The involvement of phosphatidylinositol 3-kinase /Akt signaling in high glucose-induced downregulation of GLUT-1 expression in ARPE cells

Glucose transporters have been reported to be associated with the development of diabetic retinopathy. Retinal pigment epithelial cells (RPEs) are believed to play an important role in the pathogenesis of diabetic retinopathy. However, the effect of hyperglycemia on glucose transporters in RPEs and the related signal pathways have not yet been elucidated. Therefore, we examined the effect of high glucose on the glucose transporter 1 in ARPEs and the related signal molecules. In the present study, high glucose decreased 2-deoxyglucose uptake in a time (>2 h) and dose dependent manner. In addition, we found that high glucose downregulated the expression of glucose transporter 1 (GLUT-1). The high glucose-induced downregulation of GLUT-1 was blocked by Wortmanin, LY 294002 (PI-3 kinase inhibitors) and Akt (Akt inhibitor). The high glucose increased stimulation of Akt activation in a time dependent manner. We also investigated the upstream regulator of Akt activation. The high glucose-induced phosphorylation of Akt was blocked by bisindolymaleimide I, H-7, staurosporine (protein kinase C [PKC] inhibitors), vitamin C and catalase (antioxidants). In addition, the high glucose-induced downregulation of GLUT-1 was also blocked by PKC inhibitors and antioxidants. Moreover, high glucose increased lipid peroxide formation, which was prevented by PKC inhibitors. In conclusion, high glucose downregulated GLUT-1 by Akt pathway activation mediated by the PKC-oxidative stress signaling pathway in ARPE cells.     

The early intracellular signaling pathway for the insulin/insulin-like growth factor receptor family in the mammalian central nervous system

Several studies support the idea that the polypeptides belonging to the family of insulin and insulin-like growth factors (IGFs) play an important role in brain development and continue to be produced in discrete areas of the adult brain. In numerous neuronal populations within the olfactory bulb, the cerebral and cerebellar cortex, the hippocampus, some diencephalic and brainstem nuclei, the spinal cord and the retina, specific insulin and IGF receptors, as well as crucial components of the intracellular receptor signaling pathway have been demonstrated. Thus, mature neurons are endowed with the cellular machinery to respond to insulin and IGF stimulation. Studies in vitro and in vivo, using normal and transgenic animals, have led to the hypothesis that, in the adult brain, IGF-I not only acts as a trophic factor, but also as a neuromodulator of some higher brain functions, such as long-term potentiation and depression. Furthermore, a trophic effect on certain neuronal populations becomes clearly evident in the ischemic brain or neurodegenerative disorders. Thus, the analysis of the early intracellular signaling pathway for the insulin/IGF receptor family in the brain is providing us with new intriguing findings on the way the mammalian brain is sculpted and operates.     

Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1.

Monoclonal antibodies raised against the 85-kDa subunit (p85) of bovine phosphatidylinositol (PI) 3-kinase were found to recognize uncomplexed p85 or p85 in the active PI 3-kinase. Immunoprecipitation studies of Chinese hamster ovary cells, which overexpress the human insulin receptor when treated with insulin, showed increased amounts of p85 and PI 3-kinase activity immunoprecipitable with monoclonal anti-p85 antibody and no increase in the tyrosine phosphorylation of p85. Insulin also induced an association of p85 with the tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and other phosphorylated proteins ranging in size from 100 to 170 kDa but not with the activated insulin receptor. In vitro reconstitution studies were used to show p85 in the active PI 3-kinase associated with the tyrosine-phosphorylated IRS-1 but not with the activated insulin receptor. Competition studies using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of IRS-1 revealed that phosphopeptides containing YMXM motifs inhibited this association with different potencies, whereas nonphosphorylated analogues and a phosphopeptide containing the EYYE motif had no effect. Src homology region 2 domains of p85 expressed as glutathione S-transferase fusion proteins also bound to tyrosine-phosphorylated IRS-1. These results suggest that insulin causes the association of PI 3-kinase with IRS-1 via phosphorylated YMXM motifs of IRS-1 and Src homology region 2 domains of p85.     

