Cellular mechanism of nutritionally induced insulin resistance in Psammomys obesus: overexpression of protein kinase Cepsilon in skeletal muscle precedes the onset of hyperinsulinemia and hyperglycemia.
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The sand rat (Psammomys obesus) is an animal model of nutritionally induced diabetes. We report here that several protein kinase C (PKC) isoforms (alpha, epsilon, and zeta, representing all three subclasses of PKC) are overexpressed in the skeletal muscle of diabetic animals of this species. This is most prominent for the epsilon isotype of PKC. Interestingly, increased expression of PKCepsilon could already be detected in normoinsulinemic, normoglycemic (prediabetic) animals of the diabetes-prone (DP) line when compared with a diabetes-resistant (DR) line. In addition, plasma membrane (PM)-associated fractions of PKCalpha and PKCepsilon were significantly increased in skeletal muscle of diabetic animals, suggesting chronic activation of these PKC isotypes in the diabetic state. The increased PM association of these PKC isotypes revealed a significant correlation with the diacylglycerol content in the muscle samples. Altered expression/activity of PKCepsilon, in particular, may thus contribute to the development of diabetes in these animals; along with other PKC isotypes, it may be involved in the progression of the disease. This may possibly occur through inhibition of insulin receptor (IR) tyrosine kinase activity mediated by serine/threonine phosphorylation of the IR or insulin receptor substrate 1 (IRS-1). However, overexpression of PKCepsilon also mediated down-regulation of IR numbers in a cell culture model (HEK293), resulting in attenuation of insulin downstream signaling (reduced protein kinase B [PKB]/Akt activity). In accordance with this, we detected decreased 125I-labeled insulin binding, probably reflecting a downregulation of IR numbers, in skeletal muscle of Psammomys animals from the DP line. The number of IRs was inversely correlated to both the expression and PM-associated levels of PKCepsilon. These data suggest that overexpression of PKCepsilon may be causally related to the development of insulin resistance in these animals, possibly by increasing the degradation of IRs. (+info)
Use of functional proteomics to investigate PKC epsilon-mediated cardioprotection: the signaling module hypothesis.
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The characterization of biological processes on the basis of alterations in the cellular proteins, or "proteomic" analysis, is a powerful approach that may be adopted to decipher the signaling mechanisms that underlie various pathophysiological conditions, such as ischemic heart disease. This review represents a prospectus for the implementation of proteomic analyses to delineate the myocardial intracellular signaling events that evoke cardioprotection against ischemic injury. In concert with this, the manifestation of a protective phenotype has recently been shown to involve dynamic modulation of protein kinase C-epsilon (PKC epsilon) signaling complexes (Ping P, Zhang J, Pierce WM Jr, and Bolli R. Circ Res 88: 59--62, 2001). Accordingly, "the signaling module hypothesis" is formulated as a plausible mechanism by which multipurpose stress-activated proteins and signaling kinases may function collectively to facilitate the genesis of cardioprotection. (+info)
Regulation of B(2)-kinin receptors by glucose in vascular smooth muscle cells.
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The development of vascular disease is accelerated in hyperglycemic states. Vascular injury plays a pivotal role in the progression of atherosclerotic vascular disease in diabetes, which is characterized by increased vascular smooth muscle cell (VSMC) proliferation and extracellular matrix accumulation. We previously reported that diabetes alters the activity of the kallikrein-kinin system and results in the upregulation of kinin receptors in the vessel wall. To determine whether glucose can directly influence the regulation of kinin receptors, the independent effect of high glucose (25 mM) on B(2)-kinin receptors (B2KR) in VSMC was examined. A threefold increase in B2KR protein levels and a 40% increase in B2KR surface receptors were observed after treatment with high glucose after 24 h. The mRNA levels of B2KR were also significantly increased by high glucose as early as 4 h later. To elucidate the cellular mechanisms by which glucose regulates B2KR, we examined the role of protein kinase C (PKC). High glucose increased total PKC activity and resulted in the translocation of conventional PKC isoforms (beta(1) and beta(2)), novel (epsilon), and atypical (zeta) PKC isoforms into the membrane. Inhibition of PKC activity prevented the increase in B2KR levels induced by ambient high glucose. These findings provide the first evidence that glucose regulates the expression of B(2) receptors in VSMC and provide a rationale to further study the interaction between glucose and kinins on the pathogenesis of atherosclerotic vascular disease in diabetes. (+info)
Cis-polyunsaturated fatty acids stimulate beta1 integrin-mediated adhesion of human breast carcinoma cells to type IV collagen by activating protein kinases C-epsilon and -mu.
