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Anabolic ornithine transcarbamoylase (aOTC) catalyzes the reaction between carbamoyl phosphate (CP) and L-ornithine (ORN) to form L-citrulline and phosphate in the urea cycle and L-arginine biosynthesis. The crystal structure of unliganded aOTC from Campylobacter jejuni (Cje aOTC) was determined at 2.7 Å resolution and refined to an R(work) of 20.3% and an R(free) of 24.0%. Cje aOTC is a trimer that forms a head-to-head pseudohexamer in the asymmetric unit. Each monomer is composed of an N-terminal CP-binding domain and a C-terminal ORN-binding domain joined by two interdomain helices. The Cje aOTC structure presents an open conformation of the enzyme with a relatively flexible orientation of the ORN-binding domain respective to the CP-binding domain. The conformation of the B2-H3 loop (residues 68-78), which is involved in binding CP in an adjacent subunit of the trimer, differs from that seen in homologous proteins with CP bound. The loop containing the ORN-binding motif (DxxxSMG, residues 223-230) has a conformation that is different from those observed in unliganded OTC structures from other species, but is similar to those in structures with bound ORN analogs. The major differences in tertiary structure between Cje aOTC and human aOTC are described.

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.

Nutrient intake during gestation has an impact on gestation parameters and subsequent lactation performance. The objectives of this experiment were to determine the impact of feeding two levels of amino acids in gestation on sow BW changes in gestation and lactation, and litter size, and to evaluate a factorial method for determining daily energy requirements. At mating, 419 sows (Camborough 15; Pig Improvement Canada, Acme, AB) were assigned randomly within Parities 1, 2 or 3+ to a gestation diet containing either 0.44% (low lysine) or 0.55% (high lysine) total lysine and 3,100 kcal DE/kg; other indispensable amino acids were adjusted to lysine based on ideal protein ratios. Feed allowance in gestation was determined factorially using estimated DE requirements for maintenance, maternal gain, and conceptus growth. Sows were allowed free access to the lactation diet. Gestation BW gain from d 0 to 110 was affected by parity (61.2, 60.0, and 42.3 kg for Parity 1, 2, and 3+, respectively; P < 0.05) but not (P > 0.10) by gestation lysine level. Sow BW changes from d 0 of lactation to weaning were affected by parity (0.5, 6.8, and 5.8 kg for Parity 1, 2, and 3+, respectively; P < 0.01) and gestation BW gain (P < 0.01), but not by gestation lysine level (5.0 vs 3.8 kg for low and high lysine, respectively; P > 0.10). Total piglets born was affected by parity (11.5, 12.1, and 12.5, for Parity 1, 2, and 3+, respectively; P < 0.01) and increased with increasing sow BW gain (P < 0.05). Total piglets born alive (mean = 11.2) was increased with increasing sow BW gain (P < 0.05). Total litter weight born alive was affected by parity (15.9, 18.6, and 19.4 kg for Parities 1, 2, and 3+, respectively; P < 0.01) and gestation BW gain (P < 0.05). The model used to determine daily energy intake requirements resulted in an average BW gain of 10.6 kg above the targets set by the model. Total lysine intakes greater than 10.6 g/d in gestation did not improve sow productivity. Setting target weight gains in gestation and feeding to meet these targets may not always provide predictable results due to a number of factors that affect the energy requirement in the sow.

Reproductive performance is steadily increasing within the pork industry; logically, amino acid requirements need to be redefined for sows producing larger litters. The objective of this study was to determine the threonine requirement of the high-producing lactating sow and to determine the effect of lysine on this requirement. A total of 419 PIC C-15 sows were assigned randomly to treatment within parity groups (1, 2, and 3+) and gestation treatment at d 110 of gestation. Lactation diets were formulated to contain 0.80% total lysine (tLYS) with 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, or 0.65% total threonine (tTHR) or 1.06% tLYS with 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, or 0.70% tTHR. Litters were standardized to a minimum of 11 piglets within 48 h after farrowing, and sows had free access to feed throughout lactation (lactation length = 20.1 +/- 0.1 d). Sow ADFI exceeded expectation, averaging 6.90, 7.40, and 7.20 kg/d for Parities 1, 2, and 3+, respectively. Daily tLYS intake was 58 g/d (47 g of apparent ileal digestible lysine [dLYS] per day) and 74 g/d (59 g dLYS/d) for the low- and high-lysine group, respectively. Lysine intake did not affect sow or litter performance (P > 0.10). Sows gained an average of 4.8 kg in lactation. Using regression analysis, BW gain was maximized at 0.54% tTHR for all parity groups (quadratic; P < 0.05). Litter weaning weight (67.1, 67.9, and 66.2 kg for Parities 1, 2, and 3+, respectively) and litter weight gain (2.49, 2.53, and 2.44 kg/d for Parities 1, 2, and 3+, respectively) were maximized at 0.53% tTHR using regression analysis, for all parity groups (quadratic; P < 0.05). Based on regression analysis, plasma urea nitrogen on d 10 and 18 was minimized at 0.54% tTHR (P < 0.05). Lysine levels in excess of 58 g of tLYS/d did not benefit sow or litter performance. The requirement for threonine to minimize sow tissue mobilization was 37, 40, and 38 g tTHR/d (28, 30, and 30 g of apparent ileal digestible threonine [dTHR] per day) for Parities 1, 2, and 3+ sows, respectively. The threonine required to maximize litter performance was 36, 39, and 38 g of tTHR/d (28, 30, and 29 g of dTHR/d) for Parities 1, 2, and 3+ sows, respectively. Alternatively, the requirement can be expressed as 14.3 g tTHR (11.8 g dTHR) per kilogram of litter gain.

