Endothelin-1 induces lipolysis in 3T3-L1 adipocytes

Endothelin-1 (ET-1) affects glucose uptake in adipocytes and may play an important role in adipose physiology. One of the principal functions of adipose tissue is the provision of energy substrate through lipolysis. In the present study, we investigated the effects of ET-1 on lipolysis in 3T3-L1 adipocytes. When glycerol release in the culture medium was measured as an index of lipolysis, the results showed that ET-1 caused a significant increase that was time and dose dependent. With a concentration of 10 nM ET-1, stimulation of glycerol release plateaued after 4 h of exposure. This effect was inhibited by the ETA receptor antagonist BQ-610 (10 microM) but not by the ETB receptor antagonist BQ-788 (10 microM). To further explore the underlying mechanisms of ET-1 action, we examined the involvement of the cAMP-dependent protein kinase A-mediated, phospholipase A2 (PLA2)-mediated, protein kinase C (PKC)-mediated, phosphatidylinositol 3 (PI 3)-kinase-mediated, and the mitogen-activated protein kinase (MAPK)-mediated pathways. Inhibition of adenylyl cyclase activation by SQ-22536 (100 microM) did not block ET-1-induced lipolysis. Pretreatment of adipocytes with the PLA2 inhibitor dexamethasone (100 nM), the PKC inhibitor H-7 (6 microM), or the PI 3-kinase inhibitor wortmannin (100 nM) also had no effect. ET-1-induced lipolysis was blocked by inhibition of extracellular signal-regulated kinase (ERK) activation using PD-98059 (75 microM), whereas a p38 MAPK inhibitor (SB-203580; 20 microM) had no effect. Results of Western blot further demonstrated that ET-1 induced ERK phosphorylation. These data show that ET-1 induces lipolysis in 3T3-L1 adipocytes via a pathway that is different from the conventional cAMP-dependent pathway used by isoproterenol and that involves ERK activation.     

Effect of tumor necrosis factor-alpha on insulin signal transduction in rat adipocytes: relation to PKCbeta and zeta translocation

Although much evidence has been accumulated suggesting that tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance, the precise mechanism involved is still unclear. Recently, it has been reported that insulin-induced glucose uptake is mediated by activation of second messengers such as insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and diacylglycerol (DG)-protein kinase C (PKC). We have examined the effect of TNF-alpha on insulin-induced glucose uptake and activations of tyrosine kinase, IRS-1, PI3K and PKC in rat adipocytes. Pretreatment with 0.1-100 nM TNF-alpha for 60 min resulted in a significant decrease in 10 nM insulin- or 1 microM 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced [3H]2-deoxyglucose uptake without affecting basal glucose uptake. 10 nM insulin-stimulated activation of tyrosine kinase, IRS-1 and PI3K was suppressed by preincubation with 0.1-10 nM TNF-alpha for 60 min. 10 nM TNF-alpha pretreatment also suppressed 10 nM insulin- and 1 microM TPA-induced increases in membrane-associated PKCbeta and PKCzeta. Furthermore, 10 nM TNF-alpha, by itself, altered PKCbeta translocation from the membrane to cytosol. These results suggest that TNF-alpha inhibits insulin-stimulated activation of both the tyrosine kinase-IRS-1-PI3K-PKCzeta pathway and DG-PKC pathway. Finally, TNF-alpha contributes to insulin resistance in rat adipocytes.     

Regulation of leptin secretion from white adipocytes by free fatty acids

Norepinephrine stimulates lipolysis and concurrently inhibits insulin-stimulated leptin secretion from white adipocytes. To assess whether there is a cause-effect relationship between these two metabolic events, the effects of fatty acids were investigated in isolated rat adipocytes incubated in buffer containing low (0.1%) and high (4%) albumin concentrations. Palmitic acid (1 mM) mimicked the inhibitory effects of norepinephrine (1 microM) on insulin (10 nM)-stimulated leptin secretion, but only at low albumin concentrations. Studies investigating the effects of the chain length of saturated fatty acids [from butyric (C4) to stearic (C18) acids] revealed that only fatty acids with a chain length superior or equal to eight carbons effectively inhibited insulin-stimulated leptin secretion. Long-chain mono- and polyunsaturated fatty acids constitutively present in adipocyte triglyceride stores (oleic, linoleic, gamma-linolenic, palmitoleic, eicosapentanoic, and docosahexanoic acids) also completely suppressed leptin secretion. Saturated and unsaturated fatty acids inhibited insulin-stimulated leptin secretion with the same potency and without any significant effect on basal secretion. On the other hand, inhibitors of mitochondrial fatty acid oxidation (palmoxirate, 2-bromopalmitate, 2-bromocaproate) attenuated the stimulatory effects of insulin on leptin release without reversing the effects of fatty acids or norepinephrine, suggesting that fatty acids do not need to be oxidized by the mitochondria to inhibit leptin release. These results demonstrate that long-chain fatty acids mimic the effects of norepinephrine on leptin secretion and suggest that they may play a regulatory role as messengers between stimulation of lipolysis by norepinephrine and inhibition of leptin secretion.     

