Mitochondrial storage forms of acetyl CoA carboxylase: mobilization/activation accounts for increased activity of the enzyme in liver of genetically obese Zucker rats

In earlier reports, we have described a previously unrecognized mechanism which regulates the activity of acetyl CoA carboxylase in rat liver by the control of its distribution between relatively inactive mitochondrial and active cytosolic forms. In this study, the activity, total quantity and the subcellular distribution of acetyl CoA carboxylase were determined in liver of fed and fasted (48 h) homozygous obese (fa/fa) zucker rats and homozygous lean (Fa/Fa) littermates. The results indicate that neither diet nor genetic obesity affected the total quantity of acetyl CoA carboxylase per unit weight of liver. Instead, increased activity of this enzyme in the liver of the Zucker rat was primarily due to a shift in the subcellular distribution away from relatively inactive mitochondrial forms toward active cytosolic forms. Thus, the Zucker rat appears to be yet another example illustrating the physiological importance of regulating the activity of acetyl CoA carboxylase by controlling its subcellular distribution.     

Fatty acid synthesis in testes of fat-deficient and fat-supplemented rats.

Fatty acid synthesis was studied in testes of rats fed a fat-free or fat-supplemented diet. Testes of fat-deficient rats incorporated nearly twice as much intratesticularly injected [1-14C]acetate into total fatty acids (primarily into palmitic acid) as did supplemented rats. To determine the mechanism for the increased synthesis, the activities of the following enzymes were determined in the cytoplasmic fraction of testicular homogenates: fatty acid synthetase, acetyl CoA carboxylase [EC 6.4.1.2], citrate-cleavage [EC 4.1.3.8], malic [EC 1.1.1.38], and the glucose-l-phosphate dehydrogenase [EC 1.1.1.49]: 6-phosphogluconate dehydrogenase pair [EC 1.1.1.44]. Although the activity of fatty acid synthetase did increase in livers from fat-deficient rats, no change was observed in corresponding testes. No difference between the two groups could be demonstrated in testicular activity of citrate-cleavage enzyme, malic enzyme, or the glucose-6-phosphate dehydrogenase: 6-phosphogluconate dehydrogenase pair. However, the activity of cytoplasmic acetyl CoA carboxylase in testes of rats fed the fat-deficient diet was 1.4 times higher than the activity in testes of rats fed the supplemented diet. Fat deficiency did not affect the specific activity of the testicular microsomal elongation system, assayed by incubation with 14C-malonyl CoA. The concentration of unesterified fatty acids was lower in testes of the fat-deficient compared to supplemented rats, indicating that decreased inhibition of acetyl CoA carboxylase in the fat-deficient rats testes might have been responsible for the observed increased de novo synthesis of palmitic acid.     

In vivo effects of glucagon on fatty acid synthesis in fasted and refed rats.

Since altered nutritional states evoke compensatory changes in systemic levels of several hormones, the present study was conducted to determine in vivo effects of glucagon and insulin on hepatic and adipose tissue lipogenesis in fed, fasted (3-days) and refed (3-days) rats. Compared to the fed controls, glucagon reduced hepatic fatty acid synthesis and acetyl CoA carboxylase activity by 62% and 65% in fed rats, and by 51% and 48%, respectively, in refed rats. In contrast, glucagon had no effect on fatty acid synthesis or acetyl CoA carboxylase activity in adipose tissue of any of the three experimental groups. Exogenous insulin antagonized the glucagon effects and restored hepatic fatty acid synthesis and enzyme activity in fed or refed rats. No glucagon or insulin effects were observed in fasting rats. In addition, glucagon reduced in vivo incorporation of acetate into hepatic cholesterol by about 33% and into fatty acids of the liver, and heart and the kidney by 33%, 77% and 30%, respectively. The hormone had no effect on fatty acid synthesis in the muscle.     

Effect of dietary cholesterol on hepatic lipogenesis and plasma insulin and free fatty acid levels in rats.

