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• W2080932392 abstract "Protein-tyrosine phosphatase 1B (PTP1B) is an important negative regulator of insulin and leptin signaling in vivo. Mice lacking PTP1B (PTP1B–/– mice) are hyper-responsive to insulin and leptin and resistant to diet-induced obesity. The tissue(s) that mediate these effects of global PTP1B deficiency remain controversial. We exploited the high degree of hepatotropism of adenoviruses to assess the role of PTP1B in the liver. Liver-specific re-expression of PTP1B in PTP1B–/– mice led to marked attenuation of their enhanced insulin sensitivity. This correlated with, and was probably caused by, decreased insulin-stimulated tyrosyl phosphorylation of the insulin receptor (IR) and IR substrate 2-associated phosphatidylinositide 3-kinase activity. Analysis using phospho-specific antibodies for the IR revealed preferential dephosphorylation of Tyr-1162/1163 compared with Tyr-972 by PTP1B in vivo. Our findings show that the liver is a major site of the peripheral action of PTP1B in regulating glucose homeostasis. Protein-tyrosine phosphatase 1B (PTP1B) is an important negative regulator of insulin and leptin signaling in vivo. Mice lacking PTP1B (PTP1B–/– mice) are hyper-responsive to insulin and leptin and resistant to diet-induced obesity. The tissue(s) that mediate these effects of global PTP1B deficiency remain controversial. We exploited the high degree of hepatotropism of adenoviruses to assess the role of PTP1B in the liver. Liver-specific re-expression of PTP1B in PTP1B–/– mice led to marked attenuation of their enhanced insulin sensitivity. This correlated with, and was probably caused by, decreased insulin-stimulated tyrosyl phosphorylation of the insulin receptor (IR) and IR substrate 2-associated phosphatidylinositide 3-kinase activity. Analysis using phospho-specific antibodies for the IR revealed preferential dephosphorylation of Tyr-1162/1163 compared with Tyr-972 by PTP1B in vivo. Our findings show that the liver is a major site of the peripheral action of PTP1B in regulating glucose homeostasis. Insulin action is mediated by a complex network of signaling events (reviewed in Refs. 1Lizcano J.M. Alessi D.R. Curr. Biol. 2002; 12: R236-R238Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar and 2Nandi A. Kitamura Y. Kahn C.R. Accili D. Physiol. Rev. 2004; 84: 623-647Crossref PubMed Scopus (211) Google Scholar). Upon binding to the insulin receptor (IR), 1The abbreviations used are: IR, insulin receptor; PTP, proteintyrosine phosphatase; IRS, insulin receptor substrate; ITT, insulin tolerance test; AST, aspartate aminotransferase; WT, wild type; PI3K, phosphatidylinositide 3-kinase; hPTP1B, human PTP1B; KO, knockout. insulin induces autophosphorylation of several tyrosyl residues, leading to the recruitment and phosphorylation of insulin receptor substrates (IRSs), Gab family proteins, and the adapter Shc. These serve as docking sites for Src hyomology 2 domain-containing signal relay molecules, such as phosphatidylinositide 3-kinase (PI3K). PI3K is a major mediator of the metabolic actions of insulin, including its ability to promote glycogen synthesis and stimulate glucose transport (3Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (841) Google Scholar). Regulation of insulin action requires a balance between IR phosphorylation and dephosphorylation. Several PTPs have been implicated as negative regulators of insulin signal transduction. Chief among these is protein-tyrosine phosphatase 1B (PTP1B), a ubiquitously expressed, nonreceptor PTP localized on the endoplasmic reticulum (4Frangioni J.V. Beahm P.H. Shifrin V. Jost C.A. Neel B.G. Cell. 1992; 68: 545-560Abstract Full Text PDF PubMed Scopus (508) Google Scholar, 5Woodford-Thomas T.A. Rhodes J.D. Dixon J.E. J. Cell Biol. 1992; 117: 401-414Crossref PubMed Scopus (154) Google Scholar, 6Haj F.G. Verveer P.J. Squire A. Neel B.G. Bastiaens P.I. Science. 