Insulin inhibition of apolipoprotein B mRNA translation is mediated via the PI-3 kinase/mTOR signaling cascade but does not involve internal ribosomal entry site (IRES) initiation

Although insulin normally activates global mRNA translation, it has a specific inhibitory effect on translation of apolipoprotein B (apoB) mRNA. This suggests that insulin induces a unique signaling cascade that leads to specific inhibition of apoB mRNA translation despite global translational stimulation. Recent studies have revealed that insulin functions to regulate apoB mRNA translation through a mechanism involving the apoB mRNA 5' untranslated region (5' UTR). Here, we further investigate the role of downstream insulin signaling molecules on apoB mRNA translation, and the mechanism of apoB mRNA translation itself. Transfection studies in HepG2 cells expressing deletion constructs of the apoB 5' UTR showed that the cis-acting region responding to insulin was localized within the first 64 nucleotides. Experiments using chimeric apoB UTR-luciferase constructs transfected into HepG2 cells followed by treatment with wortmannin, a PI-3K inhibitor, and rapamycin, an mTOR inhibitor, showed that signaling via PI-3K and mTOR pathways is necessary for insulin-mediated inhibition of chimeric 5' UTR-luciferase expression. In vitro translation of chimeric cRNA confirmed that the effects observed were translational in nature. Furthermore, using RNA-EMSA we found that wortmannin pretreatment blocked insulin-mediated inhibition of the binding of RNA-binding factor(s), migrating near the 110 kDa marker, to the 5' UTR. Radiolabeling studies in HepG2 cells also showed that insulin-mediated control of the synthesis of endogenously expressed full length apoB100 is mediated via the PI-3K and mTOR pathways. Finally, using dual-cistronic luciferase constructs we demonstrate that apoB 5' UTR may have weak internal ribosomal entry (IRES) translation which is not affected by insulin stimulation, and may function to stimulate basal levels of apoB mRNA translation.     

Mitogenic signaling via endogenous kappa-opioid receptors in C6 glioma cells: evidence for the involvement of protein kinase C and the mitogen-activated protein kinase signaling cascade

As reports on G protein-coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous kappa-opioid receptors in C6 glioma cells, an astrocytic model system. We find that the kappa-opioid receptor-selective agonist U69,593 stimulates phospholipase C activity, extracellular signal-regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593-stimulated extracellular signal-regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin-sensitive G proteins, L-type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen-activated protein or extracellular signal-regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant-negative or sequestering mutants, we provide evidence that extracellular signal-regulated kinase 1/2 phosphorylation is Ras-dependent and transduced by Gbetagamma subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous kappa-opioid receptor in C6 glioma cells.     

蛋白激酶C对胰岛素抵抗骨骼肌细胞的影响

目的探讨蛋白激酶C(Protein Kinase C,PKC)在棕榈酸(Palmitic Acid,PA)诱导的骨骼肌细胞胰岛素抵抗(Isulin Resistance,IR)中的作用。方法免疫荧光鉴定原代大鼠骨骼肌细胞,氧化酶-过氧化物酶偶联法(GOD-POD法)检测培养液中葡萄糖浓度。设立对照组、棕榈酸组(PA组)、罗格列酮组(Rosiglitazone,Ros组),每组一分为二,分别加PKC抑制剂白屈莱红碱(Chelerythrine Chloride,CC)与正常培养液作用1h,Western Blot检测PKB及P-Ser473 PKB表达水平。结果 90%以上的细胞-αsarcometric actin免疫荧光染色呈阳性反应,表明培养的细胞为骨骼肌细胞;0.6mmol/L的PA作用24h可诱导骨骼肌细胞产生胰岛素抵抗;PA组与对照组相比P-Ser473 PKB水平显著降低,与本组未加CC相比显著升高。同时,罗格列酮组及本组加CC中P-Ser473PKB水平均高于PA组。结论在PA诱导的骨骼肌细胞IR方面PKC起重要作用,罗格列酮与PKC抑制剂CC均能改善PA引起的IR。     

Organo-vanadium compounds are potent activators of the protein kinase B signaling pathway and protein tyrosine phosphorylation: mechanism of insulinomimesis

