R V Farese

Potassium and angiotensin II increase the concentrations of phosphatidic acid, phosphatidylinositol, and polyphosphoinositides in rat adrenal capsules in vitro.

We examined the effects of K+ and angiotensin II, the major regulators of aldosterone secretion, on phospholipid metabolism during incubation of glomerulosa-rich, adrenal capsules. Addition of increasing amounts of K+ and angiotensin II to the incubation media elicited progressive increases in corticosterone production and capsular concentrations of phosphatidic acid, phosphatidyl-inositol, and polyphosphoinositides. These effects are similar to those previously reported for ACTH in the whole adrenal cortex. A common mechanism, i.e., activation of the phosphatidate-polyphosphoinositide cycle, may be operative in the action of steroidogenic agents in their target tissues.

SULFONYLUREAS ACTIVATE GLUCOSE TRANSPORT AND PROTEIN KINASE C IN RAT ADIPOCYTES

Glyburide and tolbutamide, at concentrations of 20 to 40 mumol/L and 1 to 2 mmol/L, respectively, stimulated glucose transport in rat adipocytes. Concomitantly, protein kinase C was activated, as evidenced by translocation of immunoreactive enzyme from cytosol to membranes. Glucose transport effects of the sulfonylureas were blocked by three inhibitors of protein kinase C (H-7, staurosporine, and sangivamycin), and by phorbol ester-induced down-regulation of protein kinase C. These findings suggest that sulfonylureas may stimulate glucose transport in rat adipocytes through activation of protein kinase C.

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.

Further observations on the increases in inositide phospholipids after stimulation by ACTH, cAMP and insulin, and on discrepancies in phosphatidylinositol mass and 32PO4-labeling during inhibition of hormonal effects by cycloheximide

Abstract ACTH-induced increases in adrenal polyphosphoinositides were demonstrable after extraction by acid or non-acid methods, and after purification by unidimensional or two-dimensional thin layer chromatography. ACTH-induced increases in phosphatidylinositol mass were apparent, both as increases in measurable phosphorus and inositol. ACTH and insulin also increased 32PO4 incorporation into adrenal and adipose tissue polyphosphoinositides which were acid-extracted and purified by two-dimensional chromatography. Cyclic-AMP increased the mass of phosphatidylinositol, but decreased 32PO4 incorporation into this and other phospholipids; the cause for this decrease in specific activity was not evident. Increasing doses of cycloheximide progressively inhibited ACTH- or insulin-induced increases in the mass of phosphatidylinositol, but 32PO4 incorporation therein followed a bimodal curve, with inhibition at lower doses and stimulation at higher doses; these divergent changes in phosphatidylinositol mass and 32P-labeling at higher concentrations raise the possibility that cycloheximide may activate phospholipases in hormone-stimulated tissues. These results offer further support for our contention that ACTH and insulin increase inositide phospholipid concentrations in their target tissues by a cycloheximide-sensitive mechanism.