Joseph Triscari

Effect of (−)-hydroxycitrate upon the accumulation of lipid in the rat: I. Lipogenesis

The purpose of these investigations was to ascertain the effect of (−)-hydroxycitrate on the accumulation of lipid in the meal fed rat by examining the rates of lipogenesis after acute and chronic treatment. Oral administration of (−)-hydroxycitrate depressed significantly the in vivo lipogenic rates in a dose-dependent manner in the liver, adipose tissue, and small intestine. The hepatic inhibition was significant for the 8 hr period, when control animals demonstrated elevated rates of lipid synthesis. The kinetics of this reduction of in vivo hepatic lipogenesis were identical after acute or chronic administration of (−)-hydroxycitrate. However, in vitro rates of lipogenesis were elevated after chronic administration of (−)-hydroxycitrate for 30 days. Rats receiving (−)-hydroxycitrate consumed less food than the untreated controls; however, this decreased caloric intake was not responsible for the drug induced depression of hepatic lipogenesis, as shown by studies using pair fed rats.

Effect of (−)-hydroxycitrate upon the accumulation of lipid in the rat: II. Appetite

These studies were designed to determine the effect of (−)-hydroxycitrate upon the accumulation of lipid in the rat by examining appetite, wt gain, and total body lipid profiles. The chronic oral administration of a nontoxic dose of (−)-hydroxycitrate to growing rats for 11–30 days caused a significant reduction in body wt gain, food consumption, and total body lipid. The administration of equimolar amounts of citrate did not alter wt gain, appetite, or body lipid. No increase in liver size or liver lipid content occurred with either treatment. Pair feeding studies demonstrated that the reduction in food intake accounted for the decrease in wt gain and body lipid observed with (−)-hydroxycitrate treatment.

Abnormal sympatho-adrenal function and plasma catecholamines in obese Zucker rats

The functional integrity of the peripheral sympathetic nervous system and adrenal medulla was assessed in homozygous, lean and obese, 7--8 month old male Zucker rats by the changes in plasma catecholamines during cold and immobilization stresses. Five of eight obese, but no lean rats died during a 24 hr cold stress (4--7 degrees C) from hypothermia. While both lean and obese rats had decreased rectal temperatures after 4 hr of cold stress, the obese had lower temperatures, relatively less of an increase of plasma norepinephrine (NE) and epinephrine (E) than the lean rats, and were unable to consistently maintain their temperatures even during intravenous NE infusions. Obese rats had lower rectal temperatures and higher plasma NE and dopamine levels at 21--22 degrees C ambient temperature, a relative failure to increase plasma NE and E levels after 1 hr of immobilization, but normal or supranormal plasma catecholamine levels after decapitation compared to the lean rats. These results suggest that the obese Zucker rat has abnormalities of both peripheral sympatho-adrenal function and thermoregulation, which may play roles in the development and/or maintenance of many of the physiological and metabolic defects in this animal model of genetic obesity.

The effect of diet and chronic obesity on brain catecholamine turnover in the rat

Male Sprague-Dawley rats were fed a high calorie, high fat diet for 3 months to produce chronic diet-induced obesity (DIO) in which they gained 70% more weight than chow-fed controls. Thirty-six percent of the rats fed the DIO diet resisted the development of obesity (DR), gaining no more weight than chow-fed controls but serving as a comparison for the effects of the diet alone on the metabolism of brain catecholamines. The major influence of dietary composition was upon norepinephrine (NE) metabolism. Both DIO and DR rats had increased turnover of NE (107-217%) and/or shorter NE half-lives (42-67%) than controls in the hypothalamic paraventricular (PVN) and dorsomedial (DMN) nuclei and the median eminence (ME); dopamine (DA) turnover was similarly accelerated in the PVN. The DR rats alone exhibited decreased NE levels with increased disappearance of NE in frontal cortex and increased disappearance of DA in the ventromedial hypothalamic nucleus (VMH). The major effect of chronic obesity alone was a 31-33% decrease in DMN DA turnover and an 80% decrement in ME DA turnover associated with a 61% decrease in DA levels as compared to chow-fed controls. Therefore, the major effect of a high calorie, high fat diet was a diffuse acceleration of brain NE and DA turnover while chronic obesity led to decreased DA turnover in the DMN and ME.

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.

Changes in Lipid Metabolism in Diet-Induced Obesity

Mature male Sprague-Dawley rats fed a powdered Purina Chow diet containing corn oil and condensed milk (CM) were compared to rats fed a Purina Chow diet (control). CM rats gained more weight and consumed more calories over a 73-day period than the control rats. The increased weight gain and body fat in CM rats was accompanied by increased cell number in retroperitoneal and inguinal but not epididymal fat pads while cell size was unchanged in all three pads. After obesity had developed there was an increase in insulin levels, lipolysis, hepatic fatty acid synthesis, and fatty acid oxidation. While CM rats demonstrated hyperinsulinemia and hyperglycerolemia, they maintained normal glucagon and glucose levels. They demonstrated higher rates of fatty acid synthesis in isolated hepatocytes but not in vivo, suggesting that a greater potential for fatty acid synthesis in CM rats was masked in vivo by the inhibitory action of dietary lipids. Beta-oxidation of (1-14C) palmitate in vivo and in vitro, and in vivo ketogenesis were greater in CM than in chow fed rats. These studies demonstrate that, after the development of obesity, CM rats, like genetically obese Zucker rats, are hyperinsulinemic and have elevated levels of fatty acid synthesis. However, unlike obese Zucker rats, CM rats displayed an increase in beta-oxidation. These studies suggest that increased insulin levels and hepatic fatty acid synthesis may contribute to dietary obesity (as they do to genetic obesity), whereas increased fatty acid oxidation in dietary obesity may be a compensatory response to maintain a lower body weight.

Defective catecholamine metabolism in peripheral organs of genetically obese zucker rats

Catecholamine (CA) metabolism in peripheral organs of lean and obese, 3-4 and 7-8 month (mo) old male Zucker rats was studied to define further the known abnormalities of peripheral sympatho-adrenal functions in the obese rat. Norepinephrine (NE) levels in all sympathetically innervated organs from obese rats (aorta, heart, pancreas, brown adipose tissue and white adipose tissue) were decreased to 9-55% of those in lean rats at 3-4 mo. NE turnover, measured by inhibition of tyrosine hydroxylase (TH) with a-methyl-p-tyrosine, was also decreased in these same organs. NE levels and turnover were also decreased (by 50-95%) in many, but not all organs assayed of 7-8 mo old obese rats, while there were inconsistent changes in organ dopamine and epinephrine levels at both ages. Decreased NE metabolism was associated with decreased dopamine-beta-hydroxylase (D beta H) activity in every organ assayed from obese rats at 3-4 mo and 7-8 mo of age except in the superior cervical ganglia of 7-8 mo old rats. There were no consistent changes in TH or phenylethanolamine-N-methyltransferase activities. In 3-4 mo old obese rats, decreased D beta H activity was associated with decreased immunoprecipitable enzyme protein in the hearts but not in the adrenal glands, where differences in the affinity for substrate appeared to explain the activity differences. These results suggest that the previously reported defect in stress-induced plasma NE levels in obese rats could be explained by decreased D beta H activity in nerve terminals of their sympathetic nervous system and that, in this case, D beta H may play an important role in the regulation of NE synthesis.

Metabolic consequences of fasting in old lean and obese Zucker rats.

The effects of fasting on lipid and carbohydrate metabolism and plasma insulin and glucagon levels were compared in lean and obese Zucker rats. Sixteen-month-old female and male rats were fasted for periods of 2, 4, 6 and 12 days. Fasting produced significant decreases in hepatic rates of lipid, cholesterol, and glycogen synthesis, as well as circulating levels of triglycerides, cholesterol, phospholipids, and insulin. Significant increases in hepatic lipid levels and serum free fatty acids were noted. When compared to lean rats, obese rats had elevated rates of hepatic lipid and glycogen synthesis, hepatic lipid and glycogen stores, serum triglycerides, cholesterol, phospholipids, and plasma insulin. Lean rats had higher plasma glucagon levels. Sex differences in several parameters were observed. Females demonstrated higher levels of lipid and cholesterol synthesis and serum free fatty acids, whereas serum cholesterol levels and hepatic glycogen stores were higher in males. Following a 12-day fast, carcass fat and protein content were decreased in both lean and obese rats, but the obese animals maintained an obese body composition. It is concluded that fasting results in qualitatively similar metabolic and hormonal changes in both lean and obese rats, but that abnormalities in carbohydrate and lipid metabolism persist in obese rats even after a 12-day fast.

Comparative effects of (--)-hydroxycitrate and (+)-allo-hydroxycitrate on acetyl CoA carboxylase and fatty acid and cholesterol synthesis in vivo.

Abstract(−)-Hydroxycitrate and (+)-allo-hydroxycitrate were investigated for their effects on lipid synthesis in vivo under conditions of either high carbohydrate feeding or 24 hr fasting. Changes in rates of lipid synthesis resulting from the oral administration of these compounds were monitored with the use of radiolabeled H2O, alanine, and acetate. In the fed rat, (−)-hydroxycitrate significantly reduced the incorporation of H2O and alanine into fatty acids and cholesterol. An increased incorporation of labeled H2O into fatty acids but no change in cholesterol synthesis in the fasted rat suggested that (−)-hydroxycitrate may be an activator of acetyl CoA carboxylase. With (−)-hydroxycitrate administration, acetate incorporation into fatty acids and cholesterol was subject to pool dilution effects under fed or fasted states. (+)-allo-Hydroxycitrate was ineffective in modulating the rates of fatty acid synthesis under either nutritional condition. Both (−)-hydroxycitrate and (+)-allo-hydroxycitrate were shown to be in vitro activators of acetyl CoA carboxylase, the former being a much stronger activator than the latter. Thus, stereospecificity of the hydroxycitrate isomers was demonstrated in both the inhibition of lipid synthesis (previously shown to occur at adenosine triphosphate citrate lyase) and the stimulation of fatty acid synthesis (possibly occurring at acetyl CoA carboxylase).

Comparative effects of amphetamine and fenfluramine on lipid biosynthesis and absorption in the rat.

Abstract The effects of amphetamine and fenfluramine on lipid biosynthesis and intestinal absorption of triglycerides in the rat were observed independently of the anorectic activity of these drugs. Amphetamine and fenfluramine significantly reduced de novo lipogenesis and cholesterogenesis from 3 H 2 O and [ 14 C] alanine in isolated hepatocytes. Fenfluramine was four times more potent an inhibitor than amphetamine. At high concentrations (above 2 mM), fenfluramine inhibited 14 CO 2 production from [ 14 C] alanine in isolated hepatocytes while amphetamine stimulated 14 CO 2 production at all concentrations tested (0.25 to 8 mM). Lipogenesis in liver and adipose tissue was not reduced in vivo by the acute administration of amphetamine. It, however, reduced lipogenesis in the small intestine. Fenfluramine significantly depressed in vivo lipogenesis in liver, small intestine and adipose tissue at 10 and 20 mg/kg, i.p. Hepatic cholesterogenesis was depressed significantly in vivo by both drugs. Fenfluramine increased serum free fatty acids while amphetamine produced no change. Triglycerides were increased significantly by fenfluramine only. Serum cholesterol and phospholipids were unchanged. Fenfluramine at 40 and 60 mg/kg, p.o., diminished significantly the intestinal absorption of triglycerides and depressed the accumulation of lipid in the liver. Analogous effects were not observed with amphetamine. Amphetamine was a weak inhibitor of rat pancreatic lipase (EC 3.1.1.3) ( K i = 21 mM) and fenfluramine was a stronger inhibitor ( K i = 3.3 mM).