Agonist-independent Gαz activity negatively regulates beta-cell compensation in a diet-induced obesity model of type 2 diabetes
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.015585
ISSN1083-351X
AutoresMichael D. Schaid, Cara L. Green, Darby Peter, Shannon J. Gallagher, Erin Guthery, Kathryn A. Carbajal, Jeffrey M. Harrington, Grant M. Kelly, Austin Reuter, Molly L. Wehner, Allison L. Brill, Joshua C. Neuman, Dudley W. Lamming, Michelle E. Kimple,
Tópico(s)Diabetes and associated disorders
ResumoThe 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 WT 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 with WT HFD-fed mice, with entire gene pathways not only being more strongly upregulated or downregulated versus 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 the study end. The protection of Gαz-null mice from HFD-induced diabetes is beta-cell autonomous, as beta cell–specific Gαz-null mice phenocopy the full-body KOs. The glucose-stimulated and incretin-potentiated insulin secretion response of islets from HFD-fed beta cell–specific Gαz-null mice is significantly improved as compared with islets from HFD-fed WT 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 cAMP production and downstream beta-cell function, with both activities being dependent on the presence of beta-cell Gαz. 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 WT 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 with WT HFD-fed mice, with entire gene pathways not only being more strongly upregulated or downregulated versus 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 the study end. The protection of Gαz-null mice from HFD-induced diabetes is beta-cell autonomous, as beta cell–specific Gαz-null mice phenocopy the full-body KOs. The glucose-stimulated and incretin-potentiated insulin secretion response of islets from HFD-fed beta cell–specific Gαz-null mice is significantly improved as compared with islets from HFD-fed WT 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 cAMP production and downstream beta-cell function, with both activities being dependent on the presence of beta-cell Gαz. A crucial factor in the development of type 2 diabetes (T2D) is failure of beta cells to adapt their secretory phenotype and/or mount a compensatory increase in mass in the face of peripheral insulin resistance (1Alejandro E.U. Gregg B. Blandino-Rosano M. Cras-Meneur C. Bernal-Mizrachi E. Natural history of beta-cell adaptation and failure in type 2 diabetes.Mol. Aspects Med. 2015; 42: 19-41Crossref PubMed Scopus (113) Google Scholar). In the beta cell, cAMP is a critical second messenger essential for proper beta-cell function (2Tengholm A. Gylfe E. cAMP signalling in insulin and glucagon secretion.Diabetes Obes. Metab. 2017; 19 Suppl 1: 42-53Crossref PubMed Scopus (62) Google Scholar). Synthesis of cAMP is primarily regulated by G protein–coupled receptors (GPCRs) and their associated heterotrimeric G protein complex. GPCR action and cAMP signaling are essential in glucose-stimulated insulin secretion (GSIS) response and ability of the beta cell to compensate under stress (2Tengholm A. Gylfe E. cAMP signalling in insulin and glucagon secretion.Diabetes Obes. Metab. 2017; 19 Suppl 1: 42-53Crossref PubMed Scopus (62) Google Scholar). A variety of T2D therapeutics bind to and activate the glucagon-like peptide 1 receptor (GLP1R), which acts through Gs-subfamily proteins to increase the catalytic activity of adenylyl cyclase and downstream cAMP production (3Skugor M. Medical treatment of diabetes mellitus.Cleve Clin. J. Med. 2017; 84: S57-S61Crossref PubMed Scopus (3) Google Scholar). Conversely, inhibition of adenylyl cyclase by G proteins of the Gi/o subfamily reduces cAMP levels and negatively regulates beta-cell function (4Kimple M.E. Neuman J.C. Linnemann A.K. Casey P.J. Inhibitory G proteins and their receptors: emerging therapeutic targets for obesity and diabetes.Exp. Mol. Med. 2014; 46: e102Crossref PubMed Scopus (32) Google Scholar). Yet, no T2D drug that targets inhibitory GPCRs has ever been identified for widespread clinical use. We have previously demonstrated a role for the alpha subunit of the unique Gi/o subfamily member, Gz (G protein alpha-subunit [Gαz]), in beta-cell biology (5Brill A.L. Wisinski J.A. Cadena M.T. Thompson M.F. Fenske R.J. Brar H.K. Schaid M.D. Pasker R.L. Kimple M.E. Synergy between galphaz deficiency and GLP-1 analog treatment in preserving functional beta-cell mass in experimental diabetes.Mol. Endocrinol. 