Long-term effects of tumor necrosis factor-alpha treatment on insulin signaling pathway in HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB

Tumor necrosis factor-alpha (TNF-alpha) mediated attenuation of insulin signaling pathway is an important cause in several disorders like obesity, obesity linked diabetes mellitus. TNF-alpha actions vary depending upon concentration and time of exposure in various cells. In the present study, the effects of long-term TNF-alpha (1 ng/ml) exposure on the components of insulin signaling pathway in HepG2 and HepG2 cells overexpressing constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB) have been investigated. In parental HepG2 cells, TNF-alpha treatment for 24 h reduced the phosphorylation of Akt1/PKB-alpha and GSK-3beta and under these conditions cells also showed reduced insulin responsiveness in terms of Akt1/PKB-alpha and GSK-3beta phosphorylation. TNF-alpha pre-incubated HepG2-CA-Akt/PKB cells showed lower reduction in Akt1/PKB-alpha and GSK-3beta phosphorylation and insulin responsiveness after 24 h as compared to parental HepG2 cells. We report that the long-term TNF-alpha pre-incubation in both parental HepG2 and HepG2-CA-Akt/PKB-alpha cells leads to the reduction in the levels of IRS-1 without altering the levels of IRS-2. In order to understand the reason for the differential insulin resistance in both the cell types, the effect of long-term TNF-alpha treatment on the proteins upstream to Akt/PKB was investigated. TNF-alpha pre-incubation also showed reduced insulin-stimulated Tyr phosphorylation of insulin receptor (IR-beta) in both the cell types, moreover hyperphosphorylation of IRS-1 at Ser 312 residue was observed in TNF-alpha pre-incubated cells. As hyperphosphorylation of IRS-1 at Ser 312 can induce its degradation, it is possible that reduced insulin responsiveness after long-term TNF-alpha pre-incubation observed in this study is due to the decrease in IRS-1 levels.     

Vanadium salts stimulate mitogen-activated protein (MAP) kinases and ribosomal S6 kinases

Effect of several vanadium salts, sodium orthovanadate, vanadyl sulfate and sodium metavanadate on protein tyrosine phosphorylation and serine/threonine kinases in chinese hamster ovary (CHO) cells overexpressing a normal human insulin receptor was examined. All the compounds stimulated protein tyrosine phosphorylation of two major proteins with molecular masses of 42 kDa (p42) and 44 kDa (p44). The phosphorylation of p42 and p44 was associated with an activation of mitogen activated protein (MAP) kinase as well as increased protein tyrosine phosphorylation of p42^mapk and p44^mapk. Vanadinm salts also activated the 90 kDa ribosomal s6 kinase (p90^rsk) and 70 kDa ribosomal s6 kinase (p70^s6k). Among the three vanadium salts tested, vanadyl sulfate appeared to be slightly more potent than others in stimulating MAP kinases and p70^s6k activity. It is suggested that vanadium-induced activation of MAP kinases and ribosomal s6 kinases may be one of the mechanisms by which insulin like effects of this trace element are mediated.Abbreviations eIF-4 eukaryotic protein synthesis initiation factor-4 - GRB-2 growth factor receptor bound protein-2 - GSK-3 Glycogen Synthase Kinase-3 - IRS-1 insulin receptor substrate-1 - ISPK insulin stimulated protein kinase - MAPK mitogen activated protein kinase, also known as - ERK extracellular signal regulated kinase - MAPKK mitogen activated protein kinase kinase, also known as-MEK, MAPK or ERK kinase - PHAS-1 phosphorylated heat and acid stable protein regulated by insulin - PI3K phosphatidyl inositol 3-kinase - PP1-G protein phosphatase-glycogen bound form - PTK protein tyrosine kinase - PTPase protein tyrosine phosphatase - rsk ribosomal s6 kinases - shc src homology domain containing protein - SOS son of sevenless     

