Sylvia A. McCune

Short-term Hormonal Control of Hepatic Lipogenesis

Lipids are essential components of all living cells, functioning as an energy store and playing an important role in all biologic membranes. Triacylglycerols, the storage form and most abundant of the glycerolipids, represent a highly concentrated form of energy, yielding twice as many calories per gram as carbohydrate or protein. The phospholipids are amphipathic compounds which, along with cholesterol, are found in membranes where they interact to provide a polar-faced, hydrophobic continuum of critical importance for proper cell structure and function. A major function of lipogenesis is to store (as triacylglycerols) the chemical energy or foodstuffs ingested above the immediate requirements of an organism. Contrary to protein, which is not stored, and to carbohydrate, which can only be stored in limited quantities, the capacity to store triacylglycerols is almost unlimited. The ability to keep large amounts of triacylglycerols on board would not be useful, however, if it were not possible to quickly and efficiently stop diverting foodstuffs into triacylglycerol formation and to quickly and efficiently start utilizing these compounds to provide the energy necessary to synthesize ATP. For this reason, mechanisms have evolved which finely tune the synthesis and degradation of triacylglycerol to the ever changing demand for ATP. In contrast, phospholipid and cholesterol synthesis must be, at least, partially maintained (or their degradation curtailed), even in the face of limited food intake or frank starvation, to provide sufficient quantities of these lipids to fulfill their crucial role in biologic membranes. In rodents the proportion of total body fatty acids synthesized by the liver ranges from 3% (fasted animals) to 50%

Comparative studies on fatty acid synthesis, glycogen metabolism, and gluconeogenesis by hepatocytes isolated from lean and obese Zucker rats.

Hepatocytes isolated from genetically obese female Zucker rats and lean female Zucker rats were compared. Hepatocytes from fed obese rats exhibited greater rates of fatty acid synthesis, more extensive accumulation of lactate and pyruvate from their glycogen stores, increased rates of net glucose utilization but produced less ketone bodies from exogenous fatty acids and had lower citrate levels than hepatocytes from lean rats. Lipogenesis was not as sensitive to dibutyryl cyclic AMP (DBcAMP) inhibition in hepatocytes from obese rats but glycogenolysis was stimulated to the same extent by this nucleotide in both preparations. Ketogenesis was less sensitive to stimulation by DBcAMP in hepatocytes from obese rats. A difference in sensitivity of lipogenesis to DBcAMP was not found when lactate plus pyruvate was added to the incubation medium, suggesting that a greater rate of glycolysis by hepatocytes from obese rats accounts for their relative insensitivity to DBcAMP. Citrate levels were elevated by DBcAMP to a greater extent in hepatocytes from obese rats. Hepatocytes prepared from lean rats starved for 48 hr were glycogen depleted and lacked significant capacity for lipogenesis and glycogen synthesis. In contrast, hepatocytes isolated from starved obese rats retained considerable amounts of liver glycogen and exhibited detectable rates of lipogenesis and glycogen synthesis. Hepatocytes prepared from starved lean rats gave faster apparent rates of lactate gluconeogenesis than hepatocytes prepared from starved obese rats. Thus, hepatocytes prepared from obese Zucker rats are more glycogenic, glycolytic, and lipogenic but less ketogenic and glucogenic than hepatocytes prepared from lean rats.

Inhibition of hepatic gluconeogenesis and lipogenesis by benzoic acid, p-tert.-butylbenzoic acid, and a structurally related hypolipidemic agent SC-33459.

Abstract Benzoic acid, p-tert. -butylbenzoic acid, and a structurally related hypolipidemic agent SC-33459 were found to inhibit glucose synthesis by hepatocytes isolated from 48-h fasted rats as well as fatty acid synthesis by hepatocytes isolated from meal-fed rats. Glucose synthesis was less sensitive than fatty acid synthesis. Benzoic acid was the least effective inhibitor of both processes; SC-33459 and p-tert. -butylbenzoic acid were very potent inhibitors with similar efficacy. Glycine prevented the inhibition of fatty acid synthesis caused by benzoic acid, but had no effect on that caused by p-tert. -butylbenzoic acid. Octanoate opposed the inhibitory effects of both benzoic acid and p-tert. -butylbenzoic acid. Oxidation of [1- 14 C]oleate to ketone bodies and acid-soluble radioactive products was inhibited by both p-tert. -butylbenzoic acid and SC-33459. Preincubation of hepatocytes with SC-33459 was required for the latter effect, suggesting catabolism of this compound may be involved. SC-33459 is a p-tert. -butylphenyl derivative which should be readily converted to p-tert. -butylbenzoic acid by β oxidation. Both p-tert. -butylbenzoic acid and SC-33459 decreased citrate levels dramatically. All three compounds reduced CoA and acetyl-CoA levels and increased medium-chain acyl-CoA ester levels. p-tert. -Butylbenzoic acid and SC-33459 also increased long-chain acyl-CoA ester levels. The increase in medium-chain acyl-CoA levels presumably reflects benzoyl-CoA formation from benzoic acid and p-tert. -butylbenzoyl-CoA formation from p-tert. -butylbenzoic acid and SC-33459. Inhibition of glucose and fatty acid synthesis by these compounds may be due to effects on specific enzymes or to CoA sequestration.

Inhibition of hepatic lipogenesis by 2-tetradecylglycidic acid.

Abstract2-Tetradecylglycidic acid (TDGA), a hypoglycemic agent, has been found to be a very effective inhibitor of de novo fatty acid synthesis by isolated hepatocytes. A comparison was made between the effectiveness of TDGA and 5-(tetradecyloxy)-2-furoic acid (TOFA), a hypolipidemic agent, on the metabolic processes of isolated hepatocytes. These compounds are structurally related and both inhibit fatty acid synthesis; however, they have opposite effects from each other on the oxidation and esterification of fatty acids. TDGA inhibits whereas TOFA stimulates fatty acid oxidation. TDGA stimulates whereas TOFA inhibits fatty acid esterification.

