Insulin Receptor Substrate 1 Regulation of Sarco-endoplasmic Reticulum Calcium ATPase 3 in Insulin-secreting β-Cells
2003; Elsevier BV; Volume: 278; Issue: 13 Linguagem: Inglês
10.1074/jbc.m209521200
ISSN1083-351X
AutoresPrabhakar D. Borge, Bryan A. Wolf,
Tópico(s)Diabetes and associated disorders
ResumoWe have previously characterized an insulin receptor substrate 1 (IRS-1)-overexpressing β-cell line. These β-cells demonstrated elevated fractional insulin secretion and elevated cytosolic Ca2+ levels compared with wild-type and vector controls. This effect of IRS-1 may be mediated via an interaction with the sarco-endoplasmic reticulum calcium ATPase (SERCA). Here we demonstrate that IRS-1 and IRS-2 localize to an endoplasmic reticulum (ER)-enriched fraction in β-cells using subcellular fractionation. We also observe co-localization of both IRS-1 and IRS-2 with ER marker proteins using immunofluorescent confocal microscopy. Furthermore, immuno-electron microscopy studies confirm that IRS-1 and SERCA3b localize to vesicles derived from the ER. In Chinese hamster ovary-T (CHO-T) cells transiently transfected with SERCA3b alone or together with IRS-1, SERCA3b co-immunoprecipitates with IRS-1. This interaction is enhanced with insulin treatment. SERCA3b also co-immunoprecipitates with IRS-1 in wild-type and IRS-1-overexpressing β-cell lines. Ca2+uptake in ER-enriched fractions prepared from wild-type and IRS-1-overexpressing cell lines shows no significant difference, indicating that the previously observed decrease in Ca2+uptake by IRS-1-overexpressing cells is not the result of a defect in SERCA. Treatment of wild-type β-cells with thapsigargin, an inhibitor of SERCA, resulted in an increase in glucose-stimulated fractional insulin secretion similar to that observed in IRS-1-overexpressing cells. The colocalization of IRS proteins and SERCA in the ER of β-cells increases the likelihood that these proteins can interact with one another. Co-immunoprecipitation of IRS-1 and SERCA in CHO-T cells and β-cells confirms that these proteins do indeed interact directly. Pharmacological inhibition of SERCA in β-cells results in enhanced secretion of insulin. Taken together, our data suggest that interaction between IRS proteins and SERCA is an important regulatory step in insulin secretion. We have previously characterized an insulin receptor substrate 1 (IRS-1)-overexpressing β-cell line. These β-cells demonstrated elevated fractional insulin secretion and elevated cytosolic Ca2+ levels compared with wild-type and vector controls. This effect of IRS-1 may be mediated via an interaction with the sarco-endoplasmic reticulum calcium ATPase (SERCA). Here we demonstrate that IRS-1 and IRS-2 localize to an endoplasmic reticulum (ER)-enriched fraction in β-cells using subcellular fractionation. We also observe co-localization of both IRS-1 and IRS-2 with ER marker proteins using immunofluorescent confocal microscopy. Furthermore, immuno-electron microscopy studies confirm that IRS-1 and SERCA3b localize to vesicles derived from the ER. In Chinese hamster ovary-T (CHO-T) cells transiently transfected with SERCA3b alone or together with IRS-1, SERCA3b co-immunoprecipitates with IRS-1. This interaction is enhanced with insulin treatment. SERCA3b also co-immunoprecipitates with IRS-1 in wild-type and IRS-1-overexpressing β-cell lines. Ca2+uptake in ER-enriched fractions prepared from wild-type and IRS-1-overexpressing cell lines shows no significant difference, indicating that the previously observed decrease in Ca2+uptake by IRS-1-overexpressing cells is not the result of a defect in SERCA. Treatment of wild-type β-cells with thapsigargin, an inhibitor of SERCA, resulted in an increase in glucose-stimulated