Tissue-dependent activation of protein kinase C in fructose-induced insulin resistance

Rats fed a fructose-enriched diet develop increases in blood pressure and resistance to insulin-mediated glucose disposal, but the underlying biochemical alterations have not been clearly defined. Since protein kinase C (PKC) has been implicated in the pathogenesis of insulin resistance, as well as blood pressure (BP) regulation, the present study was initiated to see whether changes in PKC signaling are present in rats with fructose-induced insulin resistance and hypertension. Consequently, liver, muscle, and adipose tissues were collected from fructose (n = 13) and chow (n = 12) fed Sprague-Dawley rats. PKC enzyme activity, and expression of classical PKC isozymes, were measured in cytosol and membrane fractions, and 1, 2-diacylglycerol (DAG), an endogenous stimulator of PKC, was measured by radio-enzymatic assay. Fructose feeding was associated with significant increases in fasting plasma insulin (140%) and triglyceride (400%) levels, and increased BP (20 mmHg). PKC activity was increased in the membrane fraction of adipose tissue (234 ± 38 (SE)vs 85 ± 30 pmol/min/mg protein,P< 0.007), without evidence of increased translocation or activation by DAG. Thus, fructose-induced insulin resistance has no effect on conventional PKC activity and subcellular distribution in liver and muscle, but the 3-fold increase in membraneassociated kinase activity in fat may be relevant to the mechanism of hypertriglyceridemia associated with fructose feeding.     

Diacylglycerol kinase delta regulates protein kinase C and epidermal growth factor receptor signaling

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG) to terminate its signaling. To study DGKdelta, we disrupted its gene in mice and found that DGKdelta deficiency reduced EGF receptor (EGFR) protein expression and activity. Similar to EGFR knockout mice, DGKdelta-deficient pups were born with open eyelids and died shortly after birth. PKCs are activated by DAG and phosphorylate EGFR to reduce its expression and activity. We found DAG accumulation, increased threonine phosphorylation of EGFR, enhanced phosphorylation of other PKC substrates, and increased PKC autophosphorylation in DGKdelta knockout cells, indicating that DGKdelta regulates EGFR by modulating PKC signaling.     

Growth hormone stimulates the formation of sn-1,2-diacylglycerol in rat hepatocytes

The transmembrane signaling events of GH were investigated in the liver, a major target organ of GH action. Recombinant human GH when added to freshly isolated rat hepatocytes rapidly stimulated the production of sn-1,2-diacylglycerol (DAG). The generation of DAG was biphasic with the first transient peak observed at 2 min and the second peak at 15 min (1.2-fold and 1.4-fold over control, respectively). Levels of DAG continued to be elevated above those in control cells at 30 min. The response was dose-dependent with an EC50 of 0.15 nM. Both bovine GH and rat GH, which bind to the rat GH receptor but not to the PRL receptor, also stimulated DAG production. Similarly, human PRL, which binds to the PRL but not GH receptor, stimulated DAG formation to a comparable extent. These results suggest that production of DAG may be an early signaling event mediated by hormone stimulation of both the GH and PRL receptors.     

Tissue and isoform-selective activation of protein kinase C in insulin-resistant obese Zucker rats - effects of feeding.

The mechanisms of insulin resistance in the obese Zucker rat have not been clearly established but increased diacylglycerol-protein kinase C (DAG-PKC) signalling has been associated with decreased glucose utilisation in states of insulin resistance and non-insulin-dependent diabetes mellitus. The purpose of this study was to characterise tissue- and isoform-selective differences in DAG-PKC signalling in insulin-sensitive tissues from obese Zucker rats, and to assess the effects of feeding on DAG-PKC pathways. Groups of male obese (fa/fa, n=24) and lean (fa/-, n=24) Zucker rats were studied after baseline measurements of fasting serum glucose, triglycerides, insulin and oral glucose tolerance tests. Liver, epididymal fat and soleus muscle samples were obtained from fed and overnight-fasted rats for measurements of DAG, PKC activity and individual PKC isoforms in cytosol and membrane fractions. Obese rats were heavier (488+/-7 vs 315+/-9 g) with fasting hyperglycaemia (10.5+/-0.8 vs 7.7+/-0.1 mM) and hyperinsulinaemia (7167+/-363 vs 251+/-62 pM) relative to lean controls. In fasted rats, PKC activity in the membrane fraction of liver was significantly higher in the obese group (174+/-16 vs 108+/-12 pmol/min/mg protein, P<0.05) but there were no differences in muscle and fat. The fed state was associated with increased DAG levels and threefold higher PKC activity in muscle tissue of obese rats, and increased expression of the major muscle isoforms, PKC-theta and PKC-epsilon: e.g. PKC activity in the membrane fraction of muscle from obese animals was 283+/-42 (fed) vs 107+/-20 pmol/min/mg protein (fasting) compared with 197+/-27 (fed) and 154+/-21 pmol/min/mg protein (fasting) in lean rats. In conclusion, hepatic PKC activity is higher in obese rats under basal fasting conditions and feeding-induced activation of DAG-PKC signalling occurs selectively in muscle of obese (fa/fa) rats due to increased DAG-mediated activation and/or synthesis of PKC-theta and PKC-epsilon. These changes in PKC are likely to exacerbate the hyperglycaemia and hypertriglyceridaemia associated with obesity-induced diabetes.     

