Hormonal regulation of fatty acid synthetase, acetyl-CoA carboxylase and fatty acid synthesis in mammalian adipose tissue and liver.

The major objectives of this study were to define the roles of adrenal glucocorticoids and glucagon in the long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase of mammalian adipose tissue and liver. Particular emphasis was given to elucidation of the mechanisms whereby these hormones produce their regulatory effects on enzymatic activity. To dissociate mental manipulation, nutritional conditions were ridgidly controlled in the experiments described. Administration of glucocorticoids to adult rats led to a marked reductionin activities of fatty acid synthetase and carboxylase in adipose in adipose tissue but no change occurred in liver. Adrenalectomy produced an increase in activities of these lipogenic enzymes in adipose tissure, but, again, no change was noted in liver. The decrease in enzymatic activities in adipose tissue with glucocorticoid administration correlated well with a decrease in fatty acid synthesis, determined in vivo by the 3-H2O method. The mechanisms whereby glucocorticoids led to a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the decrease in fatty acid synthetase activity observed in adipose tissue was shown to reflect a decrease in content of enzyme, and not a change in catalytic efficiency. The mechanism underlying the decrease in enzyme content is a decrease in synthesis of the enzyme. The relation of the effects of glucocorticoids to the effects of certain other hormones involved in regulation of lipogenesis was investigated in hypophysectomized and in diabetic animals. Thus, the observation that the glucocorticoid effect on synthetase and carboxylase occurred in adipose tissue of hypophysectomized rats indicated that alterations in levels of other pituitary-regulated hormones were not necessary for the effect. That glucocorticoids play some role in regulation of synthetase and carboxylase in liver, at lease in the diabetic state, was shown by the observation that the low activities of these enzymes in diabetic animals could be restored to normal by adrenalectomy. An even more pronounced restorative effect was apparent in adipose tissue of adrenalectomized, diabetic animals. Administration of glucagon during the refeeding of starved rats resulted in a marked reduction in the induction of fatty acid synthetase, acetyl-CoA carboxylase and in the rate of incorporation of 3-H from 3-H2O into fatty acids in liver, but no change in these parameters occurred in adipose tissue. Administration of theophylline resulted in intermediate reduction in liver. The mechanisms whereby glucagon led tto a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the changes in fatty acid synthetase activity were shown to reflect reductions in content of enzyme. The mechanism underlying these reductions in content is reduced synthesis of enzyme.     

Stimulation of the synthesis of hepatic fatty acid synthesizing enzymes of hypophysectomized rats by 3,5,3'-l-triiodothyronine.

The levels of hepatic fatty acid synthesizing enzymes, acetyl-CoA carboxylase and fatty acid synthetase, are lowered to about one-tenth of the controls in hypophysectomized animals, whereas the lung enzymes decrease by only 25–30%. Administration of 3,5,3′-l-triiodothyronine to the hypophysectomized animals returns the hepatic and lung enzyme activities to the control values. Optimum levels are achieved at a dose of about 150 μg/100 g body wt and 3–4 days after triiodothyronine administration. The triiodothyronine response can be reduced by 80% with actinomycin-D or cycloheximide but not with hydrocortisone hemisuccinate. Antibody-antigen titrations and measurements of the rate of synthesis of fatty acid synthetase are indicative of increased synthesis of fatty acid synthetase and not of activation of the preexisting inactive species. These measurements provide evidence for the involvement of hormones other than insulin in the control of synthesis of fatty acid synthesizing enzymes.     

Acyltransferase activities in adult rat type II pneumocyte-derived subcellular fractions

