Adenovirus-mediated expression of a naturally occurring Asp905Tyr variant of the glycogen-associated regulatory subunit of protein phosphatase-1 in L6 myotubes.

AIMS/HYPOTHESIS: The glycogen-associated protein phosphatase-1 (PP1G) is thought to play an important part in the regulation of skeletal muscle glycogen content. We have previously identified an Asp905Tyr polymorphism of the glycogen-associated regulatory subunit of the protein phosphatase 1 (PPP1R3) gene which among healthy subjects was associated with decreased insulin stimulated non-oxidative glucose metabolism, i.e. primary glycogen synthesis. In this study, the functional effect of the polymorphism was examined in vitro. METHODS: Wild type (PPP1R3-Asp905) and mutant (PPP1R3-Tyr905) PPP1R3 were expressed in L6 myotubes using adenovirus-mediated gene transfer. Basal and insulin-stimulated glucose uptake and glycogen synthesis were measured. Furthermore, the sensitivity of glycogen synthesis to a cyclic AMP agonist was measured. RESULTS: Compared with green fluorescent protein-transduced myotubes and non-transduced myotubes, overexpression of PPP1R3-Asp905 and PPP1R3-Tyr905 increased both basal and insulin-stimulated glycogen synthesis approximately twofold. Treatment of both non-transduced and PPP1R3-transduced L6 myotubes with a cAMP agonist decreased both basal and insulin-stimulated glycogen synthesis by about 40%. Overexpression of PPP1R3 did not affect either basal or insulin-stimulated 2-deoxy-D-glucose uptake compared with green fluorescent protein-transduced cells. CONCLUSION/INTERPRETATION: Results obtained from L6 myotubes transduced with PPP1R3-Asp905 or PPP1R3-Tyr905 showed no statistically significant difference. Therefore, the Asp905Tyr variant alone is unlikely to account for the decreased insulin stimulated non-oxidative glucose metabolism observed in the human study reported previously.

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.

Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway. In adipose tissue and muscle of obese humans and rodents, PTP1B expression is increased, which led to the hypothesis that PTP1B plays a role in the pathogenesis of insulin resistance. Consistent with this, mice in which the PTP1B gene was disrupted exhibit increased insulin sensitivity. To test whether increased expression of PTP1B in an insulin-sensitive cell type could contribute to insulin resistance, we overexpressed wild-type PTP1B in 3T3L1 adipocytes using adenovirus-mediated gene delivery. PTP1B expression was increased approximately 3-5-fold above endogenous levels at 16 h, approximately 14-fold at 40 h, and approximately 20-fold at 72 h post-transduction. Total protein-tyrosine phosphatase activity was increased by 50% at 16 h, 3-4-fold at 40 h, and 5-6-fold at 72 h post-transduction. Compared with control cells, cells expressing high levels of PTP1B showed a 50-60% decrease in maximally insulin-stimulated tyrosyl phosphorylation of IR and insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) activity associated with IRS-1 or with phosphotyrosine. Akt phosphorylation and activity were unchanged. Phosphorylation of p42 and p44 MAP kinase (MAPK) was reduced approximately 32%. Overexpression of PTP1B had no effect on basal, submaximally or maximally (100 nm) insulin-stimulated glucose transport or on the EC(50) for transport. Our results suggest that: 1) insulin stimulation of glucose transport in adipocytes requires

Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2.

The nontransmembrane protein tyrosine phosphatase SHP-2 plays a critical role in growth factor and cytokine signaling pathways. Previous studies revealed that a fraction of SHP-2 moves to focal contacts upon integrin engagement and that SHP-2 binds to SHP substrate 1 (SHPS-1)/SIRP-1alpha, a transmembrane glycoprotein with adhesion molecule characteristics (Y. Fujioka et al., Mol. Cell. Biol. 16:6887-6899, 1996; M. Tsuda et al., J. Biol. Chem. 273:13223-13229). Therefore, we asked whether SHP2-SHPS-1 complexes participate in integrin signaling. SHPS-1 tyrosyl phosphorylation increased upon plating of murine fibroblasts onto specific extracellular matrices. Both in vitro and in vivo studies indicate that SHPS-1 tyrosyl phosphorylation is catalyzed by Src family protein tyrosine kinases (PTKs). Overexpression of SHPS-1 in 293 cells potentiated integrin-induced mitogen-activated protein kinase (MAPK) activation, and potentiation required functional SHP-2. To further explore the role of SHP-2 in integrin signaling, we analyzed the responses of SHP-2 exon 3(-/-) and wild-type cell lines to being plated on fibronectin. Integrin-induced activation of Src family PTKs, tyrosyl phosphorylation of several focal adhesion proteins, MAPK activation, and the ability to spread on fibronectin were defective in SHP-2 mutant fibroblasts but were restored upon SHP-2 expression. Our data suggest a positive-feedback model in which, upon integrin engagement, basal levels of c-Src activity catalyze the tyrosyl phosphorylation of SHPS-1, thereby recruiting SHP-2 to the plasma membrane, where, perhaps by further activating Src PTKs, SHP-2 transduces positive signals for downstream events such as MAPK activation and cell shape changes.

