The inhibition of phosphoenolpyruvate carboxykinase (guanosine triphosphate) gene expression by insulin is not mediated by protein kinase C

The role protein kinase C plays in the regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin and phorbol esters was studied in H4IIE hepatoma cells (ATCC CRL 1548). The combined effects of phorbol 12-myristate 13-acetate (PMA) and insulin on the suppression of mRNA coding for PEPCK (mRNAPEPCK) synthesis were additive. A potent inhibitor of both cyclic nucleotide-dependent protein kinases and protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, inhibited the cAMP and PMA-mediated regulation of mRNAPEPCK synthesis, but did not affect the action of insulin. Desensitization of the protein kinase C pathway by exposure to PMA for 16 h abolished the subsequent action of the phorbol ester, but did not affect insulin- or cAMP-mediated regulation of PEPCK gene expression. We conclude that insulin suppresses PEPCK gene expression independently from the protein kinase C-mediated pathway used by phorbol esters.     

Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin.

The regulation of 3-O-methyl-D-glucose (OMG) uptake by insulin and phorbol esters was studied in cultured human skin fibroblasts. Insulin rapidly stimulated OMG uptake through a mechanism independent of new protein synthesis. Maximal insulin effect was reached in 30 min and remained constant up to 12 h. The protein kinase C activators 12-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PdBU) promoted an initial rapid stimulation followed by a secondary long-term rise of OMG influx. This latter effect of phorbol esters on OMG influx began after 1 h, reached a maximum in 12-15 h, and was prevented by the simultaneous addition of protein synthesis inhibitors, suggesting that phorbol esters increased the synthesis of new glucose transporters. In accord with this interpretation, phorbol esters, but not insulin, increased mRNA levels for two distinct glucose transporters (GLUT1 and GLUT3) in human fibroblasts. Both the rapid and the long-term effects of phorbol esters on OMG influx were dose-dependent and half-maximal stimulations occurred at 15 nM for both PdBU and TPA. Kinetic analysis of OMG uptake indicated that both effects of phorbol esters were associated with an increase in the Vmax of the transport process, with no significant changes of the Km (4-6 mM). These results suggest that, in human fibroblasts, phorbol esters, unlike insulin, produce a long-term stimulation of OMG uptake, which is dependent upon protein synthesis and is associated with increased levels of GLUT1 and GLUT3 mRNA.     

Insulin-like growth factor 1 and insulin reduce epidermal growth factor binding to Swiss 3T3 cells by an indirect mechanism that is apparently independent of protein kinase C

Insulin-like growth factor 1 and insulin reduced the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells by 15-20% at 37 degrees C, but not at 4 degrees C. Scatchard analysis indicated that IGF-1 and insulin affected the higher-affinity component of EGF binding, an effect previously associated with the activation of protein kinase C. However, the inhibition of 125I-EGF binding by IGF-1 and insulin was increased, not reduced, when the cells were treated with high concentrations of phorbol esters to down-modulate protein kinase C. We suggest that IGF-1 and insulin activate a protein kinase with similar or overlapping specificity to that of protein kinase C.     

Contribution of the C1A and C1B domains to the membrane interaction of protein kinase C

