Regulatory enzymes of lipid metabolism in LA/N-cp rats

Hepatic activities of rate limiting enzymes in fatty acid and cholesterol synthesis and cholesterol degradation were determined in lean and obese LA/N-cp rats. The hepatic activities of acetyl-CoA carboxylase and fatty acid synthetase, the key enzymes of fatty acid synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase (the rate limiting enzyme in cholesterol synthesis), were increased 2-fold in the obese rats as compared with their lean littermates. In contrast, the activity of cholesterol 7alpha-hydroxylase, the rate limiting enzyme of cholesterol degradation to bile acids, was significantly decreased by 28% in the obese group as compared with the control group. Significantly, compared with the control group, the obese animals exhibited similar magnitudes of differences in the activities of the above enzymes even when they were pair-fed with the control animals. Thus these differences in the obese group are not due to hyperphagia but possibly to hypersecretion of the lipogenic hormone, insulin in this strain. These results indicate that the LA/N-cp obese rat has twice the capacity to synthesize body fat and cholesterol but has a reduced capacity to degrade the cholesterol, leading to increased accumulation of cholesterol and fat.     

Purification and characterization of ascamycin-hydrolysing aminopeptidase from Xanthomonas citri.

Intact obese rats were hyperinsulinaemic, had higher rates of whole-body fatty acid synthesis, higher activities of hepatic acetyl-CoA carboxylase and tyrosine aminotransferase and a higher hepatic glycogen concentration than intact lean animals. Adrenalectomy abolished all these factors of the obese phenotype. Treatment of adrenalectomized rats with corticosterone for 24 h increased the rate of whole-body fatty acid synthesis to the same extent in both phenotypes, but caused a larger increase in glycogen concentration, tyrosine aminotransferase activity and plasma insulin concentration in obese rats.     

Evidence for a high fatty acid synthesis activity in interscapular brown adipose tissue of genetically obese Zucker rats.

Obese (fa/fa) rats (30 days old) exhibited a 50% increase in the weight of interscapular brown adipose tissue compared with their lean (Fa/fa) littermates. The tissue weight increase was accounted for by an increased fat content. Lipogenesis in vivo, as assessed by the incorporation of 3H from 3H2O into lipid, was increased 5-fold in brown adipose tissue of obese as compared with lean rats. Accordingly, acetyl-CoA carboxylase, fatty acid synthetase, citrate-cleavage enzyme and malic enzyme in this tissue were 4-8 times more active in obese than in lean rats.     

Development of fatty acid-synthetic capacity in interscapular brown adipose tissue during suckling in genetically obese Zucker rats.

The development of the lipogenic capacity in brown adipose tissue was studied in suckling lean (Fa/fa) and obese (fa/fa) Zucker pups aged from 7 to 22 days. In both lean and obese pups, activities of the two key lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, and of citrate cleavage enzyme rose from the early to the late suckling period. Compared with lean pups, 7-day-old fa/fa pups showed a 35% increase in fat accumulation in interscapular brown adipose tissue and a 25% increase in fatty acid synthetase activity. By 10 days of age, fat deposition, lipogenesis in vivo (assessed by the incorporation of 3H from 3H2O into fatty acids) and fatty acid synthetase activity were 1.5-2-fold higher in pre-obese than in lean pups. Compared with lean pups, the increased lipogenesis in vivo observed in brown adipose tissue of 10-day-old pre-obese pups could not entirely account for the difference in fat deposition observed in this tissue, suggesting that additional mechanisms are at play to explain the increased fat content of this tissue.     

Isolation and characterization of dynein ATPase from bull spermatozoa.

To determine whether the abnormal insulin-secretory activity encountered in obese mice is due to an anomaly in the production of cyclic AMP, islets of lean and obese mice were incubated with forskolin under various conditions. Our data show that, in addition to the well-known quantitative differences in insulin-secretory activity between islets of lean and obese mice, there are important qualitative differences. The islets of obese mice accumulated less cyclic AMP than did those of lean mice in response to given doses of forskolin, yet their insulin secretion was enhanced to much higher values. In the islets of obese mice, but not in those of lean mice, the stimulatory effect of forskolin on insulin secretion was evident even at non-stimulatory concentrations of glucose or in Ca2+-deprived incubation media, showing that the islet of the obese mouse is less dependent on a primary stimulus (glucose) and on the provision of normal concentrations of Ca2+ in the bathing medium than is the islet of the lean mouse.     