Targeting of constitutively active phosphoinositide 3-kinase to GLUT4-containing vesicles in 3T3-L1 adipocytes.

Constitutive activation of phosphoinositide 3-kinase (PI3K) stimulates glucose transport and GLUT4 glucose transporter translocation to the plasma membrane in adipocytes. To determine whether a direct interaction of PI3K with GLUT4-containing vesicles (hereafter called GLUT4 vesicles) is important for the effect of insulin on GLUT4 translocation, we targeted constitutively active PI3K to GLUT4 vesicles. We fused the inter-Src homology region 2 of the regulatory p85alpha subunit of PI3K (iSH2) either to a C-terminal sequence of GLUT4 (G4c, amino acids 406-509) or to this region and the N-terminal tail of GLUT4 (G4n, amino acids 1-19), resulting in the fusion proteins iSH2-G4c and G4n-iSH2-G4c, respectively. Coexpression of the fusion proteins or untargeted iSH2 with the catalytic p110alpha subunit of PI3K (p110) in 3T3-L1 adipocytes by adenovirus-mediated gene transfer increased total PI3K activity in homogenates 5.0-6.7-fold over nontransduced cells or cells transduced with adenovirus encoding beta-galactosidase. In contrast, PI3K activity in GLUT4 vesicles increased 11-13-fold with expression of either targeted construct and p110 but only 2-fold with the untargeted iSH2 and p110, indicating successful targeting of PI3K to GLUT4 vesicles. Both targeted and nontargeted constructs stimulated DNA synthesis to levels greater than insulin, demonstrating that both types of constructs had biologic activity in intact cells. Despite this, untargeted iSH2/p110 coexpression was more effective in stimulating 2-deoxyglucose uptake (6-fold) than either iSH2-G4c/p110 or G4n-iSH2-G4c/p110 coexpression (both 2-fold). Only iSH2/p110 coexpression led to a significant GLUT4 translocation to the plasma membrane. Insulin-stimulated glucose transport was unaffected by any construct. Thus, a direct interaction between PI3K and GLUT4 vesicles is either not required or not sufficient for GLUT4 translocation and stimulation of glucose transport.

Evidence against a direct effect of leptin on glucose transport in skeletal muscle and adipocytes.

Recently, it has been proposed that leptin, the ob gene product, influences some steps in the insulin-signaling cascade. The purpose of the present study was to determine whether leptin exerts direct effects on glucose transport in insulin target tissues. Epitrochlearis muscles or isolated adipocytes from male SD rats were incubated in the absence or presence of recombinant leptin (3-1,000 ng/ml), and in the absence or presence of submaximal or maximal insulin concentrations. In skeletal muscle, insulin increased 3-O-methylglucose transport (1.88 +/- 0.21, 4.06 +/- 0.59, and 9.35 +/- 1.90 micromol x ml-1 x h-1, for 0, 0.6, and 12.0 nmol/l insulin, respectively). Leptin exposure (300 ng/ml) for 2 h did not alter the basal, submaximal, or maximal response of glucose transport to insulin in skeletal muscle (1.50 +/- 0.14, 4.76 +/- 0.58, and 9.04 +/- 1.09 micromol x ml-1 x h-1 for 0, 0.6, and 12.0 nmol/l insulin, respectively). Insulin increased glucose transport in rat adipocytes (0.194 +/- 0.007, 1.059 +/- 0.029, and 3.367 +/- 0.143 pmol [14C]glucose x 0.5 ml-1 cell suspension x min-1 for 0, 0.8, and 80 nmol/l insulin, respectively); in vitro exposure to leptin (300 ng/ml) did not alter glucose transport (0.220 +/- 0.006, 1.269 +/- 0.046, and 3.221 +/- 0.285 pmol [14C]glucose x 0.5 ml-1 cell suspension x min-1 for 0, 0.8, and 80 nmol/l insulin, respectively). Similar to our findings in the epitrochlearis muscle, leptin had no direct effect on basal or insulin-stimulated glucose uptake in soleus muscle from ob/ob or lean mice or adipocytes from normal mice. In summary, in vitro exposure of skeletal muscle or adipocytes to recombinant leptin did not alter glucose transport in the absence of insulin, nor did it affect the sensitivity or responsiveness of the glucose transport system to insulin.

Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes.

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for many insulin-induced metabolic and mitogenic responses. However, it is unclear whether PI3K activation is sufficient for any of these effects. To address this question we increased PI3K activity in differentiated 3T3-L1 adipocytes by adenovirus-mediated expression of both the inter-SH2 region of the regulatory p85 subunit of PI3K (iSH2) and the catalytic p110 alpha subunit (p110). Coexpression resulted in PI3K activity that exceeded insulin-stimulated activity by two- to fivefold in cytosol, total membranes, and the low density microsome (LDM) fraction, the site of greatest insulin stimulation. While insulin increased glucose transport 15-fold, coexpression of iSH2-p110 increased transport (5.2-) +/- 0.7-fold with a parallel increase in GLUT4 translocation to the plasma membrane. Constitutive activation of PI3K had no effect on maximally insulin-stimulated glucose transport. Neither basal nor insulin-stimulated activity of glycogen synthase or mitogen-activated protein kinase was altered by iSH2-p110 coexpression. DNA synthesis was increased twofold by insulin in control 3T3-L1 adipocytes transduced with beta-galactosidase-encoding recombinant adenovirus, while iSH2-p110 coexpression increased DNA synthesis fivefold. These data indicate that (i) increased PI3K activity is sufficient to activate some but not all metabolic responses to insulin, (ii) activation of PI3K to levels exceeding the effect of insulin in adipocyte LDM results in only a partial stimulation of glucose transport, and (iii) increased PI3K activity in the absence of growth factor or oncoprotein stimulation is a potent stimulus of DNA synthesis.

Adenovirus-mediated gene transfer of dominant negative ras(asn17) in 3T3L1 adipocytes does not alter insulin-stimulated P13-kinase activity or glucose transport.

Recent studies suggest that the ras-map kinase and PI3-kinase cascades converge. We sought to determine whether PI3-kinase is downstream of ras in insulin signaling in a classic insulin target cell. We generated a recombinant adenovirus encoding dominant negative ras by cloning the human H-ras cDNA with a ser to asn substitution at amino acid 17 (ras(asn17)) into the pACCMVpLpA vector and cotransfecting 293 cells with the pJM17 plasmid containing the adenoviral genome. Efficiency of gene transfer was assessed by infecting fully differentiated 3T3L1 adipocytes with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 70% of cells were infected. Infection of adipocytes with ras(asn17) resulted in 10-fold greater expression than endogenous ras. This high efficiency gene transfer allowed biochemical assays. Insulin stimulation of ras-GTP formation was inhibited in ras(asn17)-expressing cells. Map kinase gel mobility shift revealed that insulin (1 UM) or epidermal growth factor (100 ng/ml) resulted in the appearance of a hyperphosphorylated species of p42 map kinase in uninfected cells and those expressing beta-gal but not in cells expressing ras(asn17). In contrast, insulin increased IRS-1-associated PI3-kinase activity approximately 10-fold in control cells and high level overexpression of ras(asn17) did not impair this effect. Similarly, insulin and epidermal growth factor activation of total (no immunoprecipitation) PI3-kinase activity in both cytosol and total cellular membranes and insulin stimulation of glucose transport were not affected by expression of dominant negative ras. Thus, adenovirus-mediated gene transfer is effective for studying insulin signaling in fully differentiated insulin target cells. Inhibition of ras activation abolishes insulin-stimulated phosphorylation of map kinase but does not affect insulin stimulation of PI3-kinase activity. In normal cell physiology, PI3-kinase does not appear to be downstream of ras in mediating the actions of insulin.

Protein kinase C isoforms epsilon, eta, delta and zeta in murine adipocytes: expression, subcellular localization and tissue-specific regulation in insulin-resistant states.

The Ca(2+)-insensitive protein kinase C (PKC) isoforms epsilon, eta, delta and zeta are possible direct downstream targets of phosphatidylinositol 3-kinase (P13-K), and might therefore be involved in insulin signalling. Although isoform-specific changes in PKC expression have been reported for skeletal muscle and liver in insulin-resistant states, little is known about these isoforms in adipocytes. Therefore we studied (1) expression and subcellular localization of these isoforms in murine adipocytes, (2) translocation of specific isoforms to membranes in response to treatment with insulin and phorbol 12-myristate 13-acetate (PMA) and (3) regulation of expression in insulin-resistant states. The PKC isoforms epsilon, eta, delta and zeta are expressed in adipocytes. Immunoreactivity for all isoforms is higher in the membranes than in the cytosol, but subcellular fractionation by differential centrifugation shows an isoform-specific distribution within the membrane fractions. PMA treatment of adipocytes induces translocation of PKC-epsilon and -delta from the cytosol to the membrane fractions. Insulin treatment does not alter the subcellular distribution of any of the isoforms. 3T3-L1 adipocytes express PKC-epsilon and -zeta, and PKC-epsilon expression increases with differentiation from preadipocytes to adipocytes. PKC-epsilon expression decreases in an adipose-specific and age/obesity-dependent manner in two insulin-resistant models, the brown-adipose-tissue-deficient mouse and db/db mouse compared with control mice. We conclude that, although none of the isoforms investigated seems to be activated by insulin, the decrease in PKC-epsilon expression might contribute to metabolic alterations in adipocytes associated with insulin resistance and obesity.

