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R V Farese

Publications and source records attributed to R V Farese.

At least 145 records · Page 8Linked to original sources
PubMed1990

Pertussis toxin-sensitive and -insensitive mechanisms for diacylglycerol-protein kinase C signalling during insulin action in BC3H-1 myocytes.

The findings reported herein indicate that insulin rapidly perturbs phospholipid metabolism and consequent intracellular signalling, in its target tissues by two fully separable mechanisms. One of these mechanisms involves a pertussis toxin-sensitive Gi alpha, which probably serves to couple the insulin receptor to a PI-glycan phospholipase C, which, in turn, leads to the release of HGM and consequent activation of de novo PA synthesis. The second mechanism is PC hydrolysis, which is pertussis toxin-insensitive. Both mechanisms serve as important sources of DAG during insulin action, and PKC appears to be activated by DAG derived from both pathways. Although DAG may be derived from each of these signalling pathways, it is clear that PI-glycan HGM will only be derived from pertussis toxin-sensitive PI-glycan hydrolysis. These findings may help to explain why some, but not all, insulin effects are inhibited by pertussis toxin and are therefore apparently dependent upon Gi alpha. Whether or not other G-proteins are important in other phospholipid signalling pathways during insulin action, e.g., PC hydrolysis, remains to be determined.

Animals↗
PubMed1989

Insulin stimulates the translocation of protein kinase C in rat adipocytes.

Insulin-induced changes in protein kinase C were examined in cytosol and membrane fractions of rat adipocytes enzymatically after Mono Q column chromatography and by immunoblotting. During a 5-20 min period of insulin treatment, cytosolic protein kinase C decrease by approximately 50%, whereas membrane protein kinase C increased nearly 2-fold. These findings suggest that insulin stimulates the translocation of protein kinase C in rat adipocytes.

Adipose Tissue↗
PubMed1989

Insulin-like effects of epidermal growth factor and insulin-like growth factor-I on [3H]2-deoxyglucose uptake, diacylglycerol generation and protein kinase C activation in BC3H-1 myocytes.

Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) were found to provoke increases in [3H]2-deoxyglucose uptake, diacylglycerol (DAG) generation and membrane-bound protein kinase C activity in BC3H-1 myocytes. These effects were similar to those provoked by insulin. The increases in DAG did not appear to be derived from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol, but may have been derived from synthesis of phosphatidic acid de novo, and hydrolysis of phosphatidylcholine, as revealed by studies with [3H]glycerol and [3H]choline respectively. Accordingly, both EGF and IGF-I increased acute [3H]glycerol labelling of DAG (and other lipids) and [3H]choline labelling of phosphocholine. These labelling responses were similar in time course, suggesting that they are closely coupled. Our findings suggest that EGF and IGF-I, like insulin, increase DAG-protein kinase C signalling, apparently by activating co-ordinated lipid-synthesis and -hydrolysis responses, which are distinctly different from the PIP2-hydrolysis response.

Animals↗
PubMed1989

Glucose-induced synthesis of diacylglycerol de novo is associated with translocation (activation) of protein kinase C in rat adipocytes.

Addition of glucose (5-20 mM) to rat adipocytes provoked dose-related increases in diacylglycerol, without increasing production of [3H]inositol phosphates. Cytosolic protein kinase C enzyme activity and immunoreactivity decreased within 1-5 min of 5 mM glucose addition, and further over 20 min. Membrane protein kinase C increased stoichiometrically during the first 5 min and then decreased. Higher concentrations (10 and 20 mM) of glucose provoked greater and more rapid decreases of cytosolic and membrane protein kinase C. Our findings suggest that glucose stimulates diacylglycerol production by providing substrate for phosphatidic acid synthesis de novo, and this is associated with translocative activation of protein kinase C.

Adipose Tissue↗
PubMed1989

Retention of specific protein kinase C isozymes following chronic phorbol ester treatment in BC3H-1 myocytes.

Since insulin effects on glucose transport persist in phorbol ester "desensitized" or "down-regulated" BC3H-1 myocytes, we reexamined the evidence for protein kinase C (PKC) depletion. After 24 hrs of 5 microM 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment, PKC-directed histone phosphorylation and acute TPA effects on glucose transport were lost, but PKC-dependent vinculin phosphorylation was still evident. Hydroxylapatite (HAP) chromatography revealed loss of a type III, but not a type II, PKC-dependent vinculin phosphorylation. Immunoblots of cytosolic preparations of PKC-"depleted" myocytes confirmed the retention of PKC. Our findings indicate that TPA "down-regulated" BC3H-1 myocytes contain immunoreactive and functionally active PKC. The latter may explain the continued effectiveness of both insulin and diacylglycerol (DiC8) for stimulating glucose transport in "down-regulated" cells.

