Reversible inactivation by noradrenaline of long-chain fatty acyl-CoA synthetase in rat adipocytes.

Incubation of rat adipocytes with the same range of noradrenaline concentrations that stimulate lipolysis caused a rapid and stable decrease in the activity of fatty acyl-CoA synthetase. Corticotropin, glucagon and dibutyryl cyclic AMP also decreased the activity of the enzyme. The effect of noradrenaline was apparent over a wide range of concentrations for the three substrates of the enzyme. A novel fluorescence assay of fatty acyl-CoA synthetase using (1,N6-etheno)-CoA is described. The effect of noradrenaline was not abolished by inclusion of albumin in homogenization buffers, persisted through subcellular fractionation and isolation of microsomes (microsomal fractions) and even survived treatment of microsomes with Triton X-100. The effect of noradrenaline was rapidly reversed within cells by the subsequent addition of insulin or propranolol. The inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline. Additions of cyclic AMP-dependent protein kinase to adipocyte microsomes caused considerable phosphorylation of microsomal protein by [gamma-32P]ATP, but did not affect the activity of fatty acyl-CoA synthetase.     

Regulation by noradrenaline of the mitochondrial and microsomal forms of glycerol phosphate acyltransferase in rat adipocytes.

Incubation of rat adipocytes with 1 microM-noradrenaline caused a decrease in both the N-ethylmaleimide-sensitive (microsomal) and N-ethylmaleimide-insensitive (mitochondrial) glycerol phosphate acyltransferase activities measured in homogenates from freeze-stopped cells. The effects of noradrenaline on glycerol phosphate acyltransferase activity were apparent over a wide range of concentrations of glycerol phosphate and palmitoyl-CoA. The effect of noradrenaline was reversed within cells by the subsequent addition of insulin or propranolol. Inclusion of albumin in homogenization buffers abolished the effect of noradrenaline on the N-ethylmaleimide-sensitive activity. The effect of noradrenaline on the N-ethylmaleimide-insensitive (mitochondrial) activity was, however, not abolished by inclusion of albumin in buffers for preparation of homogenates from freeze-stopped cells. Inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline. The inactivating effect of noradrenaline persisted through the subcellular fractionation procedures used to isolate adipocyte microsomes (microsomal fractions). The effect of noradrenaline on mitochondrial glycerol phosphate acyltransferase did not persist through subcellular fractionation. Noradrenaline treatment of cells significantly decreased the Vmax. of glycerol phosphate acyltransferase in isolated microsomes without changing the activity of NADPH-cytochrome c reductase. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells is unstable, being rapidly lost on incubation at 30 degrees C. Bivalent metal ions (Mg2+, Ca2+) or post-microsomal supernatant protected against this inactivation. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells could not be re-activated by incubation with either alkaline phosphatase or phosphoprotein phosphatase-1. Addition of cyclic AMP-dependent protein kinase catalytic subunits to adipocyte microsomes incubated with [gamma-32P]ATP considerably increased the incorporation of 32P into microsomal protein, but did not cause inactivation of glycerol phosphate acyltransferase. These findings provide no support for the proposal that inactivation of adipocyte microsomal glycerol phosphate acyltransferase by noradrenaline is through a phosphorylation type of covalent modification.     

Lipogenesis in rat brown adipocytes. Effects of insulin and noradrenaline, contributions from glucose and lactate as precursors and comparisons with white adipocytes.

