Regulation of glucose transport by pioglitazone in cultured muscle cells.

Recent evidence suggests that pioglitazone, a thiazolidinedione hypoglycemic agent, acts by increasing insulin responsiveness at the peripheral level. We studied the effect of pioglitazone (1 to 50 micrograms/mL) on the glucose transporter and glucose transport in BC3H-1 cells, a continuously cultured skeletal muscle cell line lacking the myoD transcription factor required for cell fusion. Glucose-fed cells (25 mmol/L) responded to insulin with a more than twofold increase in 2-deoxyglucose (2-DOG) uptake as compared with baseline. Treating these cells with pioglitazone alone for 24 hours resulted in a dose-dependent increase in hexose uptake, reaching twofold at 50 micrograms/mL. Combining long-term pioglitazone (10 micrograms/mL for 24 hours) and short-term insulin treatment resulted in an additive effect on 2-DOG uptake over a wide range of insulin concentrations (0.1 to 100 nmol/L) without the desensitization to 2-DOG uptake seen in other systems following long-term insulin administration. To determine the basis of the increased glucose uptake response, the level of specific mRNA and immunoreactive glucose transporter protein was determined. Northern and Western blot studies on glucose-treated cells (25 mmol/L) showed that glucose transporter mRNA and protein increased in parallel following treatment with either pioglitazone or insulin alone. The combination of insulin with pioglitazone resulted in an additive stimulation of glucose transporter mRNA and protein. In summary, pioglitazone stimulates hexose uptake both independently and in combination with insulin in BC3H-1 myocytes. These effects are largely accounted for by increases in glucose transporter mRNA and protein, indicating its potential efficacy in the treatment of non-insulin-dependent diabetes mellitus (NIDDM).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the GLUT1 glucose transporter in cultured myocytes: total number and subcellular distribution as determined by photoaffinity labelling.

We have used the impermeant photoaffinity label 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-[2-3H] 1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) to identify and quantify the glucose transporters on the surface of BC3H-1 cells, a continuously cultured skeletal-muscle cell line lacking the MyoD transcription factor required for cell fusion. ATB-[2-3H]BMPA was used in combination with immunoprecipitation of the GLUT1 glucose transporter, the only isoform expressed in these cells. The total cellular GLUT1 content was also determined by photolabelling and immunoprecipitation after cell permeabilization with digitonin (0.025%). In glucose-starved cells, 85% of the glucose transporters were present at the cell surface in the basal state, with little change in response to insulin (200 nM), correlating with lack of additional 2-deoxyglucose uptake in response to insulin. Feeding the cells with glucose (25 mM) for 24 h resulted in an 80% decrease in the total GLUT1 content relative to starved cells, of which only 25% were present on the cell surface. This was associated with an 85% decrease in 2-deoxyglucose uptake. In addition, acute stimulation of the fed cells with insulin or phorbol 12-myristate 13-acetate (PMA) led to an increase in GLUT1 at the cell surface, and, in correspondence, an increase in 2-deoxyglucose uptake by approx. 2- and 4-fold respectively. We conclude that exofacial photoaffinity labelling of glucose transporters with ATB-[2-3H]BMPA in the presence and absence of digitonin, followed by specific immunoprecipitation, provides an accurate measure of total and cell-surface glucose transporters in differentiated BC3H-1 muscle cells. This technique demonstrates that glucose pre-feeding (1) decreases the total number of GLUT1 and (2) redistributes the majority of the remaining transporters to an intracellular site, where they can now be translocated to the cell surface in response to insulin and PMA.

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.

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)

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.

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.

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)

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.

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.

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.

Insulin-glycerolipid mediators and gene expression.

Insulin is an anabolic polypeptide hormone with pleiotrophic effects. During the decades since the initial description by Banting and Best, the acute effects of insulin have been widely studied with particular focus on the mechanism or mechanisms of insulin activation of hexose transport and regulation of metabolic enzyme activity. However, recently there has been a major expansion of investigation to include insulin regulation of gene expression with multiple insulin-sensitive specific mRNAs now reported. In this review, we explore the involvement of insulin-induced changes in plasma membrane glycerolipid metabolism in the transmembrane signaling process required for insulin regulation of mRNA levels. Insulin increases diacylglycerol levels in insulin-responsive cells, and synthetic diacylglycerols or their phorbol ester diacylglycerol analogs, such as 4 beta,9 alpha,12 beta,13 alpha, 20-pentahydroxytiglia-1,6-dien-3-one 12 beta-myristate 13-acetate (TPA), mimic insulin regulation of ornithine decarboxylase mRNA, c-fos mRNA, and phosphoenolpyruvate carboxykinase mRNA levels. This suggests that insulin regulation of specific mRNA levels may be mediated by insulin-induced changes in phospholipid metabolism and that diacylglycerol may play a pivotal role in insulin regulation of gene expression.

