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• W1884174564 abstract "Insufficient plasma insulin levels caused by deficits in both pancreatic β-cell function and mass contribute to the pathogenesis of type 2 diabetes. This loss of insulin-producing capacity is termed β-cell decompensation. Our work is focused on defining the role(s) of guanine nucleotide-binding protein (G protein) signaling pathways in regulating β-cell decompensation. We have previously demonstrated that the α-subunit of the heterotrimeric Gz protein, Gαz, impairs insulin secretion by suppressing production of cAMP. Pancreatic islets from Gαz-null mice also exhibit constitutively increased cAMP production and augmented glucose-stimulated insulin secretion, suggesting that Gαz is a tonic inhibitor of adenylate cyclase, the enzyme responsible for the conversion of ATP to cAMP. In the present study, we show that mice genetically deficient for Gαz are protected from developing glucose intolerance when fed a high fat (45 kcal%) diet. In these mice, a robust increase in β-cell proliferation is correlated with significantly increased β-cell mass. Further, an endogenous Gαz signaling pathway, through circulating prostaglandin E activating the EP3 isoform of the E prostanoid receptor, appears to be up-regulated in insulin-resistant, glucose-intolerant mice. These results, along with those of our previous work, link signaling through Gαz to both major aspects of β-cell decompensation: insufficient β-cell function and mass. Insufficient plasma insulin levels caused by deficits in both pancreatic β-cell function and mass contribute to the pathogenesis of type 2 diabetes. This loss of insulin-producing capacity is termed β-cell decompensation. Our work is focused on defining the role(s) of guanine nucleotide-binding protein (G protein) signaling pathways in regulating β-cell decompensation. We have previously demonstrated that the α-subunit of the heterotrimeric Gz protein, Gαz, impairs insulin secretion by suppressing production of cAMP. Pancreatic islets from Gαz-null mice also exhibit constitutively increased cAMP production and augmented glucose-stimulated insulin secretion, suggesting that Gαz is a tonic inhibitor of adenylate cyclase, the enzyme responsible for the conversion of ATP to cAMP. In the present study, we show that mice genetically deficient for Gαz are protected from developing glucose intolerance when fed a high fat (45 kcal%) diet. In these mice, a robust increase in β-cell proliferation is correlated with significantly increased β-cell mass. Further, an endogenous Gαz signaling pathway, through circulating prostaglandin E activating the EP3 isoform of the E prostanoid receptor, appears to be up-regulated in insulin-resistant, glucose-intolerant mice. These results, along with those of our previous work, link signaling through Gαz to both major aspects of β-cell decompensation: insufficient β-cell function and mass. Both insulin resistance and β-cell decompensation (i.e. the failure of β-cells to maintain sufficient insulin secretion to properly regulate blood glucose levels, because of β-cell dysfunction and/or loss of β-cell mass) are essential events in the development of type 2 diabetes mellitus (T2DM) 2The abbreviations used are: T2DMtype 2 diabetes mellitusT1DMtype 1 diabetes mellitusGSISglucose-stimulated insulin secretionHFDhigh fat dietGPCRG protein-coupled receptorDIOdiet-induced obesityPGEprostaglandin EIP-GTTintraperitoneal glucose tolerance testANOVAanalysis of varianceSSTsomatostatin 28G proteinGuanine nucleotide binding proteinDAB3,3′-diaminobenzidine. (1Kahn S.E. The relative contributions of insulin resistance and β-cell dysfunction to the pathophysiology of Type 2 diabetes.Diabetologia. 2003; 46: 3-19Crossref PubMed Scopus (1639) Google Scholar, 2Bergman R.N. Finegood D.T. Kahn S.E. The evolution of β-cell dysfunction and insulin resistance in type 2 diabetes.Eur. J. Clin. Invest. 