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Enzymatic activities of thymocytes isolated from Swiss albino mice were studied at various ages from immediately post weaning until 100 weeks of age, approaching the life expectancy of these animals. Between 5 and 10 weeks of age, the activities of lactate dehydrogenase and alkaline phosphatase decreased to a level that was maintained throughout the remainder of the aging profile. Neutral beta-glycerophosphatase (pH 7.5) activity in a thymus membrane preparation was similar in all age groups. The activity of membrane-bound 5'-nucleotidase, that is, AMP-hydrolyzing activity inhibited by 100 microM alpha, beta-methyleneadenosine 5'-diphosphate, progressively increased as a function of age, indicating thymocyte population changes occurring very late in life. In thymocytes of the oldest mice examined (100 weeks of age), 5'-nucleotidase specific activity was approximately ten-fold greater than the activity found in 5-week-old mice. Thus, membrane-bound 5'-nucleotidase activity in thymocytes increased markedly as a function of age in Swiss albino mice; yet several other enzymatic activities, including alkaline phosphatase, remained relatively unchanged in mature mice.

The consequence of aging on carbohydrate and lipid metabolism of the Swiss albino mouse was examined in a complete aging profile. The profile was developed by obtaining groups of at least 10 weanling mice at 2-3 month intervals for approximately a 2-year period. Body weight plateaued at approximately 10 months-of-age. Maximal heart and liver weights were found in 3-month-old mice. Plasma glucose concentration was highest in 1-month-old mice, declined rapidly during the first 3 months-of-age and then gradually throughout the profile. Liver glycogen content spiked sharply in 2-month-old mice, decreasing sharply to a level maintained in mature animals. A second peak in hepatic glycogen occurred in 15-month-old mice with a precipitous decrease taking place in older animals. Plasma concentrations of glycerol, 3-hydroxybutyrate and triacylglycerols, indices of lipid metabolism, were remarkably constant and showed no consistent trend with age. The level of triacylglycerols in the heart peaked in 2-month-old mice and then decreased gradually in older animals. The maximal content of triacylglycerols in the liver was found in 10- to 20-month-old mice. Hepatic levels of triacylglycerols decreased precipitously in senescent Swiss albino mice.

Efficiency of energy utilization was evaluated temporally in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 50 micrograms/kg)-treated male their pair-fed counterparts, and a group with ad libitum access to ground feed. TCDD-treated rats exhibited a progressive reduction in feed intake and body weight. The weight loss of vehicle-treated rats, pair-fed to the TCDD-treated group, was comparable to that found in rats receiving TCDD. Following treatment, rats administered TCDD were as efficient in absorbing feed energy from the gut as control rats. This was evidenced by similar relative relative digestible energy values in TCDD-treated rats, their pair-fed partners, and a group with ad libitum access to feed. Equivalent decreases in oxygen consumption and carbon dioxide production in TCDD-treated rats and their pair-fed counterparts, relative to rats with ad libitum access to feed, suggested that the decrease in both of these parameters in TCDD-treated rats was secondary to hypophagia and/or weight loss. Decline of respiratory quotient (RQ) to almost 0.7 in both TCDD-treated rats and their pair-fed counterparts is indicative of fat combustion. By Day 17 post-treatment, RQ increased significantly in the TCDD-treated and pair-fed groups possibly due to a limitation in the availability of lipid stores. Also, TCDD-treated rats and their pair-fed partners diminished their water intake to a similar extent without reducing urine output. Likewise, urinary excretion of both energy and urea was decreased to the same extent in rats treated with TCDD as it was in their pair-fed counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Obese mice (C57BL/6J ob/ob) and their lean littermates were studied at various ages from immediately post weaning until 62 weeks of age, at which mortality increased markedly. Several age-related changes were noted. 1) Plasma glucose levels were elevated in obese mice 5-20 weeks and 62 weeks of age, but were similar to those in the lean mice at 20-60 weeks of age. Plasma insulin levels were elevated in obese mice, and there were no age-related differences. 2) Brain serotonin was elevated in obese mice at all ages and increased with age in both obese and lean animals. 3) Pituitary contents of ACTH and beta-endorphin were elevated in young obese mice and increased further as these mice approached their life expectancy. 4) The ratios of ACTH to beta-endorphin immunoreactivities were similar in obese and lean mice, except in obese mice over 50 weeks of age where this ratio was increased. We conclude that: 1) the obese mouse is characterized by hyperinsulinemia and hyperadrenocorticism throughout its life; 2) the insulin resistance of the obese mouse improves at 20 weeks of age, yet deteriorates as its life expectancy is approached; 3) the obese mouse has an elevated brain serotonin content similar to previously described elevations of the putative neurotransmitters dopamine and norepinephrine in these mice; and 4) as the obese mouse approaches its life expectancy, abnormalities may occur in the synthesis, processing, or secretion of ACTH and/or beta-endorphine.