Insulin regulates the inclusion of the exon encoding protein kinase C (PKC) betaII mRNA. In this report, we show that insulin regulates this exon inclusion (alternative splicing) via the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway through the phosphorylation state of SRp40, a factor required for insulin-regulated splice site selection for PKCbetaII mRNA. By taking advantage of a well known inhibitor of PI 3-kinase, LY294002, we demonstrated that pretreatment of L6 myotubes with LY294002 blocked insulin-induced PKCbetaII exon inclusion as well as phosphorylation of SRp40. In the absence of LY294002, overexpression of SRp40 in L6 cells mimicked insulin-induced exon inclusion. When antisense oligonucleotides targeted to a putative SRp40-binding sequence in the betaII-betaI intron were transfected into L6 cells, insulin effects on splicing and glucose uptake were blocked. Taken together, these results demonstrate a role for SRp40 in insulin-mediated alternative splicing independent of changes in SRp40 concentration but dependent on serine phosphorylation of SRp40 via a PI 3-kinase signaling pathway. This switch in PKC isozyme expression is important for increases in the glucose transport effect of insulin. Significantly, insulin regulation of PKCbetaII exon inclusion occurred in the absence of cell growth and differentiation demonstrating that insulin-induced alternative splicing of PKCbetaII mRNA in L6 cells occurs in response to a metabolic change.

Acetate kinase, an enzyme widely distributed in the Bacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the alpha-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.

Protein kinases and phosphatases are likely targets for the development of therapeutic drugs since they are involved in specific signaling pathways which regulate cell functions such as metabolism, cell cycle progression, cell adhesion, vascular function and angiogenesis. Protein phosphorylation and dephosphorylation serve as molecular switches for modulating these processes and the level and duration of each is a highly regulated process in normal cells. Several compounds that inhibit the activity of tyrosine kinases are being evaluated as cancer therapeutic agents in clinical trials. Diabetes and complications of diabetes also involve deregulated levels of protein kinases. New approaches for regulating kinase gene expression include specific antisense oligonucleotides for inhibiting post-transcriptional processing of the messenger RNA, naturally occurring products and their chemical derivatives to inhibit kinase activity, and monoclonal antibodies to inhibit receptor linked kinases. Inhibition of phosphatases also serves to alter the duration of phosphorylation by kinases. Considerations for development of effective inhibitors include non-specific actions of compounds, cellular uptake, multiple intracellular targets that can dilute the effective cellular concentration of an agent, and tissue specificity. Kinase inhibitors may allow other therapeutic agents additional time to become effective and they may act synergistically with current treatments.

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.

Protein kinase C (PKCtheta) is a key enzyme in regulating a variety of cellular functions, including growth and differentiation. PKCtheta is the most abundant PKC isoform expressed in skeletal muscle; however, its role in differentiation and metabolism is not clear. We examined the effect of muscle cell differentiation on PKCtheta expression in human skeletal muscle cells from normal and type 2 diabetic subjects. Low levels of PKCtheta messenger RNA (mRNA) and protein were detected in human myoblasts from both types of subjects. Upon differentiation into myotubes, PKCtheta mRNA and protein were increased 12-fold in myotubes from normal subjects. In human skeletal muscle cells obtained from type 2 diabetic subjects, increases in PKCtheta mRNA and protein were not observed upon differentiation into myotubes although expression of other markers of differentiation and fusion increased. Cells from type 2 diabetic subjects also exhibited decreased insulin-stimulated glycogen synthase activity. To determine whether the up-regulation of PKCtheta was important for the metabolic actions of insulin, PKCtheta was overexpressed in L6 rat skeletal muscle cells. Increased expression of PKCtheta occurred with differentiation of skeletal muscle myoblasts to myotubes. Glycogen synthase activity was further increased in L6 myotubes stably transfected with the complementary DNA for PKCtheta. The decreased expression of PKCtheta found in cells from type 2 diabetic subjects may be linked to insulin resistance and decreased glycogen synthase activity.

Bone marrow stromal cells (BMSC) normally give rise to bone, cartilage, and mesenchymal cells. Recently, bone marrow cells have been shown to have the capacity to differentiate into myocytes, hepatocytes, and glial cells. We now demonstrate that human and mouse BMSC can be induced to differentiate into neural cells under experimental cell culture conditions. BMSC cultured in the presence of EGF or BDNF expressed the protein and mRNA for nestin, a marker of neural precursors. These cultures also expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN). When labeled human or mouse BMSC were cultured with rat fetal mesencephalic or striatal cells, a small proportion of BMSC-derived cells differentiated into neuron-like cells expressing NeuN and glial cells expressing GFAP., (Copyright 2000 Academic Press.)