Acyl-CoA: lysophosphatidylcholine, acyl-CoA: lysophosphatidylethanolamine, and lysophosphatidylcholine:lysophosphatidylcholine acyltransferases were investigated using subcellular fractions derived from adult rat type II pneumocytes in primary culture. Acyl-CoA:lysophospholipid acyltransferase activities were determined to be microsomal, while lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity was found to be cytosolic. Total palmitoyl CoA:lysophosphatidylcholine acyltransferase activity was 30-fold greater than lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity, indicating that the former enzyme is more important in the synthesis of dipalmitoyl phosphatidylcholine. Palmitoyl-CoA and oleoyl-CoA lysophosphatidylcholine acyltransferase activities were approximately equal under optimal substrate conditions. Specific activities of the enzyme using arachidoyl-CoA and arachidonoyl-CoA were 46% and 18%, respectively, of those with palmitoyl-CoA. Acyl-CoA:lysophosphatidylethanolamine acyltransferase showed a preference for palmitoyl-CoA as opposed to oleoyl-CoA under optimal conditions. However, when equimolar concentrations of either palmitoyl-CoA and oleoyl-CoA or palmitoyl-CoA and arachidoyl-CoA were assayed together, the relative utilization of the two substrates was found to be dependent on total acyl-CoA concentration. At higher concentrations, the incorporation of palmitoyl-CoA into phosphatidylcholine was less than other acyl-CoAs. However, at lower concentrations palmitoyl-CoA was utilized quite selectively. Whole lung microsomes did not show as marked a preference for palmitoyl-CoA as did type II pneumocyte microsomes under these same conditions. In similar experiments, low total acyl-CoA concentrations produced greater incorporation of oleoyl-CoA into phosphatidylethanolamine. For both enzymes total activity at the lowest concentrations used was at least 45% that at optimal conditions. This demonstrates that the type II pneumocyte acyltransferase system(s) can selectively utilize palmitoyl-CoA. No evidence for direct exchange of palmitoyl-CoA with 1-saturated-2-unsaturated phosphatidylcholine in subcellular fractions from type II pneumocytes was found.     

Regulation of hepatic fatty acid-synthesizing enzymes of diabetic animals by thyroid-hormone

Subcutaneous administration of l-triiodothyronine (T₃) to diabetic rats restored hepatic acetyl-CoA carboxylase and fatty acid synthetase enzymes to normal levels. T₃ stimulated the fatty acid-synthesizing enzymes of diabetic animals by two different mechanisms. Between 4 and 12 h after T₃ administration, carboxylase and synthetase increased slowly, after which both the enzyme activities increased at faster rate. Carboxylase and synthetase induction could be inhibited by cycloheximide or actinomycin D during the first 12 h. The incorporation of [¹⁴C]pantothenate into the fatty acid synthetase during 4–12 h followed the same pattern as the development of the enzyme activity. Moreover, liver supernatants from T₃-treated diabetic rats were able to compete with pure fatty acid synthetase for antibody binding sites, the degree of competition increased with increasing period of T₃ treatment. The results suggest that enzymatically inactive precursors of synthetase in the diabetic livers are converted to enzymatically active enzyme as a result of T₃ treatment. The second part of T₃-mediated stimulation (24 to 72 h following T₃ treatment) was inhibited by cycloheximide and actinomycin D. Antibody-antigen titration and measurement of rate of protein synthesis suggest that the increased activity of hepatic synthetase is due to enhanced synthesis of the enzyme for that period. These results indicate that T₃ might play a significant regulatory role in hepatic fatty acid synthesis.     

Effect of growth hormone on rat liver N-acetyl-L-glutamate

Earlier studies have revealed, upon hypophysectomy, a specific increase in mitochondrial urea cycle enzymes, namely carbamyl phosphate synthetase and ornithine transcarbamylase. Administration of growth hormone to hypophysectomized rats brought these enzyme activities back to normal. Since growth hormone plays a role in the formation of citrulline and ultimately urea, in the present study its effect on the levels of N-acetyl-L-glutamate, an allosteric activator of carbamyl phosphate synthetase has been investigated. A significant increase in N-acetyl-L-glutamate concentration in rat liver on hypophysectomy and its reversal back to normal levels on growth hormone administration was reported. These results suggest that the lack of growth hormone tends to amplify urea production by the liver.     

Oxygen-induced changes in pulmonary phospholipids in the rat.

The composition and synthesis of alveolar and lung tissue phospholipids were investigated in normal and oxygen-poisoned rat lungs. Sixty-hour exposure to oxygen increased the total amount of phospholipids in the endobronchial extracts and lung tissue. Phosphatidyl glycerol was identified in both endobronchial extracts and lung tissue. The amount of unsaturated fatty acids in surfactant lecithin and phosphatidyl glycerol was slightly increased in oxygen-poisoned lungs whereas the composition of phospholipids in the endobronchial extracts was not affected by oxygen. After intraperitoneal administration of [32P]phosphate the specific activities of surfactant lecithin and phosphatidyl glycerol were clearly lower in oxygen-treated animals whereas the specific activities of lung tissue lecithin and phosphatidyl glycerol remained unaffected. The synthesis of lecithin from [14C]methionine through N-methyltransferase pathway was markedly depressed in lung slices but increased in liver tissue taken from oxygen-poisoned rats and incubated under oxygen indicating a difference between lung and liver methyltransferase enzymes. In conclusion, the present work suggests impaired synthesis and removal of alveolar phospholipids in oxygen-poisoned rats.     

