Protein kinase C downregulation?
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Enzyme Activation↗
Biomedical subjects
D R Cooper
Publications and source records attributed to D R Cooper.
Effects of insulin and phorbol esters on subcellular distribution of protein kinase C isoforms in rat adipocytes.
Effects of insulin and phorbol esters on subcellular distribution of protein kinase C (PKC) isoforms were examined in rat adipocytes. Both agonists provoked rapid decreases in cytosolic, and/or increases in membrane, immunoreactive PKC-alpha, PKC-beta, PKC-gamma, and PKC-epsilon. Effects of phorbol esters on PKC-alpha redistribution to the plasma membrane, however, were much greater than those of insulin. In contrast, insulin, but not phorbol esters, stimulated the translocation of PKC-beta to the plasma membrane, and provoked changes in PKC-zeta redistribution. Neither agonist altered subcellular distribution of PKC-delta, which was detected only in membrane fractions. Our findings indicate that insulin and phorbol esters have overlapping and distinctly different effects on the subcellular redistribution of specific PKC isoforms.
Adipose Tissue↗
Direct evidence for protein kinase C involvement in insulin-stimulated hexose uptake.
Insulin has been reported to translocate protein kinase C (PKC) in rat adipocytes, and activation of PKC by phorbol esters is known to increase hexose uptake in these cells (1.2). To test the hypothesis that PKC may participate in insulin-stimulated hexose uptake, adipocytes were partially depleted of protein kinase C by overnight phorbol ester treatment, thereby impairing insulin effects on hexose uptake. Purified PKC was then introduced into these PKC-depleted adipocytes by electropermeabilization, and this fully restored insulin-stimulated hexose uptake. These findings provide direct evidence that PKC is required for insulin-stimulated hexose uptake.
Adipose Tissue↗
Insulin stimulates novel protein kinase C in rat adipocytes.
Insulin is known to rapidly stimulate and/or translocate Ca2+/phospholipid-dependent protein kinase (conventional PKC; cPKC) in rat adipocytes. Presently we examined whether insulin also stimulates/translocates Ca(2+)-independent, phospholipid-dependent protein kinase (novel PKC; nPKC). Total Mono Q column-elutable nPKC (like cPKC) activities were decreased in cytosolic and increased in membrane fractions with insulin treatment. Immunoblot study of novel PKC epsilon also showed insulin-induced translocation of immunoreactive PKC from cytosol to membrane, similar to the translocation of cPKC, PKC beta. These results suggest that nPKC has an important role in insulin-induced signal transduction.
Adipose Tissue↗
The role of protein kinase C in insulin action.
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Animals↗
Effects of insulin on diacylglycerol/protein kinase-C signalling and glucose transport in rat skeletal muscles in vivo and in vitro.
Insulin treatment in vivo provoked rapid dose-related increases in diacylglycerol content and/or translocation of protein kinase-C (PKC) from cytosol to membranes in rat soleus and gastrocnemius muscles. These effects were apparent with 1) insulin doses that provoked submaximal and maximal increases in glucose utilization, and 2) glucose-stimulated endogenous insulin secretion. Insulin-stimulated PKC translocation was evident when PKC was assayed by 1) histone or protamine phosphorylation after PKC purification by Mono Q column chromatography, and 2) immunoblotting for PKC beta and PKC epsilon. Dose-related effects of insulin on PKC translocation were also observed in the rat soleus in vitro, and this was associated with increased phosphorylation of 40- and 80-kilodalton proteins, which were also phosphorylated by phorbol ester treatment. A role for diacylglycerol-PKC signalling in insulin-stimulated glucose transport was suggested by studies of [3H]2-deoxyglucose ([3H]2-DOG) uptake in the rat soleus in vitro in that 1) PKC translocation and 2-DOG uptake were correlated; and 2) stimulatory effects of insulin and phorbol esters on 2-DOG uptake were apparently nonadditive.
Animals↗
Protein kinase C(19-31) pseudosubstrate inhibition of insulin action in rat adipocytes.
Treatment of intact adipocytes with the autoregulatory PKC pseudosubstrate PKC(19-31) inhibited insulin-stimulated hexose uptake and lipogenesis, with no effect on basal values. The effect was dose-dependent with respective IC50 values of 30 microM and 600 microM for insulin-stimulated hexose uptake in electroporated and intact adipocytes. These studies indicate that PKC may play a role in the mediation of insulin action in adipocytes.
Adipose Tissue↗
Sulfonylureas activate glucose transport and protein kinase C in rat adipocytes.
