Transcription factor AP-2beta inhibits expression and secretion of leptin, an insulin-sensitizing hormone, in 3T3-L1 adipocytes.

BACKGROUND: We have previously reported an association between the activator protein-2beta (AP-2beta) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with a disease-susceptible allele of AP-2beta showed stronger AP-2beta expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta led to lipid accumulation and induced insulin resistance in 3T3-L1 adipocytes. RESULT: We found that overexpression of AP-2beta in 3T3-L1 adipocytes decreased the promoter activity of leptin, and subsequently decreased both messenger RNA (mRNA) and protein expression and secretion. Furthermore, knockdown of endogenous AP-2beta by RNA-interference increased mRNA and protein expression of leptin. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed specific binding of AP-2beta to leptin promoter regions in vitro and in vivo. In addition, site-directed mutagenesis of the AP-2-binding site located between position +34 and +42 relative to the transcription start site abolished the inhibitory effect of AP-2beta. Our results clearly showed that AP-2beta directly inhibited insulin-sensitizing hormone leptin expression by binding to its promoter. CONCLUSION: AP-2beta modulated the expression of leptin through direct interaction with its promoter region.

Chronic endothelin-1 treatment leads to heterologous desensitization of insulin signaling in 3T3-L1 adipocytes.

We recently reported that insulin and endothelin-1 (ET-1) can stimulate GLUT4 translocation via the heterotrimeric G protein G alpha q/11 and through PI3-kinase--mediated pathways in 3T3-L1 adipocytes. Because both hormones stimulate glucose transport through a common downstream pathway, we determined whether chronic ET-1 pretreatment would desensitize these cells to acute insulin signaling. We found that ET-1 pretreatment substantially inhibited insulin-stimulated 2-deoxyglucose uptake and GLUT4 translocation. Cotreatment with the ETA receptor antagonist BQ 610 prevented these effects, whereas inhibitors of G alpha i or G beta gamma were without effect. Chronic ET-1 treatment inhibited insulin-stimulated tyrosine phosphorylation of G alpha q/11 and IRS-1, as well as their association with PI3-kinase and blocked the activation of PI3-kinase activity and phosphorylation of AKT: In addition, chronic ET-1 treatment caused IRS-1 degradation, which could be blocked by inhibitors of PI3-kinase or p70 S6-kinase. Similarly, expression of a constitutively active G alpha q mutant, but not the wild-type G alpha q, led to IRS-1 degradation and inhibited insulin-stimulated phosphorylation of IRS-1, suggesting that the ET-1-induced decrease in IRS-1 depends on G alpha q/11 and PI3-kinase. Insulin-stimulated tyrosine phosphorylation of SHC was also reduced in ET-1 treated cells, resulting in inhibition of the MAPK pathway. In conclusion, chronic ET-1 treatment of 3T3-L1 adipocytes leads to heterologous desensitization of metabolic and mitogenic actions of insulin, most likely through the decreased tyrosine phosphorylation of the insulin receptor substrates IRS-1, SHC, and G alpha q/11.

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.

Co-expression of mutant and normal human insulin receptors in COS 7 cells.

In order to assess the interference of the mutant insulin proreceptor on normal receptor function and formation of proreceptor-receptor heterotrimers (alpha beta-proreceptor), COS 7 cells were transfected with the same amount of expression plasmid (pGEM3SV) containing wild-type, a mutant proreceptor cDNA and both, using the DEAE-dextran method. Scatchard analysis of insulin binding data revealed that there was an approx. 50-fold higher receptor concentration in the transfected cells than in untransfected cells. After 0.025% trypsin treatment, insulin binding to the cells expressed with wild-type, proreceptor and both increased by 1-fold, 2.9-fold and 1.5-fold of the untreated cells, respectively. In the presence of 167 nM insulin, the amounts of phosphate incorporated into the 95 kDa protein beta-subunits and 210 kDa proreceptors from co-transfected cells, were identical to those of an in vitro mixture of the wild-type and the mutant receptors. At 10 nM insulin, the proreceptors from co-transfected cells normally autophosphorylated by insulin stimulation, whereas those mixed in vitro did not (73.3 +/- 9.3 vs. 29.6 +/- 2.6% of the maximal effect, n = 4, P < 0.01). However, at a similar concentration of insulin, the phosphate incorporation into Glu-80/Tyr-20 polymers by receptors from co-transfected cells was decreased when compared with a in vitro mixture (9.0 +/- 2.6 vs. 22.5 +/- 6.7% of the maximal effect at 4 nM, n = 6, P < 0.01), although the basal and maximally stimulated phosphate incorporation were comparable among these groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The acute and chronic stimulatory effects of endothelin-1 on glucose transport are mediated by distinct pathways in 3T3-L1 adipocytes.