2016; 30: 543-556Crossref PubMed Scopus (14) Google Scholar, 6Fenske R.J. Cadena M.T. Harenda Q.E. Wienkes H.N. Carbajal K. Schaid M.D. Laundre E. Brill A.L. Truchan N.A. Brar H. Wisinski J. Cai J. Graham T.E. Engin F. Kimple M.E. The inhibitory G protein alpha-subunit, galphaz, promotes type 1 diabetes-like pathophysiology in NOD mice.Endocrinology. 2017; 158: 1645-1658Crossref PubMed Scopus (7) Google Scholar, 7Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Galphaz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 9Kimple M.E. Nixon A.B. Kelly P. Bailey C.L. Young K.H. Fields T.A. Casey P.J. A role for G(z) in pancreatic islet beta-cell biology.J. Biol. Chem. 2005; 280: 31708-31713Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Islets from transgenic KO mice lacking Gαz throughout their body have elevated pancreatic islet cAMP levels and secrete more insulin in response to glucose, accelerating glucose clearance (7Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Galphaz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). C57BL/6N (B6N) mice subjected to extended high-fat diet (HFD) feeding become morbidly obese and insulin resistant, with fasting hyperglycemia and glucose intolerance mimicking T2D. Loss of Gαz throughout the body is sufficient to protect from these aspects the phenotype, even in the face of obesity and insulin resistance mirroring WT HFD controls (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Coupled with the extremely limited tissue distribution of Gαz protein (found only in the brain, platelets, retina, and pancreatic islets (10Kimple M.E. Manning D. G protein alpha z. UCSD Nature Molecule Pages.1999https://doi.org/10.1038/mp.a000011.01Crossref Google Scholar, 11Kimple M.E. Hultman R.C. Casey P.J. Signaling through Gz.in: Bradshaw R.A. Dennis E. Handbook of Cell Signaling. 2nd Ed. Cambridge, MA2009: 1649-1653Google Scholar)), this phenotype fits best with a beta cell–centric model. Previous work from our laboratory has implicated prostaglandin EP3 receptor (EP3), whose most abundant native ligand is the arachidonic acid metabolite, prostaglandin E2 (PGE2), in mediating the inhibitory effects of Gαz on islet cAMP production and GSIS (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 9Kimple M.E. Nixon A.B. Kelly P. Bailey C.L. Young K.H. Fields T.A. Casey P.J. A role for G(z) in pancreatic islet beta-cell biology.J. Biol. Chem. 2005; 280: 31708-31713Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Yet, the primary effects of EP3 on beta-cell biology have been suggested to be on proliferation, not function (12Carboneau B.A. Allan J.A. Townsend S.E. Kimple M.E. Breyer R.M. Gannon M. Opposing effects of prostaglandin E2 receptors EP3 and EP4 on mouse and human beta-cell survival and proliferation.Mol. Metab. 2017; 6: 548-559Crossref PubMed Scopus (24) Google Scholar, 13Ceddia R.P. Lee D. Maulis M.F. Carboneau B.A. Threadgill D.W. Poffenberger G. Milne G. Boyd K.L. Powers A.C. McGuinness O.P. Gannon M. Breyer R.M. The PGE2 EP3 receptor regulates diet-induced adiposity in male mice.Endocrinology. 2016; 157: 220-232Crossref PubMed Scopus (34) Google Scholar). Furthermore, extra-islet EP3 signaling is crucial in maintaining glucose homeostasis (13Ceddia R.P. Lee D. Maulis M.F. Carboneau B.A. Threadgill D.W. Poffenberger G. Milne G. Boyd K.L. Powers A.C. McGuinness O.P. Gannon M. Breyer R.M. 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Zhang X.Y. et al.Prostaglandin E2 receptor EP3 regulates both adipogenesis and lipolysis in mouse white adipose tissue.J. Mol. Cell. Biol. 2016; 8: 518-529Crossref PubMed Scopus (19) Google Scholar). Therefore, many questions remain as to the precise molecular and cellular mechanisms by which Gαz loss protects from hyperglycemia and glucose intolerance and whether beta-cell EP3 is actually a viable target for T2D therapeutics. In this study, we use islet transcriptomic analysis and beta-cell–specific Gαz-null (βKO) mice in the C57BL/6J (B6J) background to demonstrate, in the absence of alterations in the overall EP3 expression or activity of agonist-dependent EP3 variants, Gαz has differential effects on beta-cell function and mass depending on the pathophysiological context of the model. We have previously shown that male B6N mice deficient in a protein-coding exon of Gnaz, the gene encoding for Gαz, are fully protected from hyperglycemia and glucose intolerance, even after up to 26 to 30 weeks of consuming a 45 kcal% fat diet (HFD) starting at 11 weeks of age (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). HFD feeding in Gαz KO mice approximately doubles functional beta-cell mass, independent of any changes in the weight gain, food intake, or insulin resistance as compared with WT HFD-fed