Hyperammonemia decreases body fat content in rat

We have developed an animal model of hyperammonemia consisting of feeding rats a diet containing 20% (w/w) ammonium acetate. Ingestion of this diet markedly affects carcass composition, with a 46% reduction in lipid content. The ammonium diet alters levels of several key compounds involved in lipid metabolism. Long-chain acylcarnitine is increased in liver by approx. 60% while free carnitine and acetylcarnitine are unaffected. The hepatic content of acetyl-CoA increases by approx. 50%. The level of ketone bodies in blood increases by 32% but remains unchanged in liver. Our data indicate that hyperammonemia alters lipid metabolism and results in a significant decrease in body lipid content.     

Time course of insulin-receptor binding and insulin-induced lipogenesis in isolated rat fat cells.

1. Isolated rat fat cells were incubated at 37 degrees with [U-14C]-glucose 0.55 mM and 125I-labeled insulin. The amount of receptor-bound 125I-labeled insulin and the rate of insulin-induced 14C-lipid synthesis were assessed during association and dissociation of 125I-labeled insulin. 2. The rate of 14C-lipid synthesis was constant from zero time in the absence of insulin and in the presence of insulin in a high concentration (0.7 muM). With insulin in a low concentration (56 pM) the insulin-induced rate of 14C-lipid synthesis was proportional to the receptor occupancy; the receptor binding reached equilibrium and the rate of 14C-lipid synthesis reached a constant value after 30 to 45 min. With insulin in a concentration of 0.7 nM the rate of 14C-lipid synthesis reached a steady state before equilibrium of the receptor binding was obtained. 3. Ater preincubation with 56 pM 125I-labeled insulin followed by removal of extracellular insulin the decrease in the rate of insulin induced 14C-lipid synthesis followed the decrease in receptor occupancy with a half-time of about 10 min. After preincubation with insulin in concentrations of 0.28, 0.56, and 1.4 nM a maximum rate of 14C-lipid synthesis was maintained for about 8, 15, and 30 min, respectively. 4. The following model is suggested. Binding of insulin to the previously described receptors with a dissociation constant of about 3 nM (Gammeltoft, S., and Gliemann, J. (1973) Biochim. Biophys Acta 320, 16-32) represents the first step in the action of insulin on lipid synthesis from glucose. The receptor occupancy is rate-determining at low concentrations of insulin, i.e. when the occupancy is small (about 2 percent or less). At higher insulin concentrations some other step becomes rate-determining and the higher occupancy at equilibrium therefore causes no further increase in the steady state lipogenesis. However, a high receptor occupancy causes a prolonged maintenance of a maximal (or near-maximal) effect after removal of insulin from the medium.     

Influence of red pepper and capsaicin on body composition and lipogenesis in rats

Inclusion of red pepper or its active principle ‘capsaicin’ in the diet led to a lowering of total lipids, particularly triglycerides in the liver. The total body fat was lowered in animals fed red pepper or capsaicin but not in animals fed paprika powder which had negligible capsaicin content. Hyperlipogensis and hypertriglyceridemia caused by fructose feeding were significantly were decreased in capsaicin-fed animals. Activities of the key lipogenic enzymes were reduced as reflected by decreased lipogenesis.     

Obesity genes and the regulation of body fat content

Physiological investigation has demonstrated that the central nervous system monitors body composition and adjusts energy intake and expenditure to stabilize total adipose tissue mass. Genetic variations in the signalling molecules involved in this regulatory system account for the heritable component of body fat content. The application of molecular techniques to rodent models of Mendelian obesity has resulted in the characterization of five loci at which mutations produce an abnormal accumulation of body fat. The genes at these loci include agouti, which encodes a molecule that antagonizes the binding of alpha melanocyte-stimulating hormone to its receptor; fat, which encodes carboxypeptidase E; tubby, which encodes a putative phosphodiesterase; obese, which encodes a circulating satiety protein; and diabetes, which encodes the receptor for the obese gene product. A more detailed understanding of the functional interrelationships of these genes should lead to important new insights into the causes and potential therapies for human obesity.     

Analysis of lines of mice selected for fat content. 2. Correlated responses in the activities of enzymes involved in lipogenesis

Estimates of the activities (Vmax) of six enzymes involved in de novo fat synthesis were made in replicated lines of mice differing in fat content. These lines had been selected high and low for 20 generations with three replicates each of Fat, Control and Lean lines and for a further eight generations high and low as an unreplicated line. The activities of ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), fatty acid synthetase (FAS), cytoplasmic malate dehydrogenase (MDH), malic enzyme (ME) and pyruvate kinase (PK) were determined in vitro in both liver and gonadal fatpad tissues taken at ages five and ten weeks. The activities of ACL, ACC, FAS and ME were significantly higher in the Fat than the Lean lines, and the differences were more pronounced at the earlier age and in the gonadal fatpad where activities in the Fat lines were higher by factors of 3.5, 2.4, 2.5 and 3.5 respectively. The activity of PK was unchanged in each tissue. MDH activity was significantly lower in adipose tissue in the Fat lines than the Lean lines at age ten weeks but not at age five weeks or in liver tissue. Results from replicates indicated that random genetic drift affected enzyme activities but nevertheless significant changes in activity were associated with the direction of selection. The changes in enzyme activity reported here are similar to those known to be associated with major mutations causing obesity in mice.     

