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Functional adipocyte glucose disposal is a key component of global glucose homeostasis. PKCbetaII is involved in rat skeletal muscle cell ISGT. Western blot analysis and real-time PCR revealed 3T3-L1 cells developmentally regulated PKCbeta splicing such that PKCbetaI was downregulated and PKCbetaII was upregulated during the course of differentiation. An initial glucose uptake screen using PKC inhibitor LY379196 pointed to a PKC isozyme other than PKCzeta mediating 3T3-L1 adipocyte ISGT. Subsequent use of PKCbetaII inhibitor CGP53353 pointed to a role for PKCbetaII in ISGT. Western blot analysis showed that CGP53353 specifically inhibited phosphorylation of PKCbetaII Serine 660. Subcellular fractionation and immunofluorescence demonstrated that PKCbetaII regulates GLUT4 translocation. Further Western blot, immunofluorescence and co-immunoprecipitation analysis reveal that PKCbetaII inhibition does not affect mTORC2 activity yet abrogates phosphorylation of Akt Serine 473. PKCbetaII regulates GLUT4 translocation by regulating Akt phosphorylation and thus activity.

Aims/hypothesis: Insulin stimulates phosphorylation cascades, including phosphatidylinositol-3-kinase (PI3K), phosphatidylinositol-dependent kinase (PDK1), Akt, and protein kinase C (PKC). Myristoylated alanine-rich C-kinase substrate (MARCKS), a PKCbetaII substrate, could link the effects of insulin to insulin-stimulated glucose transport (ISGT) via phosphorylation of its effector domain since MARCKS has a role in cytoskeletal rearrangements., Methods: We examined phosphoPKCbetaII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCbetaII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCbetaII and calmodulin inhibitors, respectively, before insulin treatment, using western blots. ISGT was examined after cells had been treated with inhibitors, small inhibitory RNA (siRNA) for MARCKS, or transfection with MARCKS mutated at a PKC site. MARCKS, PKCbetaII, GLUT4 and insulin receptor were immunoblotted in subcellular fractions with F-actin antibody immunoprecipitates to demonstrate changes following insulin treatment. GLUT4 membrane insertion was followed after insulin with or without CG53353., Results: Insulin increased phosphoPKCbetaII(Ser660 and Thr641); LY294002 blocked this, indicating its activation by PI3K. Insulin treatment increased cytosolic phosphoMARCKS, decreased membrane MARCKS and increased membrane phospholipase D1 (PLD1), a protein regulating glucose transporter vesicle fusion resulted. PhosphoMARCKS was attenuated by CG53353 or MARCKS siRNA. MARCKS siRNA blocked ISGT. Association of PKCbetaII and GLUT4 with membrane F-actin was enhanced by insulin, as was that of cytosolic and membrane MARCKS. ISGT was attenuated in myocytes transfected with mutated MARCKS (Ser152Ala), whereas overproduction of wild-type MARCKS enhanced ISGT. CG53353 blocked insertion of GLUT4 into membranes of insulin treated cells., Conclusions/interpretation: The results suggest that PKCbetaII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.

Accelerated vascular smooth muscle cell (VSMC) proliferation contributes to the formation of atherosclerotic lesions. To investigate protein kinase C (PKC)-betaII functions with regard to glucose-induced VSMC proliferation, human VSMC from aorta (AoSMC), a clonal VSMC line of rat aorta (A10), and A10 cells overexpressing PKC-betaI (betaI-A10) and PKC-betaII (betaII-A10) were studied with the use of three techniques to evaluate glucose effects on aspects affecting proliferation. High glucose (25 mM) increased DNA synthesis and accelerated cell proliferation compared with normal glucose (5.5 mM) in AoSMC and A10 cells, but not in betaI-A10 and betaII-A10 cells. The PKC-betaII specific inhibitor CGP-53353 inhibited glucose-induced cell proliferation and DNA synthesis in AoSMC and A10 cells. In flow cytometry analysis, high glucose increased the percentage of A10 cells at 12 h after cell cycle initiation but did not increase the percentage of betaI-A10 or betaII-A10 cells entering S phase. PKC-betaII protein levels decreased before the peak of DNA synthesis, and high glucose further decreased PKC-betaII mRNA and protein levels in AoSMC and A10 cells. These results suggest that high glucose downregulates endogenous PKC-betaII, which then alters the normal inhibitory role of PKC-betaII in cell cycle progression, resulting in the stimulation of VSMC proliferation through acceleration of the cell cycle.