A new strategy for studying protein kinase B and its three isoforms. Role of protein kinase B in phosphorylating glycogen synthase kinase-3, tuberin, WNK1, and ATP citrate lyase

Protein kinase B appears to play a key role in insulin signaling and in the control of apoptosis, although the precise targets of PKB are incompletely understood. PKB exists as three isoforms (alpha, beta, and gamma) that may have unique as well as common functions within the cell. To facilitate understanding the precise roles of PKB and its isoforms, novel tools of widespread applicability are described. These tools are antisense oligonucleotide probes that enable the specific and potent knock down of endogenous PKB alpha, beta, or gamma isoforms, individually or in various combinations, including concurrent removal of all three isoforms. The probes were applied to dissect the role of PKB in phosphorylating glycogen synthase kinase-3 (GSK-3), a critical mediator in multiple responses, and other potentially key targets. Triple antisense knock down of PKB alpha, beta, and gamma so that total PKB was <6% blocked insulin-stimulated phosphorylation of endogenous GSK-3alpha and GSK-3beta isoforms by 67% and 45%, respectively, showing that GSK-3alpha and GSK-3beta are controlled by endogenous PKB. Each PKB isoform contributed to GSK-3alpha and GSK-3beta phosphorylation, with PKBbeta having the predominant role. Knock down of total PKB incompletely blocked insulin-stimulated phosphorylation of GSK-3alpha and GSK-3beta, and a pathway involving atypical PKCs, zeta/lambda, was shown to contribute to the signal. Triple antisense knock down of PKB alpha, beta, and gamma abrogated the insulin-stimulated phosphorylation of WNK1, ATP citrate lyase, and tuberin. However, antisense-mediated knock down of PKB alpha, beta, and gamma had no effect on insulin-stimulated DNA synthesis in 3T3-L1 adipocytes, indicating that pathways other than PKB mediate this response in these cells. Finally, our PKB antisense strategy provides a method of general usefulness for further dissecting the precise targets and roles of PKB and its isoforms.     

Serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling

The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1 1-320 and PTB-2 1-350, we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser332 is a GSK-3 phosphorylation site and that Ser336 serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser332 confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser332 as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.     

Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase C betaII in the diabetic fat tissue

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is an important negative modulator of insulin signaling. Previously, we showed that glycogen synthase kinase-3 (GSK-3) phosphorylates IRS-1 at Ser(332). However, the fact that GSK-3 requires prephosphorylation of its substrates suggested that Ser(336) on IRS-1 was the "priming" site phosphorylated by an as yet unknown protein kinase. Here, we sought to identify this "priming kinase" and to examine the phosphorylation of IRS-1 at Ser(336) and Ser(332) in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser(336). Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKCalpha or PKCbetaII isoforms in cells enhanced IRS-1 phosphorylation at Ser(336) and Ser(332), and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser(336). The expression level and activation state of PKCbetaII, but not PKCalpha, were remarkably elevated in the fat tissues of diabetic ob/ob mice and in high-fat diet-fed mice compared with that from lean animals. Elevated levels of PKCbetaII were also associated with enhanced phosphorylation of IRS-1 at Ser(336/332) and elevated activity of GSK-3beta. Finally, adenoviral mediated expression of PKCbetaII in adipocytes enhancedphosphorylation of IRS-1 at Ser(336). Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCbetaII and GSK-3 at Ser(336) and Ser(332). Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.     

Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism

Glycogen synthase kinase 3 comprises two isoforms (GSK-3alpha and GSK-3beta) that are implicated in type II diabetes, neurodegeneration, and cancer. GSK-3 activity is elevated in human and rodent models of diabetes, and various GSK-3 inhibitors improve glucose tolerance and insulin sensitivity in rodent models of obesity and diabetes. Here, we report the generation of mice lacking GSK-3alpha. Unlike GSK-3beta mutants, which die before birth, GSK-3alpha knockout (GSK-3alpha KO) animals are viable but display enhanced glucose and insulin sensitivity accompanied by reduced fat mass. Fasted and glucose-stimulated hepatic glycogen content was enhanced in GSK-3alpha KO mice, whereas muscle glycogen was unaltered. Insulin-stimulated protein kinase B (PKB/Akt) and GSK-3beta phosphorylation was higher in GSK-3alpha KO livers compared to wild-type littermates, and IRS-1 expression was markedly increased. We conclude that GSK-3 isoforms exhibit tissue-specific physiological functions and that GSK-3alpha KO mice are insulin sensitive, reinforcing the potential of GSK-3 as a therapeutic target for type II diabetes.     

Cytokine-stimulated phosphorylation of GSK-3 is primarily dependent upon PKCs, not PKB.

The regulation of glycogen synthase kinase-3 (GSK-3) by phosphorylation at inhibitory sites has been well documented. In many, but not all, cases, the phosphatidylinositol 3-kinase pathway, and particularly the downstream kinase protein kinase B (PKB)/akt, have been shown to be responsible for GSK-3 phosphorylation. Given that no studies have ever reported cytokine-mediated phosphorylation of GSK-3, we investigated the phosphorylation of this kinase in several hemopoietic cell types in response to either interleukin (IL)-3, IL-4 or granulocyte-macrophage colony stimulating factor (GM-CSF). Each of the cytokines was able to stimulate phosphorylation of the isoforms GSK-3alpha and GSK-3beta. However, only in the case of IL-4 stimulation was there any dependence on PKB for this phosphorylation. We were clearly able to show that PKB was capable of phosphorylating GSK-3 in these cells, but studies using inhibitors of the protein kinase C (PKC) family of kinases have shown that these enzymes are more likely to play a key role in GSK-3 phosphorylation. Cytokine-mediated generation of diacylglycerol was demonstrated, supporting the possible activation of PKC family members. Thus, cytokine-dependent GSK-3 phosphorylation in hemopoietic cells proceeds primarily through PKB independent pathways.     

IRS-4 mediates protein kinase B signaling during insulin stimulation without promoting antiapoptosis

Insulin receptor substrate (IRS) proteins are tyrosine phosphorylated and mediate multiple signals during activation of the receptors for insulin, insulin-like growth factor 1 (IGF-1), and various cytokines. In order to distinguish common and unique functions of IRS-1, IRS-2, and IRS-4, we expressed them individually in 32D myeloid progenitor cells containing the human insulin receptor (32D(IR)). Insulin promoted the association of Grb-2 with IRS-1 and IRS-4, whereas IRS-2 weakly bound Grb-2; consequently, IRS-1 and IRS-4 enhanced insulin-stimulated mitogen-activated protein kinase activity. During insulin stimulation, IRS-1 and IRS-2 strongly bound p85alpha/beta, which activated phosphatidylinositol (PI) 3-kinase, protein kinase B (PKB)/Akt, and p70(s6k), and promoted the phosphorylation of BAD. IRS-4 also promoted the activation of PKB/Akt and BAD phosphorylation during insulin stimulation; however, it weakly bound or activated p85-associated PI 3-kinase and failed to mediate the activation of p70(s6k). Insulin strongly inhibited apoptosis of interleukin-3 (IL-3)-deprived 32D(IR) cells expressing IRS-1 or IRS-2 but failed to inhibit apoptosis of cells expressing IRS-4. Consequently, 32D(IR) cells expressing IRS-4 proliferated slowly during insulin stimulation. Thus, the activation of PKB/Akt and BAD phosphorylation might not be sufficient to inhibit the apoptosis of IL-3-deprived 32D(IR) cells unless p85-associated PI 3-kinase or p70(s6k) are strongly activated.     

