Deletion of G�Z Protein Protects against Diet-inducedGlucose Intolerance via Expansion of �-Cell Mass*□S

Received for publication, March 6, 2012, and in revised form, March 26, 2012 Published, JBC Papers in Press, March 28, 2012, DOI 10.1074/jbc.M112.359745

Michelle E. Kimple‡1, Jennifer B. Moss§, Harpreet K. Brar‡, Taylor C. Rosa§¶, Nathan A. Truchan‡, Renee L. Pasker‡,Christopher B. Newgard§¶�, and Patrick J. Casey¶�

From the ‡Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin School ofMedicine and Public Health, Madison, Wisconsin 53705 and the §Sarah W. Stedman Nutrition and Metabolism Center, Departmentof Medicine, and Departments of ¶Pharmacology and Cancer Biology and �Biochemistry, Duke University Medical Center,Durham, North Carolina 27704

Background: G�z can block insulin secretion, but no one had looked at its role in glucose intolerance.Results:Mice that do not express G�z mice do not develop glucose intolerance when fed a high fat diet.Conclusion: A G�z signaling pathway contributes to the pathophysiology of glucose intolerance.Significance: This is the first work describing the specific role(s) of G�z in glucose intolerance.

Insufficient plasma insulin levels caused by deficits in bothpancreatic�-cell function andmass contribute to the pathogen-esis of type 2 diabetes. This loss of insulin-producing capacity istermed�-cell decompensation.Ourwork is focused on definingthe role(s) of guanine nucleotide-binding protein (G protein)signaling pathways in regulating �-cell decompensation. Wehave previously demonstrated that the �-subunit of the hetero-trimeric Gz protein, G�z, impairs insulin secretion by suppress-ing production of cAMP. Pancreatic islets from G�z-null micealso exhibit constitutively increased cAMPproduction and aug-mented glucose-stimulated insulin secretion, suggesting thatG�z is a tonic inhibitor of adenylate cyclase, the enzyme respon-sible for the conversion of ATP to cAMP. In the present study,we show that mice genetically deficient for G�z are protectedfrom developing glucose intolerance when fed a high fat (45kcal%) diet. In these mice, a robust increase in �-cell prolifera-tion is correlated with significantly increased �-cell mass. Fur-ther, an endogenousG�z signaling pathway, through circulatingprostaglandin E activating the EP3 isoform of the E prostanoidreceptor, appears to be up-regulated in insulin-resistant, glu-cose-intolerantmice. These results, along with those of our pre-vious work, link signaling through G�z to both major aspects of�-cell decompensation: insufficient �-cell function and mass.

Both insulin resistance and �-cell decompensation (i.e. thefailure of �-cells to maintain sufficient insulin secretion toproperly regulate blood glucose levels, because of �-cell dys-function and/or loss of �-cell mass) are essential events in thedevelopment of type 2 diabetes mellitus (T2DM)2 (1–5). Con-

sistent with this idea, recent genome-wide association studieshave shown that the majority of small nucleotide polymor-phisms that associate with T2DM susceptibility are linked to�-cell decompensation and not insulin resistance (6).

The regulation of insulin secretion has been studied inten-sively for more than three decades, yet much is left to learnabout this process. We have previously shown that the hetero-trimeric Gi protein �-subunit, G�z, modulates an endogenoussignaling pathway that is inhibitory to glucose-stimulated insu-lin secretion (GSIS) in a rat �-cell-derived cell line (7). Themechanism for G�z action appears to be a tonic negative regu-lation of adenylate cyclase, which when lost leads to constitu-tively increased cyclic AMP production (7, 8). This work wasamong the first to define a physiologic role for endogenousG�z,a protein that was first described in 1988 (9, 10) and character-ized biochemically in 1990 (11, 12).The availability of G�z-null mouse lines provided the oppor-

tunity to study the role of this G protein in normal tissue func-tion and in disease pathophysiology. In our initial experimentswith young, lean Balb/c mice, G�z-null mice secreted moreinsulin in response to glucose andhadmore rapid glucose clear-ance (8), as might be predicted from the loss of an inhibitor ofGSIS. This effect was cell autonomous, because islets isolatedfrom G�z-null mice had an increased response to stimulatoryglucose (8). The in vivo and in vitro phenotypes of G�z-nullislets fit with the loss of a negative regulator of cAMP produc-tion, because cAMP is a ubiquitous secondmessenger involvedin the glucose-dependent potentiation ofGSIS. Taken together,these results suggested that deletion or suppression of G�zmight be protective against the development of �-celldysfunction.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 (13),whereas Balb/c mice are less susceptible. Because our prior

* This work was supported, in whole or in part, by National Institutes of HealthGrants DK080845 (to M. E. K.), DK042583 (to C. B. N.), and DK076488 (toP. J. C.).

□S This article contains supplemental Figs. S1–S3.1 To whom correspondence should be addressed: Dept. of Medicine, Div. of

Endocrinology, Diabetes, and Metabolism, 4th Floor, Medical FoundationCentennial Bldg., Madison, WI 53705. Tel.: 608-263-7780; Fax: 608-263-9983; E-mail: mkimple@medicine.wisc.edu.

