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Departments of Medicine and Physiology (N.T.L., J.T.L, A.T.C., C.T.L., J.T., T.J.K.), University of Alberta, Edmonton, Alberta, Canada T6G 2S2; Veterans Administration Medical Center (J.W.), University of Tennessee, Memphis, Tennessee 38103; Division of Endocrinology, Diabetes and Hypertension (M.B.-A.), School of Medicine, University of California Los Angeles, Los Angeles, California 90095; and Department of Medicine, Louisiana State University School of Medicine (J.K.K.), New Orleans, Louisiana 70112
Address all correspondence and requests for reprints to: Timothy J. Kieffer, Department of Physiology, 2146 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3. E-mail: tim.kieffer{at}ubc.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Because early steps in the insulin signaling cascade involve phosphorylation of tyrosyl residues on cellular proteins, the efficiency of insulin action depends on the balance between the competing action of a spectrum of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). PTPs such as leukocyte common antigen-related phosphatase (LAR), Src homology 2-containing phosphatase-2 (SHP2), receptor protein tyrosine phosphatase-
, and protein tyrosine phosphatase 1B (PTP1B) have been identified in all the major target tissues of insulin, including muscle, liver, and adipose tissue (13). Not all PTPs appear to be negative regulators of insulin signaling. For example, SHP2 acts to positively regulate components of the insulin signaling cascade in Chinese hamster ovary cells (14) and in Xenopus oocytes (15). Although PTPs have potential for being important modulators of insulin sensitivity, the correlation between PTPs and the insulin-resistant state, in vivo, is controversial. Despite reports of increased PTP activity in adipose tissue (16) and muscle (17, 18) from obese insulin-resistant human subjects, other studies have described a reduction of PTP activity in tissues of rodent models of insulin resistance (19) and type 2 diabetic patients (20, 21, 22, 23). Due to these discrepancies, the roles of PTPs in normal physiology and the pathophysiology of insulin resistance and diabetes remain unclear.
One specific PTP, PTP1B, has been implicated as an important negative regulator of insulin signaling (24, 25, 26, 27, 28, 29). PTP1B antisense oligonucleotide treatment of ob/ob and db/db mice resulted in increased phosphorylation of IR, IRS-1, and IRS-2 in liver tissue. Furthermore, PTP1B knockout mice show improved insulin sensitivity with notably enhanced insulin-induced phosphorylation of IR and IRS-1 in liver and muscle and glucose uptake in muscle (28, 29). Therefore, down-regulation of PTP1B is a potential mechanism by which leptin could improve insulin sensitivity. We explored this possibility in ob/ob mice and, somewhat surprisingly, we found that leptin acutely increased insulin sensitivity in liver, despite increased expression of PTP1B.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). After 2 d of treatment, body weights of mice in all four treatment groups did not change significantly. Therefore, as previously demonstrated (30), a treatment period of 2 d of leptin (0.5 µg/g, twice daily) was insufficient to reduce body weight in ob/ob mice. PBS-treated ob/ob mice showed no significant difference in food consumption after 2 d of treatment. In comparison, leptin-treated ob/ob mice consumed slightly less food after 1 d of treatment, which did not reach statistical significance (P = 0.12, d 1 vs. d 0). After 2 d of leptin treatment, food intake was significantly reduced in the ob/ob mice (P < 0.01, d 2 vs. d 0) but, when compared with the food consumed by PBS-treated ob/ob mice on d 2, the food intake by leptin-treated mice was not significantly different (P = 0.07). Leptin treatment similarly reduced food consumption in +/+ mice after 2 d (P < 0.05, d 2 vs. d 0).
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30-fold) and glucose (2-fold) compared with their lean counterparts (Table 1
). After d 1, leptin-treated mice ob/ob mice exhibited approximately 40% reduction in plasma glucose. Following the second day, glucose levels were essentially normalized by leptin in the ob/ob mice to levels observed in the lean mice. In comparison, PBS treatment did not reduce plasma glucose concentrations in ob/ob mice. Whereas insulin levels in ob/ob mice treated with PBS remained elevated over the 2 d of treatment, leptin dramatically reduced plasma insulin levels in ob/ob mice by about 55% after 1 d and by approximately 75% after d 2 (Table 1). Leptin treatment of +/+ mice had no significant effect on either plasma glucose or insulin levels.