Genistein, a plant-derived isoflavone, counteracts the antilipolytic action of insulin in isolated rat adipocytes

Genistein is a phytoestrogen exerting numerous biological effects. Its direct influence on adipocyte metabolism and leptin secretion was previously demonstrated. This study aimed to determine whether genistein antagonizes the antilipolytic action of insulin in rat adipocytes. Freshly isolated adipose cells were incubated for 90 min with epinephrine, epinephrine with insulin and epinephrine with a specific inhibitor of protein kinase A (H-89) at different concentrations of genistein (0, 6.25, 12.5, 25, 50 and 100 μM). Genistein failed to affect epinephrine-induced glycerol release, however, the inhibitory action of insulin on epinephrine-induced lipolysis was significantly abrogated in cells exposed to the phytoestrogen (12.5–100 μM). The increase in insulin concentration did not suppress the genistein effect. Its inhibitory influence on the antilipolytic action of insulin was accompanied by a substantial rise in cAMP in adipocytes. This rise appeared despite the presence of 10 nM insulin in the incubation medium. Further experiments, in which insulin was replaced by H-89, revealed that the antilipolytic action of protein kinase A inhibitor on epinephrine-induced lipolysis was not affected by genistein. This means that genistein counteracted the antilipolytic action of insulin due to the increase in cAMP levels and activation of protein kinase A in adipocytes. The observed attenuation of the inhibitory effect of insulin on triglyceride breakdown evoked by genistein was not related to its estrogenic activities, as evidenced in experiments employing the intracellular estrogen receptor blocker, ICI 182,780. Moreover, it was found that genistein-induced impairment of the antilipolytic action of insulin was not accompanied by changes in the proportion between fatty acids and glycerol released from adipocytes. The ability of genistein to counteract the antilipolytic action of insulin may contribute to the decreased triglyceride accumulation in adipose tissue.     

Short-term fasting and lipolytic activity in rat adipocytes.

The aim of this experiment was to study the influence of 18-hour food deprivation on basal and stimulated lipolysis in adipocytes obtained from young male Wistar rats. Fat cells from fed and fasted rats were isolated from the epididymal adipose tissue by collagenase digestion. Adipocytes were incubated in Krebs-Ringer buffer (pH 7.4, 37 degrees C) without agents affecting lipolysis and with different lipolytic stimulators (epinephrine, forskolin, dibutyryl-cAMP, theophylline, DPCPX, amrinone) or inhibitors (PIA, H-89, insulin). After 60 min of incubation, glycerol and, in some cases, also fatty acids released from adipocytes to the incubation medium were determined. Basal lipolysis was substantially potentiated in cells of fasted rats in comparison to adipocytes isolated from fed animals. The inhibition of protein kinase A activity by H-89 partially suppressed lipolysis in both groups of adipocytes, but did not eliminate this difference. The agonist of adenosine A (1) receptor also did not suppress fasting-enhanced basal lipolysis. The epinephrine-induced triglyceride breakdown was also enhanced by fasting. Similarly, the direct activation of adenylyl cyclase by forskolin or protein kinase A by dibutyryl-cAMP resulted in a higher lipolytic response in cells derived from fasted animals. These results indicate that the fasting-induced rise in lipolysis results predominantly from changes in the lipolytic cascade downstream from protein kinase A. The antagonism of the adenosine A (1) receptor and the inhibition of cAMP phosphodiesterase also induced lipolysis, which was potentiated by food deprivation. Moreover, the rise in basal and epinephrine-stimulated lipolysis in adipocytes of fasted rats was shown to be associated with a diminished non-esterified fatty acids/glycerol molar ratio. This effect was presumably due to increased re-esterification of triglyceride-derived fatty acids in cells of fasted rats. Comparing fed and fasted rats for the antilipolytic effect of insulin in adipocytes revealed that short-term food deprivation resulted in a substantial deterioration of the ability of insulin to suppress epinephrine-induced lipolysis.     