2 The abbreviations used are: T2DM, type 2 diabetes mellitus; T1DM, type 1diabetes mellitus; GSIS, glucose-stimulated insulin secretion; HFD, high fat

diet; GPCR, G protein-coupled receptor; DIO, diet-induced obesity; PGE,prostaglandin E; IP-GTT, intraperitoneal glucose tolerance test; ANOVA,analysis of variance; SST, somatostatin 28; G protein, Guanine nucleotidebinding protein; DAB, 3,3�-diaminobenzidine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 24, pp. 20344 –20355, June 8, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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work on G�z-null mice was performed in the Balb/c back-ground, we utilized theG�z-null mice in a C57Bl/6 backgroundfor the current studies (14). Wild-type and G�z-null C57Bl/6mice were challenged with HFD feeding for up to 30 weeks andthen phenotyped for insulin and glucose tolerance. HFD-fedG�z-null mice were completely protected from fasting hyper-glycemia and glucose intolerance, even though they wereequally insulin resistant as wild-type HFD-fed mice. Thisimpact on glucose tolerance was largely independent of protec-tion of �-cell function but was instead associated with a dra-matic and somewhat unexpected impact of G�z loss on �-cellproliferation, as measured by Ki67 immunofluorescence offixed pancreas sections. This increased proliferation correlatedpositively with increased islet volume and�-cell mass. SelectiveG protein-coupled receptor (GPCR) agonists were utilized todelineate the signaling pathway(s) linked specifically to G�z.Similar to our previous work with a rat �-cell line (7), inC57Bl/6 islets we confirmed a role for the E prostanoid receptorin transmitting signals to G�z. Further, we suggest a linkbetween the pathophysiology of glucose intolerance and a con-stitutively activated prostaglandin E2 signaling pathway in the�-cell, which is relieved in the absence of G�z protein. Theresults of this study, along with those of our previous works (7,8), define the role of G�z in regulating �-cell function andmassin normal and pathological conditions, providing insight intothe mechanisms of �-cell decompensation.

MATERIALS AND METHODS

Mouse Care, Husbandry, and Diet-induced Obesity (DIO)Study—C57Bl/6 mice containing a genomic insertion of apGKneor cassette 160 base pairs downstream of the translationstart site of theG�z gene (gene symbol: gnaz) were developed bythe IanHendry lab atAustralianNationalUniversity and shownto be completely deficient in G�z protein expression as com-pared with wild-type controls in numerous tissues (15–19).Straws containing frozen sperm from confirmed G�z-nullC57Bl/6 mice were purchased from the Australian NationalUniversity Phenomics Facility (Canberra, Australia) (14). TheC57Bl/6 G�z-null line was regenerated at Duke Universityusing in vitro fertilization and intrauterine implantation intowild-type C57Bl/6 females (Charles River Laboratories, Wilm-ington, MA). Adult mice were placed into breeding triads on a12-h light/dark cycle with ad libitum access to breeder chow(5058 PicoLab Mouse Diet 20; LabDiet, Brentwood, MO).G�z-null and wild-type control mice were generated by

heterozygous matings to produce littermate pairs. Upon wean-ing, the male mice were housed five or fewer per cage with adlibitum access to low fat control chow (5053 PicoLab RodentDiet 20, LabDiet). Before sexual maturity, the mice were placedby 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 dietcontaining 45 kcal% fat (D12451; Research Diets, New Bruns-wick, NJ) or the appropriate low fat control diet (D12450B;Research Diets; 10 kcal% fat). Food was weighed weekly duringthe pilot study and replaced weekly for both the pilot and fullstudy. Experimental parameters recorded were: initial glucosetolerance, weight change, final insulin tolerance, final glucosetolerance, final adiposity, and final pancreas weight (wet).

Adiposity was determined from dual emission x-ray absorp-tiometry scanning and/or subgonadal fat pad weight (wet).Dual emission x-ray absorptiometry scans were performed andanalyzed on ketamine/xylazine-anesthetized mice using aLunar PIXImus II and associated software (GE HealthcareLunar, Madison, WI). Gonadal fat pad weights were eitherrecorded as the sum of both fat pads combined or just a singlefat pad; therefore, in each instance, the weights were normal-ized to those of the mean of the wild-type control diet-fed miceto obtain meaningful comparisons for the whole set. Not allparameters were recorded for all mice. The animals were han-dled in accordance with the principles and guidelines estab-lished by the Duke University Animal Care and UseCommittee.Antibodies and Reagents—Guinea pig anti-insulin (1:500),

rabbit anti-glucagon (1:300), mouse anti-Ki67 (1:100), antibodydiluent, citrate pH6 target retrieval solution, Protein Block, and10�wash buffer were purchased fromDako (Carpinteria, CA).Highly cross-absorbed Alexa Fluor� 488 goat anti-guinea pigIgG, Alexa Fluor� 680 donkey anti-mouse IgG, Alexa Fluor�568 donkey anti-rabbit IgG, ProLong� Gold antifade reagentwith DAPI, and sterile D-glucose in PBS were from Invitrogen.Insulin (Humulin�R) and sterile insulin diluent were fromLilly(Indianapolis, IN). Superfrost� Plus microscope slides werefrom Fisher Scientific (Hampton, NH), and fluorescence qual-ity 1.5-mm coverslips were fromCorning Life Sciences (Lowell,MA). The rat/mouse insulin ELISA kit was from Crystal ChemInc. (Downers Grove, IL). The prostaglandin E (PGE) metabo-lite kit was from Cayman Chemical (Ann Arbor, MI). ThecAMPBiotrak� enzyme immunoassay kit was fromGEHealth-care. CGS-12066A, BW-72386, sulprostone, 3-isobutyl-1-methylxanthine, pertussis toxin fromBordetella pertussis (buff-ered aqueous glycerol solution), andCellytic�Mcell lysis bufferwere from Sigma-Aldrich. Complete� EDTA-free proteaseinhibitor mixture tablets were from Roche Applied Sciences(Indianapolis, IN). The BCA protein assay was from ThermoFisher Scientific (Indianapolis, IN).Glucose Tolerance Tests—The mice were fasted for 4–6 h,

