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R V Farese

Publications and source records attributed to R V Farese.

At least 127 records · Page 7Linked to original sources
PubMed1991

Tissue-specific regulation of lipoprotein lipase activity by insulin/glucose in normal-weight humans.

Eight normal-weight subjects (four men, four women) were studied to determine the relative activities of lipoprotein lipase (LPL) in adipose tissue (ATLPL) and vastus lateralis skeletal muscle (SMLPL), both in the fasting state and in response to a 6-hour insulin/glucose infusion. Mean fasting levels of ATLPL and SMLPL were not statistically different. After 6 hours of insulin/glucose infusion, mean ATLPL activity was significantly greater than the fasting level (P less than .01), while mean SMLPL activity decreased from basal (P less than .05). These tissue-specific changes in LPL responsiveness (0 to 6 hours) were significantly different (P less than .01). No differences between men and women were observed. These divergent tissue-specific LPL responses to insulin/glucose would serve to direct lipoprotein triglyceride-derived fatty acids away from muscle and to adipose tissue for storage.

Adipose Tissue↗
PubMed1991

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↗
PubMed1991

Insulin administration in vivo increases 1,2-diacylglycerol in rat skeletal muscle.

Based on in vitro studies, an insulin-mediated increase in muscle 1,2-diacylglycerol (DAG) content has been proposed as a signal for the insulin induced stimulation of glucose transport. A recent study [Turinsky, J., Bayly, B.P. and O'Sullivan, D.M. (1990) J. Biol. Chem. 265, 7933-7938] challenged this hypothesis because no increase in muscle 1,2-diacylglycerol was observed after in vivo infusions of insulin at doses which markedly stimulated muscle glucose transport. We observed a 30-45% increase in DAG in rat gastrocnemius and diaphragm muscles, 5-15 min after intramuscular or intravenous injections of 1-3 U of insulin per rat, doses which would be expected to activate insulin receptors more fully. The effects on DAG were similar whether or not hypoglycemia was prevented by co-injection of glucose.

Animals↗
PubMed1991

Interrelated effects of insulin and glucose on diacylglycerol-protein kinase-C signalling in rat adipocytes and solei muscle in vitro and in vivo in diabetic rats.

The effects of insulin and glucose, alone and combined, on diacylglycerol (DAG), protein kinase-C (PKC), and glucose transport were compared in rat adipocytes and solei incubated in medium containing 0-20 mM glucose. In both tissues insulin rapidly stimulated [3H]DAG production from [3H]glycerol; extracellular glucose masked this effect in adipocytes, but not in solei. [3H]Glucose was avidly converted to DAG in adipocytes, and this conversion was enhanced by insulin. In contrast, [3H]glucose was poorly converted to DAG in solei. Glucose alone (5-20 mM) stimulated PKC translocation in adipocytes, but not in solei. Insulin stimulated PKC translocation in both tissues at all glucose concentrations. However, glucose modulated this effect of insulin in adipocytes by 1) decreasing cytosolic PKC and the absolute amount of PKC translocated, and 2) promoting apparent turnover of membrane PKC. In contrast, in solei, glucose did not affect PKC levels or translocation responses to insulin. In keeping with DAG-PKC signalling, the relative glucose transport effects of insulin were influenced by extracellular glucose in adipocytes, but not in solei. These results suggest that 1) glucose-induced PKC translocation requires metabolism of glucose to DAG; 2) glucose activates DAG-PKC signalling in adipocytes, but not in solei; 3) insulin activates DAG-PKC signalling in both tissues at all glucose levels; and 4) glucose may modulate the effects of insulin on DAG-PKC signalling in adipocytes, but not in solei. Consistent with in vitro results, in solei taken directly from diabetic rats, membrane PKC was decreased, and cytosolic PKC was increased, presumably reflecting diminished PKC translocation due to hypoinsulinemia. In contrast, in adipose tissue, cytosolic PKC was decreased, presumably reflecting hyperglycemia-induced PKC translocation. Accordingly, DAG levels were increased in adipose tissue, but not in solei, in diabetic rats, and insulin increased DAG in both tissues.

Adipose Tissue↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1990

Insulin activates glycerol-3-phosphate acyltransferase (de novo phosphatidic acid synthesis) through a phospholipid-derived mediator. Apparent involvement of Gi alpha and activation of a phospholipase C.

We studied the mechanism whereby insulin activates de novo phosphatidic acid synthesis in BC3H-1 myocytes. Insulin rapidly activated glycerol-3-phosphate acyltransferase (G3PAT) in intact and cell-free preparations of myocytes in a dose-related manner. The apparent Km of the enzyme was decreased by treatment with insulin, whereas the Vmax was unaffected. No activation was found by ACTH, insulin-like growth factor-I, angiotensin II, or phenylephrine, but epidermal growth factor, which, like insulin, is known to activate de novo phosphatidic acid synthesis in intact myocytes, also stimulated G3PAT activity. In homogenates or membrane fractions, the effect of insulin on G3PAT was fully mimicked by nonspecific or phosphatidylinositol (PI)-specific phospholipase C (PLC). An antiserum raised against PI-glycan-PLC completely blocked the effect of insulin on G3PAT. Although the above findings suggested involvement of a PLC in insulin-induced activation of G3PAT, neither diacylglycerol nor protein kinase C activation appeared to be involved. On the other hand, insulin stimulated the release of a cytosolic factor, which activated membrane-associated G3PAT. This cytosolic factor had a molecular weight of less than 5K as determined by Sephadex G-25 chromatography. NaF, a phosphatase inhibitor, blocked the activation of G3PAT by insulin, suggesting involvement of a phosphatase. Insulin-induced activation of G3PAT was also blocked by pretreatment of intact myocytes with pertussis toxin and by prior addition, to homogenates, of an antiserum that recognizes the C-terminal decapeptide of Gi alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

External calcium is required for activation of phospholipase C by angiotensin II in adrenal glomerulosa cells.