We have recently shown that pretreatment with endothelin-1 (ET-1) for 20 min stimulates GLUT4 translocation in a PI3-kinase-dependent manner in 3T3-L1 adipocytes (Imamura, T. et al., J Biol Chem 274:33691-33695). This study presents another pathway by which ET-1 potentiates glucose transport in 3T3-L1 adipocytes. ET-1 treatment (10 nM) leads to approximately 2.5-fold stimulation of 2-deoxyglucose (2-DOG) uptake within 20 min, reaching a maximal effect of approximately 4-fold at approximately 6 h, and recovering almost to basal levels after 24 h. Insulin treatment (3 ng/ml) results in an approximately 5-fold increase in 2-DOG uptake at 1 h, and recovering to basal levels after 24 h. The ETA receptor antagonist, BQ 610, inhibited ET-1 induced glucose uptake both at 20 min and 6 h, whereas the ETB receptor antagonist, BQ 788, was without effect. Interestingly, ET-1 stimulated 2-DOG uptake at 6 h, not at 20 min, was almost completely blocked by the protein-synthesis inhibitor, cycloheximide and the RNA-synthesis inhibitor, actinomycin D, suggesting that the short-term (20 min) and long-term (6 h) effects of ET-1 involve distinct mechanisms. GLUT4 translocation assay showed that 20 min, but not 6 h, exposure to ET-1 led to GLUT4 translocation to the plasma membrane. In contrast, 6 h, but not 20 min, exposure to ET-1 increased expression of the GLUT1 protein, without affecting expression of GLUT4 protein. ET-1 induced 2-DOG uptake and GLUT1 expression at 6 h were completely inhibited by the MEK inhibitor, PD 98059, and partially inhibited by the PI3-kinase inhibitor, LY 294002, and the G alpha i inhibitor, pertussis toxin. The PLC inhibitor, U 73122, was without effect. These findings suggest that ET-1 induced GLUT1 protein expression is primarily mediated via MAPK, and partially via PI3K in 3T3-L1 adipocytes.

Src homology 2 domains of protein tyrosine phosphatase are associated in vitro with both the insulin receptor and insulin receptor substrate-1 via different phosphotyrosine motifs.

To clarify the role of protein tyrosine phosphatase containing Src homology 2 (SH2) regions on insulin signaling, we investigated the interactions among the insulin receptor, a pair of SH2 domains of SH-PTP2 coupled to glutathione-S-transferase (GST) and insulin receptor substrate-1 (IRS-1)-GST fusion protein (amino-portion, IRS-IN; carboxyl portion, IRS-1C). GST-SH2 protein of SH-PTP2 bound to the wild type insulin receptor, but not to that with a carboxyl-terminal mutation (Y/F2). Furthermore, even though Y/F2 receptors were used, the SH2 protein was also co-immunoprecipitated with IRS-IC, but not with IRS-IN. These results indicate that SH2 domains of SH-PTP2 can directly associate with the Y1322TXM motif on the carboxyl terminus of insulin receptors and also may bind to the carboxyl portion of IRS-1, possibly via the Y1172IDL motif in vitro.

Clinical and laboratory characteristics in the families with diabetes and a mitochondrial tRNA(LEU(UUR)) gene mutation.

