The inhibitory G protein alpha subunit, Gαz, is an important modulator of beta-cell function. Full-body Gαz-null mice are protected from hyperglycemia and glucose intolerance after long-term high-fat diet (HFD) feeding. In this study, at a time point in the feeding regimen where wild-type mice are only mildly glucose intolerant, transcriptomics analyses reveal islets from HFD-fed Gαz KO mice have a dramatically altered gene expression pattern as compared to WT HFD-fed mice, with entire gene pathways not only being more strongly up- or down-regulated vs. control-diet fed groups, but actually reversed in direction. Genes involved in the “Pancreatic Secretion” pathway are the most strongly differentially regulated: a finding that correlates with enhanced islet insulin secretion and decreased glucagon secretion at study end. The protection of Gαz-null mice from HFD-induced diabetes is β-cell autonomous, as β-cell-specific Gαz-null (βKO) mice phenocopy the full-body knockouts. The glucose-stimulated and incretin-potentiated insulin secretion response of islets from HFD-fed βKO mice is significantly improved as compared to islets from HFD-fed wild-type controls, which, along with no impact of Gαz loss or HFD feeding on beta-cell proliferation or surrogates of beta-cell mass supports a secretion-specific mechanism. Gαz is coupled to the Prostaglandin EP3 receptor in pancreatic beta-cells. We confirm the EP3γ splice variant has both constitutive and agonist-sensitive activity to inhibit cyclic AMP production and downstream β-cell function, with both activities being dependent on the presence of beta-cell Gαz.
Elevated islet production of prostaglandin E2 (PGE2), an arachidonic acid metabolite, and expression of prostaglandin E2 receptor subtype EP3 (EP3) are well-known contributors to the β-cell dysfunction of type 2 diabetes (T2D). Yet, many of the same pathophysiological conditions exist in obesity, and little is known about how the PGE2 production and signaling pathway influences nondiabetic β-cell function. In this work, plasma arachidonic acid and PGE2 metabolite levels were quantified in a cohort of nondiabetic and T2D human subjects to identify their relationship with glycemic control, obesity, and systemic inflammation. In order to link these findings to processes happening at the islet level, cadaveric human islets were subject to gene expression and functional assays. Interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) mRNA levels, but not those of EP3, positively correlated with donor body mass index (BMI). IL-6 expression also strongly correlated with the expression of COX-2 and other PGE2 synthetic pathway genes. Insulin secretion assays using an EP3-specific antagonist confirmed functionally relevant upregulation of PGE2 production. Yet, islets from obese donors were not dysfunctional, secreting just as much insulin in basal and stimulatory conditions as those from nonobese donors as a percent of content. Islet insulin content, on the other hand, was increased with both donor BMI and islet COX-2 expression, while EP3 expression was unaffected. We conclude that upregulated islet PGE2 production may be part of the β-cell adaption response to obesity and insulin resistance that only becomes dysfunctional when both ligand and receptor are highly expressed in T2D.
Objective: Signaling through Prostaglandin E3 Receptor (EP3), a G protein-coupled receptor for E series prostaglandins such as prostaglandin E2 (PGE2), has been linked to the beta-cell dysfunction and loss of beta-cell mass in type 2 diabetes (T2D). In the beta-cell, EP3 is specifically coupled to the unique cAMP-inhibitory G protein, Gz. Divergent effects of EP3 agonists and antagonists or Gαz loss on beta-cell function, replication, and survival depending on whether islets are isolated from mice or humans in the lean and healthy, type 1 diabetic, or T2D state suggest a divergence in biological effects downstream of EP3/Gαz dependent on the physiological milieu in which the islets reside. Methods: We determined the expression of a number of genes in the EP3/Gαz signaling pathway; PGE2 production pathway; and the beta-cell metabolic, proliferative, and survival responses to insulin resistance and its corresponding metabolic and inflammatory derangements in a panel of 80 islet preparations from non-diabetic human organ donors spanning a BMI range of approximately 20-45. In a subset of islet preparations, we also performed glucose-stimulated insulin secretion assays with and without the addition of an EP3 agonist, L798,106, and a glucagon-like peptide 1 receptor agonist, exendin-4, allowing us to compare the gene expression profile of each islet preparation with its (1) total islet insulin content (2), functional responses to glucose and incretin hormones, and (3) intrinsic influence of endogenous EP3 signaling in regulating these functional responses. We also transduced two independent islet preparations from three human organ donors with adenoviruses encoding human Gαz or a GFP control in order to determine the impact of Gαz hyperactivity (a mimic of the T2D state) on human islet insulin content and functional response to glucose. Results: In contrast to results from islets isolated from T2D mice and human organ donors, where PGE2-mediated EP3 signaling actively contributes to beta-cell dysfunction, PGE2 production and EP3 expression appeared positively associated with various measurements of functional beta-cell compensation. While Gαz mRNA expression was negatively associated with islet insulin content, that of each of the Gαz-sensitive adenylate cyclase (AC) isoforms were positively associated with BMI and cyclin A1 mRNA expression, suggesting increased expression of AC1, AC5, and AC6 is a compensatory mechanism to augment beta-cell mass. Human islets over-expressing Gαz via adenoviral transduction had reduced islet insulin content and secretion of insulin in response to stimulatory glucose as a percent of content, consistent with the effects of hyperactivation of Gαz by PGE2/EP3 signaling observed in islets exposed to the T2D physiological milieu. Conclusions: Our work sheds light on critical mechanisms in the human beta-cell compensatory response, before the progression to frank T2D.
