A family of insulinomimetic zinc(II) complexes of amino ligands with Zn(Nn) (n=3 and 4) coordination modes

Several metal ions and their complexes have been known to mimic the action of insulin in in vitro and in vivo systems. We prepared a family of Zn(II) complexes derived from amino ligands with Zn(Nn) (n=3 and 4) coordination modes, the insulinomimetic activity being estimated by an inhibitory effect of free fatty acid release from isolated rat adipocytes treated with epinephrine. In comparison with the positive controls VOSO(4) and ZnSO(4), Zn(II)-amine complexes with stability constants (log beta) lower than 11.5 exhibited higher insulinomimetic activities. Among them, a bis(2-aminomethyl pyridinato)Zn(II) (Zn(2-ampy)(2)(2+)) complex with the highest insulinomimetic activity and a higher stability constant but lower than 11.5 was selected, and subjected to in vivo evaluation in KK-A(y) mice with a genetically type 2 diabetes mellitus. The high blood glucose level of the mice was lowered by daily intraperitoneal injections of Zn(2-ampy)(2)(2+) at a dose of 2 mg Zn/kg body weight for 14 days. Based on the results, Zn(2-ampy)(2)(2+) with Zn(N(4)) coordination mode was proposed to have both a high in vitro insulinomimetic activity and an in vivo blood glucose lowering effect.     

A new type of orally active anti-diabetic Zn(II)-dithiocarbamate complex

In order to find orally active Zn(II) complexes that can treat diabetes mellitus (DM) at low doses, four new Zn(II)-dithiocarbamate complexes with Zn(II)-sulfur coordination bonds were prepared and their in vitro insulinomimetic activity and in vivo anti-diabetic ability were evaluated. Among the Zn(II)-dithiocarbamate complexes, the bis(pyrrolidine-N-dithiocarbamate)zinc(II) (Zn(pdc)(2)) complex was found to be the most effective in terms of inhibiting free fatty acid-release and enhancing glucose-uptake in adipocytes. After oral administration of the Zn(pdc)(2) complex to KK-A(y) mice with obesity and type 2 DM, we observed that the high blood glucose levels in the mice were lowered from approximately 500 mg/dL to 350 mg/dL within 6 days, and the effect was maintained during the administration period. Also, indicators of insulin resistance such as serum insulin, leptin, and triglyceride levels were also reduced compared with those in untreated mice. Moreover, the Zn(pdc)(2) complex improved not only the hypertension in the mice, but also the adiponectin level in the serum. On the basis of the results, the Zn(pdc)(2) complex is proposed to improve hyperglycemia and insulin resistance in type 2 DM animals on daily oral administrations.     

Regulation of insulin signaling and glucose transporter 4 (GLUT4) exocytosis by phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase, skeletal muscle, and kidney enriched inositol polyphosphate phosphatase (SKIP)

The glucose transporter 4 (GLUT4) is responsible for glucose uptake in the skeletal muscle. Insulin-induced translocation of GLUT4 to the plasma membrane requires phosphatidylinositol 3-kinase activation-mediated generation of phosphatidylinositol 3,4,5-trisphosphate PIP(3) and subsequent activation of Akt. Previous studies suggested that skeletal muscle and kidney enriched inositol polyphosphate phosphatase (SKIP) has negative effects on the regulation of insulin signaling in the skeletal muscle cells. Here, we compared its effects on insulin signaling by selective inhibition of SKIP, SHIP2, and phosphatase and tensin homologue on chromosome 10 (PTEN) by short interfering RNA in the C2C12 myoblast cells. Suppression of SKIP significantly increased the insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate levels and Akt phosphorylation. Furthermore, silencing of SKIP, but not of PTEN, increased the insulin-dependent recruitment of GLUT4 vesicles to the plasma membrane. Taken together, these results imply that SKIP negatively regulates insulin signaling and glucose uptake by inhibiting GLUT4 docking and/or fusion to the plasma membrane.     

