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• W1978759169 abstract "Increasing evidence suggests that endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of type 2 diabetes mellitus. SEL1L is an ER membrane protein that is highly expressed in the pancreatic islet and acinar cells. We have recently reported that a deficiency of SEL1L causes systemic ER stress and leads to embryonic lethality in mice. Here we show that mice with one functional allele of Sel1l (Sel1l+/−) are more susceptible to high fat diet (HFD)-induced hyperglycemia. Sel1l+/− mice have a markedly reduced β-cell mass as a result of decreased β-cell proliferation. Consequently, Sel1l+/− mice are severely glucose-intolerant and exhibit significantly retarded glucose-stimulated insulin secretion. Pancreatic islets from Sel1l+/− mice stimulated with a high concentration of glucose in vitro express significantly higher levels of unfolded protein response genes than those from wild-type control mice. Furthermore, dominant-negative interference of SEL1L function in insulinoma cell lines severely impairs, whereas overexpression of SEL1L efficiently improves protein secretion. Taken together, our results indicate that haploid insufficiency of SEL1L predispose mice to high fat diet-induced hyperglycemia. Our findings highlight a critical and previously unknown function for SEL1L in regulating adult β-cell function and growth. Increasing evidence suggests that endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of type 2 diabetes mellitus. SEL1L is an ER membrane protein that is highly expressed in the pancreatic islet and acinar cells. We have recently reported that a deficiency of SEL1L causes systemic ER stress and leads to embryonic lethality in mice. Here we show that mice with one functional allele of Sel1l (Sel1l+/−) are more susceptible to high fat diet (HFD)-induced hyperglycemia. Sel1l+/− mice have a markedly reduced β-cell mass as a result of decreased β-cell proliferation. Consequently, Sel1l+/− mice are severely glucose-intolerant and exhibit significantly retarded glucose-stimulated insulin secretion. Pancreatic islets from Sel1l+/− mice stimulated with a high concentration of glucose in vitro express significantly higher levels of unfolded protein response genes than those from wild-type control mice. Furthermore, dominant-negative interference of SEL1L function in insulinoma cell lines severely impairs, whereas overexpression of SEL1L efficiently improves protein secretion. Taken together, our results indicate that haploid insufficiency of SEL1L predispose mice to high fat diet-induced hyperglycemia. Our findings highlight a critical and previously unknown function for SEL1L in regulating adult β-cell function and growth. Type 2 diabetes mellitus (T2DM) 3The abbreviations used are: T2DMtype 2 diabetes mellitusERendoplasmic reticulumHFDhigh fat dietSel1lsuppressor enhancer Lin12 1-likeGSISglucose-stimulated insulin secretionrtTAreverse tet transactivator4-PBAphenylbutyric acidNCnormal chowUPRunfolded protein response. is a major chronic disease currently affecting over 24 million Americans with annual costs exceeding $170 billion (1American Diabetes Association Diabetes Care. 2008; 31: 596-615Crossref PubMed Scopus (1352) Google Scholar). Deficit in β-cell mass and intra-islet amyloid deposition are hallmark features of the islet in T2DM (2Butler A.E. Janson J. Bonner-Weir S. Ritzel R. Rizza R.A. Butler P.C. Diabetes. 2003; 52: 102-110Crossref PubMed Scopus (3331) Google Scholar). Obesity is a well characterized risk factor for the development of T2DM (3Carey D.G. Jenkins A.B. Campbell L.V. Freund J. Chisholm D.J. Diabetes. 