2002; 32: 35-45Crossref PubMed Scopus (239) Google Scholar, 3Greenberg A.S. McDaniel M.L. Identifying the links between obesity, insulin resistance and beta-cell function. Potential role of adipocyte-derived cytokines in the pathogenesis of type 2 diabetes.Eur. J. Clin. Invest. 2002; 32: 24-34Crossref PubMed Scopus (254) Google Scholar, 4Gerich J.E. Redefining the clinical management of type 2 diabetes. Matching therapy to pathophysiology.Eur. J. Clin. Invest. 2002; 32: 46-53Crossref PubMed Scopus (28) Google Scholar, 5Muoio D.M. Newgard C.B. Mechanisms of disease. Molecular and metabolic mechanisms of insulin resistance and β-cell failure in type 2 diabetes.Nat. Rev. Mol. Cell Biol. 2008; 9: 193-205Crossref PubMed Scopus (897) Google Scholar). Consistent with this idea, recent genome-wide association studies have shown that the majority of small nucleotide polymorphisms that associate with T2DM susceptibility are linked to β-cell decompensation and not insulin resistance (6Billings L.K. Florez J.C. The genetics of type 2 diabetes. What have we learned from GWAS?.Ann. N.Y. Acad. Sci. 2010; 1212: 59-77Crossref PubMed Scopus (292) Google Scholar). type 2 diabetes mellitus type 1 diabetes mellitus glucose-stimulated insulin secretion high fat diet G protein-coupled receptor diet-induced obesity prostaglandin E intraperitoneal glucose tolerance test analysis of variance somatostatin 28 Guanine nucleotide binding protein 3,3′-diaminobenzidine. The regulation of insulin secretion has been studied intensively for more than three decades, yet much is left to learn about this process. We have previously shown that the heterotrimeric Gi protein α-subunit, Gαz, modulates an endogenous signaling pathway that is inhibitory to glucose-stimulated insulin secretion (GSIS) in a rat β-cell-derived cell line (7Kimple M.E. Nixon A.B. Kelly P. Bailey C.L. Young K.H. Fields T.A. Casey P.J. A role for Gz in pancreatic islet β-cell biology.J. Biol. Chem. 2005; 280: 31708-31713Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The mechanism for Gαz action appears to be a tonic negative regulation of adenylate cyclase, which when lost leads to constitutively increased cyclic AMP production (7Kimple M.E. Nixon A.B. Kelly P. Bailey C.L. Young K.H. Fields T.A. Casey P.J. A role for Gz in pancreatic islet β-cell biology.J. Biol. Chem. 2005; 280: 31708-31713Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). This work was among the first to define a physiologic role for endogenous Gαz, a protein that was first described in 1988 (9Fong H.K. Yoshimoto K.K. Eversole-Cire P. Simon M.I. Identification of a GTP-binding protein α subunit that lacks an apparent ADP-ribosylation site for pertussis toxin.Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 3066-3070Crossref PubMed Scopus (185) Google Scholar, 10Matsuoka M. Itoh H. Kozasa T. Kaziro Y. Sequence analysis of cDNA and genomic DNA for a putative pertussis toxin-insensitive guanine nucleotide-binding regulatory protein α subunit.Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 5384-5388Crossref PubMed Scopus (154) Google Scholar) and characterized biochemically in 1990 (11Casey P.J. Fong H.K. Simon M.I. Gilman A.G. Gz, a guanine nucleotide-binding protein with unique biochemical properties.J. Biol. Chem. 1990; 265: 2383-2390Abstract Full Text PDF PubMed Google Scholar, 12Hinton D.R. Blanks J.C. Fong H.K. Casey P.J. Hildebrandt E. Simons M.I. Novel localization of a G protein, Gz-α, in neurons of brain and retina.J. Neurosci. 