Delayed accumulation of diacylglycerol in platelets as a mechanism for regulation of onset of aggregation and secretion

An important mechanism of platelet regulation is the formation of the second messenger diacylglycerol (DAG) and the activation of protein kinase C (PKC). Our previous studies suggested that the DAG/PKC pathway plays an important role in the induction of secretion and secondary aggregation rather than the earlier events of shape change and primary aggregation. We therefore examined the hypothesis that the delayed effects of PKC on platelets may result from delayed accumulation of DAG. The kinetics of DAG formation in human platelets were determined. When platelets were stimulated with gamma-thrombin, the largest phase of DAG accumulation was delayed for 0.6 to 0.8 minutes and DAG mass levels remained elevated for at least 2 minutes. In platelets stimulated with collagen, DAG accumulation was delayed for 1.0 to 1.2 minutes and DAG mass levels remained elevated for at least 3 minutes after stimulation. Sustained DAG production was also associated with sustained activation of PKC as monitored by phosphorylation of the 40-Kd substrate of PKC. The physiologic significance of delayed DAG accumulation was investigated using the cell-permeable DAG analog, dioctanoylglycerol (diC8). In synergy with subthreshold gamma-thrombin or collagen, exogenous diC8 reconstituted platelet activation. The optimal timing of addition of diC8 was 0.5 minutes after stimulation with gamma-thrombin or collagen. These kinetics were similar to those of endogenous DAG accumulation. These studies underscore the importance of a delayed accumulative phase of DAG generation as a mechanism controlling the onset of platelet secretion and irreversible aggregation.     

In situ protein Kinase C activity is increased in cultured fibroblasts from Type 1 diabetic patients with nephropathy

AIMS/HYPOTHESIS: To verify whether individual susceptibility to diabetic nephropathy resides in an intrinsic difference in Protein Kinase C (PKC) activity. METHODS: We compared the effect of different glucose concentrations on PKC activity, PKC isoform expression and diacylglycerol (DAG) content in cultured fibroblasts from 14 Type 1 diabetic patients who developed nephropathy with those in cells from 14 patients without nephropathy. We recruited 14 normal subjects as control patients. Forearm skin fibroblasts were cultured in either normal (5 mmol/l) or high (20 mmol/l) glucose concentrations. RESULTS: In normal glucose, in situ PKC activity was higher in Type 1 patients with nephropathy (10.1+/-1.4 pmol/min/mg protein; p<0.01) than in those without (6.8+/-0.8) and the normal control subjects (6.3+/-0.5). This difference was due to increased concentrations of PKCalpha isoform in the membrane fraction of fibroblasts from patients with nephropathy. DAG content was also higher in cells from Type 1 patients with nephropathy. Incubation in high glucose concentration caused a further increase in PKC activity and DAG content in quiescent fibroblasts from patients with diabetic nephropathy, with no significant changes in cells from diabetic patients without nephropathy and normal control subjects. CONCLUSION/INTERPRETATION: Differences in PKC activation could contribute to the individual susceptibility to renal damage in Type 1 diabetic patients.     

Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts.