1. Brown adipocytes were isolated from the interscapular depot of male rats maintained at approx. 21 degrees C. In some experiments parallel studies were made with white adipocytes from the epididymal depot. 2. Insulin increased and noradrenaline decreased [U-14C]glucose incorporation into fatty acids by brown adipocytes. Brown adipocytes differed from white adipocytes in that exogenous fatty acid (palmitate) substantially decreased fatty acid synthesis from glucose. Both noradrenaline and insulin increased lactate + pyruvate formation by brown adipocytes. Brown adipocytes converted a greater proportion of metabolized glucose into lactate + pyruvate and a smaller proportion into fatty acids than did white adipocytes. 3. In brown adipocytes, when fatty acid synthesis from [U-14C]glucose was decreased by noradrenaline or palmitate, incorporation of 3H2O into fatty acids was also decreased to an extent which would not support proposals for extensive recycling into fatty acid synthesis of acetyl-CoA derived from fatty acid oxidation. 4. In the absence of glucose, [U-14C]lactate was a poor substrate for lipogenesis in brown adipocytes, but its use was facilitated by glucose. When brown adipocytes were incubated with 1 mM-lactate + 5 mM-glucose, lactate-derived carbon generally provided at least 50% of the precursor for fatty acid synthesis. 5. Both insulin and noradrenaline increased [U-14C]glucose conversion into CO2 by brown adipocytes (incubated in the presence of lactate) and, in combination, stimulation of glucose oxidation by these two agents showed synergism. Rates of 14CO2 formation from glucose by brown adipocytes were relatively small compared with maximum rates of oxygen consumption by these cells, suggesting that glucose is unlikely to be a major substrate for thermogenesis. 6. Brown adipocytes from 6-week-old rats had considerably lower maximum rates of fatty acid synthesis, relative to cell DNA content, than white adipocytes. By contrast, rates of fatty acid synthesis from 3H2O in vivo were similar in the interscapular and epididymal fat depots. Expressed relative to activities of fatty acid synthase or ATP citrate lyase, however, brown adipocytes synthesized fatty acids as effectively as did white adipocytes. It is suggested that the cells most active in fatty acid synthesis in the brown adipose tissue are not recovered fully in the adipocyte fraction during cell isolation. Differences in rates of fatty acid synthesis between brown and white adipocytes were less apparent at 10 weeks of age.     

Comparison of triacylglycerol synthesis in rat brown and white adipocytes. Effects of hypothyroidism and streptozotocin-diabetes on enzyme activities and metabolic fluxes.

1. Adipocytes were isolated from the interscapular brown fat and the epididymal white fat of normal, streptozotocin-diabetic and hypothyroid rats. 2. Measurements were made of the maximum rate of triacylglycerol synthesis by monitoring the incorporation of [U-14C]glucose into acylglycerol glycerol in the presence of palmitate (1 mM) and insulin (4 nM) and of the activities of the following triacylglycerol-synthesizing enzymes: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), dihydroxyacetonephosphate acyltransferase (DHAPAT), monoacylglycerol phosphate acyltransferase (MGPAT), Mg2+-dependent phosphatidate phosphohydrolase (PPH) and diacylglycerol acyltransferase (DGAT). 3. FAS activity in brown adipocytes was predominantly localized in the mitochondrial fraction, whereas a microsomal localization of this enzyme predominated in white adipocytes. Subcellular distributions of the other enzyme activities in brown adipocytes were similar to those shown previously with white adipocytes [Saggerson, Carpenter, Cheng & Sooranna (1980) Biochem. J. 190, 183-189]. 4. Relative to cell DNA, brown adipocytes had lower activities of triacylglycerol-synthesizing enzymes and showed lower rates of metabolic flux into acylglycerols than did white adipocytes isolated from the same animals. 5. Diabetes decreased both metabolic flux into acylglycerols and the activities of triacylglycerol-synthesizing enzymes in white adipocytes. By contrast, although diabetes decreased metabolic flux into brown-adipocyte acylglycerols by 80%, there were no decreases in the activities of triacylglycerol-synthesizing enzymes, and the activity of PPH was significantly increased. 6. Hypothyroidism increased metabolic flux into acylglycerols in both cell types, and increased activities of all triacylglycerol-synthesizing enzymes in brown adipocytes. By contrast, in white adipocytes, although hypothyroidism increased the activities of FAS, microsomal GPAT and DGAT, this condition decreased the activities of mitochondrial GPAT and PPH. 7. It was calculated that the maximum capabilities for fatty acid oxidation and esterification are approximately equal in brown adipocytes. In white adipocytes esterification is predominant by approx. 100-fold. 8. Diabetes almost abolished incorporation of [U-14C]glucose into fatty acids in both adipocyte types. Hypothyroidism increased fatty acid synthesis in white and brown adipocytes by 50% and 1000% respectively.     

The inactivation of rat adipocyte Mg2+-dependent phosphatidate phosphohydrolase by noradrenaline.