Direct effects of sulfonylurea agents on glucose transport in the BC3H-1 myocyte.

The actions of sulfonylurea agents to increase peripheral glucose disposal have been classically ascribed to an ability to potentiate insulin action. However, in the BC3H-1 cultured muscle cell, tolbutamide, glipizide, and glyburide directly provoked more than a twofold increase in 2-deoxyglucose (2-DG) uptake in a dose-dependent manner in the absence of insulin. Tolbutamide (3 mM) enhanced 2-DG uptake by 130% in the presence or absence of insulin and did not significantly change insulin binding or the sensitivity of the insulin response. The onset of tolbutamide-stimulated hexose transport was seen after 30 min and reached a plateau after 12 h. Tolbutamide-stimulated glucose transport was associated with a twofold increase in the Vmax of 2-DG uptake and was completely blocked by 50 microM cytochalasin B, indicating that this action is mediated by increase in cell membrane glucose transporters. We show that sulfonylureas at therapeutic concentrations directly increase glucose transport into muscle cells. Because muscle is the major peripheral target tissue for glucose disposal, these results provide the basis for the therapeutic effect of these agents in improving peripheral glucose disposal in insulin-resistant type II (non-insulin-dependent) diabetes mellitus.

Phorbol ester inhibition of insulin-stimulated deoxyribonucleic acid synthesis in BC3H-1 myocytes.

Insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA) each acutely stimulates hexose transport, amino acid uptake, and pyruvate dehydrogenase activity in the BC3H-1 myocyte in a nonadditive fashion, suggesting that the acute effects of insulin and TPA are mediated through a common mechanism of action. Here we have demonstrated that while chronic incubation with insulin stimulated DNA synthesis by 3- to 6-fold, TPA, in contrast, did not stimulate DNA synthesis and, indeed, caused a 70% inhibition of insulin-stimulated DNA synthesis in a dose-dependent fashion. In differentiated myocytes, insulin maximally stimulated hydroxyurea-sensitive [3H]thymidine incorporation into DNA at 200-400 nM with an ED50 of 5-8 nM, suggesting that insulin stimulates DNA synthesis via the insulin receptor rather than through growth factor receptors. Phorbol ester inhibition of insulin-stimulated DNA synthesis was specific for the active tumor-promoting phorbols and the synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol. Maximal TPA inhibition of insulin-stimulated DNA synthesis was observed at 100 nM with an ID50 of 30 nM TPA, values analogous to those required for TPA stimulation of hexose transport in the myocyte. Chronic incubation with TPA did not inhibit insulin-stimulated protein synthesis, acute K+ flux, K+ accumulation, cytosolic thymidine levels, or insulin binding, indicating that TPA inhibits a specific intracellular event mediating DNA synthesis and suggesting that the acute and chronic effects of insulin in BC3H-1 myocytes are regulated by distinct pathways.

Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis.

The mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes were examined. When [3H]arachidonate labeling of phospholipids was used as an indicator of phospholipase C activation, transient increases in [3H]diacylglycerol were observed between 0.5 and 10 minutes after the onset of insulin treatment. With [3H]glycerol labeling as an indicator of de novo phospholipid synthesis, [3H]diacylglycerol was increased maximally at 1 minute and remained elevated for 20 minutes. [3H]Glycerol-labeled diacylglycerol was largely derived directly from phosphatidic acid. Insulin increased de novo phosphatidic acid synthesis within 5 to 10 seconds; within 1 minute, this synthesis was 60 times greater than that of controls. Thus, the initial increase in diacylglycerol is due to both increased hydrolysis of phospholipids and a burst of de novo phosphatidic acid synthesis. After 5 to 10 minutes, de novo phosphatidic acid synthesis continues as a major source of diacylglycerol. Both phospholipid effects of insulin seem important for generating diacylglycerol and other phospholipid-derived intracellular signaling substances.

Insulin but not phorbol ester treatment increases phosphorylation of vinculin by protein kinase C in BC3H-1 myocytes.

Insulin was found to increase protein kinase C activity in BC3H-1 myocytes as determined by in vitro phosphorylation of both a lysine-rich histone fraction (histone III-S) and vinculin. TPA treatment for 20 min or 18 h provoked an apparent loss of histone-directed but not vinculin-directed phosphorylation by cytosolic C-kinase. Thus, chronic TPA-induced 'desensitization' or 'depletion' of cellular protein kinase C is more apparent than real, and is not a valid means for evaluating the role of C-kinase in hormone action.