The hallmark for protein kinase C activation is its "translocation" to membranes following generation of lipid second messengers. This translocation is mediated by the C1 and C2 domains, two membrane-targeting modules, whose engagement on membranes provides the energy for an activating conformational change in which an autoinhibitory pseudosubstrate sequence is released from the active site. Novel and conventional protein kinase C isozymes contain a tandem repeat of C1 domains, the C1A and C1B, which each contain a binding pocket for phorbol esters/diacylglycerol. This study addresses the contribution of the C1A and C1B domains in the regulation of protein kinase C's membrane interaction using bisfunctional (dimeric) phorbol myristate acetate (PMA) molecules. We show that dimeric bisphorbols are an order of magnitude more effective at recruiting full-length PKC betaII to membranes compared with monomeric PMA and that the effectiveness of the interaction depends on the nature and length of the cross-link between the PMA moieties. Most effective were dimeric phorbol 12-acetate 13-esters linked at the 13 position with a 14 carbon spacer. The increased potency of dimeric phorbol esters is reduced if either the C1A or C1B domains are mutated so that they are unable to bind PMA, if one moiety of the dimer contains a nonfunctional phorbol, or if the binding to the isolated C1B domain is measured. Thus, the increased potency of the dimeric phorbol esters results primarily from their ability to engage, to a limited extent, both C1 modules on the same molecule. Although dimeric phorbols were more potent than monomeric phorbol esters in recruiting protein kinase C to membranes, the magnitude of the increase was still several orders of magnitude lower than what would be predicted on the basis of the reduction in dimensionality that occurs when the first C1 domain is engaged on the membrane. Thus, engaging both domains can be forced but is highly unfavored. In summary, our data reveal that both C1 domains are oriented for potential membrane interaction but only one C1 domain binds ligand in a physiological context.     

Rapid effects of phorbol esters on isolated rat adipocytes. Relationship to the action of protein kinase C

Treatment of isolated rat adipocytes with tumor-promoting phorbol esters, caused a fivefold stimulation of glucose oxidation, determined as 14CO2 production from [1-14C]glucose and a fivefold increase in the rate of lipid synthesis from [14C]glucose. Treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate increased the rate of 86Rb+ uptake into the cells. Also phospholipase C was able to stimulate the rate of glucose oxidation; phospholipase C and 12-O-tetradecanoylphorbol 13-acetate stimulated glucose oxidation in a non-synergistic fashion, indicating a common mechanism for their action. Active phorbol esters and, in part, also phospholipase C, caused a translocation of protein kinase C activity from the soluble to the particulate fraction of the adipocytes. This process was rapid, being complete 30 s after the addition of phorbol ester, and resulted in the appearance of the kinase mainly in the mitochondrial and plasma membrane fractions. A comparison between the binding characteristics of adipocyte protein kinase C and the metabolic effects of the phorbol esters on the adipocytes revealed that the dose-response relationship did not correlate with binding of the phorbol esters, but, rather, a correlation was observed between the dose of phorbol esters required for translocation of protein kinase C and the intracellular effects. The results indicate that the intracellular translocation of protein kinase C might be a trigger for the effects of phorbol esters on the adipocyte and that binding of the esters to protein kinase C is not a sufficient event to cause this effect. Furthermore, it is suggested that activation of protein kinase C might be partly the action of hormones, such as insulin, on the fat cells.     

Insulin and phorbol ester stimulate phosphorylation of a 15,000 dalton membrane protein in rat diaphragm in a similar manner

The effects of insulin on the phosphorylation of a 15 kilodalton (kDa) membrane protein in rat diaphragm in situ have been investigated. Incubation of the diaphragm with insulin or tumor-promoting phorbol ester increased the 32P-labelling of the 15 kDa protein at serine residues by 50 +/- 8% and 64 +/- 11%, (mean +/- S.E.), respectively. Thermolytic peptide mapping of the 15 kDa protein after insulin treatment of the diaphragm yielded two major phosphopeptides, one of which was absent from digests from control diaphragms. The same two phosphopeptides were identified after incubation of the diaphragm with phorbol ester and after phosphorylation of sarcolemma in vitro with [gamma-32P]ATP and protein kinase C. Additional experiments indicated that pretreatment of diaphragms with insulin or phorbol ester both increased the state of phosphorylation of the 15 kDa sarcolemma protein on phosphorylation sites regulated by protein kinase C. The stimulatory effect of insulin was decreased by staurosporine or by preincubation of the diaphragms with phorbol esters. These results indicate that the insulin-induced increases in protein kinase C activity previously found in rat diaphragm (Walaas et al. (1987) FEBS Lett. 220, 311-318) may be involved in insulin-mediated regulation of phosphorylation of the 15 kDa protein in situ.     