The inactivation of rat adipocyte Mg2+-dependent phosphatidate phosphohydrolase by noradrenaline [Cheng & Saggerson (1978) FEBS Lett. 87, 65--68; Cheng & Saggerson (1978) FEBS Lett. 93, 120--124] persists for at least 40 min in crude defatted homogenates kept at 0 degrees C or 20 degrees C, but is diminished at 37 degrees C. The effect of noradrenaline persists through the isolation of post-105000 g supernatants and is then stable in these preparations at 0 degrees C and 37 degrees C. Inclusion of albumin (10--20 mg/ml) in homogenization buffers abolishes the effect of noradrenaline. The effect of noradrenaline is not removed by dialysis of extracts or by raising the concentrations of Mg2+ or phosphatidate in assays.     

Triacylglycerol biosynthesis in the adipose tissue of the obese-hyperglycaemic mouse.

Obesity in obese-hyperglycaemic mouse is associated with an increase in number and size of adipocytes. Adipocytes from the obese mouse showed increased incorporation of [14C]acetate and[14C]glucose into triacylglycerol. This increased capacity of triacylglycerol formation was correlated with increased activities of various triacylglycerol-forming enzymes measured in the microsomal fraction of adipose tissue from obese mice. Microsomal fractions from lean and obese mice contained sn-glycerol 3-phosphate acyltransferase, phosphatidate phosphohydrolase and diacylglycerol acyltransferase. Phosphatidate phosphohydrolase was also detected in the soluble fraction. In the presence of Mg2+, the phosphatidate phsophohydrolase from the soluble and the microsomal fractions was active towards membrane-bound phosphatidate. Among the three enzymes studied here, the increase in Mg2+-dependent phosphatidate phosphohydrolase was most prominent in adipose tissue of obese mice.     

Measurements of glycolytic flux rate in brown adipocytes. Effects of insulin, noradrenaline and streptozotocin diabetes

Glycolytic flux was estimated in brown adipocytes by [3-3H]-glucose detritiation. Without insulin the process was slightly stimulated by noradrenaline or palmitate. Insulin stimulated glucose detritiation by 4-fold. Noradrenaline stimulated the process in the presence of insulin and synergism between these hormones was observed. Palmitate did not stimulate glucose detritiation in the presence of insulin suggesting that the effect of noradrenaline is not secondary to stimulation of lipolysis. With insulin, cells from streptozotocin-diabetic rats showed lower rates of glucose detritiation. Extracts from these cells also had lower maximum activities of phosphofructokinase.     

Metabolism of palmitate in perfused rat liver. Isolation of subcellular fractions containing triacylglycerol.

1. The metabolism of [1-14C]palmitate in rat liver was studied in a single-pass perfusion system at concentrations of 0.2 or 1 mM. 2. After the perfusion the liver was homogenized and the floating fat was isolated. The incorporation of [1-14C]palmitate into triacylglycerol in this pool increased 9-fold when the palmitate concentration in the medium was increased from 0.2 to 1 mM. In time studies with 1 mM-[1-14C]palmitate 75% of the total accumulation of triacylglycerol occurred in this pool. Our results support the concept that the floating-fat fraction contains the storage pool of triacylglycerol, i.e. the cytoplasmic lipid droplets. 3. In a particulate preparation consisting mainly of mitochondria and microsomal fraction the incorporation of [1-14C]palmitate into triacylglycerol was proportional to the fatty acid concentration. Triacylglycerol in the perfusate medium and in the particulate fraction was in isotopic equilibrium, which indicates that the particulate fraction contained the precursor pool for secreted triacylglycerol, i.e. the pool in endoplasmic reticulum and Golgi apparatus. 4. The oxidation to labelled water-soluble products and to CO2 was increased 14-fold by the 5-fold increase in palmitate concentration.     

Storage, mobilization and secretion of cytosolic triacylglycerol in hepatocyte cultures. The role of insulin.