2002; 295: 1708-1711Crossref PubMed Scopus (371) Google Scholar). Overexpression of PTP1B in various tissue culture cells decreases IR and IRS1 tyrosyl phosphorylation and reduces IRS1-associated PI3K activity (7Ahmad F. Li P.M. Meyerovitch J. Goldstein B.J. J. Biol. Chem. 1995; 270: 20503-20508Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 8Venable C.L. Frevert E.U. Kim Y.B. Fischer B.M. Kamatkar S. Neel B.G. Kahn B.B. J. Biol. Chem. 2000; 275: 18318-18326Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 9Kenner K.A. Anyanwu E. Olefsky J.M. Kusari J. J. Biol. Chem. 1996; 271: 19810-19816Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). The IR and possibly IRS1 are direct PTP1B substrates (10Calera M.R. Vallega G. Pilch P.F. J. Biol. Chem. 2000; 275: 6308-6312Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 11Seely B.L. Staubs P.A. Reichart D.R. Berhanu P. Milarski K.L. Saltiel A.R. Kusari J. Olefsky J.M. Diabetes. 1996; 45: 1379-1385Crossref PubMed Google Scholar, 12Bandyopadhyay D. Kusari A. Kenner K.A. Liu F. Chernoff J. Gustafson T.A. Kusari J. J. Biol. Chem. 1997; 272: 1639-1645Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 13Dadke S. Kusari J. Chernoff J. J. Biol. Chem. 2000; 275: 23642-23647Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 14Goldstein B.J. Bittner-Kowalczyk A. White M.F. Harbeck M. J. Biol. Chem. 2000; 275: 4283-4289Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), and structural and kinetic studies suggest that PTP1B preferentially dephosphorylates the double phosphotyrosyl motif Tyr-1162/1163 in the IR (15Salmeen A. Andersen J.N. Myers M.P. Tonks N.K. Barford D. Mol. Cell. 2000; 6: 1401-1412Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). The physiological relevance of these observations was dramatically verified by mice lacking PTP1B (PTP1B–/– mice), which exhibit markedly increased insulin sensitivity and enhanced insulin signaling, with substantially increased IR and IRS1 tyrosyl phosphorylation in liver and muscle (16Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C.C. Ramanchandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1926) Google Scholar, 17Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1130) Google Scholar). Furthermore, hyperinsulinemic-euglycemic clamp studies reveal enhanced whole body glucose disposal in PTP1B–/– mice (17Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1130) Google Scholar). Interestingly, this is due to elevated insulin-stimulated glucose uptake in skeletal muscle but not in white adipose tissue. PTP1B–/– mice also showed a trend toward increased insulin-evoked suppression of hepatic glucose production (17Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1130) Google Scholar). In addition to these effects on insulin signaling and glucose homeostasis, which are consistent with the earlier ex vivo studies, PTP1B–/– mice also display an unanticipated decrease in adiposity and resistance to high fat diet-induced obesity (16Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C.C. Ramanchandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1926) Google Scholar, 17Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1130) Google Scholar). Subsequent studies showed that PTP1B could regulate leptin signaling ex vivo, most likely by dephosphorylating Jak2, and PTP1B–/– mice showed increased hypothalamic leptin sensitivity (18Zabolotny J.M. Bence-Hanulec K.K. Stricker-Krongrad A. Haj F. Wang Y. Minokoshi Y. Kim Y.B. Elmquist J.K. Tartaglia L.A. Kahn B.B. Neel B.G. Dev. Cell. 2002; 2: 489-495Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 19Cheng A. Uetani N. Simoncic P.D. Chaubey V.P. Lee-Loy A. McGlade C.J. Kennedy B.P. Tremblay M.L. Dev. Cell. 2002; 2: 497-503Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). However, other studies indicate that, also by dephosphorylating Jak2, PTP1B negatively regulates hepatic growth hormone signaling, potentially providing a peripheral (non-central nervous system) explanation for decreased adiposity and resistance to diet-induced obesity (20Gu F. Dube N. Kim J.W. Cheng A. Ibarra-Sanchez Mde J. Tremblay M.L. Boisclair Y.R. Mol. Cell. Biol. 2003; 23: 3753-3762Crossref PubMed Scopus (118) Google Scholar). Furthermore, PTP1B antisense oligonucleotides, which lower PTP1B expression only in liver and fat, reportedly enhance insulin sensitivity in animal models of insulin resistance (21Gum R.J. Gaede L.L. Heindel M.A. Waring J.F. Trevillyan J.M. Zinker B.A. Stark M.E. Wilcox D. Jirousek M.R. Rondinone C.M. Ulrich R.G. Mol. Endocrinol. 