controls (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). To determine potential molecular pathways by which Gαz might influence the beta-cell compensation response, we performed a shorter (16-week) diet study—a feeding regimen chosen to limit potential confounding effects of long-term uncontrolled hyperglycemia—and isolated islets for exon array analyses. As with our previous cohort (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), there were no differences in baseline blood glucose or insulin tolerance by genotype (Fig. 1, A–B). After 16 weeks of control diet (CD) or HFD feeding, both HFD-fed groups were significantly heavier than CD-fed groups, and there were no differences in the body weight by genotype (Fig. 1C). The 4- to 6-h fasting blood glucose levels of WT HFD-fed mice were mildly elevated as compared with CD-fed animals (Fig. 1D), and Gαz loss essentially ameliorated the mild fasting hyperglycemia and oral glucose intolerance of male B6N mice after 16 weeks of HFD feeding (Fig. 1E). Gαz loss again had little-to-no effect on the development of insulin resistance, whether quantified by two-way ANOVA of the percentage baseline blood glucose curves (Fig. 1F, left) or by one-way ANOVA of the area under the curves from Y = 0 (Fig. 1F, right). Total islet RNA was subjected to exon-array gene chip analysis. Islet gene expression results from 5 WT CD-fed mice, 5 KO CD-fed mice, 4 WT HFD-fed mice, and 3 KO HFD-fed mice passed quality control tests and were used for downstream analysis. Unsupervised hierarchical clustering revealed samples from each group segregated together, with islets from KO HFD-fed mice having the greatest differentiating gene expression profile among the four groups. The top 100 most significantly differentially expressed (SDE) genes across all comparisons are shown in Figure 2A, and the gene symbols according to their placement in the dendrogram can be found in Table S1. Of 16,291 expressed genes, 18 genes were SED (adjusted p-value < 0.05) between WT and KO CD-fed mice, 30 genes were SDE between WT CD-fed and HFD-fed mice, 4573 genes were SDE between WT and KO HFD-fed mice, and 7312 genes were SDE between KO CD-fed and HFD-fed mice (Fig. 2B). We applied a principal component analysis to all expressed genes and found the first two principal components accounted for approximately 50% of the variance in the levels of gene expression (Fig. 2C). The complete data set can be found in the NCBI gene expression omnibus database as accession ID GSE154325. To validate the microarray results by quantitative real-time PCR (qRT-PCR), we selected from among the top 50+ upregulated and downregulated SDE genes between WT HFD and KO HFD (Tables S2 and S3, respectively) based on potential mechanistic relevance to a beta-cell secretion or the replication phenotype. These included genes encoding GPCRs (cholecystokinin A receptor; melanocortin 5 receptor; P2Y purinoceptor 14), regulators of GPCRs or G proteins (arrestin domain containing 4; regulator of G protein signaling 16), secreted peptides (Galanin; hepcidin antimicrobial protein; trefoil factor 2), metabolic enzymes producing secreted or excreted peptides or other factors (gamma-glutamyltransferase 1, phospholipase A2 group 1b), and cell-cycle genes (cyclin E2 [Ccne2]; cyclin-dependent kinase 10). The directionality of expression change between the two groups was validated for each gene, with many of these changes being statistically significant (Fig. 2, D–E). Notably, Ccne2 and cyclin-dependent kinase 10 were actually lower in islets from KO HFD-fed mice, with Ccne2 being significantly so (Fig. 2E, right bars). We identified pathways in which SDE genes were involved using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Although there were no pathways in either analysis that were significantly different in islets from WT versus KO CD-fed mice, islets from HFD-fed mice showed significant changes in gene expression across several pathways when compared with diet or genotype controls (Fig. 3A). Focusing specifically on KEGG pathways altered between islets of HFD-fed WT versus KO mice, several pathways had decreased gene expression in KO mice, including protein-turnover related pathways such as those regulating the proteasome, ribosome biogenesis, RNA polymerase, RNA degradation, protein export, proteolysis, ubiquitin-mediated proteolysis, spliceosome, N-glycan biosynthesis, protein processing in the endoplasmic reticulum, autophagy, mitophagy, and lysosome handling (Fig. 3, A–B and Table S4). (As nearly all the genes in the "metabolism" and "sphingolipid metabolism" pathways were components