Organo-vanadium compounds (OVC) have been shown to be more effective than inorganic vanadium compounds in ameliorating glucose homeostasis and insulin resistance in rodent models of diabetes mellitus. However, the precise molecular mechanism of OVC efficiency remains poorly defined. Since inorganic vanadium compounds have been found to activate several key components of the insulin signaling cascade, such as protein kinase B (PKB), the objective of the present study was to investigate if stimulation of PKB and its downstream target glycogen synthase kinase-3 (GSK-3), are responsible for the more potent insulinomimetic effects of OVC. Among several vanadium compounds tested, vanadium (IV) oxo bis (acetylacetonate) and vanadium (IV) oxo bis(maltolato) markedly induced the phosphorylation of PKB as well as GSK-3beta compared to vanadyl sulfate (VS), an inorganic vanadium salts in Chinese hamster ovary cells overexpressing the insulin receptor (IR). Furthermore, the OVC were stronger inhibitors of protein tyrosine phosphatase (PTPase) activity than VS. The higher PTPase inhibitory potential of the OVC was associated with more robust tyrosine phosphorylation of several cellular proteins, including the IRbeta subunit and insulin receptor substrate-1 (IRS-1). In addition, greater IRS-1/p85alpha interaction was elicited by the OVC than by VS. These data indicate that the higher PTPase inhibitory potential of OVC translates into greater phosphorylation of PKB and GSK-3beta, which, in turn, may contribute to a more potent effect of OVC on glucose homeostasis.     

Protein kinase C isoforms: mediators of reactive lipid metabolites in the development of insulin resistance

The role of protein kinase C (PKCs) isoforms in the regulation of glucose metabolism by insulin is complex, partly due to the large PKC family consisting of three sub-groups: conventional, novel and atypical. Activation of some conventional and novel PKCs in response to increased levels of diacylglycerol (DAG) have been shown to counteract insulin signalling. However, roles of atypical PKCs (aPKCs) remain poorly understood. aPKCs act as molecular switches by promoting or suppressing signalling pathways, in response to insulin or ceramides respectively. Understanding how DAG- and ceramide-activated PKCs impair insulin signalling would help to develop treatments to fight insulin resistance.     

Oleic acid induces endothelin-1 expression through activation of protein kinase C and NF-kappa B

This study investigated the effect of oleic acid on the expression levels of endothelin-1 (ET-1) and on the signaling pathways mediating it in human aortic endothelial cells (HAECs). ET-1 mRNA expression was significantly increased by oleic acid in a dose- and time-dependent manner. Elevation of ET-1 expression in response to oleic acid was inhibited by the protein kinase C (PKC) inhibitor, GF109203X, or the NF-kappa B inhibitor, pyrrolidine dithiocarbamate. In addition, both PKC and NF-kappa B activities were significantly increased by oleic acid. Immunoblot analysis revealed that conventional PKCs (PKC-alpha and -beta II isoforms) were significantly increased in the membranous fractions of HAECs treated with oleic acid. PKC inhibitor completely abolished oleic acid-induced NF-kappa B activation, suggesting that PKC activation is upstream of NF-kappa B activation in oleic acid-induced ET-1 expression. These data suggest that elevated plasma oleic acid levels observed in obese, insulin-resistant subjects result in endothelial dysfunction, at least in part, through an increase in ET-1 expression.     

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 μM) and the Src inhibitor PP2 (10 μM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca²⁺ transients were reduced by imatinib and/or PP2. Ca²⁺ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca²⁺ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCα catalytic activity and PKCα co-immunoprecipitated with Bcr/Abl.Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca²⁺ influx was reduced by complexing extracellular Ca²⁺ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca²⁺ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.     

Structure and function of the protein kinase C gene family

Protein kinase C is a serine/threonine protein kinase which is activated in the cell in response to production of diacylglycerol. Gene cloning has revealed the presence of a highly related family of enzymes, which can be sub-divided into groups on the basis of sequence conservation. Differences are seen in both isoform distribution and associated biochemical activity, for example in substrate specificity and activator requirements. Comparison of the protein sequences andin vitro activities of the protein kinase C isoforms has identified regions important for particular aspects of kinase function. Some of these regions are also found associated with other proteins, allowing confirmation of the assigned activity. Site-directed mutagenesis has confirmed the presence of an autoinhibitory sequence involved in protein kinase C regulation and generated constitutively activated proteins which can be used to study differential isoform function. These same sequences have been shown to play a role in substrate selection, perhaps by competition for binding to the active site. Protein kinase C is known to be a phosphoprotein and the identification of regulatory sites phosphorylated by a ‘PKC-kinase’ suggest a possible alternative route for regulation of protein kinase C activity.     

Induction of PER1 mRNA expression in immortalized gonadotropes by gonadotropin-releasing hormone (GnRH): Involvement of protein kinase C and MAP kinase signaling