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.)

The paradigm for the response to hypoxia is erythropoietin gene expression; activation of hypoxia-inducible factor-1 (HIF-1) results in erythropoietin production. Previously, we found that oxygen deprivation induced tissue factor, especially in mononuclear phagocytes, by an early growth response (Egr-1)-dependent pathway without involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C., Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8298-8303). Now, we show that cultured monocytes subjected to hypoxia (pO2 approximately 12 torr) displayed increased Egr-1 expression because of de novo biosynthesis, with a approximately 10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase constructs localized elements responsible for hypoxia-enhanced expression to -424/-65, a region including EBS (ets binding site)-SRE (serum response element)-EBS and SRE-EBS-SRE sites. Further studies with each of these regions ligated to the basal thymidine kinase promoter and luciferase demonstrated that EBS sites in the element spanning -424/-375 were critical for hypoxia-enhanceable gene expression. These data suggested that an activated ets factor, such as Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced activation of Elk-1, and suppression of Elk-1 blocked up-regulation of Egr-1 transcription. The signaling cascade preceding Elk-1 activation in response to oxygen deprivation was traced to activation of protein kinase C-betaII, Raf, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase and mitogen-activated protein kinases. Comparable hypoxia-mediated Egr-1 induction and activation were observed in cultured hepatoma-derived cells deficient in HIF-1beta and wild-type hepatoma cells, indicating that the HIF-1 and Egr-1 pathways are initiated independently in response to oxygen deprivation. We propose that activation of Egr-1 in response to hypoxia induces a different facet of the adaptive response than HIF-1, one component of which causes expression of tissue factor, resulting in fibrin deposition.

Primary cells of renal proximal tubule epithelium (S1 segment) of human kidney (HRPTE cells) up-regulate aquaporin-1 (AQP-1) expression in response to hyperosmolarity. NaCl and D(+)-raffinose increased (2-2.5 fold) AQP-1 expression when medium osmolarity was 400 and 500 mOsm/kg.H2O. Urea did not have this effect. Unlike our previous findings with mIMCD-3 cells, vasopressin (10(-8)M) did not affect AQP-1 expression in HRPTE cells in isosmolar or NaCl-enriched hyperosmolar conditions. Furthermore, HRPTE cells increased (3-4 fold) AQP-1 expression when exposed to hyperosmolar Reno-60 and Hypaque-76 (diatrizoates, ionic) contrast agents at 400 and 500 mOsm/kg.H2O. Isosmolar (290 mOsm/kg H2O) Visipaque (iodixanol, non-ionic) at 10% (v/v) concentrations also increased AQP-1 expression, and 25% v/v of Visipaque rendered morphological alterations of HRPTE cells and a 3-fold increase in AQP-1 expression after 24h exposure. Finally, semi-quantitative RT-PCR of HRPTE cells subjected to various isosmolar or hyperosmolar conditions demonstrated up-regulation of AQP-1 mRNA and protein levels. Our results suggest AQP-1 up-regulation in HRPTE cells exposed to environmental stresses such as hyperosmolarity and high doses of isosmolar contrast agents., (Copyright 1999 Academic Press.)

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.

The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity-purified protein was identical to that of the RNA-binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCbetaII-dependent FUS phosphorylation. Moreover, suppression of PKCbetaII activity in BCR/ABL-expressing cells by treatment with the PKCbetaII inhibitor CGP53353, or by expression of a dominant-negative PKCbetaII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) beta-chain expression, and accelerated G-CSF-stimulated differentiation. Downregulation of FUS expression in BCR/ABL-expressing 32Dcl3 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression.

The role of protein kinase C (PKC) on proliferation of A10 vascular smooth muscle cells (VSMC) was studied by overexpressing specific PKC-beta I and -beta II isozymes. PKC-beta I and -beta II are derived from alternative splicing of the exon encoding the carboxy-terminal (C-terminal) 50 or 52 amino acids, respectively. The differential functions of the two isozymes with regard to cell proliferation, DNA synthesis, and the cell cycle were investigated in A10 cells, a clonal cell line of VSMC from rat aorta, and in A10 cells overexpressing PKC-beta I and PKC-beta II (beta I-A10 and beta II-A10). PKC levels were increased three- to fourfold in heterogeneous cultures of stably transfected cells. Although doubling time of A10 cells was 36 h, the cell doubling time in beta I-A10 cells decreased by 12 h, and, in contrast, the doubling time of beta II-A10 cells increased by 12 h compared to A10 cells. The increase of [3H]thymidine (TdR) incorporation was accelerated and increased in beta I-A10 cells, but slowed and diminished in beta II-A10 cells compared to A10 and control cells transfected with empty vector. Cell cycle analysis of beta I-A10 cells showed an acceleration of S phase entry while beta II-A10 cells slowed S phase entry. These results suggest that PKC-beta I and PKC-beta II regulate cell proliferation bidirectionally and that PKC-beta I and PKC-beta II may have distinct and opposing functions as cell cycle check point mediators during late G1 phase and may regulate S phase entry in A10 VSMC.