fractional insulin secretion similar to that observed in IRS-1-overexpressing cells. The colocalization of IRS proteins and SERCA in the ER of β-cells increases the likelihood that these proteins can interact with one another. Co-immunoprecipitation of IRS-1 and SERCA in CHO-T cells and β-cells confirms that these proteins do indeed interact directly. Pharmacological inhibition of SERCA in β-cells results in enhanced secretion of insulin. Taken together, our data suggest that interaction between IRS proteins and SERCA is an important regulatory step in insulin secretion. insulin receptor insulin receptor substrate(s) sarco-endoplasmic reticulum calcium ATPase endoplasmic reticulum intracellular membrane high speed pellet Dulbecco's modified Eagle's medium plasma membrane pellet secretory vesicle and mitochondria pellet cytosol Krebs-Ringer buffer Texas Red cyanine 2 phosphate-buffered saline bovine serum albumin Tris-buffered saline with Tween 20 fetal bovine serum 4-morpholineethanesulfonic acid The pancreatic β-cell plays a key role in glucose homeostasis by secretion of the hormone insulin. The first step in insulin secretion is the metabolism of glucose in β-cells (1Hedeskov C.J. Physiol. Rev. 1980; 60: 442-509Google Scholar, 2Matschinsky F.M. Diabetes. 1996; 45: 223-241Google Scholar, 3Newgard C.B. McGarry J.D. Annu. Rev. Biochem. 1995; 64: 689-719Google Scholar). Glucose enters the β-cell through GLUT2 transporters on the plasma membrane. Glucose metabolism begins with glucokinase, the β-cell glucose sensor (4Matschinsky F. Liang Y. Kesavan P. Wang L. Froguel P. Velho G. Cohen D. Permutt M.A. Tanizawa Y. Jetton T.L. Niswender K. Magnuson M.A. J. Clin. Invest. 1993; 92: 2092-2098Google Scholar), and results in an increase in the intracellular ATP/ADP ratio. This causes closure of ATP-dependent K+ channels and β-cell plasma membrane depolarization. The depolarization event leads to Ca2+ influx through voltage-gated L-type Ca2+ channels (5Dukes I.D. Philipson L.H. Diabetes. 1996; 45: 845-853Google Scholar, 6Henquin J.C. Jonas J.C. Gilon P. Diabetes Metab. 1998; 24: 30-36Google Scholar). Increased cytosolic Ca2+ stimulates insulin exocytosis by the β-cell through Ca2+-dependent protein kinase pathways. Insulin acts on a variety of tissues by binding to the insulin receptor (IR).1 Insulin binds to the dimerized insulin receptor and causes activation of the catalytic tyrosine kinase in the IR β-subunit. The IR tyrosine kinase then autophosphorylates intracellular tyrosine residues on the insulin receptor itself, which act as docking sites for insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and Shc. These proteins are then phosphorylated by the insulin receptor and interact with several downstream effectors including phosphoinositide 3-kinase and Grb2. These effectors mediate glucose transport, cell growth, and various other important cellular functions. A growing body of evidence has confirmed that the insulin receptor-signaling pathway is active in the pancreatic β-cell and is involved in regulating key cellular processes (7Xu G.G. Gao Z.Y. Borge Jr., P.D. Wolf B.A. J. Biol. Chem. 1999; 274: 18067-18074Google Scholar, 8Rothenberg P.L. Willison L.D. Simon J. Wolf B.A. Diabetes. 1995; 44: 802-809Google Scholar, 9Harbeck M.C. Louie D.C. Howland J. Wolf B.A. Rothenberg P.L. Diabetes. 