Indices of both carbohydrate and lipid metabolism were examined in obese (C57BL/6J ob/ob) mice and lean controls at 4-5 months of age in both the fed and fasted condition. Swiss albino mice (1.5-2 months in age), weight-matched to the inbred lean mice, were also investigated but differences in the metabolic parameters between these groups of mice were confounded by the dissimilarity in age. The concentrations of lactate and alanine in plasma were significantly higher in ob/ob mice than the lean controls in the fed state but this difference disappeared with fasting. Plasma levels of FFA and glycerol were similar in all three groups of mice in both the fed and fasted states. The significantly lower concentrations of plasma ketone bodies in ob/ob mice than the lean controls after a 24 h fast would suggest that the transition from the fed to fasted condition in the hepatic metabolism of the obese mouse does indeed occur more slowly. Yet after a 48 h fast, ob/ob mice had significantly higher levels of hepatic glycogen than comparably fasted lean control. Likewise, plasma glucose values decreased in ob/ob mice during food deprivation but remained significantly higher than those of the lean controls after 48 h fast. Excessive accumulation of triacylglycerols in liver and heart found in ob/ob mice in the fed condition was maintained during a fast of 48 h even though their level in plasma was significantly lower than in the comparably fasted lean control. We propose that an increase in the concentration of triacylglycerols in liver, heart and skeletal muscle may be an intracellular index of decreased insulin effectiveness in the genetically obese mouse.

Obese mice (C57BL/6J ob/ob) and their lean controls were studied longitudinally from immediately post-weaning until 62 wk of age, at which time the experiment was terminated. The dynamic nature of the metabolic aberrations of the obese mouse syndrome was clearly demonstrated. Obese mice were hyperinsulinemic at all ages yet the concentration of glucose in plasma was elevated only at 5-20 wk and 63 wk of age, but was similar to that of lean mice at 20-60 wk of age. Triacylglycerols accumulated in the liver of obese mice between 5 and 18 wk of age to a level that was 20-fold greater than that found in the age-matched lean control. A decreased concentration of DNA/g of liver was also found in 5-18 wk-old obese mice, indicative of an enlarged hepatocyte. With the exception of 5-wk-old animals, total DNA per liver was increased in obese mice when compared to the lean control throughout the profile. Following the peak in 18-wk-old mice, the hepatic content of triacylglycerols precipitously fell so that at 45 wk of age its concentration in obese mice was similar to that of the lean control. Plasma free fatty acid levels as well as liver glycogen content were comparable in obese mice and their lean controls throughout the profile. In obese mice older than 45 wk of age, the content of triacylglycerols in plasma was significantly lower than that of the age-matched lean control while an accumulation of liver triacylglycerols was again found in obese mice. Myocardial triacylglycerols were elevated in obese mice when compared to the lean control at all ages. The longitudinal metabolic profile of the obese mouse developed in the present study clearly demonstrates the dynamic nature of the deviations in carbohydrate and lipid metabolism in this animal model of human obesity and insulin resistance.

Genetically obese mice (C57BL/6J-ob/ob), fed ad libitum, demonstrated a precipitous increase in the spontaneous death rate after 50 weeks. The first signs of morbidity were a ruffled hair coat and a progressive motor ataxia. Necropsy revealed that obese mice had pale and fatty livers, urolithiasis and grossly distended bladders. Microscopically, the hepatocellular changes observed in all aged obese mice included: a loss of orientation of hepatocytes, an enormous variability in the size of both hepatocytes and their nuclei, and an extensive deposition of both large and small lipid droplets, confirmed by an increase content of triacylglycerols. A subacute-to-chronic, multifocal, necrotizing hepatitis was also present. Kidneys from aged obese mice contained hypertrophied glomeruli and increased PAS-stained material. Tubular dilation with compaction of the tubular cells was also seen. There were no significant alterations in the microanatomy or mineralization of femurs from obese mice, yet there was a significant increase in plasma alkaline phosphatase activity. In obese mice at 62-63 weeks of age, hyperglycemia was present even in spite of hyperinsulinemia. Pituitary immunoreactive ACTH and its molar ratio to pituitary immunoreactive beta-endorphin were also increased in obese mice at this age. Even though the etiology of the decreased lifespan of genetically obese mice remains uncertain, the possibility is discussed that an overall defect in the central nervous system may be involved.