Effect of Leptin on Whole-Body Insulin Sensitivity
Insulin sensitivity in mice was assessed by ip insulin tolerance tests (ITT). Upon injection of insulin, plasma glucose levels were significantly reduced to 50% of fasting glucose levels by 20 min in leptin-treated ob/ob mice, whereas this effect was not attained until 90 min after the insulin challenge in the PBS-treated ob/ob mice (Fig. 1A). Glucose levels were significantly different between the two groups by 20 min (P < 0.001) and remained different at 120 min post insulin. Whereas glucose levels in ob/ob mice receiving PBS reached the nadir at 50% of fasting levels, glucose levels were reduced to 23% of fasting levels in leptin-treated ob/ob mice, indicating improved insulin sensitivity with leptin treatment.
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). However, in response to a glucose challenge, both the ob/ob leptin and ob/ob control groups had similar area under the glucose curve values (AUC above the value at time 0; 1220 ± 104 vs. 1074 ± 153 mM·min, ob/ob leptin vs. ob/ob PBS, P = 0.36) (Fig. 1C). Leptin had little effect on basal glucose concentrations (9.8 ± 0.4 vs. 9.0 ± 0.4 mM; +/+ leptin vs. +/+ PBS, P = 0.26), or AUC in lean mice (AUC; 223 ± 28 vs. 286 ± 24 mM·min; +/+ leptin vs. +/+ PBS, P = 0.13). The finding that 2 d of leptin reduces fasting plasma glucose concentrations but has no effect on glucose disposal in ob/ob mice suggests that leptin may specifically target the liver and the regulation of hepatic glucose production in the basal state.
Leptin Receptor Expression and Signaling in Transfected Chinese Hamster Ovary (CHO) Cells and Hepatocytes
To determine whether the in vivo effects of leptin in ob/ob mice could be due to direct action in the liver, we first evaluated whether leptin receptors are present on hepatocytes in these animals. Iodocarbocyanine-coupled leptin (Cy3-leptin) was generated and tested for binding ability to leptin receptors after incubation with untransfected CHO cells and CHO cells stably transfected with the OBR gene (CHO-OBR). Although no fluorescence was detected in untransfected CHO cells exposed to Cy3-leptin, distinct membrane localization of Cy3-leptin was visualized in CHO-OBR cells (Fig. 2A). Cy3-leptin was administered via tail-vein to C57Bl6 mice for the extraction of tissue to assess the distribution of leptin receptor binding in liver. In mice that received uncoupled Cy3, fluorescence was minimal and nonspecific. However, Cy3-leptin was distributed with distinct localization to hepatocyte cellular membranes (Fig. 2B). We next looked at leptin’s capability of signaling in hepatocytes. It is believed that most of the physiological effects observed with leptin is through its signaling via OBRb. The 304-amino acid intracellular domain of OBRb contains putative motifs for Janus protein tyrosine kinase (JAK) and STAT (signal transducers and activators of transcription) binding. Upon binding of JAK, the intracellular domain of the receptor becomes phosphorylated, which then allows for binding, phosphorylation, and activation of STAT proteins. Dimerization of STAT proteins causes their nuclear translocation, where they regulate gene transcription. CHO cells transfected with the OBRb gene (CHO-OBRb) and rat liver (FAO) hepatoma cells were treated with leptin, and phosphorylation of STAT3 was evaluated. In both cell types, STAT3 phosphorylation was increased in a dose-dependent manner (Fig. 2C). Further supporting the presence of functional leptin receptors in hepatocytes, nuclear STAT3 translocation upon leptin treatment was visualized in CHO-OBRb and FAO cells (Fig. 2D). Collectively, these observations provide support for the presence of functional leptin receptor signaling in liver cells.
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). In liver and muscle, IR phosphorylation was reduced in ob/ob mice compared with wild-type lean mice consistent with insulin resistance in the ob/ob mice. Treatment with leptin (0.5 µg/g) twice daily for 2 d increased IR phosphorylation in liver but not skeletal muscle or adipose tissue [pTyr/IR densitometric ratios for ob/ob leptin vs. ob/ob PBS: liver (1.26 vs. 0.63, P < 0.01), muscle (1.18 vs. 0.95, P = 0.48), adipose tissue (0.24 vs. 0.21, P = 0.80)] (Fig. 3). Downstream phosphorylation of hepatic IRS-1 was similarly increased in leptin-treated ob/ob mice (0.41 vs. 0.82, P < 0.05; ob/ob leptin vs. ob/ob PBS) (Fig. 3B). Leptin had no effect on IR or IRS-1 phosphorylation on tissues from +/+ mice (data not shown). To determine whether the improvement in IR phosphorylation may have been a direct effect of leptin on hepatocytes, we treated human hepatoma cells, HepG2, with leptin (100 ng/ml) for various periods. IR phosphorylation appeared to increase within 3 h of leptin treatment (Fig. 4). Leptin thus appears to acutely improve insulin sensitivity in liver potentially via direct effects on hepatocytes.