1990; 10: 2763-2770Crossref PubMed Google Scholar). The availability of Gαz-null mouse lines provided the opportunity to study the role of this G protein in normal tissue function and in disease pathophysiology. In our initial experiments with young, lean Balb/c mice, Gαz-null mice secreted more insulin in response to glucose and had more rapid glucose clearance (8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), as might be predicted from the loss of an inhibitor of GSIS. This effect was cell autonomous, because islets isolated from Gαz-null mice had an increased response to stimulatory glucose (8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The in vivo and in vitro phenotypes of Gαz-null islets fit with the loss of a negative regulator of cAMP production, because cAMP is a ubiquitous second messenger involved in the glucose-dependent potentiation of GSIS. Taken together, these results suggested that deletion or suppression of Gαz might be protective against the development of β-cell dysfunction. C57Bl/6 mice are susceptible to the development of obesity and metabolic derangements after high fat diet (HFD) feeding, resulting in the development of glucose intolerance (13Winzell M.S. Magnusson C. Ahrén B. Temporal and dietary fat content-dependent islet adaptation to high-fat feeding-induced glucose intolerance in mice.Metab. Clin. Exp. 2007; 56: 122-128Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), whereas Balb/c mice are less susceptible. Because our prior work on Gαz-null mice was performed in the Balb/c background, we utilized the Gαz-null mice in a C57Bl/6 background for the current studies (14Kelleher K.L. Matthaei K.I. Hendry I.A. Targeted disruption of the mouse Gz-α gene. A role for Gz in platelet function?.Thromb. Haemost. 2001; 85: 529-532Crossref PubMed Scopus (16) Google Scholar). Wild-type and Gαz-null C57Bl/6 mice were challenged with HFD feeding for up to 30 weeks and then phenotyped for insulin and glucose tolerance. HFD-fed Gαz-null mice were completely protected from fasting hyperglycemia and glucose intolerance, even though they were equally insulin resistant as wild-type HFD-fed mice. This impact on glucose tolerance was largely independent of protection of β-cell function but was instead associated with a dramatic and somewhat unexpected impact of Gαz loss on β-cell proliferation, as measured by Ki67 immunofluorescence of fixed pancreas sections. This increased proliferation correlated positively with increased islet volume and β-cell mass. Selective G protein-coupled receptor (GPCR) agonists were utilized to delineate the signaling pathway(s) linked specifically to Gαz. Similar to our previous work with a rat β-cell line (7Kimple M.E. Nixon A.B. Kelly P. Bailey C.L. Young K.H. Fields T.A. Casey P.J. A role for Gz in pancreatic islet β-cell biology.J. Biol. Chem. 2005; 280: 31708-31713Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), in C57Bl/6 islets we confirmed a role for the E prostanoid receptor in transmitting signals to Gαz. Further, we suggest a link between the pathophysiology of glucose intolerance and a constitutively activated prostaglandin E2 signaling pathway in the β-cell, which is relieved in the absence of Gαz protein. The results of this study, along with those of our previous works (7Kimple M.E. Nixon A.B. Kelly P. Bailey C.L. Young K.H. Fields T.A. Casey P.J. A role for Gz in pancreatic islet β-cell biology.J. Biol. Chem. 2005; 280: 31708-31713Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), define the role of Gαz in regulating β-cell function and mass in normal and pathological conditions, providing insight into the mechanisms of β-cell decompensation. C57Bl/6 mice containing a genomic insertion of a pGKneor cassette 160 base pairs downstream of the translation start site of the Gαz gene (gene symbol: gnaz) were developed by the Ian Hendry lab at Australian National University and shown to be completely deficient in Gαz protein expression as compared with wild-type controls in numerous tissues (15Hendry I.A. Kelleher K.L. Bartlett S.E. Leck K.J. Reynolds A.J. Heydon K. Mellick A. Megirian D. Matthaei K.I. Hypertolerance to morphine in G(z α)-deficient mice.Brain Res. 2000; 870: 10-19Crossref PubMed Scopus (64) Google Scholar, 16Leck K.J. Bartlett S.E. Smith M.T. Megirian D. Holgate J. Powell K.L. Matthaei K.I. Hendry I.A. Deletion of guanine nucleotide binding protein α z subunit in mice induces a gene dose dependent tolerance to morphine.Neuropharmacology. 2004; 46: 836-846Crossref PubMed Scopus (19) Google Scholar, 17Leck K.J. Blaha C.D. Matthaei K.I. Forster G.L. Holgate J. Hendry I.A. Gz proteins are functionally coupled to dopamine D2-like receptors in vivo.Neuropharmacology. 