Alkaline phosphatase activity in obese mice (C57BL/6J ob/ob) was significantly increased from 18 to 63 weeks of age when compared to that of their lean controls (C57BL/6J +/?). In 5 week old animals, the earliest age examined, the circulating activity of alkaline phosphatase was similar in both obese mice and their lean counterparts. To characterize the circulating alkaline phosphatase activity in the obese mouse and its lean counterpart, the response of the enzyme to fasting, various inhibitors, heat inactivation, and urea denaturation was examined and compared. L-homoarginine and L-p-bromotetramisole inhibited to a large extent the circulating activity of alkaline phosphatase in both obese mice and their lean controls in the fed state, while L-phenylalanine had essentially no effect. Even though the response of alkaline phosphatase in plasma to several inhibitors was similar, the rate of denaturation by urea of enzyme activity in plasma was significantly slower in obese mice than in their lean controls in the fed state. While the rate of inactivation of alkaline phosphatase activity in plasma for the initial two minutes at 56 degrees C was similar in obese mice and their lean counterparts, the subsequent rate of heat inactivation was significantly slower in the plasma from obese mice. Thus, both obese and lean mice in the fed state have a circulating activity of alkaline phosphatase in plasma with a greater contribution from a skeletal isoenzyme and a lesser one of intestinal origin.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of age and cellularity on lipolysis have been investigated in isolated epididymal fat cells from both Swiss albino mice and Sprague-Dawley rats. No significant lipolytic response to glucagon could be demonstrated with adipocytes from either young or old mice, while glycerol output was increased by this hormone with fat cells from young rats. Larger adipocytes from older mice showed significantly greater isoproterenol-stimulated lipolysis than those from younger animals if the glycerol output was expressed on a per cell basis. However, the lipolytic response per cell appeared to be equivalent in young and old rat adipocytes with either isoproterenol or ACTH-(1-24). In a complete aging study, relationships between body weight, epididymal fat pad weight and cellularity were examined covering the life span of the mouse. ACTH-(1-24)- and dibutyryl cyclic AMP-stimulated lipolysis increased with age and cell size but fell at senescence when adipocyte size diminished. Although an effect of aging per se cannot be ruled out with the experimental techniques used in the present study, a dominant influence of adipocyte size on the lipolytic process was demonstrated.

Liver protein kinase was determined in the absence and presence of cAMP4. Experimental alloxan diabetes resulted in a decrease in total protein kinase (+cAMP) and an increase in the activity ratio (-cAMP) divided by (+cAMP) in liver. Insulin treatment of diabetic rats reversed the observed changes in protein kinase in liver. Glucagon administered in vivo to normal rats caused an increase in the activity ratio and a decrease in total protein kinase activity in liver. The changes are similar to those in diabetes. A decrease in the ratio of insulin to glucagon in diabetes may account for the changes in protein kinase observed.

The activities of phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase were examined in liver as a function of age in Swiss albino mice. The hepatic activity of phosphofructokinase and total pyruvate dehydrogenase peaked in mice between 8 and 12 weeks of age and then decreased to a value that remained stable in mature animals older than 24 weeks of age. Yet, the activity of pyruvate kinase and pyruvate dehydrogenase in the active form in liver remained unchanged in mice up to 12 weeks of age. As mice matured, a progressive increase in the activity of both pyruvate kinase and the active form of pyruvate dehydrogenase in liver was observed while phosphofructokinase was unaltered. The pyruvate dehydrogenase complex, both total activity and the proportion of the enzyme in the active form, in the epididymal fat pad of the mouse showed no consistent age trend. The observed increase in the activity of both pyruvate kinase and the active form of pyruvate dehydrogenase should provide an augmented capacity for the generation of acetyl-CoA units for de novo fatty acid synthesis in livers of mature mice.