2003; 17: 1131-1143Crossref PubMed Scopus (47) Google Scholar, 22Gum R.J. Gaede L.L. Koterski S.L. Heindel M. Clampit J.E. Zinker B.A. Trevillyan J.M. Ulrich R.G. Jirousek M.R. Rondinone C.M. Diabetes. 2003; 52: 21-28Crossref PubMed Scopus (184) Google Scholar, 23Rondinone C.M. Trevillyan J.M. Clampit J. Gum R.J. Berg C. Kroeger P. Frost L. Zinker B.A. Reilly R. Ulrich R. Butler M. Monia B.P. Jirousek M.R. Waring J.F. Diabetes. 2002; 51: 2405-2411Crossref PubMed Scopus (150) Google Scholar, 24Zinker B.A. Rondinone C.M. Trevillyan J.M. Gum R.J. Clampit J.E. Waring J.F. Xie N. Wilcox D. Jacobson P. Frost L. Kroeger P.E. Reilly R.M. Koterski S. Opgenorth T.J. Ulrich R.G. Crosby S. Butler M. Murray S.F. McKay R.A. Bhanot S. Monia B.P. Jirousek M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11357-11362Crossref PubMed Scopus (404) Google Scholar). Thus, although there is general agreement that PTP1B is a major regulator of insulin sensitivity and body weight, the tissue(s) that mediate these effects have remained unclear. We utilized the high degree of hepatotropism of adenoviruses to assess the effects of restoring PTP1B expression only in the livers of PTP1B–/– mice. PTP1B–/– mice expressing human PTP1B (hPTP1B) in the liver (at activity levels ∼6-fold greater than in WT mice) exhibited dramatically decreased IR tyrosyl phosphorylation and IRS2-associated PI3K activity and reversal of their enhanced insulin sensitivity. We observed preferential site-specific dephosphorylation of the IR at Tyr-1162/1163 by PTP1B in vivo, consistent with earlier in vitro studies (15Salmeen A. Andersen J.N. Myers M.P. Tonks N.K. Barford D. Mol. Cell. 2000; 6: 1401-1412Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). Our findings confirm that PTP1B is an important negative regulator of glucose homeostasis and strongly suggest that the liver is a major site of PTP1B action in the periphery. Antibodies—Rabbit polyclonal antibodies against IR and IRS1/2 were kindly provided by Drs. C. R. Kahn (Joslin Diabetes Center, Boston, MA) and M. White (Children's Hospital Boston, MA), respectively. Rabbit polyclonal phosphospecific antibodies against Tyr-1162/1163 and Tyr-972 of the IR were purchased from BIOSOURCE (Camarillo, CA). Monoclonal anti-phosphotyrosine (4G10) and anti-human PTP1B (FG6) antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit polyclonal anti-SHP2 (C-18) and anti-human PTP1B antibodies (H-135) were from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoprecipitation and Immunoblotting—The tissues were lysed in a modified radioimmune precipitation assay buffer (50 mm Tris-Cl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS and 5 mm EDTA), containing 2 mm sodium orthovanadate and a protease inhibitor mixture (final concentrations were 20 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml of pepstatin, and 1 μg/ml of antipain). The lysates were clarified by centrifugation at 13,000 rpm for 10 min, and protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce). For immunoprecipitations, the lysates were incubated with the appropriate antibodies at 4 °C for 3 h to overnight. Immune complexes were collected onto protein A-Sepharose beads, washed extensively, resolved by SDS-PAGE, and transferred onto Immobilon-P membranes (Millipore, Bedford, MA), which were blocked in 10 mm Tris-Cl, pH 7.4, 150 mm NaCl, 0.05% Tween 20 with either 5% bovine serum albumin or 5% Carnation nonfat dry milk. After incubation with appropriate primary and secondary antibodies (used at the concentrations recommended by their