1996; 45: 711-717Google Scholar, 10Aspinwall C.A. Lakey J.R. Kennedy R.T. J. Biol. Chem. 1999; 274: 6360-6365Google Scholar, 11Kulkarni R.N. Bruning J.C. Winnay J.N. Postic C. Magnuson M.A. Kahn C.R. Cell. 1999; 96: 329-339Google Scholar, 12Kwon G. Xu G. Marshall C.A. McDaniel M.L. J. Biol. Chem. 1999; 274: 18702-18708Google Scholar, 13Leibiger I.B. Leibiger B. Moede T. Berggren P.O. Mol. Cell. 1998; 1: 933-938Google Scholar, 14Marshall B.A. Tordjman K. Host H.H. Ensor N.J. Kwon G. Marshall C.A. Coleman T. McDaniel M.L. Semenkovich C.F. J. Biol. Chem. 1999; 274: 27426-27432Google Scholar, 15Xu G. Kwon G. Marshall C.A. Lin T.A. Lawrence J.C.J. McDaniel M.L. J. Biol. Chem. 1998; 273: 28178-28184Google Scholar, 16Xu G. Marshall C.A. Lin T.A. Kwon G. Munivenkatappa R.B. Hill J.R. Lawrence J.C.J. McDaniel M.L. J. Biol. Chem. 1998; 273: 4485-4491Google Scholar). Insulin stimulation of the β-cell IR results in tyrosine phosphorylation of the catalytic IR β-subunit and IRS proteins (8Rothenberg P.L. Willison L.D. Simon J. Wolf B.A. Diabetes. 1995; 44: 802-809Google Scholar). Mice with a pancreatic β-cell-specific knockout of IR exhibit hyperinsulinemia and impaired glucose tolerance that develops after 6 months (11Kulkarni R.N. Bruning J.C. Winnay J.N. Postic C. Magnuson M.A. Kahn C.R. Cell. 1999; 96: 329-339Google Scholar). In addition, these mice lose their acute first-phase glucose-stimulated insulin secretion response. The loss of IRS-1 leads to mild insulin resistance, hyperinsulinemia, and β-cell hyperplasia but no overt diabetic phenotype (17Withers D.J. Gutierrez J.S. Towery H. Burks D.J. Ren J.M. Previs S. Zhang Y. Bernal D. Pons S. Shulman G.I. Bonner-Weir S. White M.F. Nature. 1998; 391: 900-904Google Scholar, 18Araki E. Haag III, B.L. Kahn C.R. Biochim. Biophys. Acta. 1994; 1221: 353-356Google Scholar, 19Kahn B.B. Cell. 1998; 92: 593-596Google Scholar, 20Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Nature. 1994; 372: 182-186Google Scholar). Islets and β-cells derived from these IRS-1 knockout mice show decreased insulin content of 51 and 55%, respectively, and a lower glucose-stimulated insulin secretion response (21Kulkarni R.N. Winnay J.N. Daniels M. Bruning J.C. Flier S.N. Hanahan D. Kahn C.R. J. Clin. Invest. 1999; 104: R69-R75Google Scholar). In contrast, the loss of IRS-2 in mice causes early insulin resistance and eventually a decrease in β-cell mass, β-cell failure, and overt type II diabetes (17Withers D.J. Gutierrez J.S. Towery H. Burks D.J. Ren J.M. Previs S. Zhang Y. Bernal D. Pons S. Shulman G.I. Bonner-Weir S. White M.F. Nature. 1998; 391: 900-904Google Scholar, 22Withers D.J. Burks D.J. Towery H.H. Altamuro S.L. Flint C.L. White M.F. Nat. Genet. 1999; 23: 32-40Google Scholar). Analysis of islets and β-cells from IRS-2 knockout mice revealed no secretory defects, but altered β-cell growth (23Bonner-Weir S. Withers D.J. Weir G.C. Jonas J.C. Diabetes. 1999; 48: A3Google Scholar). This difference in phenotype of IRS knockouts indicates that these substrates mediate different functions in the β-cell. The mechanisms by which IRS-1 and IRS-2 accomplish these roles remain unclear. Two studies that examined the role of IRS-1 in insulin secretion from isolated pancreatic β-cells and β-cell lines found that IRS-1 also has an effect on calcium homeostasis. In one study, the overexpression of IRS-1 in the βTC6-F7 cell line (β-IRS1) lead to an increase in the glucose-stimulated fractional insulin secretion (ratio of secreted insulin/total insulin content) and, interestingly, an increase in cytosolic Ca2+ (7Xu G.G. Gao Z.Y. Borge Jr., P.D. Wolf B.A. J. Biol. Chem. 1999; 274: 18067-18074Google Scholar). This increase in cytosolic Ca2+ was also seen in a cell line overexpressing the IR, but not in a cell line overexpressing a kinase-deficient mutant of IR. Further investigation revealed that the increase in cytosolic Ca2+ was the result of inhibition of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), a protein responsible for Ca2+ uptake into the ER lumen. When the control β-cell line was treated with thapsigargin, a SERCA inhibitor, the cytosolic Ca2+ levels increased