The protein kinase Cbeta (PKCbeta) gene encodes two isoforms, PKCbetaI and PKCbetaII, as a result of alternative splicing. The unique mechanism that underlies insulin-induced alternative splicing of PKCbeta pre-mRNA was examined in L6 myotubes. Mature PKCbetaII mRNA and protein rapidly increased >3-fold following acute insulin treatment, while PKCbetaI mRNA and protein levels remained unchanged. Mature PKCbetaII mRNA resulted from inclusion of the PKCbetaII-specific exon rather than from selection of an alternative polyadenylation site. Increased PKCbetaII expression was also not likely accounted for by transcriptional activation of the gene or increased stabilization of the PKCbetaII mRNA, and suggest that PKCbetaII expression is regulated primarily at the level of alternative splicing. Insulin effects on exon inclusion were observed as early as 15 min after insulin treatment; by 20 min, a new 5'-splice site variant of PKCbetaII was also observed. After 30 min, the longer 5'-splice site variant became the predominate species through activation of a downstream 5' splice site. Similar results were obtained using IGF-I. Although the role of this new PKCbetaII mRNA species is presently unknown, inclusion of either PKCbetaII-specific exon results in the same PKCbetaII protein.

The in-vitro effects of human interferon alpha-2b (HuIFN alpha-2b), protein kinase C (PKC) agonist [TPA (12-0-tetra-decanoyl-phorbol-13-acetate)] and PKC inhibitor (calphostin C) on human glioma (U-373 MG) PKC activity, cell proliferation and cell cycle were compared. HuIFN alpha-2b and TPA increased PKC activity, elevated the number of cells in DNA synthesis (S) phase and decreased cell proliferation by similar magnitudes. Calphostin C inhibited PKC activity, increased the number of cells in S phase and produced strong cytotoxic effects (IC50 150 nM). Higher concentrations of calphostin C with or without serum induced an additional block in gap2 and mitosis. We conclude that HuIFN alpha-2b's mode of action may be directly or indirectly affecting PKC. The response produced by HuIFN alpha-2b is similar to TPA (potent PKC activation and S phase arrest).

Alternative splicing of pre-mRNA encoding the carboxy-terminal (C-terminal) exons of protein kinase C beta (PKC beta) leads to the expression of two protein isoforms, PKC beta 1 and PKC beta II, with the potential for different functions. PKC beta II expression is regulated by insulin via alternative mRNA splicing. A physiological consequence of its activation was investigated in L6 rat skeletal muscle cells expressing GLUT4 transporters, a cell line in which PKC is involved in glucose transport. We examined the contribution of PKC beta II for insulin-stimulated [3H]2-deoxyglucose uptake by constructing three PKC beta II C-terminal deletion mutants designated M216, M217, and M218. When transiently expressed in COS1 cells, M217, with nine amino acids deleted, demonstrated autophosphorylation activity 10-fold less than full-length PKC beta II. The mutants M218, with 13 amino acids deleted, and M216, with 52 amino acids deleted, demonstrated no autophosphorylation activity and are kinase negative. When transiently expressed in L6 myotubes, M217 inhibited insulin-stimulated 2-deoxyglucose uptake by 45% (with a 45% transfection efficiency) whereas M216 and M218, kinase-negative mutants, had no effect compared with cells transfected with control plasmid. Cotransfection of full-length PKC beta II with M217 was able to rescue the inhibition of insulin-stimulated 2-deoxyglucose uptake as compared with cotransfection of M217 with the control plasmid, suggesting that M217 acts as a dominant-negative. In contrast, cotransfection of full-length PKC beta I, the other alternatively spliced form, did not rescue inhibition of insulin-stimulated 2-deoxyglucose uptake by M217. To further demonstrate the involvement of PKC, specifically PKC beta II, in insulin-stimulated 2-deoxyglucose uptake, we used two inhibitors, CG41251 (a specific PKC inhibitor) and CG53353 (a PKC beta II-specific inhibitor at 1 microM). Both inhibited insulin-stimulated 2-deoxyglucose uptake 50-60% in L6 myotubes. We conclude that M217 may act as a specific PKC beta II dominant-negative and that PKC beta II is more specific for insulin-stimulated 2-deoxyglucose uptake in these cells than PKC beta I.

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.