suppliers), the blots were visualized using ECL (Amersham Biosciences). Quantification was carried out by using National Institutes of Health Image Pro Software version 1.62. Adenovirus Preparation and Animal Experiments—Adenoviruses encoding human PTP1B (AdPTP1B) and LacZ (AdLacZ) (8Venable C.L. Frevert E.U. Kim Y.B. Fischer B.M. Kamatkar S. Neel B.G. Kahn B.B. J. Biol. Chem. 2000; 275: 18318-18326Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) were purified twice using the CsCl method and then titrated using cell viability assays, as described previously (25Mittal S.K. McDermott M.R. Johnson D.C. Prevec L. Graham F.L. Virus Res. 1993; 28: 67-90Crossref PubMed Scopus (131) Google Scholar). The viruses were suspended in 200 μl of phosphate-buffered saline and injected through the tail veins of 9–12-week-old male mice at 3 × 108 plaque-forming units/g of body weight. PTP1B–/– and wild type (WT) mice were weaned onto a low fat (chow) diet (product 5010; Purina, St. Louis, MO) and maintained on this diet for ∼6 weeks. One week prior to virus injection, the mice were switched onto a high fat diet (product TD 93075; Harlan-Teklad, Madison, WI) and maintained on that diet until they were sacrificed. The low fat diet has 11.3% of calories from fat (physiological fuel value is 3.41 Kcal/g), whereas the high fat diet has 54% of calories from fat (physiological fuel value is 4.8 Kcal/g). Body weight and food intake were measured daily. For insulin tolerance tests (ITTs), the mice were fasted for 4 h and then injected with 0.65 milliunits/g (body weight) human insulin (Novo Nordisk Pharmaceuticals, Princeton, NJ). Blood glucose values were measured immediately before and at 15, 30, 45, 60, and 90 min after insulin injection. To enable data comparison from different experiments, the values were expressed as percentages of change in blood glucose levels. For signaling experiments, the mice were fasted for 12–14 h and then injected intraperitoneally with 10 milliunits of insulin/g of body weight. The mice were sacrificed 10 min after injection, and insulin-responsive tissues were removed and snap frozen. Plasma insulin levels were determined by radioimmunoassay (Crystal Chem. Inc.; catalog number 90060). Plasma aspartate aminotransferase (AST) levels were measured at the Department of Laboratory Medicine (Children's Hospital, Boston, MA). Because the AST assay requires 100 μlof serum, serial measurements were not possible. Thus, the AST levels were determined only at the end of the study. Adenoviral infection typically produces a mild hepatitis in mice. Accordingly, several injected animals had elevated AST levels. We excluded all infected mice (n = 6 mice for KO AdPTP1B and n = 1 mouse for WT AdLacZ) whose transaminases were more than three times elevated above the mean normal value (saline-injected WT mice). However, it should be noted that if all of the excluded mice were included, the results would not be changed, indicating that adenoviral infection of the liver itself did not alter glucose homeostasis. Generally high AST levels in KO AdPTP1B most likely reflect a role for PTP1B in mediating sensitivity to adenovirus-induced hepatitis. All of the experiments were approved by the Harvard Medical School Center for Animal Resources and Comparative Medicine and were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for Care and Use of Laboratory Animals. Enzyme Activity Assays—PTP1B activity assays were carried out using reduced, carboxamidomethylated, and maleyated lysozyme phosphorylated with [γ-32P]ATP, as described (26Tonks N.K. Diltz C.D. Fischer E.H. Methods Enzymol. 