Effect of pertussis toxin on insulin-induced signal transduction in rat adipocytes and soleus muscles

It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC zeta activation and insulin-induced tyrosine phosphorylation of Gialpha to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01 approximately 1 ng/ml PTX for 2 hours, followed by stimulation with 10-100 nM insulin or 1 microM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [3H]2-DOG uptake, without affecting basal [3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC zeta translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa alpha-subunit of G-protein (Gialpha-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Gialpha-2 antibody. Insulin-induced tyrosine phosphorylation of Gialpha-2 was found by immunoblot analysis with anti-Gialpha-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Gialpha-2 with GTP and PI 3-kinase-PKC zeta signalling by releasing of Gbetagamma via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.     

Insulin and phorbol ester stimulate phosphorylation of acetyl-CoA carboxylase at similar sites in isolated adipocytes. Lack of correspondence with sites phosphorylated on the purified enzyme by protein kinase C

1. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) stimulates fatty acid synthesis from glucose in isolated adipocytes with a half-maximal effect at 0.72 microM. In seven batches of cells, the maximal effects of TPA and insulin were 8.5 +/- 1.1-fold and 27.1 +/- 2.1-fold respectively. Insulin also stimulated fatty acid synthesis from acetate 8.9 +/- 0.5-fold (three experiments), but TPA did not significantly increase fatty acid synthesis from this precursor. 2. In contrast to insulin, TPA treatment of isolated adipocytes did not produce an activation of acetyl-CoA carboxylase which was detectable in crude cell extracts. 3. The total phosphate content of acetyl-CoA carboxylase, isolated from adipocytes in the presence of protein phosphatase inhibitors, was estimated by 32P-labelling experiments to be 2.6 +/- 0.1 (5), 3.4 +/- 0.2 (5), and 3.8 +/- 0.2 (3) mol/mol subunit for enzyme from control, insulin- and TPA-treated cells respectively. Insulin and TPA stimulated phosphorylation within the same two tryptic peptides. 4. Purified acetyl-CoA carboxylase is phosphorylated in vitro by protein kinase C at serine residues which are recovered in three tryptic peptides, i.e. peptide T1, which appears to be identical with the peptide Ser-Ser(P)-Met-Ser-Gly-Leu-His-Leu-Val-Lys phosphorylated by cyclic-AMP-dependent protein kinase, and peptides Ta and Tb, which have the sequences Ile-Asp-Ser(P)-Gln-Arg and Lys-Ile-Asp-Ser(P)-Gln-Arg respectively, and which appear to be derived from a single site by alternative cleavages. None of these correspond to the peptides whose 32P-labelling increase in response to insulin or TPA. Peptides Ta/Tb are not significantly phosphorylated in isolated adipocytes, even after insulin or TPA treatment. Peptide T1 is phosphorylated in isolated adipocytes, but this phosphorylation is not altered by insulin or TPA. 5. These results show that TPA mimics the effect of insulin on phosphorylation, but not activation, of acetyl-CoA carboxylase, i.e. that these two events can be dissociated. In addition, phorbol ester stimulates phosphorylation of acetyl-CoA carboxylase in isolated adipocytes, but this is not catalyzed directly by protein kinase C, and acetyl-CoA carboxylase does not appear to be a physiological substrate for this kinase.     

Effects of inhibitors on aldolase breakdown after its microinjection into HeLa cells.

The tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces insulin secretion from isolated pancreatic islets, and this suggests a potential role for protein kinase C in the regulation of stimulus-secretion coupling in islets. In the present study, the hypothesis that the insulinotropic effect of TPA is mediated by activation of protein kinase C in pancreatic islets has been examined. TPA induced a gradual translocation of protein kinase C from the cytosol to a membrane-associated state which correlated with the gradual onset of insulin secretion. The pharmacologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not mimic this effect. TPA also induced a rapid time-dependent decline of total protein kinase C activity in islets and the appearance of a Ca2+- and phospholipid-independent protein kinase activity. Insulin secretion induced by TPA was completely suppressed (IC50 approximately 10 nM) by staurosporine, a potent protein kinase C inhibitor. Staurosporine also inhibited islet cytosolic protein kinase C activity at similar concentrations (IC50 approximately 2 nM). In addition, staurosporine partially (approximately 60%) inhibited glucose-induced insulin secretion at concentrations (IC50 approximately 10 nM) similar to those required to inhibit TPA-induced insulin secretion, suggesting that staurosporine may act at a step common to both mechanisms, possibly the activation of protein kinase C. However, stimulatory concentrations of glucose did not induce down-regulation of translocation of protein kinase C, and the inhibition of glucose-induced insulin release by staurosporine was incomplete. Significant questions therefore remain unresolved as to the possible involvement of protein kinase C in glucose-induced insulin secretion.     