VMP1 is a new player in the regulation of the autophagy-specific phosphatidylinositol 3-kinase complex activation

We have elucidated a novel mechanism through which the autophagy-specific class III phosphatidylinositol 3-kinase (PtdIns3K) complex can be recruited to the PAS in mammalian cells, through the interaction between BECN1 and the vacuole membrane protein 1 (VMP1), an integral autophagosomal membrane protein. This interaction involves the binding between the C-terminal 20 amino acids of the VMP1 hydrophilic domain, which we have named the VMP1 autophagy-related domain (VMP1-AtgD), and the BH3 domain of BECN1. The association between these two proteins allows the formation of the autophagy-specific PtdIns3K complex, which activity favors the generation of phosphatidylinositol-3-phosphate (PtdIns3P) and the subsequent association of the autophagy-related (ATG) proteins, including ATG16L1, with the phagophore membranes. Therefore, VMP1 regulates the PtdIns3K activity on the phagophore membrane through its interaction with BECN1. Our data provide a novel model describing one of the key steps in phagophore assembly site (PAS) formation and autophagy regulation, and positions VMP1 as a new interactor of the autophagy-specific PtdIns3K complex in mammalian cells.     

The phosphatidylinositol/AKT/atypical PKC pathway is involved in the improved insulin sensitivity by DHEA in muscle and liver of rats in vivo

DHEA improves insulin sensitivity and has anti-obesity effect in animal models and men. However, the molecular mechanisms by which DHEA improves insulin action have not been clearly understood. In the present study, we examined the protein levels and phosphorylation state of insulin receptor (IR), IRS-1 and IRS-2, the association between IRSs and PI3K and SHP2, the insulin-induced IRSs associated PI 3-kinase activities, and the phosphorylation status of AKT and atypical PKCzeta/lambda in the liver and the muscle of 6 month-old Wistar rats treated with DHEA. There was no change in IR, IRS-1 and IRS-2 protein levels in both tissues of treated rats analysed by immunoblotting. On the other hand, insulin-induced IRS-1 tyrosine phosphorylation was increased in both tissues while IRS-2 tyrosyl phosphorylation was increased in liver of DHEA treated group. The PI3-kinase/AKT pathway was increased in the liver and the PI3K/atypical PKCzeta/lambda pathway was increased in the muscle of DHEA treated rats. These data indicate that these regulations of early steps of insulin action may play a role in the intracellular mechanism for the improved insulin sensitivity observed in this animal model.     

Motility enhancement by tumor-derived mutant E-cadherin is sensitive to treatment with epidermal growth factor receptor and phosphatidylinositol 3-kinase inhibitors

Diffuse-type gastric and lobular breast cancers are characterized by frequent mutations in the cell adhesion molecule E-cadherin. Here we report that tumor-associated mutations of E-cadherin enhanced random cell movement of transfected MDA-MB-435S mammary carcinoma cells as compared to wild-type (wt) E-cadherin-expressing cells. The mutations included in frame deletions of exons 8 or 9 and a point mutation in exon 8 which all affect putative calcium-binding sites within the linker region of the second and third extracellular domain. Motility enhancement by mutant E-cadherin was investigated by time-lapse laser scanning microscopy. Increased cell motility stimulated by mutant E-cadherin was influenced by cell-matrix interactions. The motility-increasing activity of mutant E-cadherin was blocked by application of pharmacological inhibitors of epidermal growth factor receptor and phosphatidylinositol (PI) 3-kinase. Investigation of the activation status of PI 3-kinase and the downstream signaling molecules Akt/protein kinase B and MAP kinase p44/42 showed that these kinases are not more strongly activated in mutant E-cadherin-expressing cells than in wt E-cadherin-expressing cells. Instead, the basal level of PI 3-kinase is necessary for mutant E-cadherin-enhanced cell motility. Our data suggest a critical role of E-cadherin mutations for the fine tuning of tumor cell motility.     

The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex

Phosphoinositide (PI) 3-kinase is required for most insulin and insulin-like growth factor (IGF) 1-dependent cellular responses. The p85 regulatory subunit of PI 3-kinase is required to mediate the insulin-dependent recruitment of PI 3-kinase to the plasma membrane, yet mice with reduced p85 expression have increased insulin sensitivity. To further understand the role of p85, we examined IGF-1-dependent translocation of p85alpha by using a green fluorescence protein (GFP)-tagged p85alpha (EGFP-p85alpha). In response to IGF-1, but not to PDGF signaling, EGFP-p85alpha translocates to discrete foci in the cell. These foci contain the insulin receptor substrate (IRS) 1 adaptor molecule, and their formation requires the binding of p85 to IRS-1. Surprisingly, monomeric p85 is preferentially localized to these foci compared with the p85-p110 dimer, and these foci are not sites of phosphatidylinositol-3,4,5-trisphosphate production. Ultrastructural analysis reveals that p85-IRS-1 foci are cytosolic protein complexes devoid of membrane. These results suggest a mechanism of signal down-regulation of IRS-1 that is mediated by monomeric p85 through the formation of a sequestration complex between p85 and IRS-1.     