Animals↗
PubMed1989

Immunological evidence that insulin activates protein kinase C in BC3H-1 myocytes.

Effects of insulin on immunoreactive protein kinase C were examined in BC3H-1 myocytes. Insulin provoked rapid dose-dependent decreases in cytosolic enzyme, and transient increases and subsequent decreases in membrane-associated enzyme. Phorbol esters provoked similar changes. Our findings suggest that insulin provokes the translocative activation of protein kinase C.

Blotting, Western↗
PubMed1989

Angiotensin II increases diacylglycerol in calf adrenal glomerulosa cells by activating de novo phospholipid synthesis.

Effects of angiotensin II (AII) on diacylglycerol (DAG) synthesis were examined in calf adrenal glomerulosa cells. AII provoked rapid increases in [3H]glycerol-labeling and content of DAG. Effects on [3H]glycerol-labeling of DAG were observed both in cells prelabeled with [3H]glycerol for 60 minutes, and when AII and [3H]glycerol were added simultaneously. Increases in [3H] DAG labeling were associated with increases in total glycerolipid labeling, and in simultaneous addition experiments, were preceded by increased [3H] phosphatidic acid (PA) labeling. Labeling of glycerol-3-PO4, on the other hand, was not increased by AII, suggesting that increases in lipid labeling were not due to prior increases in precursor specific activity. ACTH, which does not increase the hydrolysis of inositol-phospholipids appreciably in this tissue, provoked increases in content and [3H]glycerol-labeling of DAG, which were only slightly less than those provoked by AII. Thus, part of the AII-induced increase in DAG may also be derived from sources other than inositol-phospholipids. Moreover, AII-induced increases in DAG appear to be at least partly derived from increased de novo synthesis of PA.

Adrenocorticotropic Hormone↗
PubMed1988

ACTH stimulates turnover of the phosphatidylinositol-glycan.

Primary cultures of calf adrenal glomerulosa cells were prelabeled for 3 days with [3H]inositol or [3H]glucosamine and stimulated with 10 nM ACTH. Labeled phosphatidylinositol (PI), polyphosphoinositides (PIP and PIP2) and a novel phosphatidylinositol-glycan (PI-glycan) were measured after separation by TLC. [3H]-Inositol labeling of PI, PIP and PIP2 increased rapidly, whereas labeling of the PI-glycan showed an initial decrease at 1 minute followed by a subsequent increase. Similar results were obtained when cells were prelabeled with [3H]glucosamine, viz. the PI-glycan label decreased at 1 min and subsequently increased. These results suggest that ACTH provokes (a) coordinated increases in the synthesis of PI, PIP, PIP2 and the PI-glycan, and (b) the increase in PI-glycan synthesis is preceded by initial decrease, presumably reflecting hydrolysis of this lipid.

Adrenocorticotropic Hormone↗
PubMed1988

Phospholipid signaling systems in insulin action.

Insulin is known to control a number of anabolic metabolic processes in a variety of target tissues through activation of cell surface receptors. It is clear that insulin receptor activation provokes increases in tyrosine kinase activity and autophosphorylation of the insulin receptor, but subsequent events have not been elucidated. Recently, it has become clear that insulin provokes the following rapid changes in phospholipid metabolism, which result in the generation of several intercellular signaling substances (or mediators): (1) hydrolysis of a phosphatidylinositol-glycan; (2) stimulation of de novo synthesis of phosphatidic acid; and (3) hydrolysis of phosphatidylcholine by a phospholipase C and/or D. Hydrolysis of the phosphatidylinositol-glycan leads to the release of polar headgroups, which serve as mediators to activate phosphatases, and may thereby account for a number of insulin effects on carbohydrate metabolism, lipid metabolism, and regulation of cyclic nucleotide metabolism. All three phospholipid effects of insulin also generate diacylglycerol, which activates protein kinase C, and this may contribute to insulin effects on glucose transport, ion and amino acid transport, protein synthesis, and gene expression (messenger RNA synthesis). Combined, the headgroup mediators and diacylglycerol-protein kinase C signaling systems may account for many, or perhaps most, of insulin's actions. Moreover, the three phospholipid effects of insulin appear to be coordinated, and may function as an integrated cycle to ensure the continued synthesis of lipids, which are the sources of the signaling substances during insulin action.

Animals↗
PubMed1988

Epidermal growth factor and insulin-like growth factor I stimulate the hydrolysis of the insulin-sensitive phosphatidylinositol-glycan in BC3H-1 myocytes.

BC3H-1 myocytes contain a phospholipid(s) which is labeled with [3H]inositol, [3H]glucosamine, and [3H]myristate [a phosphatidylinositol-glycan (PI-glycan)], and which is hydrolyzed by insulin induced activation of a specific phospholipase C. Similarly, epidermal growth factor and insulin-like growth factor-I provoke rapid increases in the hydrolysis of this PI-glycan, suggesting that derived signaling substances may be important in the action of agonists which activate tyrosine kinase type receptors.