Clearing-factor lipase in obese–hyperglycaemic mice (ob/ob)

1. Clearing-factor lipase was assayed in acetone-ether-dried powders of heart and epididymal fat-pads of lean and genetically obese mice (ob/ob). In both tissues the enzyme activity in the adult was higher in the obese mice. 2. In heart the enzyme activity was unchanged from 8 to 48 weeks of age in lean mice, but in obese mice it increased between 8 and 12 weeks of age and remained elevated. 3. Starvation produced changes in the heart clearing-factor lipase activity in obese, but not lean, mice. 4. The clearing-factor lipase activity of epididymal fat-pads decreased rapidly during 24h starvation in both lean and obese mice, but the activity in the obese mice remained higher than that in lean mice. 5. Plasma triglyceride and cholesterol concentrations were determined in both lean and obese mice. Triglyceride concentrations were not greatly different, but the obese mice were hypercholesterolaemic. Plasma cholesterol concentrations were not correlated with changes in clearing-factor lipase activity.     

Neotomodon alstoni mice present sex differences between lean and obese in daily hypothalamic leptin signaling

This article compared the effects of spontaneous obesity on the daily profile in the relative amount of the leptin receptor (LepRb), and its output. That is the precursor Pro-opiomelanocortin (POMC) over a 24-hour period and compared with differences in locomotion and food intake in periods of artificial light. Differences between lean and obese mice were examined, as were sex differences. Body weight, food intake and locomotor activity were monitored in freely moving lean and obese mice. Hypothalamic tissue was collected at 5 h, 10 h, 15 h, 19 h and 24 h. Samples were analyzed by western blotting to determine the relative presence of protein for LepRb, STAT3 phosphorylation (by pSTAT3/STAT3 ratio) and POMC. Obese mice were 60% less active in locomotion than lean mice during the night. While both locomotor activity and food intake were noticeably greater during the day in obese mice than in lean mice, the hypothalamus in obese mice showed a lower relative abundance of POMC and reduced pSTAT3/STAT3 ratio and leptin receptors. Behavioral and biochemical differences were more evident in obese females than in obese males. These results indicate that obesity in N. alstoni affects hypothalamic leptin signaling according to sex.     

Possible role of acetyl-CoA-carboxylase in biosynthesis of mevalonic acid and sterols in rat liver]

Effect of citrate on acetyl-CoA incorporation into mevalonic acid, sterols and fatty acids after preliminary incubation of rat liver extracts under conditions optimal for acetyl-CoA carboxylase activation, was studied. 30 min preincubation with the citrate at 37 degrees C results in a 2--3-fold stimulation of the mevalonic acid biosynthesis from acetyl-CoA in the microsomal and soluble (140 000 g) fraction, and in that of sterols precipitated by digitonin or isolated by TLC in the mitochondria--free fraction. 2-14C-malonyl-CoA incorporation into the mevalonic acid and sterols and biosynthesis of sterols from 2-14C-mevalonic acid were not stimulated under those conditions. A correlation was shown to exist between the activity of acetyl-CoA carboxylase and the rate of acetyl-CoA incorporation into mevalonate and sterols; the activity of beta-hydroxy-beta-methylglutaryl-CoA reductase, limiting the rate of the sterol biosynthesis, was not changed. The stimulating effect of citrate was found to depend on the concentration of acetyl-CoA and NADPH in the medium. The data obtained suggest that the mevalonic acid biosynthesis in rat liver may occur in the presence of acetyl-CoA carboxylase through the formation of malonyl-CoA.     

Initiation of lipogenic enzyme activities in rat mammary glands.

The activities of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthetase remained low until parturition at 22 days of gestation and increased significantly within 1 day post partum. Administration of progesterone on days 20 and 21 and at parturition abolished the increases for at least 48 h after parturition. Removal of the pups of normal rats prevented the increases in activities of acetyl-CoA carboxylase and ATP citrate-lyase, but not of fatty acid synthetase, and administration of prolactin corticosterone or insulin did not stimulate activity. Tissue from suckled glands in which the ducts had been ligated at parturition showed no increase in the activities of acetyl-CoA carboxylase and ATP citrate-lyase within 24 h, whereas fatty acid synthetase activity was similar to that in the sham-operated contralateral glands. Foetoplacentectomy on day 18 increased the activity of fatty acid synthetase but not of acetyl-CoA carboxylase and ATP citrate-lyase; suckling of these dams by foster pups increased both acetyl-CoA carboxylase and ATP citrate-lyase.     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

Maternal Obesity Reduces Milk Lipid Production in Lactating Mice by Inhibiting Acetyl-CoA Carboxylase and Impairing Fatty Acid Synthesis