and intraperitoneal glucose tolerance tests (IP-GTTs) wereperformed before the administration of the high fat or controldiet (11 weeks of age) and after 21–25 weeks on the high fat orcontrol diet (32–36 weeks of age). Glucose readings were takenfrom tail blood using an Ascensia Breeze 2meter (Bayer Diabe-tes Care, Tarrytown, NY) before glucose injection (t � 0) and25, 60, 120, and 180 min after glucose injection. The glucosereadings were averaged within genotypes at each time point,giving the means � S.E. (n � 17–25mice for each group). Dur-ing the IP-GTTs, blood samples were collected at 0 and 10 mininto EDTA-coated tubes to generate plasma samples for insulinELISA conducted as previously described (8). One to three rep-licates of each plasma samplewere performeddepending on thetotal sample volume available; the insulin values for all of thereplicates of a single samplewere averaged, and the averages forall mice of the same treatment group were used to calculate themeans � S.E.Plasma PGE Determination—PGE metabolite levels were

measured in plasma samples collected at t � 0 of the IP-GTTsusing a specific ELISA kit and manufacturer’s protocol (Cay-

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man Chemical). Derivatized samples were diluted 1:50 intoassay buffer to obtain readings in the linear range of the assay.The samples were run in duplicate, the PGE metabolite valueswere averaged, and the averages for all mice of the same treat-ment group were used to calculate the means � S.E.Insulin Tolerance Tests—Insulin tolerance tests were per-

formed essentially as described for the IP-GTTs, but instead ofinjecting glucose, the mice were injected intraperitoneally with0.75 units/kg regular insulin in sterile insulin diluent. Glucosereadings were taken at 0, 30, 60, and 90min postinjection. Insu-lin tolerance tests were performed at least 2 weeks before and 2weeks after the IP-GTTs. The results for eachmousewere aver-aged to give as close a picture of insulin tolerance at the time ofthe IP-GTT as possible. These averaged glucose readings wereaveraged within groups at each time point, giving the means �S.E.Pancreatic Islet Isolation and Morpohometric Analysis—

Mouse pancreatic islets were isolated essentially as previouslydescribed (8). To determine islet volume, isolated islets werecultured overnight in RPMI 1640 with 8.4mM glucose and pen-icillin/streptomycin to recover from the isolation procedure.Islets were swirled to themiddle of the culture dish for imaging,the image calibrated by taking a picture of a ruler, and the diam-eter of every islet in the culture was measured to calculate theislet volume. To determine islet protein content, islets werecultured overnight as described. The entire islet culture washand-picked into 1.5-mlmicrocentrifuge tubes containing 1mlof sterile PBS in batches of 50–100/tube. The islets were pel-leted two times by pulsing to 10,000 � g, washed with 1 ml ofPBS, and resuspended in Cellytic� M lysis reagent containingprotease inhibitors. The protein content was determined byBCA assay according to the manufacturer’s protocol.GSIS Assays and cAMP Production Assays—GSIS assays and

cAMP production assays were performed essentially as previ-ously described (8).With bothGSIS and cAMP production, theislets were cultured overnight in RPMI medium containingsubmaximal stimulatory glucose (8.4 mM for insulin secretionand 11.1mM for cAMP).On the day of the assays, the islets werewashed once with Krebs-Ringer bicarbonate buffer containing1.7 mM glucose and then preincubated for 45 min in the samebuffer before being transferred to the desired stimulatory bufferfor 45 min.cAMP production assays were conducted in the presence of

100 �M 3-isobutyl-1-methylxanthine to block cAMP degrada-tion. The cAMP production for each sample was normalized toits protein content using BCA assay. Insulin secreted into thecAMP stimulation mediumwas also measured and normalizedto protein content. For GSIS assays, the insulin secreted intothe medium was normalized to the total insulin content. ForeachGSIS and cAMP assay, the normalized technical replicateswere averaged, and the averages fromall experimentswere usedto calculate the standard error.Immunofluorescence and Immunohistological Assays—Pan-

creata were dissected, fixed, and sectioned as previouslydescribed, with minor modifications (7). For immunofluores-cence experiments, the slides were deparaffinized and sub-jected to antigen retrieval. The slides were then washed andblocked for 1 h at room temperature. For fluorescence detec-

tion, a primary antibody mix (anti-insulin, anti-glucagon, andanti-Ki67) was added to the slide after blocking, covered, andincubated overnight at 4 °C. The slides were washed exten-sively and then incubated with secondary antibodies at1:4000 for 1 h at room temperature. The slides were imagedusing a Zeiss Axioplan2 fluorescence microscope with aQimaging Exi camera driven by Openlab software. Eachchannel was imaged separately, and the final images wereoverlaid 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 1:500guinea pig anti-insulin primary antibody. The slides werelightly counterstained with hematoxylin, and six to eight inde-pendent sections from two or three mice of each group wereprocessed. The slides were imaged using the Qimaging cam-era on color settings using a 1.25� lens. �-Cell fractionalarea was calculated by processing the resulting RGB imagesin open source National Institutes of Health ImageJ/64 soft-ware using shading correction and the H DAB color decon-volution vector.Statistical Analyses—The data were analyzed using GraphPad

Prism v. 5 (GraphPad Software Inc., San Diego, CA). A t test ortwo-way ANOVA was used to determine the p value as indi-cated in the figure legends. p� 0.05 was considered significant.