Integrins from normal human renal cortex epithelial cells (RCEC) and from four renal carcinoma lines (metastatic Caki-1, non-metastatic Caki-2, metastatic ACHN, and non-metastatic 769-P) were compared by immunoprecipitation with specific anti-integrin antibodies. Integrin alpha 2 was present in normal RCEC, but absent in all four tumor lines. There was a 2.0-3.0 fold decrease of alpha 3 and beta 1 in localized tumor lines, and a further 5.0-7.0 fold decrease in metastatic lines over their expression in normal renal cells. No alpha V was detected in Caki-1 cells. The greatest adhesion of all cells occurred in the presence of a stimulatory anti-alpha 3 antibody, mediated by specific matrix proteins employed as substrates, while anti-beta 1 treatment dramatically inhibited cell attachment on collagen IV, plasma fibronectin, laminin and merosin substrates. In addition, the mRNA expression of focal adhesion kinase (p125FAK) and paxillin were up-regulated (2.0-2.5 fold increase) in the metastatic Caki-1 cells over normal RCEC. The alteration of integrin subunits alpha 2, alpha 3, alpha V, beta 1, as well as p125FAK and paxillin may contribute to the pathogenicity and/or metastatic propensity of renal epithelial tumors. The up-regulation of paxillin independently or in concert with p125FAK as shown in this study indicates its significant role as a potential marker of metastasis in renal carcinoma cells.

Transmission electron microscopy was used to determine how immunogold labeling of PKC-alpha or -beta is modulated by the antitumor drug IFN (HuIFN alpha-2b) in the cytoplasm, membrane structures, and nucleus of rapidly dividing and confluent human glioma U-373 cells. Results showed that except for nuclear localization, there were no specific cytoplasmic organelles that PKC-alpha or -beta translocated to following HuIFN alpha-2b treatment. Electron micrographs of PKC-beta in proliferating cells depicted 1.34-fold more PKC-beta in the nucleus than in the cytoplasm and a 1-min HuIFN alpha-2b (500 units/ml) treatment transiently increased PKC-beta immunoreactivity in the cytoplasm (1.95-fold) and nucleus (1.97-fold). In confluent cells, incubation with HuIFN alpha-2b for 2 min significantly decreased cytoplasmic PKC-beta immunoreactivity by 37%, and no change was observed in nuclear PKC-beta labeling. PKC-alpha labeling in proliferating cells showed similar immunoreactivity in both control cytoplasm and nucleus. Treatment of proliferating cells with HuIFN alpha-2b for 2 min decreased PKC-alpha in the cytoplasm (59%) and nucleus (44%). In confluent cells, cytoplasmic PKC-alpha labeling decreased 59% at 1 min, 61% at 2 min, and 76% at 10 min of HuIFN alpha-2b treatment. Nuclear PKC-alpha decreased by 65% at 1 min, 80% at 2 min, and 62% at 10 min after HuIFN alpha-2b treatment. Western blots of total PKC-alpha in proliferating and confluent cells and PKC-beta in confluent cells showed similar results. However, Western blots of total PKC-alpha and -beta in proliferating cells did not demonstrate any significant changes in either PKC-alpha or -beta immunoreactivity following 1-min HuIFN alpha-2b treatment. These results suggest that treatment of proliferating U-373 cells with HuIFN alpha-2b for 1 min unfolds and exposes PKC-beta antigenic sites (hinge region) and increases in situ PKC-beta immunogold labeling.

Protein kinase C (PKC) is an important intracellular signalling enzyme. Numerous studies have suggested that alterations in this enzyme occur in aging and dementia. The objective of this study was to examine PKC in the cerebral microcirculation in aging and Alzheimer's disease. PKC activity, amount, and isoform distribution were analyzed in microvessels from adult and aged rodents as well as from Alzheimer patients and nondemented elderly controls. PKC activity was lower in Alzheimer vessels than in vessels from control brains, despite the presence of similar levels of PKC enzyme. In contrast, both activity and enzyme levels in young and aged rats were comparable. The beta-isoform was present in both rat and human microvessels and there were no age- or disease-related alterations. The loss in activity in cerebromicrovascular PKC in Alzheimer's suggest that perturbations in phosphorylation signalling cascades may exist at the Alzheimer blood-brain barrier.

Insulin regulates a diverse array of cellular signaling processes involved in the control of growth, differentiation, and cellular metabolism. Insulin increases glucose transport via a protein kinase C (PKC)-dependent pathway in BC3H-1 myocytes, but the function of specific PKC isozymes in insulin action has not been elucidated. Two isoforms of PKC beta result via alternative splicing of precursor mRNA. As now shown, both isoforms are present in BC3H-1 myocytes, and insulin induces alternative splicing of the PKC beta mRNA thereby switching expression from PKC beta I to PKC beta II mRNA. This effect occurs rapidly (15 min after insulin treatment) and is dose-dependent. The switch in mRNA is reflected by increases in the protein levels of PKC beta II. High levels of 12-0-tetradecanoylphorbol-13-acetate, which are commonly used to deplete or down-regulate PKC in cells, also induce the switch to PKC beta II mRNA following overnight treatment, and protein levels of PKC beta II reflected mRNA increases. To investigate the functional importance of the shift in PKC beta isoform expression, stable transfectants of NIH-3T3 fibroblasts overexpressing PKC beta I and PKC beta II were established. The overexpression of PKC beta II but not PKC beta I in NIH-3T3 cells significantly enhanced insulin effects on glucose transport. This suggests that PKC beta II may be more selective than PKC beta I for enhancing the glucose transport effects of insulin in at least certain cells and, furthermore, that insulin can regulate the expression of PKC beta II by alternative mRNA splicing.