Nutritional and hormonal control of lung and liver fatty acid synthesis.

During starvation and in streptozotocin-induced diabetes, the total activities of rat lung acetyl CoA carboxylase and fatty acid synthetase are reduced to one-third of the normal values. Refeeding of the starved animals or administration of insulin to diabetic animals restores the levels to the original values. The insulin effect is dose and time dependent. These data contrast with those in the liver, where a 30- to 50-fold depression of these enzymes is observed in the diabetic state and administration of insulin is actually followed by doubling of the activity over normal controls. Fat-free high-fructose diet (containing 60% fructose by weight) enhances the activities of liver enzymes 3- to 6-fold over the values of controls on laboratory diet but has no effect on the lung enzymes. Long-term feeding of fructose diet also increases the activities of liver enzymes from diabetic animals to twice the value of normal controls on laboratory diet. Insulin administration to fructose-fed diabetic animals restores the enzyme activities to those obtained with fructose-fed normal controls. However, the stimulation of lung enzymes of diabetic animals can be effected either by fructose or by insulin. Antigen-antibody titrations and measurements of the rate of protein synthesis show that the increased activity of the lung and liver fatty acid synthetase is due to enhanced content rather than increased specific activity. These data suggest that insulin or fructose effects on fatty acid-synthesizing enzymes are mediated through intermediate(s) whose concentration is affected in the experimental diabetes. Furthermore, all tissues may not have stringent insulin requirements since the lung enzymes can be stimulated by fructose alone.     

Adaptive synthesis of fatty acid synthetase and acetyl-CoA carboxylase by isolated rat liver cells.

Hepatocytes were isolated at specified times from livers of diabetic and insulin-treated diabetic rats during the course of a 48-h refeeding of a fat-free diet to previously fasted rats. The rates of synthesis of fatty acid synthetase and acetyl-CoA carboxylase in the isolated cells were determined as a function of time of refeeding by a 2-h incubation with l-[U-¹⁴C]leucine. Immunochemical methods were employed to determine the amount of radioactivity in the fatty acid synthetase and acetyl-CoA carboxylase proteins. The amount of radioactivity in the fatty acid synthetase synthesized by the isolated cells was also determined following enzyme purification of the enzyme to homogeneity. Enzyme activities of the fatty acid synthetase and acetyl-CoA carboxylase in the cells were measured by standard procedures. The results show that isolated liver cells obtained from insulintreated diabetic rats retain the capacity to synthesize fatty acid synthetase and acetyl-CoA carboxylase. The rate of synthesis of the fatty acid synthetase in the isolated cells was similar to the rate found in normal refed animals in in vivo experiments [Craig et al. (1972) Arch. Biochem. Biophys. 152, 619–630; Lakshmanan et al. (1972) Proc. Nat. Acad. Sci. USA69, 3516–3519]. In addition the relative rate of synthesis of fatty acid synthetase was stimulated greater than 20-fold in the diabetic animals treated with insulin. Immunochemical assays, when compared with enzyme activities, indicated the presence of an immunologically reactive, but enzymatically inactive, form or “apoenzyme” for both the fatty acid synthetase and acetyl-CoA carboxylase. The synthesis of these immunoreactive and enzymatically inactive species of protein, as well as the synthesis of the “holoenzyme” forms of both enzymes, requires insulin.     

