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Decreased Insulin Receptor (IR) Autophosphorylation in Fibroblasts from Patients with PCOS: Effects of Serine Kinase Inhibitors and IR Activators

University of California at San Francisco (M.L., J.F.Y., I.D.G., B.A.M.), Mount Zion Medical Center, San Francisco, California 94143; Northwestern University Medical School (A.D.), Chicago, Illinois 60611; Department of Molecular Endocrinology (B.B.Z.), Merck Research Laboratories, Rahway, New Jersey 07065; and Telik, Inc. (J.L.E.), South San Francisco, California 94080

Address all correspondence and requests for reprints to: Joseph L. Evans, Ph.D., Medical Research Institute, 1001 Bayhill Drive, Suite 208, San Bruno, California 94066. E-mail: . jevansphd{at}earthlink.net

Abstract

Insulin resistance is characteristic of many patients with polycystic ovary syndrome (PCOS). Several studies have suggested that a decrease in insulin receptor (IR) autophosphorylation is a significant component of this resistance. In this study, we have used a highly sensitive ELISA to measure IR tyrosine phosphorylation in fibroblasts from patients with PCOS and healthy control women. After the stimulation of intact fibroblasts with insulin, IR tyrosine phosphorylation in cells from the PCOS patients was decreased by approximately 40% when compared with controls. However, when IR were first immunocaptured from fibroblasts and then stimulated with insulin, neither basal nor insulin-stimulated IR autophosphorylation was different between the two groups, suggesting that a factor independent of the IR was involved. To examine the role of increased serine kinase activity in decreased IR autophosphorylation in PCOS, fibroblasts from PCOS patients were pretreated with inhibitors of serine kinases before insulin stimulation. Pretreatment with H7, a nonspecific protein kinase inhibitor, completely reversed the decrease in insulin-stimulated IR autophosphorylation. Pretreatment with H89, an inhibitor of protein kinase A, partially reversed this function, whereas pretreatment with Gö6983, an inhibitor of protein kinase C, was without effect. We next studied the effects of two small molecule activators of the IR tyrosine kinase: TLK16998 and Merck L7. Both TLK16998 and Merck L7 were able to reverse the impaired insulin-stimulated IR autophosphorylation. In summary, a factor(s) extrinsic to the IR cause impaired IR signaling in fibroblasts from patients with PCOS. Reversal of the impaired IR signaling by inhibitors of serine kinase activity suggests that serine kinase-mediated pathways may be involved in the insulin resistance. Moreover, the observation that TLK16998 and Merck L7 improved IR tyrosine phosphorylation in fibroblasts from patients with PCOS suggests that specific pharmacological therapies might be developed to treat the insulin resistance in PCOS.

POLYCYSTIC OVARY SYNDROME (PCOS) is a common and complex endocrine disorder with unknown etiology (1, 2, 3). PCOS is characterized by hyperandrogenism, chronic anovulation, and, frequently, profound insulin resistance (3, 4). Insulin resistance in PCOS is secondary to a postbinding defect in insulin receptor (IR) signaling (5, 6, 7). The cellular response to insulin is mediated through the IR, which is a tetrameric protein consisting of two identical extracellular -subunits that bind to insulin and two identical transmembrane ß-subunits that have intracellular tyrosine kinase activity (8, 9, 10). When insulin binds to the -subunit of the receptor, the ß-subunit tyrosine kinase undergoes autophosphorylation, resulting in the activation of the IR tyrosine kinase activity (11, 12). Once activated, the IR tyrosine kinase phosphorylates a number of intracellular targets, including the IR substrate (IRS) family of proteins, triggering a cascade of events ultimately leading to increased glucose use (13, 14, 15).

Dunaif et al. (16) reported that IR autophosphorylation was decreased in fibroblasts in approximately 50% of patients with PCOS (16). These investigators attributed this diminished IR function to increased serine phosphorylation of the IR by an unknown serine kinase activity. Increased serine phosphorylation of the IR or IRS decreases the extent of tyrosine phosphorylation and leads to impaired insulin action (11, 17, 18). Because these effects were observed in cultured fibroblasts, the data suggested that these abnormalities in IR function were intrinsic to the cell and not due to the metabolic state of the patients. Moreover, when the IRs were purified from associated proteins, IR autophosphorylation was normal, and serine kinase activity was not detected. These observations suggested that the serine kinase involved was not a component of the IR (19).