to the same level as the β-IRS1 cell line. Furthermore, ER calcium uptake in a digitonin-permeabilized cell system was reduced in the β-IRS1 cells compared with controls. In another study, insulin exocytosis and intracellular Ca2+levels of single β-cells from IRS-1-deficient mouse islets and IRS-1 null β-cell lines were measured (24Aspinwall C.A. Qian W.J. Roper M.G. Kulkarni R.N. Kahn C.R. Kennedy R.T. J. Biol. Chem. 2000; 275: 22331-22338Google Scholar). In the absence of IRS-1, insulin exocytosis and cytosolic calcium were decreased. Taken together, these data indicated that some mechanism mediated by the activation of IRS-1 through the insulin receptor affects the influx of Ca2+ into the ER, resulting in changes in Ca2+homeostasis and insulin secretion. One potential explanation for this effect is that IRS-1 interacts directly with SERCA and inhibits its function. For this to occur, activated IRS-1 must first be in the same vicinity as SERCA. Previous studies examining the subcellular distribution of IRS proteins in 3T3-L1 adipocyte cell lines have shown that both IRS-1 and IRS-2 distribute to the cytosol and intracellular membranes (IM) of cells, but the majority is located within the IM (25Kublaoui B. Lee J. Pilch P.F. J. Biol. Chem. 1995; 270: 59-65Google Scholar, 26Wang B. Balba Y. Knutson V.P. Biochem. Biophys. Res. Commun. 1996; 227: 27-34Google Scholar, 27Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Google Scholar, 28Ricort J.M. Tanti J.F. Van Obberghen E. Marchand-Brustel Y. Eur. J. Biochem. 1996; 239: 17-22Google Scholar, 29Kelly K.L. Ruderman N.B. J. Biol. Chem. 1993; 268: 4391-4398Google Scholar, 30Yang J. Clarke J.F. Ester C.J. Young P.W. Kasuga M. Holman G.D. Biochem. J. 1996; 313: 125-131Google Scholar, 31Nave B.T. Haigh R.J. Hayward A.C. Siddle K. Shepherd P.R. Biochem. J. 1996; 318: 55-60Google Scholar, 32Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Google Scholar, 33Anai M. Ono H. Funaki M. Fukushima Y. Inukai K. Ogihara T. Sakoda H. Onishi Y. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1998; 273: 29686-29692Google Scholar). Studies using ultracentrifugation, sucrose density gradients, and detergent precipitation, demonstrate that IRS proteins localize to a high speed pellet (HSP) fraction that contains the endoplasmic reticulum and the trans-Golgi network (31Nave B.T. Haigh R.J. Hayward A.C. Siddle K. Shepherd P.R. Biochem. J. 1996; 318: 55-60Google Scholar, 32Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Google Scholar, 33Anai M. Ono H. Funaki M. Fukushima Y. Inukai K. Ogihara T. Sakoda H. Onishi Y. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1998; 273: 29686-29692Google Scholar, 34Phung T.L. Roncone A. Jensen K.L. Sparks C.E. Sparks J.D. J. Biol. Chem. 1997; 272: 30693-30702Google Scholar, 35Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Google Scholar, 36VanRenterghem B. Morin M. Czech M.P. Heller-Harrison R.A. J. Biol. Chem. 1998; 273: 29942-29949Google Scholar, 37Clark S.F. Molero J.C. James D.E. J. Biol. Chem. 2000; 275: 3819-3826Google Scholar). Upon stimulation with insulin, IRS-1 is preferentially tyrosine-phosphorylated in the intracellular membrane compartment, where it remains for several minutes before translocating to the cytosol (32Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Google Scholar). The distribution of IRS-1 to the same intracellular membrane as SERCA, namely the ER, lends credence to the idea that these two proteins can interact. In addition, there is evidence for a direct interaction of IRS and SERCA proteins. In rat muscle extracts, IRS-1 and IRS-2 were shown to co-immunoprecipitate with SERCA1 and SERCA2a, the skeletal and cardiac muscle isoforms of SERCA (38Algenstaedt P. Antonetti D.A. Yaffe M.B. Kahn C.R. J. Biol. Chem. 1997; 272: 23696-23702Google Scholar). Insulin stimulation of the rats resulted in a 2–6-fold increase in the association of IRS proteins with SERCA. IRS-1 and IRS-2 also bound to the ubiquitously expressed SERCA2b isoform, which is also co-expressed with SERCA3 in pancreas (39Váradi A. Molnár E. Östenson C.G. Ashcroft S.J.H. Biochem. J. 1996; 319: 521-527Google Scholar, 40Xu G.G. Gao Z.Y. Borge Jr., P.D. Jegier P.A. Young R.A. Wolf B.A. Biochemistry. 