help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wu, X.
Right arrow Articles by Goldstein, B. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, X.
Right arrow Articles by Goldstein, B. J.
The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 12 5973-5980
Copyright © 2001 by The Endocrine Society

Other Original Articles

Depot-Specific Variation in Protein-Tyrosine Phosphatase Activities in Human Omental and Subcutaneous Adipose Tissue: A Potential Contribution to Differential Insulin Sensitivity

Xiangdong Wu, Johan Hoffstedt, Wasim Deeb, Reetu Singh, Natalia Sedkova, Assaf Zilbering, Li Zhu, Pauline K. Park, Peter Arner and Barry J. Goldstein

Dorrance H. Hamilton Research Laboratories (X.W., W.D., R.S., N.S., A.Z., L.Z., P.K.P., B.J.G.), Division of Endocrinology and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; and the Department of Medicine at Karolinska Institute (J.H., P.A.), Huddinge University Hospital, S-141 86 Stockholm, Sweden

Address all correspondence and requests for reprints to: Barry J. Goldstein, M.D., Ph.D., Director, Division of Endocrinology and Metabolic Diseases, Jefferson Medical College, Room 349 Alumni Hall, 1020 Locust Street, Philadelphia, Pennsylvania 19107-6799. E-mail: Barry.Goldstein{at}mail.tju.edu

Abstract

Compared with the sc depot, omental (om) adipose tissue is relatively resistant to the metabolic actions of insulin. Protein-tyrosine phosphatases (PTPases) modulate receptor kinase activation and signal transduction in insulin-sensitive tissues, and their activity is dependent on the reduced state of the cysteine thiol required for catalysis. Using a novel anaerobic technique to avoid air oxidation, we found that the mean endogenous PTPase activity was 2.1-fold higher in om compared with paired samples of sc adipose tissue (P < 0.003). The specific activity of PTP1B isolated under anaerobic conditions was also 41% higher in om adipose tissue (P < 0.001). Interestingly, the total PTPase activity from both adipose depots and the specific activity of PTP1B was increased by 42–71% after reduction in vitro with dithiothreitol, indicating that a major fraction of the cellular PTPase activity can be reactivated by sulfhydryl reduction. The mass of the insulin receptor ß-subunit and the PTPases PTP1B and leukocyte antigen related was not significantly different between the two adipose depots. These studies provide the first demonstration that endogenous PTPase activity, including PTP1B, is increased in om adipose tissue and may contribute to the relative insulin resistance of this fat depot. The finding that a substantial fraction of PTPase activity in human adipose tissue is present in a latent, oxidized form also suggests a potential means of in vivo regulation of these important cellular enzymes that modulate the insulin signaling cascade.

OBESITY HAS LONG been recognized as a major contributor to the pathogenesis of insulin resistance and type 2 diabetes mellitus (1). However, more prominent than the degree of overall body adiposity, the anatomic distribution of fat tissue involving increased visceral abdominal fat has been repeatedly shown to be associated with abnormalities in insulin signaling, glucose intolerance, dyslipidemia, and significantly increased risk for vascular disease (2). Compared with the sc depot, omental (om) fat is relatively resistant to the antilipolytic action of insulin (3, 4), and expansion of the om fat depot raises free fatty acid levels and contributes to insulin resistance in liver and skeletal muscle by several potential mechanisms (5, 6, 7). The cellular alterations that determine the relative insulin sensitivity of various adipose tissue depots and underlie their differential effects on systemic insulin resistance are not well understood. However, recent studies suggest that the reduced ability of insulin to stimulate the phosphorylation of several proteins involved in intracellular signaling might explain why insulin is less antilipolytic in visceral compared with sc fat cells (8).

Protein-tyrosine phosphatases (PTPases) modulate the activation state of the insulin receptor kinase and postreceptor signaling in insulin responsive tissues (9, 10). Among a number of PTPases that have been identified in insulin-sensitive cells (9), PTP1B, a single-domain intracellular PTPase, has been implicated to the greatest extent in the negative regulation of insulin receptor autophosphorylation and postreceptor insulin signaling in cellular studies (11, 12, 13, 14, 15, 16). Recently, a physiological role of PTP1B in insulin action has been further supported by demonstration of enhanced insulin sensitivity and potentiation of insulin-stimulated protein-tyrosine phosphorylation in PTP1B knock-out mice from two laboratories (17, 18). Changes in the intracellular enzymatic activity of PTP1B may also affect insulin action in adipose tissue from obese subjects (19). Leukocyte antigen related (LAR), a transmembrane, tandem-domain PTPase, has also been shown to affect insulin signaling in cultured cell systems as well as in a knock-out mouse model (20, 21, 22, 23, 24).

In addition to changes in protein abundance that may affect the tissue activity of specific PTPases, recent data have indicated that cellular PTPase enzymes are subjected to oxidative reactions in the cell that can inhibit enzyme activity in a stepwise fashion (25, 26). This susceptibility to oxidative inactivation resides in the characteristic PTPase active site sequence motif that requires the catalytic cysteine thiol to be in a reduced state (27). Because of its spatial coordination, the catalytic thiol hydrogen has a pKa more than 3 U lower than that found in a typical cysteine protein side chain, and this facilitates its oxidation and derivatization at physiological pH (28, 29). The stepwise oxidation of cellular PTPases to progressively more inert forms appears to constitute a major regulatory mechanism for PTPases within the cellular environment.