1996; 45: 633-638Crossref PubMed Google Scholar, 4DeFronzo R.A. Bonadonna R.C. Ferrannini E. Diabetes Care. 1992; 15: 318-368Crossref PubMed Scopus (1903) Google Scholar). The molecular mechanisms underlying the β-cell failure in T2DM and by which obesity promotes β-cell failure and intracellular amyloidogenesis remain elusive. type 2 diabetes mellitus endoplasmic reticulum high fat diet suppressor enhancer Lin12 1-like glucose-stimulated insulin secretion reverse tet transactivator phenylbutyric acid normal chow unfolded protein response. Accumulating evidence suggests that stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of T2DM (5Eizirik D.L. Cardozo A.K. Cnop M. Endocr. Rev. 2008; 29: 42-61Crossref PubMed Scopus (911) Google Scholar, 6Scheuner D. Kaufman R.J. Endocr. Rev. 2008; 29: 317-333Crossref PubMed Scopus (433) Google Scholar). The ER is a specialized organelle where nearly all secreted and membrane proteins undergo post-translational modifications such as glycosylation, disulfide bond formation, folding, and multimeric protein complex assembling (7Voeltz G.K. Rolls M.M. Rapoport T.A. EMBO Rep. 2002; 3: 944-950Crossref PubMed Scopus (387) Google Scholar, 8English A.R. Zurek N. Voeltz G.K. Curr. Opin. Cell Biol. 2009; 21: 596-602Crossref PubMed Scopus (139) Google Scholar). Properly folded/assembled proteins travel through the secretory pathway and reach various cellular destinations, whereas nonnative or unassembled proteins are retained in the ER lumen and eventually retrotranslocated into the cytosol for degradation by the proteasomal system (9Hampton R.Y. Curr. Opin. Cell Biol. 2002; 14: 476-482Crossref PubMed Scopus (404) Google Scholar). Disequilibrium of ER protein load and folding capacity results in accumulation of unfolded/misfolded proteins in the ER lumen, leading to ER stress (10Ma Y. Hendershot L.M. Cell. 2001; 107: 827-830Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). In response to ER stress, cells activate at least three intracellular signal transduction pathways, cumulatively referred to as the unfolded protein response (11Sundar Rajan S. Srinivasan V. Balasubramanyam M. Tatu U. Indian J. Med. Res. 2007; 125: 411-424PubMed Google Scholar). The unfolded protein response, coordinated by three distinct ER stress signal transducers, PERK, IRE1a, and ATF6 (12Ron D. Walter P. Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4929) Google Scholar), result in a general attenuation of protein translation (13Harding H.P. Novoa I. Bertolotti A. Zeng H. Zhang Y. Urano F. Jousse C. Ron D. Cold Spring Harbor Symp. Quant. Biol. 2001; 66: 499-508Crossref PubMed Scopus (41) Google Scholar) and an increased expression of ER chaperones and ER-associated degradation machinery (9Hampton R.Y. Curr. Opin. Cell Biol. 2002; 14: 476-482Crossref PubMed Scopus (404) Google Scholar, 14Brodsky J.L. Biochem. J. 2007; 404: 353-363Crossref PubMed Scopus (125) Google Scholar). Consequently, there is a reduced production of proteins that enter the ER, and there is an increase in the capacity of the ER to handle unfolded proteins. Together, these adaptive measures provide a multifaceted mechanism to maintain ER homeostasis under temporary and reversible ER stress conditions. ER homeostasis is particularly important for the function and viability of professional secretory cells such as the pancreatic β-cell (15Ortsäter H. Sjöholm A. Mol. Cell. Endocrinol. 2007; 277: 1-5Crossref PubMed Scopus (29) Google Scholar, 16van Anken E. Braakman I. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 269-283Crossref PubMed Scopus (56) Google Scholar). The unique function of the β-cell is to integrate nutrient signals into an appropriate insulin secretory rate to maintain normal glucose homeostasis. In response to serum glucose stimulus, β-cells secrete insulin from a readily available pool while activating proinsulin biosynthesis in the ER (17Straub S.G. Sharp G.W. Diabetes Metab. Res. Rev. 2002; 18: 451-463Crossref PubMed Scopus (324) Google Scholar). Proinsulin undergoes protein folding and disulfide bond formation in the lumen of the ER (17Straub S.G. Sharp G.W. Diabetes Metab. Res. Rev. 2002; 18: 451-463Crossref PubMed Scopus (324) Google Scholar). Properly folded proinsulin is then released to the Golgi apparatus and packaged into secretory granules, where conversion of proinsulin into mature insulin takes place (18Orci L. Ravazzola M. Amherdt M. Madsen O. Perrelet A. Vassalli J.D. Anderson R.G. J. Cell Biol. 1986; 103: 2273-2281Crossref PubMed Scopus (234) Google Scholar). Because of the rapid fluctuation of serum glucose levels in animals, it is essential that β-cells control proinsulin folding in the ER with exquisite sensitivity (19Lipson K.L. Fonseca S.G. Urano F. Curr. Mol. Med. 2006; 6: 71-77Crossref PubMed Scopus (57) Google Scholar). It has been suggested that conditions either evoking an overload of newly synthesized proinsulin or compromising protein folding capacity in the ER may negatively affect the homeostasis of β-cells, leading to ER stress and onset of T2DM (6Scheuner D. Kaufman R.J. Endocr. Rev. 2008; 29: 317-333Crossref PubMed Scopus (433) Google Scholar). Consistent with this, markers of ER stress have been shown to be up-regulated in the islets from T2DM patients (20Boden G. Duan X. Homko C. Molina E.J. Song W. Perez O. Cheung P. Merali S. Diabetes. 2008; 57: 2438-2444Crossref PubMed Scopus (358) Google Scholar, 21Huang C.J. Lin C.Y. Haataja L. Gurlo T. Butler A.E. Rizza R.A. Butler P.C. Diabetes. 2007; 56: 2016-2027Crossref PubMed Scopus (341) Google Scholar) and from several animal models of obesity and diabetes (22Laybutt D.R. Preston A.M. Akerfeldt M.C. Kench J.G. Busch A.K. Biankin A.V. Biden T.J. Diabetologia. 2007; 50: 752-763Crossref PubMed Scopus (660) Google Scholar, 23Matveyenko A.V. Gurlo T. Daval M. Butler A.E. Butler P.C. Diabetes. 2009; 58: 906-916Crossref PubMed Scopus (77) Google Scholar, 24Oyadomari S. Koizumi A. Takeda K. Gotoh T. Akira S. Araki E. Mori M. J. Clin. Invest. 2002; 109: 525-532Crossref PubMed Scopus (794) Google Scholar, 25Ozcan U. Cao Q. Yilmaz E. Lee A.H. Iwakoshi N.N. Ozdelen E. Tuncman G. Görgün C. Glimcher L.H. Hotamisligil G.S. Science. 2004; 306: 457-461Crossref PubMed Scopus (3010) Google Scholar). Suppressor enhancer lin12 1-like (Sel1l) is highly expressed in the developing and matured pancreatic cells (26Donoviel D.B. Donoviel M.S. Fan E. Hadjantonakis A. Bernstein A. Mech. Dev. 1998; 78: 203-207Crossref PubMed Scopus (47) Google Scholar, 27Biunno I. Appierto V. Cattaneo M. Leone B.E. Balzano G. Socci C. Saccone S. Letizia A. Della Valle G. Sgaramella V. Genomics. 1997; 46: 284-286Crossref PubMed Scopus (44) Google Scholar). Sel1l encodes an ER membrane protein (type I) with a complex domain structure (28Biunno I. Cattaneo M. Orlandi R. Canton C. Biagiotti L. Ferrero S. Barberis M. Pupa S.M. Scarpa A. Ménard S. J. Cell. Physiol. 2006; 208: 23-38Crossref PubMed Scopus (36) Google Scholar). Previous biochemical and molecular studies in vitro showed that SEL1L nucleates an ER membrane protein complex that is required for dislocation of unfolded or misfolded proteins from the ER lumen into the cytosol for degradation (29Cattaneo M. Otsu M. Fagioli C. Martino S. Lotti L.V. Sitia R. Biunno I. J. Cell. Physiol. 2008; 215: 794-802Crossref PubMed Scopus (35) Google Scholar, 30Cormier J.H. Tamura T. Sunryd J.C. Hebert D.N. Mol. Cell. 2009; 34: 627-633Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 31Hosokawa N. Wada I. Nagasawa K. Moriyama T. Okawa K. Nagata K. J. Biol. Chem. 2008; 283: 20914-20924Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 32Mueller B. Klemm E.J. Spooner E. Claessen J.H. Ploegh H.