GSK-3beta regulation in skeletal muscles by adrenaline and insulin: evidence that PKA and PKB regulate different pools of GSK-3

We have recently shown that while adrenaline alone has no effect on the activation of Protein Kinase B (PKB) in rat soleus muscle, it greatly potentiates the effects of insulin (Brennesvik et al., Cellular Signalling 17: 1551-1559, 2005). In the current study we went on to investigate whether this was paralleled by a similar effect on GSK-3, which is a major PKB target. Surprisingly adrenaline alone increased phosphorylation of GSK-3beta Ser9 and GSK-3alpha Ser21 and adrenaline's effects were additive with those of insulin but did not synergistically potentiate insulin action. Dibutyryl-cAMP (5 mM) and the PKA specific cAMP analogue N6-Benzoyl-cAMP (2 mM) increased GSK-3beta Ser9 phosphorylation, whereas the Epac specific cAMP analogue 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (1 mM) did not. Wortmannin (PI 3-kinase inhibitor; 1 microM) blocked insulin-stimulated GSK-3 phosphorylation completely, but adrenaline increased GSK-3beta Ser9 phosphorylation in the presence of wortmannin. The PKA inhibitor H89 (50 microM) reduced adrenaline-stimulated GSK-3beta Ser9 phosphorylation but did not influence the effects of insulin. Insulin-stimulated GSK-3 Ser9 phosphorylation was paralleled by decreased glycogen synthase phosphorylation at the sites phosphorylated by GSK-3 as expected. However, adrenaline-stimulated GSK-3 Ser9 phosphorylation was paralleled by increased glycogen synthase phosphorylation indicating this pool of GSK-3 may not be directly involved in phosphorylation of glycogen synthase. Our results indicate the existence of at least two distinct pools of GSK-3beta in soleus muscle, one phosphorylated by PKA and another by PKB. Further, we hypothesise that each of these pools is involved in the control of different cellular processes.     

Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C zeta activation by insulin in L6hIR cells

We have investigated glycogen synthase (GS) activation in L6hIR cells expressing a peptide corresponding to the kinase regulatory loop binding domain of insulin receptor substrate-2 (IRS-2) (KRLB). In several clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors was accompanied by a block of IRS-2, but not IRS-1, phosphorylation, and insulin receptor binding. GS activation by insulin was also inhibited by >70% in these cells (p < 0.001). The impairment of GS activation was paralleled by a similarly sized inhibition of glycogen synthase kinase 3 alpha (GSK3 alpha) and GSK3 beta inactivation by insulin with no change in protein phosphatase 1 activity. PDK1 (a phosphatidylinositol trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no difference in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKC zeta in B2 and F4 cells. In L6hIR, inhibition of PKC zeta activity by either a PKC zeta antisense or a dominant negative mutant also reduced by 75% insulin inactivation of GSK3 alpha and -beta (p < 0.001) and insulin stimulation of GS (p < 0.002), similar to Akt/PKB inhibition. In L6hIR, insulin induced protein kinase C zeta (PKC zeta) co-precipitation with GSK3 alpha and beta. PKC zeta also phosphorylated GSK3 alpha and -beta. Alone, these events did not significantly affect GSK3 alpha and -beta activities. Inhibition of PKC zeta activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3 alpha and -beta by >80% (p < 0.001) and prevented full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle cells. In these cells, insulin inhibition of GSK3 alpha and -beta requires dual phosphorylation by both Akt/PKB and PKC zeta.     

Sequential phosphorylation of insulin receptor substrate-2 by glycogen synthase kinase-3 and c-Jun NH2-terminal kinase plays a role in hepatic insulin signaling

Serine phosphorylation of insulin receptor substrate (IRS) proteins is a potential inhibitory mechanism in insulin signaling. Here we show that IRS-2 is phosphorylated by glycogen synthase kinase (GSK)-3. Phosphorylation by GSK-3 requires prior phosphorylation of its substrates, prompting us to identify the "priming kinase." It was found that the stress activator anisomycin enhanced the ability of GSK-3 to phosphorylate IRS-2. Use of a selective c-Jun NH(2)-terminal kinase (JNK) inhibitor and cells overexpressing JNK implicated JNK as the priming kinase. This allowed us to narrow down the number of potential GSK-3 phosphorylation sites within IRS-2 to four regions that follow the motif SXXXSP. IRS-2 deletion mutants enabled us to localize the GSK-3 and JNK phosphorylation sites to serines 484 and 488, respectively. Mutation at serine 488 reduced JNK phosphorylation of IRS-2, and mutation of each site separately abolished GSK-3 phosphorylation of IRS-2. Treatment of H4IIE liver cells with anisomycin inhibited insulin-induced tyrosine phosphorylation of IRS-2; inhibition was reversed by pretreatment with the JNK and GSK-3 inhibitors. Moreover, overexpression of JNK and GSK-3 in H4IIE cells reduced insulin-induced tyrosine phosphorylation of IRS-2 and its association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Finally, both GSK-3 and JNK are abnormally upregulated in the diabetic livers of ob/ob mice. Together, our data indicate that IRS-2 is sequentially phosphorylated by JNK and GSK-3 at serines 484/488 and provide evidence for their inhibitory role in hepatic insulin signaling.     

The high-fat-fed lean Zucker rat: a spontaneous isocaloric model of fat-induced insulin resistance associated with muscle GSK-3 overactivity

High-fat feeding (HFF) is a well-accepted model for nutritionally-induced insulin resistance. The purpose of this investigation was to assess the metabolic responses of female lean Zucker rats provided regular chow (4% fat) or a high-fat chow (50% fat) for 15 wk. HFF rats spontaneously adjusted food intake so that daily caloric intake matched that of chow-fed (CF) controls. HFF animals consumed more (P < 0.05) calories from fat (31.9 +/- 1.2 vs. 2.4 +/- 0.2 kcal/day) and had significantly greater final body weights (280 +/- 10 vs. 250 +/- 5 g) and total visceral fat (24 +/- 3 vs. 10 +/- 1 g). Fasting plasma insulin was 2.3-fold elevated in HFF rats. Glucose tolerance (58%) and whole body insulin sensitivity (75%) were markedly impaired in HFF animals. In HFF plantaris muscle, in vivo insulin receptor beta-subunit (IR-beta) and insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and phosphorylation of Akt Ser473 and glycogen synthase kinase-3beta (GSK-3beta) Ser9, relative to circulating insulin levels, were decreased by 40-59%. In vitro insulin-stimulated glucose transport in HFF soleus was decreased by 54%, as were IRS-1 tyrosine phosphorylation (26%) and phosphorylation of Akt Ser473 (38%) and GSK-3beta Ser9 (25%), the latter indicative of GSK-3 overactivity. GSK-3 inhibition in HFF soleus using CT98014 increased insulin-stimulated glucose transport (28%), IRS-1 tyrosine phosphorylation (28%) and phosphorylation of Akt Ser473 (38%) and GSK-3beta Ser9 (48%). In summary, the female lean Zucker rat fed a high-fat diet represents an isocaloric model of nutritionally-induced insulin resistance associated with moderate visceral fat gain, hyperinsulinemia, and impairments of skeletal muscle insulin-signaling functionality, including GSK-3beta overactivity.     

Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function.