The purpose of this study was to compare the metabolism and antiketogenic properties of fructose, glyceraldehyde, and sorbitol. Fructose, glyceraldehyde, and sorbitol were readily metabolized and exhibited an antiketogenic effect in both blood and liver when injected intramuscularly to starved (forty-eight hours) rats. Sorbitol had the most pronounced antiketogenic effect and produced an 80 to 90 per cent decrease in the blood ketone bodies sixty minutes after administration. Fructose and glyceraldehyde were equally effective and produced about a 60 to 70 per cent decrease in ketone bodies. Fructose, glyceraldehyde, and sorbitol caused a significant decrease in the concentration of hepatic ketone bodies. In liver, sorbitol was found to be most effective in its antiketogenic action. The concentration of plasma free fatty acids remained unchanged after injection of all three antiketogenic substrates. Fructose, glyceraldehyde, or sorbitol caused increased blood lactate and pyruvate concentrations, and fructose was the most effective of the three substrates. Fructose administration resulted in a significant decrease in hepatic lactate/pyruvate and beta-OH-butyrate/acetoacetate concentration ratios, whereas sorbitol caused an increase in the concentration ratio of these two substrat pairs. Decreases in blood and liver ketone body levels were associated with lowering of liver acetyl-CoA concentration . However, the decrease in hepatic acetyl-CoA produced upon the administration of antiketogenic substrates was not pronounced. Sorbitol administration resulted in the most pronounced increase in hepatic alpha-glycerophosphate concentration. Fructose or glyceraldehyde also caused an increase in alpha-glycerophosphate content. Administration of each of the three antiketogenic substrates produced an increase in hepatic dihydroxyacetone phosphate concentration. All three antiketogenic compounds increased liver glycogen and blood glucose concentrations. No significant changes were observed in hepatic ATP, ADP, or AMP concentrations sixty minutes after the injections of any of the antiketogenic substrates. Although decreased liver acetyl-CoA levels were associated with the antiketogenic effects of the compounds tested, the increased liver alpha-glycerophosphate content best explains the differences between fructose or glyceraldehyde and sorbitol.

Effects of glucagon and ethanol on hepatic metabolism were examined with in situ perfused livers from fasted Swiss albino mice. In the absence of added gluconeogenic substrate, the presence of glucagon with ethanol effectively restored hepatic glucose production to a rate found with livers perfused with the hormone alone. When lactate was added to the perfusate, the presence of ethanol almost completely suppressed glucose formation but glucagon only partially overcame this inhibition. Ethanol and/or glucagon inhibited hepatic alpha-amino acid N release both in the absence and presence of added gluconeogenic substrate. Ketone body formation was increased three-fold when both glucagon and ethanol were added together to the medium of livers perfused in the absence of exogenous gluconeogenic precursor, yet neither agent alone had an effect. Ethanol utilization by the perfused liver was markedly inhibited by glucagon in the absence of added lactate. The presence of exogenous lactate also decreased ethanol removal by the liver and the addition of glucagon with lactate diminished ethanol clearance further. Thus, interrelationships between the consequences of glucagon and ethanol on hepatic metabolism have been delineated in the perfused mouse liver.

Swiss albino mice displayed the highest activity of alkaline phosphatase at 4-6 weeks with a precipitous decline by 18 weeks of age to a value seen in the mature animal. Circulating activity of alkaline phosphatase was significantly higher in the rat than the mouse in the fed state. With fasting, enzyme activity declined in the rat yet increased in the mouse. The net result was significantly higher plasma alkaline phosphatase activity in the mouse than the rat after the 48 hr fast. L-Phenylalanine inhibition of plasma alkaline phosphatase was greater in plasma from the rat than the mouse in the fed state. Yet in the fed condition, L-homoarginine and L-p-bromotetramisole inhibited alkaline phosphatase activity in plasma from mice to a greater extent than in rats. Heat inactivation as well as urea denaturation of alkaline phosphatase was significantly faster with plasma of the mouse than the rat in the fed state. Thus, it appears that the alkaline phosphatase isoenzyme of skeletal origin contributes a greater proportion of the circulating activity in the fed Swiss albino mouse than occurs in the Sprague-Dawley rat in which the intestinal isoenzyme plays a greater role in the fed condition.

Withdrawal of exogenous insulin and a subsequent fast (24 h) of alloxan diabetic rats stimulated rates of gluconeogenesis, urogenesis, ketogenesis, and amino acid release by in situ perfused livers when compared to those from normal, fasted rats. The contribution of liver glycogen to the high rates of gluconeogenesis observed with the diabetic liver could be excluded. Perfusate lactate concentrations remained constant during the period when the elevated rate of gluconeogenesis was observed with diabetic liver. Addition of insulin as a bolus (750 mU) and continuous infusion (12.5 mU/min) to the perfusion medium of diabetic livers resulted in constant perfusate levels of glucose, urea and alpha-amino nitrogen indicating a suppression of the catabolic processes present in the fasted, diabetic liver. The rate of ketogenesis was also slowed by insulin to about half the rate prior to addition of the hormone. These data indicate that insulin has an immediate anti-catabolic effect in the perfused, diabetic liver.