1991; 201: 442-451Crossref PubMed Scopus (21) Google Scholar). The liver samples were lysed in 1% Triton X-100, 0.6 m KCl, 50 mm dithiothreitol, and the protease inhibitor mixture. Phosphatase assays were initiated by the addition of 10 μl of radiolabeled carboxamidomethylated and maleyated lysozyme (10 μm) and incubated at 37 °C for 5 min. The reactions were terminated by the addition of 750 μl of ice-cold acidic charcoal mixture (0.9 m NaCl, 90 mm sodium pyrophosphate, 2 mm NaH2PO4 and 4% (v/v) Norit A). After centrifugation for 1 min, the amount of radioactivity in 400 μl of supernatant was measured in a liquid scintillation counter. The data are represented as fold change compared with WT mice. For PI3K assays, the tissue lysates (1.5 mg protein) were subjected to immunoprecipitation with polyclonal antibodies to IRS1 or IRS2, and immune complex PI3K activity was determined, as described previously (27Kim Y.B. Nikoulina S.E. Ciaraldi T.P. Henry R.R. Kahn B.B. J. Clin. Invest. 1999; 104: 733-741Crossref PubMed Scopus (368) Google Scholar). Immunohistochemistry—Paraffin-embedded sections (5 μm) were dewaxed twice in Xylene and then rehydrated through a series of 100, 95, 80, and 70% ethanol washes for 5 min each. The sections were boiled for 25 min in 1 mm EDTA for antigen retrieval, blocked with 2% normal donkey serum and 1% bovine serum albumin in phosphate-buffered saline, and then incubated in rabbit anti-human PTP1B antibodies (H-135) at 4 °C overnight. Following washing, the sections were incubated with biotin-conjugated donkey anti-rabbit secondary antibodies (Jackson Immuno Research, West Grove, PA) for 1 h. For detection of bound antibodies, the sections were incubated in ABC complex (Vector Laboratories, Burlingame, CA) for 30 min, washed, and developed in 3,3′-diaminobenzidine for 5 min. The sections were then dehydrated, mounted, and dried overnight before observation. Statistical Analysis—The data are expressed as the means ± S.E. The statistical analyses were performed using the Statview program (Abacus Concepts, Berkeley, CA). ITTs were analyzed by repeated measures analysis of variance. Post hoc differences were considered significant at p ≤ 0.05 and highly significant at p ≤ 0.01 using a Fisher's protected least square difference. All of the other data were analyzed using analysis of variance. Re-expression of PTP1B in PTP1B–/– Mice by Adenovirus-mediated Gene Transfer—To assess the role of PTP1B in the liver, we selectively re-expressed hPTP1B in the livers of PTP1B–/– mice by injecting them intravenously with recombinant Ad5 category viruses (28Li Q. Kay M.A. Finegold M. Stratford-Perricaudet L.D. Woo S.L. Hum. Gene Ther. 1993; 4: 403-409Crossref PubMed Scopus (295) Google Scholar, 29Futagawa Y. Okamoto T. Ohashi T. Eto Y. Res. Exp. Med. 2000; 199: 263-274Crossref PubMed Scopus (5) Google Scholar, 30Ueki K. Yamauchi T. Tamemoto H. Tobe K. Yamamoto-Honda R. Kaburagi Y. Akanuma Y. Yazaki Y. Aizawa S. Nagai R. Kadowaki T. J. Clin. Invest. 2000; 105: 1437-1445Crossref PubMed Scopus (55) Google Scholar). Male mice (9–12 weeks old) on a low fat diet were switched to a high fat diet for 7 days, injected with the appropriate adenovirus, and then studied for an additional 7 days (Fig. 1A). hPTP1B protein expression in tissue lysates was assessed by immunoblotting with monoclonal antibodies against hPTP1B (FG6), which have low affinity for mouse PTP1B (31Zabolotny J.M. Haj F.G. Kim Y.B. Kim H.J. Shulman G.I. Kim J.K. Neel B.G. Kahn B.B. J. Biol. Chem. 2004; 279: 24844-24851Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) (Fig. 1B). As expected, and consistent with the hepatotropism of these viruses, hPTP1B protein expression was present in the liver and absent from all other insulin-responsive tissues (25Mittal S.K. McDermott M.R. Johnson D.C. Prevec L. Graham F.L. Virus Res. 1993; 28: 67-90Crossref PubMed Scopus (131) Google Scholar, 28Li Q. Kay M.A. Finegold M. Stratford-Perricaudet L.D. Woo S.L. Hum. Gene Ther. 1993; 4: 403-409Crossref PubMed Scopus (295) Google Scholar, 29Futagawa Y. Okamoto T. Ohashi T. Eto Y. Res. Exp. Med. 2000; 199: 263-274Crossref PubMed Scopus (5) Google Scholar) (Fig. 1B). Immunohistochemical analysis (using H-135 anti-hPTP1B antibodies) revealed hPTP1B expression in a large number of hepatocytes, wherein it displayed the intracellular, reticular staining pattern characteristic of PTP1B (Fig. 1C, left panel) (4Frangioni J.V. Beahm P.H. Shifrin V. Jost C.A. Neel B.G. Cell. 1992; 68: 545-560Abstract Full Text PDF PubMed Scopus (508) Google Scholar, 6Haj F.G. Verveer P.J. Squire A. Neel B.G. Bastiaens P.I. Science. 