Energy restriction reduces metabolic rate in adult male Fisher-344 rats.

Energy restriction, without malnutrition, prolongs the maximum life span of laboratory rodents. A reduction in metabolic rate has been proposed as a potential mechanism for increased longevity. The present study examines changes in metabolic rate of adult rats after a 6-wk period of energy restriction. Two groups (n = 6) of 6-mo-old male Fisher-344 rats were studied. Restricted rats were pair-fed a diet equivalent in vitamins and minerals but restricted to 60% of energy consumed by rats eating ad libitum. Average and basal metabolic rates were measured by direct calorimetry over a 24-h period without food. Fat mass and lean body mass were determined by NMR spectroscopy. After 6 wk of restriction, when expressed per kilogram of lean body mass the average metabolic rate was reduced by 14% and basal metabolic rate by 12% compared with the ad libitum diet rats (P < or = 0.01). Reduction of metabolic rate did not seem to be a transient effect of chronic energy restriction in mature rats.

Formation of crystalline tripalmitin-rich spicules in isolated hepatocytes.

Conditions were developed for rapid deposition of triglyceride in isolated rat hepatocytes. Liver cells from fasted rats were incubated for 90 min at 37 degrees C with 3.0 mM palmitic or oleic acid, 4% bovine serum albumin, 20 mM glucose, 10 mM lactate, and 1 mM pyruvate. When oleic acid was used, numerous cytoplasmic lipid droplets were produced. When hepatocytes were incubated with palmitic acid, similar amounts of triglyceride were synthesized but instead of lipid droplets, a vast accumulation of peculiar spicules permeated the cytoplasm. These inclusions appeared in myriads of swirled threads, thick elongated angular plates, and needles, some of which exhibited longitudinal osmiophilic bands of 250 A thickness. These structures were associated with smooth endoplasmic reticulum. The cells appeared otherwise normal. Polarized light microscopy at 37 degrees C revealed a multiplicity of brilliant white inclusions between crossed polars in cells incubated with palmitic acid. These birefringent structures exhibited 90 degrees periodicity between both maximum brilliance and extinction, indicative of anisotropic crystalline deposits. Molecular species analysis of triglycerides in cells incubated with palmitic acid, together with data on [1-14C]palmitic acid incorporation, demonstrated an almost exclusive synthesis of tripalmitin. Spicules isolated from homogenized hepatocytes displayed needles containing longitudinal single and double osmiophilic bands of 110 A and 260 A thickness, respectively, and lipid spicular aggregates. The isolated spicules were almost pure tripalmitin by analysis. These observations document the formation and development of crystalline triglyceride in living cells and may provide a unique system for the study of cellular lipid synthesis, transport and deposition.

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 alpha 1-adrenergic inhibition (alpha 1-inhibition) 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 alpha 1-adrenergic inhibitor (alpha 1-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 alpha 1-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.

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 tetradecyglycidic 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 alpha 1-adrenoceptor activation suppresses hepatic triglyceride mobilization. The weak stimulatory effects of beta-adrenoceptor activation on hepatocyte triglyceride mobilization compared with the potent stimulation by DBcAMP is probably attributable to the relatively low level of beta-adrenoceptors in hepatocytes from the mature male rats used in these studies. Further characterization of a possible mutually antagonistic interaction between alpha 1- and beta 2-adrenoceptors in the regulation of hepatic triglyceride mobilization might be better accomplished in a species with hepatocytes that have a higher proportion of beta-adrenoceptors, e.g., guinea pig, rabbit, and dog.

Nocturnal regulation of free fatty acids in healthy young and elderly men.

To determine the effects of age on nocturnal fuel regulation, we measured spontaneous plasma glucose and free fatty acid (FFA) levels as well as counterregulatory hormones in healthy young (n = 9, mean age 26 +/- 3 years) and old (n = 10, mean age 69 +/- 3 years) men from midnight to 8 AM. FFA levels rose from midnight (660 +/- 80 mEq/L for young subjects, 545 +/- 55 mEq/L for old) to a peak mean level of 866 +/- 110 mEq/L at 3 AM in young and 713 +/- 120 mEq/L at 1:30 AM in old (P less than .05). FFA levels declined thereafter for both groups. FFA levels were lower in older subjects (P less than .05) but integrated glucose (P less than .05) and insulin (P less than .05) levels were higher. FFA levels were inversely related to integrated insulin (r = -0.46, P less than .05) and glucose concentrations (r = -0.66, P less than .05). Integrated insulin levels were significantly higher in older subjects, which may explain the lower FFA levels as may lower growth hormone levels in the older subjects. While fasting glucose responsivity to endogenous insulin is impaired in healthy older men, the FFA response appears to be preserved.

Effects of alpha 1-inhibition on lipid metabolism in the rat.

Prazosin or doxazosin, selective alpha 1-adrenergic receptor inhibitors used in the treatment of hypertension, are known from clinical studies to lower plasma lipids. When diets and dosing regimens are carefully controlled, both agents produce similar effects on plasma lipids in rats, i.e., decreases in triglycerides and in the non-high density lipoprotein (non-HDL) fraction of cholesterol. In order to conduct these studies, models of rats partially fasting (PF) and eating a high-sucrose diet were developed. The effects of prazosin 2.3 mg/kg/day and doxazosin 20 mg/kg/day on plasma levels of triglycerides, total and HDL cholesterol, free fatty acids (FFA), ketones, and other metabolites were measured in rats in the fed, fasting, or PF states, at various times after a meal, and with a high-cholesterol diet. The pattern of responses leads to the hypothesis that, under conditions of partial or complete fasting, alpha 1-adrenergic receptor inhibition can alter intrahepatic FFA metabolism, causing increased ketogenesis and diminished triglyceride synthesis, possibly through potentiation of beta-adrenergic or related pathways. If these concepts prove to be valid in humans, they suggest that factors such as the rate of very-low-density lipoprotein (VLDL) production or the state of sympathetic nervous activity may influence the changes in lipids induced by these agents.