RESULTS

Adult G�z-null HFD-fed Mice Retain Glucose Tolerance—Male wild-type and G�z-null C57Bl/6 mice were fed either acontrol diet containing 10% of calories as fat, or a diet com-posed of 45% of calories as fat (HFD). Male C57Bl/6 mice areknown to respond toHFD feeding by becoming obese and insu-lin-resistant and developing glucose intolerance (8). A pilotDIO study was conducted first with three groups of 8-week-oldmice (wild type: control diet, n � 11; wild type: HFD, n � 11;G�z-null: HFD, n � 12) to determine whether 1) there was asignificant difference in food intake and/or weight gain basedon genotype and 2) to design appropriate parameters for the fullstudy. The results of this pilot study indicated that both thewild-type and G�z-null mice became obese when fed on HFDcomparedwith thewild-type control diet groups, with theG�z-null HFD-fed mice exhibiting a greater initial weight gain thatultimately plateaued at the same level as the wild-type HFDmice (supplemental Fig. S1A). Of note, the G�z-null HFDmiceate significantly more as normalized to body weight than eitherof the other two groups at week 1 and tended to eat more untilweek 6 of the pilot study, at which point there was no differencebetween the food eaten (kcal/g body weight; supplemental Fig.S1B). This increased food intake seemed to explain theincreased initial rate of weight gain of the G�z-null mice andsuggested possible changes to the experimental protocol. Spe-cifically, the G�z-null mice are known to be runted because of afailure to thrive phenotype, with catch-up growth postweaning(15). We hypothesized that the increased initial food intake ofthe G�z-null mice was a compensation for a decreased bodyweight during a period where the mouse is still developing.Thus, for the full experimental set, 17–25 mice of each geno-

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type were placed on the control or HFD at 11 weeks of age,when the G�z-null mice are closer in weight to their wild-typelittermates and are essentially fully developed (13).The G�z-null mice at 11 weeks of age were still somewhat

lighter than their wild-type littermates (24.14 � 0.096 (wildtype, n � 49) versus 21.64 � 0.083 (G�z-null, n � 34); p �0.001), although dual emission x-ray absorptiometry scanningrevealed no significant differences in their initial body compo-sition (n� 4mice of each genotype; p� 0.2073 (data not shownand Ref. 8). There were also no significant differences in theglucose tolerance of wild-type and G�z-null C57Bl/6 mice at adose of 1 g/kg glucose (Fig. 1A), although the glucose tolerance

of the G�z-null mice trended toward a lower area under thecurve, consistent with our previous observations using 2 g/kgglucose in Balb/c mice (8). The lack of difference in glucosetolerance was also reflected in the lack of significant differencesbetween the plasma insulin levels of the wild-type andG�z-nullmice at t � 0 or t � 10 min; both genotypes had similar andsignificant 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 thecontrol diet-fedmice, and the weight gain did not vary by geno-type (Fig. 1C). The insulin tolerance of obese HFD-fed mice, asrecorded as the average insulin tolerance before and after the

FIGURE 1. Initial glucose tolerance, adiposity, and insulin resistance among mice subjected to the DIO study. A, blood glucose values recorded duringIP-GTTs (1 g/kg glucose) of 11-week-old lean wild-type (n � 40) and G�z-null mice (n � 23) mice. The data were analyzed by two-way repeated measuresANOVA followed by Bonferroni post-test. B, plasma insulin values recorded at t � 0 and t � 10 min during the IP-GTT after 1 g/kg glucose in both the wild-type(n � 23) and G�z-null (n � 21) mice. The data were analyzed by paired t test for the effect of time within genotype and unpaired t test for the effect of genotypeat each time point. C, body weight measurements taken during the course of the DIO experiment (wild type: control diet, n � 25; wild type: HFD, n � 24;G�z-null: control diet, n � 17; G�z-null: HFD, n � 17). The data were analyzed by two-way repeated measures ANOVA followed by Bonferroni post-test. D,decrement in blood glucose recorded during intraperitoneal insulin tolerance tests (0.75 units/kg insulin) of mice fed the control diet or HFD (wild type: controldiet, n � 21; wild type: HFD, n � 20; G�z-null: control diet, n � 16; G�z-null: HFD, n � 14). The data were analyzed by two-way repeated measures ANOVAfollowed by Bonferroni post-test. E, gonadal fat pad weights recorded from representative mice from each group. The data were analyzed by unpaired t test.*, p � 0.05; **, p � 0.01; ***, p � 0.001. ctrl, control.

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IP-GTT, was significantly worse than the control diet-fed miceand did not differ by genotype (Fig. 1D). This confirms that G�zloss does not induce insulin resistance, which was not surpris-ing based on our previous work (8). Furthermore, the adiposityof the G�z-null mice did not differ from that of the wild-typemice in either treatment group, although gonadal fat pad massmeasurements revealed that the HFD-fed mice had signifi-cantly increased adiposity as compared with the appropriatecontrol 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 ofage. As expected, HFD-fed wild-type C57Bl/6 mice exhibitedimpaired glucose clearance compared with wild-type mice fedon the control diet (Fig. 2A). The area under the curve for bloodglucose was significantly elevated in HFD-fed wild-type miceversus control diet-fed mice, which in turn was significantlyhigher than that observed in the 11-week-old mice (Fig. 2B).Furthermore, the fasting glucose level for HFD-fed wild-typemice was significantly higher than that observed at 11 weeks orin the control diet-fed mice (Fig. 2C). Elevated fasting plasmaglucose and impaired glucose tolerance are consistent with thedevelopment of prediabetes in obese C57Bl/6 mice (20).The elevated fasting plasma insulin levels as recorded during