We are using a Rat-6 fibroblast cell line that stably overexpresses the beta 1 isozyme of protein kinase C (PKC) to study regulation of phospholipid hydrolysis by PKC. Stimulation of control (R6-C1) or overexpressing (R6-PKC3) cells with phorbol ester results in an increase in diacylglycerol (DAG) mass with no increase in inositol phosphates, indicating that DAG is not formed by inositol phospholipid breakdown. A more dramatic DAG increase occurs in R6-PKC3 cells (4.0-fold over basal) compared to R6-C1 cells (1.5-fold over basal). To further define the source of DAG, phosphatidylcholine (PC) pools were labeled with [3H]myristic acid or with [3H]- or [32P]alkyllyso-PC and formation of labeled phosphatidylethanol, an unambiguous marker of phospholipase D activation, was monitored. Phorbol ester-stimulated phosphatidylethanol formation is 5-fold greater in the R6-PKC3 cell line. Formation of radiolabeled phosphatidic acid (PA) is also enhanced by PKC overexpression. In cells double-labeled with [3H]- and [32P]-alkyl-lysoPC, the 3H/32P ratio of PA and PC are identical 15 min after stimulation, suggesting that a phospholipase D mechanism predominates. In support of this, the PA phosphohydrolase inhibitor propranolol decreased phorbol 12-myristate 13-acetate-stimulated DAG formation by 72%. Increases in DAG and phosphatidylethanol were inhibited by the PKC inhibitors K252a and staurosporine. These results indicate that phospholipase D is regulated by the action of PKC. Enhanced phospholipase D activity may contribute to the growth abnormalities seen in PKC-overexpressing cells.     

Liver tissue from lactating rats produces a factor that stimulates prolactin release and gene expression

Liver tissue from nursing rats produces a substance, termed liver lactogenic factor (LLF), that potently stimulates casein release from isolated mammary cells. Inasmuch as the production of LLF is dependent on PRL, we decided to determine whether it could influence the release of the hormone by dissociated pituitary cells in culture. This was accomplished by measuring PRL release with a reverse hemolytic plaque assay and PRL gene expression with a DNA probe complementary to PRL mRNA. Treatment of pituitary cells from day 10 lactating rats with liver slice incubates from the same type of animal caused a 35.3 +/- 4.3% increase in PRL release during a 3-h incubation. Likewise, the same dose of LLF activity markedly increased (3.5-fold) the steady state levels of PRL mRNA. The responses were reasonably specific for PRL, since neither GH plaque development nor gene expression was affected by identical treatment. Taken together these results demonstrate that LLF can act directly at the pituitary level to exert positive feedback effects on both PRL release and gene expression.     

钒酸盐纠正了糖尿病大鼠心肌组织内活化的DAG—PKC信息传导

目的 探讨糖尿病大鼠心肌细胞内传导与心功能降低的关系。方法 检测糖尿病大鼠心肌二酰基甘油含量,蛋白激酶活性和心功能变化及胃饲钒酸钠的影响。结果 糖尿病大鼠心肌ＤＡＧ含量和胞膜ＰＫＣ活性显著升高,心功能显著降低,胃饲ＳＶ后明显改善。结论ＤＡＧ－ＰＫＣ通路激活是形成糖尿病心肌病的一个重要发病机制。     

Activation of the Ca(2+)-activated nonselective cation channel by diacylglycerol analogues in rat cardiomyocytes

INTRODUCTION: Cardiac hypertrophy is associated with changes in electrophysiologic properties due to ionic channel modifications and increases in protein kinase C (PKC) activity and diacylglycerol (DAG) content. These changes may contribute to an increased propensity for arrhythmia. Similar electrophysiologic modifications have been reported in adult rat cardiomyocytes undergoing dedifferentiation in primary culture. METHODS AND RESULTS: Single-channel measurements on such cells identified the appearance of a Ca(2+)-activated nonselective cation channel (NSC(Ca)) during the dedifferentiation process. The current study investigated the sensitivity of this channel to PKC and DAG analogues. In the cell-attached configuration, channel conductance was 20.2 pS under physiologic conditions. Perfusion with the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG, 0.1 mM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 0.5 microM) increased the channel normalized open probability (nPo), whereas in the presence of the PKC inhibitor calphostin C (1 microM), only OAG retained this effect. In the inside-out configuration, perfusion of both DAG analogues OAG (0.1 mM) and 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG, 10 microM) on the inside of the membrane increased nPo. These results indicate that DAG regulates the NSC(Ca) channel via both the PKC pathway and by a direct interaction. CONCLUSION: DAG content, PKC activity, and channel expression increased during hypertrophy. This indicates that the NSC(Ca) channel exhibits high activity in this condition and, therefore, is a candidate for the genesis of arrhythmias in ventricular cardiomyocytes. In addition, regulation of the channel by DAG and PKC contributes to current understanding of the physiologic role of this channel, which shares properties with the cloned TRPM4b channel.     

Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation.