We identified three families having a mutation in the mitochondrial tRNA(LEU(UUR)) gene at bp 3243 in 300 patients with non-insulin dependent diabetes mellitus (NIDDM), who had first degree relatives of patients with NIDDM. We found six individuals with diabetes, one with impaired glucose tolerance (IGT), and five with normal glucose tolerance (NGT) among three families. Insulin secretory response to oral glucose load was impaired in six diabetics, but was normal in IGT and NGT, and the proportion of mutant DNA in the blood did not always associate with the severity of glucose intolerance. Furthermore, both gender and obesity may influence the clinical expression of diabetes in three pairs with an age-matched brother-sister relationship with similar high mutation rate in blood samples. Thus, although patients with mitochondrial gene mutation had a high frequency of diabetes, the proportion of mutant DNA evaluated by blood samples may not necessarily indicate glucose intolerance in the members with the mutation. Unidentified factors including gender, aging, and obesity may alter the clinical manifestation of diabetes.

Membrane localization of protein-tyrosine phosphatase 1B is essential for its activation of sterol regulatory element-binding protein-1 gene expression.

Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor in stimulating lipogenesis in the liver. Protein-tyrosine phosphatase 1B (PTP1B) induces SREBP-1 gene expression via protein phosphatase 2A (PP2A) activation. PTP1B is reported to be anchored on the endoplasmic reticulum (ER) via its C-terminal tail, and change in intracellular localization of PTP1B by C-terminal-truncation did not alter its inhibitory effects on insulin signaling. In this study, we investigated whether the change in intracellular localization of PTP1B could influence SREBP-1 gene expression. Overexpression of C-terminal truncated PTP1B (PTP1BdeltaCT) in rat Fao cells did not induce SREBP-1 gene expression. Furthermore, PTP1BdeltaCT failed to bind PP2A, resulting in impaired PP2A activation, whereas overexpression of wild-type PTP1B (PTP1BWT) associated with PP2A. Moreover, a membrane-targeted PTP1BDeltaCT activated PP2A with restored PP2A binding, despite the absence of its C-terminal region. Finally, overexpression of PTP1BdeltaCT into mouse primary cultured hepatocytes failed to enhance SREBP-1c mRNA, whereas membrane-targeted PTP1BdeltaCT led to enhanced SREBP-1c mRNA in hepatocytes as well as PTP1BWT. In conclusion, membrane localization of PTP1B is essential for PP2A activation, which is crucial for its enhancement of SREBP-1 gene expression.

Insulin signaling and its regulation of system A amino acid uptake in cultured rat vascular smooth muscle cells.

Hyperinsulinemia has been recognized as an independent risk factor for atherosclerosis. However, its exact mechanisms are still unclear. In our previous work, we showed that 10 nmol/L insulin stimulated neither mitogen-activated protein kinase (MAP kinase) activity nor [3H]thymidine incorporation but did stimulated S6 kinase through the specific insulin receptors in cultured rat vascular smooth muscle cells (VSMCs). In this study, we observed that > or = 1 nmol/L insulin stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and activated IRS-1-dependent phosphatidylinositol 3'-kinase (PI 3'-kinase) and p70 S6 kinase (p70S6K) but not MAP kinase (extracellular signal-regulated kinase 2) and p90 S6 kinase (p90RSK). However, 10 nmol/L insulin-like growth factor I stimulated all these pathways. Finally, 10 nmol/L insulin stimulated alpha-amino-isobutyric acid (AIB) uptake, and wortmannin (100 nmol/L) completely inhibited insulin-stimulated AIB uptake, whereas rapamycin (20 nmol/L) had no such effect. Furthermore, cycloheximide (10 micrograms/mL) completely inhibited insulin-stimulated AIB uptake, but actinomycin D (5 micrograms/mL) failed to inhibit this. Thus, we reached the following conclusions: (1) Insulin (1 nmol/L) induced phosphorylation of IRS-1 and activated the PI 3'-kinase and p70S6K pathways in VSMCs, even though 10 nmol/L insulin did not significantly stimulate MAP kinase or p90RSK. (2) Stimulation of AIB uptake by insulin was regulated at the translational level via wortmannin-sensitive pathways but not p70S6K pathways.

Expression of dominant negative mutant SHPTP2 attenuates phosphatidylinositol 3'-kinase activity via modulation of phosphorylation of insulin receptor substrate-1.