The activation of protein kinase G by daphnane, ingenane and tigliane diterpenoid esters

The activation of protein kinase C by daphnane, ingenane and tigliane diterpenoid eaters. In this review, the mechanism of action of phorbol esters and related diterpenes is described. These compounds have been shown to stimulate a Ca^{2 +} and phospholipid dependent protein kinase, termed kinase C. Phorbol esters activate protein kinase C by substituting for the natural effector, the second messenger, diacylglycerol. The various known protein substrates of this enzyme are described. Many of these substrates are involved in regulation of protein synthesis, DNA expression, cell transformation etc. This provides the explanation for the tumour promotion effects of some phorbol esters. Evidence for the biochemical mechanisms of action of phorbol esters that have other biological effects are also described. Recent evidence from our laboratories indicates that phorbol esters with limited biological effects, e.g. inflammatory but not tumour promoting, also act through this protein kinase. These phorbol esters appear to stimulate the phosphorylation of a different range of substrate proteins in vivo.     

Insulin increases membrane protein kinase C activity in rat diaphragm

Calcium/phospholipid-dependent protein kinase activity (protein kinase C) was identified in rat diaphragm membrane and cytosol fractions by means of in vitro phosphorylation either of histones or of a specific 87 kDa protein substrate, combined with phosphopeptide-mapping techniques. Both insulin and tumor-promoting phorbol ester treatment of the diaphragm preparations led to increased protein kinase C activity in the membrane fractions. In contrast to the phorbol ester, however, insulin did not induce a concomitant decrease in cytosolic activity, indicating that translocation of the enzyme had not taken place. Thus, insulin appears to increase specifically membrane protein kinase C activity in rat skeletal muscle, possibly through a mechanism not identical to that induced by phorbol esters.     

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.     

Phorbol ester effects on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells

Tumor promoting phorbol esters stimulate Ca++ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for 125I-HEAT binding to alpha 1-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate alpha 1-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating alpha 1-adrenergic receptor functional properties.     

Potentiation of specific association of insulin with HepG2 cells by phorbol esters.

The effects of tumour-promoting phorbol esters on the receptor-mediated endocytosis of insulin were investigated in the human hepatoma cell line HepG2. Treatment of these cells with the biologically active phorbol 12-O-tetradecanoylphorbol 13-acetate (TPA), but not with the non-tumour-promoting analogue 4 alpha-phorbol 12,13-didecanoate, resulted in dramatic morphological changes, which were accompanied by a 1.5-2.5-fold increase in specific 125I-insulin association with the cells at 37 degrees C. This increase in insulin binding was not observed when the binding reaction was performed at 4 degrees C. The potentiation of 125I-insulin association with TPA-treated cells at 37 degrees C could be completely accounted for by an increase in the intracellular pool of internalized insulin; there was no concomitant increase in cell-surface insulin binding. Dissociation studies showed that the enhanced internalization of insulin by cells after treatment with TPA resulted from a decrease in the rate of intracellular processing of the insulin after receptor-mediated endocytosis. The phorbol-ester-induced enhancement of internalized insulin in HepG2 cells was additive with the potentiation of endocytosed insulin induced by both the lysosomotropic reagent chloroquine and the ionophore monensin; this indicates that TPA affects the intracellular processing of the insulin receptor at a point other than those disrupted by either of these two reagents. The potentiation of insulin receptor internalization by tumour-promoting phorbol esters could be completely mimicked by treatment with phospholipase C, but not with phospholipase A, and partially mimicked by treatment with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol. By these criteria, the effects of phorbol esters on the insulin receptor in HepG2 cells appear to be mediated through protein kinase C. These results support the concept that the activation of protein kinase C by treatment with phorbol esters causes a perturbation of the insulin-receptor-mediated endocytotic pathway in HepG2 cells, reflected in a long-term decreased rate of dissociation of internalized insulin by the phorbol-ester-treated cells.     

