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)     

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 increases the intracellular concentration of total oxalyl thiolesters in BC3H-1 myocytes: potential anti-insulin mediators.

Oxalyl thiolesters (RS-CO-COOH) may represent negative intracellular messengers for insulin action. Using a reverse-phase, ion-pair high pressure liquid chromatographic technique, total intracellular oxalyl thiolesters were measured in insulin-sensitive BC3H-1 myocytes after the addition of insulin. The total oxalyl thiolester concentration increased to a maximum of 2.9 times the basal concentration by 30 min after the addition of 100 microU/ml insulin and decreased to 1.8 times by 180 min. Insulin's stimulation of pyruvate dehydrogenase as measured by lactate oxidation ([1-14C]-lactate --> 14CO2) in intact BC3H-1 myocytes reached a maximum at 15-30 min and returned to basal activity during the 60-90 min measurement interval. These results suggest that oxalyl thiolesters are increased in concentration following insulin-induced signal transduction to reverse insulin-stimulated metabolic events.     

Inositolphosphoglycans and diacylglycerol are possible mediators in the glycogenic effect of GLP-1(7-36)amide in BC3H-1 myocytes

A potent glycogenic effect of GLP-1(7-36)amide has been found in rat hepatocytes and skeletal muscle, and specific receptors for this peptide, which do not seem to be associated with the adenylate cyclase—cAMP system, have been detected in these tissue membranes. On the other hand, inositolphosphoglycan molecules (IPGs) have been implicated as second messengers of the action of insulin. In this work, we have found, in differentiated BC3H-1 myocytes, specific binding of [¹²⁵I]GLP-1(7-36)amide, and a stimulatory effect of the peptide on glycogen synthesis, confirming the findings in rat skeletal muscle. Also, GLP-1(7-36)amide modulates the cell content of radiolabelled glycosylphosphatidylinositols (GPIs) and increases the production of diacylglycerol (DAG), in the same manner as insulin acts, indicating hydrolysis of GPIs and an immediate and short-lived generation of IPGs. Thus, IPGs and DAG could be mediators in the glycogenic action of GLP-1(7-36)amide in skeletal muscle.     

Effects of insulin and protein synthesis inhibitors on phospholipid metabolism, diacylglycerol levels, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes

BC3H-1 myocytes were cultured with 32PO4 for 3 days to label phospholipids to constant specific activity. Subsequent treatment with physiological concentrations of insulin provoked 40-70% increases in 32PO4 levels (reflecting increases in mass) in phosphatidic acid, phosphatidylinositol, and polyphosphoinositides, and, lesser, 20-25% increases in phosphatidylserine and the combined chromatographic area containing phosphatidylethanolamine plus phosphatidylcholine plus phosphatidylcholine. Insulin-induced increases in phospholipids were significant within 5 min and near-maximal at 15-30 min. Comparable rapid insulin-induced increases in [3H]phosphatidylinositol were observed in myocytes prelabeled with [3H]inositol. These insulin effects (as per prolonged pulse-chase experiments) were due to increase phospholipid synthesis rather than decreased phospholipid degradation. Cycloheximide (and puromycin) pretreatment prevented insulin-induced increases in phospholipids and rapidly reversed ongoing insulin effects on phospholipids and pyruvate dehydrogenase activity. Insulin also rapidly increased diacylglycerol levels. These findings suggest that: (a) insulin provokes rapid increases in de novo synthesis of phosphatidic acid and its derivatives, e.g. phosphoinositides and diacylglycerol; (b) protein synthesis inhibitors diminish phospholipid levels in insulin-treated (but not control) tissues by increasing phospholipid degradation (?phospholipase(s) activation); and (c) changes in phospholipids and diacylglycerol may be important for changes in pyruvate dehydrogenase and other enzymatic activities during treatment with insulin and/or protein synthesis inhibitors.     

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.     