Epigallocatechin-3-O-gallate (EGCG) protects the insulin sensitivity in rat L6 muscle cells exposed to dexamethasone condition

The tea polyphenol epigallocatechin-3-O-gallate (EGCG) displays some antidiabetic effects; however the mechanisms are incompletely understood. In the present study, the investigation of the effects of EGCG on insulin resistance was performed in rat L6 cells treated with dexamethasone. We found that dexamethasone increased Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1) and reduced phosphorylation of AMPK and Akt. Furthermore, glucose uptake and glucose transporter (GLUT4) translocation were inhibited by dexamethasone. However, the treatment of EGCG improved insulin-stimulated glucose uptake by increasing GLUT4 translocation to plasma membrane. Furthermore, we also demonstrated these EGCG effects essentially depended on the AMPK and Akt activation. Together, our data suggested that EGCG inhibited dexamethasone-induced insulin resistance through AMPK and PI3K/Akt pathway.     

Molecular mechanism of antidiabetic zinc–allixin complexes: regulations of glucose utilization and lipid metabolism

We previously reported new zinc complexes of allixin [bis(allixinato)zinc] and its derivative bis(thioallixin-N-methyl)zinc that demonstrated excellent antidiabetic activity in type 2 diabetic mellitus KKA(y) mice. However, the molecular mechanism of these complexes is not fully understood. Thus, we attempted to reveal the intracellular mechanism of these complexes in 3T3-L1 adipocytes. Both zinc complexes induced Akt/protein kinase B (Akt/PKB) phosphorylation. The phosphorylation of Akt/PKB enhanced glucose transporter 4 translocation to the plasma membrane; this in turn enhanced the glucose utilization in a dose- and time-dependent manner. Glucose utilization by the complexes depended on the intracellular zinc concentration. Moreover, zinc complexes suppressed the cyclic AMP dependent protein kinase mediated phosphorylation of hormone-sensitive lipase (HSL), leading to the inhibition of free fatty acid release from the 3T3-L1 adipocytes. Such responses were inhibited by wortmannin, suggesting that the suppression of HSL by zinc complexes was dependent in the phosphoinositide 3-kinase-Akt/PKB signaling cascade. On the basis of these results, we proposed that both zinc complexes activated the Akt/PKB-mediated insulin-signaling pathway and improved both glucose utilization and lipid metabolism.     

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.     

Expression of constitutively active Akt/protein kinase B signals GLUT4 translocation in the absence of an intact actin cytoskeleton

The actin cytoskeleton has been shown to be required for insulin-dependent GLUT4 translocation; however, the role that the actin network plays is unknown. Actin may play a role in formation of an active signaling complex, or actin may be required for movement of vesicles to the plasma membrane surface. To distinguish between these possibilities, we examined the ability of myr-Akt, a constitutively active form of Akt that signals GLUT4 translocation to the plasma membrane in the absence of insulin, to signal translocation of an HA-GLUT4-GFP reporter protein in the presence or absence of an intact cytoskeleton in 3T3-L1 adipocytes. Expression of myr-Akt signaled the redistribution of the GLUT4 reporter protein to the cell surface in the absence or presence of 10 microm latrunculin B, a concentration sufficient to completely inhibit insulin-dependent redistribution of the GLUT4 reporter to the cell surface. These data suggest that the actin network plays a primary role in organization of the insulin-signaling complex. To further support this conclusion, we measured the activation of known signaling proteins using a saturating concentration of insulin in cells pretreated without or with 10 microm latrunculin B. We found that latrunculin treatment did not affect insulin-dependent tyrosine phosphorylation of the insulin receptor beta-subunit and IRS-1 but completely inhibited activation of Akt/PKB enzymatic activity. Phosphorylation of Akt/PKB at Ser-473 and Thr-308 was inhibited by latrunculin B treatment, indicating that the defect in signaling lies prior to Akt/PKB activation. In summary, our data support the hypothesis that the actin network plays a role in organization of the insulin-signaling complex but is not required for vesicle trafficking and/or fusion.     