In order to further investigate the metabolic alterations in the liver of cholesterol-fed rats, the following parameters were determined: (a) the activities of hepatic glucose-6-phosphate dehydrogenase, NADP-malate dehydrogenase, citrate cleavage enzyme, acetyl CoC carboxylase, and fatty acid synthetase; (b) the rate of hepatic fatty acids synthesis in vivo or in vitro; and (c) the concentration of immunoreactive insulin, free fatty acids, and glucose in the plasma. The experimental diets usually contained 1.5% cholesterol and 0.5% cholic acid. Cholesterol feeding resulted in a three- to fourfold decrease in the activities of hepatic glucose-6-phosphate dehydrogenase, NADP-malate dehydrogenase, and citrate cleavage enzyme and up to a two-fold decrease in the activities of acetyl CoA carboxylase and fatty acid synthetase. The rate of fatty acid synthesis was not sifnigicantly decreased when rats were fed the cholesterol-supplemented diets for only 2 to 4 weeks, despite marked decreases in the activities of the lipogenic enzymes. But when cholesterol feeding was continued for periods longer than 5 weeks, there was a significant decrease in the rate of fatty acid synthesis in the liver. Cholesterol feeding decreased the levels of circulating insulin and elevated plasma free fatty acid levels. Plasma glucose levels were not significantly changed. Cholesterol feeding can result in a wide range of metabolic alterations. These metabolic alterations may have some impact on the development of hypercholesterolemic-related metabolic disorders.     

Dietary biotin intake modulates the pool of free and protein-bound biotin in rat liver

The current studies were undertaken to analyze the relationships among dietary biotin intake, hepatic free biotin and hepatic protein-bound biotin in rats. The biotin status of rats was manipulated through dietary intervention to model moderate biotin deficiency, adequacy, supplementation and pharmacologic biotin supplementation (0, 0.06, 0.6 and 100 mg/kg, respectively). Urinary biotin excretion was directly related to biotin intake, but no difference between biotin-adequate and biotin-supplemented rats was detected. In contrast, plasma biotin was directly and significantly regulated by biotin intake at every intake level. A hepatic free biotin pool was directly demonstrated in these studies, and like plasma, its size was directly related to dietary biotin intake. The relationship between dietary biotin intake and protein-bound biotin was also analyzed. Moderate biotin deficiency markedly decreased the abundance of each biotinylated polypeptide in rat liver. Biotin supplementation did not significantly elevate the abundance of biotinylated pyruvate, propionyl CoA, methylcrotonyl CoA or acetyl CoA carboxylase 1. The abundance of biotinylated acetyl CoA carboxylase 2, however, was significantly higher in biotin-supplemented rats. Pharmacologic biotin intake significantly reduced the abundance of biotinylated propionyl CoA and methylcrotonyl CoA carboxylase. These results indicate the following: 1) moderate biotin deficiency reduces free and protein bound biotin; 2) biotin intakes in rats that mimic the currently recommended daily value (DV) do not result in full protein biotinylation; and 3) pharmacologic supplementation may reduce the abundance of functional carboxylases.     

Neutral lipid accumulation in yeast due to inositol deficiency: kinetic studies on the reciprocal regulation by fructose bisphosphate and citrate of yeast acetyl CoA carboxylase.

Neutral lipids, especially triacylglycerols, accumulated due to myo-inositol deficiency both in the cells of Saccharomyces carlsbergensis (Hayashi et al. (1976) J. Biol. Chem., 251, 5759--5769) and in the liver of the rat (Hayashi et al. (1974) Biochim. Biophys. Acta, 360, 134--155). The accumulation of triacylglycerols in the deficient yeast resulted, at least partly, from an enhancement of acetyl CoA carboxylase activity. The activation of the enzyme reflected the fluctuation due to the deficiency in the levels of fructose bisphosphate and citrate (Hayashi et al. (1978) Biochim. Biophys. Acta, 540, 231--237). Thus, the kinetics of the regulation of acetyl CoA carboxylase by these intermediates was studied. In physiological concentrations fructose bisphosphate sigmoidally activated acetyl CoA carboxylase from yeast with the Hill coefficient of 3, while citrate counteracted the fructose bisphosphate activation in a sigmoidal manner with the Hill coefficient of 2. Fructose bisphosphate markedly increased the apparent Vmax value of acetyl CoA carboxylase for the substrate, ATP and slightly decreased the apparent Km value. Citrate greatly decreased the apparent Vmax value increased by fructose bisphosphate.     