of other KEGG pathways, these were excluded in the heat map shown in Fig. 3A for clarity). Only four KEGG pathways were upregulated in HFD-fed KO islets versus HFD-fed WT islets—glycerolipid metabolism, protein digestion and absorption, fat digestion and absorption, and pancreatic secretion (Fig. 3, A–B and Table S4). Interestingly, these are the same four pathways significantly downregulated in WT CD versus WT HFD islets (Fig. 3C and Table S5). KO mice showed the inverse relationship, in that fat digestion and pancreatic secretion, as well as olfactory transduction, were upregulated by HFD feeding (Fig. 3C and Table S6). In all three comparisons, pancreatic secretion was the most significant differentially regulated KEGG pathway in HFD feeding (downadjusted p-value = 5.027 × 10−10, WT CD versus WT HFD; upadjusted p-value = 3.71 × 10−9, WT HFD versus KO HFD; and upadjusted p-value = 1.0 × 10−2 KO HFD versus KO CD). Figure 3D shows a diagram of the Log2 fold-change of the SDE genes in the pancreatic secretion pathway in WT HFD versus KO HFD, with a number of these as known regulators or effectors of cAMP signaling in the beta cell. These gene expression changes after 16 weeks of HFD feeding are associated with significantly higher ex vivo islet insulin secretion after 26 to 30 weeks of HFD feeding (Fig. 3E), at the same time that glucagon secretion is lower in islets from HFD-fed KO mice as than in WT mice (Fig. 3F). Therefore, the resistance of Gαz KO mice to HFD-induced glucose intolerance appears due to enhanced insulin secretory capacity and not a primary effect of Gαz loss on beta-cell replication. Furthermore, owing to the wholesale shift in pancreatic secretion pathways, many of which are associated with cAMP signaling, it is unlikely any one gene or handful of genes is responsible for this phenotype. Gαz protein expression is limited to the brain, retina, platelets, and pancreatic islets (10Kimple M.E. Manning D. G protein alpha z. UCSD Nature Molecule Pages.1999https://doi.org/10.1038/mp.a000011.01Crossref Google Scholar, 11Kimple M.E. Hultman R.C. Casey P.J. Signaling through Gz.in: Bradshaw R.A. Dennis E. Handbook of Cell Signaling. 2nd Ed. Cambridge, MA2009: 1649-1653Google Scholar). To prove the effect of Gαz loss on protection from glucose intolerance is beta cell specific, we repeated the original extended HFD feeding study using a previously generated and validated βKO transgenic line in the B6J background (6Fenske R.J. Cadena M.T. Harenda Q.E. Wienkes H.N. Carbajal K. Schaid M.D. Laundre E. Brill A.L. Truchan N.A. Brar H. Wisinski J. Cai J. Graham T.E. Engin F. Kimple M.E. The inhibitory G protein alpha-subunit, galphaz, promotes type 1 diabetes-like pathophysiology in NOD mice.Endocrinology. 2017; 158: 1645-1658Crossref PubMed Scopus (7) Google Scholar). Like the full-body Gαz KO mice in the B6N substrain, 12-week-old βKO had nearly identical glucose and insulin tolerance as their WT controls (Fig. 4, A–B). After 28 weeks of CD or HFD feeding, both HFD-fed groups were significantly heavier than CD-fed mice, with no differences in weight by genotype (Fig. 4C). As with the original B6N study (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), fasting blood glucose and glucose tolerance were severely impacted by HFD feeding in WT B6J mice: effects that were significantly decreased in βKO mice (Fig. 4, D–E). Yet, in contrast to our previous study, but consistent with prior B6J HFD studies (17Hogan M.F. Ravnskjaer K. Matsumura S. Huising M.O. Hull R.L. Kahn S.E. Montminy M. Hepatic insulin resistance following chronic activation of the CREB coactivator CRTC2.J. Biol. Chem. 2015; 290: 25997-26006Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 18Hull R.L. Willard J.R. Struck M.D. Barrow B.M. Brar G.S. Andrikopoulos S. Zraika S. High fat feeding unmasks variable insulin responses in male C57BL/6 mouse substrains.J. Endocrinol. 2017; 233: 53-64Crossref PubMed Scopus (23) Google Scholar), insulin sensitivity was only mildly affected by long-term HFD feeding and did not differ by genotype (Fig. 4F). Experimental evidence of the increased insulin sensitivity of lean B6J mice comes from the fact several young mice or CD-fed mice, regardless of the genotype, had to be rescued with glucose injection 30, 45, or 60 min after insulin injection (noted by ˆ in Fig. 4, B–F). In all of our previous studies in the B6N substrain, we have never had to rescue a mouse because of hypoglycemia during insulin tolerance tests (ITTs) (M.E.K., personal observations). Gαz KO mice in the B6N background display a synergistic increase in beta-cell replication with HFD