Protein kinase C (PKC)-theta is a newly recognized major PKC isoform in skeletal muscle. In this study we found that insulin provoked rapid biphasic increases in membrane-associated immunoreactive PKC-theta, as well as PKC-alpha, PKC-beta and PKC-epsilon, in rat soleus muscles incubated in vitro. Effects of insulin on PKC isoforms in the soleus were comparable in magnitude with those of phorbol esters. Increases in membrane-associated PKC-theta, PKC-alpha, PKC-beta and PKC-epsilon were also observed in rat gastrocnemius muscles after insulin treatment in vivo. Our findings suggest that PKC-theta, like other diacylglycerol-sensitive PKC isoforms (alpha, beta and epsilon), may play a role in insulin action in skeletal muscles.

We evaluated the role of protein kinase-C (PKC) during insulin action in HIRC-B cells. Insulin provoked rapid increases in 1) diacylglycerol; 2) translocation of PKC epsilon, but not PKC alpha, PKC delta, or PKC zeta, from the cytosol to the membrane fraction; 3) membrane PKC enzyme activity; and 4) phosphorylation of immunoprecipitable 80-kilodalton (kDa) myristylated alanine-rich C-kinase substrate (MARCKS) protein and heat-stable 80-kDa protein (also probably MARCKS). Phorbol esters stimulated the translocation of PKC alpha and PKC delta as well as PKC epsilon, but not PKC zeta. The effects of phorbol esters on 80-kDa MARCKS phosphorylation were approximately 4 times as strong as those of insulin. Treatment of HIRC-B cells with phorbol esters for 20-24 h resulted in complete loss of immunoreactive PKC alpha and PKC delta in cytosol and membrane fractions, but substantial amounts of PKC epsilon were persistently translocated to the membrane fraction of down-regulated cells. This persistently translocated, residual PKC epsilon in down-regulated cells was associated with increased basal hexose uptake, but this was not due to PKC activation, as it was not inhibited by the PKC inhibitor, RO 31-8220. Acute insulin treatment, on the other hand, increased hexose uptake in down-regulated cells, and this insulin-stimulated uptake was inhibited by RO 31-8220 in down-regulated cells as well as in nondown-regulated cells. Insulin also stimulated the phosphorylation of the heat-stable 80-kDa protein in down-regulated cells, suggesting that the residual PKC epsilon in these cells can be activated by insulin.

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats.

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.

The presence of ANP prohormone immunoreactivity in rat GI tract suggests that it may be an extracardiac site of ANP synthesis. The aim of this study was to investigate the expression of ANP mRNA in the adult rat GI tract. ANP mRNA was detected by ribonuclease protection analysis in stomach, small and large intestines, and rectum/anus. The highest concentrations of ANP transcripts were found in the proximal stomach, antrum, proximal colon, and rectum/anus at levels that ranged from 1 to 10% of that found in cardiac ventricle. Northern blot analysis of total RNA from these tissues identified a single 0.9 kb ANP transcript similar to that detected in heart. Gel filtration chromatography of tissue extracts provided evidence for the presence of the complete ANP prohormone in proximal stomach, antrum, proximal colon and rectum/anus. These results demonstrate that the gene for ANP is expressed in specific regions of the rat GI tract, suggesting that tissue-specific differential regulation of ANP synthesis occurs within the GI tract.

In this paper we examine whether sodium orthovanadate activates diacylglycerol (DAG)/protein kinase C (PKC) signaling systems that are activated by insulin in BC3H-1 myocytes. Like insulin, sodium orthovanadate provoked increases in membrane DAG, PKC enzyme activity, and immunoreactive PKC-beta. Concomitantly, both PKC enzyme activity and immunoreactive PKC-beta decreased in the cytosol, suggesting that sodium orthovanadate, like insulin, stimulated the translocation of PKC-beta from the cytosol to the membrane fraction. Sodium orthovanadate was also found to activate phospholipid signaling pathways that were previously reported to be activated by insulin, viz., inositol-lipid synthesis/turnover; phosphatidylcholine hydrolysis; and de novo phospholipid synthesis by activation of glycerol-3-PO4 acyltransferase. Our findings suggest that vanadate mimics insulin in the activation of specific phospholipid/DAG/PKC signaling pathways.

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.