To test whether alterations in PTPase specific activity or abundance might contribute to the differential action of insulin in adipose depots, we measured PTPase activity in lysates of sc and om adipose tissue obtained during elective surgery in 12 nondiabetic subjects. In these studies, we employed our recently reported technique involving strict anaerobic conditions for cell lysate preparation and PTPase assays to evaluate the overall activity of endogenous PTPases by preventing the oxidation and inhibition of cellular PTPases that occurs on exposure to air. Performing the PTPase assays in the presence and absence of added dithiothreitol (DTT) enabled us to measure the proportion of endogenous PTP activity that is latent, in a partially oxidized or thiol-conjugated form that is activated by incubation with the strong reducing agent. Finally, we tested whether the endogenous PTPase activity in the different cell depots correlated with the body mass index (BMI). Similar measures were also performed specifically for PTP1B, assayed after immunoprecipitation with a monoclonal antibody that retains the catalytic activity of the enzyme.

Materials and Methods

Study subjects

Following institutionally approved informed consent procedures, paired 8–10 g samples of sc and om adipose tissue were obtained at elective benign surgery (for hysterectomy, ovarian cysts or cholecystectomy) or during gastric banding for obesity at Huddinge Hospital in Stockholm, Sweden, or Thomas Jefferson University Hospital in Philadelphia, PA. The study participants included 9 female and 3 male nondiabetic subjects, age 46 ± 4 yr (range 30–70 yr) and with BMI values of 45 ± 3 kg/m2 (range 24–54 kg/m2). The subjects did not have fasting hyperglycemia and none was on regular medication. Two women were menopausal. The adipose tissue was snap frozen and maintained at -85 C until use. Before surgery, all subjects fasted overnight, and only saline was infused iv until adipose tissue was removed, which was done at the beginning of surgery.

Materials

Protein assay reagents were from Bio-Rad Laboratories, Inc. (Hercules, CA). Enhanced chemiluminescence reagents were from NEN Life Science Products (Boston, MA). Wheat germ agglutinin was from Vector Laboratories, Inc. (Burlingame, CA). Immobilized protein G was from Pierce Chemical Co. (Rockford, IL). Monoclonal antihuman PTP1B was from Oncogene Research Products (Cambridge, MA), polyclonal anti-LAR was generated from the recombinant rat protein (21) by Cocalico Biologicals (Reamstown, PA), polyclonal antihuman insulin receptor was from Transduction Laboratories (Lexington, KY), and monoclonal anti-ß-tubulin was from Sigma (St. Louis, MO). Horseradish peroxidase-conjugated secondary antimouse, antirabbit IgG, and {gamma}-[32P]ATP was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Unless otherwise indicated, reagents were of the highest available grade and obtained from Sigmaor Fisher Scientific (Pittsburgh, PA).

Anaerobic experimental conditions

Tissue homogenization and enzyme activity assay were performed in an enclosed anaerobic work station (Forma Scientific, Model No. 901024) as we recently described (30). This chamber uses a palladium catalyst and desiccant wafers to maintain strict anaerobic to less that 10 ppm O2, using high purity N2 for purging. The working anaerobic gas mixture was N2:H2:CO2 proportioned at 85:10:5. For anaerobic conditions, the tissue samples were introduced into the chamber in a frozen state, and all buffer solutions exposed to the tissue samples were deoxygenated within the chamber before use.

Preparation of adipose tissue homogenates

Approximately 4 g of adipose tissue from each subject was homogenized in 16 ml of ice-cold deoxygenated homogenization buffer containing 10% glycerol, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 25 mM benzamidine, 10 µM leupeptin, 2.5 µmol/liter pepstatin A, and 50 U/ml aprotinin in 10 mM Tris-HCl (pH 7.0), with four up/down strokes at Setting No. 3 using a Polytron (Brinkmann Instruments, Inc., Westbury NY). The crude homogenate was centrifuged at 3,000 x g for 15 min, and the fat cake was discarded. The infranate was made up to 1% (vol/vol) Triton X-100 to solubilize PTPase enzymes from the particulate compartment into the tissue homogenate. The supernatant resulting from centrifugation at 15,000 x g for 20 min at 4 C was stored in aliquots at -80 C. The protein concentration of each sample was estimated by the Bio-Rad Laboratories, Inc. protein dye reagent according to the manufacturer’s protocol.

PTPase enzyme activity using para-nitrophenylphosphate (pNPP) as substrate

Using strictly anaerobic working conditions, samples containing 20 µg of lysate protein were incubated with 180-µl reaction buffer including 10 mM pNPP, 20 mM 2-(N-morpholino)ethanesulfonic acid at pH 6.0, and with or without 2 mM DTT at 37 C. The reaction time course was established to determine the incubation time that enzyme activity was in the linear reaction period. The reactions were quenched by the addition of 0.2 M NaOH, and the absorption was determined at 410 nm (16). The enzyme activity was expressed as the products of pNPP hydrolysis per gram protein per hour.

Preparation of [32P]-reduced, carboxamidomethylated, maleyated (RCM) lysozyme

Recombinant human insulin receptors from transfected Chinese hamster ovary cells (31) were partially purified on wheat germ lectin-agarose as described (32). RCM lysozyme (Sigma) was phosphorylated using the insulin receptor preparation as described (33). The reaction was initiated with the addition of 1 mg of RCM lysozyme in the reaction buffer and incubated at 25 C for 12–18 h. The reaction buffer included 40 mM imidazole hydrochloride, 50 mM NaCl, 12 mM magnesium acetate, 4 mM MnCl2, 0.2 mM EGTA, 0.05% (vol/vol) Triton X-100, 3% glycerol, 0.1 mM ammonium molybdate, 0.1 mM sodium vanadate, 30 mM N-acetylglucosamine, 0.2% (wt/vol) sodium deoxycholate, 2 mM DTT, 4 mM ATP, 300 nM insulin, 400 µg insulin receptor protein from the wheat germ lectin column eluate, and [{gamma}-32P]ATP. The reaction was terminated with the addition of trichloroacetic acid to a final concentration of 20% (wt/vol) and centrifuged at 27,000 x g for 15 min at 4 C. The pellet was washed 3 times with 20% trichloroacetic acid, suspended in 2 M Tris base and dialyzed against 50 mM imidazole-HCl, pH 7.2.