Enhancement of insulin signaling pathway in adipocytes by oxovanadium(IV) complexes

Recently, we have found that some oxovanadium(IV) complexes are potent insulin-mimetic compounds for treating both type I and type II diabetic animals. However, the functional mechanism of oxovanadium(IV) complexes is not fully understood. In this report, we have shown that oxovanadium(IV)-picolinate complexes such as VO(pa)(2), VO(3mpa)(2), and VO(6mpa)(2) act on the insulin signaling pathway in 3T3-L1 adipocytes. Among them, VO(3mpa)(2) was found to be the highest potent activator in inducing not only the phosphotyrosine levels of both IRbeta and IRS but also the activation of downstream kinases in the insulin receptor, such as Akt and GSK3beta, which in turn translocated the insulin-dependent GLUT4 to the plasma membrane. Then, we examined whether or not oxovanadium(IV)-picolinates exhibit the hypoglycemic activity in STZ-induced diabetic mice, and found that VO(3mpa)(2) is more effective than the others in improving the hyperglycemia of the animals. Our present data indicate that both activation of insulin signaling pathway, which follows the GLUT4 translocation to the plasma membrane, and enhancement of glucose utilization by oxovanadium(IV) complexes cause the hypoglycemic effect in diabetic animals.     

Myosin 5a is an insulin-stimulated Akt2 (protein kinase Bbeta) substrate modulating GLUT4 vesicle translocation

Phosphatidylinositol 3-kinase activation of Akt signaling is critical to insulin-stimulated glucose transport and GLUT4 translocation. However, the downstream signaling events following Akt activation which mediate glucose transport stimulation remain relatively unknown. Here we identify an Akt consensus phosphorylation motif in the actin-based motor protein myosin 5a and show that insulin stimulation leads to phosphorylation of myosin 5a at serine 1650. This Akt-mediated phosphorylation event enhances the ability of myosin 5a to interact with the actin cytoskeleton. Small interfering RNA-induced inhibition of myosin 5a and expression of dominant-negative myosin 5a attenuate insulin-stimulated glucose transport and GLUT4 translocation. Furthermore, knockdown of Akt2 or expression of dominant-negative Akt (DN-Akt) abolished insulin-stimulated phosphorylation of myosin 5a, inhibited myosin 5a binding to actin, and blocked insulin-stimulated glucose transport. Taken together, these data indicate that myosin 5a is a newly identified direct substrate of Akt2 and, upon insulin stimulation, phosphorylated myosin 5a facilitates anterograde movement of GLUT4 vesicles along actin to the cell surface.     

The action mechanism of zinc(II) complexes with insulinomimetic activity in rat adipocytes

Zinc (Zn), an essential trace element, and its complexes have recently been known to exhibit insulinomimetic activities. However, the action mechanism of Zn(II) has yet been obscure. The purpose of the present study was to estimate the action mechanism of the Zn(II) complexes. We found first that Zn given in the chemical forms such as Zn(maltolate)2 and Zn(threoninate)2 complexes is highly uptaken in the isolated rat adipocytes compared with that of Zn(picolinate)2. Then, the action mechanism for the insulinomimetic activities was examined in terms of free fatty acid release from the adipocytes. Four Zn(II) compounds, ZnSO4, Zn(picolinate)2, Zn(maltolate)2, and Zn(threoninate)2, inhibited the free fatty acid release from the adipocytes treated with epinephrine (adrenaline). By using several inhibitors for fatty acids and glucose metabolisms in the adipocytes, the following results were obtained. (1) Zn(picolinic acid)2 complex acts on the insulin receptor and PI3-k, which relate to the glucose uptake, as indicated by the experiments using hydroxy-2-naphthalenylmethyl phosphonic acid tris acetoxy methyl ester (HNMPA-(AM)3) and wortmannin, respectively. (2) ZnSO4, and Zn(maltolate)2 and Zn(threoninate)2 complexes affect a glucose transporter 4 (GLUT 4), which is involved in the glucose uptake as indicated by the results using cytochalasin B. (3) Four Zn(II) compounds affect the activation of the phosphodiesterase as indicated by the experiments using cilostamide. These results indicate that the Zn(II) compounds promote the glucose uptake into the adipocytes by affecting at least three sites in the adipocytes, which in turn normalize the blood glucose levels in the experimental diabetic animals.     