Hypoglycemic effect of Rehmannie Radix Preparata (Sookjihwang) extract in streptozotocin-induced diabetic rats

Rhemannie Radix Preparata (RRP) has been previously employed in traditional oriental medicine as a treatment for diabetic thirst and improving blood flow. The aim of this study was to evaluate its hypoglycemic control by assaying the activities of key enzymes of carbohydrate metabolism in streptozotocin-(STZ)-induced diabetic rats. Further, RRP extracts were prepared in water (RRPW), in 50% ethanol (RRP50), and in 100% ethanol (RRP100), respectively, and compared for their actions in diabetic rats. The oral treatment of RRP (5 mg/kg b.w./d) to diabetic rats for 21 days resulted in a significant decline in blood glucose by 67% compared to diabetic control rats (P < 0.05). The altered activities of glucokinase, glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and acetyl CoA carboxylase (ACC) in the livers of diabetic rats were reversed significantly to near-normal levels by the administration of RRP (P < 0.05). Among the three RRP extracts, RRP100 was the most effective in terms of hypoglycemic action. However, the administration of RRP to diabetic rats did not improve insulin production. The modulatory effects of RRP100 on the attenuation of carbohydrate enzyme activities appear to hold promise for widespread use for the treatment of diabetes in the future.     

The abundance and function of biotin-dependent enzymes are reduced in rats chronically administered carbamazepine

The effect of dietary antiepileptic drug administration on the metabolism and function of the water-soluble vitamin biotin was analyzed in a physiologically relevant rat model of biotin nutriture. Administration of carbamazepine (CBZ) in semipurified rat diet at 1.5 and 2.9 g/kg for 19 d did not reduce growth rate or food intake. After this dietary treatment, brain lactic acid and ammonia concentrations were significantly elevated, but no changes in these metabolites occurred in the liver. Urinary biotin excretion was altered and the concentrations of biotin sulfoxides and biocytin in the serum were elevated. Brain biotin was unaffected, but concentrations of bisnorbiotin and biocytin were significantly reduced by dietary administration of CBZ. The relative abundance of hepatic acetyl CoA carboxylase 1 and 2, pyruvate carboxylase (PC), methylcrotonyl CoA carboxylase and propionyl CoA carboxylase was significantly reduced by CBZ, whereas the relative abundance of biotinylated PC was significantly reduced in the brain. In agreement with the carboxylase abundance data, the activity of hepatic PC was significantly reduced in rats consuming CBZ-containing diets. These data demonstrate that administration of the antiepileptic medication CBZ, even with food, reduces the abundance and function of biotin-dependent enzymes in the liver and brain, partially accounting for the metabolic alterations, including organic acidemia, that are observed clinically.     

Time sequence of lipogenic changes in adipose tissue of rats when converted from ad libitum feeding to meal-eating.

This study was undertaken to establish the time sequence of lipogenic changes in adipose tissue of rats when converted from ad libitum feeding to meal-eating. Rats were fed a high carbohydrate diet 2 hours/day for 0 to 10 days (meal-eating). The high speed supernatant fraction from homogenized epididymal fat pads was assayed for citrate cleavage enzyme, acetyl CoA carboxylase, fatty acid synthetase and malic enzyme activities. The effects of meal-feeding on in vitro and in vivo rates of fatty acid synthesis in adipose tissue as well as the amounts of glycogen deposited in the adipose tissue were measured. During the first 10 days of meal-feeding, the lipogenic enzyme activities were actually decreased or unchanged in the meal-fed rats but during this time the in vitro and in vivo rates of fatty acid synthesis were progressively increased in the meal-fed rats. Glycogen levels in the adipose tissue of meal-fed rats were greater than the levels in the nibblers. The initial hyperlipogenesis observed in the meal-fed rat appears to be due to changes in substrate uptake by the adipose tissue and/or to alterations in enzyme activation in the adipose tissue rather than to changes in the quantity of enzyme present in the tissue.     