In the present study, we have measured protein kinase C (PKC) specific activities and total diacylglycerol (DAG) level in the aorta and heart of rats, which showed that after 2 weeks of streptozotocin (STZ)-induced diabetes, membranous PKC specific activity and total DAG content were increased significantly by 88% and 40% in the aorta and by 21% and 72% in the heart, respectively. Hyperglycemia was identified as being a causal factor since elevated glucose levels increased DAG levels in cultured aortic endothelial and smooth muscle cells. Analysis by immunoblotting revealed that only alpha and beta II PKC isoenzymes are detected in these two tissues and vascular cells among those studied. In STZ-induced diabetic rats, beta II isoenzyme is preferentially increased in both aorta and heart, whereas PKC alpha did not change significantly. The increases in membranous PKC specific activity and DAG level are observed in both spontaneous diabetes-prone diabetic BB rats as well as in STZ-induced diabetic BB and Sprague-Dawley rats, which persisted for up to 5 weeks. After 2 weeks of diabetes without treatment, the normalization of blood glucose levels for up to 3 weeks with islet cell transplants in STZ-induced diabetic BB rats reversed the biochemical changes only in the heart, but not in the aorta. These results suggest that PKC activity and DAG level may be persistently activated in the macrovascular tissues from diabetic animals and indicate a possible role for these biochemical parameters in the development of diabetic chronic vascular complications.     

Dopamine infusion into the third ventricle increases gene expression of hypothalamic vasoactive intestinal peptide and pituitary prolactin and luteinizing hormone beta subunit in the turkey

Turkey prolactin (PRL) secretion is controlled by vasoactive intestinal peptide (VIP) neurons residing in the infundibular nuclear complex (INF) of the hypothalamus. The VIPergic activity is modulated by dopamine (DA) via stimulatory D(1) DA receptors. DA (10 nmol/min for 40 min) was infused into the third ventricle of laying turkey hens to study its effect on circulating PRL, hypothalamic VIP and pituitary PRL and LHbeta subunit mRNA levels. Plasma PRL was significantly elevated after 20 min of DA infusion and remained elevated 30 min after cessation of infusion. Hypothalamic VIP mRNA content was significantly greater in the INF of DA-infused birds than it was in the INF of vehicle-infused control birds. No increase in VIP mRNA due to DA infusion was noted in the preoptic area. Pituitary PRL and LHbeta subunit mRNAs were increased in DA-infused hens as compared to vehicle-infused controls but the rate of increase was more in PRL than LHbeta subunit. This study demonstrates that exogenous DA activates hypothalamic VIP gene expression and this increased expression is limited exclusively to the avian INF. The increased VIP mRNA in the INF is correlated with increased levels of circulating PRL and PRL and LHbeta mRNAs in the anterior pituitary.     

磷脂酰乙醇胺N-甲基转移酶2过表达对大鼠肝癌细胞不同亚型蛋白激酶C表达及转位的影响

目的 探讨转染磷脂酰乙醇胺N-甲基转移酶2-（PEMT2）基因抑制大鼠肝癌CBRH-7919细胞增殖的机制。方法 采用免疫细胞化学和蛋白质印迹法观察Pemt2过表达对肝癌细咆不矧亚型蛋白激酶C-（PKC）表达及在细胞内转位的影响，同时采用高效薄板层忻技术对细胞内二脂酰甘油（DAG）的水平进行检测。结果 转染PEMT2使细胞cPKC-α表达降低，cPKC-β2表达增加，并由胞浆向质膜转位。同时细胞内DAG水平下降。转染PEMT2对其它PKC亚型的表达及细胞内转位无显著影响。结论 转染PEMT2对不同亚型PKC表达及细胞内转位的影响可能与其抑制细胞增殖、诱导凋亡的机制有关。     

Diacylglycerol kinase α exacerbates cardiac injury after ischemia/reperfusion

Early coronary reperfusion of the ischemic myocardium is a desired therapeutic goal for the preservation of myocardial function. However, reperfusion itself causes additional myocardium injuries. Activation of the diacylglycerol–protein kinase C (DAG–PKC) cascade has been implicated in the cardioprotective effects occurring after ischemia/reperfusion (I/R). DAG kinase (DGK) controls cellular DAG levels by converting DAG to phosphatidic acid, and may act as an endogenous regulator of DAG–PKC signaling. In the present study, we examined the functional role of DGKα in cardiac injury after I/R in in vivo mouse hearts. We generated transgenic mice with cardiac-specific overexpression of DGKα (DGKα-TG). The left anterior descending coronary artery was transiently occluded for 20 min and reperfused for 24 h in DGKα-TG mice and wild-type littermate (WT) mice. The levels of phosphorylation activity of PKCε, extracellular-signal regulated kinase (ERK) 1/2, and p70 ribosomal S6 kinase (p70S6K) were increased after I/R in WT mouse hearts. However, in DGKα-TG mice, activation of PKCε, ERK1/2, and p70S6K was attenuated compared to WT mice. After 24 h, Evans blue/triphenyltetrazolium chloride double staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed that DGKα-TG mice had significantly larger myocardial infarctions and larger numbers of TUNEL-positive cardiomyocytes than WT mice. Echocardiography and cardiac catheterization revealed that left ventricular systolic function was more severely depressed in DGKα-TG mice than in WT mice after I/R. These findings suggest that DGKα exacerbates I/R injury by inhibiting the cardioprotective effects of PKCε, ERK1/2, and p70S6K activation.     

Interrelated effects of insulin and glucose on diacylglycerol-protein kinase-C signalling in rat adipocytes and solei muscle in vitro and in vivo in diabetic rats

The effects of insulin and glucose, alone and combined, on diacylglycerol (DAG), protein kinase-C (PKC), and glucose transport were compared in rat adipocytes and solei incubated in medium containing 0-20 mM glucose. In both tissues insulin rapidly stimulated [3H]DAG production from [3H]glycerol; extracellular glucose masked this effect in adipocytes, but not in solei. [3H]Glucose was avidly converted to DAG in adipocytes, and this conversion was enhanced by insulin. In contrast, [3H]glucose was poorly converted to DAG in solei. Glucose alone (5-20 mM) stimulated PKC translocation in adipocytes, but not in solei. Insulin stimulated PKC translocation in both tissues at all glucose concentrations. However, glucose modulated this effect of insulin in adipocytes by 1) decreasing cytosolic PKC and the absolute amount of PKC translocated, and 2) promoting apparent turnover of membrane PKC. In contrast, in solei, glucose did not affect PKC levels or translocation responses to insulin. In keeping with DAG-PKC signalling, the relative glucose transport effects of insulin were influenced by extracellular glucose in adipocytes, but not in solei. These results suggest that 1) glucose-induced PKC translocation requires metabolism of glucose to DAG; 2) glucose activates DAG-PKC signalling in adipocytes, but not in solei; 3) insulin activates DAG-PKC signalling in both tissues at all glucose levels; and 4) glucose may modulate the effects of insulin on DAG-PKC signalling in adipocytes, but not in solei. Consistent with in vitro results, in solei taken directly from diabetic rats, membrane PKC was decreased, and cytosolic PKC was increased, presumably reflecting diminished PKC translocation due to hypoinsulinemia. In contrast, in adipose tissue, cytosolic PKC was decreased, presumably reflecting hyperglycemia-induced PKC translocation. Accordingly, DAG levels were increased in adipose tissue, but not in solei, in diabetic rats, and insulin increased DAG in both tissues.     

Phosphorylation of RasGRP3 on threonine 133 provides a mechanistic link between PKC and Ras signaling systems in B cells

B-cell receptor (BCR) signaling activates a number of intracellular signaling molecules including phospholipase C-gamma2 (PLC-gamma2), which generates membrane diacylglycerol (DAG). DAG recruits both protein kinase C (PKC) and RasGRP family members to the membrane and contributes to their activation. We have hypothesized that membrane colocalization facilitates activation of RasGRP3 by PKC. Here we demonstrate that PKC phosphorylates RasGRP3 on Thr133 in vitro, as determined by mass spectrometry. RasGRP3 with a Thr133Ala substitution is a poor PKC substrate in vitro and a poor Ras activator in vivo. Antiphosphopeptide antibodies recognize Thr133-phosphorylated RasGRP3 in B cells after BCR stimulation or DAG analog treatment, but much less so in resting cells. PKC inhibitors block RasGRP3 Thr133 phosphorylation and Ras-extracellular signal-related kinase (Erk) signaling with a similar pattern. After stimulation of T-cell receptor (TCR) or DAG analog treatment of T cells, PKC-catalyzed phosphorylation of RasGRP1 occurs on the homologous residue, Thr184. These studies shed light on the proposed "PKC-Ras pathway" and support the hypothesis that RasGRP phosphorylation by PKC is a mechanism that integrates DAG signaling systems in T and B cells. PKC-mediated regulation of RasGRPs in lymphocytes may generate cooperative signaling in response to increases in DAG. The mast- and myeloid-selective family member RasGRP4 is regulated by different means.