Protein kinase C is not required for insulin stimulation of hexose uptake in muscle cells in culture.

The L6 skeletal muscle cell line has been identified as a suitable model to study the action of insulin on glucose uptake in muscle [Klip, Li & Logan (1984) Am. J. Physiol. 247, E291-E296]. The signals that transfer information from occupied insulin receptors to glucose transporters remain unknown. Here we report that activation of protein kinase C by exogenous phorbol esters results in stimulation of glucose uptake. Protein C kinase activity was induced to migrate from the cytosolic fraction to the microsomal fraction after 40 min of exposure of intact cells to 4 beta-phorbol 12,13-dibutyrate. In contrast, incubation with insulin did not alter the subcellular distribution of the kinase. Prolonged preincubation of L6 cells with phorbol esters resulted in depletion of kinase C activity, whereas neither the basal rate of glucose uptake nor its stimulation by insulin were affected. This suggests that protein kinase C is expressed in L6 cells, and that insulin stimulation of hexose transport does not involve protein kinase C.     

The binding of PRAS40 to 14-3-3 proteins is not required for activation of mTORC1 signalling by phorbol esters/ERK

PRAS40 binds to the mTORC1 (mammalian target of rapamycin complex 1) and is released in response to insulin. It has been suggested that this effect is due to 14-3-3 binding and leads to activation of mTORC1 signalling. In a similar manner to insulin, phorbol esters also activate mTORC1 signalling, in this case via PKC (protein kinase C) and ERK (extracellular-signal-regulated kinase). However, phorbol esters do not induce phosphorylation of PRAS40 at Thr(246), binding of 14-3-3 proteins to PRAS40 or its release from mTORC1. Mutation of Thr(246) to a serine residue permits phorbol esters to induce phosphorylation and binding to 14-3-3 proteins. Such phosphorylation is apparently mediated by RSKs (ribosomal S6 kinases), which lie downstream of ERK. However, although the PRAS40(T246S) mutant binds to 14-3-3 better than wild-type PRAS40, each inhibits mTORC1 signalling to a similar extent. Our results show that activation of mTORC1 signalling by phorbol esters does not require PRAS40 to be phosphorylated at Thr(246), bind to 14-3-3 or be released from mTORC1. It is conceivable that phorbol esters activate mTORC1 by a distinct mechanism not involving PRAS40. Indeed, our results suggest that PRAS40 may not actually be involved in controlling mTORC1, but rather be a downstream target of mTORC1 that is regulated in response only to specific stimuli, such as insulin.     

Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells

Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.     

Chimaerins, novel non-protein kinase C phorbol ester receptors, associate with Tmp21-I (p23): evidence for a novel anchoring mechanism involving the chimaerin C1 domain.

The regulation and function of chimaerins, a family of "non-protein kinase C" (PKC) phorbol ester/diacylglycerol receptors with Rac-GAP activity, is largely unknown. In a search for chimaerin-interacting proteins, we isolated Tmp21-I (p23), a protein localized at the perinuclear Golgi area. Remarkably, phorbol esters translocate beta2-chimaerin to the perinuclear region and promote its association with Tmp21-I in a PKC-independent manner. A deletional analysis revealed that the C1 domain in chimaerins is required for the interaction with Tmp21-I, thereby implying a novel function for this domain in protein-protein associations in addition to its role in lipid and phorbol ester binding. Our results support the emerging concept that multiple pathways transduce signaling by phorbol esters and revealed that, like PKC isozymes, chimaerins are subject to a positional regulation. In this setting, Tmp21-I serves as an anchoring protein that determines the intracellular localization of these novel phorbol ester receptors.     