To clarify the role of protein-tyrosine phosphatase (PTPase) containing Src homology 2 regions (SHPTP2) in insulin signaling, either wild-type or mutant SHPTP2 (delta PTP; lacking full PTPase domain) was expressed in Rat 1 fibroblasts overexpressing human insulin receptors. In response to insulin, phosphorylation of insulin receptor substrate 1 (IRS-1), IRS-1-associated PTPase activities and phosphatidylinositol (PI) 3'-kinase activities were slightly enhanced in wild-type cells when compared with those in the parent cells transfected with hygromycin-resistant gene alone. In contrast, introduction of delta PTP inhibited insulin-induced association of IRS-1 with endogenous SHPTP2 and impaired both insulin-stimulated phosphorylation of IRS-1 and activation of PI 3'-kinase. Furthermore, decreased content of p85 subunit of PI 3'-kinase was also found in mutant cells. Consistently, the insulin-stimulated mitogen-activated protein kinase activities and DNA synthesis were also enhanced in wild-type cells, but impaired in mutant cells. Thus, the interaction of SHPTP2 with IRS-1 may be associated with modulation of phosphorylation levels of IRS-1, resulting in the changes of PI 3'-kinase and mitogen-activated protein kinase activity. Furthermore, an impaired insulin signaling in mutant cells may be partly reflected in a decreased content of p85 protein of PI 3'-kinase.

Endothelin-1-induced GLUT4 translocation is mediated via Galpha(q/11) protein and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes.

Endothelin-1 (ET-1) can stimulate insulin-responsive glucose transporter (GLUT4) translocation in 3T3-L1 adipocytes (Wu-Wong, J. R., Berg, C. E., Wang, J., Chiou, W. J., and Fissel, B. (1999) J. Biol. Chem. 274, 8103-8110), and in the current study, we have evaluated the signaling pathway leading to this response. First, we inhibited endogenous Galpha(q/11) function by single-cell microinjection using anti-Galpha(q/11) antibody or RGS2 protein (a GTPase activating protein for Galpha(q)) followed by immunostaining to quantitate GLUT4 translocation in 3T3-L1 adipocytes. ET-1-stimulated GLUT4 translocation was markedly decreased by 70 or 75% by microinjection of Galpha(q/11) antibody or RGS2 protein, respectively. Pretreatment of cells with the Galpha(i) inhibitor (pertussis toxin) or microinjection of a Gbetagamma inhibitor (glutathione S-transferase-beta-adrenergic receptor kinase (GST-BARK)) did not inhibit ET-1-induced GLUT4 translocation, indicating that Galpha(q/11 )mediates ET-1 signaling to GLUT4 translocation. Next, we found that ET-1-induced GLUT4 translocation was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitors wortmannin or LY294002, but not by the phospholipase C inhibitor U-73122. ET-1 stimulated the PI 3-kinase activity of the p110alpha subunit (5.5-fold), and microinjection of anti-p110alpha or PKC-lambda antibodies inhibited ET-stimulated GLUT4 translocation. Finally, we found that Galpha(q/11) formed immunocomplexes with the type-A endothelin receptor and the 110alpha subunit of PI 3-kinase and that ET-1 stimulation enhances tyrosine phosphorylation of Galpha(q/11). These results indicate that: 1) ET-1 signaling to GLUT4 translocation is dependent upon Galpha(q/11) and PI 3-kinase; and 2) Galpha(q/11) can transmit signals from the ET(A) receptor to the p110alpha subunit of PI 3-kinase, as does insulin, subsequently leading to GLUT4 translocation.

Pioglitazone ameliorates high glucose induced desensitization of insulin receptor kinase in Rat 1 fibroblasts in culture.

A new oral agent, pioglitazone, increases insulin sensitivity by activating receptor kinase in insulin-resistant rats. To clarify the mechanism, we studied in vitro effects of glucose and pioglitazone on the insulin receptor function using Rat 1 fibroblasts which expressed human insulin receptors. Insulin receptor kinase activity was impaired by incubating cells for 4 days in the presence of 27mM D-glucose. The glucose effect was time- and dose-dependent and also specific for D-glucose, since D-raffinose incubation had no effect. Pioglitazone treatment did not have any effect on intact receptor kinase. However, exposure of both 27mM D-glucose and 0.1 microM pioglitazone to the cells completely prevented the glucose-induced impairment of insulin receptor kinase activity, suggesting that pioglitazone might reverse the processes which are critical for the glucose-induced desensitization of insulin receptor kinase.