2002; 295: 1708-1711Crossref PubMed Scopus (371) Google Scholar). Thus, hPTP1B is expressed in the expected location in infected hepatocytes. A similar staining pattern was observed using hPTP1B monoclonal antibodies (FG6) (data not shown), whereas no immunoreactivity was detected in the livers of AdLacZ-injected PTP1B–/– mice (Fig. 1C, right panel). Because there are no antibodies available that recognize mouse and human PTP1B proteins equally, we could not easily compare the level of expressed hPTP1B to endogenous mouse PTP1B in WT mice by immunoblotting. Instead, we assessed PTP activity in liver lysates from WT AdLacZ, KO AdLacZ, and KO AdPTP1B mice. The overall PTP activity was ∼20% less in liver of KO AdLacZ mice compared with WT mice. If we assume that all of the “missing” PTP activity is PTP1B, these data indicate that PTP1B normally accounts for ∼20% of the total PTP activity in liver of WT mice. Upon re-expressing PTP1B in liver of the KO animals, the overall PTP activity was about 100% greater than that in WT mice. If all of this “extra” activity is due to (re-expressed) PTP1B, then the total PTP1B activity in the reconstituted mice is 120% of WT levels. In other words, the restored activity is 6-fold higher (120/20) than normal levels of activity. Effects of Liver PTP1B Re-expression on Body Weight—We were interested in the potential effects of liver PTP1B on both glucose homeostasis and body mass. It is optimal to determine the effects of genetic manipulations on glucose homeostasis after puberty. Because PTP1B–/– mice have lower weight than WT littermates as early as 2 weeks post-weaning onto a high fat diet, by puberty such mice would weigh considerably less than WT controls. Such a weight difference would confound assessment of the effects of liver PTP1B on glucose homeostasis. Adenovirus-mediated gene expression in the liver is sustained for a relatively short time; thus, a long term study of the effect of hepatic PTP1B re-expression on weight gain is not feasible. Therefore to facilitate detection of possible differences in body weight caused by hepatic PTP1B expression, mice were placed on low fat diet after weaning, then switched to a high fat diet 1 week before virus administration, and maintained on that diet for the rest of the study (Fig. 1A). The mice were housed individually, and food intake and body weights were measured daily. Prior to viral injection (during the first 7 days post-switch to high fat diet), no significant differences in body weights of the mice were detected. Within a few days after viral injection, a significant difference was detected between KO AdLacZ and WT mice, but for most part there was no significant weight difference between mice in any of the virally injected groups (Fig. 2A). As expected, there also was no significant difference in food intake between WT and PTP1B–/– mice prior to infection (17Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1130) Google Scholar, 18Zabolotny J.M. Bence-Hanulec K.K. Stricker-Krongrad A. Haj F. Wang Y. Minokoshi Y. Kim Y.B. Elmquist J.K. Tartaglia L.A. Kahn B.B. Neel B.G. Dev. Cell. 2002; 2: 489-495Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar) (Fig. 2B). Viral infection had a mild but significant anorexogenic effect on both WT and PTP1B–/– mice. Interestingly, the duration of this suppressed food intake appeared to be longer in KO AdLacZ mice than in KO mice re-expressing PTP1B. However, all of these differences were small and had little effect on body mass and thus are unlikely alone to account for altered glucose homeostasis. Re-expression of PTP1B in Liver of PTPB–/– Mice Attenuates Insulin Sensitivity—PTP1B–/– mice have increased insulin sensitivity (16Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C.C. Ramanchandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1926) Google