Inhibition of hepatocyte proteolysis and lactate gluconeogenesis by chloroquine.

Abstract Chloroquine (50 μ m ) is rapidly taken up by isolated hepatocytes in a temperature-dependent manner. It inhibits glucose synthesis from lactate, but not from pyruvate or dihydroxyacetone. The inhibition is reversed by lysine or ammonia but not by oleate or carnitine. Ammonia inhibits chloroquine uptake by the hepatocytes but lysine does not. Chloroquine also inhibits urea synthesis, the release of ninhydrin-reacting substances, the accumulation of amino acids, and the lactate-dependent accumulation of glutamate. Ethanol oxidation in the presence of lactate is also inhibited, and this too is reversed by lysine. Chloroquine increases the redox state of the cytosolic compartment, as evidenced by lactate-to-pyruvate ratios, of hepatocytes prepared from both 48-h fasted and meal-fed rats. The above findings are consistent with chloroquine entering the lysosomes of the hepatocytes and inhibiting proteolysis by raising the lysosomal pH. Isolated hepatocytes are deficient in amino acids and, chloroquine inhibition of proteolysis prevents replenishment of the amino acid pools. Thus, chloroquine prevents reconstitution of the malate-aspartate shuttle required for the movement of reducing equivalents into the mitochondrion during lactate gluconeogenesis, ethanol oxidation, and glycolysis. The metabolic competency of freshly isolated hepatocytes, therefore, depends on the replenishment of amino acid pools by lysosomal breakdown of endogenous protein. Furthermore, chloroquine uptake may be an index of lysosomal function with isolated hepatocytes.

Neoplastic transformation-linked alterations in arginyl-tRNA synthetase activity

The behavior of the activity of arginyl-tRNA synthetase (L-arginine : tRNAArg Ligase(AMP-forming), EC 6.1.1.19) was determined in extracts of rat liver: normal adult, normal proliferating (from developing and from partially hepatectomized rats), and neoplastic (hepatomas of different growth rates) and in extracts of rat kidney cortex and transplantable kidney tumors. The Km values for arginine, ATP, and tRNA of the enzyme of the rapidly growing hepatoma 3924A were the same as those of the enzyme from the liver of control rats. The pH optima of the control and neoplastic livers were in the same range of 7.25-8.0. Taking the hepatic specific activity for arginyl-tRNA synthetase as 100%, deep layer of gut, thymus and testis had higher activity; renal cortex and spleen had the same activity; and skeletal muscle, brain, heart, lung, superficial layer of gut and adipose tissue had lower activity. In a wide spectrum of hepatomas of different growth rates, a significant increase of 1.4-2.4-fold in arginyl-tRNA synthetase activity was observed when compared with that of liver of control normal rats. This elevation in enzyme activity in hepatomas appears to be specific to neoplasia, since it is unaltered in regenerating and low in differentiating liver. The increase in arginyl-tRNA synthetase in the liver tumors appears to be transformation-linked, since the activity was increased in all hepatomas, even in the slowest growing ones. Furthermore, the increase in enzyme activity was not limited to hepatic neoplasms, since a rise was also observed in transplantable rat kidney tumors. Thus, the reprogramming of gene expression in neoplastic tissue entails an increase in arginine-tRNA synthetase activity.

[41] 5-(Tetradecyloxy)-2-furoic acid

Publisher Summary This chapter discusses the preparation and effects of 5-(tetradecyloxy)-2-furoic acid (TOFA). TOFA reduces plasma triglyceride levels of both rats and monkeys, inhibits hepatic fatty acid synthesis both in vivo and in vitro, inhibits lipogenesis by isolated acini of lactating mammary gland, and inhibits cholesterolgenesis by isolated hepatocytes. The compound stimulates hepatic fatty acid oxidation and ketone body formation in vitro and inhibits glucose utilization by isolated hepatocytes and isolated acini. It is possible to prepare 10 mM solutions of TOFA in 20 mM NaOH, but the solution must be maintained at a high temperature. Aliquots of this solution can then be added to solutions containing albumin without the precipitation of TOFA. The effects of 50 μM TOFA on some of the metabolic processes of isolated hepatocytes are shown. Utilization of glucose carbon obtained by glycogenolysis is inhibited by TOFA, as is de novo fatty acid synthesis. TOFyl-CoA formation within intact hepatocytes can also be demonstrated. The inhibition of acetyl-CoA carboxylase by TOFyI-CoA is also elaborated.

A semiautomated assay procedure for the determination of aminoacyl--tRNA synthetase activity.

Abstract A semiautomated assay procedure for the determination of aminoacyl-tRNA synthetase activity is described. The assay system employs a Technicon AutoAnalyzer with a continuous filter paper attachment. A larger number of samples can be measured with greater accuracy, reproducibility, and more rapidly than the manual filter paper disk assay system.

A genetic study of erythrocyte arginine-tRNA synthetase activity in man.

To search for evidence of genetic variation among the aminoacyl-tRNA synthetases, a semi-automated assay procedure employing a Technicon AutoAnalyzer was used to measure erythrocyte arginine-tRNA synthetase activity in samples obtained from normal human twins of various ages. Variation in enzyme activity within the older DZ twins was five times that of the MZ twins suggesting the existence of genetically determined variation in enzyme activity. Higher enzyme activity was observed in newborn DZ unlike-sexed twins than in like-sexed twins for either zygosity. Possible explanations for this observation are discussed.