Animals↗
PubMed1988

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes.

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)

Arachidonic Acid↗
PubMed1988

Insulin provokes co-ordinated increases in the synthesis of phosphatidylinositol, phosphatidylinositol phosphates and the phosphatidylinositol-glycan in BC3H-1 myocytes.

BC3H-1 myocytes were cultured in the presence of [3H]inositol or [3H]glucosamine during their entire growth cycle to ensure that all lipids containing inositol and glucosamine were labelled to isotopic equilibrium or maximal specific radioactivity. After such labelling, a lipid (or group of lipids), which was labelled with both inositol and glucosamine, was observed to migrate between phosphatidylinositol 4-phosphate and phosphatidylinositol (PI) in two different t.l.c. systems. Insulin provoked rapid, sizeable, increases in the inositol-labelling of this lipid (presumably a PI-glycan), and these increases were similar to those observed in PI and PI phosphates. Our results indicate that insulin provokes co-ordinated increases in the net synthesis de novo of PI and its derivatives, PI phosphates and the PI-glycan, in BC3H-1 myocytes. This increase in synthesis of PI may serve as the mechanism for replenishing the PI-glycan during stimulation of its hydrolysis by insulin. Moreover, increases in the content of the PI-glycan may contribute to increases in the generation of head-group 'mediators' during insulin action.

Cell Line↗
PubMed1988

Effects of insulin and phorbol esters on diacylglycerol generation and synthesis and hydrolysis of phosphatidylcholine in BC3H-1 myocytes.

Insulin was found to provoke simultaneous, rapid, biphasic increases in [3H]choline-labeling of phosphatidylcholine and phosphocholine in BC3H-1 myocytes. Phorbol esters increased [3H]choline-labeling of phosphocholine, but not phosphatidylcholine. Both agonists increased diacylglycerol production. These results suggest that: (a) insulin provokes coordinated increases in the synthesis and hydrolysis of PC; and, (b) insulin-induced activation of protein kinase C may activate a PC-specific phospholipase.

Animals↗
PubMed1988

Insulin-induced glycerolipid mediators and the stimulation of glucose transport in BC3H-1 myocytes.

We have previously demonstrated that insulin stimulates glycerolipid synthesis and phospholipid hydrolysis in BC3H-1 myocytes, resulting in the generation of membrane diacylglycerol, a known cellular mediator. This led us to the original proposal that diacylglycerol may contribute to the mediation of insulin action, especially stimulation of glucose transport. The fact that agents such as phenylephrine and phorbol esters, which increase or act as membrane diacylglycerols, are fully active in stimulating glucose transport in this tissue lent further support to this proposal. In this paper, we demonstrate that the diacylglycerol analogues PMA (4 beta-phorbol 12-myristate 13-acetate) and mezerein (both possessing 12 beta- and 13 alpha-O-linked substituents as well as a 4 beta-hydroxyl group) each increase the Vmax of the glucose transporter as does insulin. Diacylglycerol generated by the addition of phospholipase C also stimulates glucose uptake to a maximum which is equal and nonadditive to that of insulin, while addition of the narrowly active phosphatidylinositol-specific phospholipase C which generates the putative phosphoinositol-glycan mediator of Saltiel et al. (Saltiel, A., Fox, J., She Lin, P., and Cutrecasas, P. (1986) Science 233, 967-972) stimulates pyruvate dehydrogenase in these cells without any effect on glucose uptake. Pretreatment of the myocytes with PMA resulted in desensitization of subsequent glucose uptake to stimulation by phenylephrine, but had no effect on stimulation of glucose uptake by phospholipase C or by insulin, indicating that PMA pretreatment primarily desensitizes agonist-induced polyphosphoinositide hydrolysis which, as we have previously shown, is not involved in the insulin-induced generation of diacylglycerol. This was confirmed by the absence of intracellular Ca2+ mobilization during insulin administration, as measured by the sensitive fluorescent probe fura-2 in attached monolayer BC3H-1 myocytes. Furthermore, we have shown that insulin-generated diacylglycerol satisfies several criteria for a mediator of insulin action, including the demonstration that insulin-stimulated endogenous diacylglycerol generation is antecedent to glucose transport and has an identical insulin dose-response curve and moreover that the magnitude and time course of subsequent stimulation of glucose transport is reproduced by the addition of the simple exogenous diacylglyerol, dioctanoylglycerol, in the complete absence of the hormone. These results establish a central role for insulin-induced glycerolipid metabolism in mediating insulin-stimulated glucose transport in BC3H-1 myocytes.

Animals↗