Herman Hernandez

Effects of Insulin on Diacylglycerol–Protein Kinase C Signaling in Rat Diaphragm and Soleus Muscles and Relationship to Glucose Transport

Insulin was found to provoke rapid increases in diacylglycerol (DAG) content and [3H]glycerol incorporation into DAG and other lipids during incubations of rat hemidiaphragms and soleus muscles. Insulin also rapidly increased phosphatidic acid and total glycerolipid labeling by [3H]glycerol, suggesting that insulin increases DAG production at least partly through stimulation of the de novo pathway. Increased DAG production may activate protein kinase C (PKC) as reported previously in the rat diaphragm. We also observed apparent insulininduced translocation of PKC from cytosol to membrane in the rat soleus muscle. The importance of insulin-induced increases in DAG-PKC signaling in the stimulation of glucose transport in rat diaphragm and soleus muscles was suggested by 1) PKC activators phorbol esters and phospholipase C stimulation of [3H]-2-deoxyglucose (DOG) uptake and 2) PKC inhibitors staurosporine and polymixin B inhibition of insulin effects on [3H]-2-DOG uptake. Although phorbol ester was much less effective than insulin in the diaphragm, phospholipase C provoked increases in [3H]-2-DOG uptake that equaled or exceeded those of insulin. In the soleus muscle, phorbol ester, like phospholipase C, was only slightly but not significantly less effective than insulin. Similar variability in effectiveness of phorbol ester has also been noted previously in rat adipocytes (weak) and BC3HI myocytes (strong), whereas DAG, added exogenously or generated by phospholipase C treatment, stimulates glucose transport to a degree that is quantitatively more comparable to that of insulin in each of the four tissues. Differences in effectiveness of phorbol ester and DAG could not be readily explained by postulating that the latter acts independently of PKC, because DAG provoked the apparent translocation of the enzyme from cytosol to membranes in rat adipocytes, and effects of DAG on [3H]-2-DOG uptake were blocked by inhibitors of PKC in both rat adipocytes and BC3H1 myocytes. Collectively, our findings provide further support for the hypothesis that insulin increases DAG production and PKC activity, and these processes are important in the stimulation of glucose transport in rat skeletal muscle and other tissues.

Insulin increases the synthesis of phospholipid and diacylglycerol and protein kinase C activity in rat hepatocytes.

The effects of insulin on phospholipid metabolism and generation of diacylglycerol (DAG) and on activation of protein kinase C in rat hepatocytes were compared to those of vasopressin and angiotension II. Insulin provoked increases in [3H]glycerol labeling of phosphatidic acid (PA), diacylglycerol (DAG), and other glycerolipids within 30 s of stimulation. Similar increases were also noted for vasopressin and angiotensin II. Corresponding rapid increases in DAG mass also occurred with all three hormones. As increases in [3H]DAG (and DAG mass) occurred within 30-60 s of the simultaneous addition of [3H]glycerol and hormone, it appeared that DAG was increased, at least partly, through the de novo synthesis of PA. That de novo synthesis of PA was increased is supported by the fact that [3H]glycerol labeling of total glycerolipids was increased by all three agents. Increases in [3H]glycerol labeling of lipids by insulin were not due to increased labeling of glycerol 3-phosphate, and were therefore probably due to activation of glycerol-3-phosphate acyltransferase. Unlike vasopressin, insulin did not increase the hydrolysis of inositol phospholipids. Insulin- and vasopressin-induced increases in DAG were accompanied by increases in cytosolic and membrane-associated protein kinase C activity. These findings suggest that insulin-induced increases in DAG may lead to increases in protein kinase C activity, and may explain some of the insulin-like effects of phorbol esters and vasopressin on hepatocyte metabolism.

Induction of presenilins in the rat brain after middle cerebral arterial occlusion.

In the present study, we have examined the expression of both presenilins in the rat hippocampus, cortex, striatum, and cerebellum after middle cerebral artery occlusion (MCA-O), an animal model of ischemia. The cortex showed the greatest increase in PS mRNA levels (7-10-fold) at 4 and 8 days posttreatment. Presenilin-1 (PS-1) levels in the contralateral cortex were significantly increased 1 day after MCA-O. In comparison, PS mRNA content was only modestly elevated in the hippocampus and striatum at 4 and 8 days after MCA-O (30-100% changes). Other Alzheimers disease (AD)-related genes, amyloid precursor protein and apolipoprotein E, are induced in brain injury suggesting that these AD-related genes may well be components of a brain-injury response. Thus, a breakdown in this response via cerebrovascular disease and/or genetic mutation may contribute to AD pathology.

Downregulation of Protein Kinase C and Insulin-Stimulated 2-Deoxyglucose Uptake in Rat Adipocytes by Phorbol Esters, Glucose, and Insulin

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.

Direct evidence for protein kinase c involvement in insulin-stimulated hexose uptake

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.

Differential effect of aging on protein kinase C activity in rat adipocytes and soleus muscle

Protein kinase C (PKC) has been postulated to play an important role in glucose transport in insulin-sensitive tissues such as rat adipocytes and skeletal muscle. Since glucose transport decreases in old rats, we examined age-related changes in PKC. Cytosolic PKC-dependent histone-phosphorylating enzyme activity and PKC-beta immunoreactivity of both adipocytes and soleus muscles increased progressively with age (or weight) in rats weighing less than 400 g. In comparing PKC enzyme activity and PKC-beta immunoreactivity in young rats (180 +/- 32 g; mean +/- SE, body weight) versus old rats (658 +/- 108 g), both cytosolic and membrane-associated PKC were greater in adipocytes of old rats (relative to adipocytes of young rats), whereas in the soleus muscle of old rats cytosolic PKC activity was diminished and membrane-associated PKC was increased (relative to solei of young rats). The latter redistribution of soleus PKC may be due to endogenous hyperinsulinemia, which is known to occur in old rats and which may have stimulated the translocation of PKC from cytosol to membrane in the soleus. Whatever the cause, decreases in cytosolic PKC in the soleus muscle may limit acute PKC translocation responses to insulin or other agents in old rats.

Preferential activation of microsomal diacylglycerol/protein kinase C signaling during glucose treatment (De Novo phospholipid synthesis) of rat adipocytes.

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.