2000; 39: 14912-14919Google Scholar). In this current study, we have employed several different techniques to show that IRS-1 and IRS-2 proteins localize to intracellular membranes in pancreatic β-cell lines. More specifically, IRS-1 co-localizes with SERCA3b, one of two SERCA isoforms present in β-cells, to endoplasmic reticulum-derived microsomes prepared from β-cells. SERCA3b has also been shown to co-immunoprecipitate with IRS-1 in a model cell line and β-cell lines. This interaction is enhanced with acute insulin stimulation of the cells. Examination of in vitro ER Ca2+ uptake in both wild-type and IRS-1-overexpressing β-cells demonstrates that the β-cell SERCA isoforms function normally and respond to inhibition with thapsigargin in the same fashion. Finally, treatment of wild-type β-cells with thapsigargin resulted in an increase in glucose-stimulated fractional insulin secretion comparable with the increase observed for untreated IRS-1-overexpressing β-cells. These results provide a functional link between the direct IRS-1/SERCA interaction and regulation of calcium homeostasis and insulin secretion. The β-IRS1 cell line and clonal mouse β-cell line βTC6-F7 were cultured as previously described (7Xu G.G. Gao Z.Y. Borge Jr., P.D. Wolf B.A. J. Biol. Chem. 1999; 274: 18067-18074Google Scholar). In brief, cells were maintained in high glucose DMEM (25 mm glucose; Invitrogen) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 units/ml penicillin, 50 μg/ml streptomycin (complete DMEM) and incubated at 37 °C in a 10% CO2, 90% air humidified incubator. Both cell lines were passaged every 7–10 days and seeded in new T-175 flasks (Falcon, catalog no. 353112) at a cell density of 1:10 (1.0–2.0 × 106 cells/T-175 flask) as follows. The cell lines were washed with 10 ml of Versene and then incubated with 2 ml of 0.05% trypsin-EDTA (Invitrogen, catalog no. 25300-054) for 5 min at 37 °C in a 10% CO2, 90% air humidified incubator. The cells were then rinsed with complete DMEM, pelleted, and re-suspended in complete DMEM. Cells were counted and seeded into T-175 flasks as described above. The Chinese hamster ovary-T cell line (CHO-T), which stably expresses the human insulin receptor, was a kind gift from Dr. R. Roth (Stanford University School of Medicine, Stanford, CA). This cell line was cultured in F-12 (Ham's) medium (Invitrogen) supplemented with 5% fetal bovine serum, 100 units/ml penicillin, 50 μg/ml streptomycin (complete F-12) and incubated at 37 °C in a 5% CO2, 95% air humidified incubator. The pMT2-SERCA3b expression plasmid was a kind gift from Dr. F. Wuytack (Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Leuven, Belgium). The pCMV-IRS1 was constructed as described previously (7Xu G.G. Gao Z.Y. Borge Jr., P.D. Wolf B.A. J. Biol. Chem. 1999; 274: 18067-18074Google Scholar). A rabbit polyclonal antibody directed against the C terminus of SERCA3b was prepared as follows. A C-terminal peptide of SERCA3b (sequence =C-TGKKGPEVNPGSRGES) that is unique to the SERCA3b splice variant was synthesized by the Howard Hughes Medical Institute Biopolymer Facility at Yale University (W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, CT) containing an additional N-terminal cysteine (in bold). The