2006; 51: 597-605Crossref PubMed Scopus (18) Google Scholar, 18Oleskevich S. Leck K.J. Matthaei K. Hendry I.A. Enhanced serotonin response in the hippocampus of Gαz protein knock-out mice.Neuroreport. 2005; 16: 921-925Crossref PubMed Scopus (15) Google Scholar, 19Yang J. Wu J. Jiang H. Mortensen R. Austin S. Manning D.R. Woulfe D. Brass L.F. Signaling through Gi family members in platelets. Redundancy and specificity in the regulation of adenylyl cyclase and other effectors.J. Biol. Chem. 2002; 277: 46035-46042Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Straws containing frozen sperm from confirmed Gαz-null C57Bl/6 mice were purchased from the Australian National University Phenomics Facility (Canberra, Australia) (14Kelleher K.L. Matthaei K.I. Hendry I.A. Targeted disruption of the mouse Gz-α gene. A role for Gz in platelet function?.Thromb. Haemost. 2001; 85: 529-532Crossref PubMed Scopus (16) Google Scholar). The C57Bl/6 Gαz-null line was regenerated at Duke University using in vitro fertilization and intrauterine implantation into wild-type C57Bl/6 females (Charles River Laboratories, Wilmington, MA). Adult mice were placed into breeding triads on a 12-h light/dark cycle with ad libitum access to breeder chow (5058 PicoLab Mouse Diet 20; LabDiet, Brentwood, MO). Gαz-null and wild-type control mice were generated by heterozygous matings to produce littermate pairs. Upon weaning, the male mice were housed five or fewer per cage with ad libitum access to low fat control chow (5053 PicoLab Rodent Diet 20, LabDiet). Before sexual maturity, the mice were placed by pairs of the same genotype into a new cage. At 8 (pilot study) or 11 weeks of age (full study), the chow was changed to a diet containing 45 kcal% fat (D12451; Research Diets, New Brunswick, NJ) or the appropriate low fat control diet (D12450B; Research Diets; 10 kcal% fat). Food was weighed weekly during the pilot study and replaced weekly for both the pilot and full study. Experimental parameters recorded were: initial glucose tolerance, weight change, final insulin tolerance, final glucose tolerance, final adiposity, and final pancreas weight (wet). Adiposity was determined from dual emission x-ray absorptiometry scanning and/or subgonadal fat pad weight (wet). Dual emission x-ray absorptiometry scans were performed and analyzed on ketamine/xylazine-anesthetized mice using a Lunar PIXImus II and associated software (GE Healthcare Lunar, Madison, WI). Gonadal fat pad weights were either recorded as the sum of both fat pads combined or just a single fat pad; therefore, in each instance, the weights were normalized to those of the mean of the wild-type control diet-fed mice to obtain meaningful comparisons for the whole set. Not all parameters were recorded for all mice. The animals were handled in accordance with the principles and guidelines established by the Duke University Animal Care and Use Committee. Guinea pig anti-insulin (1:500), rabbit anti-glucagon (1:300), mouse anti-Ki67 (1:100), antibody diluent, citrate pH 6 target retrieval solution, Protein Block, and 10× wash buffer were purchased from Dako (Carpinteria, CA). Highly cross-absorbed Alexa Fluor® 488 goat anti-guinea pig IgG, Alexa Fluor® 680 donkey anti-mouse IgG, Alexa Fluor® 568 donkey anti-rabbit IgG, ProLong® Gold antifade reagent with DAPI, and sterile d-glucose in PBS were from Invitrogen. Insulin (Humulin® R) and sterile insulin diluent were from Lilly (Indianapolis, IN). Superfrost® Plus microscope slides were from Fisher Scientific (Hampton, NH), and fluorescence quality 1.5-mm coverslips were from Corning Life Sciences (Lowell, MA). The rat/mouse insulin ELISA kit was from Crystal Chem Inc. (Downers Grove, IL). The prostaglandin E (PGE) metabolite kit was from Cayman Chemical (Ann Arbor, MI). The cAMP Biotrak® enzyme immunoassay kit was from GE Healthcare. CGS-12066A, BW-72386, sulprostone, 3-isobutyl-1-methylxanthine, pertussis