Rapid stimulation of liver palmitoyl-CoA synthetase, carnitine palmitoyltransferase and glycerophosphate acyltransferase compared to peroxisomal beta-oxidation and palmitoyl-CoA hydrolase in rats fed high-fat diets

Key enzymes involved in oxidation and esterification of long-chain fatty acids were investigated in male rats fed different types and amounts of oil in their diet. A diet with 20% (w/w) fish oil, partially hydrogenated fish oil (PHFO) and partially hydrogenated soybean oil (PHSO) was shown to stimulate the mitochondrial and microsomal palmitoyl-CoA synthetase activity (EC 6.2.1.3) compared to soybean oil-fed animals after 1 week of feeding. Rapeseed oil had no effect. Partially hydrogenated oils in the diet resulted in significantly higher levels of mitochondrial glycerophosphate acyltransferase compared to unhydrogenated oils in the diet. Rats fed 20% (w/w) rapeseed oil had a decreased activity of this mitochondrial enzyme, whereas the microsomal glycerophosphate acyltransferase activity was stimulated to a comparable extent with 20% (w/w) rapeseed oil, fish oil or PHFO in the diet. Increasing the amount of PHFO (from 5 to 25% (w/w)) in the diet for 3 days led to increased mitochondrial and microsomal palmitoyl-CoA synthetase and microsomal glycerophosphate acyltransferase activities with 5% of this oil in the diet. The mitochondrial glycerophosphate acyltransferase was only marginally affected by increasing the oil dose. Administration of 20% (w/w) PHFO increased rapidly the mitochondrial and microsomal palmitoyl-CoA synthetase, carnitine palmitoyltransferase and microsomal glycerophosphate acyltransferase activities almost to their maximum value within 36 h. In contrast, the glycerophosphate acyltransferase and palmitoyl-CoA hydrolase (EC 3.1.2.2) activities of the mitochondrial fraction and the peroxisomal beta-oxidation reached their maximum activities after administration of the dietary oil for 6.5 days. This sequence of enzyme changes (a) is in accordance with the proposal that an increased cellular level of long-chain acyl-CoA species act as metabolic messages for induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase, i.e., these enzymes are regulated by a substrate-induced mechanism, and (b) indicates that, with PHFO, a greater part of the activated fatty acids are directed from triacylglycerol esterification and hydrolysis towards oxidation in the mitochondria. It is also conceivable that the mitochondrial beta-oxidation is proceeding before the enhancement of peroxisomal beta-oxidation.     

Renal enzymes during experimental diabetes mellitus in the rat. Role of insulin, carbohydrate metabolism, and ketoacidosis

The activities of various ammoniagenic, gluconeogenic, and glycolytic enzymes were measured in the renal cortex and also in the liver of rats made diabetic with streptozotocin. Five groups of animals were studied: normal, normoglycemic diabetic (insulin therapy), hyperglycemic, ketoacidotic, and ammonium chloride treated rats. Glutaminase I, glutamate dehydrogenase, glutamine synthetase, phosphoenolpyruvate carboxykinase (PEPCK), hexokinase, phosphofructokinase, fructose-1,6-diphosphatase, malate dehydrogenase, malic enzyme, and lactate dehydrogenase were measured. Renal glutaminase I activity rose during ketoacidosis and ammonium chloride acidosis. Glutamate dehydrogenase in the kidney rose only in ammonium chloride treated animals. Glutamine synthetase showed no particular variation. PEPCK rose in diabetic hyperglycemic animals and more so during ketoacidosis and ammonium chloride acidosis. It also rose in the liver of the diabetic animals. Hexokinase activity in the kidney rose in diabetic insulin-treated normoglycemic rats and also during ketoacidosis. The same pattern was observed in the liver of these diabetic rats. Renal and hepatic phosphofructokinase activities were elevated in all groups of experimental animals. Fructose-1,6-diphosphatase and malate dehydrogenase did not vary significantly in the kidney and the liver. Malic enzyme was lower in the kidney and liver of the hyperglycemic diabetic animals and also in the liver of the ketoacidotic rats. Lactate dehydrogenase fell slightly in the liver of diabetic hyperglycemic and NH4Cl acidotic animals. The present study indicates that glutaminase I is associated with the first step of increased renal ammoniagenesis during ketoacidosis. PEPCK activity is influenced both by hyperglycemia and ketoacidosis, acidosis playing an additional role. Insulin appears to prevent renal gluconeogenesis and to favour glycolysis. The latter would seem to remain operative in hyperglycemic and ketoacidotic diabetic animals.     

Correlation between the cellular level of long-chain acyl-CoA, peroxisomal beta-oxidation, and palmitoyl-CoA hydrolase activity in rat liver. Are the two enzyme systems regulated by a substrate-induced mechanism?