Ketone body utilization for lipogenesis in the perfused liver of the obese Zucker rat

The purpose of these studies was to determine if the utilization of ketone bodies as a carbon source for lipogenesis could account for the decreased ketone body production by livers of obese Zucker rats, as well as contribute to the enhanced rates of fatty acid synthesis observed in these animals. Ketone body production was decreased from 822 mumol/liver in the lean to 538 mumol/liver in the obese genotype (P less than 0.05). The incorporation of ketone bodies into fatty acids was significantly greater in the obese rat liver (lean, 1.95 mumol of ketone bodies/liver, versus obese, 35.22 mumol/liver; P less than 0.025). The relative contribution of this pathway to the overall rate of fatty acid synthesis was not affected by genotype and accounted for only 3 to 4% of the fatty acids synthesized. The incorporation of ketone bodies into digitonin precipitable sterols was similar in the two genotypes (lean, 4.5 mmol/liver, versus obese 4.7 mumol/liver; NS). This accounted for 9.2 and 6.3% of the total sterol synthesis in lean and obese rat livers, respectively. The total incorporation of ketone bodies into lipid was 7.5 mumols in the lean rat livers and 42.0 mumoles in the obese (P less than 0.025). The net increase was 35 mumoles incorporated, whereas the net decrease in ketogenesis was 284 mumoles. Thus, although ketone body carbon utilization for lipid synthesis was increased in the liver of the obese rats, this pathway could only account for a fraction of the genotypic difference in ketone body production and was of relatively minor importance as a source of carbon for hepatic fatty acid synthesis in both lean and obese rats.     

Effects of fat content in the diet on hepatic peroxisomes of the rat

Effects of fat content in the diet on rat liver peroxisomes was examined. In the livers of rats fed for one week on the high-fat diet containing 30% fat, the cyanide-insensitive palmitoyl-CoA oxidation was accelerated to eight times that of control and the enzymic activities of catalase, carnitine acetyltransferase and carnitine palmitoyltransferase were elevated by the factors of 1.3, 5 and 2, respectively. In contrast, the activities of D-amino acid oxidase in addition to the three enzymes mentioned above were all lowered by 20% when the animals were maintained on a fat-free diet for the same period of time. It appears that the high-fat diet-induced increase in the activity of carnitine palmitoyltransferase is a result of the raised activity of this enzyme in mitochondria only while the apparent high activity reflects stimulation of carnitine acetyltransferase in all the subcellular fractions. Another notable effect of the high-fat diet was a remarkable increase in the quantity of a peroxisome-associated polypeptide which was separable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is noteworthy that this effect of the high-fat diet resemble that of clofibrate. If the diet was deprived of fat, however, this polypeptide species, with an estimated molecular weight of 80 000, decreased to a level slightly lower than normal. On the basis of the electron micrographic criteria, the high-fat diet provoked a marked proliferation of hepatic peroxisomes.     

Effects of 2,4-dihydroxybutyrate on lipogenesis in rat hepatocytes.

Hepatocytes were prepared from rats fasted 2 days and refed a high carbohydrate diet for 1 day. These cells contained very high levels of glycogen (about half the defatted dry weight) and carried out high rates of lipogenesis (up to 800 micron at tritium incorporation from 3HOH/g (defatted dry weight)/h), even in the absence of added substrates. Pentose cycle flux was estimated by a method involving the use of [1-14C]galactose (Rognstad, R. (1976) Int. J. Biochem. 7, 221-228). In hepatocytes from normal fasted refed rats, the amount of NADPH produced by the pentose cycle was sufficient for about one-half to three-fourths of that required for fatty acid synthesis. 2,4-Dihydroxybutyrate, a malic enzyme inhibitor (Schimerlik, M.I. & Cleland, W.W. (1977) Biochemistry 16, 565-570) markedly depressed the randomization of 14C in lactate from [6-14C]hexoses, indicating an inhibition of the pyruvate cycle. 2,4-Dihydroxybutyrate (10 mM) had only a slight inhibitory effect on overall lipogenesis, but increased the rate of the pentose cycle by 40 to 90%.