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). The difference in PTP1B expression appeared to be a phosphatase-specific event, because there were no differences in the expression of SHP2 or LAR in ob/ob mice treated with leptin compared with those treated with PBS (Fig. 5B). Liver tissue extracted from ob/ob mice treated with leptin over a period of 2 wk also displayed increased PTP1B expression, suggesting that leptin’s effects after 2 d are retained even after 2 wk (Fig. 5C). Although leptin dramatically increased PTP1B expression in livers of ob/ob mice, it did not elevate PTP1B levels in +/+ mice (data not shown). To determine whether the effects of leptin on PTP1B may have been direct, HepG2 cells were treated with leptin for various periods and immunoblotted for PTP1B. Similar to its effects on IR phosphorylation, leptin increased PTP1B expression within 4 h of leptin treatment and reached a maximal value at 6 h (Fig. 6). Therefore, leptin-induced increases in PTP1B expression in liver of ob/ob mice after 2 d of treatment may be due to direct effects in hepatocytes.
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). In control, nontransduced cells, insulin stimulation for 2 min increased IR phosphorylation by approximately 2-fold. Adß-gal expression had no significant effect on basal or insulin-stimulated IR phosphorylation. In PTP1B-overexpressing cells, basal and insulin-stimulated IR phosphorylation was reduced by about 50% (Fig. 7B).
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DISCUSSION |
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The reduction in plasma glucose and concurrent decrease in plasma insulin indicate that insulin sensitivity was improved upon leptin treatment in the ob/ob mice. In support, insulin tolerance tests performed after the second day of treatment indicated that ob/ob mice were extremely resistant to the actions of insulin, whereas leptin-treated ob/ob mice had significantly improved responses to insulin. Although leptin-treated mice appeared to be more insulin sensitive and basal glucose levels were normalized by leptin, oral glucose tolerance tests performed on the second day of treatment showed that glucose disposal after an oral glucose challenge remained impaired. During the short treatment period, leptin was capable of affecting fasting but not postprandial glucose homeostasis, which may reflect tissue-specific leptin action and/or sensitivity. Indeed, whereas leptin markedly increased insulin-stimulated IR phosphorylation in liver after 2 d of treatment, insulin-induced IR phosphorylation remained reduced in muscle and adipose tissue. In vivo, leptin has been reported to enhance insulin’s action to inhibit hepatic glucose production (10, 37, 38). A reduction in high glucose production after leptin treatment is consistent with our observations. Although leptin action was observed in the liver, our data do not preclude the possibility that leptin may similarly affect skeletal muscle and adipose tissue after a longer treatment period or with a higher dose of leptin.
Our observations of leptin binding sites on hepatocytes, leptin-induced translocation of STAT3, and increased insulin-stimulated IR phosphorylation suggest that leptin-mediated changes in glucose homeostasis may be due, in part, to direct actions of leptin on hepatocytes. This does not exclude the possibility that the in vivo effects observed in the liver are mediated centrally (38, 39, 40, 41, 42) or via other tissues. Because leptin curtails insulin secretion from pancreatic ß-cells (43), it is possible that the increase in hepatic IR phosphorylation after leptin treatment resulted indirectly from the reduction in plasma insulin levels and subsequent improvements in insulin sensitivity. However, in agreement with our in vitro studies, leptin has been reported to stimulate JAK2 phosphorylation in liver (44) and interact directly with the insulin signaling cascade, both augmenting and antagonizing insulin signaling in hepatocytes (8, 9, 45).