Cytosolic triacylglycerol labelled from [3H]oleate accounted for almost 50% (57 +/- 22 nmol/mg of protein) of the total cellular triacylglycerol which was newly synthesized by cultured hepatocytes during a 24 h incubation. Insulin decreased the export of triacylglycerol as very-low-density lipoprotein (VLDL) during this period. This resulted in a sequestration of newly synthesized triacylglycerol in the cytosol, rather than in the particulate fraction of the cell. Longer periods of incubation with [3H]oleate resulted in increased concentrations of newly synthesized triacylglycerol within the cell, most of which (78 +/- 3% after 48 h; 80 +/- 3% after 72 h) was located within the cytosolic fraction. The quantity of newly synthesized triacylglycerol in the cell cytosol was further increased by insulin. During these periods there were decreases in the amounts of triacylglycerol associated with the particulate fraction of the cell, irrespective of the presence or absence of insulin. In no case was a decrease in VLDL triacylglycerol secretion in response to insulin accompanied by an increased triacylglycerol content in the particulate fraction of the cell. In some experiments, the fate of the cytosolic triacylglycerol was studied by pulse labelling with [3H]oleate. In these cases, when insulin was removed from the medium of cells to which they had previously been exposed, more newly synthesized triacylglycerol was secreted compared with cells which had not been exposed to insulin. This extra triacylglycerol was mobilized from the cytosolic rather than from the particulate fraction of the cell. Subsequent addition of insulin to the medium prevented the mobilization of cytosolic triacylglycerol. These results suggest that insulin enhances the storage of hepatocellular triacylglycerol in a cytosolic pool. Deficiency of insulin in the medium stimulates the mobilization of this pool which is channelled into the secretory pathway, entering the extracellular medium as VLDL.     

Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes.

Myocardial triacylglycerol hydrolysis is subject to product inhibition. After hydrolysis of endogenous triacylglycerols, the main proportion of the liberated fatty acids is re-esterified to triacylglycerol, indicating the importance of fatty acid re-esterification in the regulation of myocardial triacylglycerol homoeostasis. Therefore, we characterized phosphatidate phosphohydrolase (PAP) and diacylglycerol acyltransferase (DGAT) activities, enzymes catalysing the final steps in the re-esterification of fatty acids to triacylglycerols in the isolated rat heart. The PAP activity was mainly recovered in the microsomal and soluble cell fractions, with an apparent Km of 0.14 mM for both the microsomal and the soluble enzyme. PAP was stimulated by Mg2+ and oleic acid. Oleic acid, like a high concentration of KCl, stimulated the translocation of PAP activity from the soluble to the particulate (microsomal) fraction. Myocardial DGAT had an apparent Km of 3.8 microM and was predominantly recovered in the particulate (microsomal) fraction. Both enzyme activities were significantly increased after acute streptozotocin-induced diabetes, PAP from 15.6 +/- 1.1 to 28.1 +/- 3.6 m-units/g wet wt. (P less than 0.01) and DGAT from 2.23 +/- 0.11 to 3.01 +/- 0.11 m-units/g wet wt. (P less than 0.01). In contrast with diabetes, low-flow ischaemia during 30 min did not affect PAP and DGAT activity in rat hearts. Perfusion with glucagon (0.1 microM) during 30 min did not affect total PAP activity, but changed the subcellular distribution. More PAP activity was recovered in the particulate fraction. DGAT activity was lowered by glucagon treatment from 0.37 +/- 0.03 to 0.23 +/- 0.02 m-unit/mg of microsomal protein (P less than 0.05). The role of PAP and DGAT activity and PAP distribution in the myocardial glucose/fatty acid cycle is discussed.     

Regulation of fructose 2,6-bisphosphate concentration in white adipose tissue.

Injection of insulin to fed rats diminished the concentration of fructose 2,6-bisphosphate in white adipose tissue. Incubation of epididymal fat-pads or adipocytes with insulin stimulated lactate release and sugar detritiation and also decreased fructose 2,6-bisphosphate concentration. Such a decrease was, however, not observed in fat-pads from starved or alloxan-diabetic rats. Incubation of adipocytes from fed rats with various concentrations of glucose or fructose led to a dose-dependent rise in fructose 2,6-bisphosphate which correlated with lactate output and detritiation of 3-3H-labelled sugar. In adipocytes from fed rats, palmitate stimulated the detritiation of [3-3H]glucose without affecting lactate production and fructose 2,6-bisphosphate concentration. Incubation of epididymal fat-pads from fed rats in the presence of antimycin stimulated lactate output but decreased fructose 2,6-bisphosphate concentration. Changes in lipolytic rates brought about by noradrenaline, insulin, adenosine and corticotropin in adipocytes from fed rats were not related to changes in fructose 2,6-bisphosphate or to rates of lactate output. In fed rats, the activity of 6-phosphofructo-2-kinase was not changed after treatment of adipocytes with insulin, noradrenaline or adenosine. It is suggested that the decrease in fructose 2,6-bisphosphate concentration observed after insulin treatment can be explained by the increase in sn-glycerol 3-phosphate, an inhibitor of 6-phosphofructo-2-kinase.     