Markers of bone metabolism during short-term administration of thyroxine in healthy volunteers.

We investigated the influence of L-thyroxine (L-T4) treatment over 3 weeks on biochemical markers of bone turnover in 12 healthy young men (age 25.6 +/- 1.4 years, BMI: 22.6 +/- 2.5 kg/m2). Serum parameters indicating bone formation [bone Gla protein (BGP), carboxyterminal propeptide of type I procollagen (PICP), and bone-specific alkaline phosphatase (BAP)] and bone resorption [cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and the urinary excretion of pyridinoline (Pyr) and deoxypyridinoline (D-Pyr)] were measured before and after three weeks of treatment with 300 micrograms L-T4/d. T3 and T4 significantly increased and TSH decreased to almost undetectable levels even when measured with a third generation TSH assay. Markers of bone formation showed variable responses with a small but significant increase in BGP but not in PICP or BAP. In contrast, all parameters of bone resorption increased significantly with a good correlation between D-Pyr excretion and the serum parameter ICTP (r = 0.78, p < 0.0001). These changes in bone-turnover markers were not necessarily paralleled by comparable increments of other markers of tissue thyrotoxicosis (SHBG, pulse rate, VO2), suggesting a variability in tissue sensitivity. These rapid responding parameters, especially in the easily obtainable serum parameter ICTP, might be valuable tools in the evaluation of several states of thyroxine excess.

Studies on the subcellular mechanisms mediating the negative estradiol effect on GnRH-induced LH-release by rat pituitary cells in culture.

Cultured pituitary cells from adult female rats were treated for 4 or 24 h in the absence or presence of E2 (10(-9) mol/l) with increasing concentrations of the transcription inhibitor actinomycin-D or the translation inhibitor puromycin. During the last 4 h of incubation, LH release was stimulated with 5 X 10(-10) mol/l GnRH. The positive E2 effect observed after 24 h treatment with the steroid was clearly abolished by actinomycin-D at concentrations greater than or equal to 10(-10) mol/l and by puromycin at concentrations greater than or equal to 10(-5) mol/l. These findings indicate that the positive E2 effect on GnRH-induced LH release is fully dependent on intact mRNA and protein synthesis. The negative E2 effect observed after 4 h treatment with the steroid was not affected by actinomycin-D and abolished by puromycin only at concentrations greater than or equal to 10(-4) mol/l. Similar results were obtained, when cells had been treated for 4 h with actinomycin-D or puromycin before the 4 h E2 treatment started. Thus, the negative E2 effect seems to be independent of mRNA synthesis and dependent on protein synthesis to a lesser extent than the positive E2 effect. In an attempt to identify positively the subcellular mechanism via which E2 exerts its negative effect, several steps in the GnRH stimulus secretion coupling mechanism were checked whether or not they are modulated by E2. The negative effects of E2 (10(-9) mol/l) on LH release induced by GnRH (10(-10), 10(-9) mol/l) and by the activators of voltage dependent Ca2+ channels K+ (64 mmol/l) or veratridine (3.3 X 10(-5) mol/l) were comparable to those of the calcium antagonist verapamil (10(-6) mol/l). These findings supported the speculation that E2 might act on the Ca2+ channels. The LH release induced by the Ca2+ ionophores A 23 187 (10(-4) mol/l) or ionomycin (6.6 X 10(-5) mol/l), however, was also significantly reduced by 10(-9) mol/l E2, indicating that the steroid modulated a mechanism secondary to the increase of intracellular Ca2+. Also GnRH (10(-9), 10(-8) mol/l) induced accumulation of [3H]inositol phosphates was not influenced by E2 (10(-9) mol/l) treatment, though the steroid exerted a significant negative effect on the LH release by these cells, indicating that phosphatidylinositol-4,5-biphosphate breakdown is not the point of attack for the estrogen.(ABSTRACT TRUNCATED AT 400 WORDS)