The initiation and maintenance of reproductive function in mammals is critically dependent on the pulsatile secretion of gonadotropin-releasing hormone (GnRH). This peptide drives the pulsatile release of FSH and LH from the pituitary pars distalis via signaling pathways that are activated by the type I GnRH receptor (GnRH-R). Recently, a microarray analysis study reported that a number of genes, including mPer1, are induced by GnRH in immortalized gonadotrope cells. In view of these data, we have begun to analyze in detail the signaling pathways mediating the action of GnRH on mPer1 expression in these cells. Using quantitative real-time polymprose cho read (PCR), we could confirm that exposure of immortalized gonadotropes (LβT2 cells) to the GnRH analog, buserelin, markedly induces mPer1 (but not mPer2) expression. Consistent with GnRH receptor signaling via the protein kinase (PK)-C pathway, exposure of the cells to phorbol 12,13-dibutyrate rapidly elevates both mPer1 and LHβ subunit mRNA levels, while pharmacological inhibition of PKC prevents the mPer1 and LHβ response to buserelin. As GnRH is known to regulate gonadotropin synthesis via activation of p42/44 mitogen-activated protein kinase (MAPK) signaling pathways, we then examined the involvement of this pathway in regulating mPer1 expression in gonadotropes. Our data reveal that GnRH-induced mPer1 expression is blocked following acute exposure to a MAPK kinase inhibitor. Although the involvement of this signaling mechanism in the regulation of mPer1 is known in neurons, e.g., in the suprachiasmatic nuclei, the induction of mPer1 in gonadotropes represents a novel mechanism of GnRH signaling, whose functional significance is still under investigation.     

Activation of protein kinase C sensitizes the cyclic AMP signalling system of T51B rat liver cells

Activation of protein kinase C (PKC) bu phorbol esters (TPA) results in a modification of the cyclic AMP system leading to either attenuation or amplification of the cyclic AMP signal. In the non-neoplastic T51B rat live cell line, TPA, when added to intact cells, had no effect on the basal level of cyclic AMP synthesis but caused a 1.5 fold amplification of the stimulation induced by β-adrenergic agents, cholera toxin and forskolin. The effect appeared to be mediated by PKC since diacylglycerols caused the same amplification as did TPA while inactive phorbol esters were without effect. Phosphorylation of Gs or the catalytic subunit of adenylate cyclase by PKC is likely to be responsible for the enhancement of cyclic AMP synthesis. TPA also caused translocation of PKC; however, the time course of the translocation was loner than the time course of the enhancement of adenylate cyclase activity. Thus, the ability of TPA to amplify cyclic AMP synthesis is probably mediated by activation of PKC that is already present in the membrane.     

Gastrin''s trophic effect in the colon: Identification of a signaling pathway mediated by protein kinase C

In previous studies we have reported that gastrin exerts a trophic effect on rat colonic epithelial cells in vitro. The effect of gastrin appeared to be mediated through a protein kinase C mechanism. In this study, we have characterized the role of protein kinase C in the gastrin-induced stimulation. Gastrin, in a time- and dose-dependent manner, increased protein kinase C translocation from the cytosol to the membrane, an index of enzyme activation. Maximum translocation occurred in 1 to 2 min following exposure to gastrin (10⁻⁸ M), before declining back to baseline level within 5 min. Gastrin did not change total protein kinase C activity in the colonic cells. Staurosporine, an inhibitor of protein kinase C, totally abolished the basal as well as the gastrin-stimulated activity of protein kinase C. The tumor promoter phorbol 12-myristate 13-acetate also stimulated colonic epithelial protein kinase C. However, prolonged treatment of cells with phorbol inhibited their subsequent response to gastrin stimulation. The response to gastrin was also prevented by the gastrin receptor antagonist proglumide. These observations suggest that protein kinase C mediates the stimulatory effect of gastrin on colonic epithelial cells, possibly through a receptor mechanism.     

Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes

A strong correlation between intramyocellular lipid concentrations and the severity of insulin resistance has fueled speculation that lipid oversupply to skeletal muscle, fat, or liver may desensitize these tissues to the anabolic effects of insulin. To identify free fatty acids (FFAs) capable of inhibiting insulin action, we treated 3T3-L1 adipocytes or C2C12 myotubes with either the saturated FFA palmitate (C16:0) or the monounsaturated FFA oleate (C18:1), which were shown previously to be the most prevalent FFAs in rat soleus and gastrocnemius muscles. In C2C12 myotubes, palmitate, but not oleate, inhibited insulin-stimulation of glycogen synthesis, as well as its activation of Akt/Protein Kinase B (PKB), an obligate intermediate in the regulation of anabolic metabolism. Palmitate also induced the accrual of ceramide and diacylglycerol (DAG), two lipid metabolites that have been shown to inhibit insulin signaling in cultured cells and to accumulate in insulin resistant tissues. Interestingly, in 3T3-L1 adipocytes, neither palmitate nor oleate inhibited glycogen synthesis or Akt/PKB activation, nor did they induce ceramide or DAG synthesis. Using myotubes, we also tested whether other saturated fatty acids blocked insulin signaling while promoting ceramide and DAG accumulation. The long-chain fatty acids stearate (18:0), arachidate (20:0), and lignocerate (24:0) reproduced palmitate's effects on these events, while saturated fatty acids with shorter hydrocarbon chains [i.e., laurate (12:0) and myristate (14:0)] failed to induce ceramide accumulation or inhibit Akt/PKB activation. Collectively these findings implicate excess delivery of long-chain fatty acids in the development of insulin resistance resulting from lipid oversupply to skeletal muscle.     