Kinetic and physical approaches have been employed to investigate the binding of acetoacetyl-CoA to hydroxymethylglutaryl-CoA synthase. The enzyme has an apparent Km for acetoacetyl-CoA (0.35 microM) which is more than an order of magnitude lower than the Ki (6--10 microM) measured for substrate inhibition by this metabolite. Hepatic acetoacetyl-CoA concentration, as measured by a sensitive and highly specific radioactive assay appears to be in the 1--10 microM range; the concentration decreases during diabetic ketoacidosis. Total hepatic activity of hydroxymethylglutaryl-CoA synthase and levels of mitochondrial enzyme protein, determined by radioimmunoassay, are not appreciably different in livers from control or ketoacidotic animals. In contrast to the decrease in hepatic acetoacetyl-CoA concentration observed during ketoacidosis, myocardial acetoacetyl-CoA levels are increased by at least tenfold when compared to controls. Elevated acetoacetyl-CoA levels may serve to inhibit fatty acid utilization by the heart. Thus, a consideration of the multiple interactions of acetoacetyl-CoA with the enzymes involved in ketone body production and utilization may be useful in evaluating the metabolic significance of this intermediate.

Alterations in lipid metabolism were examined in adult male Sprague-Dawley rats seven days after a single intraperitoneal injection of perfluorodecanoic acid (PFDA; 20, 40 or 80 mg/kg). Because PFDA treatment caused a dose-related reduction in feed intake, the response of vehicle-treated rats pair-fed to those receiving PFDA was monitored to distinguish direct effects of the perfluorinated fatty acid from those secondary to hypophagia. Carcass content of lipid phosphorus and free cholesterol decreased in dose-dependent fashion in both PFDA-treated and pair-fed rats. Carcass triacylglycerols diminished in a similar manner, yet PFDA-treated rats at each dose had a higher concentration of neutral acylglycerols than their vehicle-treated, pair-fed counterparts. In vehicle-treated, pair-fed rats at the 80 mg/kg dose level, lipid phosphorus and free cholesterol as a proportion of carcass fat increased, whereas the share of the triacylglycerols declined. Because of the higher concentration of triacylglycerols in the carcass of rats treated with 80 mg/kg PFDA, enrichment of lipid phosphorus and free cholesterol in carcass fat was less than in their pair-fed partners. The amount of lipid phosphorus and free cholesterol per hepatocyte was similar in both PFDA-treated rats and their pair-fed partners. Liver triacylglycerols were markedly increased in PFDA-treated rats. A similar but less extensive augmentary effect of PFDA on hepatic esterified cholesterol was found. Concentration of triacylglycerols in plasma was not elevated in PFDA-treated rats, in spite of hepatic accumulation of esterified compounds. Also, the plasma level of free fatty acids and 3-hydroxybutyrate was similar in all treatment groups, including those receiving PFDA.(ABSTRACT TRUNCATED AT 250 WORDS)

A sensitive micromethod for the colorimetric determination of free fatty acids (FFA) in plasma has been developed. FFA are extracted with chloroform-heptane-methanol with silicic acid being added to eliminate interfering phospholipids. Diphenylcarbazide containing diphenylcarbazone has been chosen as color-developing reagent, since the chromophore formed in the presence of FFA-Cu complex is stable and sensitive. Comparison of the proposed method and a non-routine reference procedure involving, among other steps, isolation of FFA on thin-layer chromatography would indicate that the efficiency of extraction of FFA in the proposed method was 90% that of the reference procedure. The proposed method offers the sensitivity, convenience, and accuracy needed for the determination of FFA in situations where the size of sample available is limiting, as with small rodents.