Insulin regulates a diverse array of signaling pathways involved in the control of growth, differentiation, proliferation, and metabolism. Insulin increases in glucose uptake via a protein kinase C-dependent pathway in target tissues such as fat and muscle are well documented. Insulin-regulated events, however, occur in all cells. The utilization of glucose as a preferred energy source is a ubiquitous event in eukaryotic cells. In NIH-3T3 fibroblasts, insulin treatment increased levels of the cPKC and nPKC activator, diacylglycerol. Insulin-responsive 2-[(3)H]deoxyglucose uptake was stimulated in a dose-dependent manner. The overexpression of protein kinase C (PKC)betaI, -betaII, -delta, -epsilon, and -zeta was used to investigate the specificity of PKC isozymes for insulin-sensitive glucose uptake. The stable overexpression of PKCbetaII, -delta, and -epsilon resulted in increases in insulin-stimulated 2-[(3)H]deoxyglucose uptake compared to vector control cells, while basal 2-deoxyglucose uptake levels were not elevated. Overexpression of PKCbetaI and PKCzeta isozymes had no further effect on basal or insulin-stimulated 2-deoxyglucose uptake. The PKC-specific inhibitor, CGP41251, blocked insulin effects on 2-deoxyglucose uptake but not its effects on tyrosine phosphorylation of cellular substrates. Insulin-stimulated 3-O-methylglucose uptake was also greater in cells overexpressing PKCbetaII, -delta, and -epsilon, compared to control cells. The increased responsiveness was not accompanied by conversion of 3T3 cells to the adipocyte phenotype or the increased expression of insulin receptors or glucose transporters (GLUT1-type). Insulin-stimulated recruitment of GLUT1 to plasma membranes of cells overexpressing PKCbetaII, -delta, and -epsilon, was greater than that in control cells. The data suggest that more than one PKC isozyme is involved in insulin signaling pathways in fibroblasts, resulting in increased GLUT1 transporter recruitment to cell membranes., (Copyright 1999 Academic Press.)

Glucose concentration may be an important factor in breast cancer cell proliferation because the prevalence of breast cancer is high in diabetic patients. To determine the role of protein kinase C (PKC)-betaII in regulating MCF-7 cell proliferation at different glucose concentrations, the effects of glucose and a PKC-betaII-specific inhibitor (CGP53353) were examined in cultures of MCF-7 human breast cancer cell line and its multidrug resistant variant (MCF-7/ADR). Cell proliferation and DNA synthesis of MCF-7 were increased when glucose concentration in the culture medium was increased from normal (5.5 mM) to high (25 mM) levels. However, MCF-7/ADR cell proliferation and DNA synthesis were unaffected by the increase in glucose. PKC-betaII protein and the corresponding mRNA levels were 4- to 5-fold higher in MCF-7/ADR than in MCF-7 cells. High glucose-induced decreases of PKC-betaII protein and mRNA levels were observed during the DNA synthesis phase in MCF-7 but not in MCF-7/ADR cells. Decreases in PKC-betaII mRNA and protein levels below a critical threshold in response to high glucose levels may account for glucose-stimulated proliferation of MCF-7 cells. Cultures of multidrug resistant MCF-7/ADR cells reach maximal cell density in medium containing normal (5.5 mM) glucose levels and are not induced to grow further in response to high (25 mM) glucose. Our results demonstrate a link between high glucose-induced PKC-betaII isozyme down-regulation with concomitant acceleration of cell cycle progression in MCF-7 cells., (Copyright 1999 Wiley-Liss, Inc.)

Acute hyperglycemia may contribute to the progression of atherosclerosis by regulating protein kinase C (PKC) isozymes and by accelerating vascular smooth muscle cell (VSMC) proliferation. We investigated acute glucose regulation of PKCbeta gene expression in A10 cells, a rat aortic smooth muscle cell line. Western blot analysis showed that PKCbetaII protein levels decreased with high glucose (25 mM) compared to normal glucose (5.5 mM), whereas PKCbetaI levels were unaltered. PKCbeta mRNA levels were depleted by 60-75% in hyperglycemic conditions. To elucidate whether high glucose regulated PKCbeta expression via the common promoter for PKCbetaI and PKCbetaII, deletion constructs of the PKCbeta promoter ligated to CAT as reporter gene were transfected into A10 cells. Construct D (-411 to +179CAT) showed quenching in high glucose (25 mM) and suggested the involvement of a carbohydrate response element in the 5' promoter region of the PKCbeta gene. In actinomycin D-treated A10 cells, a 60% decrease in PKCbeta mRNA with high glucose treatment indicated that posttranscriptional destabilization by glucose was also occurring. We have demonstrated that glucose-induced posttranscriptional destabilization of PKCbetaII message is mediated via a nuclease activity present in the cytosol. The specificity of glucose to posttranscriptionally destabilize PKCbetaII mRNA, but not the PKCbetaI mRNA, was confirmed in both A10 cells and primary cultures from human aorta.