Data obtained in earlier studies with rats fed diets containing high doses of peroxisome proliferators (niadenate, tiadenol, clofibrate, or nitotinic acid) are used to look for a quantitative relationship between peroxisomal beta-oxidation, palmitoyl-CoA hydrolase, palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities, and the cellular concentration of their substrate and reaction products. The order of the hyperlipidemic drugs with regard to their effect on CoA derivatives and enzyme activities was niadenate greater than tiadenol greater than clofibrate greater than nicotinic acid. Linear regression analysis of long-chain acyl-CoA content versus palmitoyl-CoA hydrolase and peroxisomal beta-oxidation activity showed highly significant linear correlations both in the total liver homogenate and in the peroxisome-enriched fractions. A dose-response curve of tiadenol showed that carnitine palmitoyltransferase and palmitoyl-CoA synthetase activities and the ratio of long-chain acyl-CoA to free CoASH in total homogenate rose at low doses before detectable changes occurred in the peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. A plot of this ratio parallelled the palmitoyl-CoA synthetase activity. The specific activity of microsomally localized carnitine palmitoyl-transferase was low and unchanged up to a dose where no enhanced peroxisomal beta-oxidation was observed, but over this dose the activity increased considerably so that the specific of the enzyme in the mitochondrial and microsomal fractions became comparable. The mitochondrial palmitoyl-CoA synthetase activity decreased gradually. The correlations may be interpreted as reflecting a common regulation mechanism for palmitoyl-CoA hydrolase and peroxisomal beta-oxidation enzymes, i.e., the cellular level of long-chain acyl-CoA acting as the metabolic message for peroxisomal proliferation resulting in induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. The findings are discussed with regard to their possible consequences for mitochondrial fatty acid oxidation and the conversion of long-chain acyl-L-carnitine to acyl-CoA derivatives.     

Effects of aging on contributions of dietary fat and triiodothyronine treatment to lipogenic enzyme induction

Although lipogenic enzyme inductions are reduced by fat feeding, this reduction decreases with aging and is particularly detectable in the case of acetyl-CoA carboxylase and fatty acid synthetase activities. On the other hand, the fat-dependent reductions of malic enzyme and acetyl-CoA carboxylase were consistently relieved by triiodothyronine (T3) treatment. The effects of T3 treatment on these enzyme inductions were greater in 10-month-old rats than in 1-month-old rats, while the carbohydrate-dependent induction and the fat-dependent reduction of the enzymes decreased with aging. In these animals, alterations in malic enzyme mRNA translational activities were roughly in parallel to the enzyme activities. Therefore, the age-dependent alterations in effects of T3 treatment and fat on malic enzyme induction do not appear to occur in post-translation.     

Biochemical changes in rat liver after 18.5 days of spaceflight

The effect of "weightlessness" on liver metabolism was examined using tissue from rats flown in earth orbit for 18.5 days aboard the Soviet Cosmos 936 biosatellite. Changes in the activities of certain carbohydrate and lipid enzymes were noted. Of the 28 hepatic enzyme activities assayed, two, palmitoyl-CoA desaturase and lactate dehydrogenase, increased, whereas five, glycogen phosphorylase, 6-phosphogluconate dehydrogenase, both acyltransferases which act on alpha-glycerolphosphate and diglycerides, and aconitate hydratase decreased. The remaining enzyme activities measured were unchanged. In addition, increased levels of liver glycogen and palmitoleate were noted which probably resulted from the lowered glycogen phosphorylase and increased palmitoyl-CoA desaturase activities, respectively, in those animals that experienced weightlessness. These changes caused by weightlessness were transient since all of the aforementioned alterations returned to normal values when measured in the livers of other rats which had flown in the biosatellite 25 days after recovery.     

Binding of specific ATP citrate lyase and fatty acid synthetase antibodies to heavy populations of rat liver polysomes.

The polysome fractions involved in the synthesis of the rat-liver inducible lipogenic enzymes, ATP citrate lyase and fatty acid synthetase, were identified by their binding of radioiodinated specific antibodies to enzyme. Both of these populations of specific polysomes were shown to be markedly heavier than specific polysomes involved in albumin synthesis. The quanity of antibody bound to the lipogenic enzyme-related polysomes was markedly affected by the dietary status of the animal. A dietary regimen which induced ipogenesis resulted in a tenfold increase in the hepatic activities of these enzymes found in normally fed animals. The radioactivity bound to hepatic polysomes of induced rats was likewise greater than tenfole higher, presumably reflecting an increase in the number of polysomes active in enzyme synthesis. The fasting state resulted in lower hepatic enzyme activity than normal and correspondingly less binding of ATP citrate lyase and fatty acid synthetase antibodies to the heavy polysomes of the sucrose gradient.     

