Harold J. Fallon

Glycerolipid formation from sn-glycerol-3-phosphate by rat liver cell fractions. The role of phosphatidate phosphohydrolase.

Abstract The rate of sn-glycerol-3-phosphate incorporation into glycerolipids by rat liver microsomal and homogenate preparations was compared with the activity of the individual reactions in triacylglycerol biosynthesis. Microsomal phosphatidate phosphohydrolase was the least active reaction in this pathway when measured with either an aqueous dispersion of phosphatidate or microsomal bound phosphatidate. When supernatant phosphatidate phosphohydrolase was added to microsomes, diacylglycerol and triacylglycerol increased but the ratio of diacylglycerol and triacylglycerol to phosphatidate remained less than one. These results indicate that phosphatidate phosphohydrolase may be the rate limiting reaction in diacylglycerol and triacylglycerol formation by liver cells. The characteristics of liver microsomal and supernatant phosphatidate phosphohydrolase were studied further. The results show (I) microsomal activity was higher with an aqueous dispersion of phosphatidate while the supernatant enzyme was more active with microsomal bound substrate, (2) Mg2+ activates microsomal but not supernatant phosphatidate phosphohydrolase, (3) optimum microsomal bound phosphatidate substrate levels were 5-fold greater for microsomes than supernatant, (4) most divalent cations inhibit phosphatidate phosphohydrolase activity in both fractions.

Inhibition of hepatic triglyceride formation by clofibrate

The effect of clofibrate (CPIB) on hepatic glycerolipid formation has been studied in vivo and in vitro in the rat. Feeding 0.25% CPIB in laboratory chow significantly reduced serum triglyceride levels by 6 hr and concomitantly decreased the rate of glycerol-(14)C incorporation into hepatic and serum glycerides, in vivo. These changes persisted for at least 14 days. A similar decrease in serum triglyceride and glycerol incorporation into hepatic glycerides was observed in rats fed high glucose diets containing 0.25% CPIB. Serum glycerol was reduced by feeding CPIB for 14 days. The formation of diglyceride and triglyceride from (14)C-sn-glycerol-3-P by rat liver homogenates was inhibited by addition of 1-40 mM CPIB to the reaction mixture. These results suggest that CPIB reduces hepatic glycerolipid synthesis, possibly by inhibition of one or more reactions in the esterification of sn-glycerol-3-P. This change may account for the early fall in serum triglyceride. At later time periods, serum glycerol levels fall and in some experiments, hepatic triglyceride content increases. Therefore, it is likely that additional metabolic alterations may contribute to the sustained hypotriglyceridemic effects of CPIB.

An enzymatic explanation for dietary induced alterations in hepatic glycerolipid metabolism

Abstract Diets containing 30, 50 and 75 % glucose and fructose were fed to rats for various time periods. Each diet increased formation of triacylglycerol by several liver cell fractions in the presence of sn-[I,3-14C]glycerol-3-P, ATP, palmitate and CoA. The enzymatic basis for the increased triacylglycerol synthesis was studied using liver microsomal preparations. The activity of two enzymes, sn-glycerol-3-P acyltransferase and phosphatidate phosphohydrolase, was increased by the high fructose and glucose diets. Diets rich in either sugar produced marked increases in the ratio of neutral lipid to polar lipid formed by microsomes or whole liver homogenates indicating that the effect on phosphatidate phosphohydrolase was predominate. Phosphatidate phosphohydrolase activity was also recovered in a fraction of the particle free supernatant precipitated by 40 % (NH4)2SO4. The high glucose and fructose diets increased the activity of the supernatant phosphohydrolase three fold. The increase in triacylglycerol formation by various cell preparations correlated temporally with the increase in microsomal and supernatant phosphatidate phosphohydrolase activity and with an acceleration in liver triacylglycerol formation as previously estimated in vivo. It is concluded that phosphatidate phosphohydrolase activity is important in the regulation of endogenous triacylglycerol formation under these dietary conditions.

Effects of ethanol on hepatic microsomal drug-metabolizing enzymes in the rat.