Pertussis toxin treatment attenuates some effects of insulin in BC3H-1 murine myocytes

The effects of pertussis toxin (PT) treatment on insulin-stimulated myristoyl-diacylglycerol (DAG) generation, hexose transport, and thymidine incorporation were studied in differentiated BC3H-1 myocytes. Insulin treatment caused a biphasic increase in myristoyl-DAG production which was abolished in myocytes treated with PT. There was no effect of PT treatment on basal (nonstimulated) myristoyl-DAG production. Insulin-stimulated hydrolysis of a membrane phosphatidylinositol glycan was blocked by PT treatment. ADP-ribosylation of BC3H-1 plasma membranes with [32P]NAD revealed a 40-kDa protein as the major PT substrate in vivo and in vitro. The time course and dose dependence of the effects of PT on diacylglycerol generation correlated with the in vivo ADP-ribosylation of the 40-kDa substrate. Pertussis toxin treatment resulted in a 71% attenuation of insulin-stimulated hexose uptake without effect on either basal or phorbol ester-stimulated uptake. The stimulatory effects of insulin and fetal calf serum on [3H]thymidine incorporation into quiescent myocytes were attenuated by 61 and 59%, respectively, when PT was added coincidently with the growth factors. Nonstimulated and EGF-stimulated [3H]thymidine incorporation was unaffected by PT treatment. These data suggest that a PT-sensitive G protein is involved in the cellular signaling mechanisms of insulin.     

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.     

Insulin stimulates generation of intracellular mediators in rat heart

Insulin treatment of rats results in an increased amount or activity of insulin mediators in heart muscle. The mediators stimulated mitochondrial pyruvate dehydrogenase and inhibited glucagon-stimulated adenylate cyclase. The mediators were copurified by ultrafiltration, ethanol extraction, Dowex cation-exchange, and QAE-Sephadex anion-exchange chromatography. The activities of the two mediators were separated by Sephadex G-10 chromatography. Fasting rats for 72 h diminished the mediator response to insulin treatment. These results, taken together with previous reports, indicate that insulin generates a number of mediators which have a ubiquitous tissue distribution. The activity of these mediators, like insulin responsiveness, is altered by the metabolic state of the animal.     

A pertussis toxin-sensitive G-protein mediates some aspects of insulin action in BC3H-1 murine myocytes.

The involvement of G-proteins in the insulin signal transduction system has been studied in detail using the murine BC3H-1 myocyte system. Pertussis toxin (PT) treatment, previously shown to attenuate some of the metabolic effects of insulin in this cell line (Luttrell, L.M., Hewlett, E.L., Romero, G., and Rogol, A.D. (1988) J. Biol. Chem. 263, 6134-6141), abolished insulin-induced generation of diacylglycerol and inositolglycan mediators with no effects on either the autophosphorylation of the insulin receptor or the phosphorylation of the major endogenous substrates for insulin-stimulated tyrosine kinase activity (pp185 and pp42-45). In vitro ADP-ribosylation and immunoblotting studies suggest that the major PT substrate is a 40-kDa protein of the G alpha family. This protein band did not exhibit detectable tyrosine phosphorylation upon stimulation of either intact cells or cell membranes with insulin. In the presence of low concentrations of GTP, insulin treatment of isolated myocyte plasma membranes resulted in a small (30-40%) but significant stimulation of GTP hydrolysis. This effect was best observed in the presence of small concentrations of sodium dodecyl sulfate. The rate of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to BC3H-1 membranes was also significantly increased in the presence of insulin. The effects of insulin on GTP hydrolysis and GTP gamma S binding were found to be dependent on the concentration of insulin. These effects were not detected in plasma membranes prepared from PT-pretreated BC3H-1 myocytes. In contrast, pretreatment with the B (inactive) subunit of PT did not alter the response of myocyte membranes to insulin. High affinity binding of [125I]iodoinsulin to myocyte plasma membranes was reduced by 60-70% in the presence of guanine nucleotides. Similar effects on insulin binding were produced by PT pretreatment of the cells. In contrast, adenine nucleotides had no effect on insulin binding. Scatchard analysis of the binding data showed that the observed effects of guanine nucleotides and PT on insulin binding resulted either from a reduction in the number of high affinity insulin binding sites or from a significant reduction of the affinity of insulin for its receptor. Low affinity binding sites did not appear to be affected by either guanine nucleotides nor PT pretreatment. These results provide substantial evidence suggestive of a noncovalent interaction between the insulin receptor and a regulatory G-protein system during the process of insulin signaling.     