Mice lacking the PTP1B gene exhibit significantly enhanced whole-body insulin sensitivity and are protected from diet-induced obesity (28, 29). The improvement in insulin sensitivity is correlated with enhanced and/or sustained tyrosyl phosphorylation of IR and IRS-1 in response to insulin stimulation in liver and muscle but not in adipose tissue (28). Based on these studies, we hypothesized that leptin improved insulin sensitivity by reducing PTP1B expression. In liver, skeletal muscle, and adipose tissue from ob/ob mice, we observed relatively low levels of PTP1B protein expression, which appeared to be counterintuitive given the insulin-resistant state of these mice. However, this finding is consistent with the observations of reduced PTP1B protein expression in muscle biopsies from patients with type 2 diabetes in comparison with nondiabetic control subjects (21, 22). Worm et al. (23) hypothesized that the associated down-regulation of PTP activity in type 2 diabetic patients was an attempt to compensate for the inability of the tissues to sense insulin. Alternatively, it has been proposed that although PTP1B levels are decreased, there may be a larger reduction in IR abundance. This would result in a higher ratio of PTP1B to IR in ob/ob in comparison with wild-type mice, thereby explaining the relative increase in insulin resistance in the ob/ob mice (25). In our study, however, we did not observe any difference in liver IR protein abundance between ob/ob and wild-type mice. This suggests that insulin resistance in ob/ob mice is unlikely to be attributed to overexpression of PTP1B.
Contrary to our original hypothesis that leptin could improve insulin sensitivity by reducing PTP1B expression, leptin treatment of ob/ob mice resulted in an increase in PTP1B protein expression in liver, the same tissue in which insulin sensitivity had evidently been restored. This effect of leptin appeared to be tissue-specific, as leptin did not increase PTP1B expression in skeletal muscle or adipose tissue. Furthermore, this effect of leptin may be mediated through direct effects on hepatocytes as leptin treatment of HepG2 cells also increased PTP1B protein levels. Although PTP1B expression may have been elevated in response to increased insulin signaling after leptin treatment in vivo (46), this does not account for our findings in the HepG2 cells that were treated with leptin in the absence of insulin. Therefore, leptin increases IR activation in the liver and can independently up-regulate PTP1B expression. Given the metabolic and growth-promoting functions of insulin (47, 48), the ability of PTP1B to counterregulate the IR tyrosine kinase activity may be physiologically relevant. As insulin sensitivity is improved by leptin, a subsequent corresponding increase in PTP1B expression may serve to provide a "brake," and thereby intervene to attenuate undesirable insulin effects in cells. A negative-feedback loop is supported by the demonstration of PTP1B as a negative regulator of leptin signaling as well as insulin signaling (49, 50). This autoregulatory mechanism might allow for more dynamic control over the down-regulation of both insulin and leptin signaling.
In summary, our findings demonstrate that leptin is capable of improving whole-body glucose homeostasis and increasing insulin sensitivity in liver of ob/ob mice independent of changes in body weight. The mechanism by which leptin enhances insulin sensitivity remains to be elucidated, as it does not appear to involve a reduction of PTP1B protein levels. Instead, PTP1B expression is reduced in the insulin-resistant state and is elevated when insulin sensitivity is restored. We propose that the increase in PTP1B in response to improved insulin sensitivity is part of a negative feedback loop designed to provide an autoregulatory means by which insulin can attenuate its own signaling cascade. Further understanding of the roles of leptin and PTP1B and their interaction with the insulin signaling pathway may provide additional insight into the regulation of insulin sensitivity and the pathogenesis of insulin resistance associated with obesity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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20 wk old) were administered, by ip injections, either PBS or 0.5 µg/g recombinant murine leptin (PeproTech Inc., Rocky Hill, NJ), twice daily for 2 d or 2 wk. Body weight, chow consumption, plasma glucose, and insulin were measured daily. For the 2-d cohort, after the 2 d of leptin treatment, mice were fasted for 4 h after the final leptin dose, and then subjected to either an ip ITT or an OGTT. For the ITT, mice received an ip injection of 50 U/kg of human insulin (Novo Nordisk A/S, Bagsværd, Denmark). For the OGTT, mice were given 1.5 mg/g glucose as a 50% solution by gavage. Tail vein blood was collected at time 0 and at 10, 20, 30, 60, 90, and 120 min after the insulin injection for the