Long-chain fatty acids and their acyl-CoA esters cause the translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction of rat liver

A translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction was promoted in cell-free extracts of rat liver by oleate and palmitate and their CoA esters. Oleate was more potent in this respect than palmitate and the CoA esters were more effective than the unesterified acids. Octanoate, octanoyl-CoA and CoA did not cause the translocation. It is proposed that the interaction of phosphatidate phosphohydrolase with the membranes that synthesize glycerolipids causes it to become metabolically active. This enables the liver to increase its capacity for triacylglycerol synthesis in response to an increased supply of fatty acids.     

Insulin and noradrenaline independently stimulate the translocation of glucose transporters from intracellular stores to the plasma membrane in mouse brown adipocytes.

The mechanism of the effect of noradrenaline on the transport of 3-O-methyl-D-[14C]glucose ([14C]-MG) was studied in mouse brown adipocytes. When cells were exposed to low concentrations (< 10(-8) M) of insulin, the [14C]-MG uptake by cells was enhanced by noradrenaline additively. The action of noradrenaline was mimicked by isoproterenol, and was completely blocked by propranolol. Exposing cells to noradrenaline induced both an increase in the transport activity of plasma membrane fractions and a decrease in that of microsomal fractions similar to insulin exposure, indicating that noradrenaline also induces the translocation of glucose transporters to the plasma membrane. The ratio of an increase in the transport activity of plasma membrane fraction to a decrease in the activity of microsomal fraction was lower in cells exposed to noradrenaline than in cells exposed to insulin. This quantitative disagreement suggests that there are at least two different modes involved in the regulation of the translocation of glucose transporters in mouse brown adipocytes.     

Sensitivity of adipocyte lipolysis to stimulatory and inhibitory agonists in hypothyroidism and starvation.

The responsiveness of lipolysis to the stimulatory agonists noradrenaline, corticotropin and glucagon and to the inhibitory agonists N6-phenylisopropyladenosine, prostaglandin E1 and nicotinic acid was investigated with rat white adipocytes incubated with a high concentration of adenosine deaminase (1 unit/ml). The cells were obtained from fed or 48 h-starved euthyroid animals or from fed or starved animals rendered hypothyroid by 4 weeks of treatment with low-iodine diet and propylthiouracil. Hypothyroidism increased sensitivity to and efficacy of all three inhibitory agonists in their opposition of noradrenaline-stimulated lipolysis. Starvation decreased sensitivity to all three inhibitory agonists when opposing basal lipolysis. Hypothyroidism decreased sensitivity to noradrenaline, glucagon and corticotropin by 37-, 4- and 4-fold respectively and decreased the maximum response to these agonists by approx. 50%, 50% and 75% respectively. Starvation reversed decreases in maximum response to these agonists in hypothyroidism. Starvation in the euthyroid state increased sensitivity to glucagon and noradrenaline, but did not alter sensitivity to corticotropin. Cells from hypothyroid rats were relatively insensitive to Bordetella pertussis toxin, which substantially increased basal lipolysis in the euthyroid state.     

Rapid modulation of adipocyte phosphofructokinase activity by noradrenaline and insulin.

1. Alterations in phosphofructokinase properties can be reproducibly seen in tissue extracts prepared and rapidly assayed after exposure of rat adipocytes to hormones. 2. Noradrenaline, corticotropin or isoprenaline (isoproterenol; beta-adrenergic agonist) decreased the activity measured with high fructose 6-phosphate concentrations (3--6 mM), but increased activity measured with lower concentrations of this substrate (0.3--0.9 mM). Noradrenaline decreased the Vmax. and the concentration of fructose 6-phosphate that gave half the Vmax.. 3. Insulin opposed the actions of noradrenaline and itself increased phosphofructokinase activity. 4. The effect of noradrenaline appeared to be exerted through a beta- rather than an alpha-type of adrenoceptor. 5. The effects of noradrenaline to decrease phosphofructokinase activity at high [fructose 6-phosphate] and to increase activity at low [fructose 6-phosphate] could be rapidly reversed in cells by addition of the beta-blocker propranolol. 6. The effect of noradrenaline seen at low [fructose 6-phosphate] could be abolished by homogenization of cells in buffer containing albumin or reversed by brief incubation of tissue extracts with albumin, suggesting that this effect of the hormone is due to the association of some ligand with the enzyme.     