TISSUE LEVELS AND SYNTHESIS OF COENZYME A IN DIFFERENT METABOLIC STATES

The concentration of total CoA in rat liver was increased by fasting and alloxan diabetes. Feeding rats one meal of glucose after an overnight fast reduced greatly the incorporation of ¹⁴-C-pantothenate into liver CoA. Rapid changes in CoA synthesis can alter fatty acid metabolism in liver.     

Regulation of acetyl CoA carboxylase and carnitine palmitoyl transferase-1 in rat adipocytes

OBJECTIVE: Acetyl CoA carboxylase (ACC) is a key enzyme in energy balance. It controls the synthesis of malonyl-CoA, an allosteric inhibitor of carnitine palmitoyltransferase-1 (CPT-I). CPT-I is the gatekeeper of free fatty acid (FFA) oxidation. To test the hypothesis that both enzymes play critical roles in regulation of FFA partitioning in adipocytes, we compared enzyme mRNA expression and specific activity from fed, fasted, and diabetic rats. RESEARCH METHODS AND PROCEDURES: Direct effects of nutritional state, insulin, and FFAs on CPT-I and ACC mRNA expression were assessed in adipocytes, liver, and cultured adipose tissue explants. We also determined FFA partitioning in adipocytes from donors exposed to different nutritional conditions. RESULTS: CPT-I mRNA and activity decreased in adipocytes but increased in liver in response to fasting. ACC mRNA and activity decreased in both adipocytes and liver during fasting. These changes were not caused directly by fasting-associated changes in plasma insulin and FFA concentrations because insulin suppressed CPT-I mRNA and did not affect ACC mRNA in vitro, whereas exogenous oleate had no effect on either. Despite the decrease in adipocyte CPT-I mRNA and specific activity, CO(2) production from endogenous FFAs increased, suggesting increased FFA transport through CPT-I for beta-oxidation. DISCUSSION: Stimulation of FFA transport through CPT-I occurs in both tissues, but CPT-I mRNA and specific activity correlate with FFA transport in liver and not in adipocytes. We conclude that the mechanism responsible for increasing FFA oxidation in adipose tissue during fasting involves mainly allosteric regulation, whereas altered gene expression may play a central role in the liver.     

Possible role of insulin status in the increased lipogenic enzyme activity by dietary medium-chain triglyceride in rat liver

The possible role of insulin status in the increase in liver lipogenic enzyme activities upon feeding medium-chain triglyceride (MCT) was investigated with streptozotocin-induced diabetic rats and insulin-treated diabetic rats. Rats were fed synthetic diets that contained either 2% corn oil (control), fat free, 13% MCT +2% corn oil, or 13% lard +2% corn oil, respectively. Feeding the MCT diet for 3 days increased serum ketone bodies in both the normal and diabetic rats. Insulin levels of MCT-fed rats tended to be higher than in normal animals. MCT feeding caused an enhancement of fatty acid synthetase (FAS) and malic enzyme (ME) in the liver of normal rats, whereas diabetic rats failed to register an increase in those activities due to MCT feeding. Administration of insulin to diabetic rats resulted in a recovery of the level of those enzyme activities to about the same degree as in each of the normal rat groups. It was interesting that diabetic MCT-fed rats with insulin treatment maintained higher enzyme activities in comparison to the lard and control groups. These results suggest that the increase in lipogenic enzyme activities caused by dietary MCT is presumably dependent on differences in insulin status.     

Hepatic cholesterol and fatty acid synthesis in pregnant and fetal rats: effect of maternal dietary fat and cholestyramine.