toxin from Bordetella pertussis (buffered aqueous glycerol solution), and Cellytic® M cell lysis buffer were from Sigma-Aldrich. Complete® EDTA-free protease inhibitor mixture tablets were from Roche Applied Sciences (Indianapolis, IN). The BCA protein assay was from Thermo Fisher Scientific (Indianapolis, IN). The mice were fasted for 4–6 h, and intraperitoneal glucose tolerance tests (IP-GTTs) were performed before the administration of the high fat or control diet (11 weeks of age) and after 21–25 weeks on the high fat or control diet (32–36 weeks of age). Glucose readings were taken from tail blood using an Ascensia Breeze 2 meter (Bayer Diabetes Care, Tarrytown, NY) before glucose injection (t = 0) and 25, 60, 120, and 180 min after glucose injection. The glucose readings were averaged within genotypes at each time point, giving the means ± S.E. (n = 17–25 mice for each group). During the IP-GTTs, blood samples were collected at 0 and 10 min into EDTA-coated tubes to generate plasma samples for insulin ELISA conducted as previously described (8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). One to three replicates of each plasma sample were performed depending on the total sample volume available; the insulin values for all of the replicates of a single sample were averaged, and the averages for all mice of the same treatment group were used to calculate the means ± S.E. PGE metabolite levels were measured in plasma samples collected at t = 0 of the IP-GTTs using a specific ELISA kit and manufacturer's protocol (Cayman Chemical). Derivatized samples were diluted 1:50 into assay buffer to obtain readings in the linear range of the assay. The samples were run in duplicate, the PGE metabolite values were averaged, and the averages for all mice of the same treatment group were used to calculate the means ± S.E. Insulin tolerance tests were performed essentially as described for the IP-GTTs, but instead of injecting glucose, the mice were injected intraperitoneally with 0.75 units/kg regular insulin in sterile insulin diluent. Glucose readings were taken at 0, 30, 60, and 90 min postinjection. Insulin tolerance tests were performed at least 2 weeks before and 2 weeks after the IP-GTTs. The results for each mouse were averaged to give as close a picture of insulin tolerance at the time of the IP-GTT as possible. These averaged glucose readings were averaged within groups at each time point, giving the means ± S.E. Mouse pancreatic islets were isolated essentially as previously described (8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). To determine islet volume, isolated islets were cultured overnight in RPMI 1640 with 8.4 mm glucose and penicillin/streptomycin to recover from the isolation procedure. Islets were swirled to the middle of the culture dish for imaging, the image calibrated by taking a picture of a ruler, and the diameter of every islet in the culture was measured to calculate the islet volume. To determine islet protein content, islets were cultured overnight as described. The entire islet culture was hand-picked into 1.5-ml microcentrifuge tubes containing 1 ml of sterile PBS in batches of 50–100/tube. The islets were pelleted two times by pulsing to 10,000 × g, washed with 1 ml of PBS, and resuspended in Cellytic® M lysis reagent containing protease inhibitors. The protein content was determined by BCA assay according to the manufacturer's protocol. GSIS assays and cAMP production assays were performed essentially as previously described (8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). With both GSIS and cAMP production, the islets were cultured overnight in RPMI medium containing submaximal stimulatory glucose (8.4 mm for insulin secretion and 11.1 mm for cAMP). On the day of the assays, the islets were washed once with Krebs-Ringer bicarbonate buffer containing 1.7 mm glucose and then preincubated for 45 min in the same buffer before being transferred to the desired stimulatory buffer for 45 min. cAMP