Novel form of lipolysis induced by leptin.

Hyperleptinemia causes disappearance of body fat without a rise in free fatty acids (FFA) or ketones, suggesting that leptin can deplete adipocytes of fat without releasing FFA. To test this, we measured FFA and glycerol released from adipocytes obtained from normal lean Zucker diabetic fatty rats (+/+) and incubated for 0, 3, 6, or 24 h in either 20 ng/ml recombinant leptin or 100 nM norepinephrine (NE). Whereas NE increased both FFA and glycerol release from adipocytes of +/+ rats, leptin increased glycerol release in +/+ adipocytes without a parallel increase in FFA release. In adipocytes of obese Zucker diabetic fatty rats (fa/fa) with defective leptin receptors, NE increased both FFA and glycerol release, but leptin had no effect on either. Leptin significantly lowered the mRNA of leptin and fatty acid synthase of adipocytes (FAS) (p < 0.05), and up-regulated the mRNA of peroxisome proliferator-activated receptor (PPAR)-alpha, carnitine palmitoyl transferase-1, (CPT-1), and acyl CoA oxidase (ACO) (p < 0.05). NE (100 nM) also lowered leptin mRNA (p < 0.05) but did not affect FAS, PPARalpha, ACO, or CPT-1 expression. We conclude that in normal adipocytes leptin directly decreases FAS expression, increases PPARalpha and the enzymes of FFA oxidation, and stimulates a novel form of lipolysis in which glycerol is released without a proportional release of FFA.     

Effect of tyramine, a dietary amine, on glycerol and lactate release by isolated adipocytes from old rats

Amine degradation by adipocyte amine oxidases leads to the production of metabolites that interact with lipid and glucose metabolisms and their hormonal regulations. To further investigate these interactions, we determined the effect of a dietary amine, tyramine (TYR), on glycerol and lactate releases, respectively taken as indices of lipolytic and glycolytic activities of isolated adipocytes. Old male Wistar rats were used to prepare adipocytes by collagenase dissociation of retroperitoneal fat pads. The two tested doses of tyramine (10 microM and 1 mM) had no effect on basal glycerol release. On the other hand, TYR, at the highest dose tested (1 mM), weakly but significantly increased basal lactate release, which was elevated in adipocytes from old rats. Norepinephrine (NE), highly stimulated adipocyte lipolysis with a submaximal effect at 1 microM which was slightly but significantly inhibited by TYR 1 mM. Insulin 1 nM (INS) also poorly inhibited the NE-stimulated lipolysis in adipocytes isolated from old rats. TYR was able to potentiate the poor antilipolytic efficiency of INS. Under similar conditions, a high dose of NE greatly reduced lactate production and TYR (1 mM) reversed this inhibition of lactate release. INS was also able to totally reverse the inhibitory effect of NE on lactate release, but there was no potentiation between insulin and tyramine effects. It can be concluded that high doses of TYR interact with norepinephrine and insulin, at least on the control of glycerol and lactate release, by counteracting catecholamine effects and by mimicking insulin actions.     

Insulin stimulates the translocation of protein kinase C in rat adipocytes

Insulin-induced changes in protein kinase C were examined in cytosol and membrane fractions of rat adipocytes enzymatically after Mono Q column chromatography and by immunoblotting. During a 5-20 min period of insulin treatment, cytosolic protein kinase C decrease by approximately 50%, whereas membrane protein kinase C increased nearly 2-fold. These findings suggest that insulin stimulates the translocation of protein kinase C in rat adipocytes.     