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12325-12330Crossref PubMed Scopus (189) Google Scholar, 33Mueller B. Lilley B.N. Ploegh H.L. J. Cell Biol. 2006; 175: 261-270Crossref PubMed Scopus (160) Google Scholar, 34Oresic K. Mueller B. Tortorella D. Biosci. Rep. 2009; 29: 173-181Crossref PubMed Scopus (16) Google Scholar). We recently reported that mice homozygous for a gene trap mutation in Sel1l develop systemic ER stress and die during mid-gestation (35Francisco A.B. Singh R. Li S. Vani A.K. Yang L. Munroe R.J. Diaferia G. Cardano M. Biunno I. Qi L. Schimenti J.C. Long Q. J. Biol. Chem. 2010; 285: 13694-13703Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In addition, we revealed that SEL1L deficiency impairs the growth and differentiation of pancreatic epithelial cells during early mouse embryonic development (36Li S. Francisco A.B. Munroe R.J. Schimenti J.C. Long Q. BMC Dev. Biol. 2010; 10: 19Crossref PubMed Scopus (17) Google Scholar). These genetic data are consistent with the hypothesis that SEL1L regulates ER homeostasis in mammalian cells by facilitating ER-associated degradation of unfolded proteins (32Mueller B. Klemm E.J. Spooner E. Claessen J.H. Ploegh H.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12325-12330Crossref PubMed Scopus (189) Google Scholar, 33Mueller B. Lilley B.N. Ploegh H.L. J. Cell Biol. 2006; 175: 261-270Crossref PubMed Scopus (160) Google Scholar). The physiological role of SEL1L in adult islets remains unclear. Here, we show that mice heterozygous for the gene-trap mutation in Sel1l (Sel1l+/−) are more susceptible to diet-induced hyperglycemia. Sel1l+/− mice are glucose intolerant and have a reduced β-cell mass. Cultured islets from Sel1l+/− mice show significantly higher expression of unfolded protein response marker genes than wild-type control islets. Thus, our data suggest that SEL1L is a critical regulator of β-cell function and growth in adult mice. Generation of Sel1l gene trap mice (Sel1l+/−) was described previously (35Francisco A.B. Singh R. Li S. Vani A.K. Yang L. Munroe R.J. Diaferia G. Cardano M. Biunno I. Qi L. Schimenti J.C. Long Q. J. Biol. Chem. 2010; 285: 13694-13703Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). For physiological studies, Sel1l+/− mice were back-crossed to C57/B6 mice for five generations and then intercrossed to generate wild-type and Sel1l+/− mice. All mice were weaned at 3 weeks of age, and genotyping was done by PCR using the following primers: F1-Sel1l, 5′-TGGGACAGAGCGGGCTTGGAAT-3′; R1-Sel1l, 5′-CACCAGGAGTCAAAGGCATCACTG-3′; R-βGeo, 5′-ATTCAGGCTGCGCAACTGTTGGG-3′. For high fat diet feeding experiments, wild-type and heterozygous male mice at 6–8 weeks of age were put on a TD.06414 diet (Harlan, Madison, WI), which contains 23.5, 27.3, and 34.3% protein, carbohydrate, and fat, respectively. All animal experiments were performed in accordance with the Cornell Animal Care and Use Guidelines. Fasting blood glucose was measured using an Ascensia Elite XL glucometer (Bayer). Glucose and insulin tolerance tests and glucose-stimulated insulin secretion (GSIS) were performed essentially as described (37Sachdeva M.M. Claiborn K.C. Khoo C. Yang J. Groff D.N. Mirmira R.G. Stoffers D.A. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 19090-19095Crossref PubMed Scopus (165) Google Scholar). For the glucose tolerance test, mice were fasted for 6–8 hours and then injected intraperitoneal with 2 g of d-glucose/kg body weight. Blood glucose was measured at 0, 5, 15, 30, 60, and 120 min after glucose injection. For the insulin tolerance test, mice were fasted for 6 hours and then injected intraperitoneally with 1.0 unit of regular human insulin (Eli Lilly, Indianapolis, IN)/kg body weight dissolved in phosphate-buffered saline (PBS). Blood glucose was measured at 0, 5, 15, 30, 60, and 120 min post-insulin injection. 