Immediate and sustained signal transduction is involved in mediating phorbol ester-induced changes in growth and differentiation. Activation of protein kinase C (PKC) is the initial step in phorbol ester-induced signal transduction. By virtue of preferential down-regulation of individual isoforms and generation of proteolytically derived kinase activities, the signal transduced by sustained activation of this pathway may differ substantially from that generated initially upon application of the phorbol ester. To examine the effect of chronic phorbol ester-induced activation of this pathway, the relationship between PKC activity/content and AP-1 binding activity and gene expression was studied in the U937 cell. Phorbol ester-induced differentiation of the U937 cell into a monocyte/macrophage-like cell requires sustained activation of the PKC pathway. AP-1 binding activity was enhanced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and in a temporally dependent manner, with conversion of a high to low mobility band shift occurring after a 12-h exposure to TPA. After a 72-h exposure, AP-1 binding activity was maximally increased by 1 nM TPA and remained elevated to a similar degree even after treatment with 600 nM TPA. Enhanced AP-1 binding activity was dependent upon continuous exposure to TPA and was not secondary to differentiation. A 72-h treatment with one nM TPA maximally increased expression of c-jun, krox-24, and jun-B mRNA transcripts. Exposure to higher TPA concentrations decreased the content of these transcripts. Maximal expression of collagenase and plasminogen activator receptor transcripts required exposure to much higher TPA concentrations (100 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of protein kinase C is a key event in the transduction of receptor-mediated extracellular signals. Little is known about the role of protein kinase C in the microcirculation of the brain. In this study, we examined protein kinase C in isolated cerebral microvessels. A technique for partial purification of protein kinase C from microvessels was employed, using Q-Sepharose batch adsorption and single-step salt elution in microfuge tubes. This procedure greatly reduced variability and increased protein kinase C specific activity in both the cytosolic and particulate fractions by nearly 50-fold. The identity of the enzyme was confirmed by its inhibition by staurosporine and bisindolylmaleimide and by its translocation in response to phorbol ester. The level of protein kinase C was assessed by [3H]phorbol ester binding and the endogenous substrates evaluated by in vitro phosphorylation studies. Finally, western blot analysis of protein kinase C isoforms indicated that the beta-isoform was present in both cytosolic and particulate fractions. The alpha-isoform was present at low levels in the cytosolic fraction, whereas the gamma-isoform was not detected.

Skeletal muscle is one of the first tissues to become insulin resistant in genetically obese rodents. The activation of protein kinase-C (PKC) in rat skeletal muscle is mediated by insulin stimulation of diacylglycerol (DAG) levels. Defects in the activation of PKC in the heart and liver of obese Zucker rats indicate that an abnormality in either stimulation of DAG or PKC occurs in obese tissues. DAG levels were significantly increased in soleus muscle from 15- to 19-week-old obese (fa/fa) Zucker rats. PKC activity was diminished in soleus muscle from fa/fa rats. Decreased levels of PKC alpha and -beta activity wer enoted after resolution of common PKC isozymes (Ca2+ and phospholipid dependent) by hydroxyapatite chromatography. Immunoreactivity of PKC-alpha, -beta, and -epsilon also indicated that their levels are diminished in fa/fa soleus muscle by 70-90%. To determine at which level down-regulation occurs (i.e. gene expression or protein turnover), mRNA levels were examined by Northern blot analysis of total RNA. PKC alpha and -beta levels were diminished in Zucker obese soleus muscle compared to soleus from Zucker lean control (fa/-) animals, and PKC epsilon mRNA was not detected on the same blots. The transcript size for PKC beta mRNA in Zucker soleus muscle was unique. Both lean and obese Zucker muscle tissues expressed three transcripts that hybridized with the full-length PKC beta cDNA probes, with sizes ranging between 2.5-1.7 kilobases. Levels of all PKC beta transcripts were significantly decreased in obese Zucker tissues. Thus, levels of common PKC isozyme mRNA, protein, and enzyme activity in soleus muscle of the obese Zucker rat are decreased even though levels of the endogenous PKC activator DAG are elevated. The decreased levels of PKC may be related to the etiology of insulin resistance in skeletal muscle.

To evaluate the question of whether or not insulin activates protein kinase C (PKC), we compared the effects of insulin and phorbol esters on the phosphorylation of the PKC substrate, i.e. myristoylated alanine-rich C-kinase substrate (MARCKS). In rat adipocytes, rat soleus muscle and BC3H-1 myocytes, maximally effective concentrations of insulin and phorbol esters provoked comparable, rapid, 2-fold (on average), non-additive increases in the phosphorylation of immunoprecipitable MARCKS. These effects of insulin and phorbol esters on MARCKS phosphorylation in intact adipocytes and soleus muscles were paralleled by similar increases in the phosphorylation of an exogenous, soluble, 85 kDa PKC substrate (apparently a MARCKS protein) during incubation of post-nuclear membrane fractions in vitro. Increases in the phosphorylation of this 85 kDa PKC substrate in vitro were also observed in assays of both plasma membranes and microsomes obtained from rat adipocytes that had been treated with insulin or phorbol esters. These insulin-induced increases in PKC-dependent phosphorylating activities of adipocyte plasma membrane and microsomes were associated with increases in membrane contents of diacylglycerol, PKC-beta 1 and PKC-beta 2. Our findings suggest that insulin both translocates and activates PKC in rat adipocytes, rat soleus muscles and BC3H-1 myocytes.