Arachidonate activation of protein kinase C may be involved in the stimulation of protein synthesis by insulin in L6 myoblasts

Insulin stimulated protein synthesis in L6 myoblasts but did not increase the labelling of DAG or the release of phosphocholine from phosphatidylcholine. The DAG lipase inhibitor, RHC 80267, more than doubled the amount of label appearing in DAG but did not stimulate protein synthesis. Even in the presence of the DAG lipase inhibitor insulin failed to have any effect on DAG labelling, and conversely RHC 80267 did not modify the insulin-induced increase in protein synthesis. These results suggest that endogenous DAG production is not involved in the stimulation of protein synthesis by insulin. However, exogenous diacylglycerols (1-oleoyl-2-acetyl glycerol and 1-stearoyl-2-arachidonoyl glycerol) both stimulated protein synthesis in L6 myoblasts. The efficacy of the former (arachidonatefree) DAG suggested that their action was by activation of protein kinase C rather than by arachidonate release and prostaglandin formation. Ibuprofen, an inhibitor of cyclo-oxygenase failed to block the effects of insulin whereas a second cyclo-oxygenase inhibitor, indomethacin had only a partial inhibitory effect. The protein kinase C (PKC) inhibitor, RO-31-8220, totally blocked the effect of insulin. Since indomethacin is also recognised to inhibit phospholipase A₂, the data suggests that insulin acts on protein synthesis in myoblasts by arachidonate activation of PKC.     

Phosphorylation of troponin I by protein kinase C: Mechanism of inhibition by calmodulin and troponin C

The mechanism by which calmodulin and troponin C influence phosphorylation of troponin I (TnI) by protein kinase C was investigated. The phosphorylation of TnI by protein kinase C requires the presence of acidic phospholipid, calcium and diacylglycerol. Light scattering intensity and fluorescence intensity experiments showed that TnI associated with the phospholipid membranes and caused extensive aggregation. In the presence of Ca²⁺, TnI-phospholipid interactions were prevented by approximately stoichiometric amounts of either troponin C or calmodulin. Troponin C was shown to completely inhibit phosphorylation of TnI by either protein kianse C or by phosphorylase b kinase. In contrast, calmodulin completely inhibited phosphorylation of TnI by protein kinase C, but had only little effect on TnI phosphorylation by phosphorylase b kinase. Inhibition by calmodulin did not appear to be due to interaction with PKC, since calmodulin mildly increased protein kinase C phosphorylation of histone III-S. The ratio of phosphoserine to phosphothreonine in protein kinase C-phosphorylated TnI remained approximately constant for reactions inhibited by up to 90% by clamodulin. TnI interactions with phospholipid and phosphorylation of TnI by PKC were also prevented by high salt concentrations. However, salt concentrations adequate to inhibit phosphorylation were sufficient to dissociate only TnI, but not protein kinase C from the membrane. These results suggest that the binding of TnI to phospholipid is required for phosphorylation by protein kinase C and that prevention of this binding by any means completely inhibited phosphorylation of TnI by protein kinase C.     

Production and characterization of antibodies against C-terminal peptide of protein F1: A novel phosphorylation at serine 209 of the peptide by protein kinase C

Protein F1 (GAP-43, B-50, neuromodulin, P-57), a neural tissue-specific phosphoprotein enriched in the growth cones of elongating neurites, is suggested to be involved in synaptic plasticity, neuronal development, and neurotransmitter release. In this study, a 21 amino acid polypeptide (AKPKES^* ARQDEGKEDPEADQE) that corresponds to the C-terminus sequence of protein F1 (from position 204–224) was synthesized and used to produce anti-protein F1 antibodies. Immunoblot analysis has demonstrated that the prepared antibodies recognized intact protein F1. Protein F1 and the synthesized F1 peptide were phosphorylated in vitro by PKC. Furthermore, phosphorylated protein F1 was immunoprecipitated by anti-F1 peptide antibodies demonstrating that these antibodies recognized both native, non-phosphorylated and phosphorylated protein. The anti-protein F1 antibodies also stained the plasma membranes of cell bodies and neurities of mouse neuronal cultures obtained from 14-day old spinal embryonic tissue. By contrast, no glial cells were stained. These data suggest that serine 209 at the C-terminus of protein F1 may be a substrate for PKC phosphorylation in vivo. In addition, antibodies raised against F1 peptide revealed protein F1 immunoreactivity that outlined all neurites of cultured mouse spinal neurons.Abbreviations used IgG immunoglobulin G - KLH keyhole limpet haemocyanin - OAG L--1-oleoyl-2-acetoyl-sn-3-glycerol - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PKC protein kinase C - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid