Joseph A. Ontko

Hypolipidemic activity of (−)-hydroxycitrate

The influence of (−)-hydroxycitrate, a potent competitive inhibitor of adenosine triphosphate (ATP) citrate lyase, on serum triglyceride and cholesterol levels, and in vitro and in vivo rates of hepatic fatty acid and cholesterol synthesis was investigated in normal and hyperlipidemic rat model systems. (−)-Hydroxycitrate reduced equivalently the biosynthesis of triglycerides, phospholipids, cholesterol, diglycerides, cholesteryl esters, and free fatty acids in isolated liver cells. In vivo hepatic rates of fatty acid and cholesterol synthesis determined in meal-fed normolipidemic rats were suppressed significantly by the oral administration of (−)-hydroxycitrate for 6 hr, when control animals exhibited maximal rates of lipid synthesis; serum triglyceride and cholesterol levels were significantly reduced by (−)-hydroxycitrate. In two hypertryglyceridemic models—the genetically obese Zucker rat and the fructose-treated rat—elevated triglyceride levels were due, in part, to enhanced hepatic rates of fatty acid synthesis. (−)-Hydroxycitrate significantly reduced the hypertriglyceridemia and hyperlipogenesis in both models. The marked hypertriglyceridemia exhibited by the triton-treated rat was only minimally due to increased hepatic lipogenesis; (−)-hydroxycitrate significantly inhibited both serum triglyceride levels and lipogenesis in this model.

Regulation of the mitochondrial oxidation-reduction state in intact hepatocytes by calcium ions.

Abstract The mitochondrial NADH:NAD ratio of isolated intact liver cells incubated in calcium-free Hanks solution, in the endogenous state or with lactate, alanine, α-ketoglutarate, glutamate, fumarate, malate, or albumin-bound palmitate, was elevated by 1 mM CaCl2. The chloride salts of Ba+2, Cd+2, Cu+2, Mn+2, Sr+2, Zn+2, Al+3, Ce+3 and La+3 caused no such change. In contrast, calcium decreased the mitochondrial NADH:NAD ratio of hepatocytes incubated with succinate. Calcium did not affect the NADH:NAD ratio in the liver cell cytosol or the energy charge. The calcium-induced elevation in the mitochondrial NADH:NAD ratio was reversed by the uncoupler 1799. These observations demonstrate a specific effect of calcium ions in the regulation of the mitochondrial oxidation-reduction state in intact liver cells.

Reciprocal effects of energy utilization on palmitate oxidation and esterification in hepatocytes of fed rats

The effects of the energy-dependent process of urea synthesis from NH4Cl on the partition of [1-14C]palmitate between oxidation and esterification were examined in hepatocytes of fed rats. A high rate of urea formation from NH4Cl resulted in stimulation of total palmitate oxidation by 25 and 15% at 0.2 and 1 mM fatty acid, respectively. The stimulation of palmitate oxidation was reciprocally correlated with diminished palmitate incorporation into lipids, mainly triacylglycerols. This relationship was almost stoichiometric. NH4Cl increased the palmitate oxidation/esterification ratio from 0.72 to 1.13 and from 0.94 to 1.36 in the presence of 0.2 mM and 1 mM palmitate, respectively. The transaminase inhibitor, aminooxyacetate, strongly inhibited urea synthesis from NH4Cl, had little effect on the low beta-hydroxybutyrate/acetoacetate ratio in the presence of NH4Cl, completely reversed the changes in palmitate metabolism caused by NH4Cl and did not affect palmitate metabolism in the absence of NH4Cl. Therefore, the increased utilization of energy for urea synthesis was the causative factor by which NH4Cl stimulated total palmitate oxidation and led in consequence to its decreased esterification into lipids. Accordingly, these observations indicate that in liver cells the rate of ATP utilization is one of the determinants of triacylglycerol synthesis.

Regulation of triglyceride mobilization in isolated hepatocytes by dibutyryl cyclic AMP and epinephrine.

Regulation of the mobilization of endogenous fatty acids from lipid droplet triglyceride was investigated in isolated hepatocytes from normal-fed rats. Mobilized fatty acid was entirely accounted for in oxidation products (CO2 plus acid-soluble products). Dibutyryl cyclic AMP (DBcAMP) stimulated the mobilization of endogenous fatty acids by over 50% although no change in free fatty acid (FFA) levels was observed. In the presence of tetrade-cylglycidic acid (TDGA), a specific inhibitor of mitochondrial fatty acid oxidation, DBcAMP was unable to promote endogenous fatty acid oxidation; instead, the nucleotide increased the FFA level. This effect was blocked by the lysosomal inhibitor, chloroquine, supporting the concept that a lysosomal lipase is involved in the degradation of endogenous triglyceride. These findings suggest that cyclic AMP stimulates lysosomal lipolysis by a mechanism that is independent of changes in fatty acid oxidation. Epinephrine alone slightly suppressed triglyceride mobilization. Epinephrine plus doxazosin slightly increased lipid mobilization. Epinephrine plus propranolol suppressed endogenous fatty acid oxidation to a level significantly below that induced by epinephrine plus doxazosin. These results suggest that α1-adrenoceptor activation suppresses hepatic triglyceride mobilization. The weak stimulatory effects of β-adrenoceptor activation on hepatocyte triglyceride mobilization compared with the potent stimulation by DBcAMP is probably attributable to the relatively low level of β-adrenoceptors in hepatocytes from the mature male rats used in these studies. Further characterization of a possible mutually antagonistic interaction between α1 and β2-adrenoceptors in the regulation of hepatic triglyceride mobilization might be better accomplished in a species with hepatocytes that have a higher proportion of β-adrenoceptors, e.g., guinea pig, rabbit, and dog. It is suggested that adrenergic regulation of hepatic triglyceride mobilization may be involved in the antiatherosclerotic serum lipid changes induced by selective α1-inhibition and the proath-erosclerotic serum lipid changes induced by β-blockade.

Direct effects of fructose metabolism on fatty acid oxidation in a recombined rat liver mitochondria-high speed supernatant system

The effects of fructose on the oxidation of [1-(14)C]palmitate in a rat liver mitochondria-high speed supernatant system have been investigated. This model system permitted study of the direct effects of fructose and the metabolism of fructose on fatty acid oxidation in the near absence of fatty acid esterification. Fructose inhibited the utilization of albumin-bound [1-(14)C] palmitate in the mitochondria-supernatant system, but did not affect fatty acid utilization by isolated liver mitochondria. Although fructose decreased the ATP content in the mitochondrial-supernatant system, the level of ATP throughout the incubation period was sufficient for maximal fatty acid activation. Fructose decreased the conversion of [1-(14)C]palmitate to 14CO2 and depressed the formation of total labeled oxidation products (14CO2 + 14C-labeled ketone bodies) in this system. The results suggest that fructose metabolism inhibited fatty acid oxidation in the mitochondria-supernatant system by competitive substrate oxidation and thereby decreased utilization of the added [1-(14)C]palmitate. The ihibition of L-[L-(14)C]palmitoylcarnitine oxidation, fructose was in all respects similar to its inhibition of palmitate oxidation, indicating that the site of fructose interaction was within the beta-oxidation sequence. These observations support the concept (Ontko, J.A. [1972] J. Biol. Chem. 247, 1788-1800) that the reciprocal changes in esterification and oxidation of palmitate caused by fructose in liver cells are primarily mediated via inhibitory effects on long-chain fatty acid oxidation.

Influence of doxazosin on lipid transport in rats and hamsters.

A variety of nutritional conditions were investigated to identify those most responsive in terms of selective α1-adrenergic inhibition (α1-innibition) on the serum lipid concentrations in rats and hamsters. In rats fasted for 24 h and then refed a lipogenic diet for the same period, serum triglycerides were markedly elevated. Inclusion of the selective α1-adrenergic inhibitor (α1-inhibitor) doxazosin in the diet decreased the intensity of this response. Serum cholesterol was not appreciably altered by the drug in these animals. Although dietary doxazosin did not affect serum lipids in rats fed chow ad libitum, in chow-fed hamsters (which have much higher serum lipid levels than rats) consumption of the drug for 4 days substantially decreased serum levels of both triglyceride and cholesterol. Very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol were all decreased by doxazosin. It is suggested that selective (α1-inhibition influences lipoprotein metabolism at several sites, including both formation and removal processes. The hamster may be especially useful in studies designed to define these sites and the underlying mechanisms involved.

Cholesterol content as a possible determinant in the removal of chylomicron triglyceride by the liver

The nature of chylomicron removal from the circulation and the role of the liver in this process have been investigated by measuring the removal of chylomicron triglyceride and cholesterol from the plasma of normal and functionally hepatectomized rats. The rate of removal of chylomicron triglyceride exceeded that of both free and esterified cholesterol in all animals. Whereas chylomicrons were completely removed from the circulation of normal rats soon after intravenous injection, chylomicron remnant particles accumulated in the plasma very low density lipoprotein and chylomicron fractions of hepatectomized rats. The rate of removal of chylomicron triglyceride from the blood of the hepatectomized rats decreased when the cholesterol content of the chylomicrons was elevated. In addition, more chylomicron triglyceride remained in the circulation of the hepatectomized rats when the chylomicrons were enriched with cholesterol and the remnant particles derived from these chylomicrons were of lower average density. These observations indicate that the cholesteryl ester content of the chylomicrons influences the degree of triglyceride removal in extrahepatic tissue capillaries and thereby the amount of residual triglyceride in the remnant particles subsequently removed by the liver.