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We have investigated the effects of various fatty acids (FAs) on integrin-mediated MDA-MB-435 breast carcinoma cell adhesion to type IV collagen (collagen IV) in vitro. Arachidonic acid (AA) and linoleic acid both induced a dose-dependent increase in cell adhesion to collagen IV with no significant increase in nonspecific adhesion to polylysine and BSA. Oleic acid (a monounsaturated FA), AA methyl ester, and linoelaidic acid (a trans-isomer of linoleic acid) failed to stimulate adhesion to collagen IV, suggesting that these effects required cis-polyunsaturation and a free carboxylic moiety and that they were not due to membrane perturbations. Calphostin C, a protein kinase C (PKC) inhibitor, blocked cis-polyunsaturated FA (cis-PUFA)-induced cell adhesion in a dose-dependent manner, suggesting a role for a calcium-dependent PKC in this signal transduction pathway. Immunoblotting revealed that cis-PUFAs induced the translocation of PKCepsilon and PKCmu, two of the novel PKC isozymes, from the cytosol to the membrane. In contrast, a conventional PKC isozyme, PKCalpha, as well as the atypical isozymes, PKCzeta and PKCiota, did not translocate after cis-PUFA treatment. Function-blocking antibodies specific for alpha1, alpha2, and beta1, integrin subunits inhibited cell adhesion to collagen IV, whereas antibodies to alpha3 and alpha5 did not. No increase in the expression of these integrins on the cell surface was detected after the incubation of cells with cis-PUFAs, suggesting that there is an increase in the activity, but not in the amount, of these beta1, integrins. Altogether, these data suggest that cis-PUFAs enhance human breast cancer cell adhesion to collagen IV by selectively activating specific PKC isozymes, which leads to the activation of beta1 integrins. (+info)
Ischemic preconditioning upregulates vascular endothelial growth factor mRNA expression and neovascularization via nuclear translocation of protein kinase C epsilon in the rat ischemic myocardium.
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Ischemic preconditioning (IP) exerts cardioprotection through protein kinase C (PKC) activation, whereas myocardial ischemia enhances vascular endothelial growth factor (VEGF) mRNA expression. However, the IP effect or the involvement of PKC on the VEGF expression is unknown in myocardial infarction. We investigated whether IP enhances VEGF gene expression and angiogenesis through PKC activation in the in vivo myocardial infarction model. Sprague-Dawley rats were assigned into the following 3 groups: the sham group; the IP group, which underwent 3 cycles of 3 minutes of ischemia and 5 minutes of reperfusion (IP procedure); and the non-IP group. The latter 2 groups were subsequently subjected to left anterior descending coronary artery occlusion. To examine the involvement of PKC, the PKC inhibitor chelerythrine (5 mg/kg) or bisindolylmaleimide (1 mg/kg) was injected intravenously before the IP procedures. PKCepsilon was translocated to the nucleus after 10 minutes of ischemia after the IP procedure but was not translocated in the non-IP and the sham groups. VEGF mRNA expression 3 hours after infarction was significantly higher in the IP group than in the non-IP and the sham groups. Capillary density in the infarction was significantly higher, whereas the infarct size was smaller in the IP group than in the non-IP group at 3 days of infarction. Chelerythrine but not bisindolylmaleimide blocked all of the IP effects on the nuclear translocation of PKCepsilon, enhancement of VEGF mRNA expression and angiogenesis, and infarct size limitation. These results show that IP may enhance VEGF gene expression and angiogenesis through nuclear translocation of PKCepsilon in the infarcted myocardium. (+info)
Localization and kinetics of protein kinase C-epsilon anchoring in cardiac myocytes.