feeding, resulting in significantly increased beta-cell mass (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Yet, although βKO mice in the B6J background have identical T2D protection as full-body Gαz KO mice in the B6N background, there is no individual or synergistic effect of HFD feeding or Gαz loss on the percentage of Ki67-positive beta cells (Fig. 5A). Although the mean Ki67 positivity of HFD-fed βKO beta cells is lower than that of the other three groups, this is not statistically significant (Fig. 5A, right), and, when the results are plotted on the same axis as those from the original HFD study in the B6N background (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), none of the data points exceed the mean Ki67 positivity of even CD-fed B6N beta cells (Fig. 5B). This phenotypic difference is also apparent in a lack of enhancement of the beta-cell fractional area in B6J mice by Gαz loss or HFD feeding (Fig. 5C). Although we did not record pancreas weights in the present study to calculate beta-cell mass, our original study found the greater influence of HFD feeding on beta-cell mass was the islet size/volume (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar): a value that may be hidden in beta-cell fractional area calculations if the volume of the pancreas is greater. Therefore, to be able to directly compare the results of our two studies, we normalized the insulin-positive slide area from densiometry analyses to the mean of their own WT control group, revealing the significant enhancement in Gαz KO insulin-positive area by HFD feeding in the B6N substrain is lost in the B6J substrain (Fig. 5D). A lack of effect of Gαz loss on beta-cell mass in the B6J substrain is further supported by quantifying islet insulin content, which is unchanged in HFD-fed βKO B6J mice (Fig. 5E), as compared with the 2-fold enhancement found in HFD-fed KO B6N mice (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Of all of the metabolic phenotypes tested (weight gain, insulin sensitivity, fasting blood glucose, and glucose tolerance), the only phenotype that was significantly different between our two studies was the apparent effect of HFD feeding on insulin sensitivity. Using the blood glucose levels at baseline and 30, 60, and 90 min after insulin challenge—the time points consistent among all of our studies—we calculated insulin areas under the curve (AUCs). Even at baseline (11 weeks of age), B6N mice are much less insulin sensitive than B6J mice, with insulin AUCs approximately one-third greater (Fig. 5F). Long-term CD feeding had little effect on insulin AUCs in either substrain, and, while the mean ITT AUC was higher in both HFD-fed groups than their own baseline, this was only statistically significant in the B6N background (Fig. 5F). Finally, long-term HFD feeding in the B6N background approximately doubled the ITT AUC, representative of the severe insulin resistance characterized in our previous work (8Kimple M.E. Moss J.B. Brar H.K. Rosa T.C. Truchan N.A. Pasker R.L. Newgard C.B. Casey P.J. Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.J. Biol. Chem. 2012; 287: 20344-20355Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). These substrain-specific differences are not a factor of beta-cell Cre expression, as insulin sensitivity of Cre-negative littermates was nearly identical (data not shown). Islets isolated from WT or Gαz βKO mice after 28 weeks of CD or HFD feeding were treated ex vivo with basal (1.7 mM) or stimulatory (16.7 mM) concentrations of glucose, the latter with or without 10-nM exendin-4 (Ex4), which acts through the cAMP-stimulatory GLP1R to potentiate GSIS (see model in Fig. 6G). Islets from all groups secreted more insulin in response to 16.7-mM glucose versus 1.7-mM glucose, although this difference was not statistically significant in islets from WT HFD-fed mice (Fig. 6A, solid versus dotted bars). Islets from both CD-fed groups had a strong (∼3-fold) potentiating response to 10-nM Ex4, whether shown as the ng/ml secreted insulin (Fig. 6A, solid versus diagonally striped bars) or the proportion of individual islet preparations secreting more insulin in glucose + Ex4 than glucose alone (Fig. 6B). In both cases, islets from WT HFD-fed B6J mice were essentially nonresponsive to Ex4 (Fig. 6, A–B). Islets from HFD-fed Gαz βKO mice, on the other hand, had a remarkably improved potentiation of GSIS with Ex4 (Fig. 6A), and all islet preparations were Ex4 responsive (Fig. 6B). The arachidonic acid metabolite, PGE2, is the most abundant native ligand for the cAMP-inhibitory EP3. 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