested whether pancreatic islets of adult mice contain precursor beta-cells that initiate insulin synthesis during aging and after islet injury. We used bigenic mice in which the activation of an inducible form of Cre recombinase by a one-time pulse of tamoxifen results in the permanent expression of a floxed human placental alkaline phosphatase (PLAP) gene in 30% of pancreatic beta-cells. If islets contain PLAP(-) precursor cells that differentiate into beta-cells (PLAP(-)IN(+)), a decrease in the percentage of PLAP(+)IN(+) cells per total number of IN(+) cells would occur. Conversely, if islets contain PLAP(+)IN(-) precursors that initiate synthesis of insulin, the percentage of PLAP(+)IN(+) cells would increase. Confocal microscope analysis revealed that the percentage of PLAP(+)IN(+) cells in islets increased from 30 to 45% at 6 months and to 60% at 12 months. The augmentation in the level of PLAP in islets with time was confirmed by real-time PCR. Our studies also demonstrate that the percentage of PLAP(+)IN(+) cells in islets increased after islet injury and identified putative precursors in islets. We postulate that PLAP(+)IN(-) precursors differentiate into insulin-positive cells that participate in a slow renewal of the beta-cell mass during aging and replenish beta-cells eliminated by injury.
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Project description:Glucagon-like peptide-1 (GLP-1), an incretin hormone plays an important role in regulating glucose homeostasis. The therapeutic use of native GLP-1 is inadequate due to its short in vivo half-life. We recently developed a novel GLP-1 mimetics supaglutide, and demonstrated that this formulation retained native GLP-1 biological activities and possessed long-lasting GLP-1 actions. In this study, we further examined its abilities in regulating blood glucose in diabetic mice. We found that supaglutide stimulated insulin secretion in both mouse and human islets in a dose-dependent fashion. Oral glucose tolerance test conducted in normal ICR mice showed that supaglutide significantly decreased postprandial glucose excursions in a dose-dependent fashion. In type 2 diabetic db/db mice, a single-dose injection of supaglutide significantly decreased blood glucose levels, and this efficacy was lasted for at least 72 h in a dose-dependent fashion. During a 4-weeks intervention course supaglutide (twice injections per week) dose-dependently and significantly decreased fasting and random blood glucose levels in hyperglycemic db/db mice. Supaglutide, at a dose of 1.2 mg/kg, significantly reduced serum fructosamine levels. This was associated with significant enlargement of beta-cell mass, increased pancreatic insulin content, and increased plasma insulin level. Notably, during the intervention course supaglutide significantly reduced body-weight gain in these obese diabetic mice, associated with reduced fat mass (but not the lean mass), improved lipid profile, i.e., declined serum triglyceride, and free fatty acid levels compared to the placebo control. These finding reveals that supaglutide exerts beneficial effects in regulating blood glucose and lipid homeostasis in diabetic db/db mice.
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Project description:Insulin resistance is the major risk factor for Type 2 diabetes (T2D). In vulnerable individuals, insulin resistance induces a progressive loss of insulin secretion with islet pathology revealing a partial deficit of beta cells and islet amyloid derived from islet amyloid polypeptide (IAPP). IAPP is co-expressed and secreted with insulin by beta cells, expression of both proteins being upregulated in response to insulin resistance. If IAPP expression exceeds the threshold for clearance of misfolded proteins, beta cell failure occurs exacerbated by the action of IAPP toxicity to compromise the autophagy lysosomal pathway. We postulated that suppression of IAPP expression by an IAPP antisense oligonucleotide delivered to beta cells by the GLP-1 agonist exenatide (eGLP1-IAPP-ASO) is a potential disease modifying therapy for T2D. While eGLP1-IAPP-ASO suppressed mouse IAPP and transgenic human IAPP expression in mouse islets, it had no discernable effects on IAPP expression in human islets under the conditions studied. Suppression of transgenic human IAPP expression in mouse islets attenuated disruption of the autophagy lysosomal pathway in beta cells, supporting the potential of this strategy.
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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.
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Project description:Loss of functional pancreatic β-cell mass and increased β-cell apoptosis are fundamental to the pathophysiology of type 1 and type 2 diabetes. Pancreatic islet transplantation has the potential to cure type 1 diabetes but is often ineffective due to the death of the islet graft within the first few years after transplant. Therapeutic strategies to directly target pancreatic β-cell survival are needed to prevent and treat diabetes and to improve islet transplant outcomes. Reducing β-cell apoptosis is also a therapeutic strategy for type 2 diabetes. Cholecystokinin (CCK) is a peptide hormone typically produced in the gut after food intake, with positive effects on obesity and glucose metabolism in mouse models and human subjects. We have previously shown that pancreatic islets also produce CCK. The production of CCK within the islet promotes β-cell survival in rodent models of diabetes and aging. We demonstrate a direct effect of CCK to reduce cytokine-mediated apoptosis in a β-cell line and in isolated mouse islets in a receptor-dependent manner. However, whether CCK can protect human β-cells was previously unknown. Here, we report that CCK can also reduce cytokine-mediated apoptosis in isolated human islets and CCK treatment in vivo decreases β-cell apoptosis in human islets transplanted into the kidney capsule of diabetic NOD/SCID mice. Collectively, these data identify CCK as a novel therapy that can directly promote β-cell survival in human islets and has therapeutic potential to preserve β-cell mass in diabetes and as an adjunct therapy after transplant.
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Project description:During intra-portal pancreatic islet transplantation (PITx), innate immune reactions such as the instant blood mediated inflammatory reaction (IBMIR) cause an immediate loss of islets. The non-hematopoietic erythropoietin analogue cibinetide has previously shown islet-protective effects in mouse PITx. Herein, we aimed to confirm cibinetide's efficacy on human islets, and to characterize its effect on IBMIR. We cultured human islets with pro-inflammatory cytokines for 18 hours with or without cibinetide. ATP content and caspase 3/7 activity were measured. Dynamic glucose perfusion assay was used to evaluate islet function. To evaluate cibinetides effect on IBMIR, human islets were incubated in heparinized polyvinyl chloride tubing system with ABO compatible blood and rotated for 60 minutes to mimic the portal vein system. Moreover, human islets were transplanted into athymic mice livers via the portal vein with or without perioperative cibinetide treatment. The mice were sacrificed six days following transplantation and the livers were analyzed for human insulin and serum for human C-peptide levels. Histological examination of recipient livers to evaluate islet graft infiltration by CD11b+ cells was performed. Our results show that cibinetide maintained human islet ATP levels and reduced the caspase 3/7 activity during culture with pro-inflammatory cytokines and improved their insulin secreting capacity. In the PVC loop system, administration of cibinetide reduced the IBMIR-induced platelet consumption. In human islet to athymic mice PITx, cibinetide treatment showed an increased amount of human insulin in the livers and higher serum human C-peptide, while histological examination of the livers showed reduced infiltration of pro-inflammatory CD11b+ cells around islets grafts compared to the controls. In summary, Cibinetide protected isolated human islets in a pro-inflammatory milieu and reduced IBMIR related platelet consumption. It improved engraftment of human islets in athymic mice. The study confirms that cibinetide is a promising agent to be used in clinical PITx.
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