YM201636, an inhibitor of retroviral budding and PIKfyve-catalyzed PtdIns(3,5)P2 synthesis, halts glucose entry by insulin in adipocytes

Silencing of PIKfyve, the sole enzyme for PtdIns(3,5)P₂ biosynthesis that controls proper endosome dynamics, inhibits retroviral replication. A novel PIKfyve-specific inhibitor YM201636 disrupts retroviral budding at 800 nM, suggesting its potential use as an antiretroviral therapeutic. Because PIKfyve is also required for optimal insulin activation of GLUT4 surface translocation and glucose influx, we tested the outcome of YM201636 application on insulin responsiveness in 3T3L1 adipocytes. YM201636 almost completely inhibited basal and insulin-activated 2-deoxyglucose uptake at doses as low as 160 nM, with IC₅₀ = 54 ± 4 nM for the net insulin response. Insulin-induced GLUT4 translocation was partially inhibited at substantially higher doses, comparable to those required for inhibition of insulin-induced phosphorylation of Akt/PKB. In addition to PIKfyve, YM201636 also completely inhibited insulin-dependent activation of class IA PI 3-kinase. We suggest that apart from PIKfyve, there are at least two additional targets for YM201636 in the context of insulin signaling to GLUT4 and glucose uptake: the insulin-activated class IA PI 3-kinase and a here-unidentified high-affinity target responsible for the greater inhibition of glucose entry vs. GLUT4 translocation. The profound inhibition of the net insulin effect on glucose influx at YM201636 doses markedly lower than those required for efficient retroviral budding disruption warns of severe perturbations in glucose homeostasis associated with potential YM201636 use in antiretroviral therapy.     

Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.     

Hyperglycemia impairs the insulin signaling step between PI 3-kinase and Akt/PKB activations in ZDF rat liver

Akt/PKB activation is reportedly essential for insulin-induced glucose metabolism in the liver. During the hypoinsulinemic and hyperglycemic phase in the Zucker diabetic fatty (ZDF) rat liver, insulin-induced phosphorylations of the insulin receptor (IR) and insulin receptor substrate (IRS)-1/2 were significantly enhanced. Similarly, phosphatidylinositol (PI) 3-kinase activities associated with IRS-1/2 were markedly increased in ZDF rat liver compared with those in the control lean rat liver. However, interestingly, insulin-induced phosphorylation and kinase activation of Akt/PKB were severely suppressed. The restoration of normoglycemia by sodium-dependent glucose transporter (SGLT) inhibitor to ZDF rats normalized elevated PI 3-kinase activation and phosphorylation of IR and IRS-1/2 to lean control rat levels. In addition, impaired insulin-induced Akt/PKB activation was also normalized. These results suggest that chronic hyperglycemia reduces the efficiency of the activation step from PI 3-kinase to Akt/PKB kinase and that this impairment is the molecular mechanism underlying hyperglycemia-induced insulin resistance in the liver.     

Effects of streptozotocin-induced type 1 maternal diabetes on PI3K/AKT signaling pathway in the hippocampus of rat neonates

Diabetes in pregnancy impairs hippocampus development in offspring, leading to behavioral problems and learning deficits. Phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway plays a pivotal role in the regulation of neuronal proliferation, survival and death. The present study was designed to examine the effects of maternal diabetes on PKB/Akt expression and phosphorylation in the developing rat hippocampus. Wistar female rats were maintained diabetic from a week before pregnancy through parturition and male offspring was killed at first postnatal day (P1). The hippocampal expression and phosphorylation level of PKB/Akt, one of the key molecules in PI3K/AKT signaling pathway, was evaluated using real-time polymerase chain reaction (PCR) and western blot analysis. We found a significant bilateral downregulation of AKT1 gene expression in the hippocampus of pups born to diabetic mothers (p?<?0.05). Interestingly, our results revealed a marked upregulation of Akt1 gene in insulin-treated group compared with other groups (p?<?0.05). The western blot analysis also showed the reduction of phosphorylation level of all AKT isoforms in both diabetic and insulin-treated groups compared with control (p?<?0.05). Moreover, the results showed a significant increase in phosphorylation level of AKT in insulin-treated group compared with the diabetic group. These results represent that diabetes during pregnancy strongly influences the regulation of PKB/AKT in the developing rat hippocampus. Furthermore, although the control of glycemia by insulin administration is not sufficient to prevent the alterations in PKB/Akt expression, it modulates the phosphorylation process, thus ultimately resulting in a situation comparable to that found in the normal condition.     