The study by Dunaif et al. (16), therefore, strongly implicated that decreased IR function and excess serine kinase activity were key features of PCOS. These findings suggested that inhibition of serine kinase might reverse the inhibition of the IR autophosphorylation and provide information as to the nature of the kinase involved. Recently, small molecule, nonpeptide, IR activators have been developed that restore IR autophosphorylation in insulin-resistant cells, but their effects on PCOS fibroblasts have not yet been studied.

We have recently developed and validated a highly specific and sensitive ELISA to measure IR tyrosine phosphorylation in fibroblasts and other cells (20). In the present study, we have used this ELISA to investigate IR function in fibroblasts from patients with PCOS and control individuals, and we studied the effects of inhibitors of serine kinases on IR autophosphorylation. In addition, we studied the effects of two new IR activators, Merck L7 and TLK16998 (21, 22, 23, 24). In these studies, we find diminished IR autophosphorylation in PCOS fibroblasts. This effect is blocked by inhibiting cellular serine kinase activity and is reversed by the IR activators Merck L7 and TLK16998.

Subjects and Methods

Subjects

Skin fibroblast cell lines were established in seven women with PCOS and four control women aged 18–43 yr as part of previously reported studies (14, 16) at the Mt. Sinai School of Medicine (New York, NY) and at the Pennsylvania State University College of Medicine (Hershey, PA). The Institutional Review Boards of both institutions approved the studies, and all women gave written informed consent. The women were in good health and, for at least 1 month (and 3 months for oral contraceptives) before study, were off medications known to affect sex hormone, lipid, or carbohydrate metabolism. The diagnosis of PCOS was made by the presence of chronic anovulation (six or fewer menses per year) in association with elevated circulating levels of testosterone, androstenedione, and/or free and weakly bound (unbound) testosterone, and exclusion of other causes of hyperandrogenism (16). The PCOS subjects were subjects 2, 5, 6, 8, 12, 13, and 15 in our previous study of IR phosphorylation in cultured skin fibroblasts (16). Three of these subjects had impaired glucose tolerance. The control women had menses every 27–32 d, no hirsutism, and no personal history of or first-degree relative with diabetes mellitus. Their circulating androgen levels were within the normal range established for reproductively normal premenopausal women in the follicular phase of the menstrual cycle (16). The control women had participated in our previous studies of insulin action in cultured skin fibroblasts (14, 16). The clinical, reproductive, and metabolic features of the PCOS and control women have been previously reported (14, 16).

Chemicals

TLK16998 and Merck L7 were kindly provided by Telik, Inc. (South San Francisco, CA), and Merck \|[amp ]\| Co., Inc. (Rahway, NJ), respectively. H89 (N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulonamide) was purchased from Sigma (St. Louis, MO). H7 [1-(5-isoquinoline-sulfonyl)-2-methylpipelazine, 2HCL] and Gö6983 were purchased from Calbiochem (San Diego, CA). Tetramethylbenzidine (TMB) reagent kit was purchased from Kirkegaard \|[amp ]\| Perry Laboratories (Gaithersburg, MD).

Skin biopsies and fibroblast cultures

Skin fibroblast cultures were established from forearm skin punch biopsies as previously described (16). The outgrown fibroblasts from the primary biopsies of control individuals and patients with PCOS were subcultured in DMEM containing 4.5 g/liter D-glucose supplemented with 10% (vol/vol) fetal calf serum. Penicillin (10 U/ml), fungizone (0.25 µg/ml), and streptomycin (10 µg/ml) were routinely added to cultures. Cells were cultivated at 37 C in a 5% CO₂-enriched, humidified atmosphere. Cells were used for study between the sixth and ninth passage.