Previous studies have shown that external calcium (Ca2+) is required for the effects of angiotensin II (AII) on aldosterone secretion in adrenal glomerulosa zone. Using bovine adrenal glomerulosa cells prepared by collagenase dispersion, we examined whether external Ca2+ is required for the activation of phospholipase C by AII. Adrenal glomerulosa cells were exposed to Ca-EGTA buffered media to provide accurate estimates of external free Ca2+ concentrations. Phospholipase C activation was evaluated by measurement of inositol phosphates production. At 0.1 microM Ca2+ and less, sustained AII effects on inositol monophosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) were markedly inhibited. Increasing the Ca2+ concentration to 50 microM or greater fully restored AII-induced inositol phosphates production. AII-induced increases in cytosolic Ca2+ measured by Quin-2 fluorescence, were diminished at lower external Ca2+ concentrations. Treating adrenal glomerulosa cells with Chelex-100, a strong Ca2+ binding resin, blocked early activation of phospholipase C by AII. Inhibition of IP3 production was also observed when inhibitors of Ca2+ movement across the plasma membrane were used, viz., La2+, TMB-8 and nifedipine. The requirement for Ca2+ during AII-induced activation of phospholipase C may be explained, at least partly by a requirement for Ca2+ at a site between the AII receptor and Phospholipase C.

Aldosterone↗
PubMed1990

Prolactin and known modulators of rat splenocytes activate nuclear protein kinase C.

Prolactin (PRL) and other trophic factors rapidly activate a nuclear pool(s) of protein kinase C (nPKC) in purified splenocyte nuclei. The PRL also enhanced [2-3H]glycerol incorporation into nuclear mono- and triacylglycerol. An assay was devised which not only probed the ability of the hormone to activate protein kinase C (PKC) but also demonstrated the presence of nuclear substrates. Using this methodology, a biphasic concentration-response curve to PRL was observed. Heterologous species of PRL and various growth factors also activated nPKC. The PRL-induced nPKC stimulation was antagonized by various immunomodulators, G protein-coupling inhibitors, PKC inhibitors, a calmodulin inhibitor, and a peripheral benzodiazepine agonist and antagonist. A monoclonal antibody to PKC, anti-rat PRL antiserum and a monoclonal anti-rat PRL receptor antibody antagonized PRL-induced PKC-dependent nuclear phosphorylation, further implicating nPKC and a PRL receptor-mediated activation process. Nuclear PKC may be a major target for trophic regulation in response to both positive and negative growth signals.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

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↗
PubMed1990

ACTH increases de novo synthesis of diacylglycerol and translocates protein kinase C in primary cultures of calf adrenal glomerulosa cells.

Effects of ACTH on the production of diacylglycerol (DAG) and translocation of protein kinase C were studied in primary cultures of calf adrenal glomerulosa cells. To study DAG production two different labeling protocols were used: (a) cells were prelabeled for 3 days with [2-3H]glycerol before ACTH addition; (b) ACTH and [2-3H]glycerol were added simultaneously to cells. In both cases, ACTH provoked rapid increases in the labeling of DAG which were maximal in 2 min, dose-dependent, and paralleled by increases in DAG mass. ACTH also increased the labeling of total glycerolipids including phosphatidic acid (PA), phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine and triacylglycerol. In both labeling protocols, the rates of increase in the labeling of DAG and PA were greater than those of other glycerolipids. Our results indicate that ACTH rapidly increases DAG, at least partly by stimulating the de novo synthesis of PA. In addition, we found that ACTH, like phorbol esters, stimulated the apparent translocation of immunoreactive protein kinase C from the cytosol to the membrane fraction.

Adrenocorticotropic Hormone↗
PubMed1990

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↗
PubMed1990

Treatment of primary cultures of calf adrenal glomerulosa cells with adrenocorticotropin (ACTH) and phorbol esters: a comparative study of the effects on aldosterone production and ACTH signaling system.

We studied the mechanism that underlies the desensitization of calf adrenal glomerulosa cells induced by 4 h of ACTH treatment. In control cells, acute ACTH treatment provoked sizeable increases in aldosterone, cAMP, and diacylglycerol, and translocated protein kinase-C from cytosol to membrane. In desensitized cells, acute ACTH effects on aldosterone and cAMP decreased by 25-60%, and diacylglycerol levels and protein kinase-C translocation were persistently stimulated and not substantially affected by further acute ACTH treatment. After 4 h of treatment with 1 microM phorbol 12-myristate 13-acetate (PMA) there were no acute effects of ACTH on the production of aldosterone, cAMP, or diacylglycerol or on protein kinase-C, which was already strongly translocated. These results suggest that ACTH-mediated desensitization of calf adrenal glomerulosa cells may be at least partially mimicked by long term treatment with phorbol esters and could be due to ACTH-induced increases in diacylglycerol-protein kinase-C signaling.

Adrenal Glands↗
PubMed1990

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↗
PubMed1990

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↗