Scholar, 17Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1130) Google Scholar). Hyper-insulinemic euglycemic clamp studies revealed a marked enhancement of insulin-stimulated glucose uptake into skeletal muscle but also a trend toward greater suppression of hepatic glucose output in PTP1B–/– mice (17Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1130) Google Scholar). Therefore, we assessed glucose homeostasis in PTP1B–/– mice after re-expression of hPTP1B in the liver. Consistent with previous studies, before viral injection, fed glucose levels were lower in PTP1B–/– (KO), compared with WT groups (Table I). There was no significant difference in fed or fasting glucose or fed insulin levels between PTP1B–/– mice and their counterparts re-expressing PTP1B (Table I). Serum AST levels were used as indications of liver function. PTP1B–/– mice infected with the PTP1B virus generally had higher AST levels than mice infected with the LacZ control virus. Moreover, PTP1B–/– mice infected with adenovirus LacZ had significantly lower AST elevations than WT mice injected with the same virus. When PTP1B expression is restored (adenovirus PTP1B into PTP1B–/– mice), AST levels were increased (compared with PTP1B–/– mice infected with adenovirus LacZ). The higher level of AST in PTP1B–/– mice infected with adenovirus PTP1B than in WT mice infected with adenovirus LacZ is likely explained by the 6 times higher level of PTP1B expression (compared with WT) in reconstituted mice. The data suggest that liver PTP1B levels may affect sensitivity to adenoviral induced hepatitis (see “Discussion”).Table IBlood glucose, serum insulin, and AST levels in WT and PTP1B–/– mice infected with AdLacZ or AdPTP1B adenovirusParameterWTWT AdLacZKO AdLacZKO AdPTP1BBlood glucose (mg/dl)Fed before viral injection132 ± 3.8126 ± 4.65107 ± 7.06aSignificant difference between KO AdLacZ and WT AdLacZ (p ≤ 0.01).105 ± 4.61bSignificant difference between WT AdLacZ and KO AdPTP1B (p ≤ 0.05).Fed after viral injection135 ± 6.53136 ± 5.70122 ± 7.57128 ± 15.73Fasted after viral injection113 ± 7.296 ± 6.0583 ± 4.0680 ± 3.19Serum insulin (ng/ml)Fed before viral injection1.19 ± 0.141.60 ± 0.351.43 ± 0.340.71 ± 0.12bSignificant difference between WT AdLacZ and KO AdPTP1B (p ≤ 0.05).Fed after viral injection1.55 ± 0.441.87 ± 0.460.92 ± 0.211.26 ± 0.33AST (units/liter)264 ± 44303 ± 25216 ± 23cSignificant difference between KO AdPTP1B and KO AdLacZ (p ≤ 0.01).618 ± 72dSignificant difference between WT AdLacZ and KO AdPTP1B (p ≤ 0.01).a Significant difference between KO AdLacZ and WT AdLacZ (p ≤ 0.01).b Significant difference between WT AdLacZ and KO AdPTP1B (p ≤ 0.05).c Significant difference between KO AdPTP1B and KO AdLacZ (p ≤ 0.01).d Significant difference between WT AdLacZ and KO AdPTP1B (p ≤ 0.01). Open table in a new tab To directly assess insulin tolerance in vivo, mice were subjected to ITTs. Fig. 3A shows the percentage of change in blood glucose values following insulin injection from two independent experiments containing mice from all of the indicated groups. These data were collected after viral infection. Notably, KO AdPTP1B mice exhibited a significantly smaller decrease in blood glucose levels during ITT, compared with KO AdLacZ mice, indicating that insulin sensitivity is attenuated in PTP1B–/– mice re-expressing hPTP1B in the liver. Remarkably, the insulin sensitivity of KO AdPTP1B mice was similar to that of WT AdLacZ mice (Fig. 3A). The difference in the insulin response between KO AdLacZ and WT AdLacZ (and WT saline) was similar to the differences seen earlier in uninfected WT and PTP1B–/– mice (16Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C.C. Ramanchandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed S" @default.
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• W2080932392 title "Liver-specific Protein-tyrosine Phosphatase 1B (PTP1B) Re-expression Alters Glucose Homeostasis of PTP1B–/–Mice" @default.
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