Reduction of insulin-stimulated glucose uptake by peroxynitrite is concurrent with tyrosine nitration of insulin receptor substrate-1

Inducible nitric oxide synthetase plays an essential role in insulin resistance induced by a high-fat diet. The reaction of nitric oxide with superoxide leads to the formation of peroxynitrite (ONOO-), which can modify several proteins. In this study, we investigated whether peroxynitrite impairs insulin-signalling pathway. Our experiments showed that 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), a constitutive producer of peroxynitrite, dose-dependently inhibited insulin-stimulated glucose uptake. While SIN-1 did not affect the insulin receptor protein level and tyrosine phosphorylation, it reduced the insulin receptor substrate-1 (IRS-1) protein level, and IRS-1 associated phosphatidylinositol-3 kinase (PI-3 kinase) activity. Although SIN-1 did not induce Ser307 phosphorylation of IRS-1, tyrosine nitration of IRS-1 was detected in SIN-1-treated-Rat1 fibroblasts expressing human insulin receptors. Mass spectrometry showed that peroxynitrite induced at least four nitrated tyrosine residues in rat IRS-1, including Tyr939, which is critical for association of IRS-1 with the p85 subunit of PI-3 kinase. Our results suggest that peroxynitrite reduces the IRS-1 protein level and decreases phosphorylation of IRS-1 concurrent with nitration of its tyrosine residues.     

IKBKE protein activates Akt independent of phosphatidylinositol 3-kinase/PDK1/mTORC2 and the pleckstrin homology domain to sustain malignant transformation

Serine/threonine kinase Akt regulates key cellular processes such as cell growth, proliferation, and survival. Activation of Akt by mitogenic factor depends on phosphatidylinositol 3-kinase (PI3K). Here, we report that IKBKE (also known as IKKε and IKKi) activates Akt through a PI3K-independent pathway. IKBKE directly phosphorylates Akt-Thr308 and Ser473 independent of the pleckstrin homology (PH) domain. IKBKE activation of Akt was not affected by inhibition of PI3K, knockdown of PDK1 or mTORC2 complex. Further, this activation could be inhibited by Akt inhibitors MK-2206 and GSK690693 but not the compounds (perifosine and triciribine) targeting the PH domain of Akt. Expression of IKBKE largely correlates with activation of Akt in breast cancer. Moreover, inhibition of Akt suppresses IKBKE oncogenic transformation. These findings indicate that IKBKE is an Akt-Thr308 and -Ser473 kinase and directly activates Akt independent of PI3K, PDK1, and mTORC2 as well as PH domain. Our data also suggest that Akt inhibitors targeting the PH domain have no effect on the tumors in which hyperactive Akt resulted from elevated IKBKE.     

Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the beta-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission.     

Structure of a specific peptide complex of the carboxy-terminal SH2 domain from the p85 alpha subunit of phosphatidylinositol 3-kinase.

We have determined the solution structure of the C-terminal SH2 domain of the p85 alpha subunit of human phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) in complex with a phosphorylated tyrosine pentapeptide sequence from the platelet-derived growth factor receptor using heteronuclear nuclear magnetic resonance spectroscopy. Overall, the structure is similar to other SH2 domain complexes, but displays different detail interactions within the phosphotyrosine binding site and in the recognition site for the +3 methionine residue of the peptide, the side chain of which inserts into a particularly deep and narrow pocket which is displaced relative to that of other SH2 domains. The contacts made within this +3 pocket provide the structural basis for the strong selection for methionine at this position which characterizes the SH2 domains of PI3-kinase. Comparison with spectral and structural features of the uncomplexed domain shows that the long BG loop becomes less mobile in the presence of the bound peptide. In contrast, extreme resonance broadening encountered for most residues in the beta D', beta E and beta F strands and associated connecting loops of the domain in the absence of peptide persists in the complex, implying conformational averaging in this part of the molecule on a microsecond-to-millisecond time scale.     