Regulation of hepatic fatty acid synthetase in the obese-hyperglycemic mutant mouse.

Regulation of fatty acid synthetase has been studied in the obese-hyperglycemic mouse and compared with regulation in non obese, littermate control animals. The mechanisms underlying the regulatory changes were defined by immunochemical techniques. Several major conclusions are justified from the data obtained: (1) Although the hepatic specific activity of fatty acid synthetase is higher in obese than in non obese animals pair-fed chow, no difference in hepatic activities is apparent in animals pair-fed the fat-free diet; (2) The higher enzymatic activity in obese animals fed chow is related to a higher content of enzyme, and this higher content is associated with a higher rate of enzyme synthesis; (3) The decrease in hepatic synthetase activity with starvation is distinctly more striking in non obese than in obese animals, and the changes in activity reflect changes in content of enzyme; (4) With starvation there is a decrease in synthesis of enzyme in obese and non obese animals, but only in non obese animals is there also a marked increase in the rate of synthetase degradation (t1/2 = 24 h during starvation, t1/2 = 76 h during normalfeeding); (5) Refeeding starved mice a fat-free diet results in a more striking increase in hepatic synthetase activity in non obese than in obese animals; (6) Administration of triiodothyronine causes a more marked increase in hepatic synthetase activity in non obese than in obese animals. The data thus define a variety of differences in regulation of hepatic fatty acid synthetase in mutant and normal animals. The roles of enzyme synthesis and degradation in the etiology of these differences are defined, and possible mechanisms underlying regulation of synthetase synthesis and degradation in normal mammalian liver are suggested by the observations.     

Influence of pancreatic hormones on enzymes concerned with urea synthesis in rat liver

1. The activities of enzymes of the urea cycle [carbamoyl phosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinase (these last two comprising the arginine-synthetase system) and arginase] have been measured in control, alloxan-diabetic and glucagon-treated rats. In addition, measurements were made on alloxan-diabetic rats treated with protamine–zinc–insulin. 2. Treatment of rats with glucagon for 3 days results in a marked increase in the activities of three enzymes of the urea cycle (carbamoyl phosphate synthetase, argininosuccinate synthetase and argininosuccinase). The pattern of change in the alloxan-diabetic group is very similar to that of the glucagon-treated group, although the magnitude of the change was much greater. 3. Comparison was made of the actual and potential rate of urea synthesis in normal and diabetic rats. In both groups the potential rate of urea production, as measured by the activity of the rate-limiting enzyme, argininosuccinate synthetase, slightly exceeds the actual rate of synthesis by liver slices in the presence of substrates. The relative activities of the actual and potential rates were similar in the two groups of animals, this ratio being 1:0·70. 4. In the alloxan-diabetic rats treated with protamine–zinc–insulin for 2·5 or 4 days there was a marked increase in liver weight. This was associated with a rise in the total hepatic activity of the urea-cycle enzymes located in the soluble fraction of the cell (the arginine-synthetase system and arginase) after 2·5 days of treatment. After 4 days of treatment the concentration of these enzymes/g. of liver decreased, and the total hepatic content then reverted to the untreated alloxan-diabetic value. 5. No effects of glucagon or of insulin in vitro could be found on the rate of urea production by liver slices. 6. The present results are discussed in relation to how far this pattern of change is typical of conditions resulting in a high urea output, and comparison has been made with other values in the literature.     