PTPase enzyme activity using [32P]-RCM lysozyme as a substrate

Using strictly anaerobic conditions described above, PTPase activity was assayed using 20 µg protein of adipose tissue sample in reaction buffer (25 mM imidazole hydrochloride, pH 7.0, 1 mg/ml fatty acid and globulin-free BSA, with or without 2 mM DTT) and preincubated for 5 min at 30 C. The reaction was initiated by the addition of phosphotyrosyl RCM lysozyme (>10,000 cpm) and incubated at 30 C, and terminated by the addition of 0.9 ml of acidic charcoal mixture, consisting of 0.9 M NaCl, 90 mM Na pyrophosphate, 2 mM NaH2PO4 and 4% (vol/vol) Norit A (34). After centrifugation in a microfuge, the amount of radioactivity in 0.4 ml of supernatant was measured by Cerenkov counter. The tissue fraction was diluted so that less than 20% of the RCM lysozyme was hydrolyzed during the reaction period of up to 30 min. The initial rate of RCM lysozyme hydrolysis was estimated from the linear portion of the earliest time points of the enzymatic reaction.

Immunoprecipitation of PTP1B followed by PTPase enzyme activity assay

With samples maintained under strictly anaerobic conditions, aliquots containing 200 µg of adipose tissue homogenate protein were incubated with 2-µl mouse serum and 30-µl protein G beads at 4 C for 1 h in a preclearing step. The supernatant was then incubated with 2 µg monoclonal antihuman PTP1B antibody at 4 C for 12 h before the addition of 40-µl protein G beads on ice for 2 h. After washing in the homogenate buffer 3 times, protein G beads were used for enzyme activity assay using [32P]-RCM lysozyme as described above. Immunoblot analysis performed separately showed no significant difference in the amount of PTP1B protein adsorbed to the protein G beads among different samples tested (data not shown).

Immunoblot analysis

Aliquots of 20 µg protein of adipose tissue sample fractionated on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membrane using a semidry Western blotting apparatus, and immunoblotted with antibody to human PTP1B, LAR, human insulin receptor, or ß-tubulin. Proteins were visualized by enhanced chemiluminescence after incubation with horseradish peroxidase-conjugated secondary antibodies, and quantitatively analyzed on the ImageStation (Kodak Digital Science, Rochester, NY).

Statistical analyses

Quantitative data are calculated from the mean ± SE values from at least three replicate determinations. One-way ANOVA was used for comparison of group means with Bonferroni’s correction for determination of significance. t test was used for comparing two group means and a paired t test was used for samples taken from the same individuals. Correlation analysis was performed with the Pearson Product moment technique using SigmaStat software (SPSS, Inc., Chicago, IL).

Results

PTPase activities in sc and om adipose tissue

We initially measured the PTPase activity in homogenates of the two adipose depots using anaerobic experimental conditions without added DTT to the reaction buffer to reflect the endogenous level of enzyme activity (Fig. 1AGo). As a group, the mean PTPase activity using pNPP as substrate was 2.1-fold higher in the om adipose tissue compared with the sc samples (5.51 ± 0.79 vs. 2.63 ± 0.31 mol/liter product x 10-4/g·h; P = 0.003). Interestingly, when assayed in the presence of added DTT, the PTPase activity was higher in both depots, by 39% and 30% in sc and om fat, respectively. This result indicated that a significant fraction of tissue PTPase activity was present in a latent, oxidized state that can be reactivated by biochemical reduction. Even in the presence of added DTT, the PTPase activity in the om adipose tissue remained 2.0-fold higher than in the sc adipose tissue (7.17 ± 0.67 vs. 3.66 ± 0.45; P < 0.001). These results suggest that the activity of the PTPases in both tissue depots may be regulated by oxidation/reduction reactions in various states, and that the PTPase activity in the om depot is consistently higher than in the sc depot under preserved endogenous conditions or in the reduced state.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Group mean PTPase activity in lysates of sc and om adipose tissue from human subjects. Tissue homogenates were prepared from sc and om adipose tissues obtained at surgery from 12 human subjects as described in Materials and Methods. The group mean specific PTPase activity is shown using hydrolysis of pNPP (A) or 32P released from RCM-lysozyme as described in Materials and Methods (B). For each comparison between sc (SC) and om adipose tissue (OM), the P values ranged from <0.001–0.006 (**).

 
RCM lysozyme, a tyrosine phosphatase substrate that closely parallels the overall dephosphorylation of the insulin receptor in tissue extracts (35), was also used in PTPase assays under anaerobic conditions, initially without added DTT (Fig. 1BGo). For each group, the mean PTPase activity using RCM lysozyme was 45% higher in the om adipose tissue compared with the sc samples in the absence of DTT (2.36 ± 0.20 vs. 1.63 ± 0.13 cpm 32P released/µg·min; P = 0.006). As with pNPP, when assayed in the presence of added DTT, the PTPase activity with RCM lysozyme was higher in both depots, increasing by 33% and 42% in sc and om fat, respectively. Again, in the presence of added DTT, the PTPase activity in the om adipose tissue measured with RCM lysozyme was 42% higher than in the sc adipose tissue (3.08 ± 0.24 vs. 2.17 ± 0.17; P = 0.005).