Adenosine A1 receptor densities were increased in cultured LLC-PK1 and OK cells by chronic treatment with the adenosine receptor antagonists 1,3,7-trimethylxanthine (caffeine, 1 mM) and 1,3-dimethyl-8-cyclopentylxanthine [cyclopentyltheophylline (CPT), < or = 0.4 mM], respectively. The A1 receptor number per cell was increased twofold by 10-day treatments with 1 mM caffeine or 0.1 mM CPT, and the sodium-coupled glucose uptake was augmented twofold by 1 mM caffeine and sevenfold by 0.1 microM CPT (higher doses of CPT were progressively less stimulatory). Glucose uptake was blocked by acute (2-h) treatment with CPT, adenosine deaminase, or calphostin C. Caffeine (1 mM) or CPT (> or = 0.1 mM) inhibited cell proliferation for the first 10 days, then cell growth assumed a normal proliferative rate despite continued presence of antagonist. Cytosolic protein kinase C (PKC) beta-isoform immunoactivity and PKC-beta II mRNA were elevated at least twofold during 10 days of 0.1 mM CPT or 1 mM caffeine treatment. The sustained elevation in sodium-glucose symport and PKC activity observed with adenosine receptor antagonists was similar to acute (2-h) effects of the adenosine A1 agonist R(-)-N6-phenylisopropyladenosine (R-PIA, 0.1-1 microM). Moreover, cell proliferation was increased by adenosine (0.1 microM R-PIA), whereas Na-K-adenosinetriphosphatase activity was unaltered with chronic antagonist or acute adenosine treatments. Caffeine treatment may have some non-adenosine A1 receptor-mediated actions, because it slightly (30%) augmented protein kinase A activity. It is concluded that chronic exposure of proximal tubule cells to caffeine or CPT augments PKC and sodium-glucose transport but retards cell proliferation mainly via adenosine A1 receptor-mediated mechanisms.

Pertussis toxin was used to block insulin-stimulated phosphatidylinositol (PI)-glycan hydrolysis, consequent de novo synthesis of phosphatidic acid (PA) and the diacylglycerol (DAG) production that results from these two related processes in BC3H-1 myocytes. In contrast, pertussis toxin pretreatment did not inhibit insulin-stimulated hydrolysis of phosphatidylcholine (PC) which was found to be at least partly due to activation of a phospholipase D. Moreover, pertussis toxin-insensitive PC hydrolysis was accompanied by rapid biphasic increases in DAG and translocative activation of protein kinase C (PKC). Insulin-stimulated glucose transport was also insensitive to pertussis toxin pretreatment. Our findings suggest that insulin-stimulated PC hydrolysis pays an important role in DAG/PKC signalling during insulin action.

Glucose has been reported to increase the de novo synthesis of diacylglycerol (DAG) and translocate and activate protein kinase C (PKC) in rat adipocytes. Presently, we examined the major subcellular site of PKC translocation/activation in response to glucose-induced DAG. Glucose rapidly increased DAG content and PKC enzyme activity in microsomes, but not in plasma membranes or other membranes, during a 30-min treatment of rat adipocytes. This glucose-induced increase in microsomal DAG was attended by increases in immunoreactive PKC alpha, beta, and epsilon. Glucose-induced activation of DAG/PKC signaling in microsomes was not associated with a change in the translocation of Glut-4 transporters from microsomes to the plasma membrane, a biological response that is known to be stimulated by agonists, e.g., phorbol esters, which increase DAG/PKC signaling in plasma membranes, as well as in microsomes. In conclusion, an increase in de novo phospholipid synthesis, as occurs during glucose treatment of rat adipocytes, primarily activates DAG/PKC signaling in microsomes; moreover, this signaling response and biological consequences thereof may differ from those of agonists that primarily stimulate DAG/PKC signaling in the plasma membrane.