Cyclic‐adenosine monophosphate (cAMP) is an important secondary messenger in the insulin‐secreting pancreatic β‐cells. Intracellular production of cAMP is augmented by stimulatory heterotrimeric G proteins (Gs) and blocked by inhibitory heterotrimeric G proteins (Gi). Pancreatic expression of the unique Gi family member, Gαz, is restricted to the islet, which contains β‐cells and other endocrine cell types, including glucagon/GLP‐1‐secreting alpha cells and somatostatin‐secreting delta cells. We have previously shown that full body Gαz‐null mice are protected from hyperglycemia in several diabetes model systems. Here, we use a high fat diet (HFD) model of obesity and insulin resistance to explore the impact of loss of beta‐cell Gαz on the type 2 diabetes (T2D) phenotype of male C57BL/6J mice. We hypothesized that loss of beta‐cell Gαz in the context of HFD feeding would enhance insulin secretion, protecting mice from HFD‐induced glucose intolerance. To generate beta‐cell specific Gαz‐null and control mice, we bred Gαz flox/flox mice with rat insulin promoter RIP‐Creherr mice. At 12 weeks of age, transgenic Gαz‐null mice were fed a diet containing 45 kcal% fat or a 10 kcal% fat control diet for 26 weeks. At study end, mice were metabolically phenotyped and sacrificed for whole pancreas and islet phenotyping. Beta‐cell‐specific Gαz loss protected mice from HFD induced glucose intolerance due to a selective effect on islet insulin secretion, particularly in concert with a stable agonist of the Gs‐coupled GLP‐1 receptor. This protective effect was independent of changes in Gαz‐coupled EP3 receptor signaling, as neither EP3 expression nor production of its endogenous ligand, PGE2, were enhanced in islets from HFD‐fed mice. This protective effect was also beta‐cell autonomous, as both full‐body and beta‐cell‐specific Gαz‐null mice had enhanced GLP‐1‐positive alpha‐cells, albeit with no changes in active GLP‐1 secretion. Finally, gene microarray results confirm that Gαz loss and HFD feeding synergize to differentially regulate islet gene expression, specifically up‐regulating secretion‐centric genes, many of which are related to cAMP signaling. This study presents a novel inhibitory mechanism of receptor‐independent G‐protein signaling that may hold promise for developing beta‐cell targeted diabetes therapeutics.
Of the β-cell signaling pathways altered by obesity and insulin resistance, some are adaptive while others contribute to β-cell failure. Two critical second messengers are Ca 2+ and cAMP, which control the timing and amplitude of insulin secretion. Previous work has shown the importance of the cAMP-inhibitory Prostaglandin EP3 receptor (EP3) in mediating the β-cell dysfunction of type 2 diabetes (T2D). Here, we used three groups of C57BL/6J mice as a model of the progression from metabolic health to T2D: wildtype, normoglycemic Leptin Ob (NGOB), and hyperglycemic Leptin Ob (HGOB). Robust increases in β-cell cAMP and insulin secretion were observed in NGOB islets as compared to wildtype controls; an effect lost in HGOB islets, which exhibited reduced β-cell cAMP and insulin secretion despite increased glucose-dependent Ca 2+ influx. An EP3 antagonist had no effect on β-cell cAMP or Ca 2+ oscillations, demonstrating agonist-independent EP3 signaling. Finally, using sulprostone to hyperactivate EP3 signaling, we found EP3-dependent suppression of β-cell cAMP and Ca 2+ duty cycle effectively reduces insulin secretion in HGOB islets, while having no impact insulin secretion on NGOB islets, despite similar and robust effects on cAMP levels and Ca 2+ duty cycle. Finally, increased cAMP levels in NGOB islets are consistent with increased recruitment of the small G protein, Rap1GAP, to the plasma membrane, sequestering the EP3 effector, Gɑ z , from inhibition of adenylyl cyclase. Taken together, these results suggest that rewiring of EP3 receptor-dependent cAMP signaling contributes to the progressive changes in β cell function observed in the Leptin Ob model of diabetes.