Tumour-promoting phorbol esters increase basal and inhibit insulin-stimulated lipogenesis in rat adipocytes without decreasing insulin binding.

In isolated rat adipocytes, tumour-promoting phorbol esters caused (1) dose-dependent stimulation of lipogenesis in the absence of insulin and (2) inhibition of the lipogenic effect of submaximal concentrations of insulin, but without affecting insulin binding. The possible involvement of protein kinase C in insulin action is discussed.     

Phorbol esters, but not epidermal growth factor or insulin, rapidly decrease soluble protein kinase C activity in rat hepatocytes

Exposure of freshly isolated rat hepatocytes to tumor-promoting phorbol esters like phorbol 12-myristate 13-acetate resulted in a time- and concentration-dependent translocation of protein kinase C from the soluble to the particulate fraction of the cells. No such disappearance of soluble protein kinase C activity was observed with either epidermal growth factor or insulin, indicating that activation of protein kinase C is not necessarily involved in the short-term metabolic action of physiological growth factors on rat hepatocytes.     

Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways

Exposure of serum-deprived 3T3-L1 fibroblasts to phorbol 12-myristate 13-acetate (PMA), synthetic diacylglycerols, platelet-derived growth factor (PDGF), or pituitary fibroblast growth factor (FGF) resulted in stimulated phosphorylation of an acidic, multicomponent, soluble protein of Mr 80,000. Phosphorylation of this protein was promoted to a lesser extent by epidermal growth factor; however, neither insulin nor dibutyryl cAMP was effective. Phosphoamino acid analysis and peptide mapping of the Mr 80,000 32P-protein after exposure of fibroblasts to PDGF revealed identical patterns to those obtained with PMA or diacylglycerols. In contrast to the Mr 80,000 protein, proteins of Mr 22,000 (and pI 4.4) and Mr 31,000 were also phosphorylated in response to insulin as well as to PMA, diacylglycerols, epidermal growth factor, PDGF, and FGF in these cells. Similar findings were noted in fully differentiated 3T3-L1 adipocytes. Preincubation of the cells with high concentrations of active phorbol esters abolished specific [3H]phorbol 12,13-dibutyrate binding, protein kinase C activity, and immunoreactivity and also prevented stimulated phosphorylation of the Mr 80,000 protein by PMA, diacylglycerols, PDGF, or FGF, supporting the contention that this effect was mediated through protein kinase C. The stimulated phosphorylation of the Mr 22,000 and 31,000 proteins in response to PMA was also abolished by such pretreatment. In contrast, the ability of insulin, PDGF, and FGF to promote phosphorylation of the Mr 22,000 and 31,000 proteins was unaffected in the protein kinase C-deficient cells. We conclude that PDGF and FGF may exert some of their effects on these cells through at least two distinct pathways of protein phosphorylation, phorbol ester-like (P) activation of protein kinase C, and an insulin-like (I) pathway exemplified by phosphorylation of the Mr 22,000 and 31,000 proteins.     

The effects of phorbol esters on fluid transport and blood flow in the small intestine

Studies were designed to examine the effects of phorbol esters on intestinal fluid transport and blood flow in the anesthetized cat and enteropooling in the conscious rat. Intraluminal administration of phorbol ester into a segment of isolated small bowel produced a copious intestinal secretion and a concomitant mesenteric hyperemia in the cat. Net fluid movement in the intestine was converted from absorption in the control state to secretion following phorbol ester administration. Intravenous atropine reduced the phorbol ester-induced secretion by 56%; clonidine abolished the remaining secretory response. In the rat, intragastric administration of phorbol ester produced enteropooling comparable to that of other potent intestinal secretagogues. Since phorbol esters are known to activate protein kinase C, these studies suggest that activation of protein kinase C in the small intestine may lead to a full secretory response. The evidence suggests that this secretion is accompanied by a metabolic hyperemia. These results suggest that protein kinase C plays an important role in the regulation of intestinal fluid transport.