Maternal metabolic and nutrient trafficking adaptations to lactation differ among lean and obese mice fed a high fat (HF) diet. Obesity is thought to impair milk lipid production, in part, by decreasing trafficking of dietary and de novo synthesized lipids to the mammary gland. Here, we report that de novo lipogenesis regulatory mechanisms are disrupted in mammary glands of lactating HF-fed obese (HF-Ob) mice. HF feeding decreased the total levels of acetyl-CoA carboxylase-1 (ACC), and this effect was exacerbated in obese mice. The relative levels of phosphorylated (inactive) ACC, were elevated in the epithelium, and decreased in the adipose stroma, of mammary tissue from HF-Ob mice compared to those of HF-fed lean (HF-Ln) mice. Mammary gland levels of AMP-activated protein kinase (AMPK), which catalyzes formation of inactive ACC, were also selectively elevated in mammary glands of HF-Ob relative to HF-Ln dams or to low fat fed dams. These responses correlated with evidence of increased lipid retention in mammary adipose, and decreased lipid levels in mammary epithelial cells, of HF-Ob dams. Collectively, our data suggests that maternal obesity impairs milk lipid production, in part, by disrupting the balance of de novo lipid synthesis in the epithelial and adipose stromal compartments of mammary tissue through processes that appear to be related to increased mammary gland AMPK activity, ACC inhibition, and decreased fatty acid synthesis.     

Pyruvate dehydrogenase-complex activity in brown adipose tissue of gold thioglucose-obese mice.

The activity of pyruvate dehydrogenase (PDH) complex and PDH kinase were measured in brown adipose tissue (BAT) of 4-week-gold thioglucose (GTG)-obese mice. The proportion of PDH complex in the active dephosphorylated form was 2-fold higher in BAT of post-absorptive obese mice compared with lean controls. This result was consistent with the higher circulating insulin concentration observed in GTG-obese mice. In both obese and lean mice the PDH-complex activity in BAT decreased after 24 h starvation and increased in response to supraphysiological insulin injection, indicating that the PDH complex is insulin-responsive in BAT of GTG-obese mice. There was no difference in the PDH kinase activity of BAT in post-absorptive or insulin-injected lean and obese mice, suggesting that the higher PDH-complex activity in obese mice was not due to decreased PDH kinase activity. There is no evidence for a decreased activity of PDH complex contributing to insulin resistance in BAT of 4-week-GTG-obese mice.     

Pyruvate dehydrogenase activity in several tissues of genetically obese hyperglycemic mice

The active form (PDHa) and total activity of pyruvate dehydrogenase (PDH) were measured in homogenates from heart muscle, epididymal fat pads and liver of genetically obese hyperglycemic mice and compared with similar data derived from lean controls or Swiss albino mice. Both PDHa and total PDH activities were similar in heart muscle from all mice with a precipitous decrease in the PDHa upon fasting. Adipose tissue and liver of obese mice had a PDHa level that was almost two-fold higher than either lean control or Swiss albino mice. Fasting for 24 hours decreased the elevated activity of PDHa in adipose tissue and liver in obese mice to a value that was comparable to lean control or Swiss albino mice, fasted similarly. The elevation in both the active form and total activity of pyruvate dehydrogenase in livers from obese mice could explain the increased provision of acetyl-CoA units necessary for the accelerated hepatic lipogenesis observed with this mouse, a model for human obesity and insulin resistance.     

The source of malonyl-CoA in rat heart. The calcium paradox releases acetyl-CoA carboxylase and not propionyl-CoA carboxylase

The formation of malonyl-CoA in rat heart is catalyzed by cytosolic acetyl-CoA carboxylase. The existence of this enzyme in heart is difficult to prove by the abundant occurrence of mitochondrial propionyl-CoA carboxylase, which is also able to catalyze the carboxylation of acetyl-CoA. We used the calcium paradox as a tool to separate cytosolic components from the remaining heart, and found that acetyl-CoA carboxylase activity was preferentially released, like lactate dehydrogenase and carnitine, while propionyl-CoA carboxylase was almost fully retained. Acetyl-CoA carboxylase activity was determined after activation by citrate ion and Mg2+. The activity decreased to 64% by 48 h of fasting.     

Vanadate induces normolipidemia and a reduction in the levels of hepatic lipogenic enzymes in obese Zucker rat

The effects of vanadate administration on the plasma lipids and hepatic lipogenic enzymes were investigated in Zucker (fa/fa) rat, a model for obesity and non insulin-dependent diabetes. These animals were administered sodium orthovanadate through drinking water for a period of four months. The plasma levels of insulin, triacylglycerols and total cholesterol were significantly (p<0.001) elevated in untreated obese control rats as compared to the lean animals. In the livers of obese rats, the number of insulin receptors decreased by 60% and the activities of lipogenic enzymes acetyl-CoA carboxylase and ATP-citrate lyase increased by 4.7- and 5.6-folds, respectively. The messenger RNA for ATP-citrate lyase as measured by Northern blot analysis showed a parallel increase in obese control rats. Treatment of these rats with vanadate caused 56–77% decreases in the plasma levels of insulin, triacylglycerols and total cholesterol. The insulin receptor numbers in vanadate-treated obese rats increased (119%) compared to levels in untreated obese animals. The elevated activities of acetyl-CoA carboxylase and ATP-citrate lyase observed in livers of obese rats were significantly reduced by vanadate. The messenger RNA for ATP-citrate lyase also decreased in vanadate-treated obese rats back to the lean control levels. This study demonstrates that vanadate exerts potent actions on lipid metabolism in diabetic animals in addition to the recognized effects on glucose homeostasis.     