production assays were conducted in the presence of 100 μm 3-isobutyl-1-methylxanthine to block cAMP degradation. The cAMP production for each sample was normalized to its protein content using BCA assay. Insulin secreted into the cAMP stimulation medium was also measured and normalized to protein content. For GSIS assays, the insulin secreted into the medium was normalized to the total insulin content. For each GSIS and cAMP assay, the normalized technical replicates were averaged, and the averages from all experiments were used to calculate the standard error. Pancreata were dissected, fixed, and sectioned as previously described, with minor modifications (7Kimple M.E. Nixon A.B. Kelly P. Bailey C.L. Young K.H. Fields T.A. Casey P.J. A role for Gz in pancreatic islet β-cell biology.J. Biol. Chem. 2005; 280: 31708-31713Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). For immunofluorescence experiments, the slides were deparaffinized and subjected to antigen retrieval. The slides were then washed and blocked for 1 h at room temperature. For fluorescence detection, a primary antibody mix (anti-insulin, anti-glucagon, and anti-Ki67) was added to the slide after blocking, covered, and incubated overnight at 4 °C. The slides were washed extensively and then incubated with secondary antibodies at 1:4000 for 1 h at room temperature. The slides were imaged using a Zeiss Axioplan2 fluorescence microscope with a Qimaging Exi camera driven by Openlab software. Each channel was imaged separately, and the final images were overlaid in Photoshop. For insulin immunostaining, 5-micron sections 100 microns apart were stained using the EnVisionTM diaminobenzidine (DAB) reagents according to manufacturer's protocol (Dako), along with a 30-min room temperature incubation of 1:500 guinea pig anti-insulin primary antibody. The slides were lightly counterstained with hematoxylin, and six to eight independent sections from two or three mice of each group were processed. The slides were imaged using the Qimaging camera on color settings using a 1.25× lens. β-Cell fractional area was calculated by processing the resulting RGB images in open source National Institutes of Health ImageJ/64 software using shading correction and the H DAB color deconvolution vector. The data were analyzed using GraphPad Prism v. 5 (GraphPad Software Inc., San Diego, CA). A t test or two-way ANOVA was used to determine the p value as indicated in the figure legends. p < 0.05 was considered significant. Male wild-type and Gαz-null C57Bl/6 mice were fed either a control diet containing 10% of calories as fat, or a diet composed of 45% of calories as fat (HFD). Male C57Bl/6 mice are known to respond to HFD feeding by becoming obese and insulin-resistant and developing glucose intolerance (8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). A pilot DIO study was conducted first with three groups of 8-week-old mice (wild type: control diet, n = 11; wild type: HFD, n = 11; Gαz-null: HFD, n = 12) to determine whether 1) there was a significant difference in food intake and/or weight gain based on genotype and 2) to design appropriate parameters for the full study. The results of this pilot study indicated that both the wild-type and Gαz-null mice became obese when fed on HFD compared with the wild-type control diet groups, with the Gαz-null HFD-fed mice exhibiting a greater initial weight gain that ultimately plateaued at the same level as the wild-type HFD mice (supplemental Fig. S1A). Of note, the Gαz-null HFD mice ate significantly more as normalized to body weight than either of the other two groups at week 1 and tended to eat more until week 6 of the pilot study, at which point there was no difference between the food eaten (kcal/g body weight; supplemental Fig. S1B). This increased food intake seemed to explain the increased initial rate of weight gain of the Gαz-null mice and suggested possible changes to the experimental protocol. Specifically, the Gαz-null mice are known to be runted because of a failure