ELISA for intact-cell IR autophosphorylation

Fibroblasts were grown in six-well plates until confluent and then serum starved [0.1% (wt/vol) BSA] for 15 h before insulin stimulation (10 min at 37 C). After treatment, cells were washed extensively in ice-cold PBS, scraped from culture plates, and incubated for 60 min at 4 C in lysis buffer [50 mM HEPES, pH 7.6, 150 mM NaCl, 1% (vol/vol) Triton X-100, 1 mM phenylmethylsulfonylfluoride, and 2 mM sodium orthovanadate]. Lysates were centrifuged (15,000 x g for 30 min at 4 C) to remove insoluble materials. The protein content in each sample was measured using Bio-Rad protein assay dye reagent concentrate according to the manufacturer’s instructions (Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts of cell lysates (50 µg protein) were applied to a 96-well microtiter plate previously coated with an antihuman IR monoclonal antibody, MA-20 (25). After 18 h incubation at 4 C, microtiter plates were washed with 20 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% (vol/vol) Tween 20 (TBST). Subsequently, biotin-conjugated anti-phosphotyrosine antibody (Upstate Biotechnology, Inc., Lake Placid, NY) was added and followed by streptavidin-horseradish peroxidase (Pierce Chemical Co., Rockford, IL). TMB (3,3',5,5'-tetramethylbenzidine) was used as the chromogenic substrate for horseradish peroxidase. The autophosphorylation signal was detected using Microplate Reader II-Multiskan Mcc/340 (DuPont, Boston, MA) by reading absorption at 450 nm. In some experiments, fibroblasts were pretreated with serine kinase inhibitors, IR tyrosine kinase activators, or vehicle (as a control) as indicated in figure legends before stimulation with insulin. Stock solutions (20 mM in dimethylsulfoxide) of test compounds were prepared fresh before each experiment. To improve compound stability, Merck L7 was diluted to the desired concentration in cell medium containing ascorbic acid (final concentration, 200 µg/ml; also included in vehicle-treated cells). Control cells were incubated with the identical concentration of vehicle (0.1% final dimethylsulfoxide).

ELISA for IR autophosphorylation in immunocaptured receptors

Fibroblasts were cultured to confluence, serum starved for 15 h, solubilized and proteins quantified as described above. Lysates of fibroblasts containing 50 µg protein were applied to 96-well microtiter plates coated with monoclonal antihuman IR antibody, MA-20. Insulin (0–100 nM) was added to the immunocaptured IR along with 10 µM of ATP for 1 h in 96-well microtiter plates. Plates were washed in TBST and processed for IR tyrosine autophosphorylation as described above.

Statistics

Data are expressed as means ± SEM. Differences between means were assessed by the t test or one-way ANOVA using GraphPad Prism version 3.02 for Windows (GraphPad Software, Inc., San Diego, CA; www.graphpad.com). Post hoc comparisons were performed using Dunnett’s test or the Newman-Keuls test for multiple comparisons. Statistical significance was accepted at P value less than 0.05.

Results

Insulin-stimulated IR ß-subunit autophosphorylation of fibroblasts from PCOS vs. controls

Intact fibroblasts from control and PCOS patients were treated with increasing concentrations of insulin up to 100 nM, and autophosphorylation of the IR ß-subunit was measured (Fig. 1). In fibroblasts from normal individuals, an effect of insulin was detectable at 0.3 nM, the one half maximal effect at 3 nM, and the maximal effect at 10 nM. When compared with controls, fibroblasts from PCOS patients had an approximate 40% reduced response to insulin at all concentrations tested (P < 0.05 for insulin concentrations 1 nM). The slight difference in basal IR autophosphorylation was not statistically significant.

View larger version (15K): [in this window] [in a new window]   Figure 1. Autophosphorylation of IRs in fibroblasts from control subjects and patients with PCOS. Fibroblasts were incubated with increasing concentrations of insulin for 10 min at 37 C, and lysates were prepared. An equal amount of protein from each preparation was analyzed. Autophosphorylation of the IR ß-subunit was performed using an ELISA as described in Subjects and Methods. Data points represent means ± SEM for separate experiments with fibroblasts from four control subjects and seven patients with PCOS. *, P < 0.05, PCOS vs. control (t test, paired).

View larger version (12K): [in this window] [in a new window]   Figure 2. Autophosphorylation of immunopurified IRs from fibroblasts from control subjects and patients with PCOS. Cell lysis, protein determination, immunopurification of IR ß-subunit, and ELISA for autophosphorylation were performed as described in Subjects and Methods. Immunopurified IRs were incubated with increasing concentrations of insulin. Data represent means ± range for separate experiments with fibroblasts from two control subjects and two patients with PCOS. Not significant, PCOS vs. control (t test, paired).

A previous study indicated that this factor could be a serine kinase activity (16). Therefore, we investigated whether chemical inhibitors of serine kinase activity could affect IR autophosphorylation in fibroblasts from patients with PCOS. Preliminary experiments using specific enzyme assays were performed to identify the maximally effective concentration of each inhibitor (data not shown). H7 (2 µM), an inhibitor of cyclic nucleotide-dependent protein kinase and protein kinase C (PKC) (26), completely reversed the impaired IR autophosphorylation (Fig. 3). Partial reversal was observed with H89, a selective inhibitor of protein kinase A (PKA; Ref. 27). Gö6983, a selective inhibitor of PKC, at concentration (100 nM) sufficient to block PKC isoforms , ß, , , and (28, 29), did not significantly modify IR tyrosine phosphorylation of fibroblasts from the PCOS (Fig. 3). In fibroblasts from the control individuals, H7, H89, and Gö6983 did not affect insulin-stimulated IR autophosphorylation (Fig. 3).