4-Phenylbutyric acid (4-PBA) was administered into mice by gavage as previously described (38Ozcan U. Yilmaz E. Ozcan L. Furuhashi M. Vaillancourt E. Smith R.O. Görgün C.Z. Hotamisligil G.S. Science. 2006; 313: 1137-1140Crossref PubMed Scopus (2008) Google Scholar). Pancreata were fixed in 4% paraformaldehyde overnight at 4 °C and paraffin embedded. Pancreatic sections were cut at 5-μm and mounted on glass slides. Insulin immunostaining was performed by using guinea pig anti-human insulin (Linco, 1:1000). Secondary antibodies used were either Cy2- or HRP-conjugated donkey anti-guinea pig immunoglobulin (IgG) (Jackson ImmunoResearch Laboratories, 1:500). Nuclear counterstaining was performed using 4′,6-diamidino-2-phenylindole (DAPI). β-Cell mass was determined as previously described (37Sachdeva M.M. Claiborn K.C. Khoo C. Yang J. Groff D.N. Mirmira R.G. Stoffers D.A. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 19090-19095Crossref PubMed Scopus (165) Google Scholar). Briefly, total pancreatic and β-cell areas were measured using the AxioVision software (Version 4.1). β-Cell mass was calculated by multiplying the total pancreatic weight with the percentage of β-cell area. To determine β-cell proliferation, pancreatic sections were co-immunostained with a mouse monoclonal anti-Ki67 (Vector Laboratories, 1:500) and anti-insulin antibodies. Nuclei were counterstained with DAPI. The percentage of Ki67+ cells was derived by dividing the total number of counted β-cell nuclei with the number of Ki67+ nuclei. β-Cell apoptosis was assessed by TUNEL assay using the ApopTag In Situ Apoptosis detection kit (Chemicon, Temecula, CA) according to the manufacturer's instructions. All images were acquired using an Axiovert 40 microscope (Zeiss) equipped with an AxioCam camera. Pancreatic insulin content was determined as previously described (39Gupta S. McGrath B. Cavener D.R. Diabetes. 2010; 59: 1937-1947Crossref PubMed Scopus (105) Google Scholar). Briefly, pancreata were removed from mice and homogenized in 1 ml of acid ethanol (95% ethanol, 10.2 n HCl in a 50:1 ratio). The homogenized pancreata were incubated overnight at 4 °C and centrifuged. Insulin concentration in the supernatant was determined using ELISA (Crystal Chem, Downers Grove, IL) and normalized by total protein content. Mouse islet isolation was performed as previously described (40Scharp D.W. Kemp C.B. Knight M.J. Ballinger W.F. Lacy P.E. Transplantation. 1973; 16: 686-689Crossref PubMed Scopus (206) Google Scholar). Briefly, after sacrifice of each mouse, the pancreas was perfused with 1× Hanks' balanced salt solution, pH 7.4, containing 2.0 mg/ml type V collagenase (Sigma). The inflated pancreas was removed from the body and incubated at 37 °C for 20 min. The collagenase-digested pancreas was vigorously shaken for 5 s, washed 3 times with 5–7 ml of ice-cold 1× Hanks' balanced salt solution buffer, and re-suspended in 2 ml of 28% Ficoll (VWR, West Chester, PA). The islet and acinar mixture was loaded onto the top of gradient Ficoll solutions and centrifuged at 2250 rpm for 7 min at 4 °C. Islets were collected and washed three times with ice-cold 1× Hanks' balanced salt solution before processing for further analysis. GSIS for islets was performed as previously described with minor modifications (41Zhang W. Feng D. Li Y. Iida K. McGrath B. Cavener D.R. Cell Metab. 