peptide was conjugated to the Imject® maleimide-activated SuperCarrier® modulator (Pierce catalog no. 77656) according to the protocol from the manufacturer. The peptide conjugate was then injected into 2 rabbits (Covance Research Products, Inc.) at an initial dose of 0.5 mg followed by a booster dose of 0.25 mg every 21 days for the duration of the study. Bleeds were collected 10 days after each booster dose, and the sera were shipped frozen to our laboratory. The sera were screened for antibody production by Western blot analysis using CHO-T cell lysate from cells transfected with or without the pMT2-SERCA3b plasmid (described below) as positive and negative controls. Sera from bleeds containing SERCA3b antibody were affinity-purified using the Sulfo-Link® kit (Pierce catalog no. 44895) as follows. The SERCA3b peptide antigen was dissolved in Sulfo-Link® Coupling buffer (50 mm Tris, 5 mm EDTA-Na, pH 8.5) and coupled to a column containing Sulfo-Link® Coupling gel via stable disulfide bonding. Excess free sulfhydryl groups were blocked with the addition of 0.05 m cysteine in Coupling buffer to the column. After washing away excess peptide and cysteine, SERCA3b antibody-containing serum was applied to the column and incubated at room temperature for 1 h. The column was washed, and then the SERCA3b antibody was eluted with 100 mm glycine, pH 2.75. Fractions (1 ml) were collected and neutralized by adding 50 μl of 1m Tris, pH 9.5. Fractions containing the SERCA3b antibody (determined by A 280 measured spectrophotometrically and Bio-Rad protein assay) were pooled and dialyzed in PBS with 0.05% sodium azide overnight. The concentration of the SERCA3b antibody solution was determined by Bio-Rad protein assay and A 280 measurements. The solution was aliquoted and frozen at −20 °C. Subcellular fractions were prepared using a modification of the method described by Colca et al. (41Colca J.R. McDonald J.M. Kotagal N. Patke C. Fink C.J. Greider M.H. Lacy P.E. McDaniel M.L. J. Biol. Chem. 1982; 257: 7223-7228Google Scholar). Both the βTC6-F7 and β-IRS1 were seeded on 15-cm dishes (Falcon, catalog no. 353025) at a cell density of 2.0 × 106 cells/dish and cultured for 7 days as described above. Cells were homogenized on ice in 50 mmMES, pH 7.2, 250 mm sucrose, 1 mm EDTA buffer (Fractionation buffer). The homogenate was centrifuged at 600 ×g for 5 min to yield pellet PM, containing plasma membrane, nuclear material, and debris. The supernatant was then centrifuged at 20,000 × g for 20 min to yield a secretory vesicle and mitochondria-enriched pellet (SV). The resulting supernatant was centrifuged at 150,000 × g for 90 min to yield a HSP, enriched with IM and cytoskeleton. The HSP fraction was re-suspended in fractionation buffer without EDTA. The remaining supernatant, called CYT, contains cytosol. ER enrichment of the HSP was verified by comparing the activity of the ER marker enzyme, NAPDH cytochromec reductase, in the HSP to that in the homogenate. Results indicate a 4-fold enrichment in ER marker enzyme activity (Table I).Table INAPDH cytochrome c reductase activity (-fold enrichment) in β-cell fractionsCell lineHomogenatePMSVHSPCYTβTC6-F71.001.14 ± 0.1704.11 ± 1.09*1.69 ± 0.42β-IRS11.001.14 ± 0.181.39 ± 0.233.39 ± 0.54*2.23 ± 0.80*, p < 0.005. Open table in a new tab *, p < 0.005. Subcellular fractions were analyzed by Western blot using rabbit polyclonal anti-IRS-1 (Upstate Biotechnology Inc. (UBI) catalog no. 06-248), anti-IRS-2 (UBI catalog no. 06506), and anti-GLUT2 (H-67) (Santa Cruz catalog no. SC-9117) antibodies. Briefly, 40 μg of each fraction and the homogenate were run on 7.5% SDS-PAGE gels and transferred to nitrocellulose. The blots were incubated in a Tris-buffered saline (TBS-T) blocking