the IP-GTT are consistent with the development of insulinresistance in both the HFD-fed wild-type and G�z-null groups(Fig. 3A, 0min IP-GTT). In considering thewild-typemice only,neither the HFD-fed nor control diet-fed groups demonstratedan appreciable increase in plasma insulin after glucose chal-lenge (Fig. 3A, 10min IP-GTT). This is not unexpected, becausethe acute insulin response after glucose challenge tends to dete-riorate with both age andHFD feeding (13, 21). The acute insu-lin response can be better represented by normalizing theplasma insulin levels after glucose challenge to the fasting insu-lin levels for each mouse: in our case, 10-min IP-GTT over0-min IP-GTT. Again, the acute insulin responses of the wild-type mice were significantly impacted by both age and HFDfeeding, but those of theG�z-nullmicewere not (Fig. 3B). Inter-estingly, the G�z-null mice fed the control diet were the onlygroup that displayed plasma insulin levels, indicative of anabsence of both insulin resistance and glucose intolerance(Fig. 3).Loss of G�z in Adult HFD-fed Mice Induces �-Cell Prolifera-

tion and Increases �-Cell Mass—To further explore the �-cellfunction of HFD-fed G�z-null mice, pancreatic islets were iso-lated from each group of mice 30 weeks after initiation ofthe DIO study. In the pilot study, islets were isolated from fourmice of each group in two experiments, and their diameterswere measured using calibrated stereomicroscopic images. Weobserved the expected increase in size of wild-type islets frommice fed the HFD, as calculated from measuring the diametersof isolated islets and assuming a spherical volume (V �(1/6)�d3) (supplemental Figs. S1C and S2). Interestingly, a fur-ther increase in islet volume was observed in islets from G�z-null HFD-fed mice. Because the islet volume was directly cor-related with protein content, protein content per islet was usedas a surrogate measurement for islet size in the full DIO study.Again, the size of islets from HFD-fed G�z-null mice was sig-nificantly increased above the normal C57Bl/6 compensatory

mechanisms (Fig. 4A). The insulin content of islets from HFD-fed G�z-null mice also increased significantly and correlateddirectly with the increase in islet size (Fig. 4B). In vitro GSISassays were performed at several glucose concentrations todetermine the effect of genotype and diet status on �-cell func-

FIGURE 2. Loss of G�z normalizes glucose tolerance in mice fed the HFD.A, blood glucose values recorded during an IP-GTT (1 g/kg glucose) per-formed after 21–25 weeks on the HFD or control diet (wild type: control diet,n � 15; wild type: HFD, n � 17; G�z-null: control diet, n � 12; G�z-null: HFD,n � 15). The data were analyzed by two-way repeated measures ANOVAfollowed by Bonferroni post-test. B, area under the curve (AUC) measure-ments performed on IP-GTT curves at 11 weeks of age (base line; Fig. 1A) or at21–25 weeks after beginning the DIO study. The data were analyzed byunpaired t test. C, fasting glucose levels taken at t � 0 during the IP-GTTsindicate a better maintenance of base-line glycemia in the G�z-null mice withHFD feeding. The data were analyzed by unpaired t test. **, p � 0.01; ***, p �0.001. ctrl, control.

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tion (as represented by insulin secreted as a percentage of thetotal). The G�z-null HFD islets did not secrete more insulin asa percentage of total insulin content (Fig. 4C). This is in contrastto islets from G�z-null mice fed the control diet, which diddisplay an improved �-cell function (Fig. 4C), consistent withour previous observations in islets fromyoung, leanBalb/cmice(8). This suggested that G�z may have two separate roles in the�-cell and that an improved insulin response in G�z-null HFD-fed mice might be primarily due to expansion of �-cell massrather than enhanced GSIS from individual �-cells.

To explore the phenotype of increased �-cell mass in theG�z-null HFD-fedmice, �-cell fractional area wasmeasured byanalysis of anti-insulin DAB staining of formalin-fixed, paraf-fin-embedded sections (Fig. 5, A–D). Of note, the average sizeof islets from G�z-null control diet mice calculated from theDAB-stained sections was consistent with our estimate of isletsize based on total protein (Fig. 4A), suggesting a positiveimpact of G�z loss on islet size (data not shown). In quantifyingthe �-cell fractional area, we observed a trend toward anincrease in insulin-positive area in pancreas sections from the

two HFD-fed groups as compared with their respective controldiet-fed cohorts, consistent with a compensatory response toinsulin resistance (Fig. 5E). Interestingly, when the mean frac-

FIGURE 3. G�z-null mice demonstrate a better maintenance of stimula-tion of plasma insulin levels, both with age and HFD feeding. A, plasmainsulin levels recorded at t � 0 and t � 10 min during the final IP-GTT. The datawere analyzed by paired t test to determine the effect of time within a groupand by unpaired t test with Welch’s correction, which corrects for unequalvariances, among treatment groups. B, acute insulin response, which is theplasma insulin after glucose challenge (t � 10 min) as normalized to theplasma insulin levels at t � 0 min. The data were analyzed by unpaired t test.*, p � 0.05; **, p � 0.01; ***, p � 0.001. ctrl, control.

FIGURE 4. Islets from G�z-null HFD-fed mice are significantly larger thanthose of wild-type mice, but do not demonstrate increased function.A, islet protein content was used as a surrogate measurement for islet volumein the full DIO study (wild type: control diet, n � 25, wild type: HFD, n � 35;G�z-null: control diet, n � 8; G�z-null: HFD, n � 17). The data were analyzed byunpaired t test. B, insulin content of islets isolated from mice from each of thefour groups (n � 3 mice for each group). The data were analyzed by unpairedt-test. C, GSIS response of islets isolated from mice from each of the fourgroups to increasing concentrations of glucose (n � 3 mice for each group).The data were analyzed by two-way repeated measures ANOVA followed byBonferroni post-test. *, p � 0.05; **, p � 0.01; ***, p � 0.001. ctrl, control.