2006; 4: 491-497Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Briefly, isolated islets were washed twice with 1× KRBH buffer (118.5 mm NaCl, 2.54 mm CaCl2·2H2O, 1.19 mm KH2PO4, 4.74 mm KCl, 25 mm NaHCO3, 1.19 mm MgSO4·7H2O, 10 mm HEPES, 0.1% BSA, 5 mm glutamic acid, 5 mm fumaric acid, 5 mm pyruvic acid, pH 7.4) and equilibrated in the same buffer containing 2.8 mm glucose for 60 min at 37 °C. Islets were then incubated in 1× KRBH buffer containing either 2.8 or 16.8 mm glucose for 30 min at 37 °C. The low and high glucose-incubated islets were briefly centrifuged, and the supernatants were assayed for secreted insulin, which was normalized to islet insulin content. Islet insulin content was normalized to the total protein concentration. RNA isolation and real-time RT-PCR were performed as previously described (35Francisco A.B. Singh R. Li S. Vani A.K. Yang L. Munroe R.J. Diaferia G. Cardano M. Biunno I. Qi L. Schimenti J.C. Long Q. J. Biol. Chem. 2010; 285: 13694-13703Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). PCR primers were designed using the PrimerSelect program of Lasergene 7.1 Sequence Analysis Software (DNAStar, Madison, WI). Relative mRNA expression was calculated by dividing the expression value in Sel1l+/− islets (set to 1) with that in wild-type islets. Min6 cells were maintained as previously described (42Miyazaki J. Araki K. Yamato E. Ikegami H. Asano T. Shibasaki Y. Oka Y. Yamamura K. Endocrinology. 1990; 127: 126-132Crossref PubMed Scopus (1060) Google Scholar). For establishing stable cell lines, Min6 cells were grown to 50–60% confluency, trypsinized, washed three times in cold PBS, and resuspended in PBS. 5.6 × 106/ml was mixed with 10–20 μg of plasmid DNA, electroporated (0.24 kV and 500 microfarads) and plated into a 24-well plate. The electroporated Min6 cells were incubated in regular media for 48 h and then in neomycin resistance selection medium (G418 at 1 mg/ml) for 10 days. Stable Min6 clones were maintained in regular medium. GSIS for Min6 cells was performed essentially as previously described (43Dowling P. O'Driscoll L. O'Sullivan F. Dowd A. Henry M. Jeppesen P.B. Meleady P. Clynes M. Proteomics. 2006; 6: 6578-6587Crossref PubMed Scopus (48) Google Scholar). INS-1 cells were maintained as previously described (44Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (480) Google Scholar). For generation of stable cell lines, INS-1 cells were seeded at a confluency of <10% and infected overnight with lentivirus expressing the reverse tet transactivator (rtTA), rtTA plus SEL1L-GFP, or rtTA plus dSEL1L-GFP. To induced expression of transgenes, doxycycline (Sigma) was added into the culture medium of Min6 and INS-1 cells (200 μg/ml). Transient transfection and Gaussia luciferase (G-luc) assay in Min6 cells were performed as previously described (35Francisco A.B. Singh R. Li S. Vani A.K. Yang L. Munroe R.J. Diaferia G. Cardano M. Biunno I. Qi L. Schimenti J.C. Long Q. J. Biol. Chem. 2010; 285: 13694-13703Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Cell growth profile was generated, and thymidine incorporation assay was performed as previously described (45Li S. Francisco A.B. Han C. Pattabiraman S. Foote M.R. Giesy S.L. Wang C. Schimenti J.C. Boisclair Y.R. Long Q. FEBS Lett. 2010; 584: 4121-4127Crossref PubMed Scopus (15) Google Scholar). Preparation of tissue/cell lysates, protein quantification, electrophoresis, and blotting were performed as previously described (35Francisco A.B. Singh R. Li S. Vani A.K. Yang L. Munroe R.J. Diaferia G. Cardano M. Biunno I. Qi L. Schimenti J.C. Long Q. J. Biol. Chem. 2010; 285: 13694-13703Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Immunodetection was carried out using the Western blotting Luminol Reagent kit (Origene) according to the manufacturer's specifications. The primary antibodies used were: GFP (Abcam, 1:5,000), tubulin (Cell