Previous studies from this laboratory have demonstrated that 20% rather than 5% (wt/wt) safflower oil or addition of 5% (wt/wt) cholestyramine to the diet of pregnant rats leads to an increase in the activity of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme of cholesterol synthesis, in the fetal liver. Total cholesterol, however, was not altered in fetal plasma or liver. The effect of these diets on cholesterol and fatty acid synthesis in vivo was therefore studied in fetal and maternal liver. In fetuses of rats fed a reference nonpurified diet, rates of hepatic cholesterol and fatty acid synthesis decreased from gestation d 20 to 21. In contrast, total and active 3-hydroxy-3-methyl-glutaryl CoA reductase activity increased. Adding cholestyramine to the diet or modifying the quantity of safflower oil fed had no effect on fetal hepatic lipogenesis. Maternal hepatic cholesterol synthesis was greater in rats fed cholestyramine, whereas fatty acid synthesis was lower in the dams fed the diet containing 20% compared with 5% safflower oil. The results suggest near-term fetal liver 3-hydroxy-3-methylglutaryl CoA reductase activities do not reflect fetal cholesterol synthesis in vivo.     

Demonstration of a specific metabolic effect of dietary disaccharides in the rat.

Male Wistar rats were starved and refed diets containing either 40% carbohydrate as monosaccharides (glucose, fructose, invert sugar) or disaccharides (maltose, sucrose), or 42.2% carbohydrate as glucose. Induction of various liver enzymes and changes in total liver lipid levels by the different dietary sugars were studied. Liver enzymes measured included glucose-6-phosphate dehydrogenase (g6pd), 6-phosphogluconate dehydrogenase (6PGD), malic enzyme (ME), phosphofructokinase (PFK), L-alpha-glycerol phosphate dehydrogenase (LalphaGPD), pyruvate kinase (PK), citrate cleavage enzyme (CCE), acetyl CoA carboxylase (AcCoAC), and fatty acid synthetase (FAS). The responses in enzyme activity to diets containing glucose or invert sugar were used as the basal response. Enzyme responses to refeeding the carbohydrate diets fell into three categories: (1) enzyme activity increased both by the disaccharide configuration of the carbohydrate and by fructose (G6PD, PK, CCE, AcCoAC, FAS); (2) enzyme activity increased only by the disaccharide configuration of the carbohydrate (6PGD, ME); and (3) enzyme activity increased only by fructose (PFK, LalphaGPD). Total liver lipid level was increased both by the disaccharide configuration of the carbohydrate and by fructose. Refeeding diets containing equal molar amounts of glucose or maltose did not abolish the disaccharide effect. The data indicate that the disaccharide configuration of maltose and sucrose may have an effect at the gastrointestinal level, which causes an increased induction of certain enzymes in the liver.     

Rat hepatic coenzyme A is redistributed in response to mitochondrial acyl-coenzyme A accumulation.

Coenzyme A without an acyl-thioester (CoASH) is required for numerous cellular reactions, and sequestration of CoASH as acyl-CoAs may impair metabolic function. Increased total CoA protects the cell from acyl-CoA accumulation, and enhanced CoA biosynthesis may represent a compensatory response in metabolic disease. To test the hypothesis that cellular CoA is redistributed from the cytosol to the mitochondria in response to mitochondrial acyl-CoA accretion, the subcellular distribution of hepatic CoA was determined by differential centrifugation and measurement of the mitochondrial marker enzyme citrate synthase. Liver from control, clofibrate-treated and hydroxycobalamin[c-lactam] (HCCL)-treated rats were used. Clofibrate increased total hepatic CoA concentration 2.2-fold, whereas HCCL (which causes inhibition of L-methylmalonyl-CoA mutase and consequent propionyl- and methylmalonyl-CoA accumulation) increased it threefold. However, clofibrate did not affect the percentage of total CoA in the mitochondria (control: 44 +/- 3%, clofibrate: 49 +/- 5%), and HCCL-treatment induced a marked redistribution of CoA into the mitochondria (HCCL: 78 +/- 8%). Redistribution of total CoA was also induced acutely by incubation of hepatocytes from control rats with 10 mmol/L propionate. Thus, redistribution of the cellular CoA pool can help maintain CoASH availability as mitochondrial acyl-CoA accumulation occurs and may be an important compensatory response to metabolic injury.     

The effects of dietary self-selection upon the overshoot phenomenon in starved-refed rats.