Glyburide and tolbutamide, at concentrations of 20 to 40 mumol/L and 1 to 2 mmol/L, respectively, stimulated glucose transport in rat adipocytes. Concomitantly, protein kinase C was activated, as evidenced by translocation of immunoreactive enzyme from cytosol to membranes. Glucose transport effects of the sulfonylureas were blocked by three inhibitors of protein kinase C (H-7, staurosporine, and sangivamycin), and by phorbol ester-induced down-regulation of protein kinase C. These findings suggest that sulfonylureas may stimulate glucose transport in rat adipocytes through activation of protein kinase C.
Adipose Tissue↗
Antisense DNA downregulates protein kinase C isozymes (beta and alpha) and insulin-stimulated 2-deoxyglucose uptake in rat adipocytes.
Rat adipocytes were treated with antisense dimethoxytrityl pentadecadeoxynucleotides, complementary to mRNA initiation codon regions for alpha and beta isozymes of protein kinase C (PKC). This antisense treatment provoked 50-70% decreases in PKC and insulin-stimulated 2-deoxyglucose uptake, but did not inhibit insulin-stimulated diacylglycerol synthesis. Sense or nonsense oligodeoxynucleotides were without effect on PKC and 2-deoxyglucose uptake. These results suggest that: (i) PKC-alpha and PKC-beta isozymes can be specifically downregulated in rat adipocytes by antisense oligodeoxynucleotides, and (ii) insulin-stimulated glucose transport requires PKC.
Adipose Tissue↗
Adenosine stimulates phosphate and glucose transport in opossum kidney epithelial cells.
We have examined the effects of adenosine on sodium-coupled phosphate and glucose transport in cultured opossum kidney (OK) cells, a continuous cell line that resembles proximal tubule. Adenosine analogues R-(-)-N6-phenylisopropyladenosine (PIA) and 2',5'-dideoxy-adenosine (DDA) were employed as adenosine A1 receptor and P site-selective agonists, respectively. Sodium-dependent phosphate uptake activity (Na-Pi symport) increased by approximately 25% above both basal and parathyroid hormone (PTH)-inhibited levels in cells treated with PIA (0.1, 1 microM) but not in cells treated with DDA (100 microM). Adenosine (PIA) also stimulated sodium-coupled 3-O-methylglucose transport by approximately 40%. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) content was inversely related to Na-Pi symport activity in cells treated with PIA and PTH. However, changes in Na-Pi symport activity did not consistently relate to changes in intracellular cAMP. Protein kinase C was activated 15 s after treatment of OK cells with 1 microM PIA. Preincubation of cells with 3 microM staurosporine attenuated the effect of 1 microM PIA on phosphate uptake. These data suggest that Na-Pi and Na-glucose symport activities are stimulated by adenosine acting at a receptor coupled to more than one intracellular signal. It is likely that both protein kinases A and C are involved in these actions of adenosine.
Adenosine↗
Downregulation of protein kinase C and insulin-stimulated 2-deoxyglucose uptake in rat adipocytes by phorbol esters, glucose, and insulin.
Phorbol esters translocatively activate and subsequently downregulate protein kinase C and insulin-stimulated glucose uptake in rat adipocytes. This study examined the possibility that other translocative activators of protein kinase C in rat adipocytes, e.g., insulin and glucose, provoke similar downregulating effects. Pretreatment of rat adipocytes for 20-24 h with phorbol esters, 3 nM insulin, 20 mM glucose, or 3 nM insulin plus 20 mM glucose resulted in concomitant decreases in protein kinase C and insulin-stimulated (or phorbol ester-stimulated) [3H]-2-deoxyglucose uptake. Downregulating effects of glucose on protein kinase C and insulin-stimulated [3H]-2-deoxyglucose uptake were also evident within 30 min in adipocytes freshly incubated in medium containing 5-20 mM, rather than 0, glucose. These findings confirm that protein kinase C is required during insulin-stimulated glucose uptake and raise the possibility that downregulation of protein kinase C by continued translocative activation of the enzyme may contribute (along with other factors) to impaired responsiveness of the glucose transport system after prolonged insulin and/or glucose treatment.
Adipose Tissue↗
The role of diacylglycerol/protein kinase C signaling in insulin-stimulated glucose transport.
There seems to be little doubt that insulin rapidly perturbs phospholipid metabolism, and this appears to increase DAG/PKC signaling in many target tissues. Considerable new evidence further suggests that DAG/PKC signaling plays an important role in insulin-stimulated glucose transport. Further studies are needed to test this hypothesis and examine its importance in states of clinical insulin resistance.