Comparison of sc and om adipose tissue PTPase activities was also performed using a pairwise analysis of within-individual samples and a paired t test (Fig. 2Go). Using the pNPP (Fig. 2AGo) and the RCM lysozyme assay (Fig. 2BGo), the difference between the PTPase activity in the two adipose tissue depots in the absence of added DTT was highly significant, P = 0.003 and P = 0.01, respectively, for the two assay methods. Similar results were obtained for the data from samples evaluated in the presence of DTT during the enzyme assay.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 2. Pairwise analysis of PTPase activity in lysates of sc and om adipose tissue from individual human subjects. Tissue homogenates were prepared from sc and om adipose tissues obtained at surgery from 12 human subjects as described in Materials and Methods. Comparison of sc and om adipose tissue PTPase activities was performed using a pairwise analysis of within-individual samples using the pNPP assay (A) and the RCM-lysozyme assay (B) as shown in the absence of added DTT. By paired t test, the difference between the PTPase activity in the two adipose tissue depots revealed P = 0.003 and P = 0.01, respectively, for the data shown in panels A and B.

 
PTPase activity of PTP1B in sc and om adipose tissue

To evaluate whether PTP1B itself played an important role in the differential PTPase activity in homogenates of the two adipose tissue depots, we isolated PTP1B protein in an active form by immunoprecipitation and performed PTPase enzyme assays using 32P-RCM lysozyme as substrate under anaerobic conditions both with and without added DTT (Fig. 3AGo). The enzyme activity of PTP1B immunoprecipitated from the matched lysate protein samples was 41% higher in the om compared with the sc fraction in the absence of added DTT (0.065 ± 0.003 (mean ± SE) vs. 0.091 ± 0.003; P < 0.001). Interestingly, the inclusion of DTT during the enzyme assay significantly increased the PTP1B activity by 71% in the sc adipose tissue and by 52% in the om tissue, indicating that a significant fraction of the tissue PTP1B enzyme activity was present in an inactive, latent state. Even when assayed in the presence of DTT, a significant increase of 25% in the PTP1B activity in om vs. sc adipose tissue remained (Fig. 3AGo). Furthermore, by paired analysis of samples from the same individuals, there was a striking increase in PTP1B activity (Fig. 3BGo) that was highly statistically significant (P < 0.001).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. Specific activity of PT1B in sc and om adipose tissue from human subjects. Aliquots of 200 µg protein of each adipose tissue lysate were incubated with 2.0 µg monoclonal antihuman PTP1B antibody after preclearing the samples using protein G and mouse serum. PTP1B enzyme activity was measured with the adsorbed enzyme samples using tyrosine-phosphorylated RCM-lysozyme as substrate and assayed without added DTT. **, Difference in PTPase activity between om adipose cytosol compared with sc adipose cytosol was highly statistically significant (P < 0.001). A, Group mean data with and without DTT added during the enzyme assay. B, Pairwise analysis of SC and OM samples from the same individuals.

 
Correlation analysis between adipose tissue PTPase activity and BMI

In our previous work, we noted a significant correlation between the overall PTPase activity in homogenates of sc adipose tissue across a wide range of BMI values (22). In the present study, however, no significant relationship was found between BMI and the overall PTPase activity in the lysates of sc and om adipose tissue, perhaps owing to the few samples of adipose tissue from lean individuals for the present work (Fig. 4Go), compared with the previously published data. A weak correlation (R = 0.66; P = 0.05) was present between the PTP1B enzyme activity in om adipose tissue (measured with added DTT) and BMI (Fig. 4AGo). Although this correlation is lost when the single lean individual is omitted from the analysis, these initial findings suggest that, in om fat, a relationship potentially exists between the activatable PTP1B content and BMI. In contrast, the level of PTP1B enzyme activity in the sc adipose tissue compartment did not correlate significantly with BMI (Fig. 4B).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Correlation analysis between BMI and PTP1B enzyme activity in human sc and om adipose tissue. The enzyme activities of PTP1B in sc and om adipose tissue samples in the presence of DTT were plotted against the individual BMI values and correlation analysis was performed using the Pearson product-moment analysis method. The correlation coefficient between PTP1B enzyme activity in om adipose tissue and BMI was R = 0.66 with P = 0.05 (A). For sc adipose tissue, the correlation was not significant (B).

 
Quantitation of PTP1B, LAR, insulin receptor ß-subunit, and ß-tubulin protein mass in sc and om adipose tissue

Because the steady-state balance of protein tyrosine phosphorylation depends on the abundance of both the cellular PTPase enzymes as well as key substrate proteins, we measured the protein mass of PTP1B, LAR and the insulin receptor ß-subunit in the tissue lysates of the two adipose depots. ß-tubulin served as an internal control. A representative immunoblot is shown in Fig. 5Go. The expression levels of PTP1B, LAR, the insulin receptor ß-subunit, and ß-tubulin itself were not significantly different between sc and om adipose tissues within the same individual subject.



View larger version (94K):
[in this window]
[in a new window]
  		Figure 5. Representative immunoblot of PTP1B, LAR, IR-ß, and ß-tubulin protein in paired sc and om adipose tissue samples from two of the human subjects. Equal amounts of total protein (20 µg) from sc (SC) or om adipose tissue (OM), as indicated, were subjected to PAGE, transferred to nitrocellulose filters, and immunoblotted with a monoclonal antibody to human PTP1B, polyclonal antibody to human LAR, polyclonal antibody to human insulin receptor ß, and monoclonal antibody to tubulin ß as described in Materials and Methods. The migration position of full-length PTP1B was 50 kDa, LAR transmembrane subunit at 85 kDa, insulin receptor ß-subunit at 95 kDa, and ß-tubulin at 55 kDa.

 
Influence of age and gender

Correlation analysis revealed no significant relationship between age and the different PTPase measures. Data from the three men and two menopausal women showed no apparent differences due to gender or menopausal status when compared with the data obtained from the seven premenopausal female subjects.