Abstract The activities of several hepatic microsomal drug-metabolizing enzymes were determined in male and female rats after administration of 20% ethanol or an isocaloric amount of glucose in drinking water for 10–49 days. The aniline hydroxylase activity increased, whereas the activities of both pentobarbital hydroxylase and benzphetamine N -demethylase were decreased in male rats given ethanol and killed without ethanol withdrawal. Twenty-four hr after removal of ethanol, the aniline hydroxylase remained elevated but a striking increase of both pentobarbital hydroxylase and benzphetamine N -demethylase above control values occurred. Six days later, all three of these microsomal enzymes returned to normal control values. The reduction in pentobarbital hydroxylase and benzphetamine N -demethylase could not be attributed simply to high endogenous ethanol levels since: (1) addition of high concentrations of ethanol in vitro inhibited microsomal aniline hydroxylase and pentobarbital hydroxylase but did not reduce benzphetamine N -demethylase; and (2) acute administration of ethanol by gastric tube, which markedly elevated the blood ethanol level, did not result in a decline in the enzyme activities. These two findings suggest that the observed decrease in microsomal enzymes in male rats required persistent ethanol exposure. In contrast to male rats, female rats given ethanol for 28 days showed a significant increase in aniline hydroxylase activity, but the activities of pentobarbital hydroxylase and benzphetamine N -demethylase were not decreased. Moreover, administration of ethanol for 28 days to female rats did not reduce the response of these three enzyme activities to pentobarbital administration. It is concluded that the effects of chronic ethanol ingestion on the hepatic microsomal drug-metabolizing enzymes are complex and depend on sex, exposure to other agents and, most importantly, on the duration and proximity of ethanol intake.

The effects of ethanol ingestion and choline deficiency on hepatic lecithin biosynthesis in the rat.

Abstract The effects of long-term ethanol ingestion and choline deficiency on hepatic lecithin metabolism in the rat are described. Rats were given 20% ethanol in drinking water for 1 month to 1 year and changes compared to an isocalorically fed control group. Choline-deficient diets were fed for 4 weeks and results compared with a control group fed the same diet supplemented with 0.5% choline. 1. 1. Chronic ethanol ingestion resulted in a significant (10–15%) increase in hepatic total phospholipid content. An increased content of phospholipid in relation to protein was noted in the isolated microsomal fraction. An increase in both lecithin and phosphatidyl ethanolamine occurred. Triglyceride content was unchanged. 2. 2 An increase in lecithin synthesis by the sequential methylation pathway was noted in homogenates and microsomal fractions prepared from ethanol-treated rats. No changes in the CDP-choline pathway of lecithin synthesis or in triglyceride synthesis were observed. 3. 3. Choline deficiency resulted in a similar increased capacity for lecithin synthesis by sequential methylation without change in the CDP-choline pathway. However, hepatic content of total phospholipid and lecithin fell and triglyceride increased in the choline-deficient rats. Triglyceride synthesis was increased. 4. 4. Supplementation of the diet with 2% choline completely prevented changes in lecithin biosynthesis in the rats given ethanol and partially reversed the increase in phospholipid content. It is concluded that only one pathway of lecithin synthesis de novo is responsive to changes in dietary choline and to the administration of ethanol. Choline in large amounts prevents or decreases ethanol-induced changes in phospholipid metabolism.

A quantitative morphological analysis of ethanol effect upon rat liver.

Ethanol ingestion causes morphological changes in the livers of man and animals. This report describes a quantitative morphometric analysis of ethanol effects upon rat liver and a comparison with some biochemical determinations. Twenty-four rats were divided into three pair-fed groups: chow and 25% ethanol in drinking water; chow and an amount of sucrose isocaloric to the ethanol; and 25% ethanol in water and chow supplemented with 2% choline. After 35 days, a portion of liver was prepared for electron microscopy by standard techniques. Liver was analyzed for total phospholipid, triglyceride, lecithin, nitroreductase activity, and aniline hydroxylase activity. Twenty-five electron micrographs were obtained randomly from each specimen. Micrographs were analyzed by the lineal analysis method for cell and nuclear dimensions, and fractional volume of cytoplasmic components including mitochondria, peroxisomes, lysosomes, lipid bodies, and glycogen. Surface area of the mitochondrial envelope and the endoplasmic reticulum membrane and the approximate volume of mitochondria and peroxisomes were determined. Subjective analysis of electron micrographs failed to distinguish clearly the controls from the ethanol-treated rats. However, lineal analysis with statistical evaluation showed increased hepatic cell size (P

Diphenylhydantoin-Induced Hepatic Necrosis: A Case Study

A patient with post-traumatic seizure disorder developed lymphadenopathy, exfoliative dermatitis, and hepatic failure while on diphenylhydantoin therapy and died in hepatic coma. Autopsy disclosed massive hepatic necrosis. The clinical and pathological pictures are similar to the six previously reported cases of diphenylhydantoin-induced hepatic necrosis, with the exception of the time of onset of hepatic failure, which is explained. The cause of such hepatotoxicity is unknown, although hypersensitivity is postulated. It appears that studies of liver function in patients receiving diphenylhydantion are indicated to assess the true indicence of hepatocellular injury.