The activities of lipoprotein lipase and of enzymes involved in triacylglycerol synthesis in rat adipose tissue. Effects of starvation, dietary modification and of corticotropin injection.

1. The effects of dietary modification, including starvation, and of corticotropin injection on the activities of acyl-CoA synthetase, glycerol phosphate acyltransferase, dihydroxyacetone phosphate acyltransferase, phosphatidate phosphohydrolase, diacylglycerol acyltransferase and lipoprotein lipase were measured in adipose tissue. 2. Lipoprotein lipase activities in heart were increased and those in adipose tissue were decreased when rats were fed on diets enriched with corn oil or beef tallow rather than with sucrose or starch. The lipoprotein lipase activity was lower in the adipose tissue of rats fed on the sucrose rather than on the starch diet. 3. Rats fed on the beef tallow diet had slightly higher activities of the total glycerol phosphate acyltransferase in adipose tissue than did rats fed on the sucrose or starch diet. The diacylglycerol acyltransferase and the mitochondrial glycerol phosphate acyltransferase activities were higher for the rats fed on the tallow diet than for those fed on the corn-oil diet. 4. Starvation significantly decreased the activities of lipoprotein lipase (after 24 and 48 h), acyl-CoA synthetase (after 24 h) and of the mitochondrial glycerol phosphate acyltransferase and the N-ethylmaleimide-insensitive dihydroxyacetone phosphate acyltransferase (after 48 h) in adipose tissue. The activities of the microsomal glycerol phosphate acyltransferase, diacylglycerol acyltransferase and the soluble phosphatidate phosphohydrolase were not significantly changed after 24 or 48 h of starvation. 5. The activities of lipoprotein lipase and phosphatidate phosphohydrolase in adipose tissue were decreased 15 min after corticotropin was injected into rats during November to December. No statistically significant differences were found when these experiments were performed during March to September. These differences may be related to the seasonal variation in acute lipolytic responses. 6. These results are discussed in relation to the control of triacylglycerol synthesis and lipoprotein metabolism.     

Starvation-induced changes of palmitate metabolism and insulin secretion in isolated rat islets stimulated by glucose.

The influence of 48 h starvation on glucose-induced changes of palmitate metabolism and insulin release in isolated rat islets was investigated. (1) Islet insulin response to 20 mM-glucose was abolished after 48 h starvation, and it was restored by 0.25 mM-2-bromostearate, an inhibitor of fatty acid oxidation. (2) The increase in glucose concentration from 3 to 20 mM was accompanied by a 50% decrease in the oxidation rate of 0.5 mM-[U-14C]palmitate in control (fed) islets, and a concomitant increase (100%) in its incorporation into triacylglycerol and phospholipid fractions. (3) Starvation induced a higher basal (3 mM-glucose) rate of palmitate oxidation, which was resistant to inhibition by 20 mM-glucose. The latter also failed to increase palmitate incorporation into islet triacylglycerols and phospholipids. (4) 2-Bromostearate (0.25 mM) strongly inhibited the high oxidation rate of palmitate in islets of starved rats, and allowed a normal stimulation of its incorporation rate into islet lipids by 20mM-glucose. (5) The results suggest that starvation restricts islet esterification of fatty acids by inducing a higher rate of their oxidative degradation that is insensitive to regulation by glucose.     

Differential effects of palmitate on glucose uptake in rat-1 fibroblasts and 3T3-L1 adipocytes.

Non-esterified fatty acids are thought to be one of the causes for insulin resistance. However, the molecular mechanism of fatty acid-induced insulin resistance is not clearly known. In this study, we first examined the effect of palmitate on insulin signaling in 3T3-L1 adipocytes. We found that 1h treatment with 1 mmol/l palmitate had no effect on insulin binding, tyrosine phosphorylation of insulin receptors, 185 kDa proteins and Shc, and PI3 kinase activity in 3T3-L1 adipocytes. Then, the effects of palmitate on MAP kinase activity and glucose uptake in fully differentiated 3T3-L1 adipocytes were compared with those in poorly differentiated 3T3-L1 cells and in HIRc-B cells. Palmitate treatment had no effect on MAP kinase activity in fully differentiated 3T3-L1 adipocytes, while it inhibited MAP kinase in poorly differentiated 3T3-L1 cells and HIRc-B cells. Glucose transport in 3T3-L1 adipocytes treated with palmitate for 1 h, 4 h and 16 h was higher than that in control cells, but palmitate treatment caused a rightward shift of the insulin-dose responsive curve for glucose uptake in HIRc-B cells. Palmitate treatment did not significantly affect basal and insulin-stimulated GLUT4 translocation. When the cells were treated with PD98059, a specific MEK inhibitor, insulin-stimulated glucose uptake was not affected in 3T3-L1 adipocytes, while it was almost completely inhibited in HIRc-B cells. These results suggest the primary effect of palmitate on adipocytes may not involve insulin resistance of adipocytes themselves.     