Discussion

With the incidence of obesity and type 2 diabetes mellitus growing worldwide, more attention has been paid to understanding the strong influence that body adiposity exerts on glucose tolerance and insulin sensitivity. Increased body weight and BMI are correlated with an augmented risk for diabetes and cardiovascular disease, and many studies have shown an enhanced effect when the anthropomorphic distribution of body adiposity is considered (36). Increased truncal and particularly intraabdominal accumulation of body fat has been associated with higher plasma insulin and glucose levels indicative of insulin resistance (2, 37, 38). The clinical sequelae of this prevalent condition includes not only type 2 diabetes but also the "plurimetabolic" or "insulin resistance" syndrome with a heightened propensity toward atherosclerosis (39). Thus, a better understanding of the contribution of regional adipose tissue to the pathogenesis of insulin resistance in obesity will help our understanding of the clinical consequences of this disorder.

In attempts to help explain the association between abdominal adipose tissue and a variety of metabolic diseases, several studies have indicated that intraabdominal adipose tissue has a particularly high lipid turnover rate, and adipocytes from this depot demonstrate a relative resistance to the anti-lipolytic action of insulin and enhanced sensitivity to catecholamine-induced lipolysis as recently reviewed (40). The enhanced lipolysis increases the portal and systemic concentration of nonesterified fatty acids, which reduce the insulin sensitivity of glucose disposal and metabolism in skeletal muscle and liver and pancreatic insulin secretion leading to a diabetogenic state (41).

Cellular PTPases have been recently shown to play a major role in the regulation of the insulin signaling pathway by modulating the steady-state tyrosine phosphorylation of the insulin receptor and its substrate proteins in its target tissues (9, 10). In adipose tissue, insulin affects the flux of metabolites through a number of pathways, including those involving fatty acid storage and release. In our previous work, we demonstrated that PTPase activity was significantly increased in homogenates of sc adipose tissue from obese subjects with insulin resistance (22). Furthermore, it is well established that insulin sensitivity is enhanced following a modest amount of weight loss (for example, Ref. 42 , and our studies showing that weight loss is also associated with a significant reduction in PTPase activity in sc adipose tissue (43). Thus, in various pathophysiologic situations, alterations in PTPase activities may influence the sensitivity of insulin action in its target tissues.

In the present study, we tested whether alterations in adipose tissue PTPase activity might vary in the sc and om adipose depots and potentially play a role in the relative sensitivity of insulin action in these different adipose tissue depots. Using an anaerobic working environment and PTPase assays performed with and without added biochemical reductants, we were able to measure the level of endogenous overall tissue PTPase activity, the activity present after reduction and reactivation of the endogenous PTPases, and the specific activity of PTP1B under identical conditions. The present work shows that the mean endogenous PTPase specific activity in the om adipose tissue was significantly higher than in the corresponding sc adipose tissue depot in a series of 12 subjects without diabetes. These results suggest that increased PTPase activity in om fat may contribute to the relative resistance to the action of insulin that has repeatedly been shown to be characteristic of this adipose depot, as discussed above. The ability of insulin to phosphorylate its receptor, insulin receptor substrate-1, and the p85 kDa subunit of phosphatidylinositol 3'-kinase was shown by Zierath and colleagues (8) to be markedly reduced in fat cells from om compared with sc adipocytes in a cohort of subjects similar to those used in the present investigation. It is a distinct possibility that the increase in PTPase activity, in particular involving PTP1B, in om vs. sc adipose cells might help to explain some of the previously reported regional differences in the tyrosine phosphorylation of proteins in the insulin signaling pathways in human fat cells.

The data presented here are also the first to show that a substantial fraction of the enzymatic activity of PTPases in human adipose tissue in both om and sc compartments is present in a latent form that is activatable by biochemical reduction. Recent work from several laboratories has provided clear evidence that the enzymes in the PTPase superfamily are regulated in vivo by oxidation/reduction reactions that can alter the reduced state of the cysteine thiol side chain that is required for phosphotyrosine hydrolysis (27, 28). Several laboratories have recently provided evidence that reactive oxygen species, including H2O2, can oxidize and inactivate PTPases in vivo (25, 26). Because only the reduced form of the catalytic thiol is enzymatically active, stepwise and progressively irreversible oxidative inhibition is emerging as an important means by which PTPase activity can be suppressed in specific signal transduction pathways. The initial oxidation of the catalytic thiol results in a sulfenic derivative, which is reversible and amenable to reduction by cellular mechanisms or by reducing agents in vitro, which may constitute a major regulatory mechanism for PTPases within the cellular environment (44, 45). Redox regulation of PTPases involved in the regulation of the insulin signaling pathway may be of particular importance because changes in cellular oxidative stress can occur in states of diabetes with hyperglycemia (46, 47). We have also recently shown that insulin-stimulation of cultured hepatoma and 3T3-L1 adipocytes in culture leads to the rapid formation of intracellular H2O2, which is associated with significant changes in cellular PTPase activity as well as a reduction in the specific activity of PTP1B (48). Thus, regulation of the activity of PTPases by oxidation or reduction of the catalytic thiol moiety within insulin target tissues is a novel site for a control mechanism that appears to contribute significantly to the steady-state balance and propagation of the cellular insulin signal.

PTP1B, in particular, appears to be a cellular target for oxidative inactivation possibly accompanied by conjugation of the catalytic thiol side chain with glutathione (glutathiolation) (44, 45, 48, 49). PTP1B was one of the first specific PTPases be implicated in the negative regulation of insulin receptor autophosphorylation and postreceptor insulin signaling (11, 12, 14, 15, 16) and studies in PTP1B knock-out mice have provided compelling evidence for a physiological role of PTP1B in insulin action (17, 18). Changes in the intracellular enzymatic activity of PTP1B may also affect insulin action in adipose tissue from obese subjects (19). Using anaerobic experimental conditions to preserve the endogenous activity state of cellular PTPases, our finding that the activity of PTP1B in lysates of om adipose tissue was significantly higher than that found in the sc depot further implicates this homolog in the regulation of the insulin action pathway. We also previously reported that increasing body mass is associated with elevated levels of PTPase activity in homogenates of sc adipose tissue (22). Similarly, our observation here that the enzymatic activity of PTP1B may also be correlated with increasing BMI in lysates of om adipose tissue (Fig. 4AGo), suggests that this enzyme in particular may be involved in the insulin resistance of om adipose tissue in obese subjects.