In rat adipocytes and soleus muscles, 2-hydroxypropyl-beta-cyclodextrin (CD) was found to have a relatively small or no effect on basal or insulin-stimulated hexose uptake, but markedly enhanced hexose uptake effects of phorbol esters and/or diacylglycerol. In rat adipocytes, the CD-induced enhancement of hexose uptake during concurrent phorbol ester treatment was not associated with an increase in GLUT4 glucose transporter translocation to the plasma membrane, which was stimulated comparably by insulin and phorbol esters. Moreover, CD appeared to activate or facilitate the activation of glucose transporters subsequent to their translocation to the plasma membrane during ongoing phorbol ester treatment. In rat adipocytes, CD also enhanced the translocation of protein kinase C (PKC)-beta to the plasma membrane during the action of phorbol esters, which alone had little or no effect on this specific PKC translocation. Although it is uncertain how CD alters the function of plasma membranes to enhance the translocation of PKC-beta to, and the activation of glucose transporters within, this subcellular fraction during phorbol ester treatment, our findings provide direct support for a two-step model in the activation of glucose transport. In addition, it seems clear that, at least in some cell types, simple phorbol ester treatment does not necessarily serve as a ubiquitous activator of all activable PKC pools and all potential PKC-mediated responses.

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.

In rat adipocytes, chronic incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced immunoreactive protein kinase-C (PKC) beta, gamma, delta, and zeta isoforms by 40-60% and PKC alpha by 75%, but had little effect on PKC epsilon levels. In BC3H-1 myocytes, chronic TPA treatment had no effect on PKC beta, increased PKC zeta, and depleted PKC alpha. Acute treatment with insulin induced the translocation of PKC beta in the myocytes both before and after chronic TPA treatment, but had no acute effect on the alpha or zeta isoforms. In contrast, acute TPA treatment in the myocytes had little effect on PKC beta, but induced the rapid translocation of alpha and zeta. The differential effects of chronic TPA treatment on the down-regulation of PKC beta may explain why insulin continues to activate biological processes in TPA-treated BC3H-1 myocytes, but not in adipocytes.

Effects of insulin and phorbol esters on subcellular distribution of protein kinase C (PKC) isoforms were examined in rat adipocytes. Both agonists provoked rapid decreases in cytosolic, and/or increases in membrane, immunoreactive PKC-alpha, PKC-beta, PKC-gamma, and PKC-epsilon. Effects of phorbol esters on PKC-alpha redistribution to the plasma membrane, however, were much greater than those of insulin. In contrast, insulin, but not phorbol esters, stimulated the translocation of PKC-beta to the plasma membrane, and provoked changes in PKC-zeta redistribution. Neither agonist altered subcellular distribution of PKC-delta, which was detected only in membrane fractions. Our findings indicate that insulin and phorbol esters have overlapping and distinctly different effects on the subcellular redistribution of specific PKC isoforms.

Insulin has been reported to translocate protein kinase C (PKC) in rat adipocytes, and activation of PKC by phorbol esters is known to increase hexose uptake in these cells (1.2). To test the hypothesis that PKC may participate in insulin-stimulated hexose uptake, adipocytes were partially depleted of protein kinase C by overnight phorbol ester treatment, thereby impairing insulin effects on hexose uptake. Purified PKC was then introduced into these PKC-depleted adipocytes by electropermeabilization, and this fully restored insulin-stimulated hexose uptake. These findings provide direct evidence that PKC is required for insulin-stimulated hexose uptake.

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.

Insulin is known to rapidly stimulate and/or translocate Ca2+/phospholipid-dependent protein kinase (conventional PKC; cPKC) in rat adipocytes. Presently we examined whether insulin also stimulates/translocates Ca(2+)-independent, phospholipid-dependent protein kinase (novel PKC; nPKC). Total Mono Q column-elutable nPKC (like cPKC) activities were decreased in cytosolic and increased in membrane fractions with insulin treatment. Immunoblot study of novel PKC epsilon also showed insulin-induced translocation of immunoreactive PKC from cytosol to membrane, similar to the translocation of cPKC, PKC beta. These results suggest that nPKC has an important role in insulin-induced signal transduction.

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.

Treatment of intact adipocytes with the autoregulatory PKC pseudosubstrate PKC(19-31) inhibited insulin-stimulated hexose uptake and lipogenesis, with no effect on basal values. The effect was dose-dependent with respective IC50 values of 30 microM and 600 microM for insulin-stimulated hexose uptake in electroporated and intact adipocytes. These studies indicate that PKC may play a role in the mediation of insulin action in adipocytes.

Rat adipocytes were treated with antisense dimethoxytrityl pentadecadeoxynucleotides, complementary to mRNA initiation codon regions for alpha and beta isozymes of protein kinase C (PKC). This antisense treatment provoked 50-70% decreases in PKC and insulin-stimulated 2-deoxyglucose uptake, but did not inhibit insulin-stimulated diacylglycerol synthesis. Sense or nonsense oligodeoxynucleotides were without effect on PKC and 2-deoxyglucose uptake. These results suggest that: (i) PKC-alpha and PKC-beta isozymes can be specifically downregulated in rat adipocytes by antisense oligodeoxynucleotides, and (ii) insulin-stimulated glucose transport requires PKC.