GM1-induced activation of phosphatidylinositol 3-kinase: involvement of Trk receptors

The ganglioside GM1 promotes neuronal growth, differentiation, survival, phenotypic expression, and function restoration, by apparently interacting with neurotrophic factors and/or their receptors. In brain, GM1 activates the Trk receptors for neurotrophins and the Raf/MEK/ERK cascade in situ and in vivo . We have expanded these studies and explored whether GM1 recruits the phosphatidylinositol 3 (PI3)-kinase pathway in brain also. Incubating striatal slices with GM1 increased the activity of PI3-kinase in phosphotyrosine immunoprecipitates in a time- and concentration-dependent manner, and the response was blocked by the PI3-kinase inhibitors wortmannin and LY294002. PI3-kinase activation following GM1 was rapid and short lasting with an EC₅₀ of 5 μmol/L. There was a temporally parallel activation of the downstream PI3-kinase target Akt, which was prevented by PI3-kinase inhibition. PI3-kinase activity was found increased in Trk and Gab1 immunoprecipitates, and co-immunoprecipitation studies demonstrated the association of Trk and Gab1 after GM1 treatment. Enhanced PI3-kinase activity associated with Trk or Gab1 immunoprecipitates was blocked by the Trk inhibitor K252a. GM1 did not appear to transactivate Trk and did not alter the efflux of neurotrophins in striatal slices. Our findings suggest that GM1 induces activation of PI3-kinase that is, in part, mediated through Trk and Gab1.     

Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase.

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.     

Overexpression of Fer increases the association of tyrosine-phosphorylated IRS-1 with P85 phosphatidylinositol kinase via SH2 domain of Fer in transfected cells

We have reported that the protein-tyrosine kinase Fer is associated with signaling complexes containing insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI-3 kinase) in insulin-stimulated 3T3-L1 adipocytes [J. Biol. Chem. 275 (50) (2000) 38995]. We examined the subcellular localization of this complex in 3T3-L1 adipocytes and performed transfection study to know how this complex is formed. Interestingly we have detected that this complex is formed in LDM of insulin-stimulated 3T3-L1 adipocytes, which may be important for specific biological insulin effect. Based on transfection study, we have demonstrated that overexpression of both Fer and IRS-1 can induce Fer/IRS-1/P85 complexes without insulin stimulation and SH2 domain of Fer is essential for this complex. We have also demonstrated that Fer was an efficient substrate for insulin receptor kinase. Taken together, these data suggested that Fer may play a critically important role to form Fer/IRS-1/P85 complex in LDM of insulin-stimulated adipocytes and elicit biological effect through PI-3 kinase activity in LDM.     

Combination of insulinomimetic agents H2O2 and vanadate enhances insulin receptor mediated tyrosine phosphorylation of IRS-1 leading to IRS-1 association with the phosphatidylinositol 3-kinase

To analyze the mechanism of action of the insulinomimetic agents H₂O₂, vanadate, and pervanadate (H₂O₂ and vanadate), CHO cells or CHO cells that overexpress wild-type or mutant insulin receptor and/or the insulin receptor substrate (IRS-1) were used. H₂O₂ or vanadate treatment alone had little or no effect on tyrosine phosphorylation of cellular proteins; however, pevanadate treatment dramatically enhanced tyrosine phosphorylation of a number of proteins including the insulin receptor and IRS-1. However, the insulin receptor and IRS-1 coimmunoprecipitate from insulin-treated but not from pervanadate-treated cells. Pervanadate-induced tyrosine phosphorylation of the insulin receptor led to an increase in insulin receptor tyrosine kinase activity toward IRS-1 in vivo and IRS-1 peptides in vitro equal to that induced by insulin treatment. Pervanadate-enhanced phosphorylation of IRS-1 led to a fifteenfold increase in IRS-1–associated phosphatidylinositol (Ptdlns) 3-kinase activity. However, insulin receptor–associated Ptdlns 3-kinase activity from pervanadate-treated cells was not detectable, while insulin receptor–associated Ptdlns 3-kinase activity from insulin-treated cells was 20% of the IRS-1-associated activity. Thus, pervanadate but not H₂O₂ or vanadate alone under these conditions mimics many of insulin actions, but pervanadate treatment does not induce insulin receptor/IRS-1 association.     

Attenuated 5-HT1A receptor signaling in brains of suicide victims: involvement of adenylyl cyclase,phosphatidylinositol 3-kinase,Akt and mitogen-activated protein kinase

Positron emission tomography studies in major depression show reduced serotonin (5-HT)1A receptor antagonist-binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5-HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III-R criteria. Measurements of [35S]GTPgammaS binding to Galphai/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5-HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Galphai/o, Galphaq/11 or Galphas proteins, or in the activity of cAMP-dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5-HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3-kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5-triphosphate. Finally, the activation of extracellular signal-regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist-stimulated 5-HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5-HT1A receptor transduction pathways that may have implications for cell survival.     