View larger version (55K): [in this window] [in a new window]   Figure 3. Effects of serine kinase inhibitors on IR autophosphorylation in fibroblasts from control subjects and patients with PCOS. Fibroblasts were pretreated for 20 min in the absence (vehicle control) or presence of 2 µM H7, an inhibitor of cyclic nucleotide-dependent protein kinase; 20 µM H89, an inhibitor of PKA; and 100 nM Gö6983, an inhibitor of PKC. Where indicated, fibroblasts were stimulated with insulin (100 nM). Cell lysis, protein determination, and ELISA for autophosphorylation were performed as described in Subjects and Methods. Data represent means ± SEM for separate experiments with fibroblasts from three control subjects and three patients with PCOS. *, P < 0.05, compared with control subjects treated with insulin (ANOVA and Newman-Keuls post hoc test). In fibroblasts pretreated with H7 or H89 followed by insulin, there was no significant difference between PCOS compared with control (ANOVA).

Small, nonpeptide molecules have been shown to activate IR ß-subunit tyrosine kinase activity (21, 22, 23, 24). Accordingly, in fibroblasts from normal subjects, both TLK16998 and Merck L7 increased IR tyrosine kinase activity (P < 0.05; Fig. 4A). In addition, Merck L7 significantly increased basal autophosphorylation (P < 0.05). In fibroblasts from patients with PCOS, TLK16998 significantly enhanced insulin-stimulated IR ß-subunit autophosphorylation at both submaximal and maximal concentrations of insulin. Merck L7 enhanced insulin-stimulated IR ß-subunit autophosphorylation at submaximal concentrations (1 and 3 nM; P < 0.05), but not at a maximal concentration of insulin (Fig. 4B).

View larger version (26K): [in this window] [in a new window]   Figure 4. Effects of IR activators on IR autophosphorylation in fibroblasts from control subjects and patients with PCOS. Fibroblasts from control subjects (A) and patients with PCOS (B) were incubated in the absence (vehicle) or presence of Merck L7 (20 µM) or TLK16998 (20 µM) for 10 min, and then stimulated with increasing concentrations of insulin for 10 min at 37 C. Lysates were prepared, and an equal amount of protein from each preparation was analyzed. Autophosphorylation of the IR ß-subunit was performed using an ELISA as described in Subjects and Methods. In each figure, absorbance at 450 nm for the vehicle-treated fibroblasts stimulated with 100 nM insulin was set to 100%, and the other treatment conditions expressed as percentage vehicle-treated maximum. Data points represent means ± SEM for separate experiments with fibroblasts from four control subjects and four patients with PCOS. A (Control subjects), P < 0.05, Merck L7 and TLK16998 vs. vehicle (repeated measures ANOVA and Dunnett’s post hoc test). In addition, in the absence of insulin (basal), P < 0.05, Merck L7 vs. vehicle (ANOVA and Dunnett’s post hoc test). B, (Patients with PCOS), P < 0.05, Merck L7 and TLK16998 vs. vehicle (repeated measures ANOVA and Dunnett’s post hoc test). In addition, P < 0.05, Merck L7 vs. vehicle for 1 and 3 nM insulin, and TLK16998 vs. vehicle for insulin concentrations 1 nM (ANOVA and Newman-Keuls post hoc test).

The present study was performed to determine whether decreased IR autophosphorylation in fibroblasts from PCOS patients could be reversed by either inhibition of serine kinase activity or IR activation. In intact fibroblasts, using a sensitive and specific ELISA technique, we found a 40% decrease in this function. The decrease in IR autophosphorylation was prevented in the presence of inhibitors of serine kinase activity. In contrast, in isolated purified IR from fibroblasts, no difference in IR autophosphorylation could be detected. These studies support the original observations of Dunaif et al. (16) that decreased IR tyrosine kinase activity and increased serine kinase activity occur in fibroblasts from patients with PCOS.

Although the present study suggests a role for decreased IR autophosphorylation in the insulin resistance of PCOS, it is likely that other defects might be present. Book and Dunaif (30) have found additional evidence that, in fibroblasts from patients with PCOS, there is a greater defect in metabolic vs. mitogenic signaling, suggesting additional defects in downstream signaling. Ciaraldi et al. (6), using adipocytes from patients with PCOS, reported a 30% decrease in IR autophosphorylation, but an 8-fold decrease in insulin-stimulated glucose transport. They concluded, therefore, that a major postreceptor defect in IR signaling was also present.