Insulin increases membrane and cytosolic protein kinase C activity in BC3H-1 myocytes.

Insulin treatment stimulated the activity of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in both cytosolic and membrane fractions of BC3H-1 myocytes. Within 60 s of insulin treatment, membrane protein kinase C activity increased 2-fold, diminished toward control levels transiently, and then increased 2-fold again after 15 min. Cytosolic protein kinase C activity increased more gradually and steadily up to 80% over a 20-min period. Increases in protein kinase C activity were dose-dependent and were not simply a result of translocation of cytosolic enzyme (although this may have occurred), as total activity was also increased. The increase in protein kinase C activity was not inhibited by cycloheximide (which also increased protein kinase C activity and 2-deoxyglucose transport) and was still evident following anion exchange chromatography. The insulin effect was decidedly different from those of 12-O-tetradecanoylphorbol-13-acetate and phenylephrine using histone III-S as substrate. Phenylephrine decreased cytosolic protein kinase C activity while increasing membrane activity; 12-O-tetradecanoylphorbol-13-acetate only decreased cytosolic protein kinase C activity. The early insulin-induced increases in membrane protein kinase C activity may be related to increased diacylglycerol generation from de novo phosphatidic acid synthesis, as there were rapid increases in [3H]glycerol incorporation into diacylglycerol, and transient increases in phospholipid hydrolysis, as there were transient rapid increases in [3H]diacylglycerol in cells prelabeled with [3H]arachidonate. Later, sustained increases in membrane and cytosolic protein kinase C activity may reflect the continuous activation of de novo phospholipid synthesis, as there were associated increases in [3H]glycerol incorporation into diacylglycerol at later, as well as very early time points.

Further evidence implicating diacylglycerol generation and protein kinase C activation in agonist-induced increases in glucose uptake. Insulin-like effects of phenylephrine in BC3H-1 myocytes.

We have previously suggested that insulin effects on 2-deoxyglucose (2-DOG) uptake in BC3H-1 myocytes are due to increases in de novo phospholipid synthesis, diacylglycerol generation, and protein kinase C activation. To test this hypothesis further, we examined the effects of phenylephrine, an agonist that increases diacylglycerol and protein kinase C activity through phospholipase C activation. As evidence for phospholipase activation in BC3H-1 myocytes, we found that phenylephrine increased acute 32PO4 incorporation into phosphatidic acid and phosphatidylinositol, generation of [3H]inositol phosphates from prelabeled [3H]inositol phospholipids, cytosolic Ca2+, and membrane-bound protein kinase C. Phenylephrine also provoked dose-related increases in [3H]2-DOG uptake that were similar in magnitude and time course to those induced by insulin. As with insulin, phenylephrine effects on 2-DOG uptake were not apparent in myocytes that were maximally stimulated with 12-O-tetradecanoylphorbol-13-acetate, a diacylglycerol analogue that activates protein kinase C. These findings support our hypothesis that diacylglycerol generation and protein kinase C activation may be important in the stimulation of glucose uptake by agents such as phenylephrine and insulin that activate the phosphoinositide cycle.

The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+.

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated 'second messenger' which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular 'messenger' for controlling metabolic processes during insulin action.

Phorbol ester provokes insulin-like effects on glucose transport, amino acid uptake, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes.

We evaluated the possibility that diacylglycerol may function as a second messenger in insulin action. To this end, we employed 12-O-tetradecanoyl phorbol 13-acetate (TPA) to mimic diacylglycerol in BC3H-1 myocytes. Like insulin, TPA provoked rapid increases in 2-deoxyglucose transport and pyruvate dehydrogenase activity in mature insulin-responsive BC3H-1 cultured myocytes. TPA also stimulated amino acid uptake, as evidenced by uptake of alpha-methylaminoisobutyric acid; the relatively slow time course of this effect paralleled that of insulin. In contrast, the effects of TPA were not apparent in undifferentiated BC3H-1 myoblasts, which were also unresponsive to insulin. The insulin-like effects in the myocytes appeared to be specific for TPA, the biologically active phorbol diester which activates protein kinase C, as other tested phorbol derivatives were without effect. Effects of maximally effective concentrations of TPA and insulin were nonadditive. Two synthetic diacylglycerols, 1,2-diolien and 1-oleoyl-2-acetyl-sn-glycerol, also provoked insulin-like effects on 2-deoxyglucose transport. Since insulin rapidly increases diacylglycerol levels in these cells, and TPA mimics diacylglycerol biochemically, it is possible that insulin may control cellular processes through changes in diacylglycerol.