Equilibrium model for insulin-induced receptor down-regulation. Regulation of insulin receptors in differentiated BC3H-1 myocytes

The mechanism of insulin-induced down-regulation of surface membrane insulin receptors was studied in the muscle cell line BC3H-1. Down-regulation for the differentiated myocytes is dose- and time-dependent with a half-maximum response at 0.5 nM insulin and a maximum decrease of 50% in the number of surface insulin receptors following exposure to 20 nM insulin for 18 h at 37 degrees C, as confirmed by Scatchard analysis. These receptors were fully recoverable upon lysis of the down-regulated myocyte with Triton X-100, demonstrating that down-regulation is mediated solely by insulin-induced receptor internalization without detectable receptor degradation. Phospholipase C treatment of intact down-regulated cells and Triton X-100 treatment after subcellular fractionation showed that no cryptic or masked receptors were detectable within the plasma membrane. Insulin-induced receptor internalization was dependent upon cellular energy production, protein synthesis, and endocytosis, but was insensitive to agents which primarily affect lysosomal, cytoskeletal, or transglutaminase activities. The magnitude of insulin-induced down-regulation and the kinetics of down-regulation and recovery of cell surface receptors indicate that the surface and internal receptor pools are in dynamic equilibrium with each other. The kinetic data are accommodated by separate internalization rate constants for the unoccupied (0.01 h-1) and occupied (0.11 h-1) surface receptors and a single recycling rate constant (0.11 h-1) for the internalized receptors. This model also explains the previous apparently paradoxical finding in several other systems that down-regulation is more sensitive to hormone than hormone-receptor binding under physiologic conditions. Down-regulation in BC3H-1 myocytes, therefore, appears to be mediated solely by an insulin-induced increase in the receptor internalization rate constant and a consequent shift in the dynamic equilibrium between the surface and internalized receptor pools, resulting in a 50% decrease in the number of cell surface receptors. In other systems where the internalized hormone receptor is a substrate for rapid degradation, the essential role of this shift in mediating the down-regulation process may be obscured.     

Glucose regulates the expression of Gi-proteins in cultured BC3H-1 myocytes.

To examine whether glucose has regulatory effects on the expression of Gi-proteins, BC3H-1 myocytes were incubated for 24 hr in the presence of various concentrations of glucose (0-25 mM) and the amount of Gi-proteins was detected by pertussis toxin ADP-ribosylation and immunoblot analysis. Both detection methods showed a progressive decrease in the amount of Gi proteins in cells treated with increasing concentrations of glucose. A maximal reduction of 40% was observed after a 24 hr exposure to 25 mM glucose. The reduction in Gi-proteins correlated with a decrease in insulin-stimulated glucose transport.     

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.     

Protein phosphorylation and protein kinase activities in BC3H-1 myocytes. Differences between the effects of insulin and phorbol esters

To determine whether insulin activates protein kinase C in BC3H-1 myocytes, we evaluated changes in protein phosphorylation, protein kinase activities, and the intracellular translocation of protein kinase C activity in response to insulin and phorbol esters. Phorbol 12-myristate 13-acetate (PMA), but not insulin, stimulated the phosphorylation of an acidic Mr 80,000 protein which has been shown to be an apparently specific marker for protein kinase C activation. In addition, PMA, but not insulin, stimulated the rapid association of protein kinase C activity with a cellular particulate fraction. In contrast to these differences, both insulin and PMA stimulated the phosphorylation of ribosomal protein S6 and activated a ribosomal protein S6 kinase in cell-free extracts from cells exposed to these agents. In cells exposed to high concentrations of PMA for 16 h, protein kinase C activity and immunoreactivity were abolished, without changes in cellular morphology. Under these conditions, insulin, but not PMA, stimulated phosphorylation of the ribosomal protein S6 in intact cells and activated the S6 kinase in cell-free extracts derived from insulin-treated intact cells. We conclude that: insulin does not appear to activate protein kinase C in BC3H-1 myocytes, at least as assessed by phosphorylation of the Mr 80,000 protein; both insulin and PMA activate an S6 protein kinase in these cells; and insulin can promote S6 phosphorylation and activate the S6 kinase normally in protein kinase C-deficient cells. Activation of the S6 kinase by insulin and PMA, although apparently proceeding through different mechanisms, may explain some of the similar biological actions of these compounds in BC3H-1 myocytes.     

Translocation of the brain-type glucose transporter largely accounts for insulin stimulation of glucose transport in BC3H-1 myocytes.