The functional thyroid status of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats is unknown. Therefore, activities of certain thyroid-responsive enzymes were examined in the livers of adult male Sprague-Dawley rats 1 week after treatment with TCDD (6.25, 25 or 100 micrograms/kg). Activity of the thyroid-responsive flavin L-glycerol-3-phosphate dehydrogenase (per mg mitochondrial protein) was decreased slightly in livers of TCDD-treated rats, while that of succinate dehydrogenase remained unchanged. In contrast, activities (per mg supernatant protein) of three thyroid-responsive NADP-dependent cytosolic enzymes, malic enzyme, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were increased by TCDD treatment in a dose-dependent manner. Lactate dehydrogenase (activity per mg supernatant protein) was also augmented slightly 1 week after TCDD administration. Liver mass was increased by TCDD treatment in a dose-dependent manner, but DNA content per liver was similar at all doses examined. Total hepatic protein, expressed per liver or mg hepatic DNA, was increased in TCDD-treated rats when compared to their pair-fed counterparts. The decreased activity of the mitochondrial L-glycerol-3-phosphate dehydrogenase, in contrast to the increased activities of the supernatant enzymes, malic enzyme, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, is not consistent with a shift in functional thyroid status following TCDD treatment.

Mechanical responses of isolated atria to (-)-isoproterenol and activities of myocardial pyruvate kinase and citrate synthase, enzymes involved in energy metabolism, were assessed in rats 7 days after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment. Basal tension development by electrically paced left atria was significantly elevated by all doses of TCDD (6.25, 25, or 100 micrograms/kg) when compared to that of vehicle-treated rats with unlimited access to feed. The basal rate of spontaneously beating right atria was significantly depressed in rats receiving 100 micrograms/kg TCDD. In left atria from rats treated with 100 micrograms/kg TCDD, maximal inotropic responses to (-)-isoproterenol and 1-methyl-3-isobutylxanthine were enhanced to the same degree. Right and left atria from rats receiving 100 micrograms/kg TCDD had an increased sensitivity to the chronotropic and inotropic effects of (-)-isoproterenol, respectively. The augmented atrial effects caused by TCDD were not secondary to loss of body weight because pair-fed animals that lost the same amount of weight did not display the responses. The ratio of heart ventricular mass to body weight and the activities of pyruvate kinase and citrate synthase in homogenates of heart ventricular muscle were not affected by TCDD treatment. Thus, overtly toxic doses of TCDD in the rat did not depress mechanical function of the heart.

Changes of the pyruvate dehydrogenase complex in liver and epididymal fat pad were examined longitudinally in obese mice (C57BL/6J-ob/ob) and their lean controls as a function of age. Total pyruvate dehydrogenase in liver was expressed on several reference bases because of differences in hepatic cellularity and protein content between obese mice and their age-matched lean controls. When total hepatic pyruvate dehydrogenase was expressed on a protein basis, the enzyme activity was elevated in obese mice older than 28 weeks in age when compared to lean controls of a similar age. However, when expressed on a DNA basis, total pyruvate dehydrogenase activity in livers of obese mice up to 10 weeks in age was increased when compared to the age-matched lean control. The proportion of hepatic pyruvate dehydrogenase in the active form was also augmented significantly in obese mice from 5 to 28 weeks of age. In 18-week-old obese mice, the proportion of total pyruvate dehydrogenase in the active form of adipose tissue was significantly higher than that of the lean controls. When expressed on a DNA basis, total pyruvate dehydrogenase in the fat pad was also increased in obese mice up to 10 weeks in age when compared to age-matched controls. Total pyruvate dehydrogenase activity in the epididymal fat pad was higher in obese mice than the lean controls in animals as old as 32 weeks in age when the enzyme activity was expressed per 100 g body weight. The increase in the active form and total activity of pyruvate dehydrogenase in both liver and epididymal fat pad during the dynamic early phase of obesity would augment the capacity for acetyl-coenzyme A formation necessary in the support of an accelerated lipogenesis and fat deposition.

Genetically obese hyperglycemic mice (ob/ob) were compared with their nonlittermate lean controls at 4-5 months of age with regard to brain serotonin, pituitary ACTH content, and circulating levels of glucose, glucagon, insulin, TSH, T3, T4, total tryptophan and free tryptophan. Brain serotonin pituitary ACTH content, and plasma insulin, glucose, total tryptophan, and free tryptophan were all significantly higher in obese mice than in the controls. TSH, T3, and T4 were not significantly different in obese mice vs. controls, suggesting that the obese mouse is euthyroid. Fasting improved but failed to normalize the glucose and insulin levels or insulin to glucagon ratios. Since serotonin is an important neurotransmitter with regard to hypothalamic-pituitary function and since its levels in the brain are dependent on the availability of tryptophan, the findings of elevated levels of free tryptophan in the plasma and serotonin in the brain of the obese hyperglycemic mouse may help to explain some of the previously observed abnormalities of pituitary hormone secretion in these animals.