To examine the effects of hyperglycemia on insulin signaling in A-10 vascular smooth muscle cells, cells were treated with extracellular D-glucose and effects of insulin were studied on the diacylglycerol-protein kinase C signaling system. A-10 cells specifically bound 125I-insulin, and insulin-like growth factor-I did not displace the label. 125I-insulin binding was unaltered under hyperglycemic conditions. To determine if insulin receptors were coupled to other insulin-regulated processes, diacylglycerol, protein kinase C, and glucose transport were evaluated. Insulin increased cellular diacylglycerol (DAG) levels which were also increased following glucose treatment and not further stimulated by insulin. The uptake of 2-[3H]deoxy-D-glucose (2-DOG) was stimulated by insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Insulin- and TPA-stimulated 2-[3H]DOG uptake was inhibited by a protein kinase inhibitor, staurosporine. Preincubation of cells with 500 nM TPA overnight resulted in the inhibition of insulin- and TPA-stimulated 2-[3H]DOG uptake. Protein kinase C activity was translocated from cytosolic to membrane fractions following insulin treatment. Overnight glucose (25 mM) treatment resulted in a 50% decrease in protein kinase C enzyme activity and > 90% decrease in protein kinase C beta immunoreactive levels. Protein kinase C activity and levels were not affected by osmotic control media containing mannitol. A-10 cells express GLUT4-type glucose transporters. Neither insulin-regulatable glucose transporter (GLUT4) mRNA nor GLUT4 protein levels were diminished by glucose. Significant decreases in insulin- and TPA-stimulated 2-[3H]DOG uptake occurred, however, with glucose. The down-regulation of protein kinase C beta and resultant inhibition of 2-[3H]DOG uptake by chronic glucose suggests a biochemical link between hyperglycemia and DAG-protein kinase C signaling in vascular smooth muscle cells.

Insulin treatment in vivo provoked rapid dose-related increases in diacylglycerol content and/or translocation of protein kinase-C (PKC) from cytosol to membranes in rat soleus and gastrocnemius muscles. These effects were apparent with 1) insulin doses that provoked submaximal and maximal increases in glucose utilization, and 2) glucose-stimulated endogenous insulin secretion. Insulin-stimulated PKC translocation was evident when PKC was assayed by 1) histone or protamine phosphorylation after PKC purification by Mono Q column chromatography, and 2) immunoblotting for PKC beta and PKC epsilon. Dose-related effects of insulin on PKC translocation were also observed in the rat soleus in vitro, and this was associated with increased phosphorylation of 40- and 80-kilodalton proteins, which were also phosphorylated by phorbol ester treatment. A role for diacylglycerol-PKC signalling in insulin-stimulated glucose transport was suggested by studies of [3H]2-deoxyglucose ([3H]2-DOG) uptake in the rat soleus in vitro in that 1) PKC translocation and 2-DOG uptake were correlated; and 2) stimulatory effects of insulin and phorbol esters on 2-DOG uptake were apparently nonadditive.

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.

We have examined the effects of adenosine on sodium-coupled phosphate and glucose transport in cultured opossum kidney (OK) cells, a continuous cell line that resembles proximal tubule. Adenosine analogues R-(-)-N6-phenylisopropyladenosine (PIA) and 2',5'-dideoxy-adenosine (DDA) were employed as adenosine A1 receptor and P site-selective agonists, respectively. Sodium-dependent phosphate uptake activity (Na-Pi symport) increased by approximately 25% above both basal and parathyroid hormone (PTH)-inhibited levels in cells treated with PIA (0.1, 1 microM) but not in cells treated with DDA (100 microM). Adenosine (PIA) also stimulated sodium-coupled 3-O-methylglucose transport by approximately 40%. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) content was inversely related to Na-Pi symport activity in cells treated with PIA and PTH. However, changes in Na-Pi symport activity did not consistently relate to changes in intracellular cAMP. Protein kinase C was activated 15 s after treatment of OK cells with 1 microM PIA. Preincubation of cells with 3 microM staurosporine attenuated the effect of 1 microM PIA on phosphate uptake. These data suggest that Na-Pi and Na-glucose symport activities are stimulated by adenosine acting at a receptor coupled to more than one intracellular signal. It is likely that both protein kinases A and C are involved in these actions of adenosine.

Glyburide and tolbutamide, at concentrations of 20 to 40 mumol/L and 1 to 2 mmol/L, respectively, stimulated glucose transport in rat adipocytes. Concomitantly, protein kinase C was activated, as evidenced by translocation of immunoreactive enzyme from cytosol to membranes. Glucose transport effects of the sulfonylureas were blocked by three inhibitors of protein kinase C (H-7, staurosporine, and sangivamycin), and by phorbol ester-induced down-regulation of protein kinase C. These findings suggest that sulfonylureas may stimulate glucose transport in rat adipocytes through activation of protein kinase C.

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.

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 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.

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.