Glycerolipid biosynthesis in rat adipose tissue. Influence of adipocyte size.

A simple one-step filtration method is described to separate larger adipocytes from the smaller ones by using nylon screen (52 microM pore size). Adipocytes retained on the screen were larger (60-90 micrometers) compared with those that passed through the screen. By using this separation technique, activities of various enzymes involved in triacylglycerol formation from sn-glycerol 3-phosphate were measured in the larger and smaller adipocytes isolated from gonadal fat-depots. The homogenates from larger adipocytes were more active in lipid formation compared with those derived from small adipocytes. This was evident from the increased activities of sn-glycerol 3-phosphate acyltransferase. Mg2+-dependent phosphatidate phosphohydrolase and diacylglycerol acyltransferase in the larger adipocytes. The activities of these enzymes were also measured in the adipocytes isolated from gonadal, perirenal and subcutaneous fat-depots. Subcutaneous adipocytes were smaller and were less active in lipid formation than gonadal and perirenal adipocytes. These measurements in the activities of individual enzymes provide evidence that the entire pathway of esterification via sn-glycerol 3-phosphate is accelerated in the larger adipocytes.     

Phosphatase activity in rat adipocytes: effects of insulin and insulin resistance

Insulin regulates the activity of both protein kinases and phosphatases. Little is known concerning the subcellular effects of insulin on phosphatase activity and how it is affected by insulin resistance. The purpose of this study was to determine insulin-stimulated subcellular changes in phosphatase activity and how they are affected by insulin resistance. We used an in vitro fatty acid (palmitate) induced insulin resistance model, differential centrifugation to fractionate rat adipocytes, and a malachite green phosphatase assay using peptide substrates to measure enzyme activity. Overall, insulin alone had no effect on adipocyte tyrosine phosphatase activity; however, subcellularly, insulin increased plasma membrane adipocyte tyrosine phosphatase activity 78 +/- 26% (n = 4, P < 0.007), and decreased high-density microsome adipocyte tyrosine phosphatase activity 42 +/- 13% (n = 4, P < 0.005). Although insulin resistance induced specific changes in basal tyrosine phosphatase activity, insulin-stimulated changes were not significantly altered by insulin resistance. Insulin-stimulated overall serine/threonine phosphatase activity by 16 +/- 5% (n = 4, P < 0.005), which was blocked in insulin resistance. Subcellularly, insulin increased plasma membrane and crude nuclear fraction serine/threonine phosphatase activities by 59 +/- 19% (n = 4, P < 0. 005) and 21 +/- 7% (n = 4, P < 0.007), respectively. This increase in plasma membrane fractions was inhibited 23 +/- 7% (n = 4, P < 0. 05) by palmitate. Furthermore, insulin increased cytosolic protein phosphatase-1 (PP-1) activity 160 +/- 50% (n = 3, P < 0.015), and palmitate did not significantly reduce this activity. However, palmitate did reduce insulin-treated low-density microsome protein phosphatase-1 activity by 28 +/- 6% (n = 3, P < 0.04). Insulin completely inhibited protein phosphatase-2A activity in the cytosol and increased crude nuclear fraction protein phosphatase-2A activity 70 +/- 29% (n = 3, P < 0.038). Thus, the major effects of insulin on phosphatase activity in adipocytes are to increase plasma membrane tyrosine and serine/threonine phosphatase, crude nuclear fraction protein phosphatase-2A, and cytosolic protein phosphatase-1 activities, while inhibiting cytosolic protein phosphatase-2A. Insulin resistance was characterized by reduced insulin-stimulated serine/threonine phosphatase activity in the plasma membrane and low-density microsomes. Specific changes in phosphatase activity may be related to the development of insulin resistance.