Hypernatremia induces hyperlipemia and a fatty liver.

Hypernatremic states, often the result of hypothalamic osmoreceptor dysfunction in humans, are sometimes accompanied by hyperlipemia. To investigate whether hypernatremia could cause hyperlipemia we induced hypernatremia in three groups of rats with their respective controls: Group A rats received hypertonic saline alone intragastrically; group B animals were pair-fed and tap water was substituted for hypertonic saline in the treated group; in group C the rats were again fed intragastrically with a liquid diet mixed with hypertonic saline. Rats receiving excess salt had mean serum Na+ concentrations exceeding 159 mmoles/l. While the serum triglyceride values were significantly higher in all hypernatremic rats, hepatic triglyceride content was greater only in group C rats (p less than .01). Serum free fatty acids and ketone bodies were also higher in group C rats (p less than .01) as compared to controls. These data suggest that hypernatremia by itself leads to hyperlipemia and a fatty liver.

Short-term influences of dichloroacetate on genetically hyperlipemic rats.

The effects of short-term (7 days) administration of dichloroacetate (DCA) on carbohydrate and lipid metabolism in the Zucker obese and lean rat were investigated. Metabolic effects of the drug were more pronounced in the obese than in the lean rat. DCA decreased fasting blood glucose concentrations in both lean and obese rats, but more so in the fat animals, probably because of higher initial levels. The hypoglycemic action of DCA is likely attributable to a direct effect on liver and peripheral tissues and not to an indirect action caused by a decrease in the glucagon-to-insulin ratio because the drug induced just the opposite effect. DCA decreased plasma triglycerides (TG) and free fatty acids (FFA) in the hyperlipemic rats but not in lean rats. Intrahepatic triglyceride content diminished after drug treatment in fat rats, suggesting decreased hepatic TG synthesis. Hyperketonemia, induced in both lean and fat rats by DCA treatment, was also greater in the obese animal. This response was probably caused by accelerated hepatic ketone body production due to increased beta-oxidation, and not to enhance FFA substrate supply. These data demonstrate that DCA is capable of correcting many of the underlying abnormalities in carbohydrate and fat metabolism in the obese Zucker rat.

The metabolism of oleic acid by the perfused rat liver in experimental diabetes induced by antiinsulin serum.

The metabolism of varying quantities of oleic acid was examined in isolated perfused livers from normal fed rats and from animals made diabetic by pretreatment with guinea pig antiinsulin serum (AIS). The data presented reemphasize the fact that the quantity of free fatty acid (FFA) coming to the liver is a necessary, but not the most important, factor affecting the subsequent metabolism of the FFA. Rates of ketogenesis and output of triglyceride and the terminal concentration of hepatic triglyceride were proportional to uptake of FFA in certain concentration ranges. For equal rates of uptake of FFA, ketogenesis was greater, and the quantity of triglyceride secreted or accumulated within the liver was less, with livers from diabetic animals than with livers from normal animals. In confirmation of previous data, the liver was observed to have a maximal capacity to secrete triglyceride. Triglyceride accumulated in livers from normal-fed and diabetic animals only when uptake of FFA was more than sufficient to saturate the secretory process. Since proportionately more FFA was catabolized by livers from AIS treated animals, greater uptake of FFA was required to produce maximal rates of output of triglyceride and accumulation in livers from diabetic than from normal animals. Rates of ketogenesis by livers from normal fed animals increased minimally with increasing uptake of FFA (up to 1.0 mM free fatty acid). Even when uptake increased considerably with FFA concentrations of approximately 2.5 mM, rates of ketogenesis by livers from normal animals were less than half those of livers from diabetic rats, and maximal rates were not achieved by the normal controls. It is evident that changes in hepatic metabolism of FFA in the intact diabetic animal result from simultaneous alterations of supply of FFA and hormonally induced metabolic changes in the liver. Moreover, although hepatic secretion and accumulation of triglyceride is greater in isolated perfused livers from normal rats than from diabetic animals when the livers are exposed to equal quantities of FFA, the diabetic livers can accumulate more triglyceride, secrete more triglyceride, and oxidize more FFA to ketone bodies than can the normal under conditions in which considerably more substrate is available to the diabetic rather than to the normal livers. These differences might also be expected to occur in the acutely insulin deficient intact animal, in which changes in hormonal status and substrate (FFA) availability occur simultaneously, and might, in part, explain the ketonemia, hypertriglyceridemia, and hepatic steatosis often observed in vivo.

Effects of anti-insulin serum, insulin, and glucose on output of triglycerides and on ketogenesis by the perfused rat liver.

The rate of change of the concentration of various metabolites in blood in vivo and of the metbolism of free fatty acids by the perfused liver in vitro was sutidied as a function of time after the induction of acute insulin deficiency in rats by administration of guinea pig anti-insulin serum; the rate of reversal of these changes afte treatment of the anti-insulinserum diabetic ratss with insulin was also investigated. The concentrations of blood glucose and ketonebodies, and plasma-free fatty acids increased rapidly after injection of anti-insuli serum, while plasma triglycerides increased more slowly. These alterations were restored rapidly toward normal after treatment of the diabetic animals with insulin...