Tumor necrosis factor (TNF) interferes with insulin signaling through the p55 TNF receptor death domain

Tumor necrosis factor (TNF) contributes to insulin resistance by binding to the 55kDa TNF receptor (TNF-R55), resulting in serine phosphorylation of proteins such as insulin receptor (IR) substrate (IRS)-1, followed by reduced tyrosine phosphorylation of IRS-1 through the IR and, thereby, diminished IR signal transduction. Through independent receptor domains, TNF-R55 activates a neutral (N-SMase) and an acid sphingomyelinase (A-SMase), that both generate the sphingolipid ceramide. Multiple candidate kinases have been identified that serine-phosphorylate IRS-1 in response to TNF or ceramide. However, due to the fact that the receptor domain of TNF-R55 mediating inhibition of the IR has not been mapped, it is currently unknown whether TNF exerts these effects with participation of N-SMase or A-SMase. Here, we identify the death domain of TNF-R55 as responsible for the inhibitory effects of TNF on tyrosine phosphorylation of IRS-1, implicating ceramide generated by A-SMase as a downstream mediator of inhibition of IR signaling.     

Reduced expression of PGC-1 and insulin-signaling molecules in adipose tissue is associated with insulin resistance

Peroxisome proliferator-activated receptor gamma (PPAR gamma) co-activator 1 (PGC-1) regulates glucose metabolism and energy expenditure and, thus, potentially insulin sensitivity. We examined the expression of PGC-1, PPAR gamma, insulin receptor substrate-1 (IRS-1), glucose transporter isoform-4 (GLUT-4), and mitochondrial uncoupling protein-1 (UCP-1) in adipose tissue and skeletal muscle from non-obese, non-diabetic insulin-resistant, and insulin-sensitive individuals. PGC-1, both mRNA and protein, was expressed in human adipose tissue and the expression was significantly reduced in insulin-resistant subjects. The expression of PGC-1 correlated with the mRNA levels of IRS-1, GLUT-4, and UCP-1 in adipose tissue. Furthermore, the adipose tissue expression of PGC-1 and IRS-1 correlated with insulin action in vivo. In contrast, no differential expression of PGC-1, GLUT-4, or IRS-1 was found in the skeletal muscle of insulin-resistant vs insulin-sensitive subjects. The findings suggest that PGC-1 may be involved in the differential gene expression and regulation between adipose tissue and skeletal muscle. The combined reduction of PGC-1 and insulin signaling molecules in adipose tissue implicates adipose tissue dysfunction which, in turn, can impair the systemic insulin response in the insulin-resistant subjects.     

PKCtheta is a key player in the development of insulin resistance

Activation of PKCtheta is associated with lipid-induced insulin resistance and PKCtheta knockout mice are protected from the lipid-induced defects. However, the exact mechanism by which PKCtheta contributes to insulin resistance is not known. To investigate whether an increase in PKCtheta expression leads to insulin resistance, C2C12 skeletal muscle cells were transfected with PKCtheta DNA and treated with different concentrations of insulin for 10 min. PKCtheta overexpression induced reduction of IRS-1 protein levels with a decrease in insulin-induced p85 binding to IRS-1, phosphorylation of PKB and its substrates, p70 and GSK3. Pretreatment of these cells with GF-109203X (a non-specific PKC inhibitor, IC50 for PKCtheta = 10 nM) recovered insulin signaling. PKCtheta was found to be expressed in liver and treatment of human hepatoma cells (HepG2) with high insulin and glucose resulted in an increase in PKCtheta expression that correlated with a decrease in IRS-1 protein levels and the development of insulin resistance. Reduction of PKCtheta expression using RNAi technology significantly inhibited the degradation of IRS-1 and enhanced insulin-induced IRS-1 tyrosine phosphorylation, p85 association to IRS-1 and PKB phosphorylation. In conclusion, by overexpressing PKCtheta or using RNAi technology to downregulate PKCtheta, we have demonstrated that PKCtheta has a key role in the development of insulin resistance. These findings suggest that PKCtheta mediates not only insulin resistance in muscle but also in liver, which may contribute to the development of whole body insulin resistance and diabetes.