Diabetes Volume 65, August 2016 2331 Ronadip R. Banerjee,1,2 Holly A. Cyphert,3 Emily M. Walker,3 Harini Chakravarthy,1 Heshan Peiris,1 Xueying Gu,1 Yinghua Liu,1 Elizabeth Conrad,3 Lisa Goodrich,4 Roland W. Stein,3 and Seung K. Kim1,5,6 Gestational Diabetes Mellitus From Inactivation of Prolactin Receptor and MafB in Islet b-Cells Diabetes 2016;65:2331–2341 | DOI: 10.2337/db15-1527 b-Cell proliferation and expansion during pregnancy through enhanced insulin secretion and by b-cell prolifera- are crucial for maintaining euglycemia in response to in- tion and expansion in mice (1). Failure of adaptive b-cell creased metabolic demands placed on the mother. Pro- expansion underlies dysregulated glucose homeostasis and lactin and placental lactogen signal through the prolactin progression to diabetes mellitus (2). Thus, the mechanisms receptor (PRLR) and contribute to adaptive b-cell re- underlying gestational b-cell proliferation are a focus of in- sponses in pregnancy; however, the in vivo requirement tensive ongoing investigation. fi b for PRLR signaling speci cally in maternal -cell adapta- Pregnancy hormones are known regulators of b-cell tions remains unknown. We generated a floxed allele of growth and function (3). The lactogenic hormones prolactin ISLET STUDIES Prlr b , allowing conditional loss of PRLR in -cells. In this and placental lactogen signal through the prolactin receptor study, we show that loss of PRLR signaling in b-cells re- (PRLR) and are crucial regulators of pregnancy adaptation in sults in gestational diabetes mellitus (GDM), reduced b-cell b many maternal tissues (4). PRLR is expressed in both rodent proliferation, and failure to expand -cell mass during preg- b b and human pancreatic -cells (5), and in vitro treatment of nancy. Targeted PRLR loss in maternal -cells in vivo im- b paired expression of the transcription factor Foxm1,both islets with prolactin has established it as a potent -cell mi- G /S and G /M cyclins, tryptophan hydroxylase 1 (Tph1), togen in both species (1). Although gene expression studies of 1 2 fi and islet serotonin production, for which synthesis requires islets during pregnancy identi ed strong induction of some – Tph1. This conditional system also revealed that PRLR sig- prolactin signaling targets (6 8), the mechanisms underlying b naling is required for the transient gestational expression of lactogen-stimulated changes in -cells during pregnancy are the transcription factor MafB within a subset of b-cells incompletely understood. During transient b-cell proliferation during pregnancy. MafB deletion in maternal b-cells also and expansion during gestation, prior studies have reported produced GDM, with inadequate b-cell expansion accom- increased expression of nuclear factors like FoxM1, the cyclin- panied by failure to induce PRLR-dependent target genes dependent kinases cyclin A2 and cyclin B1, and MafB (6,9,10). regulating b-cell proliferation. These results unveil molec- However, the requirement for PRLR signaling to induce ex- ular roles for PRLR signaling in orchestrating the physio- pression of these factors and the physiologic significance of logic expansion of maternal b-cells during pregnancy. the gestational MafB+ b-cell subpopulation are unknown. Studies of b-cells during pregnancy in humans are con- founded by practical and ethical challenges. Thus, animal Pregnancy is a unique acquired physiologic state of increased studies remain critical for understanding b-cell biology dur- metabolic demand, requiring increased output of insulin by ing pregnancy (11). Mouse genetic studies of PRLR support islet b-cells. This adaptive response is accomplished both aroleinb-cell development and function; the global PRLR 1Department of Developmental Biology, Stanford University School of Medicine, Corresponding author: Seung K. Kim, [email protected]. Stanford, CA Received 4 November 2015 and accepted 11 May 2016. 2Division of Endocrinology, Gerontology and Metabolism, Department of Medi- This article contains Supplementary Data online at http://diabetes cine, Stanford University School of Medicine, Stanford, CA .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1527/-/DC1. 3Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN © 2016 by the American Diabetes Association. Readers may use this article as 4Department of Neurobiology, Harvard Medical School, Boston, MA long as the work is properly cited, the use is educational and not for profit, and 5Division of Oncology, Department of Medicine, Stanford University School of the work is not altered. Medicine, Stanford, CA 6Howard Hughes Medical Institute, Stanford University School of Medicine, Stan- ford, CA 2332 b-Cell PRLR KO and Gestational Diabetes Mellitus Diabetes Volume 65, August 2016 knockout has glucose intolerance and reduced b-cell mass Laboratory Animal Care or Vanderbilt Animal Care and (12). Unfortunately, the global Prlr knockout mouse is ster- Use Program. Prlr+/2 ile, precluding pregnancy studies (13). mice studied Tolerance Testing and Serum Metabolite Analysis during pregnancy develop glucose intolerance and reduction Intraperitoneal glucose and insulin tolerance testing were in b-cell proliferation and mass expansion (14). Neverthe- performed as previously described (19). An oral glucose less, as loss of PRLR results in multiple abnormalities in tolerance test (GTT) was performed using a 22-gauge rigid other metabolic tissues that could indirectly influence b-cell gavage needle to deliver an intragastric glucose bolus of function (15), it is essential to assess the consequences of fi b 2 g/kg body weight. Blood was collected by tail vein bleed- targeted PRLR inactivation speci cally in -cells. In this ing. Blood glucose levels were determined by glucometer study, we generated a conditional Prlr allele allowing Cre (Bayer Contour; Bayer). Ad libitum fed or overnight (16 h)– recombinase–mediated genetic ablation of PRLR signaling fasted blood glucose levels were measured at 8:30 A.M.Se- and identified a requirement for PRLR in molecular, hor- rum insulin levels were performed by ELISA (Crystal Chem) monal, and proliferative adaptations by maternal b-cells following the manufacturer’sdirections. in pregnancy. Collectively, our results suggest PRLR sig- naling is a master regulator of adaptive b-cell responses Islet Isolation and Culture during pregnancy. Islets were isolated using retrograde perfusion of the pancreatic duct with collagenase, purified using density RESEARCH DESIGN AND METHODS centrifugation, and cultured as previously described (20). Prlr b Recombinant mouse prolactin (R&D Systems) was diluted Creation of the Floxed Allele, PRLRKO, and fi bMafBKO Mice in culture media to a nal concentration of 500 ng/ml. Culture media was changed daily. A targeting vector containing Prlr genomic DNA encompass- ing exons 4 through 9 were subcloned into plasmid PL253 Imaging containing a thymidine kinase cassette. Using recombineer- Microscopy was performed on a Zeiss AxioM1 Fluores- ing, loxP and FRT-neo-FRT-loxP cassettes were placed flank- cence microscope with AxioVision software (Carl Zeiss). ing exon 5. The targeting vector was electroporated into Confocal images were obtained using a Leica Sp2 micro- C57BL/6J embryonic stem cells and clones selected us- scope (Beckman Cell Sciences Imaging Facility; Leica ing G418; validated clones were injected into 129 blastocysts Microsystems). generating chimeric males (Stanford Transgenic Core). Germ- Quantifying b-Cell Mass and Proliferation Prlrf fi line transmission of allele was identi ed by brown fur After weighing, the pancreas was fixed, embedded in pups after crossing male chimeras with C57BL/6J females. optimal cutting temperature compound, and then frozen. Mouse tail genomic DNA was digested with NheI and South- Tissues were sectioned at 10-mm intervals using a Leica 9 fi ern blotting performed with a 5 external probe to con rm a 3050S Cryostat (Leica Microsystems). We immunostained fl correctly targeted allele. The FRT- anked neo cassette was tissue with anti-insulin antibody and DAPI and then an- FLP removed by crosses with eR mice (The Jackson Labora- alyzed sections separated by 200 mm. b-Cell mass was fl Prlrf/+ tory,BarHarbor,ME),generating oxed PRLR mice ( ), measured using ImageJ (National Institutes of Health) which were backcrossed with C57BL/6J mice (The Jackson to quantify insulin+ and total pancreas area in each section; Prlrf/+ Laboratory) for more than eight generations. mice b-cell area was the average of nine measured sections. The were crossed with mice with a transgene encoding Cre calculated b-cell mass = (b-cell area/total pancreas area) 3 RIP-Cre recombinase from rat insulin promoter elements ( ) (total pancreas weight). To measure proliferation, we RIP-Cre;Prlrf/+ (16). Subsequently, males were crossed with immunostained tissues using antibodies against insulin Prlrf/+ b PRLRf/f RIP-Cre females, generating PRLRKO ( ; )and and Ki-67 and the proportion of Ki-67+ b-cells calculated Prlrf/+ Prlrf/f RIP-Cre;Prlrf/+ RIP-Cre;Prlr+/+ littermate , , ,and as a percentage. At least 1,500 b-cells were counted for MafBf/f RIP-CreM controls. mice (17) were crossed with each animal; only clusters containing at least 20 b-cells mice (18) and backcrossed to B6J mice at least six gener- were counted. The total number of islets per mouse was ations (see Supplementary Table 1: genotyping primers). the sum of clusters counted in nine analyzed sections. Mouse Husbandry, Breeding, and Experimentation Antibodies used are listed in