ITT and the glucose bolus for the OGTT. Samples were centrifuged at 14,000 rpm in a microcentrifuge for 5 min at 4 C. Insulin was separated and stored at –20 C for later analysis. Insulin glucose was determined using a calorimetric enzymatic assay kit (Sigma Chemical Co., St. Louis, MO), scaled down for use in 96-well microtiter plates. The area under the curve for the OGTT above the value at time 0 was calculated with Prism 4 Software by applying the trapezoidal rule to incremental changes (GraphPad Software Inc., San Diego, CA). Insulin concentrations were measured using an ultrasensitive mouse insulin EIA kit (ALPCO Diagnostics, Windham, NH). Approximately 30 min after the OGTT, mice were anesthetized with 50 mg/kg ketamine hydrochloride/xylazine (Bimeda-MTC Animal Health Inc., Cambridge, Ontario, Bayer Inc., Toronto, Ontario), a laparotomy was performed and 25 mU/kg human insulin was infused in the portal vein using an ultrafine syringe and needle (28 G). Two minutes after the insulin infusion, the epididymal fat pads, soleus, plantaris, and gastrocnemius muscles and liver samples from the mice were excised and snap frozen. Protein extracts from tissues samples were obtained by resuspending tissue samples in lysis buffer (50 mM HEPES, 1% Triton-X, 2 mM EDTA, 200 mM NaF, 10 mM Na2P4O7, 1 mM phenyl-methylsulfonylfluoride, 1 mM Na3VO4, 10 µg/ml protease inhibitor cocktail; all reagents from Sigma-Aldrich Canada Inc., Oakville, Ontario, Canada) in lysing matrix D FastRNA tubes (Q-BIOgene, Inc., Carlsbad, CA). The 2-wk cohort of mice were administered leptin similarly to the 2-d cohort. However, the 2-wk cohort did not undergo ITT or OGTT but were monitored for 2 wk. Six hours after the last leptin dose, a laparotomy was performed under anesthesia and liver tissue was extracted. Tissues were homogenized on a FastPrep FP120 (Q-BIOgene, Inc.), and extracts were centrifuged at 14,000 rpm for 15 min to separate insoluble matter. The supernatant was removed for determination of protein concentration (DC protein assay, Bio-Rad Laboratories Canada Ltd., Mississauga, Ontario, Canada), and then used for further analysis.
Treatment of HepG2 Cells for Antiphosphotyrosine Immunoblotting of IR
HepG2 cells were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in 5 mM glucose at 37 C, 5% CO2-95% air in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin (all reagents from Life Technologies Inc., Gaithersburg, MD). Cells were grown to approximately 70% confluency, and then cultured for about 16 h in serum-free DMEM. After the 16-h starvation period, cells were treated with 100 ng/ml recombinant human leptin (PeproTech, Inc.) for 1–12 h in serum-free DMEM. After the leptin incubation period, media were removed and replaced with media containing 100 nmol/liter insulin for 2 min. At the end of the treatment period, cells were washed twice in ice-cold Dulbecco’s PBS (D-PBS), lysed in lysis buffer, harvested and measured for protein concentration, and then stored at –70 C until further analysis. For analysis of tyrosine phosphorylation of the ß-subunit of IR (IRß), 2 mg protein lysates from tissues and HepG2 cells were resuspended in 400 µl lysis buffer and immunoprecipitated by incubation at 4 C overnight with an antibody to IRß (Santa-Cruz Biotechnology, Inc., Santa Cruz, CA) and protein A-conjugated protein A/G (Pierce Chemical Co., Rockford, IL). Immunoprecipitates were washed three times with lysis buffer and then boiled in Laemmli buffer. The resulting supernatants were separated on an 8% SDS-PAGE gel and transferred overnight at 4 C to nitrocellulose membrane (Osmonics, Inc., Westborough, MA). The membranes were blocked in Tris-buffered saline buffer with Tween-20 (20 mM Tris, 150 mM NaCl, 0.1% Tween-20) containing 5% BSA (all reagents from Sigma-Aldrich Canada Inc.). Immunoblotting was performed using a monoclonal mouse antiphosphotyrosine antibody (PY99; sc-7020, Santa Cruz Biotechnology, Inc.). Blots were washed and incubated with a goat antimouse secondary antibody conjugated to horseradish peroxidase (HRP) (sc-2005, Santa Cruz Biotechnology, Inc.). Protein content was visualized by enhanced chemiluminescence (ECL detection kit, Amersham Pharmacia Biotech, Uppsala, Sweden), and densitometric values were determined with ImageQuant software (Amersham Pharmacia Biotech). IR and IRS-1 protein amounts were normalized by reblotting the membrane with rabbit-polyclonal antibodies to the carboxy terminus of IRß chain of human origin (sc-711, Santa Cruz Biotechnology, Inc.) and carboxy terminus of IRS-1 of human origin (sc-559, Santa Cruz Biotechnology, Inc.) after stripping the membrane in buffer containing 100 mM ß-mercaptoethanol, 2% sodium dodecyl sulfate, 62.5 mM Tris-HCl (pH 6.7) at 50 C for 30 min. Blots were washed and incubated with a donkey antirabbit secondary antibody conjugated to HRP (NA934, Amersham Pharmacia Biotech). Protein content was visualized as described above.
