Showing papers by "Denise R. Cooper published in 1993"
TL;DR: Levels of common PKC isozyme mRNA, protein, and enzyme activity in soleus muscle of the obese Zucker rat are decreased even though levels of the endogenous PKC activator DAG are elevated.
Abstract: Skeletal muscle is one of the first tissues to become insulin resistant in genetically obese rodents. The activation of protein kinase-C (PKC) in rat skeletal muscle is mediated by insulin stimulation of diacylglycerol (DAG) levels. Defects in the activation of PKC in the heart and liver of obese Zucker rats indicate that an abnormality in either stimulation of DAG or PKC occurs in obese tissues. DAG levels were significantly increased in soleus muscle from 15- to 19-week-old obese (fa/fa) Zucker rats. PKC activity was diminished in soleus muscle from fa/fa rats. Decreased levels of PKC alpha and -beta activity wer enoted after resolution of common PKC isozymes (Ca2+ and phospholipid dependent) by hydroxyapatite chromatography. Immunoreactivity of PKC-alpha, -beta, and -epsilon also indicated that their levels are diminished in fa/fa soleus muscle by 70-90%. To determine at which level down-regulation occurs (i.e. gene expression or protein turnover), mRNA levels were examined by Northern blot analysis of...
36 citations
TL;DR: The differential effects of chronic TPA treatment on the down-regulation of PKC beta may explain why insulin continues to activate biological processes in TPA-treated BC3H-1 myocytes, but not in adipocytes.
Abstract: In rat adipocytes, chronic incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced immunoreactive protein kinase-C (PKC) beta, gamma, delta, and zeta isoforms by 40-60% and PKC alpha by 75%, but had little effect on PKC epsilon levels. In BC3H-1 myocytes, chronic TPA treatment had no effect on PKC beta, increased PKC zeta, and depleted PKC alpha. Acute treatment with insulin induced the translocation of PKC beta in the myocytes both before and after chronic TPA treatment, but had no acute effect on the alpha or zeta isoforms. In contrast, acute TPA treatment in the myocytes had little effect on PKC beta, but induced the rapid translocation of alpha and zeta. The differential effects of chronic TPA treatment on the down-regulation of PKC beta may explain why insulin continues to activate biological processes in TPA-treated BC3H-1 myocytes, but not in adipocytes.
34 citations
TL;DR: The down-regulation of protein kinase C beta and resultant inhibition of 2-[3H]DOG uptake by chronic glucose suggests a biochemical link between hyperglycemia and DAG-protein kinases C signaling in vascular smooth muscle cells.
31 citations
TL;DR: In rat adipocytes, rat soleus muscle and BC3H-1 myocytes, maximally effective concentrations of insulin and phorbol esters provoked comparable, rapid, 2-fold (on average), non-additive increases in the phosphorylation of immunoprecipitable MARCKS.
Abstract: To evaluate the question of whether or not insulin activates protein kinase C (PKC), we compared the effects of insulin and phorbol esters on the phosphorylation of the PKC substrate, i.e. myristoylated alanine-rich C-kinase substrate (MARCKS). In rat adipocytes, rat soleus muscle and BC3H-1 myocytes, maximally effective concentrations of insulin and phorbol esters provoked comparable, rapid, 2-fold (on average), non-additive increases in the phosphorylation of immunoprecipitable MARCKS. These effects of insulin and phorbol esters on MARCKS phosphorylation in intact adipocytes and soleus muscles were paralleled by similar increases in the phosphorylation of an exogenous, soluble, 85 kDa PKC substrate (apparently a MARCKS protein) during incubation of post-nuclear membrane fractions in vitro. Increases in the phosphorylation of this 85 kDa PKC substrate in vitro were also observed in assays of both plasma membranes and microsomes obtained from rat adipocytes that had been treated with insulin or phorbol esters. These insulin-induced increases in PKC-dependent phosphorylating activities of adipocyte plasma membrane and microsomes were associated with increases in membrane contents of diacylglycerol, PKC-beta 1 and PKC-beta 2. Our findings suggest that insulin both translocates and activates PKC in rat adipocytes, rat soleus muscles and BC3H-1 myocytes.
26 citations
TL;DR: These findings provide direct support for a two-step model in the activation of glucose transport and it seems clear that, at least in some cell types, simple phorbol ester treatment does not necessarily serve as a ubiquitous activator of all activable PKC pools and all potential PKC-mediated responses.
10 citations
TL;DR: The identity of the enzyme was confirmed by its inhibition by staurosporine and bisindolylmaleimide and by its translocation in response to phorbol ester, and western blot analysis of protein kinase C isoforms indicated that the beta-isoform was present in both cytosolic and particulate fractions.
Abstract: Activation of protein kinase C is a key event in the transduction of receptor-mediated extracellular signals. Little is known about the role of protein kinase C in the microcirculation of the brain. In this study, we examined protein kinase C in isolated cerebral microvessels. A technique for partial purification of protein kinase C from microvessels was employed, using Q-Sepharose batch adsorption and singlestep salt elution in microfuge tubes. This procedure greatly reduced variability and increased protein kinase C specific activity in both the cytosolic and particulate fractions by nearly 50-fold. The identity of the enzyme was confirmed by its inhibition by staurosporine and bisindolylmaleimide and by its translocation in response to phorbol ester. The level of protein kinase C was assessed by [3H]phorbol ester binding and the endogenous substrates evaluated by in vitro phosphorylation studies. Finally, western blot analysis of protein kinase C isoforms indicated that the β-isoform was present in both cytosolic and particulate fractions. The α-isoform was present at low levels in the cytosolic fraction, whereas the ψ-isoform was not detected.
8 citations