Insulin receptor kinase phosphorylates protein tyrosine phosphatase containing Src homology 2 regions and modulates its PTPase activity in vitro.

To clarify the role of protein tyrosine phosphatase (PTPase) containing a pair of Src homology 2 (SH2) regions upon insulin signaling, we studied the interactions between the insulin receptor and SH-PTP2 coupled to glutathione-S-transferase. A full length SH-PTP2 was phosphorylated by insulin receptor kinase and associated with the insulin receptor in vitro. The N-terminal SH2 domain was more phosphorylated than the other SH2 domain of SH-PTP2. However, both SH2 domains of SH-PTP2 were necessary for association with insulin receptors. Phosphorylation of the SH2 domains of SH-PTP2 resulted in decreased PTPase activities toward the phosphorylated insulin receptor. These results indicate that the insulin receptor can negatively regulate SH-PTP2 activity by means of phosphorylating the SH2 domains.

Thiazolidine derivatives ameliorate high glucose-induced insulin resistance via the normalization of protein-tyrosine phosphatase activities.

The mechanisms for the insulin resistance induced by hyperglycemia were investigated by studying the effect of high glucose concentration (HG) and its modulation by thiazolidine derivatives, on insulin signaling using Rat 1 fibroblasts expressing human insulin receptors (HIRc). Incubating HIRc cells in 27 mM D-glucose for 4 days impaired the insulin-stimulated phosphorylation of pp185 and receptor beta-subunits. Both protein kinase C activities and phorbol dibutyrate binding to intact cells were unchanged; however, cytosolic protein-tyrosine phosphatase (PTPase) activity increased within 1 h prior to the impairment of insulin receptor kinase in HG cells (Maegawa, H., Tachikawa-Ide, R., Ugi, S., Iwanishi, M., Egawa, K., Kikkawa, R., Shigeta, Y., and Kashiwagi, A. (1993) Biochem. Biophys. Res. Commun. 197, 1078-1082). Increased PTPase activity was consistent with a 2-fold increase in the amount of PTP1B, and anti-PTP1B antibody inhibited this increment of cytosolic PTPase activity in HG cells. Co-incubating cells with pioglitazone prevented these abnormalities in cytosolic PTPase, the PTP1B content and the impaired phosphorylation of pp185 and receptor beta subunits in HG cells. Finally, HG cells had impaired insulin-stimulated alpha-amino-isobutyric acid uptake, which was ameliorated by exposure to thiazolidine derivatives. In conclusion, exposing cells to high glucose levels desensitizes insulin receptor function, and thiazolidine derivatives can reverse the process via the normalization of cytosolic PTPase, but not of protein kinase C.

Src homology 2 domains of protein tyrosine phosphatase are phosphorylated by insulin receptor kinase and bind to the COOH-terminus of insulin receptors in vitro.

To clarify the role of protein tyrosine phosphatases(PTPase) containing Src homology 2 (SH2) regions on insulin signaling, we investigated the interactions between SH2 regions of PTPase and insulin receptors. We made a pair of SH2 domains of PTP1C and SH-PTP2 fusion proteins coupled to glutathione-S-transferase (GST) using pGEX-3X expression vector. After incubating with insulin, insulin receptors were incubated with SH2 proteins in the presence of 100 mu ATP at 4 degrees C for 3 hr, and then immunoprecipitated and analyzed by SDS-PAGE. We found that SH2 domains of SH-PTP2 were phosphorylated, but not those of PTP1C by insulin receptor kinase and the SH2 domains of SH-PTP2, but not those of PTP1C, directly bound to the phosphorylated COOH-terminus of insulin receptors in vitro.

beta -Arrestin-mediated recruitment of the Src family kinase Yes mediates endothelin-1-stimulated glucose transport.

The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.

SHPTP2 serves adapter protein linking between Janus kinase 2 and insulin receptor substrates.