buffer (1% bovine serum albumin (BSA) in 10 mm Tris-Cl, pH 7.5, 100 mm NaCl, 0.1% Tween 20) for 1 h. The blots were then incubated in 1:500 solutions of anti-IRS-1 or anti-IRS-2 antibody in blocking buffer overnight and washed with TBS-T five times for 5 min each the following day. Next, the blots were incubated for 1 h with125I-labeled Protein A (Amersham Biosciences, catalog no. IM144) diluted in blocking buffer. Blots were then washed with TBS-T five times for 5 min each and air-dried. The radiolabeled blots were exposed on a phosphor screen (Amersham Biosciences) overnight and analyzed with a PhosphorImager scanner. For immunofluorescence, β-cell lines were seeded on 10-mm poly-d-lysine-coated cover slips in 24-well plates at a cell density of 5 × 104 cells/well. The cells were cultured under the conditions described above for 2 days. Cells were washed twice with 0.5 ml of Dulbecco's phosphate-buffered saline, fixed with 0.5 ml of ice-cold methanol for 10 min and incubated in blocking buffer (Dulbecco's phosphate-buffered saline supplemented with 5% FBS) for 10 min. For ER localization, the cover slips were incubated for 1 h with 50 μl of the following antibodies at the indicated dilutions in blocking buffer (5% FBS-PBS): anti-IRS-1 (1:100), anti-IRS-2 (1:100), and anti-BiP/GRP78 (Transduction Laboratories, catalog no. G73320) (1:50). The cover slips were washed with PBS and then incubated for 1 h with 50 μl of secondary antibody solutions in blocking buffer as follows: goat anti-rabbit labeled with cyanine 2 (cy2) (1:500) and/or donkey anti-mouse labeled with Texas Red (TR) (1:500) (Jackson Immunoresearch Laboratories, Inc., catalog no. 111-225-03 and 715-075-150, respectively). For Golgi localization, the same procedures were followed substituting anti-GM130 (Transduction Laboratories, catalog no. G65120) for anti-BiP/GRP78 at a 1:50 dilution. Immunofluorescence-labeled cover slips were mounted onto microscope slides with PermaFluor reagent (Immunon, catalog no. 434980) and analyzed by confocal microscopy in the University of Pennsylvania Diabetes Center Biomedical Imaging Core. Images were captured using the Nikon Eclipse E600 fluorescent microscope and employing a krypton-argon laser as a light source. The Bio-Rad MRC1024 software was used to operate the microscope and laser source. Cross-sectional images through the β-cells were collected in a series of slices 0.10–0.30 μm apart (depending on the total diameter of the cells) from the upper to the lower surface of the cells (∼25–40 image slices/cell group). Each image was collected concurrently at 506 nm (green) and 615 nm (red). The images were analyzed using Confocal AssistantTMversion 4.02 software (copyright 1994–1996, Todd Clark Brelje). Pre-coated nickel grids were floated on a solution of the IM-enriched fraction (HSP) from both βTC6-F7 and β-IRS1 cells for 10 min. The grids were then incubated on 20-μl drops of blocking buffer (1% ovalbumin, 0.2% cold water fish skin gelatin, PBS, pH 7.4) and washed four times with 50 mm Tris, 20 mmglycine for 1 min each. The grids were next incubated on 5-μl drops of the following primary antibody solutions for 60 min: rabbit polyclonal anti-IRS-1 (1:50 dilution), mouse monoclonal anti-IRS-1 (Santa Cruz, catalog no. sc8038) (1:50), anti-SERCA3b (1:500), and anti-BiP/GRP78 (1:200). The incubations were performed for each individual antibody solution alone and in the following combinations: rabbit polyclonal anti-IRS-1 + anti-BiP/GRP78, anti-SERCA3b + anti-BiP/GRP78, and mouse monoclonal anti-IRS-1 + anti-SERCA3b. Grids were washed four times with 0.1% BSA in PBS and incubated with the appropriate combination of species-specific gold-conjugated secondary antibody solutions in 