Increased serine phosphorylation of the IR or downstream signaling components such as IRS decreases the extent of their tyrosine phosphorylation and leads to impaired insulin action (31, 32, 33, 34, 35, 36, 37). For instance, serine phosphorylation of the IR via a number of mechanisms impairs its ability to undergo autophosphorylation. Moreover, serine phosphorylated forms of IRS molecules are less able to associate with the IR and downstream target molecules, especially phosphatidylinositol 3-kinase (31, 38), resulting in impaired insulin action, including protein kinase B activation, and glucose transport. Recently, several serine kinases have been implicated in insulin resistance, including a certain isoform of PKC (35) and the IB kinase (37).

We observed that H7, a nonselective protein kinase inhibitor, and H89, a relatively selective inhibitor of PKA, reversed or partially reversed, respectively, the impaired IR autophosphorylation of fibroblasts from the PCOS. In contrast, a relatively selective PKC inhibitor, Gö6983, at a concentration sufficient to block PKC isoforms , ß, , , and , did not significantly modify the insulin response in insulin-resistant PCOS fibroblasts. These data suggest that a serine kinase, perhaps associated with a PKA-regulated pathway, might be involved in the insulin resistance. This pathway, which remains to be defined, could potentially account for both receptor and postreceptor defects observed in cells from patients with PCOS.

Small-molecule activators of IR might represent a new class of antidiabetic agents (39, 40). In the present study, we found that TLK16998 increased IR autophosphorylation in fibroblasts from both control subjects and insulin-resistant patients. This increase in IR autophosphorylation is in agreement with recent studies (41), in which TLK16998 enhanced insulin-stimulated IR autophosphorylation in insulin-resistant cells. Merck L7 also increased IR autophosphorylation in control subjects. In fibroblasts from patients with PCOS, Merck L7 enhanced insulin-stimulated IR ß-subunit autophosphorylation at submaximal concentrations of insulin, but not at maximal insulin. In contrast, TLK16998 stimulated IR ß-subunit autophosphorylation at both submaximal and maximal concentrations of insulin.

TLK16998 and Merck L7 are chemically distinct compounds. TLK16998 has a polysulfonic acid moiety (24), whereas Merck L7 is a quinone-like compound (21). In a previous study, both compounds improved insulin sensitivity in cells that either overexpressed membrane glycoprotein PC-1 or were incubated with TNF-. However, only TLK16998 overcame insulin resistance in cells incubated with phorbol esters, activators of PKC (41). The present finding that both TLK16998 and Merck L7 were effective, to a varying degree, in fibroblasts from patients with PCOS is in agreement with the earlier observation (41). Insulin-sensitizing agents such as metformin, troglitazone, rosiglitazone, and D-chiroinositol produce significant improvements in the reproductive abnormalities of PCOS (42, 43, 44, 45, 46, 47, 48, 49). Specific serine kinase inhibitors or IR tyrosine kinase activators might also hold promise as therapeutic agents for not only the metabolic but also the reproductive abnormalities associated with PCOS.

In addition to the ability of serine phosphorylation to reduce IR activity (16), previous work has demonstrated that increased serine phosphorylation of cytochrome P450c17, a microsomal enzyme normally expressed in ovaries and adrenal tissue, increases its 17,20-lyase activity (50). An increase in the activity of this enzyme would promote the increased androgen production characteristic of PCOS (51). These observations have prompted the idea that an increase in a serine kinase activity targeting both the IR and P450c17 could serve as a mechanistic link between the insulin resistance and hyperandrogenism observed in PCOS (52). To explore this possibility, P450c17 was stably expressed in fibroblasts from normal individuals and patients with PCOS (a hyperphosphorylating environment), and the activity of 17,20-lyase was measured (53). This study found no correlation between 17,20-lyase activity and the clinical phenotype of the donors of cells. However, it is quite possible that the hyperphosphorylation and activation of P450c17 is mediated by the tissue-specific expression of the relevant kinase or, alternatively, by the need for accessory proteins not expressed in fibroblasts. Thus, the unifying hypothesis of the enhanced activity of a single serine kinase acting on the IR and P450c17, resulting in insulin resistance and hyperandrogenism, remains appealing but requires experimental validation.