Immunoblotting of PTPs
Protein lysates (20 µg) from tissue and HepG2 samples were boiled in Laemmli buffer, run on 10% SDS-PAGE gel, and transferred overnight at 4 C to nitrocellulose membrane. Immunoblotting was performed as described above using either a goat polyclonal antibody to PTP1B of human origin (sc-1718, Santa Cruz Biotechnology, Inc), mouse monoclonal antibody to PTP1B of human origin (PH01, Oncogene Research Products, Boston, MA), rabbit polyclonal antibody to SHP2 of human origin (sc-424, Santa Cruz Biotechnology, Inc.), and goat polyclonal antibody to LAR of rat origin (sc-1119, Santa Cruz Biotechnology, Inc). Secondary antibodies used were HRP-conjugated, rabbit antigoat (AP106P, Chemicon International Inc., Temecula, CA), goat antimouse, and donkey antirabbit, respectively.
In Vitro and in Vivo Visualization of Cy3-Leptin
Murine leptin (PeproTech, Inc.) was coupled to iodocarbocyanine (Cy3) monofunctional reactive dye (Amersham Biosciences UK Ltd) in 0.1 M bicarbonate buffer, pH 9.2, and purified on an Econo-Pac 10DG column (Bio-Rad Laboratories, Richmond, CA) in PBS. For the in vitro studies, CHO-OBRb cells were a kind gift from Dr. Takashi Murakami, Tokushima, Japan (52). Cells were cultured at 37 C, 5% CO2-95% air in Ham’s F-12K nutrient mixture with 2 mM L-glutamine and 1.5 g/liter sodium bicarbonate, supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). For the experiment, cells were seeded onto Lab-Tek chamber slides (Nalge Nunc International, Rochester, NY) in F-12 medium supplemented with 10% FBS. After overnight culture, slides were moved to 4 C, washed with ice-cold PBS, and incubated at 4 C for 1 h with approximately 40 nmol/liter Cy3-leptin in PBS. After several rinses with ice-cold PBS, cells were fixed for 10 min with ice-cold 4% paraformaldehyde, rinsed again, and coverslipped. Cells were examined on a Leica DMIRB microscope (Leica Microsystems, Wetzlar, Germany), and fluorescence was viewed under a Cy3 filter. Images were obtained with a Photometrics CoolSNAP camera (Roper Scientific, Inc., Trenton, NJ) and IPLab Spectrum analysis software (Signal Analytics, Vienna, VA). For the in vivo studies, C57Bl6 mice (20 wk old, The Jackson Laboratory, Bar Harbor, ME) were injected with 100 µl of Cy3-leptin or uncoupled Cy3 (control), via tail vein injection under anesthesia. After 5 min, a laparotomy was performed and mice were perfused via the portal vein with cold PBS (5 ml/min) for 1 min, and then cold 4% paraformaldehyde in PBS, pH 7.4 (5 ml/min), for 2 min with a multispeed infusion pump (Harvard Apparatus Co., Millis, MA). Organs were removed immediately after perfusion and stored in cold 4% paraformaldehyde in PBS and kept shielded from light until they were prepared for microscopic examination. Tissues were embedded in Tissue-Tek OCT compound (IMEB Inc., San Marcos, CA) and frozen, and then cut into 10-µm cryosections on a Reichert-Jung Cryocut 1800 (Leica Microsystems). Specimens were mounted with Fluoromount antifade medium (Electron Microscopy Sciences, Fort Washington, PA) and stored at 4 C until examined. Tissue sections were examined and images were obtained as described above.