In summary, this work is the first demonstration that the endogenous tissue PTPase activity, specifically including PTP1B, is increased in the om adipose depot and provides support for the hypothesis that increased PTPase activity contribute to the relative resistance of om fat to the action of insulin compared with the sc compartment. In addition, our finding that a substantial fraction of the endogenous PTPase activity in human adipose tissue is present in a latent form, activatable by thiol reduction, also suggests a potential means of in vivo regulation of these important cellular enzymes that affects their capacity to modulate the insulin signaling cascade.

Acknowledgments

We thank Dr. J. Chernoff (Fox Chase Cancer Center, Philadelphia, PA) for providing the human PTP1B cDNA used as a control in some of these studies.

Footnotes

This study was supported by grants from the National Institutes of Health (DK-53388 and DK-43396) to Dr. Goldstein and from the Swedish Medical Research Council, Swedish Diabetes Association, and Swedish Heart and Lung Foundation (to P.A.).

Abbreviations: BMI, Body mass index; DTT, dithiothreitol; LAR, leukocyte antigen related; om, omental; pNPP; para-nitrophenylphosphate; PTPase, protein-tyrosine phosphatase; RCM, reduced, carboxamidomethylated, maleyated.

Received September 14, 2000.

Accepted September 14, 2001.

References

  1. Kolaczynski JW, Goldstein BJ 2000 The influence of obesity on the development of non-insulin-dependent diabetes mellitus. In: Lockwood DH, Heffner TG, eds. Obesity: pathology and therapy. New York: Springer-Verlag; 91–119
  2. Kissebah AH, Vydelingum N, Murray R, Evans DJ, Hartz AJ, Kalkhoff RK, Adams PW 1982 Relation of body fat distribution to metabolic complications of obesity. J Clin Endocrinol Metab 54:254–260[Abstract/Free Full Text]
  3. Bolinder J, Kager L, Ostman J, Arner P 1983 Differences at the receptor and postreceptor levels between human om and subcutaneous adipose tissue in the action of insulin on lipolysis. Diabetes 32:117–123[Abstract]
  4. Rebuffe-Scrive M, Andersson B, Olbe L, Bjorntorp P 1989 Metabolism of adipose tissue in intraabdominal depots of nonobese men and women. Metabolism 38:453–458[CrossRef][Medline]
  5. Boden G 1999 Free fatty acids, insulin resistance, and type 2 diabetes mellitus. Proc Assoc Am Physicians 111:241–248[CrossRef][Medline]
  6. Shulman GI 2000 Cellular mechanisms of insulin resistance. J Clin Invest 106:171–176[Medline]
  7. Rossetti L 2000 Perspective: hexosamines and nutrient sensing. Endocrinology 141:1922–1925[Free Full Text]
  8. Zierath JR, Livingston JN, Thorne A, Bolinder J, Reynisdottir S, Lonnqvist F, Arner P 1998 Regional difference in insulin inhibition of non-esterified fatty acid release from human adipocytes—relation to insulin receptor phosphorylation and intracellular signalling through the insulin receptor substrate-1 pathway. Diabetologia 41:1343–1354[CrossRef][Medline]
  9. Goldstein BJ 2000 Protein-tyrosine phosphatases and the regulation of insulin action. In: LeRoith D, Olefsky JM, Taylor SI, eds. Diabetes mellitus: a fundamental and clinical text. Philadelphia: Lippincott; 206–217
  10. Kennedy BP, Ramachandran C 2000 Protein tyrosine phosphatase-1B in diabetes. Biochem Pharmacol 60:877–883[CrossRef][Medline]
  11. Cicirelli MF, Tonks NK, Diltz CD, Weiel JE, Fischer EH, Krebs EG 1990 Microinjection of a protein-tyrosine-phosphatase inhibits insulin action in Xenopus oocytes. Proc Natl Acad Sci USA 87:5514–5518[Abstract/Free Full Text]
  12. Kenner KA, Anyanwu E, Olefsky JM, Kusari J 1996 Protein-tyrosine phosphatase 1B is a negative regulator of insulin- and insulin-like growth factor-1 stimulated signaling. J Biol Chem 271:19810–19816[Abstract/Free Full Text]
  13. Ahmad F, Li PM, Meyerovitch J, Goldstein BJ 1995 Osmotic loading of neutralizing antibodies defines a role for protein-tyrosine phosphatase 1B in negative regulation of the insulin action pathway. J Biol Chem 270:20503–20508[Abstract/Free Full Text]
  14. Venable CL, Frevert EU, Kim YB, Fischer BM, Kamatkar S, Neel BG, Kahn BB 2000 Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/protein kinase B activation. J Biol Chem 275:18318–18326[Abstract/Free Full Text]
  15. Calera MR, Vallega G, Pilch PF 2000 Dynamics of protein-tyrosine phosphatases in rat adipocytes. J Biol Chem 275:6308–6312[Abstract/Free Full Text]
  16. Goldstein BJ, Bittner-Kowalczyk A, White MF, Harbeck M 2000 Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein phosphatase 1B. Possible facilitation by the formation of a ternary complex with the GRB2 adaptor protein. J Biol Chem 275:4283–4289[Abstract/Free Full Text]
  17. Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy AL, Normandin D, Cheng A, Himms-Hagen J, Chan CC, Ramachandran C, Gresser MJ, Tremblay ML, Kennedy BP 1999 Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283:1544–1548[Abstract/Free Full Text]
  18. Klaman LD, Boss O, Peroni OD, Kim JK, Martino JL, Zabolotny JM, Moghal N, Lubkin M, Kim YB, Sharpe AH, Stricker-Krongrad A, Shulman GI, Neel BG, Kahn BB 2000 Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice. Mol Cell Biol 20:5479–5489[Abstract/Free Full Text]
  19. Cheung A, Kusari J, Jansen D, Bandyopadhyay D, Kusari A, Bryer-Ash M 1999 Marked impairment of protein tyrosine phosphatase 1B activity in adipose tissue of obese subjects with and without type 2 diabetes mellitus. J Lab Clin Med 134:115–123[CrossRef][Medline]
  20. Mooney RA, Kulas DT, Bleyle LA, Novak JS 1997 The protein tyrosine phosphatase LAR has a major impact on insulin receptor dephosphorylation. Biochem Biophys Res Commun 235:709–712[CrossRef][Medline]