A procedure for the extraction, separation, and isolation of perfluorodecanoic and perfluorooctanoic acids from biological samples is described. The use of conventional lipid extraction procedures leads to substantial loss of the perfluorinated fatty acids added to tissue. The presence of sulfuric acid in aqueous saline during phase partitioning is essential for the recovery of perfluorodecanoic and perfluorooctanoic acids in the organic phase following their extraction from tissue. The perfluorinated fatty acids are co-eluted with simple lipids from silica gel columns using diethyl ether/trifluoroacetic acid (100:1, v/v). Simple lipids are separated by thin layer chromatography. By substituting trifluoroacetic acid for acetic acid in the developing solvents, perfluorodecanoic and perfluorooctanoic acids migrate with other free fatty acids.

1. Swiss albino mice and Sprague-Dawley derived rats, 6-8 weeks in age were compared in the fed condition and after a fast of 24 and 48 hr. 2. Plasma glucose was significantly higher in mice than rats in the fed state but this difference disappeared after the 48 hr fast. 3. In the fed condition, plasma FFA and glycerol levels were significantly higher in mice than rats yet did not change during fasting. With starvation, there was a progressive increase in plasma FFA in the rat but no change in plasma glycerol. 4. The content of triacylglycerols in the plasma and liver was comparable in mice and rats in the fed state. Plasma triacylglycerols declined to a similar extent in both species during a fast of 48 hr. In mice, hepatic triacylglycerols increased markedly during fasting but declined in rats. 5. Heart triacylglycerols were higher in mice than rats in the fed state and increased still further during a fast of 48 hr during which time little change was observed in the rat. Myocardial glycogen was comparable in mice and rats in the fed state. With fasting, cardiac glycogen content decreased in the mouse but remained unchanged in the rat. 6. Distinct differences in certain of the parameters of carbohydrate and lipid metabolism were found with mice and rats in the fed and fasted condition.

The role of ketone bodies and long-chain fatty acids in regulating myocardial content of acetoacetyl-CoA was examined in isolated perfused hearts from fed rats. Addition of 10 mM acetoacetate to a perfusion medium containing 5.5 mM glucose as exogenous substrate resulted in a sevenfold increase of cardiac acetoacetyl-CoA content. Inclusion of 0.75--0.94 mM palmitate in the perfusate did not augment the basal level of acetoacetyl-CoA nor did it influence the already elevated content of the metabolic intermediate shown to exist in the presence of acetoacetate alone. In addition to the observed increase in myocardial acetoacetyl-CoA, a decrease in free carnitine in hearts perfused with acetoacetate was accompanied by elevated ratios of both acetylcarnitine/carnitine and long-chain fatty acylcarnitine/carnitine. Addition of palmitate in the presence of acetoacetate resulted in a further diminution of myocardial free carnitine leading to a further elevation of the long-chain fatty acylcarnitine/carnitine ratio when compared to hearts perfused with the ketone body alone. The changes in myocardial CoA and carnitine and their intermediates in hearts perfused with acetoacetate either alone or in combination with palmitate indicate that ketone bodies can divert fatty acids away from oxidation and toward esterification as triacylglycerols. Even though the interrelationships between ketones and myocardial fatty acid metabolism have not been fully characterized, a role for ketone bodies in promoting the increase in myocardial acetoacetyl-CoA found in diabetic ketoacidosis has been clearly defined.