Straipsnyje nagrinėjama itin aktuali suaugusiųjų švietimo problema. Autorius kelia klausimus: kokia suaugusiųjų švietimo vieta šiandienėje edukologijoje ir kokį vaidmenį suaugusiųjų mokymasis visą gyvenimą atlieka postmoderniame pasaulyje. Vertinga tai, kad šiuo aspektu autorius pateikia skirtingų tyrėjų požiūrį. Kartu panaudodamas lyginamosios analizės metodą iš dalies paliečia ir Lietuvą – jos geografinį ir socialinį-ekonominį kontekstą, turintį ryšį su dabarties ir ateities visuomene bei jos plėtros prognoze. Todėl čia pateikiamos autoriaus įžvalgos gali būti naudingos plačiam skaitytojų ratui.
We and others previously reported that increased signaling through the Prostaglandin E3 Receptor (EP3), a G protein-coupled receptor (GPCR) for the arachidonic acid metabolite, prostaglandin E2 (PGE2), is associated with β-cell dysfunction of type 2 diabetes (T2D). Yet, the relationship between PGE2 production and signaling and β-cell function during the progression to T2D remains unclear. In this work, we assessed gene expression from a panel of cadaveric human islets from 40 non-diabetic donors with BMI values spanning the spectrum from lean to high-risk obesity. Interleukin-6 (gene symbol: IL6) and cyclooxygenase-2 (COX-2) (gene symbol: PTGS2) mRNA levels were positively correlated with donor body mass index (BMI), while EP3 (gene symbol: PTGER3) was not. IL6 was itself strongly correlated with PTGS2 and all but one of the other PGE2 synthetic pathway genes tested. About half of the islet preparations were used in glucose-stimulated- and incretin-potentiated insulin secretion assays using an EP3-specific antagonist, confirming functionally-relevant up-regulation of PGE2 production. Islets from obese donors showed no inherent β-cell dysfunction and were at least equally as glucose- and incretin-responsive as islets from non-obese donors. Furthermore, insulin content, a marker of islet size known to be associated with donor BMI, was also significantly and positively correlated with islet PTGS2 expression. We conclude up-regulated islet PGE2 production and signaling may be a necessary part of the β-cell adaption response, compensating for obesity and insulin resistance. Analysis of plasma PGE2 metabolite levels from a clinical cohort reveal these findings are not in conflict with the concept of further elevations in PGE2 production contributing to T2D-related β-cell dysfunction where islet EP3 expression has also been up-regulated.
Of the β-cell signaling pathways altered by non-diabetic obesity and insulin resistance, some are adaptive while others actively contribute to β-cell failure and demise. Cytoplasmic calcium (Ca2+) and cyclic AMP (cAMP), which control the timing and amplitude of insulin secretion, are two important signaling intermediates that can be controlled by stimulatory and inhibitory G protein-coupled receptors. Previous work has shown the importance of the cAMP-inhibitory EP3 receptor in the beta-cell dysfunction of type 2 diabetes. To examine alterations in β-cell cAMP during diabetes progression we utilized a β-cell specific cAMP biosensor in tandem with islet Ca2+ recordings and insulin secretion assays. Three groups of C57BL/6J mice were used as a model of the progression from metabolic health to type 2 diabetes: wildtype, normoglycemic LeptinOb, and hyperglycemic LeptinOb. Here, we report robust increases in β-cell cAMP and insulin secretion responses in normoglycemic Leptinob mice as compared to wild-type: an effect that was lost in islets from hyperglycemic Leptinob mice, despite elevated Ca2+ duty cycle. Yet, the correlation of EP3 expression and activity to reduce cAMP levels and Ca2+ duty cycle with reduced insulin secretion only held true in hyperglycemic LeptinOb mice. Our results suggest alterations in beta-cell EP3 signaling may be both adaptive and maladaptive and define β-cell EP3 signaling as much more nuanced than previously understood.
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