Antidiabetic zinc(II)-N-acetyl-L-cysteine complex: evaluations of in vitro insulinomimetic and in vivo blood glucose-lowering activities

The diabetic state is known to induce oxidative stress in its mechanism, which in turn is responsible for the complications of diabetes mellitus (DM). Recently, we found that Zn(II) complexes have in vitro insulinomimetic and in vivo blood glucose-lowering activities. During our study on the development of new Zn(II) complexes with antioxidative ligands involving L-cysteine, L-cysteine-methylester, and N-acetyl-L-cysteine (nac), we found a new (N-acetyl-L-cysteinato)Zn(II) (Zn(nac)) complex by evaluating of both its in vitro insulinomimetic and in vivo potencies. The insulinomimetic activity of Zn(nac) with respect to the inhibition of free fatty acid release in isolated rat adipocytes treated with epinephrine was higher than that of a well-known insulinomimetic VOSO4, being equivalent to that of ZnSO4. The blood glucose level of hyperglycemic KK-Ay mice with type 2 DM was reduced by daily intraperitoneal injections of Zn(nac) for 28 days. Their serum insulin, HbA1c, TCHO, and UN levels were remarkably decreased, indicating that Zn(nac) improved the insulin resistance of the mice. The improvement of DM by Zn(nac) was also confirmed by the oral glucose tolerance test. In conclusion, Zn(nac) complex is proposed to attenuate both hyperglycemia and hyperinsulinemia in KK-Ay mice by decreasing serum insulin, HbA1c, UN, and TCHO levels.     

Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells.

Angiotensin II, a hypertrophic/anti-apoptotic hormone, utilizes reactive oxygen species (ROS) as growth-related signaling molecules in vascular smooth muscle cells (VSMCs). Recently, the cell survival protein kinase Akt/protein kinase B (PKB) was proposed to be involved in protein synthesis. Here we show that angiotensin II causes rapid phosphorylation of Akt/PKB (6- +/- 0.4-fold increase). Exogenous H(2)O(2) (50-200 microM) also stimulates Akt/PKB phosphorylation (maximal 8- +/- 0.2-fold increase), suggesting that Akt/PKB activation is redox-sensitive. Both angiotensin II and H(2)O(2) stimulation of Akt/PKB are abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002 (2(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), suggesting that PI3-K is an upstream mediator of Akt/PKB activation in VSMCs. Furthermore, diphenylene iodonium, an inhibitor of flavin-containing oxidases, or overexpression of catalase to block angiotensin II-induced intracellular H(2)O(2) production significantly inhibits angiotensin II-induced Akt/PKB phosphorylation, indicating a role for ROS in agonist-induced Akt/PKB activation. In VSMCs infected with dominant-negative Akt/PKB, angiotensin II-stimulated [(3)H]leucine incorporation is attenuated. Thus, our studies indicate that Akt/PKB is part of the remarkable spectrum of angiotensin II signaling pathways and provide insight into the highly organized signaling mechanisms coordinated by ROS, which mediate the hypertrophic response to angiotensin II in VSMCs.     

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.     

Cell-type-specific roles of IGF-1R and EGFR in mediating Zn2+-induced ERK1/2 and PKB phosphorylation