Treatment of CHO-OBRb and FAO for STAT3 Phosphorylation
CHO-OBRb cells and FAO cells (kind gift from Dr. C. Ronald Kahn, Boston, MA) were cultured in 5 mmol/liter glucose at 37 C, 5% CO2-95% air in F-12 and RPMI medium, respectively, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin (all reagents from Life Technologies, Inc.). Cells were grown to approximately 70% confluency and then cultured for about 4 h in serum-free medium. Cells were treated with 100 ng/ml recombinant human leptin (PeproTech, Inc.) for 15 min in serum-free medium. After the leptin incubation period, cells were washed twice in ice-cold D-PBS, lysed in lysis buffer, harvested and measured for protein concentration, and then stored at –70 C for further analysis.
For analysis of tyrosine phosphorylation of STAT3, 750 µg protein lysates were resuspended in 400 µl lysis buffer and immunoprecipitated by incubation at 4 C overnight with an antibody to STAT3 (sc-482, Santa-Cruz Biotechnology, Inc.) and protein A conjugated protein A/G (Pierce Chemical Co.). Immunoprecipitates were washed three times with lysis buffer and then boiled in Laemmli buffer. The resulting supernatants were separated on a 10% SDS-PAGE gel and transferred overnight at 4 C to nitrocellulose membrane (Osmonics, Inc., Westborough, MA). Immunoblotting was performed, as described above, using a monoclonal mouse antiphosphotyrosine antibody (PY99; sc-7020, Santa Cruz Biotechnology, Inc.). STAT3 protein expression was determined by reblotting the membrane with a rabbit-polyclonal antibody to the carboxy terminus of STAT3 of mouse origin (sc-482, Santa Cruz Biotechnology, Inc.). Detection of signal was detected as described above.
Immunocytochemical Detection of STAT3 in CHO-OBRb, and FAO Hepatocytes
CHO-OBRb and FAO cells were cultured onto glass coverslips in 10% FBS-supplemented F-12 and RPMI media, respectively. When cells reached a confluency of approximately 70%, media were aspirated and cells were incubated in serum-free media for 4 h, at which point medium was aspirated and cells were incubated in medium containing leptin (100 ng/ml) at 37 C. After 30 min of incubation, cells were washed with ice-cold PBS and then fixed in methanol-acetone for 15 min. All subsequent steps were performed at room temperature. Cells were washed with PBS, permeabilized with PBS containing 0.1% Tween at for 30 min, and then blocked with 3% BSA in PBS for 30 min. Cells were incubated with rabbit polyclonal STAT3 antibody (sc-482, Santa Cruz Biotechnology, Inc.) for 1 h, washed with PBS, and then incubated with Cy3-conjugated AffiniPure Donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 45 min. After a final wash with PBS, coverslips were mounted onto glass microscope slides with fluoromount. Cy3 fluorescence was visualized as described above.
Construction and Propagation of Recombinant Adenovirus
The recombinant adenovirus containing PTP1B cDNA was generated as previously described (53, 54). Virus titers were determined by plaque assay. Briefly, 90% confluent HER911 cells (grown on 150-mm plates) were infected with serial dilutions of the virus stock. The infected cell monolayer was then overlaid with agarose in MEM (Life Technologies, Inc.). Plates were incubated at 37 C until visible plaque colonies were observed (6–9 d). The number of visible plaque colonies was counted to determine virus titers.
PTP1B Overexpression in HepG2 Cells
HepG2 cells were grown to about 70% confluency on 60-mm plates. Culture medium was then replaced with 0.5 ml of D-PBS with 4.3 mmol/liter calcium (D-PBS-Ca2+) containing 6.7 x 109 plaque forming units (PFU) of adenoviruses expressing either ß-gal (Adß-gal) or PTP1B (AdPTP1B) for 30 min. Fresh culture media were then added to the plates, and cells were returned to CO2 incubator at 37 C. Thirty-six hours after infection, cells were washed and incubated in serum-free DMEM for 6 h. Serum-starved cells were then treated with D-PBS or insulin (100 nmol/liter) for 2 min, after which cells were washed twice with ice-cold D-PBS, snap frozen in liquid N2, and lysed in cold lysis buffer. Measurement of IR phosphorylation and PTP1B protein expression were perfomed as described above.
Statistical Analysis
Group differences were evaluated by ANOVA analysis with Statview software (SAS Institutes Inc., Cary, NC), with P < 0.05 deemed as significant.