Animals↗
Protein kinase C activation patterns are determined by methodological variations. Studies of insulin action in BC3H-1 myocytes and rat adipose tissue.
In BC3H-1 myocytes, insulin has been reported to (a) increase diacyglycerol (DAG) production and provoke increases in protein kinase C enzyme activity of crude or DEAE-Sephacel-purified cytosol and membrane fractions in BC3H-1 myocytes (Cooper et al. (1987) J. Biol. Chem. 262, 3633-3739), but (b) decrease cytosolic, and transiently increase membrane, immunoreactive protein kinase C (Acevedo-Duncan et al. (1989) FEBS Lett. 244, 174-176). Presently, we used a Mono-Q column to purify protein kinase C and found that, similar to immunoblot findings, enzyme activity decreased in the cytosol, and increased in the membrane during insulin treatment. Similar differences in protein kinase C activation patterns were observed in rat adipose tissue: insulin stimulated cytosolic protein kinase C enzyme activity as measured after DEAE-Sephacel chromatography, but decreased cytosolic enzyme activity when measured after Mono-Q chromatography or by immunoblotting. We presently evaluated the possibility that insulin-induced increases in endogenous DAG may influence protein kinase C during assay in vitro. Crude cytosol from BC3H-1 myocytes contained 25-35% of total and [3H]glycerol-labelled DAG and insulin increased this DAG. Considerable amounts of [3H]glycerol-labelled DAG were present in insulin-stimulated protein kinase C-containing column fractions following DEAE-Sephacel chromatography of cytosol fractions, whereas lesser amounts were recovered after Mono-Q column chromatography. This difference in recovery of DAG and activation of the enzyme by this endogenous DAG may explain why we were able to discern insulin-induced (presumably translocation 'provoked') decreases in cytosolic protein kinase C in the present Mono-Q column preparations of both BC3H-1 myocytes and rat adipose tissue.
Adipose Tissue↗
Insulin provokes apparent increases in rat adipocyte M-kinase.
Insulin has been shown to stimulate the translocation of protein kinase C (PKC) in rat adipocytes. Presently, we found that decreases in cytosolic 80 kDa PKC were associated with increases in Ca++/phospholipid-independent protein kinase activity, and increases in a 50 kDa cytosolic protein recognized by anti-PKC antiserum. These findings suggest that insulin provokes increases in M-kinase in rat adipocytes.
Adipose Tissue↗
Insulin increases the synthesis of phospholipid and diacylglycerol and protein kinase C activity in rat hepatocytes.
The effects of insulin on phospholipid metabolism and generation of diacylglycerol (DAG) and on activation of protein kinase C in rat hepatocytes were compared to those of vasopressin and angiotension II. Insulin provoked increases in [3H]glycerol labeling of phosphatidic acid (PA), diacylglycerol (DAG), and other glycerolipids within 30 s of stimulation. Similar increases were also noted for vasopressin and angiotensin II. Corresponding rapid increases in DAG mass also occurred with all three hormones. As increases in [3H]DAG (and DAG mass) occurred within 30-60 s of the simultaneous addition of [3H]glycerol and hormone, it appeared that DAG was increased, at least partly, through the de novo synthesis of PA. That de novo synthesis of PA was increased is supported by the fact that [3H]glycerol labeling of total glycerolipids was increased by all three agents. Increases in [3H]glycerol labeling of lipids by insulin were not due to increased labeling of glycerol 3-phosphate, and were therefore probably due to activation of glycerol-3-phosphate acyltransferase. Unlike vasopressin, insulin did not increase the hydrolysis of inositol phospholipids. Insulin- and vasopressin-induced increases in DAG were accompanied by increases in cytosolic and membrane-associated protein kinase C activity. These findings suggest that insulin-induced increases in DAG may lead to increases in protein kinase C activity, and may explain some of the insulin-like effects of phorbol esters and vasopressin on hepatocyte metabolism.
Angiotensin II↗
Protein kinase C inhibitors block insulin and PMA-stimulated hexose transport in isolated rat adipocytes and BC3H-1 myocytes.
