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Abstract
The effects of hormones on phosphoinositide metabolism were examined in rat adipocytes prelabeled with 32Pi or [3H]inositol. Oxytocin and vasopressin produced large decreases in labeled polyphosphoinositides and increases in phosphatidic acid and inositol phosphates, whereas insulin was without effect, although it stimulated lipogenesis from glucose. Likewise, insulin did not elevate 1,2-diacylglycerol measured chemically by high pressure liquid or thin-layer chromatography in fat cells or pads. It also did not increase the radioactivity in 1,2-diacylglycerol in ghosts prepared from fat cells previously labeled with [3H]arachidonic acid, although oxytocin and vasopressin increased this. It is therefore concluded that insulin does not stimulate the breakdown of polyphosphoinositides to yield 1,2-diacylglycerol and inositol phosphates in adipocytes and that the insulin-like actions of oxytocin must be due to other changes. Insulin induced small, but significant and equal increases (40% at 30 min) in the incorporation of [3H] inositol into phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in adipocytes. The effects were not dependent upon glucose and were not evident before 15 min. Oxytocin also produced large increases in the labeling of the three phosphoinositides. Insulin stimulated the incorporation of [3H]glycerol into the three phosphoinositides and also phosphatidic acid, phosphatidylserine, and phosphatidylethanolamine by 50-100% in cells incubated without glucose. No changes in the labeling of glycerol 3-phosphate, lysophosphatidic acid, phosphatidylcholine, and triacylglycerol were detected, and there was a small increase (30%) in 1,2-diacylglycerol labeling. It is concluded that insulin increases the synthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylethanolamine, and phosphatidylserine in fat cells partly by stimulating a reaction(s) located between glycerol 3-phosphate and phosphatidic acid in the biosynthetic pathway.
Highlights
From theHoward Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt Uniuersity School of Medicine, Nashville, Tennessee37232
There have been some reports that insulin large decreases in labeled polyphosphoinositides and increases protein kinase C activity in BC3H-1 cells [10] and increases in phosphatidic acid and inositol phosphates, that some, butnot all, of theactions of insulin can be whereas insulin was withouetffect, it stimu- mimicked by phorbol esters [5, 13,14,15,16,17,18,19,20,21] or by exposure of fat lated lipogenesis from glucose
The experiments reported here can be consideredin relation to three questionsconcerning phospholipid metabolism in fat cells, namely 1)is phosphoinositide breakdown involved in the responses to insulin and oxytocin; 2) do the hormones increase DAG content; and 3) do the hormones increase the synthesis of phosphoinositides? Thedata presented show that, incontrast to oxytocin and vasopressin, which produced large effects, insulin at a maximally effective concentration had no effect on the breakdown of PIP, or PIP or on the accumulation of IPSin fat cells
Summary
After separation from buffer, fat cells were resuspended in 5 ml of ice-cold extraction medium (0.25 M sucrose, 10 mM Tris/HCl, 2 mM EGTA (pH 7.4)) and were broken by vigorous agitation in a Effect of Insulin on Lipogenesis in Rat F a t Cells-As a routine check of the responsiveness of isolated fat cells to insulin, in those experiments givingnegative results, the effect of the hormone on lipogenesiswas measured in presence or absence of 1 mM glucose. In experimentsinvolvingthe measurement of IPS,the reaction was stopped by adding 500 pl of cell suspensions (500-600 pg of protein) lipids-Fig. 1 illustrates the effects of insulin and oxytocin on the radioactivity of polyphosphoinositides and phosphatidic acid in 3ZP-prelabeledfat cells. Aliquots (500 pl) of cells were mixed with 1.5 ml [35]
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