Insulin-stimulated glucose transport was examined in BC3H-1 myocytes. Insulin treatment lead to a 2.7 +/- 0.3-fold increase in the rate of deoxyglucose transport and, under the same conditions, a 2.1 +/- 0.1-fold increase in the amount of the brain-type glucose transporter (GLUT 1) at the cell surface. It has been shown that some insulin-responsive tissues express a second, immunologically distinct, transporter, namely GLUT 4. We report here that BC3H-1 myocytes and C2 and G8 myotubes express only GLUT 1; in contrast, rat soleus muscle and heart express 3-4 times higher levels of GLUT 4 than GLUT 1. Thus translocation of GLUT 1 can account for most, if not all, of the insulin stimulation of glucose transport in BC3H-1 myocytes. On the other, hand, neither BC3H-1 myocytes nor the other muscle-cell lines are adequate as models for the study of insulin regulation of glucose transport in muscle tissue.     

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.     

The development of insulin receptors and responses in the differentiating nonfusing muscle cell line BC3H-1

We have studied the development of high affinity insulin receptors and insulin-stimulated responses in the differentiating nonfusing muscle cell line BC3H-1. In the logarithmic growth phase, these myoblasts exhibit very low levels of insulin binding and no detectable insulin-stimulated glucose or amino acid uptake. Following the cessation of cell division and subsequent spontaneous differentiation, the resulting myocytes develop a 5-fold increase in specific 125I-insulin binding and demonstrate physiologic insulin-stimulated glucose and amino acid uptake (100% increase above baseline) with half-maximum stimulation at 1-3 nM in agreement with the known in vivo and in vitro insulin sensitivity of muscle tissue. Insulin stimulation of 2-deoxyglucose uptake is detectable within 3 min, becomes maximal within 15 min, and is mediated by a rapid increase of plasma membrane transport units, as determined by D-glucose-inhibitable cytochalasin B binding, resulting in a 2-fold increase in the Vmax for 2-deoxyglucose transport with no change in Km. Myocyte insulin binding is specific, reversible, and saturable, yielding equilibrium within 18 h at 4 degrees C. Scatchard analysis identified the high affinity insulin receptor with a Kd of 0.5 nM at 4 degrees C. The myocytes also demonstrate sensitive down-regulation of cell surface insulin receptors, with a maximum decrease of 50% in cell surface insulin binding following exposure to 20 nM insulin for 18 h at 37 degrees C. Since the differentiation of this muscle cell line from myoblasts to nonfusing myocytes is accompanied by the development of high affinity insulin receptors and physiologic insulin-stimulated glucose and alpha-methylaminoisobutyric acid uptake, this continuously cultured system provides an excellent model for the study of differentiation and mechanism of insulin action in muscle, its quantitatively most significant target tissue.     

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.     

(Thr-59)-insulin-like growth factor I stimulates 2-deoxyglucose transport in BC3H1 myocytes through the insulin-like growth factor receptor, not the insulin receptor

The murine non-fusing muscle cell line contains distinct receptors for insulin and insulin-like growth factors. Pretreatment of myocytes with insulin for 20 h at 37 degrees C inhibits the binding of [125I]iodoinsulin by 60% without affecting the binding of [125I]iodoinsulin-like growth factor I. The ED50 values for down-regulation of the insulin and insulin-like growth factor receptor by their respective ligands are 1 nM and 3 nM, respectively. Insulin, (Thr-59)-insulin-like growth factor I and multiplication-stimulating activity stimulate 2-[3H]deoxyglucose transport in myocytes with ED50 values of 5 nM, 5.6 nM and 33 nM, respectively. In order to determine whether (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes via its own receptor or the insulin receptor, we determined the activity of these peptides after down-regulation of the insulin receptor. The rate of 2-[3H]deoxyglucose transport in myocytes pretreated with insulin (5 nM) is elevated but returns to control levels by 1 h after the washout of insulin. The dose-response curve for insulin-stimulated 2-[3H]deoxyglucose transport is shifted to the right (ED50 greater than 100 nM) immediately after insulin washout but is normal by 1 h after insulin washout. In contrast, the dose-response curve for (Thr-59)-insulin-like growth factor I is unchanged in insulin-pretreated cells immediately after insulin washout. These data show that (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes by acting through an insulin-like growth factor receptor and not through the insulin receptor. Since multiplication-stimulating activity is 6-fold less active than (Thr-59)-insulin-like growth factor, they both may be acting through a type 1 insulin-like growth factor receptor.