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.
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.
Incubation of rat brown adipocytes with noradrenaline in the presence of insulin and palmitate caused a decrease in the rate of triacylglycerol synthesis as measured by [U-14C]glucose incorporation into acylglycerol glycerol. Concomitantly, the ratio of [1-14C]palmitate oxidized to CO2 to that esterified was increased. This alteration in the rate of triacylglycerol synthesis by noradrenaline was not observed when fatty acid oxidation was inhibited by etomoxir. Noradrenaline did not cause any acute inactivation of enzymes of the triacylglycerol-synthesis pathway. It is suggested that the decrease in triacylglycerol synthesis seen with noradrenaline is secondary to activation of fatty acid oxidation.
1. Rats were made hypothyroid by feeding them with propylthiouracil together with a low-iodine diet for 4 weeks. 2. [U-14C]Glucose conversion into fatty acids was substantially enhanced in brown adipocytes isolated from hypothyroid rats. Incorporation of 3H2O into fatty acids in vivo was enhanced in hypothyroidism in interscapular brown fat, but not in epididymal white fat or in liver. Hypothyroidism increased the activities of fatty acid synthase and ATP citrate lyase in brown, but not in white, adipocytes. 3. Glycolytic flux in brown adipocytes, quantified by [3-3H]glucose detritiation, was increased by hypothyroidism. This change was accompanied by increased maximum activity of phosphofructokinase. In white adipocytes a large increase in phosphofructokinase maximum activity was observed in hypothyroidism, but this change was accompanied by only small increases in the rate of glucose detritiation by incubated cells. It is suggested that in the brown adipocyte the overall conversion of glucose into fatty acids is enhanced in thyroid deficiency, but that this change is muted in the white adipocyte, possibly because of limitation of glucose transport. 4. Fatty acid synthesis in brown adipocytes from hypothyroid animals was considerably less sensitive to inhibition by exogenous fatty acids than is the process in cells from euthyroid animals. Consequently, the effect of hypothyroidism to enhance lipogenesis is amplified in the presence of physiological concentrations of fatty acid.
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