In summary, the data support the concept that decreased IR tyrosine kinase activity is a feature of the insulin resistance found in patients with PCOS. In addition, serine kinases may play a role in this type of insulin resistance. In tissues from insulin-resistant women with gestational diabetes, enhanced serine kinase activity was shown to play a role in decreased IR autophosphorylation (54). Taken together, these data along with recent data from Griffin et al. (35) and Yuan et al. (37) implicating serine kinase activity in the insulin resistance of obesity suggest that further investigations into the family of serine kinases and the use of IR tyrosine kinase activators may lead to improved treatments for the insulin resistance in patients with PCOS.

Acknowledgments

Footnotes

This work was supported, in part, by the Mt. Zion Fund: Robert Domush Fund.

Present address for J.L.E.: Medical Research Institute, San Bruno, California 94066.

Abbreviations: IR, Insulin receptor; IRS, IR substrate; PCOS, polycystic ovary syndrome; PKA, protein kinase A; PKC, protein kinase C.

Received March 7, 2002.

Accepted May 7, 2002.

References

1. Legro RS, Spielman R, Urbanek M, Driscoll D, Strauss III JF, Dunaif A 1998 Phenotype and genotype in polycystic ovary syndrome. Recent Prog Horm Res 53:217–256
2. Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R 1998 Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Endocrinol Metab 83:3078–3082[Abstract/Free Full Text]
3. Dunaif A 1997 Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev 18:774–800[Abstract/Free Full Text]
4. Dunaif A, Thomas A 2001 Current concepts in the polycystic ovary syndrome. Annu Rev Med 52:401–419[CrossRef][Medline]
5. Dunaif A, Segal KR, Shelley DR, Green G, Dobrjansky A, Licholai T 1992 Evidence for distinctive and intrinsic defects in insulin action in polycystic ovary syndrome. Diabetes 41:1257–1266[Abstract]
6. Ciaraldi TP, el-Roeiy A, Madar Z, Reichart D, Olefsky JM, Yen SS 1992 Cellular mechanisms of insulin resistance in polycystic ovarian syndrome. J Clin Endocrinol Metab 75:577–583[Abstract]
7. Dunaif A, Wu X, Lee A, Diamanti-Kandarakis E 2001 Defects in insulin receptor signaling in vivo in the polycystic ovary syndrome (PCOS). Am J Physiol 281:E392–E399
8. Moller DE, Flier JS 1991 Insulin resistance: mechanisms, syndromes, and implications. N Engl J Med 325:938–948[Medline]
9. Goldfine ID 1987 The insulin receptor: molecular biology and transmembrane signaling. Endocr Rev 8:235–255[Medline]
10. Ottensmeyer FP, Beniac DR, Luo RZ, Yip CC 2000 Mechanism of transmembrane signaling: insulin binding and the insulin receptor. Biochemistry 39:12103–12112[CrossRef][Medline]
11. Kahn CR 1994 Insulin action, diabetogenes, and the cause of type II diabetes. Diabetes 43:1066–1084[Medline]
12. Rosen OM 1988 After insulin binds. Science 237:1452–1458
13. Cheatham B, Kahn CR 1995 Insulin action and the insulin signaling network. Endocr Rev 16:117–142[CrossRef][Medline]
14. White MF 1998 The IRS-signaling system: a network of docking proteins that mediate insulin and cytokine action. Recent Prog Horm Res 53:119–138
15. Kido Y, Nakae J, Accili D 2001 Clinical review 125: the insulin receptor and its cellular targets. J Clin Endocrinol Metab 86:972–979[Abstract/Free Full Text]
16. Dunaif A, Xia JR, Book CB, Schenker E, Tang ZC 1995 Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle: a potential mechanism for insulin resistance in the polycystic ovary syndrome. J Clin Invest 96:801–810
17. Chin JE, Dickens M, Tavare JM, Roth RA 1993 Overexpression of protein kinase C isoenzymes , ß I, , and in cells overexpressing the insulin receptor. Effects on receptor phosphorylation and signaling. J Biol Chem 268:6338–6347[Abstract/Free Full Text]
18. Birnbaum MJ 2001 Turning down insulin signaling. J Clin Invest 108:655–659[CrossRef][Medline]
19. Lewis RE, Wu GP, Mac Donald RG, Czech MP 1990 Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase. J Biol Chem 265:947–954[Abstract/Free Full Text]