0.1% BSA, acetylated PBS (Aurion, catalog no. 25558) for 45 min. The gold-conjugated secondary antibodies used were anti-rabbit conjugated to 18-nm gold particles and anti-mouse conjugated to 5-nm gold particles (Rockland, Gilbertsville, PA). The grids were then washed four times with 0.1% BSA in PBS for 5 min each and incubated in 1% glutaraldehyde in deionized water for 5 min to stabilize the signal. The grids were washed with deionized water, fixed for 5 min in 1% osmium (in deionized water), and rinsed with deionized water. Finally, all grids were stained with 2% aqueous uranyl acetate for 5 min and then dried. Grids were examined by electron microscopy at the University of Pennsylvania Diabetes Center Biomedical Imaging Core using the JEOL JEM1010 electron microscope (JEOL, Tokyo, Japan). Images were collected at 80.0 kV and magnification ×120,000. CHO-T cells were seeded in 15-cm dishes and cultured overnight as described above. When cells were 50–70% confluent, they were transiently transfected with pMT2-SERCA3b alone or with pMT2-SERCA3b and pCMV-IRS-1 using the FuGENETM 6 transfection reagent (Roche Molecular Biochemicals, catalog no. 1814443) in a 6:1 FuGENETM 6: total DNA ratio as follows: 90 μl of FuGENETM 6 and 15 μg of pMT-SERCA3b; 180 μl of FuGENETM 6, 15 μg of pMT-SERCA3b, and 15 μg of pCMV-IRS-1. Each reagent mixture was enough to transfect two 15-cm dishes. After 24 h of transfection, cells were incubated in starvation medium (F-12 medium, 0.1% FBS) for 3 h and then treated for 5 min with or without insulin (100 nm). The cells were lysed with Kasuga's lysis buffer (30Yang J. Clarke J.F. Ester C.J. Young P.W. Kasuga M. Holman G.D. Biochem. J. 1996; 313: 125-131Google Scholar) (20 mm Tris-Cl, pH 7.6, 1% Nonidet P-40, 10% glycerol, 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 20 mmNa4P2O7, 20 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin and aprotinin) supplemented with 100 μl/10 ml of phosphatase inhibitor mixture 2 (Sigma, catalog no. P-5726). βTC6-F7 and β-IRS1 cells were seeded and cultured to confluence as described above. The cells were washed twice and then incubated with 20 ml of Krebs-Ringer buffer (KRB) (115 mm NaCl, 24 mm NaHCO3, 5 mm KCl, 1 mm MgCl2, 2.5 mm CaCl2, 25 mm Hepes, pH 7.4) supplemented with 0.1% BSA for 30 min prior to insulin treatment. The KRB solution was changed, and then the cells were treated with insulin as described above. The protein concentration of the lysates was determined by the Bio-Rad BCA protein assay. Sample aliquots of 500 μg were pre-cleared with 20 μl of a 50% Protein A-Sepharose CL-4B slurry (Amersham Biosciences, catalog no. 71-7090-00). The samples were incubated overnight with anti-IRS-1, anti-SERCA3b, pre-immune serum, or normal rabbit serum. Immune complexes were pulled down with 40 μl of the 50% Protein A-Sepharose CL-4B slurry overnight, washed twice with Wash Buffer 1 (50 mm Hepes, pH 7.8, 1.0% Triton X-100, 0.1% SDS, 150 mm NaCl), and then washed twice with Wash Buffer 2 (50 mm Hepes, pH 7.8, 1.0% Triton X-100, 0.1% SDS). Laemmli buffer (with 3 mg/ml dithiothreitol) was added to the samples, which were then analyzed by Western blot using the anti-IRS-1 and anti-SERCA3b antibodies. Calcium uptake was measured as described previously (41Colca J.R. McDonald J.M. Kotagal N. Patke C. Fink C.J. Greider M.H. Lacy P.E. McDaniel M.L. J. Biol. Chem. 1982; 257: 7223-7228Google Scholar). 45CaCl2 (ICN Biomedicals, Inc., Catalog no. 62005) was used as a tracer of calcium uptake. Standard final assay solution conditions were as follows: 50 mm Tris, 5 mm MgCl2, 100 mm KCl, 10 mm oxalate, 68.5 μm CaCl2 total Ca2+ (10 μm free Ca2+, 1.0–2.0 μCi of 45CaCl2), ± 1.25 mm ATP, pH 6.8, at 37 °C. The assay was initiated
Referência(s)