  21. Zhang WR, Li PM, Oswald MA, Goldstein BJ 1996 Modulation of insulin signal transduction by eutopic overexpression of the receptor-type protein-tyrosine phosphatase LAR. Mol Endocrinol 10:575–584[Abstract/Free Full Text]
  22. Ahmad F, Considine RV, Goldstein BJ 1995 Increased abundance of the receptor-type protein-tyrosine phosphatase LAR accounts for the elevated insulin receptor dephosphorylating activity in adipose tissue of obese human subjects. J Clin Invest 95:2806–2812
  23. Kulas DT, Zhang WR, Goldstein BJ, Furlanetto RW, Mooney RA 1995 Insulin receptor signalling is augmented by antisense inhibition of the protein-tyrosine phosphatase LAR. J Biol Chem 270:2435–2438[Abstract/Free Full Text]
  24. Ren JM, Li PM, Zhang WR, Sweet LJ, Cline G, Shulman GI, Livingston JN, Goldstein BJ 1998 Transgenic mice deficient in the LAR protein-tyrosine phosphatase exhibit profound defects in glucose homeostasis. Diabetes 47:493–497[Abstract]
  25. Claiborne A, Yeh JI, Mallett TC, Luba J, Crane EJ, Charrier V, Parsonage D 1999 Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation. Biochemistry 38:15407–15416[CrossRef][Medline]
  26. Herrlich P, Bohmer FD 2000 Redox regulation of signal transduction in mammalian cells. Biochem Pharmacol 59:35–41[CrossRef][Medline]
  27. Zhang ZY 1998 Protein-tyrosine phosphatases— biological function, structural characteristics, and mechanism of catalysis. Crit Rev Biochem Mol Biol 33:1–52[CrossRef][Medline]
  28. Denu JM, Dixon JE 1998 Protein tyrosine phosphatases—mechanisms of catalysis and regulation. Curr Opin Chem Biol 2:633–641[CrossRef][Medline]
  29. Barford D, Das AK, Egloff MP 1998 The structure and mechanism of protein phosphatases—insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 27:133–164[CrossRef][Medline]
  30. Zhu L, Zilbering A, Wu X, Mahadev K, Joseph JI, Jabbour S, Deeb W, Goldstein BJ 2001 Use of an anaerobic environment to preserve the endogenous activity of protein-tyrosine phosphatases isolated from intact cells. FASEB J 15:1637–1639[Abstract/Free Full Text]
  31. Hashimoto N, Feener EP, Zhang WR, Goldstein BJ 1992 Insulin receptor protein-tyrosine phosphatases—leukocyte common antigen-related phosphatase rapidly deactivates the insulin receptor kinase by preferential dephosphorylation of the receptor regulatory domain. J Biol Chem 267:13811–13814[Abstract/Free Full Text]
  32. Pike LJ, Kuenzel EA, Casnellie JE, Krebs EG 1984 A comparison of the insulin- and epidermal growth factor-stimulated protein kinases from human placenta. J Biol Chem 259:9913–9921[Abstract/Free Full Text]
  33. Tonks NK, Diltz CD, Fischer EH 1991 Purification of protein-tyrosine phosphatases from human placenta. Methods Enzymol 201:427–442[Medline]
  34. Streuli M, Krueger NX, Thai T, Tang M, Saito H 1990 Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR. EMBO J 9:2399–2407[Medline]
  35. Ahmad F, Azevedo JL, Cortright R, Dohm GL, Goldstein BJ 1997 Alterations in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes. J Clin Invest 100:449–458[Medline]
  36. Montagne CT, O’Rahilly S 2000 The perils of portliness—causes and consequences of visceral adiposity. Diabetes 49:883–888[Abstract]
  37. Krotkiewski M, Bjorntorp P, Sjostrom L, Smith U 1983 Impact of obesity on metabolism in men and women. Importance of regional adipose tissue distribution. J Clin Invest 72:1150–1162
  38. Ohlson LO, Larsson B, Svardsudd K, Welin L, Eriksson H, Wilhelmsen L, Bjorntorp P, Tibblin G 1985 The influence of body fat distribution on the incidence of diabetes mellitus. 13.5 years of follow-up of the participants in the study of men born in 1913. Diabetes 34:1055–1058[Abstract]
  39. Reaven GM 1998 Insulin resistance and human disease. J Basic Clin Physiol Pharmacol 9:387–406[Medline]
  40. Arner P 1998 Not all fat is alike. Lancet 351:1301–1302[CrossRef][Medline]
  41. Bjorntorp P 1990 "Portal" adipose tissue as a generator of risk factors for cardiovascular disease and diabetes. Arteriosclerosis 10:493–496[Free Full Text]
  42. Friedman JE, Dohm GL, Leggett-Frazier N, Elton CW, Tapscott EB, Pories WP, Caro JF 1992 Restoration of insulin responsiveness in skeletal muscle of morbidly obese patients after weight loss—effect on muscle glucose transport and glucose transporter GLUT4. J Clin Invest 89:701–705
  43. Ahmad F, Considine RV, Bauer TL, Ohannesian JP, Marco CC, Goldstein BJ 1997 Improved sensitivity to insulin in obese subjects following weight loss is accompanied by reduced protein-tyrosine phosphatases in adipose tissue. Metabolism 46:1140–1145[CrossRef][Medline]
  44. Denu JM, Tanner KG 1998 Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide—evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry 37:5633–5642[CrossRef][Medline]
  45. Lee SR, Kwon KS, Kim SR, Rhee SG 1998 Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor. J Biol Chem 273:15366–15372[Abstract/Free Full Text]
  46. Ha HJ, Lee HB 2000 Reactive oxygen species as glucose signaling molecules in mesangial cells cultured under high glucose. Kidney Int 58:S19–S25
  47. Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, Nawata H 2000 High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes 49:1939–1945[Abstract]
  48. Mahadev K, Zilbering A, Zhu L, Goldstein BJ 2001 Insulin-stimulated generation of hydrogen peroxide reversibly inhibits PTP1B and enhances the early insulin action cascade. J Biol Chem 276:21938–21942[Abstract/Free Full Text]
  49. Barrett WC, DeGnore JP, Konig S, Fales HM, Keng YF, Zhang ZY, Yim MB, Chock PB 1999 Regulation of PTP1B via glutathionylation of the active site cysteine 215. Biochemistry 38:6699–6705[CrossRef][Medline]