20. Maddux BA, Goldfine ID 2000 Membrane glycoprotein PC-1 inhibition of insulin receptor function occurs via direct interaction with the receptor -subunit. Diabetes 49:13–19[Abstract]
21. Zhang BB, Salituro G, Szalkowski D, Li ZH, Zhang Y, Royo I, Vilella D, Diez MT, Pelaez F, Ruby C, Kendall RL, Mao XZ, Griffin P, Calaycay J, Zierath JR, Heck JV, Smith RG, Moller DE 1999 Discovery of a small molecule insulin mimetic with antidiabetic activity in mice. Science 284:974–977[Abstract/Free Full Text]
22. Qureshi SA, Ding V, Li Z, Szalkowski D, Biazzo-Ashnault DE, Xie D, Saperstein R, Brady E, Huskey S, Shen X, Liu K, Xu L, Salituro GM, Heck JV, Moller DE, Jones AB, Zhang BB 2000 Activation of insulin signal transduction pathway and anti-diabetic activity of small molecule insulin receptor activators. J Biol Chem 275:36590–36595[Abstract/Free Full Text]
23. Liu K, Xu L, Szalkowski D, Li Z, Ding V, Kwei G, Huskey S, Moller DE, Heck JV, Zhang BB, Jones AB 2000 Discovery of a potent, highly selective, and orally efficacious small-molecule activator of the insulin receptor. J Med Chem 43:3487–3494[CrossRef][Medline]
24. Manchem VP, Goldfine ID, Kohanski RA, Cristobal CP, Lum RT, Schow SR, Shi S, Spevak WR, Laborde E, Toavs DK, Villar HO, Wick MM, Kozlowski MR 2001 A novel small molecule that directly sensitizes the insulin receptor in vitro and in vivo. Diabetes 50:824–830[Abstract/Free Full Text]
25. Wang CC, Goldfine ID, Fujita-Yamaguchi Y, Gattner HG, Brandenburg D, De Meyts P 1988 Negative and positive site-site interactions, and their modulation by pH, insulin analogs, and monoclonal antibodies, are preserved in the purified insulin receptor. Proc Natl Acad Sci USA 85:8400–8404[Abstract/Free Full Text]
26. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y 1984 Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5036–5041[CrossRef][Medline]
27. Fujihara M, Muroi M, Muroi Y, Ito N, Suzuki T 1993 Mechanism of lipopolysaccharide-triggered junB activation in a mouse macrophage-like cell line (J774). J Biol Chem 268:14898–14905[Abstract/Free Full Text]
28. Douglas DN, Fink HS, Ridgway ND, Cook HW, Byers DM 1999 Myristoylated alanine-rich C-kinase substrate is phosphorylated and translocated by a phorbol ester-insensitive and calcium-independent protein kinase C isoform in C6 glioma cell membranes. Biochim Biophys Acta 1448:439–449[Medline]
29. Stempka L, Schnolzer M, Radke S, Rincke G, Marks F, Gschwendt M 1999 Requirements of protein kinase cdelta for catalytic function. Role of glutamic acid 500 and autophosphorylation on serine 643. J Biol Chem 274:8886–8892[Abstract/Free Full Text]
30. Book CB, Dunaif A 1999 Selective insulin resistance in the polycystic ovary syndrome. J Clin Endocrinol Metab 84:3110–3116[Abstract/Free Full Text]
31. Paz K, Hemi R, LeRoith D, Karasik A, Elhanany E, Kanety H, Zick Y 1997 A molecular basis for insulin resistance. Elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation. J Biol Chem 272:29911–29918[Abstract/Free Full Text]
32. Kellerer M, Mushack J, Seffer E, Mischak H, Ullrich A, Haring HU 1998 Protein kinase C isoforms , and require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells). Diabetologia 41:833–838[CrossRef][Medline]
33. Li J, DeFea K, Roth RA 1999 Modulation of insulin receptor substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase pathway. J Biol Chem 274:9351–9356[Abstract/Free Full Text]
34. Qiao LY, Goldberg JL, Russell JC, Sun XJ 1999 Identification of enhanced serine kinase activity in insulin resistance. J Biol Chem 274:10625–10632[Abstract/Free Full Text]
35. Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, Goodyear LJ, Kraegen EW, White MF, Shulman GI 1999 Free fatty acid-induced insulin resistance is associated with activation of protein kinase C and alterations in the insulin signaling cascade. Diabetes 48:1270–1274[Abstract]
36. Aguirre V, Uchida T, Yenush L, Davis R, White MF 2000 The c-jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J Biol Chem 275:9047–9054[Abstract/Free Full Text]
37. Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE 2001 Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of IKKß. Science 293:1673–1677[Abstract/Free Full Text]