Signaling, 1:10,000), calnexin (Assay Design, 1:10,000), p-eIF2a (Cell Signaling, 1:2000), eIF2a (Cell Signaling, 1:2,000), ERP57 (Assay Design, 1:2,000), PDI (Assay Design, 1:5,000), HRD1 (Novus Biologicals, 1:1,000), ERO1L (Novus Biologicals, 1:2,000) and GRP78 (Santa Cruz, 1:1,000). Differences between compared groups were evaluated by performing two-tailed Student's t test, and p < 0.05 is considered significant. We recently reported that mice homozygous for a gene trap mutation in Sel1l (Sel1l−/−) develop systemic ER stress and die during mid-gestation (35Francisco A.B. Singh R. Li S. Vani A.K. Yang L. Munroe R.J. Diaferia G. Cardano M. Biunno I. Qi L. Schimenti J.C. Long Q. J. Biol. Chem. 2010; 285: 13694-13703Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To determine the functional role of SEL1L in adult pancreatic β-cells, we investigated whether haploid insufficiency of SEL1L affects glucose homeostasis in mice. Wild-type and Sel1l+/− male mice at 10 weeks of age were fed with normal chow (NC) and a high fat diet (HFD) for 20 weeks (Fig. 1A). Sel1l+/− and wild-type mice showed similar body weight gain profiles in response to the two diets (Fig. 1B). Basal fasting blood glucose levels of Sel1l+/− and wild-type mice were comparable (supplemental Fig. S1A) and remained insignificantly changed in response to NC feeding (Fig. 1C). However, after 8 weeks of HFD, Sel1l+/− mice showed significantly higher blood glucose levels than the wild-type control mice (Fig. 1B). These results indicate that mice with one functional allele of Sel1l are genetically predisposed to HFD-induced hyperglycemia. Hyperglycemia in mice may be the result of a defective insulin production, action, or both. To determine the specific causes underlying the impaired glucose homeostasis in Sel1l+/− mice, we first assessed whether haploid insufficiency of SEL1L reduces insulin sensitivity in organs such as liver, skeletal muscle, and adipose tissue. Insulin tolerance tests showed that Sel1l+/− mice showed comparable insulin sensitivity to wild-type mice (Fig. 1D), suggesting that haploinsufficiency of SEL1L did not significantly affect peripheral insulin signaling. Next, we examined whether Sel1l+/− mice had an impaired β-cell function using glucose tolerance tests. Sel1l+/− and wild-type mice fed with NC showed similar glucose tolerance to wild-type control mice (supplemental Fig. S1B). However, Sel1l+/− mice fed with HFD exhibited significantly higher glucose intolerance than wild-type control mice (Fig. 1E). Administration of 4-PBA, a chemical chaperone previously shown to enhance insulin signaling (38Ozcan U. Yilmaz E. Ozcan L. Furuhashi M. Vaillancourt E. Smith R.O. Görgün C.Z. Hotamisligil G.S. Science. 2006; 313: 1137-1140Crossref PubMed Scopus (2008) Google Scholar), resulted in a significant reduction of serum glucose levels in both Sel1l+/− and wild-type mice fed with HFD (Fig. 1F). Together, these data suggest that in our current diet-induced obesity model, haploid insufficiency of Sel1l impairs insulin production in pancreatic β-cells and had little effect on insulin action in peripheral organs. We next assessed the serum insulin levels in Sel1l+/− mice fed with NC and HFD using insulin ELISA. No significant difference in serum insulin level was observed between Sel1l+/− and wild-type mice fed with NC. However, Sel1l+/− mice fed with HFD showed significantly higher serum insulin levels than wild-type control mice (Fig. 2A). The pancreatic insulin contents of wild-type and Sel1l+/− mice fed with HFD were comparable (supplemental Fig. S2A). We next carried out glucose-stimulated insulin secretion studies in Sel1l+/− mice. Although Sel1l+/− mice fed with NC showed no difference in GSIS to wild-type control