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FOOTNOTES |
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Abbreviations: AUC, Area under the curve; CHO, Chinese hamster ovary; Cy3-leptin, iodocarbocyanine-coupled leptin; D-PBS, Dulbecco’s PBS; FBS, fetal bovine serum; HRP, horseradish peroxidase; IR, insulin receptor; IRS, insulin receptor substrate; ITT, insulin tolerance test; JAK, Janus protein tyrosine kinase; LAR, leukocyte common antigen-related phosphatase; OGTT, oral glucose tolerance test; PTP, protein tyrosine phosphatase; SHP, src homology 2-containing phosphatase; STAT, signal transducer and activator of transcription.
Received for publication May 23, 2002. Accepted for publication February 11, 2004.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 J. Girard The Inhibitory Effects of Insulin on Hepatic Glucose Production Are Both Direct and Indirect Diabetes, December 1, 2006; 55(Supplement_2): S65 - S69. [Abstract] [Full Text] [PDF]
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 N T Lam, S D Covey, J T Lewis, S Oosman, T Webber, E C Hsu, A T Cheung, and T J Kieffer Leptin resistance following over-expression of protein tyrosine phosphatase 1B in liver J. Mol. Endocrinol., February 1, 2006; 36(1): 163 - 174. [Abstract] [Full Text] [PDF]
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C. Li, G. Baquiran, F. Gu, M. L. Tremblay, A. Fazel, J. J. M. Bergeron, and B. I. Posner Insulin Receptor Kinase-Associated Phosphotyrosine Phosphatases in Hepatic Endosomes: Assessing the Role of Phosphotyrosine Phosphatase-1B Endocrinology, February 1, 2006; 147(2): 912 - 918. [Abstract] [Full Text] [PDF]
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P. M. Thule, A. G. Campbell, D. J. Kleinhenz, D. E. Olson, J. J. Boutwell, R. L. Sutliff, and C. M. Hart Hepatic insulin gene therapy prevents deterioration of vascular function and improves adipocytokine profile in STZ-diabetic rats Am J Physiol Endocrinol Metab, January 1, 2006; 290(1): E114 - E122. [Abstract] [Full Text] [PDF]
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E. Watson, S. Hahm, T. M. Mizuno, J. Windsor, C. Montgomery, P. E. Scherer, C. V. Mobbs, and S. R. J. Salton VGF Ablation Blocks the Development of Hyperinsulinemia and Hyperglycemia in Several Mouse Models of Obesity Endocrinology, December 1, 2005; 146(12): 5151 - 5163. [Abstract] [Full Text] [PDF]
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M. C. Moore, K. Kimura, H. Shibata, T. Honjoh, M. Saito, C. A. Everett, M. S. Smith, and A. D. Cherrington Portal 5-hydroxytryptophan infusion enhances glucose disposal in conscious dogs Am J Physiol Endocrinol Metab, August 1, 2005; 289(2): E225 - E231. [Abstract] [Full Text] [PDF]
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F. G. Haj, J. M. Zabolotny, Y.-B. Kim, B. B. Kahn, and B. G. Neel Liver-specific Protein-tyrosine Phosphatase 1B (PTP1B) Re-expression Alters Glucose Homeostasis of PTP1B-/-Mice J. Biol. Chem., April 15, 2005; 280(15): 15038 - 15046. [Abstract] [Full Text] [PDF]
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M. C. Moore, C. A. DiCostanzo, D. Dardevet, M. Lautz, B. Farmer, and A. D. Cherrington Interaction of a selective serotonin reuptake inhibitor with insulin in the control of hepatic glucose uptake in conscious dogs Am J Physiol Endocrinol Metab, March 1, 2005; 288(3): E556 - E563. [Abstract] [Full Text] [PDF]
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N. X. Cawley, J. Zhou, J. M. Hill, D. Abebe, S. Romboz, T. Yanik, R. M. Rodriguiz, W. C. Wetsel, and Y. P. Loh The Carboxypeptidase E Knockout Mouse Exhibits Endocrinological and Behavioral Deficits Endocrinology, December 1, 2004; 145(12): 5807 - 5819. [Abstract] [Full Text] [PDF]
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R. Gutierrez-Juarez, S. Obici, and L. Rossetti Melanocortin-independent Effects of Leptin on Hepatic Glucose Fluxes J. Biol. Chem., November 26, 2004; 279(48): 49704 - 49715. [Abstract] [Full Text] [PDF]
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