A review of studies on the mode of action of clofibrate and betabenzalbutyrate

Clofibrate and betabenzalbutyrate produce a variety of metabolic alterations in vivo. These include reduction in serum triglyceride and alterations in adipose tissue uptake and release of lipids. Clofibrate displaces thyroxine from albumin binding sites and produces an enlargement of liver and changes in ultrastructure. The biochemical changes produced by clofibrate include reduction in adenyl cyclase activity, inhibition of acetyl CoA carboxylase, inhibition of cholesterol biosynthesis and inhibition of triglyceride formation. Recent studies in this laboratory have shown that the inhibition of hepatic triglyceride formation is an early metabolic consequence of clofibrate administration and precedes the fall in serum triglyceride and several of the other biochemical alterations. Moreover clofibrate and betabenzalbutyrate inhibit the esterification ofsn-glycerol-3-P by rat liver homogenate and microsomal preparations. The initial step in this pathway, acyl-CoA-sn-glycerol-3-P acyltransferase, is inhibited by both drugs, in vitro. It is possible that this in vitro inhibition explains the early hypotriglyceridemic effect of these agents.

Regulatory phenomena in mammalian serine metabolism.

Abstract The biosynthesis of serine in mammalian liver may proceed by two parallel pathways. Serine in physiological concentrations inhibits P-serine phosphatase, the last reaction in the “phosphorylated pathway”. No amino acid inhibitors of the alternative pathway have been identified. The restriction of protein intake in the rat results in a marked increase in hepatic 3-P-glycerate dehydrogenase and a lesser rise in P-serine phosphatase. Serine dehydratase, a catabolic enzyme, promptly decreases in activity on this diet. The addition of 1% cysteine to the low protein diet prevents the rise in enzyme activity but does not alter the level of serine dehydratase. Therefore, cysteine content of the diet appears of potential importance in the regulation of serine biosynthesis by the phosphorylated pathway. The nonlinear change in enzyme levels and studies with actinomycin D and cycloheximide suggest that the changes noted in enzyme activity are caused by alterations in enzyme synthesis. The degradation rate of 3-P-glycerate dehydrogenase does not appreciably change when rats are fed low protein diets. Cortisone acetate suppresses the level of 3-P-glycerate dehydrogenase and d -glycerate dehydrogenase, a unique effect for this hormone. No other factors studied altered enzyme levels in the “nonphosphorylated pathway”.

Alterations in Serine Metabolism in Rats Fed Liquid Synthetic Diets.

The liquid synthetic diet proposed by Greenstein et al, has been found to support good growth rates, reproduction and lactation in rats (1,2). Therefore, this diet has been considered nutritionally complete and the content of essential plus non-essential amino acids considered optimal. This diet has recently been used as a control diet in studies of hepatic serine biosynthesis in rats. Prompt alterations in the concentration of several enzymes important in serine metabolism were noted. This observation suggests that synthetic diets supporting normal or near normal growth rates may nevertheless require significant compensatory changes in enzyme levels to provide required metabolites. The importance of this observation to the study of alterations caused by specific dietary deficiencies is apparent. These studies also demonstrate the importance of dietary amino acids in the regulation of enzyme levels in serine metabolism. Methods. Male Osborne-Mendel rats (40-80 g) were used in these studies. Liquid synthetic diets, based on the diet of Greenstein et al{2), (Diet A) were prepared by General Biochemicals, Inc. (Diet No. 116). Identical diets were also prepared omitting serine and glycine (Diet B) or serine, glycine and cys-teine (Diet C). Rats were pair fed these liquid diets from Richter feeding tubes. Control rats were fed, ad libitum, either Purina Lab Chow or a solid 25% casein diet(3). The rats were fed the diets for 7 days, killed and enzyme preparations made from liver as described previously(3). Assays for 3-P-glyc-erate dehydrogenase, using 3-P-glycerate as substrate, serine dehydratase and D-glycerate dehydrogenase have been described(3). One unit of cnzqme activity is defined as a change in absorbency at 340 mp of 0.001 per minute. All materials were obtained from prior sources (3). Results. Weight gain in rats fed chow, 25% casein or diets A, B and C were equivalent for a 7-day period. However, an increase in hepatic 3-P-glycerate dehydrogenase concentration was noted when rats were fed the synthetic liquid diet (A), (Table I).