Effects of protein kinase C (PKC) inhibitors and "down-regulation" on insulin and PMA-stimulated 2-deoxyglucose transport were determined in isolated rat adipocytes or BC3H-1 myocytes. In both model systems, H-7, sangivamycin, and staurosporine, inhibitors of the catalytic domain of PKC, each effectively blocked insulin and PMA-stimulated hexose uptake at similar concentrations. In the myocytes, staurosporine completely blocked the insulin effect retained post-chronic phorbol myristate acetate (PMA)-induced "down-regulation." These findings indicate (1) that chronic pretreatment with PMA may not lead to a complete loss of PKC activity in the myocyte, and (2) that PKC is involved in insulin-stimulated hexose transport in both isolated rat adipocytes and BC3H-1 myocytes.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Sulfonylurea-stimulated glucose transport association with diacylglycerollike activation of protein kinase C in BC3H1 myocytes.
The extrapancreatic effects of sulfonylurea drugs include increased glucose uptake by certain peripheral tissues. To study this effect, we used BC3H1 myocytes, which are reported to respond to these drugs. Within 30 min, tolbutamide and glyburide increased [3H]-2-deoxyglucose uptake in a dose-dependent manner. The inactive analogue carboxytolbutamide had no effect on glucose transport. Because increases in glucose transport may be mediated by activation of the diacylglycerol-protein kinase C signaling system, we examined the effects of these drugs on lipid metabolism and protein kinase C activity. Unlike insulin, tolbutamide and glyburide failed to increase [3H]glycerol labeling of diacylglycerol or labeling of phospholipids by 32P. After 30 min of treatment with tolbutamide or glyburide, however, membrane-associated and cytosolic protein kinase C activity were each increased. When cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 48 h to deplete certain isoforms of protein kinase C, glyburide, tolbutamide, and acute TPA treatment failed to increase glucose uptake, suggesting that TPA and sulfonylureas operate through activation of a common pathway. The effect of glyburide was additive to TPA in stimulating glucose uptake at low but not high TPA concentrations. As with insulin and TPA, extracellular Ca2+ was not essential for sulfonylurea-stimulated glucose uptake. Staurosporine, a protein kinase C inhibitor, blocked glyburide-, tolbutamide-, and insulin-stimulated glucose uptake. In intact cells, glyburide stimulated the phosphorylation of both 80,000-Mr and 40,000-Mr proteins, which are markers for protein kinase C activation. Addition of sulfonylureas directly to the protein kinase C assay system in vitro provoked dioleinlike effects, in that sensitivity of the enzyme to Ca2+ was increased.(ABSTRACT TRUNCATED AT 250 WORDS)
Alkaloids↗
Effects of insulin on diacylglycerol-protein kinase C signaling in rat diaphragm and soleus muscles and relationship to glucose transport.
Insulin was found to provoke rapid increases in diacylglycerol (DAG) content and [3H]glycerol incorporation into DAG and other lipids during incubations of rat hemidiaphragms and soleus muscles. Insulin also rapidly increased phosphatidic acid and total glycerolipid labeling by [3H]glycerol, suggesting that insulin increases DAG production at least partly through stimulation of the de novo pathway. Increased DAG production may activate protein kinase C (PKC) as reported previously in the rat diaphragm. We also observed apparent insulin-induced translocation of PKC from cytosol to membrane in the rat soleus muscle. The importance of insulin-induced increases in DAG-PKC signaling in the stimulation of glucose transport in rat diaphragm and soleus muscles was suggested by 1) PKC activators phorbol esters and phospholipase C stimulation of [3H]-2-deoxyglucose (DOG) uptake and 2) PKC inhibitors staurosporine and polymixin B inhibition of insulin effects on [3H]-2-DOG uptake. Although phorbol ester was much less effective than insulin in the diaphragm, phospholipase C provoked increases in [3H]-2-DOG uptake that equaled or exceeded those of insulin. In the soleus muscle, phorbol ester, like phospholipase C, was only slightly but not significantly less effective than insulin. Similar variability in effectiveness of phorbol ester has also been noted previously in rat adipocytes (weak) and BC3H1 myocytes (strong), whereas DAG, added exogenously or generated by phospholipase C treatment, stimulates glucose transport to a degree that is quantitatively more comparable to that of insulin in each of the four tissues. Differences in effectiveness of phorbol ester and DAG could not be readily explained by postulating that the latter acts independently of PKC, because DAG provoked the apparent translocation of the enzyme from cytosol to membranes in rat adipocytes, and effects of DAG on [3H]-2-DOG uptake were blocked by inhibitors of PKC in both rat adipocytes and BC3H1 myocytes. Collectively, our findings provide further support for the hypothesis that insulin increases DAG production and PKC activity, and these processes are important in the stimulation of glucose transport in rat skeletal muscle and other tissues.
Adipose Tissue↗