to thrive phenotype, with catch-up growth postweaning (15Hendry I.A. Kelleher K.L. Bartlett S.E. Leck K.J. Reynolds A.J. Heydon K. Mellick A. Megirian D. Matthaei K.I. Hypertolerance to morphine in G(z α)-deficient mice.Brain Res. 2000; 870: 10-19Crossref PubMed Scopus (64) Google Scholar). We hypothesized that the increased initial food intake of the Gαz-null mice was a compensation for a decreased body weight during a period where the mouse is still developing. Thus, for the full experimental set, 17–25 mice of each genotype were placed on the control or HFD at 11 weeks of age, when the Gαz-null mice are closer in weight to their wild-type littermates and are essentially fully developed (13Winzell M.S. Magnusson C. Ahrén B. Temporal and dietary fat content-dependent islet adaptation to high-fat feeding-induced glucose intolerance in mice.Metab. Clin. Exp. 2007; 56: 122-128Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The Gαz-null mice at 11 weeks of age were still somewhat lighter than their wild-type littermates (24.14 ± 0.096 (wild type, n = 49) versus 21.64 ± 0.083 (Gαz-null, n = 34); p < 0.001), although dual emission x-ray absorptiometry scanning revealed no significant differences in their initial body composition (n = 4 mice of each genotype; p = 0.2073 (data not shown and Ref. 8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). There were also no significant differences in the glucose tolerance of wild-type and Gαz-null C57Bl/6 mice at a dose of 1 g/kg glucose (Fig. 1A), although the glucose tolerance of the Gαz-null mice trended toward a lower area under the curve, consistent with our previous observations using 2 g/kg glucose in Balb/c mice (8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The lack of difference in glucose tolerance was also reflected in the lack of significant differences between the plasma insulin levels of the wild-type and Gαz-null mice at t = 0 or t = 10 min; both genotypes had similar and significant increases in plasma insulin at t = 10 min (Fig. 1B). After being placed on the appropriate diet at 11 weeks of age, the HFD-fed mice gained significantly more weight than the control diet-fed mice, and the weight gain did not vary by genotype (Fig. 1C). The insulin tolerance of obese HFD-fed mice, as recorded as the average insulin tolerance before and after the IP-GTT, was significantly worse than the control diet-fed mice and did not differ by genotype (Fig. 1D). This confirms that Gαz loss does not induce insulin resistance, which was not surprising based on our previous work (8Kimple M.E. Joseph J.W. Bailey C.L. Fueger P.T. Hendry I.A. Newgard C.B. Casey P.J. Gαz negatively regulates insulin secretion and glucose clearance.J. Biol. Chem. 2008; 283: 4560-4567Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Furthermore, the adiposity of the Gαz-null mice did not differ from that of the wild-type mice in either treatment group, although gonadal fat pad mass measurements revealed that the HFD-fed mice had significantly increased adiposity as compared with the appropriate control mice (Fig. 1E). IP-GTTs (1 g/kg glucose) were performed 21–25 weeks after beginning the DIO study, when the mice were 32–36 weeks of age. As expected, HFD-fed wild-type C57Bl/6 mice exhibited impaired glucose clearance compared with wild-type mice fed on the control diet (Fig. 2A). The area under the curve for blood glucose was significantly elevated in HFD-fed wild-type mice versus control diet-fed mice, which in turn was significantly higher than that observed in the 11-week-old mice (Fig. 2B). Furthermore, the fasting glucose level for HFD-fed wil" @default.
• W1884174564 created "2016-06-24" @default.
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• W1884174564 date "2012-06-01" @default.
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• W1884174564 title "Deletion of GαZ Protein Protects against Diet-induced Glucose Intolerance via Expansion of β-Cell Mass" @default.
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