mice (Fig. 2C), Sel1l+/− mice fed with HFD showed a significantly lower GSIS as compared with wild-type mice (Fig. 2B). These results together with the finding that Sel1l+/− mice were glucose-intolerant (Fig. 1E) indicate that haploid insufficiency of SEL1L also causes impairment in glucose-stimulated insulin secretion. The functional β-cell mass is a critical factor in determining how much insulin is released into the blood in response to glucose stimulation. As Sel1l+/− mice showed an impaired pancreatic function, we speculated that Sel1l+/− mice fed with HFD might have a reduced β-cell mass. To test this, we carried out morphological analysis of the endocrine pancreas in Sel1l+/− fed with HFD. Wild-type and Sel1l+/− mice on HFD for 8 weeks showed no difference in β-cell masses (Fig. 2C). However, after 20 weeks of HFD-feeding, Sel1l+/− mice exhibited a markedly reduced β-cell mass as compared with that of wild-type mice (Fig. 2, D–F). We then investigated whether the reduction of β-cell mass in Sel1l+/− mice was due to an increased apoptosis or a decreased β-cell proliferation. In situ cell apoptosis assays showed that there was no significant difference in β-cell apoptosis between Sel1l+/− and wild-type mice (supplemental Fig. S2B). However, Sel1l+/− islets contained a significantly lower number of Ki67-positive β-cells than wild-type islets (Fig. 2, G–I). Together, these results indicate that Sel1l+/− mice fed with HFD for 20 weeks had a reduced β-cell mass, and the reduction of β-cell mass in Sel1l+/− mice was due at least in part to an impaired compensatory β-cell growth. SEL1L is a critical component of the ER-associated protein degradation machinery, and SEL1L deficiency results in systemic ER stress in mouse embryonic cells (35Francisco A.B. Singh R. Li S. Vani A.K. Yang L. Munroe R.J. Diaferia G. Cardano M. Biunno I. Qi L. Schimenti J.C. Long Q. J. Biol. Chem. 2010; 285: 13694-13703Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We, therefore, next investigated possible alterations of the UPR pathway in liver and pancreatic islets of Sel1l+/− mice fed with HFD. First, we cultured islets isolated from wild-type and Sel1l+/− mice in the presence of low (10 mm) and high (30 mm) glucose and performed quantitative RT-PCR analysis of UPR marker genes. Wild-type and Sel1l+/− islets treated with low glucose showed comparable expression of Bip, Herp, Chop, Xbp-1s, and p58IPK at the mRNA level (Fig. 3A). However, Bip, Herp, Chop, and Xbp-1s were significantly up-regulated in high glucose-treated Sel1l+/− islets as opposed to wild-type control islets (Fig. 3B). Treatment of Sel1l+/− pancreatic islets with 30 mm glucose in the presence of the chemical chaperon 4-PBA markedly reduced the transcription of Bip, Herp, Xbp-1s, and p58IPK (Fig. 3C). ER stress has previously been mechanistically linked to obesity-induced insulin resistance in mice (25Ozcan U. Cao Q. Yilmaz E. Lee A.H. Iwakoshi N.N. Ozdelen E. Tuncman G. Görgün C. Glimcher L.H. Hotamisligil G.S. Science. 2004; 306: 457-461Crossref PubMed Scopus (3010) Google Scholar). Because Sel1l+/− mice were modestly insulin-intolerant (Fig. 1D), we speculated that this might be due to low levels of ER stress in peripheral organs such as liver and adipose tissue. To test this, we prepared lysates from livers of Sel1l+/− and wild-type control mice fed with HFD and examined the expression of a number of UPR genes through Western blot analyses (Fig. 3D). The liver of" @default.
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• W1978759169 title "Haploid Insufficiency of Suppressor Enhancer Lin12 1-like (SEL1L) Protein Predisposes Mice to High Fat Diet-induced Hyperglycemia" @default.
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