This article has been cited by other articles:


Home page
 DiabetesHome page
I. Nieto-Vazquez, S. Fernandez-Veledo, C. de Alvaro, and M. Lorenzo
Dual Role of Interleukin-6 in Regulating Insulin Sensitivity in Murine Skeletal Muscle
Diabetes, December 1, 2008; 57(12): 3211 - 3221.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
M. Lorenzo, S. Fernandez-Veledo, R. Vila-Bedmar, L. Garcia-Guerra, C. De Alvaro, and I. Nieto-Vazquez
Insulin resistance induced by tumor necrosis factor-{alpha} in myocytes and brown adipocytes
J Anim Sci, April 1, 2008; 86(14_suppl): E94 - E104.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. M. Picha, S. S. Patel, S. Mandiyan, J. Koehn, and L. P. Wennogle
The Role of the C-terminal Domain of Protein Tyrosine Phosphatase-1B in Phosphatase Activity and Substrate Binding
J. Biol. Chem., February 2, 2007; 282(5): 2911 - 2917.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
I. Nieto-Vazquez, S. Fernandez-Veledo, C. de Alvaro, C. M. Rondinone, A. M. Valverde, and M. Lorenzo
Protein-Tyrosine Phosphatase 1B-Deficient Myocytes Show Increased Insulin Sensitivity and Protection Against Tumor Necrosis Factor-{alpha}-Induced Insulin Resistance
Diabetes, February 1, 2007; 56(2): 404 - 413.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
J. J. Hernandez-Morante, F. Milagro, J. A. Gabaldon, J. A. Martinez, S. Zamora, and M. Garaulet
Effect of DHEA-sulfate on adiponectin gene expression in adipose tissue from different fat depots in morbidly obese humans.
Eur. J. Endocrinol., October 1, 2006; 155(4): 593 - 600.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
L. Laviola, S. Perrini, A. Cignarelli, A. Natalicchio, A. Leonardini, F. De Stefano, M. Cuscito, M. De Fazio, V. Memeo, V. Neri, et al.
Insulin signaling in human visceral and subcutaneous adipose tissue in vivo.
Diabetes, April 1, 2006; 55(4): 952 - 961.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
M. Lafontan, C. Moro, C. Sengenes, J. Galitzky, F. Crampes, and M. Berlan
An Unsuspected Metabolic Role for Atrial Natriuretic Peptides: The Control of Lipolysis, Lipid Mobilization, and Systemic Nonesterified Fatty Acids Levels in Humans
Arterioscler. Thromb. Vasc. Biol., October 1, 2005; 25(10): 2032 - 2042.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. M. Zabolotny, F. G. Haj, Y.-B. Kim, H.-J. Kim, G. I. Shulman, J. K. Kim, B. G. Neel, and B. B. Kahn
Transgenic Overexpression of Protein-tyrosine Phosphatase 1B in Muscle Causes Insulin Resistance, but Overexpression with Leukocyte Antigen-related Phosphatase Does Not Additively Impair Insulin Action
J. Biol. Chem., June 4, 2004; 279(23): 24844 - 24851.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
E. Asante-Appiah and B. P. Kennedy
Protein tyrosine phosphatases: the quest for negative regulators of insulin action
Am J Physiol Endocrinol Metab, April 1, 2003; 284(4): E663 - E670.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
H. Motoshima, X. Wu, M. K. Sinha, V. E. Hardy, E. L. Rosato, D. J. Barbot, F. E. Rosato, and B. J. Goldstein
Differential Regulation of Adiponectin Secretion from Cultured Human Omental and Subcutaneous Adipocytes: Effects of Insulin and Rosiglitazone
J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5662 - 5667.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Shimizu, H. Maegawa, K. Egawa, K. Shi, M. Bryer-Ash, and A. Kashiwagi
Mechanism for Differential Effect of Protein-Tyrosine Phosphatase 1B on Akt Versus Mitogen-Activated Protein Kinase in 3T3-L1 Adipocytes
Endocrinology, December 1, 2002; 143(12): 4563 - 4569.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
B. J. Goldstein
Protein-Tyrosine Phosphatases: Emerging Targets for Therapeutic Intervention in Type 2 Diabetes and Related States of Insulin Resistance
J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2474 - 2480.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wu, X.
Right arrow Articles by Goldstein, B. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, X.
Right arrow Articles by Goldstein, B. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals