Time-dependent effects of insulin on lipid synthesis in cultured fetal rat hepatocytes: a comparison between lipogenesis and glycogenesis.

The lipogenic effect of insulin was studied in 18-day-old fetal rat hepatocytes after 2 to 3 days of culture in the presence of glucocorticoids when an acute stimulatory effect of insulin on glycogenesis was present. The rate of [1-14C]-acetate incorporation into lipids measured for 4 hours was much higher than with [U-14C]-glucose (30 v 3.8 nmol/h/mg protein). The stimulatory effect of insulin on lipid labeling remained weak (1.2-fold) and contrasted with its striking stimulatory effect on [U-14C]-glucose incorporation into glycogen (fourfold). When lipid labeling was assessed in longer experiments, increasing acetate concentrations in the medium stimulated the incorporation rate of [1-14C]-acetate into lipids (3.5-fold from 1 to 5 mmol/L after 36 hours) and decreased that of [U-14C]-glucose (by twofold). The stimulatory effect of insulin on the rate of lipid labeling developed with both precursors from 12 to 36 hours after insulin exposure (by approximately twofold) independently of acetate concentration and was not glucocorticoid-dependent, contrary to the glycogenic response. Addition of a glucose, load simultaneously with insulin increased the stimulation of lipogenesis when measured with [U-14C]-glucose (twofold to 3.7-fold). Besides contributing to an accumulation of larger and numerous lipid droplets in the cells, insulin increased fatty acid synthase activity by 26%, whereas malic enzyme was not affected. Thus, insulin-dependent lipogenesis in cultured fetal hepatocytes appears to be mostly regulated by a long-term mechanism, contrary to the glycogenic effect of insulin.

Compared roles of glucose, galactose and fructose as glycogen precursors during the acute response to insulin in cultured rat foetal hepatocytes.

1. The efficiency of the contribution of hexoses to basal- and stimulated-glycogenesis, when studied in cultured 18 day-old rat foetal hepatocytes in the presence of glucose, was as follows: galactose greater than glucose greater than fructose. 2. Glucose deprivation had opposite effects on the contributions of [14C]galactose (decreased) and [14C]fructose (increased) to glycogenesis, which occurred independently of insulin and were reversed by glucose concentrations as low as 30-100 microM. 3. The stimulation of glycogenesis by insulin measured with [14C]glucose (3.2-fold) was superior to that obtained with either [14C]galactose or [14C]fructose (2.7-fold in both cases), which revealed a specific beneficial effect of insulin on glucose contribution.

Cholesterol depletion affects the Ca2+ influx but not the Ca2+ pump in human erythrocytes.

Control and cholesterol-depleted human erythrocytes were loaded with permeant Ca2+ chelators (Benz2-AM or Quin2-AM) in order to increase their exchangeable Ca2+ pool and to measure both Ca2+ fluxes and [Ca]i (free cytoplasmic calcium concentration). The fluxes were independent of the concentration and of the nature of the intracellular chelator. The ATP content was not decreased by more than 50% under our experimental conditions. Cholesterol depletion (up to 28%) induced a decrease in both Ca2+ fluxes and [Ca]i which was proportional to the extent of the depletion. It is shown that cholesterol depletion primarily altered the properties of the system responsible for Ca2+ entry causing a diminution of the [Ca]i. This, in turn, induced a diminution of the activity of the Ca2+ pump without affecting the properties of this pump.

Phosphoinositide reorganization in human erythrocyte membrane upon cholesterol depletion.

The effect of cholesterol depletion on the activity of phosphatidylinositol/phosphatidylinositol 4-phosphate and diacylglycerol kinases and polyphosphoinositide phosphodiesterase has been studied in isolated membranes of human normal and cholesterol-depleted erythrocytes. Polyphosphoinositide synthesis (phosphatidylinositol/phosphatidylinositol 4-phosphate kinase activities) were found to depend on the permeability and sidedness characteristics of the membrane vesicles, which could limit the accessibility of ATP for the enzymes. When measured under proper conditions, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate synthesis were decreased in cholesterol-depleted membranes as compared with control membranes. The same level of synthesis could be obtained in both membranes by the addition of phosphatidylinositol (and Triton X-100) or of phosphatidylinositol 4-phosphate. Phosphatidic acid synthesis (diacylglycerol kinase activity) was also decreased in cholesterol-depleted membranes as compared with control membranes when measured in the presence of Ca2+. Addition of diolein (and Triton X-100) caused a large increase in phosphatidic acid synthesis which reached approximately the same level in both membranes. This showed that the apparent inhibition of polyphosphoinositide and phosphatidic acid synthesis was not due to a loss or to an inactivation of the kinases. Ca2+-activated polyphosphoinositide phosphodiesterase promoted the hydrolysis of 65-70% of the polyphosphoinositides in control and of only 45-55% in cholesterol-depleted membranes without changing the Ca2+ concentration for half-maximum hydrolysis (1 microM). Upon addition of sodium oleate, the extent of polyphosphoinositide hydrolysis became identical in both membranes, indicating again that there was no loss nor inactivation of the polyphosphoinositide phosphodiesterase in the cholesterol-depleted membranes. Since the concentration of the polyphosphoinositides was not changed by cholesterol depletion [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200], the reduction in both their synthesis and degradation observed here could be attributed to a reorganization of the phosphoinositides in membrane domains where they were not accessible to the kinases and phosphodiesterase. The reduction in phosphatidic acid synthesis was likely caused by a reduction in the total amount of the substrate diacylglycerol in cholesterol-depleted membranes as already shown [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200].

Changes in morphology and in polyphosphoinositide turnover of human erythrocytes after cholesterol depletion.

Human erythrocytes were cholesterol-depleted (5-25%) by incubation with phosphatidylcholine vesicles in media containing Ca2+ at different concentrations (0, 28 nM, 5 microM or 1 mM). After removal of the vesicles, the cells were reincubated with [32P]phosphate in the same media. Control (incubated in buffer alone) and cholesterol-maintained erythrocytes (incubated with cholesterol/phosphatidylcholine vesicles) were treated similarly. Cholesterol depletion induced the conversion of the cells into stomatocytes III and spherostomatocytes and decreased the turnover rate of phosphatidylinositol phosphate and of phosphatidylinositol bisphosphate. None of these effects were observed in cholesterol-maintained cells. In cholesterol-depleted cells, they occurred without changes in the ATP specific activity or in the polyphosphoinositide concentrations. Moreover, these modifications of shape and of lipid metabolism were proportional to the extent of the cholesterol depletion and were independent of the external Ca2+ concentration. In contrast, other effects of cholesterol depletion, a decrease in the turnover rate of phosphatidic acid, a decrease in diacylglycerol and in phosphatidic acid concentrations were dependent on the external Ca2+ concentration. Thus it appears that the shape change was not correlated with a change in the concentrations of these phospholipids or of diacylglycerol and therefore cannot be explained by a bilayer couple mechanism involving these phospholipids. However, the spherostomatocytic transformation was correlated with the decrease in the turnover rate of the polyphosphoinositides, but not with the turnover rate of phosphatidic acid, suggesting a role for the turnover of the polyphosphoinositides in the maintenance of the erythrocyte shape.