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Protein kinase C-epsilon (PKC-epsilon) plays a central role in cardiac cell signaling, but mechanisms of translocation and anchoring upon activation are poorly understood. Conventional PKC isoforms rely on a rapid Ca2+-mediated recruitment to cell membranes, but this mechanism cannot be employed by PKC-epsilon or other PKC isoforms lacking a Ca2+-binding domain. In this study, we used recombinant green fluorescent protein (GFP) fusion constructs and confocal microscopy to examine the localization, kinetics, and reversibility of PKC-epsilon anchoring in permeabilized rat cardiac myocytes. PKC-epsilon-GFP bound with a striated pattern that co-localized with alpha-actinin, a marker of the Z-line of the sarcomere. Binding required activation of PKC and occurred slowly but reversibly with apparent rate constants of k(on) = 4.6 +/- 1.2 x 10(3) M(-1) x s(-1) and k(off) = 1.4 +/- 0.5 x 10(-3) s(-1) (t1/2 = 8 min) as determined by fluorescence recovery after photobleaching and by perfusion experiments. A truncated construct composed of the N-terminal 144-amino-acid variable region of PKC-epsilon (epsilonV1-GFP), but not an analogous N-terminal domain of PKC-delta, mimicked the Z-line decoration and slow binding rate of the full-length enzyme. These findings suggest that the epsilonV1 domain is important in determining PKC-epsilon localization and translocation kinetics in cardiac muscle. Moreover, PKC-epsilon translocation is not a diffusion-controlled binding process but instead may be limited by intramolecular conformational changes within the V1 domain. The k(off) for epsilonV1-GFP was two- to threefold faster than for full-length enzyme, indicating that other domains in PKC-epsilon contribute to anchoring by prolonging the bound state. (+info)
Conventional protein kinase C isoforms and cross-activation of protein kinase A regulate cardiac Na+ current.
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We tested the hypothesis that specific isoforms of protein kinase C (PKC) are responsible for modulation of Na+ current (I(Na)) derived from the human cardiac Na+ channel using activators and inhibitors selective for specific PKCs. Experimental results demonstrated that I(Na) suppression was mediated by activation of conventional PKCs (cPKCs) and possibly resulted from channel internalization. In the presence of cPKC inhibition, phorbol ester application unexpectedly increased Na+ current, an effect eliminated by inhibition of protein kinase A. These findings demonstrate complex modulation of cardiac I(Na) by protein kinases and provide further evidence that PKC isoforms have distinct protein targets. (+info)
Inhibitory actions of ceramide upon PKC-epsilon/ERK interactions.
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We have previously shown that interleukin-1 receptor-generated ceramide induces growth arrest in smooth muscle pericytes by inhibiting an upstream kinase in the extracellular signal-regulated kinase (ERK) cascade. Here, we now report the mechanism by which ceramide inhibits ERK activity. Ceramide renders the human embryonic kidney 293 cells (HEK 293) resistant to the mitogenic actions of growth factors and activators of protein kinase C (PKC). A role for PKC to mediate ceramide inhibition of growth factor-induced ERK activity and mitogenesis is suggested, as exogenous ceramide directly inhibits both immunoprecipitated and recombinant PKC-epsilon activities. To confirm that PKC-epsilon is necessary for ceramide-inhibited ERK activity, HEK 293 cells were transfected with a dominant-negative mutant of PKC-epsilon (DeltaPKC-epsilon). These transfected cells respond to insulin-like growth factor I (IGF-I) with a significantly decreased ERK activity that is not further reduced by ceramide treatment. Coimmunoprecipitation studies reveal that the treatment with IGF-I induces the association of ERK with PKC-epsilon but not with PKC-zeta. Ceramide treatment significantly inhibits the IGF-I-induced PKC-epsilon interaction with bioactive phosphorylated ERK. Ceramide also inhibits IGF-I-induced PKC-epsilon association with Raf-1, an upstream kinase of ERK. Together, these studies demonstrate that ceramide exerts anti-mitogenic actions by limiting the ability of PKC-epsilon to form a signaling complex with Raf-1 and ERK. (+info)