Zn²⁺ exerts insulin-mimetic and antidiabetic effects in rodent models of insulin resistance, and activates extracellular-signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key components of the insulin signaling pathway. Zn²⁺-induced signaling has been shown to be associated with an increase in the tyrosine phosphorylation of insulin receptor (IR), as well as of insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) in several cell types. However, the specific contribution of these receptor protein tyrosine kinases (R-PTKs) in mediating Zn²⁺-induced responses in a cell-specific fashion remains to be established. Therefore, using a series of pharmacological inhibitors and genetically engineered cells, we have investigated the roles of various R-PTKs in Zn²⁺-induced ERK1/2 and PKB phosphorylation. Pretreatment of Chinese hamster ovary (CHO) cells overexpressing a human IR (CHO-HIR cells) with AG1024, an inhibitor for IR protein tyrosine kinase (PTK) and IGF-1R-PTK, blocked Zn²⁺-induced ERK1/2 and PKB phosphorylation, but AG1478, an inhibitor for EGFR, was without effect in CHO cells. On the other hand, both of these inhibitors were able to attenuate Zn²⁺-induced phosphorylation of ERK1/2 and PKB in A10 vascular smooth muscle cells. In addition, in CHO cells overexpressing tyrosine kinase deficient IR, Zn²⁺ was still able to induce the phosphorylation of these two signaling molecules, whereas the insulin effect was significantly attenuated. Furthermore, both Zn²⁺ and insulin-like growth factor 1 failed to stimulate ERK1/2 and PKB phosphorylation in IGF-1R knockout cells. Also, Zn²⁺-induced responses in CHO-HIR cells were not associated with an increase in the tyrosine phosphorylation of the IR β-subunit and insulin receptor substrate 1 in CHO-HIR cells. Taken together, these data suggest that distinct R-PTKs mediate Zn²⁺-evoked ERK1/2 and PKB phosphorylation in a cell-specific manner.     

Cluster Analysis of Insulin Action in Adipocytes Reveals a Key Role for Akt at the Plasma Membrane

The phosphatidylinositol 3-kinase/Akt pathway regulates many biological processes, including insulin-regulated GLUT4 insertion into the plasma membrane. However, Akt operates well below its capacity to facilitate maximal GLUT4 translocation. Thus, reconciling modest changes in Akt expression or activity as a cause of metabolic dysfunction is complex. To resolve this, we examined insulin regulation of components within the signaling cascade in a quantitative kinetic and dose-response study combined with hierarchical cluster analysis. This revealed a strong relationship between phosphorylation of Akt substrates and GLUT4 translocation but not whole cell Akt phosphorylation. In contrast, Akt activity at the plasma membrane strongly correlated with GLUT4 translocation and Akt substrate phosphorylation. Additionally, two of the phosphorylated sites in the Akt substrate AS160 clustered separately, with Thr(P)-642 grouped with other Akt substrates. Further experiments suggested that atypical protein kinase Cζ phosphorylates AS160 at Ser-588 and that these two sites are mutually exclusive. These data indicate the utility of hierarchical cluster analysis for identifying functionally related biological nodes and highlight the importance of subcellular partitioning of key signaling components for biological specificity.     

Inositol pyrophosphates and Akt/PKB: Is the pancreatic β-cell the exception to the rule?

The inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP₇), is thought to negatively regulate the critical insulin signaling protein Akt/PKB. Knockdown of the IP₇-generating inositol hexakisphosphate kinase 1 (IP6K1) results in a concomitant increase in signaling through Akt/PKB in most cell types so far examined. Total in vivo knockout of IP6K1 is associated with a phenotype resistant to high-fat diet, due to enhanced Akt/PKB signaling in classic insulin regulated tissues, counteracting insulin resistance. In contrast, we have shown an important positive role for IP6K1 in insulin exocytosis in the pancreatic β-cell. These cells also possess functional insulin receptors and the feedback loop following insulin secretion is a key aspect of their normal function. Thus we examined the effect of silencing IP6K1 on the activation of Akt/PKB in β-cells. Silencing reduced the glucose-stimulated increase in Akt/PKB phosphorylation on T308 and S473. These effects were reproduced with the selective pan-IP6K inhibitor TNP. The likely explanation for IP₇ reduction decreasing rather than increasing Akt/PKB phosphorylation is that IP₇ is responsible for generating the insulin signal, which is the main source of Akt/PKB activation. In agreement, insulin receptor activation was compromised in TNP treated cells. To test whether the mechanism of IP₇ inhibition of Akt/PKB still exists in β-cells, we treated them at basal glucose with an insulin concentration equivalent to that reached during glucose stimulation. TNP potentiated the Akt/PKB phosphorylation of T308 induced by exogenous insulin. Thus, the IP₇ regulation of β-cell Akt/PKB is determined by two opposing forces, direct inhibition of Akt/PKB versus indirect stimulation via secreted insulin. The latter mechanism is dominant, masking the inhibitory effect. Consequently, pharmacological strategies to knock down IP6K activity might not have the same positive output in the β-cell as in other insulin regulated tissues.