38. Paz K, Voliovitch H, Hadari YR, Roberts CT, LeRoith D, Zick Y 1996 Interaction between the insulin receptor and its downstream effectors. Use of individually expressed receptor domains for structure function analysis. J Biol Chem 271:6998–7003[Abstract/Free Full Text]
39. Zhang BB, Moller DE 2000 New approaches in the treatment of type 2 diabetes. Curr Opin Chem Biol 4:461–467[CrossRef][Medline]
40. Salituro GM, Pelaez F, Zhang BB 2001 Discovery of a small molecule insulin receptor activator. Recent Prog Horm Res 56:107–126[Abstract]
41. Li M, Youngren JF, Manchem VP, Kozlowski MR, Zhang BB, Maddux BA, Goldfine ID 2001 Small molecule insulin receptor activators potentiate insulin action in insulin resistant cells. Diabetes 50:2323–2328[Abstract/Free Full Text]
42. Nestler JE, Barlascini CO, Matt DW, Steingold KA, Plymate SR, Clore JN, Blackard WG 1989 Suppression of serum insulin by diazoxide reduces serum testosterone levels in obese women with polycystic ovary syndrome. J Clin Endocrinol Metab 68:1027–1032[Abstract]
43. Arslanian SA, Lewy V, Danadian K, Saad R 2002 Metformin therapy in obese adolescents with polycystic ovary syndrome and impaired glucose tolerance: amelioration of exaggerated adrenal response to adrenocorticotropin with reduction of insulinemia/insulin resistance. J Clin Endocrinol Metab 87:1555–1559[Abstract/Free Full Text]
44. Heard MJ, Pierce A, Carson SA, Buster JE 2002 Pregnancies following use of metformin for ovulation induction in patients with polycystic ovary syndrome. Fertil Steril 77:669–673[CrossRef][Medline]
45. Nestler JE, Jakubowicz DJ 1996 Decreases in ovarian cytochrome P450C17 activity and serum free testosterone after reduction of insulin secretion in polycystic ovary syndrome. N Engl J Med 335:617–623[Abstract/Free Full Text]
46. Azziz R, Ehrmann D, Legro RS, Whitcomb RW, Hanley R, Fereshetian AG, O’Keefe M, Ghazzi MN 2001 Troglitazone improves ovulation and hirsutism in the polycystic ovary syndrome: a multicenter, double blind, placebo-controlled trial. J Clin Endocrinol Metab 86:1626–1632[Abstract/Free Full Text]
47. Dunaif A, Scott D, Finegood D, Quintana B, Whitcomb R 1996 The insulin-sensitizing agent troglitazone improves metabolic and reproductive abnormalities in the polycystic ovary syndrome. J Clin Endocrinol Metab 81:3299–3306[Abstract]
48. Cataldo NA, Abbasi F, McLaughlin TL, Lamendola C, Reaven GM 2001 Improvement in insulin sensitivity followed by ovulation and pregnancy in a woman with polycystic ovary syndrome who was treated with rosiglitazone. Fertil Steril 76:1057–1059[CrossRef][Medline]
49. Nestler JE, Jakubowicz DJ, Reamer P, Gunn RD, Allan G 1999 Ovulatory and metabolic effects of D-chiro-inositol in the polycystic ovary syndrome. N Engl J Med 340:1314–1320[Abstract/Free Full Text]
50. Zhang LH, Rodriguez H, Ohno S, Miller WL 1995 Serine phosphorylation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome. Proc Natl Acad Sci USA 92:10619–10623[Abstract/Free Full Text]
51. Qin KN, Rosenfield RL 1998 Role of cytochrome P450c17 in polycystic ovary syndrome. Mol Cell Endocrinol 145:111–121[CrossRef][Medline]
52. Auchus RJ, Geller DH, Lee TC, Miller WL 1998 The regulation of human P450c17 activity: relationship to premature adrenarche, insulin resistance and the polycystic ovary syndrome. Trends Endocrinol Metab 9:47–50
53. Martens JW, Geller DH, Arlt W, Auchus RJ, Ossovskaya VS, Rodriguez H, Dunaif A, Miller WL 2000 Enzymatic activities of P450c17 stably expressed in fibroblasts from patients with the polycystic ovary syndrome. J Clin Endocrinol Metab 85:4338–4346[Abstract/Free Full Text]
54. Shao J, Catalano PM, Yamashita H, Ruyter I, Smith S, Youngren JF, Friedman JE 2000 Decreased insulin receptor tyrosine kinase activity and plasma cell membrane glycoprotein-1 overexpression in skeletal muscle from obese women with gestational diabetes mellitus (GDM): evidence for increased serine/threonine phosphorylation in pregnancy and GDM. Diabetes 49:603–610[Abstract]

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