Resveratrol,a naturally occurring diphenolic compound,affects lipogenesis,lipolysis and the antilipolytic action of insulin in isolated rat adipocytes

Resveratrol is a naturally occurring diphenolic compound exerting numerous beneficial effects in the organism. The present study demonstrated its short-term, direct influence on lipogenesis, lipolysis and the antilipolytic action of insulin in freshly isolated rat adipocytes. In fat cells incubated for 90 min with 125 and 250 μM resveratrol (but not with 62.5 μM resveratrol), basal and insulin-induced lipogenesis from glucose was significantly reduced. The antilipogenic effect was accompanied by a significant diminution of CO₂ release and enhanced production of lactate. The inhibition of glucose conversion to lipids found in the presence of resveratrol was not attenuated by activator of protein kinase C. However, acetate conversion to lipids appeared to be insensitive to resveratrol.In adipocytes incubated for 90 min with epinephrine, 10 and 100 μM resveratrol significantly enhanced lipolysis, especially at lower concentrations of the hormone. However, the lipolytic response to dibutyryl-cAMP, a direct activator of protein kinase A, was unchanged. Further studies demonstrated that, in cells stimulated with epinephrine, 1, 10 and 100 μM resveratrol significantly enhanced glycerol release despite the presence of insulin or H-89, an inhibitor of protein kinase A. The influence of resveratrol on epinephrine-induced lipolysis and on the antilipolytic action of insulin was not abated by the blocking of estrogen receptor and was accompanied by a significant (with the exception of 1 μM resveratrol in experiment with insulin) increase in cAMP in adipocytes. It was also revealed that resveratrol did not change the proportion between glycerol and fatty acids released from adipocytes exposed to epinephrine.Results of the present study revealed that resveratrol reduced glucose conversion to lipids in adipocytes, probably due to disturbed mitochondrial metabolism of the sugar. Moreover, resveratrol increased epinephrine-induced lipolysis. This effect was found also in the presence of insulin and resulted from the synergistic action of resveratrol and epinephrine. The obtained results provided evidence that resveratrol affects lipogenesis and lipolysis in adipocytes contributing to reduced lipid accumulation in these cells.     

Persistent activity modification of phosphatidate phosphohydrolase and fatty acyl-CoA synthetase on incubation of adipocytes with the tumour promoter 12-O-tetradecanoylphorbol 13-acetate

Incubation of rat adipocytes with the phorbol ester TPA caused a dose-dependent, persistent increase in phosphatidate phosphohydrolase activity and decrease in fatty acyl-CoA synthetase activity. Half-maximal effects were obtained with 1-2 X 10(-10) M TPA. TPA did not alter basal or noradrenaline-stimulated lipolysis. It is suggested that these two enzymes might be modified by protein kinase C.     

Protein kinase C-dependent antilipolysis by insulin in rat adipocytes

Recently, we have shown that protein kinase C (PKC) activated by phorbol 12-myristate 13-acetate (PMA) attenuates the beta1-adrenergic receptor (beta1-AR)-mediated lipolysis in rat adipocytes. Stimulation of cells by insulin, angiotensin II, and alpha1-AR agonist is known to cause activation of PKC. In this study, we found that lipolysis induced by the beta1-AR agonist dobutamine is decreased and is no longer inhibited by PMA in adipocytes that have been treated with 20 nM insulin for 30 min followed by washing out insulin. Such effects on lipolysis were not found after pretreatment with angiotensin II and alpha1-AR agonists. The rate of lipolysis in the insulin-treated cells was normalized by the PKCalpha- and beta-specific inhibitor G? 6976 and PKCbeta-specific inhibitor LY 333531. In the insulin-treated cells, wortmannin increased lipolysis and recovered the lipolysis-attenuating effect of PMA. Western blot analysis revealed that insulin slightly increases membrane-bound PKCalpha, betaI, and delta, and wortmannin decreases PKCbetaI, betaII, and delta in the membrane fraction. These results indicate that stimulation of insulin receptor induces a sustained activation of PKC-dependent antilipolysis in rat adipocytes.     

Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.     

Vasopressin and norepinephrine stimulation of inositol phosphate accumulation in rat hepatocytes are modified differently by protein f1nase C and protein kinase A

Rat hepatocytes were maintained in primary monolayer culture for 24 h in the presence of serum. Treatment of hepatocytes with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) for 5-15 min increased membrane-associated protein kinase C activity and concomitantly decreased soluble activity. Membrane protein kinase C activity returned to basal values within 1 h then decreased by more than 50% within 2 h. Prolonged (2-18 h) incubation with PMA did not further decrease protein kinase C activity. Pretreatment of hepatocytes with PMA for 5-15 min had little effect on the subsequent actions of 100 nM vasopressin but abolished the stimulation of inositol phosphate accumulation by 3 nM vasopressin and 20 microM norepinephrine. Long-term exposure (2-18 h) of hepatocytes to 1 microM PMA actually enhanced the effects of vasopressin and 20 microM norepinephrine. The stimulation by norepinephrine (20 microM) of inositol phosphate accumulation was abolished by the alpha 1-adrenergic antagonist prazosin (1 microM), whereas the beta-adrenergic antagonist propranolol (30 microM) had little effect. Addition of 8Br-cAMP (100 microM) or glucagon (10 nM) for 5 min or 8 h had no significant effect alone, but enhanced the subsequent vasopressin stimulation of inositol phosphate accumulation. There was no effect of 8Br-cAMP or glucagon on norepinephrine stimulation of phosphoinositide breakdown. These data indicate that the stimulation of phospholipase C activity in rat hepatocytes by 3 nM vasopressin is enhanced by cyclic AMP-dependent kinase but inhibited by protein kinase C. In contrast, down regulation of protein kinase C markedly enhanced the maximal phosphoinositide response due to both vasopressin and norepinephrine.     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Insulin-dependent alterations of phorbol ester binding to adipocyte subcellular constituents. Evidence for the involvement of protein kinase C in insulin action

The binding of tritiated phorbol-12,13-dibutyrate (3H-PBu2) was employed to estimate the mass of protein kinase C associated with plasma membranes and cytosol isolated from untreated and insulin-treated adipocytes. Binding of 3H-PBu2 to both plasma membranes and cytosol was rapid, achieving a steady state within minutes. Treatment of cells with physiological concentration of insulin (0.67 nM) caused a 42% increase (from 0.92 +/- 0.08 to 1.30 +/- 0.12 pmol 3H-PBu2/mg protein, p less than 0.0001) and a 27% decrease (from 0.41 +/- 0.07 to 0.30 +/- 0.05 pmol 3H-PBu2/mg protein, p less than 0.020) in phorbol ester bound to cytosol and plasma membranes, respectively. The half-maximal concentrations of unlabelled PBu2 needed to displace 3H-PBu2 bound to cytosol from control and insulin-treated cells were 54 and 13 pM, respectively. These data indicate that insulin modifies protein kinase C in adipocytes.     

Insulin inhibits lipolytic activity and degradation of beta-lipotropin in rabbit adipose tissue

beta-Lipotropin, a pituitary peptide, is a potent stimulator of lipolysis in rabbit adipose tissue in vitro and in vivo. Insulin inhibited the beta-lipotropin (1-100 nM)-stimulated glycerol release from rabbit adipocytes and fat pads significantly at concentrations of 10 and 100 microM. Both these concentrations of insulin also decreased the degradation of beta-lipotropin in intact adipose tissue to the same extent as the lipolytic activity. Furthermore, insulin reduced the degradation of beta-lipotropin in rabbit adipose tissue homogenate. Like insulin, several lysosomotropic agents also decreased significantly the degradation and the lipolytic activity of beta-lipotropin. On the other hand, insulin-like growth factor I in lower concentrations (1-100 nM) did not effect degradation and lipolytic activity of beta-lipotropin in rabbit adipose tissue. Thus, a direct influence of insulin on lysosomal enzymes degrading beta-lipotropin in rabbit adipose tissue can be suggested.     

Hormonal Regulation of Adipose S-100 Protein Release

The release of S-100 protein from epididymal fat pads was enhanced by epinephrine in vitro, and about 50% of S-100 protein in the tissue was released into the medium after 2-h incubation at 37 degrees C with 10 microM epinephrine. Similar results were obtained with the incubation of isolated adipocytes. The S-100 protein release was also enhanced by isoproterenol, norepinephrine, ACTH, and dibutyryl cyclic AMP, which all increase the lipolysis by increasing cyclic AMP levels in the tissue. Propranolol, a beta-adrenergic blocker, could block the increase of S-100 protein release by catecholamines, indicating that the release was mediated by the beta-adrenergic effect of catecholamines. However propranolol had no suppressive effect on the enhancement of S-100 protein release by ACTH or dibutyryl cyclic AMP. Insulin had an inhibitory effect on the epinephrine-enhanced S-100 protein release. Epinephrine or ACTH could not stimulate the S-100 protein release in the absence of Ca2+, whereas the epinephrine-enhanced glycerol release was not affected under the same conditions. The increase in S-100 protein release was induced by only a pretreatment of the tissue with epinephrine. However, the lipolysis in the tissue was not enhanced by the pretreatment alone. These results indicate that the release of S-100 protein from adipocytes is regulated by the hormones that have been known to control the lipolysis with a manner slightly different from that of lipolysis.