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

The purpose of this study was to investigate early biochemical changes and possible mechanisms via which alkyl(C12)thioacetic acid (CMTTD, blocked for beta-oxidation), alkyl(C12)thiopropionic acid (CETTD, undergo one cycle of beta-oxidation) and a 3-thiadicarboxylic acid (BCMTD, blocked for both omega- (and beta-oxidation) influence the peroxisomal beta-oxidation in liver of rats. Treatment of rats with CMTTD caused a stimulation of the palmitoyl-CoA synthetase activity accompanied with increased concentration of hepatic acid-insoluble CoA. This effect was already established during 12-24 h of feeding. From 2 days of feeding, the cellular level of acid-insoluble CoA began to decrease, whereas free CoASH content increased. Stimulation of [1-14C]palmitoyl-CoA oxidation in the presence of KCN, palmitoyl-CoA-dependent dehydrogenase (termed peroxisomal beta-oxidation) and palmitoyl-CoA hydrolase activities were revealed after 36-48 h of CMTTD-feeding. Administration of BCMTD affected the enzymatic activities and altered the distribution of CoA between acid-insoluble and free forms comparable to what was observed in CMTTD-treated rats. It is evident that treatment of peroxisome proliferators (BCMTD and CMTTD), the level of acyl-CoA esters and the enzyme activity involved in their formation precede the increase in peroxisomal and palmitoyl-CoA hydrolase activities. In CMTTD-fed animals the activity of cyanide-insensitive fatty acid oxidation remained unchanged when the mitochondrial beta-oxidation and carnitine palmitoyltransferase operated at maximum rates. The sequence and redistribution of CoA and enzyme changes were interpreted as support for the hypothesis that substrate supply is an important factor in the regulation of peroxisomal fatty acid metabolism, i.e., the fatty acyl-CoA species appear to be catabolized by peroxisomes at high rates only when uptake into mitochondria is saturated. Administration of CETTD led to an inhibition of mitochondrial fatty acid oxidation accompanied with a rise in the concentration of acyl-CoA esters in the liver. Consequently, fatty liver developed. The peroxisomal beta-oxidation was marginally affected. Whether inhibition of mitochondrial beta-oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.     

Enzyme site-specific changes in hepatic microsomal fatty acid chain elongation in streptozotocin-induced diabetic rats

The hepatic microsomal fatty acid chain elongation of palmitoyl-CoA and γ-linolenoyl-CoA was diminished by 40–50% in male Sprague-Dawley rats made diabetic for 2 and 4 weeks following the intravenous administration of a single dose (65 mg/kg) of streptozotocin. Analysis of the activities of the four enzymatic components showed that only one enzyme, the condensing enzyme, which catalyzes the initial and rate-limiting step in chain elongation, was altered by the diabetic state. Both chain elongation and condensation activities were depressed to the same extent, whereas β-ketoacyl-CoA reductase, β-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase activities were the same as the values obtained with non-diabetic controls. 2 week administration of 10 units of insulin per day to rats which were diabetic for a 2-week period resulted in the reversal of the reduced palmitoyl-CoA elongation and condensation activities to control values. However, neither the condensation nor the elongation of γ-linolenoyl was reversed by the insulin treatment. These results support the notion of multiple condensing enzymes or chain elongation systems.     

The hypolipidemic peroxisome-proliferating drug, bis(carboxymethylthio)-1.10 decane, a dicarboxylic metabolite of tiadenol, is activated to an acylcoenzyme A thioester

Bis(carboxymethylthio)-1.10 decane (BCMTD), a thiodicarboxylic acid, was shown to be a hypolipidemic peroxisome-proliferating drug as it: (a) decreased the total serum triacylglycerols and cholesterol; (b) induced hepatomegaly; (c) increased the peroxisomal beta-oxidation and catalase activity and the activities of the multiorganelle localized enzymes: palmitoyl-CoA synthetase, palmitoyl-CoA hydrolase, glycerophosphate acyltransferase; (d) decreased the carnitine palmitoyltransferase and urate oxidase activities; and (e) induced the bifunctional eonyl-CoA hydratase in peroxisomes. The present study has confirmed the effect of tiadenol administration on the activities of key enzymes involved in hepatic fatty acid metabolism in male rats. However, the hepatic pleiotropic response was more marked with the dicarboxylic acid than with its alcohol. In a separate dose-response study BCMTD was found to be a more potent inducer of peroxisomal beta-oxidation compared to tiadenol. BCMTD can be activated in vitro to its coenzyme A thioester by a dicarboxyl-CoA synthetase. In control and BCMTD-treated animals, the synthetase activity was found in all cellular fractions except the cytosolic. Whether the acyl-CoA thioesters of peroxisome-proliferating drugs may be mediators of peroxisomal proliferation should be considered.     

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.