The food intake, liver composition and hepatic activity of pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6P-DH), malic enzyme (ME) and acetyl CoA carboxylase (AcCoA Cx) were studied in starved-refed rats. When rats were refed a mixed diet for 3 days, food intake significantly increased (by 33%) from day 1 to 3 and the glycogen accumulation was maximal after 24 hours, but decreased significantly by day 3 (by 34%). In contrast, liver triglycerides sharply increased (10-fold) from day 1 to 3. Furthermore, during refeeding a large increase of G6P-DH, ME and AcCoA Cx was reached on day 3 when the average activity was 5.5- to 6.5-fold higher than before fasting. When rats were refed under conditions of self-selection (carbohydrates, lipids and proteins) total food intake was the same each day, but lipid and carbohydrate intakes varied reciprocally: lipid intake decreased whereas carbohydrate consumption increased during the 3 day refeeding period. Liver glycogen level was unchanged and both the triglyceride accumulation and the overshoot of lipogenic enzymes were highly attenuated: on day 3, they reached 50% of values observed in mixed diet refed rats. Administration of 8-azaguanine during refeeding under self-selecting conditions lowered food intake but had no effect on the pattern of food intake on the first day. In the following days, lipid intake fell dramatically. Azaguanine does not alter liver glycogenesis, but prevents both liver triglyceride accumulation and the overshoot of lipogenic enzymes.     

Effect of Gymnema montanum on blood glucose,plasma insulin,and carbohydrate metabolic enzymes in alloxan-induced diabetic rats

The effects of Gymnema montanum, an endangered plant used in the ancient period of India, on blood glucose, plasma insulin, and carbohydrate metabolic enzymes were studied in alloxan diabetic rats. Administration of alcoholic extract of G. montanum leaves (50, 100, 200 mg/kg body weight) to alloxan diabetic rats for 3 weeks reduced the blood glucose level. Administration of G. montanum leaf extract (GLEt) at 200 mg/kg body weight significantly decreased the blood glucose levels and significantly increased the plasma insulin levels. This clearly shows the antidiabetic efficacy of GLEt, which was better than that of glibenclamide.     

Some unexplained features of hepatic ethanol oxidation

Ethanol is an excellent substrate for the liver, competing effectively for oxidation with other substrates such as fatty acids. Using isolated liver cells from fed and starved rats, we have found that ethanol strongly inhibits Krebs cycle oxidations, so that the combustion through the cycle of acetyl CoA, derived from fatty acids, is reduced more than 50%. In contrast, fatty acid beta-oxidation to acetyl CoA is inhibited only 20% in fed and fasted states. Ethanol was not antiketogenic. In the fed state, octanoate but not palmitate inhibited ethanol oxidation whereas in cells from fasted rats palmitate inhibited ethanol oxidation. Gluconeogenesis from lactate was reduced 50% in hepatocytes from fasted rats but oxygen consumption was unaffected. This paradoxical maintenance of oxygen consumption in a state where the only overt need for ATP synthesis is depressed, suggests that ethanol oxidation may not be exclusively coupled to ATP synthesis but also can be linked to other energy transducing processes.     

Octanoate inhibits triglyceride synthesis in 3T3-L1 and human adipocytes

To understand how medium-chain fatty acids (FA) influence lipid metabolism in adipocytes, we studied the effects of octanoate on the oxidation of glucose and endogenous palmitate, cellular O(2) consumption, mitochondrial membrane potential, lipid synthesis from long-chain FA, glucose and lactate. We found that octanoate significantly suppressed the esterification of oleate into triglycerides (TG) in both 3T3-L1 and human adipocytes. Octanoate also significantly suppressed de novo FA synthesis. These effects were associated with octanoate-mediated reductions in the activities of acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) and acetyl CoA carboxylase (ACC). Cells pretreated with octanoate had reduced mRNA levels for a number of lipid metabolism genes, including of DGAT, ACC and stearoyl CoA desaturase-1. On the other hand, octanoate did not acutely perturb cellular O(2) consumption or mitochondrial membrane potential. Together, these results suggest that octanoate affected adipocyte function by reducing TG synthesis but not by enhancing oxidation.