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tional area values were used to convert total pancreatic mass to�-cell mass, the G�z-null HFD-fed mice demonstrated signifi-cantly increased �-cell mass as compared with the wild-typeHFD-fed mice (Fig. 5F).To determine a possible mechanism for increased �-cell

mass in the G�z-null HFD-fed mice, we prepared paraformal-dehyde-fixed pancreas sections from each group of mice andused an immunocytochemical assay to measure Ki67, a nuclearmarker of active cell replication. In nearly all islets from wild-type animals fed the control diet, no Ki67-positive �-cells wereobserved (Fig. 6A). Islets fromG�z-null animals fed the controldiet or fromwild-type animals fed the HFD showed a few Ki67-positive cells per islet (Fig. 6, B and C), whereas islets fromG�z-null HFD-fed mice had a clear increase in nuclear Ki67immunofluorescence (Fig. 6D and inset). Furthermore, of all ofthe Ki67-positive islet cells, only five non-�-cells were found(all �-cells, in three different treatment groups: wild-type HFD,G�z-null control diet, and G�z-null HFD), suggesting that theincreases in islet size were likely driven by the increase in �-cellreplication. In quantifying the Ki67-positive �-cell nuclei as apercentage of total�-cell nuclei, HFD feeding of wild-typemicecaused a 4-fold increase in Ki67 positive �-cells compared withcontrol diet feeding. G�z loss in the control diet backgroundalso had significant impact on the number of Ki67-positive

�-cells, equivalent to the effect of HFD feeding in the wild-typemice. Finally, �-cells from G�z-null HFD-fed mice demon-strated a 24-fold increase in Ki67 staining, larger than either ofthe other groups (Fig. 6E).The EP3 Isoform of the PGE Receptor Is Coupled Specifically

to G�z to Negatively Regulate �-Cell Function—The findingthat G�z loss alone impacts on �-cell function and mass sug-gests that, in wild-type mice, tonic activation of a G�z-coupledreceptor increases with both age and HFD feeding and that thissignaling pathway is inoperative when G�z is absent. Weshowed previously that cAMP production is constitutivelyincreased in islets isolated from G�z-null Balb/c mice (8). Todetermine whether this holds true for the C57Bl/6 line studiedin this work, we isolated islets from G�z-null or wild-typeC57Bl/6 mice and quantified their constitutive cAMP produc-tion. Just as with Balb/c islets, G�z-null C57Bl/6 islets exhibitsignificantly increased cAMP production as compared withislets from littermate controls (Fig. 7A, left panel). Further-more, the amount of insulin secreted into the cAMP stimula-tionmediumdirectly correlates with the increase in cAMP (Fig.7A, right panel).Certain GPCRs signal through inhibitory G proteins, such as

G�z, to block cAMP production. We have previously shownthat the endogenous PGE receptor in a rat�-cell line was exclu-

FIGURE 5. Improved glucose tolerance in the HFD-fed G�z-null mice may be due to increased �-cell mass. A–D, representative sections from anti-insulinstained pancreas sections (brown) counterstained with hematoxylin (blue) that were used to calculate fractional �-cell area. A, wild type, control diet; B, wildtype, HFD; C, G�z-null, control diet; D, G�z-null, HFD. Three nonconsecutive sections were imaged per pancreas, with two or three pancreata imaged per group.The black scale bars indicate 1 mm (ticks indicate 200 �m). E, �-cell fractional area was calculated from insulin-stained pancreas sections and reported as thepercentage of total pancreas area on the slide. Three nonconsecutive sections separated by at least 100 microns were quantified for each pancreas (n � 2–3mice for each group). F, the HFD-fed G�z-null mice have significantly increased calculated �-cell mass as compared with the wild-type HFD-fed mice. Pancreasweights from individual mice were transformed using the mean fractional area in A. The data were analyzed by unpaired t test. *, p � 0.05. ctrl, control.

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sively coupled to G�z to block insulin secretion (7). Of the fourisoforms of the PGE receptor, only the EP3 isoform has beenshown to couple to inhibitory G proteins. In addition, theGPCR family that has been best linked to regulation of mouse�-cell mass is the serotonin (5HT: 5-hydroxytryptamine)receptor family. Activation of the 5HT2B receptor was found tostimulate �-cell proliferation in pregnant mice, augmenting�-cell mass, while signaling through the 5HT1D receptorrestored normal �-cell mass after parturition (22). To explorethe role(s) of EP3 and 5HT receptor signaling in islets from anobese, glucose-intolerant mouse model, we isolated islets from10-week-old C57Bl/6 ob/ob mice and incubated them over-night in medium containing submaximal stimulatory glucosewith or without the addition of 200 ng/ml pertussis toxin toinactivate all G�i subfamily members save G�z (11). This over-night treatment regimen was shown to completely relieve inhi-bition of GSIS by somatostatin 28 (SST), which is known to act

through other G�i subfamily members and not G�z (supple-mental Fig. S3). After pertussis toxin treatment, the islets wereused in GSIS assays with the selective 5HT1 agonist CGS-12066A, the selective 5HT2 agonist BW-72386, the selectiveEP3 agonist sulprostone, or SST. Sulprostone and SST bothsignificantly blocked GSIS in the control treated islets, but asexpected for a G�z-coupled receptor, only the sulprostoneeffect was resistant to PTX (Fig. 7B). Neither CGS-12066A norBW-72386 had a significant effect on GSIS, regardless of PTXtreatment. This suggests that, at least in the moderate �-cellcompensation of the C57Bl/6 ob/obmice, 5HT receptor signal-ing does not play a role in regulating �-cell function.Finally, to explore potential up-regulation of the EP3 signal-

ing pathway in our DIO study, we determined the plasma PGEconcentrations before and after control- or HFD feeding. Wefound that PGE levels were significantly elevated from base line(11-weeks-old) with both age and HFD feeding, regardless of