To investigate the role of Janus kinase family (JAK1 and JAK2) in insulin signaling, we assessed their insulin-induced associations with other molecules in the cells overexpressing insulin receptors (HIRc and CHO-IR). After insulin stimulation, pp185 proteins (insulin receptor substrate, IRS) were co-immunoprecipitated with both kinases by alpha JAK1 and alpha JAK2 antibodies. However, JAK2 constitutively associated with pp95 protein (IR beta). Moreover, JAK2 also constitutively bound to a protein tyrosine phosphatase containing Src 2 regions (SHPTP2), but JAK1 did not. In HIRc cells expressing PTPase-negative mutant SHPTP2, no association of JAK2 with either pp185 or pp95 was detected. Thus, SHPTP2 might serve as an adapter protein linking between JAK2 and IRS. These results suggest that JAK1 and JAK2 behave differently and they may constitute a new regulatory component in insulin signaling.

Expression of a dominant negative SHP-2 in transgenic mice induces insulin resistance.

To elucidate the roles of SHP-2, we generated transgenic (Tg) mice expressing a dominant negative mutant lacking protein tyrosine phosphatase domain (DeltaPTP). On examining two lines of Tg mice identified by Southern blot, the transgene product was expressed in skeletal muscle, liver, and adipose tissues, and insulin-induced association of insulin receptor substrate 1 with endogenous SHP-2 was inhibited, confirming that DeltaPTP has a dominant negative property. The intraperitoneal glucose loading test demonstrated an increase in blood glucose levels in Tg mice. Plasma insulin levels in Tg mice after 4 h fasting were 3 times greater with comparable blood glucose levels. To estimate insulin sensitivity by a constant glucose, insulin, and somatostatin infusion, steady state blood glucose levels were higher, suggesting the presence of insulin resistance. Furthermore, we observed the impairment of insulin-stimulated glucose uptake in muscle and adipocytes in the presence of physiological concentrations of insulin. Moreover, tyrosine phosphorylation of insulin receptor substrate-1 and stimulation of phosphatidylinositol 3-kinase and Akt kinase activities by insulin were attenuated in muscle and liver. These results indicate that the inhibition of endogenous SHP-2 function by the overexpression of a dominant negative mutant may lead to impaired insulin sensitivity of glucose metabolism, and thus SHP-2 may function to modulate insulin signaling in target tissues.

A new antidiabetic agent (JTT-501) rapidly stimulates glucose disposal rates by enhancing insulin signal transduction in skeletal muscle.

A newly synthesized antidiabetic agent, JTT-501 is an isoxazolidinedione rather than a thiazolidinedione. An oral dose of JTT-501 (100 mg x kg(-1) x day(-1)) given to 12-week-old male Zucker fatty rats for 7 days led to the amelioration of both hyperinsulinaemia (40% of non-treated) and hypertriglyceridaemia (23% of non-treated) as well as a 2.4-fold increased insulin sensitivity as determined by a euglycaemic insulin clamp. In our study, we further evaluated the acute effect of JTT-501 on both the glucose infusion rates (GIR) and insulin signalling in skeletal muscle. Male Sprague-Dawley (SD) rats aged 10 weeks were injected intravenously with JTT-501 (5 mg/kg) and then a euglycaemic insulin clamp was initiated and glucose infusion rates monitored for 150 min. We found that this treatment increased the glucose infusion rate by 33% during the last 30 min in SD rats. After the clamp had been initiated for 30 min, the insulin-stimulated phosphatidylinositol 3-kinase (PI3-kinase) activities co-immunoprecipitated with insulin receptor substrate 1 (IRS-1) were also enhanced, resulting in increased glycogen synthase activities in the soleus muscles. Treatment with JTT-501 also enhanced the phosphorylation of insulin receptors and insulin receptor-substrate 1 rapidly as well as the phosphatidylinositol 3-kinase activities, which were stimulated by a bolus injection of insulin. Similarly, JTT-501 stimulated the glucose infusion rate by 30% and enhanced insulin signalling in Zucker fatty rats. In conclusion, a newly developed isoxazolidinedione, JTT-501, rapidly potentiates the insulin sensitivity of skeletal muscle by enhancing insulin signalling and could be useful for the treatment of insulin-resistant diabetic subjects.