Physiological regulation of acetyl-CoA carboxylase gene expression: effects of diet, diabetes, and lactation on acetyl-CoA carboxylase mRNA

We measured acetyl-CoA carboxylase mRNA levels in various tissues of the rat under different nutritional and hormonal states using a cDNA probe. We surveyed physiological conditions which are known to alter carboxylase activity, and thus fatty acid synthesis, to determine whether changes in the levels of carboxylase mRNA are involved. The present studies include the effects of fasting and refeeding, diabetes and insulin, and lactation on carboxylase mRNA levels. Northern blot analysis of liver RNA revealed that fasting followed by refeeding animals a fat-free (high carbohydrate) diet dramatically increased the amount of carboxylase mRNA compared to the fasted condition. These changes in the level of mRNA correspond to changes in the activity and amount of acetyl-CoA carboxylase. Acetyl-CoA carboxylase mRNA levels in epididymal fat tissue decreased upon fasting and increased to virtually normal levels after 72 h of refeeding, closely resembling the liver response. The amount of acetyl-CoA carboxylase mRNA decreased markedly in epididymal fat tissue of diabetic rats as compared to nondiabetic animals. However, 6 h after injection of insulin the mRNA level returned to that of the nondiabetic animals. Gestation and lactation also affected the levels of carboxylase mRNA in both liver and mammary gland. Maximum induction in both tissues occurred 5 days postpartum. These studies suggest that these diverse physiological conditions affect fatty acid synthesis in part by altering acetyl-CoA carboxylase gene expression.     

Cold‐Induced Adaptive Thermogenesis in Lean and Obese

On entering a cold environment, people react by increasing insulation and energy expenditure (EE). However, large interindividual differences exist in the relative contribution of each mechanism. Short‐term studies revealed that obese subjects increase EE (i.e., adaptive thermogenesis) less than lean subjects, which might have implications for the predisposition to obesity. In this study, we validate the differences in adaptive thermogenesis between lean and obese upon midterm mild cold exposure. Therefore, 10 lean and 10 obese subjects were exposed for 48 h to mild cold (16 °C) in a respiration chamber. The preceding 36 h they stayed in the same chamber at a neutral temperature (22 °C) for the baseline measurements. EE, physical activity, skin temperature, and core temperature have been measured for the last 24 h of both parts. Mean daytime EE increased significantly in the lean subjects (P < 0.01), but not in the obese. Physical activity decreased significantly in the lean (P < 0.01) and the obese (P < 0.001) subjects. The change in EE was related to the change in physical activity in both groups (respectively R² = 0.673, P < 0.01 and R² = 0.454, P < 0.05). Upon mild cold exposure, lean subjects decreased proximal skin temperature less, but distal skin temperature more than obese. In conclusion, the interindividual differences in cold‐induced thermogenesis were related to changes in physical activity in both lean and obese, pointing at the existence of individual variation in physical activity to compensate for cold‐induced thermogenesis. Furthermore, although a large part of the lean subjects counteracted the cold by increasing EE, most obese subjects changed temperature distribution, and therefore, increased insulation.     

Inactivation of pyruvate dehydrogenase complex in heart muscle mitochondria of gold-thioglucose-induced obese mice is not due to a stable increase in activity of pyruvate dehydrogenase kinase.

The proportion of pyruvate dehydrogenase (PDH) complex in the active dephosphorylated form was decreased (compared with fed lean control mice) in heart muscle mitochondria after the induction of obesity with gold-thioglucose (by 54%) or starvation of lean mice for 48 h (by 81%). The effects of obesity to inactivate PDH complex were demonstrable 4 weeks after administration of gold-thioglucose, and occurred despite significant hyperinsulinaemia in obese animals. Phosphorylation and inactivation of PDH complex in mouse heart muscle in starvation was attributed to a stable increase (2.7-fold) in the activity of PDH kinase as measured in extracts of mitochondria mediated by increased specific activity of a protein activator of PDH kinase (KAP) [Denyer, Kerbey & Randle (1986) Biochem. J. 239, 347-354]. In obese mice no such increase in kinase activity was observed, and we conclude that phosphorylation and inactivation of PDH complex in heart muscle in obesity is not mediated by KAP, but rather is a consequence of increased lipid oxidation.