FIGURE 6. Increased �-cell proliferation in G�z-null islets from mice fed the HFD as compared with their wild-type counterparts. A–D, pancreas sectionsfrom wild-type control diet-fed mice (A), G�z-null control diet-fed mice (B), wild-type HFD-fed mice (C), and G�z-null HFD-fed mice (D) were incubated withprimary antibodies against insulin (red), glucagon (blue), and Ki67 (green). Overlaying the images revealed �-cells that are actively replicating (examplesindicated by white arrows). E, quantification of the Ki67-positive �-cells as a percentage of the total �-cells. Three nonconsecutive sections separated by at least100 microns were quantified for each pancreas. n � 2 mice for each group. The data were analyzed by unpaired t test. **, p � 0.01; ***, p � 0.001. ctrl, control.

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genotype,withmice being fed theHFDhaving the highest levelsof all the groups (Fig. 8). These results, combined with thosefrom Fig. 7, support our hypothesis that tonic activation of aG�z-coupled receptor by an endogenous ligand limits �-cellcompensation in response to aging and nutritional stress andthat in the absence of G�z, this inhibitory pathway is broken.

DISCUSSION

The Centers for Disease Control and Prevention have pub-lished sobering statistics for the impact of T2DMon theUnitedStates population (2011 National Diabetes Fact Sheet). Cur-rently, 25.8million people are estimated to haveT2DM(8.3%ofthe total United States population). Furthermore, 1.9 millionT2DM cases were newly diagnosed in 2010. The number ofT2DM cases in the 10–19 year age group now approximatelyequals that of type 1 diabetes mellitus (T1DM), and the young-est members of this age group have an overall lifetime risk ofdeveloping T2DM of 30–50%.T2DM susceptibility genes emanating from genome-wide

association studies are almost exclusively related to�-cell func-

tion andmaintenance ofmass rather than insulin resistance (6).In light of this, many T2DM treatments under developmentaim to improve �-cell function and not simply insulin sensitiv-ity (4, 23–25). In this regard, our results with C57Bl/6 G�z-nullmice are quite intriguing, because the C57Bl/6 mouse line hasbeen suggested to be resistant to diabetes because of their�-cellcompensatory response (20, 26). Our data suggest there isuntapped potential for �-cell expansion and mass even in ananimal that has a strong intrinsic compensatory response andthat this increase in mass can substitute for a decrease infunction.Our previous studies of mice deficient in G�z demonstrated

constitutively increased cAMP production, leading toenhancedGSIS (8). Interestingly, this difference in in vitroGSISresponse was phenocopied in the older C57Bl/6 mice fed thecontrol diet, but not in the G�z-null mice fed the HFD. Thisindicates that the loss of G�z is unable to preserve normal glu-cose responsiveness in an environment of overnutrition thatincludes increased plasma levels of glucose, insulin, free fattyacids, triglycerides, cholesterol, oxidative stressors, and/orinflammatory cytokines (3, 27–30). Long term HFD feeding inC57Bl/6 mice causes functional uncoupling of Ca2� channelsfrom the secretory granules (31); this effect is mimicked bychronic exposure of isolated mouse and human islets to highlevels of palmitate (32). To the extent that this constitutes amajor mechanism of impaired insulin secretion in response toovernutrition, loss of G�z, which acts through cAMP potentia-tion of GSIS (7, 8), would not be expected to repair �-cell dys-function elicited by uncoupling of the Ca2� signal from thesecretory response.Rather than affecting function, the dramatic impact of G�z

loss on �-cell replication and �-cell mass appears to be suffi-cient to protect these mice from developing glucose intoler-ance.With the role that cAMP has been shown to play in �-cellgrowth, survival, and/or proliferation (33–43), it is possiblethat the increased cAMP production observed in G�z-nullislets (Ref. 8 and Fig. 7A) is responsible for the increase in�-cellreplication and mass. Interestingly, it appears that augmentedcAMP production is a general feature of G�z loss in tissues

FIGURE 7. G�z-null islets produce more cAMP, and the EP3 isoform of thePGE receptor is linked to G�z to negatively regulate GSIS. A, intracellularcAMP production (left panel) and secreted insulin (right panel) were measuredfrom the same islets samples isolated from wild-type and G�z-null mice. n �3 for each group. The data were analyzed by unpaired t test. *, p � 0.05. B,islets were isolated from glucose-intolerant C57Bl/6 ob/ob mice and culturedovernight in medium containing 11.1 mM glucose (glc.) with and without 200ng/ml pertussis toxin (PTX). In vitro GSIS assays were performed with the addi-tion of the 5HT1 agonist CGS-12066A (100 nM), the 5HT2 agonist BW-723C56(1 �M), the EP3 agonist sulprostone (10 �M), or SST (2 �g/ml). The data werenormalized within each group to the effect of 16.7 mM glucose � Me2SOcontrol. n � 5 for each group, except for SST (n � 3). The data were analyzedby paired t test. *, p � 0.05; **, p � 0.01; ***, p � 0.001.

FIGURE 8. Increased plasma PGE levels in C57Bl/6 mice with age and HFDsupports tonic activation of a G�z-coupled receptor. Plasma PGE metab-olite levels were recorded from plasma samples collected after a 4-h fast, att � 0 of the IP-GTTs. n � 6 mice per group. The data were analyzed byunpaired t test. *, p � 0.05; **, p � 0.01; ***, p � 0.001.