Incubation of cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins, accompanied by elevation in their Ser(P)/Thr(P) content and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, selectively prevented the increase in Ser(P)/Thr(P) content of IRS-1, its dissociation from IR, and the decrease in its Tyr(P) content following 60 min of insulin treatment. Four conserved phosphorylation sites within the phosphotyrosine binding/SAIN domains of IRS-1 and IRS-2 served as in vitro substrates for protein kinase B (PKB), a Ser/Thr kinase downstream of phosphatidylinositol 3-kinase. Furthermore, PKB and IRS-1 formed stable complexes in vivo, and overexpression of PKB enhanced Ser phosphorylation of IRS-1. Overexpression of PKB did not affect the acute Tyr phosphorylation of IRS-1; however, it significantly attenuated its rate of Tyr dephosphorylation following 60 min of treatment with insulin. Accordingly, overexpression of IRS-1(4A), lacking the four potential PKB phosphorylation sites, markedly enhanced the rate of Tyr dephosphorylation of IRS-1, while inclusion of vanadate reversed this effect. These results implicate a wortmannin-sensitive Ser/Thr kinase, different from PKB, as the kinase that phosphorylates IRS-1 and acts as the feedback control regulator that turns off insulin signals by inducting the dissociation of IRS proteins from IR. In contrast, insulin-stimulated PKB-mediated phosphorylation of Ser residues within the phosphotyrosine binding/SAIN domain of IRS-1 protects IRS-1 from the rapid action of protein-tyrosine phosphatases and enables it to maintain its Tyr-phosphorylated active conformation. These findings implicate PKB as a positive regulator of IRS-1 functions.     

Prolongation of insulin-induced activation of mitogen-activated protein kinases ERK 1/2 and phosphatidylinositol 3-kinase by vanadyl sulfate, a protein tyrosine phosphatase inhibitor

Vanadium salts such as vanadyl sulfate (VS), potent inhibitors of protein tyrosine phosphatases, have been shown to mimic, augment, and prolong insulin's action. However, the molecular mechanism of responses to these salts is not clear. In the present studies, we examined if VS-induced effects on insulin action are associated with enhancement or augmentation in the activation state of key components of the insulin signaling pathway. Treatment of insulin receptor-overexpressing cells with insulin or VS resulted in a time-dependent transient increase in phosphorylation and activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) that peaked at about 5 min, then declined rapidly to about baseline within 30 min. However, when the cells were treated with VS before stimulation with insulin, sustained ERK 1/2 phosphorylation and activation were observed well beyond 60 min. VS treatment also prolonged the insulin-stimulated activation of phosphatidylinositol 3-kinase (PI3-K), which was associated with sustained interaction between insulin receptor substrate-1 (IRS-1) and the p(85 alpha) subunit of phosphatidylinositol 3-kinase (PI3-K) in response to insulin. These data indicate that prolongation of insulin-stimulated ERK 1/2 and PI3-K activation by VS is due to a more stable complex formation of IRS-1 with the p(85 alpha) subunit which may, in turn, be responsible for its ability to enhance and extend the biological effects of insulin.     

Involvement of insulin-like growth factor type 1 receptor and protein kinase Cdelta in bis(maltolato)oxovanadium(IV)-induced phosphorylation of protein kinase B in HepG2 cells

Vanadium(IV) oxo-bis(maltolato) (BMOV), an organovanadium compound, is a potent insulinomimetic agent and improves glucose homeostasis in various models of diabetes. We have shown previously that BMOV stimulates the phosphorylation of PKB which may contribute as one of the mechanisms for the insulinomimetic effect of this compound. However, the upstream mechanism of BMOV-induced PKB phosphorylation remains elusive. Therefore, in this study, we examine the upstream events leading to BMOV-induced PKB phosphorylation in HepG2 cells. Since BMOV is an inhibitor of protein tyrosine phosphatases and through enhanced tyrosine phosphorylation may activate various protein tyrosine kinases (PTK), we have investigated the potential role of different receptor or nonreceptor PTK in mediating BMOV-induced PKB phosphorylation. Among several pharmacological inhibitors that were tested, only AG1024, a selective inhibitor of IGF-1R-PTK, almost completely blocked BMOV-stimulated phosphorylation of PKB. In contrast, AG1295 and AG1478, specific inhibitors of PDGFR and EGFR, respectively, were unable to block the BMOV response. Moreover, efficient reduction of the level of IGF-1R protein expression by antisense oligonucleotides (ASO) attenuated BMOV-induced PKB phosphorylation. BMOV-induced PKB phosphorylation was associated with an increased level of tyrosine phosphorylation of the IRbeta subunit, IGF-1Rbeta subunit, IRS-1, and p85alpha subunit of PI3-kinase. However, this response was independent of IR-PTK activity because in cells overexpressing a PTK-inactive form of IR, insulin response was attenuated while the effect of BMOV remained intact. A role of PKC in BMOV-induced response was also tested. Pharmacological inhibition with chelerythrine, a nonselective PKC inhibitor, or rottlerin, a PKCdelta inhibitor, as well as chronic treatment with PMA attenuated BMOV-induced PKB phosphorylation. In contrast, GO6976 and RO31-8220 PKCalpha/beta selective inhibitors failed to alter the BMOV effect. Taken together, these data suggest that IGF-1R and PKCdelta are required to stimulate PKB phosphorylation in response to BMOV in HepG2 cells and provide new insights into the molecular mechanism by which this compound exerts its insulinomimetic effects.     

Burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle

The molecular bases underlying burn- or critical illness-induced insulin resistance still remain unclarified. Muscle protein catabolism is a ubiquitous feature of critical illness. Akt/PKB plays a central role in the metabolic actions of insulin and is a pivotal regulator of hypertrophy and atrophy of skeletal muscle. We therefore examined the effects of burn injury on insulin-stimulated Akt/PKB activation in skeletal muscle. Insulin-stimulated phosphorylation of Akt/PKB was significantly attenuated in burned compared with sham-burned rats. Insulin-stimulated Akt/PKB kinase activity, as judged by immune complex kinase assay and phosphorylation status of the endogenous substrate of Akt/PKB, glycogen synthase kinase-3beta (GSK-3beta), was significantly impaired in burned rats. Furthermore, insulin consistently failed to increase the phosphorylation of p70 S6 kinase, another downstream effector of Akt/PKB, in rats with burn injury, whereas phosphorylation of p70 S6 kinase was increased by insulin in controls. The protein expression of Akt/PKB, GSK-3beta, and p70 S6 kinase was unaltered by burn injury. However, insulin-stimulated activation of ERK, a signaling pathway parallel to Akt/PKB, was not affected by burn injury. These results demonstrate that burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle and suggest that attenuated Akt/PKB activation may be involved in deranged metabolism and muscle wasting observed after burn injury.     

Oral treatment with vanadium of Zucker fatty rats activates muscle glycogen synthesis and insulin-stimulated protein phosphatase-1 activity

Since the glucose-lowering effects of vanadium could be related to increased muscle glycogen synthesis, we examined the in vivo effects of vanadium and insulin treatment on glycogen synthase (GS) activation in Zucker fatty rats. The GS fractional activity (GSFA), protein phosphatase-1 (PP1), and glycogen synthase kinase-3 (GSK-3) activity were determined in fatty and lean rats following treatment with bis(maltolato)oxovanadium(IV) (BMOV) for 3 weeks (0.2 mmol/kg/day) administered in drinking water. Skeletal muscle was freeze-clamped before or following an insulin injection (5 U/kg i.v.). In both lean and fatty rats, muscle GSFA was significantly increased at 15 min following insulin stimulation. Vanadium treatment resulted in decreased insulin levels and improved insulin sensitivity in the fatty rats. Interestingly, this treatment stimulated muscle GSFA by 2-fold (p < 0.05) and increased insulin-stimulated PP1 activity by 77% (p < 0.05) in the fatty rats as compared to untreated rats. Insulin resistance, vanadium and insulin in vivo treatment did not affect muscle GSK-3 activity in either fatty or lean rats. Therefore, an impaired insulin sensitivity in the Zucker fatty rats was improved following vanadium treatment, resulting in an enhanced muscle glucose metabolism through increased GS and insulin-stimulated PP1 activity.     

Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase.

Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.     

Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3beta (GSK-3beta) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.