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where G�z has been shown to have a functional role. Specifi-cally, platelets from G�z-null C57Bl/6 mice also displayincreased cAMP levels, correlating with inhibited plateletaggregation responses to ADP and epinephrine (19).G�z has been conclusively linked to five GPCRs in vivo: the

Mu opioid, D2 dopamine, E prostanoid, serotonin-1A(5HT1A), and �2A-adrenergic (7, 17, 18, 44, 45). The �2A-adrenoreceptor specifically couples to G�z in platelets (19, 46).We have previously shown that in a �-cell line, G�z is specifi-cally coupled to an E prostanoid receptor, and not the �2A-aderenoreceptor, to block cAMPaccumulation and subsequentGSIS (7). In the current study, we show that this signaling path-way is also functional in primary islets and that the specific Eprostanoid isoform is EP3 (Fig. 7B).Increased plasma PGE metabolite levels have been found to

correlate with diabetic status (47). In our HFDmodel of insulinresistance and glucose intolerance, plasma PGE levels areincreased bothwith age andHFD feeding, with the latter havingthe most significant effect (Fig. 8). The pathway from PGEs3EP33G�z3 adenylate cyclase leads to decreased cAMP pro-duction, which would negatively impact on �-cell compensa-tion. We hypothesize that the absence of G�z breaks thisendogenous signaling pathway, whichmay simply be a negativeconsequence of the insulin resistant and/or diabetic condition(see model in Fig. 9).Although the hypothesized pathway from circulating PGE to

cAMPmodulation fits very well with the phenotypic data fromG�z-null mice, there are other possible explanations for theresistance of G�z-null mice to HFD-induced glucose intoler-ance. One such explanation comes from the observation that5HT1A serotonin receptor is coupled to G�z in the hippocam-pus to negatively regulate serotonin signaling, consistent withthe finding that G�z-null mice have an enhanced response toserotonin (18). Recently, the 5HT2B receptor has been shownto act downstream of lactogen signaling to promote �-cell pro-liferation in pregnant mice, whereas 5HT1D receptor signalingreduces �-cell mass after partuition (22).We explored the roles

of these families of serotonin receptors in islets from glucose-intolerantC57Bl/6mice (Fig. 7B).Neither 5HT1nor 5HT2 ago-nism had an impact on GSIS from islets harvested from glu-cose-intolerant C57Bl/6mice, either in the presence or absenceof G�i subfamily inhibition. Caveats to this study are that sero-tonin signaling may impact only on �-cell mass and not func-tion, or serotonin receptors may only play roles at certain timepoints during the �-cell compensatory response to insulinresistance. These questions will be important to research infuture works.Finally, our results may have important implications for the

T1DM field. T1DMoccurswhen immune-mediated pancreatic�-cell destruction leads to nearly absolute insulin deficiency(48, 49). Most patients with newly diagnosed T1DM still havethe capacity to secrete insulin in amounts corresponding to20–30% of those of nondiabetics (50). Meanwhile, only �10%of pancreatic islet transplant patients remain completely insu-lin independent at 5 years after (51). The residual �-cell func-tion observed in both early T1DM and in pancreatic islet trans-plant patients indicates the presence of a pool of potentiallyexpandable �-cells. The link between the loss of �-cell mass inboth T1DM and late stage T2DM has provided a rationale fortesting T2DM treatments that act through cAMP generation inrodent models of T1DM and in human T1DM patients andpancreatic islet transplant recipients. These studies show pro-longed survival, improved glycemia, and maintenance of graftfunction for a longer duration (52–54). Therapeutic strategiesthat increase islet cell cAMP production, such as those thatbased on the G�z pathway described in this manuscript, couldbe an important tool in T1DM therapy.In conclusion, G�z loss is protective against the development

of age-related �-cell dysfunction and HFD-induced glucoseintolerance in mice, the former via effects on �-cell functionand proliferation, and the latter working predominantlythrough enhanced �-cell proliferation leading to increased�-cell mass. Taken together, our work has helped to define therole of G�z in normal and pathophysiological pathways in the

FIGURE 9. Model for G�z as a tonic negative regulator of �-cell adenylate cyclase in conditions of high circulating E prostanoids (PGEs). A, when PGElevels are high, such as with aging and/or after the extended DIO study performed in this work, the PGE receptor activates G�z, which negatively regulatesadenylate cyclase, decreasing the production of cAMP from ATP. cAMP has dual roles in promoting both GSIS and processes involved in the maintenance of�-cell mass (e.g. cell growth, proliferation, and/or survival). Therefore, the positive impact of cAMP on these processes is blocked when the E prostanoidreceptor activates G�z. B, when G�z is lost, there is no inhibitory G protein coupled to the E prostanoid receptor. Therefore, the tonic inhibition on adenylatecyclase is lost, allowing for increased cAMP production, stimulation of GSIS, and stimulation of proliferation.

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�-cell and lent credence to the further study of this pathway inadditional models of diabetes mellitus.

Acknowledgments—We thank the former and present members of theKimple, Casey, and Newgard labs, in particular Everett McCook andMissy Infante, for expert technical assistance, and Patrick Fueger,SamStephens, HansHohmeier, and Jeff Tessem for productive discus-sions. Finally, we thank Alan Attie at University of Wisconsin-Madi-son for providing the environment and encouragement to completethis study.

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G�z Negatively Regulates �-Cell Mass

JUNE 8, 2012 • VOLUME 287 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 20355

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Truchan, Renee L. Pasker, Christopher B. Newgard and Patrick J. CaseyMichelle E. Kimple, Jennifer B. Moss, Harpreet K. Brar, Taylor C. Rosa, Nathan A.

-Cell MassβExpansion of Protein Protects against Diet-induced Glucose Intolerance viaZαDeletion of G

doi: 10.1074/jbc.M112.359745 originally published online March 28, 20122012, 287:20344-20355.J. Biol. Chem. 

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