Perfluorodecanoic acid (PFDA) alters the circulating level of thyroid hormones, but the physiological significance of this change at the target tissue remains to be defined. To this end, the activities of thyroid-responsive hepatic enzymes were examined in adult male rats 1 week after treatment with a single dose of PFDA (20, 40 or 80 mg/kg). Since PFDA treatment caused a dose-related reduction in feed intake, vehicle-treated rats pair-fed to their counterparts receiving PFDA were used to determine if any of the PFDA-induced alterations in enzyme activity were secondary to hypophagia. Following the administration of PFDA, L-glycerol-3-phosphate dehydrogenase, a liver mitochondrial enzyme sensitive to thyroid status, exhibited a modest increase in activity, whereas that of succinate dehydrogenase, a constitutive mitochondrial marker enzyme, was similar in both PFDA-treated rats and their pair-fed counterparts at all dose levels examined. Activity of cytosolic lactate dehydrogenase was also augmented modestly in livers of rats receiving PFDA. In contrast, activity of cytosolic malic enzyme, a thyroid-responsive enzyme, was increased markedly in PFDA-treated rats. Hepatic activity of glucose-6-phosphate dehydrogenase, which also responds to alterations in thyroid status, exhibited a modest increase with 20 and 40 mg/kg PFDA but was similar in both PFDA-treated rats and their pair-fed counterparts at the 80 mg/kg dose level. Absolute and relative liver mass was elevated in PFDA-treated rats at all dose levels in comparison to the appropriate vehicle-treated pair-fed animals. Total hepatic content of DNA was maintained in PFDA-treated rats at all dose levels, whereas a significant decrease in liver DNA was found in the vehicle-treated rats pair-fed to animals receiving 80 mg/kg PFDA. Following administration of PFDA, protein content per total liver was similar to that of their pair-fed counterparts. Thus, the pattern of activity of thyroid-responsive hepatic enzymes was not compatible with a functional shift toward a lessened thyroid status in rats treated with PFDA.

Activities of key enzymes in hepatic glucose utilization were compared between obese (C57BL/6J ob/ob) mice, their lean controls and outbred Swiss albino mice in the fed condition and during fasting. As liver hyperplasia and hepatocyte hypertrophy were present in the ob/ob mice at 4-5 months of age and changes in hepatic cellularity did occur with fasting, enzyme activity was expressed on the basis of protein, DNA, and wet weight. In the fed state, activities of glucokinase + hexokinase (glucose phosphorylating capability), phosphofructokinase and pyruvate kinase were significantly greater in livers of ob/ob mice when compared to those of the lean control. Glucokinase + hexokinase activities in livers of ob/ob mice remained significantly higher throughout the 48 h fast yet the activities of hepatic phosphofructokinase and pyruvate kinase, when expressed per g wet wt or mg protein, decreased so that a statistical difference from the fasted lean control was no longer detected. When expressed per 100 g body weight, hepatic glucokinase + hexokinase as well as phosphofructokinase and pyruvate kinase activities in obese mice were higher both in the fed and fasted states when compared to lean controls in the comparable nutritional condition. This increased capacity of key enzyme activities in hepatic glucose utilization can be attributed to liver hyperplasia found in ob/ob mice in both the fed and fasted condition. While higher hepatic glucose phosphorylating capability was maintained during fasting, the elevated specific activities of hepatic phosphofructokinase and pyruvate kinase in the obese mouse in the fed state decreased with starvation to values found in the lean control.

The lipolytic response of isolated adipocytes from genetic obese (C57/BL/64 ob/ob) and lean (C57BL/6J +/?) mice to ACTH-(1-24), isoproterenol and glucagon has been studied. The mean cell idameter of adipocytes form ob/ob mice was approximately twice that of lean controls. The adipocytes from obese mice contained on the average approximately six times the amount of triacylglycerol present in the smaller lean mouse adipocyte. Lipolysis was calculated both on a per cell basis (10(5) cells) and per mu mole of triacylglycerol and when expressed on a cell number basis, the larger adipocytes from obese mice showed an ACTH-(1-24) stimulated glycerol release which was quantitatively similar to that of smaller adipocytes from lean mice. When expressed per mu mole of triacylglycerol, the smaller cells from lean animals appeared to be dramatically more responsive to either isoproterenol or ACTH-(1-24). On either basis, ACTH-(1-24) stimulated glycerol release from obese mouse cells was greater than the isoproterenol response. The obese mouse of adipocyte showed selective loss of response to isoproterenol compared to its lean control.

Energy metabolism and body composition were investigated in perfluorodecanoic acid (PFDA)-treated rats (single i.p. dose of 20, 40 or 80 mg/kg) and their respective pair-fed counterparts 7 days after dosing. Cumulative feed intake and body weight were decreased in a dose-dependent manner. However, PFDA-treated rats either gained less weight or lost more weight (dependent on the dose administered) than their pair-fed, vehicle-treated counterparts, even though feed intake in these two treatment groups was similar. Energy expenditure, determined indirectly by quantifying oxygen consumption and carbon dioxide production, decreased in a dose-dependent fashion, yet it was similar in PFDA-treated rats and their pair-fed counterparts at a given dose. Body composition analysis indicated a dose-dependent decrease in carcass water and protein content in both PFDA-treated rats and pair-fed partners, while total amount of ash remained unchanged in all treatment groups. These alterations in body composition are compatible with a negative energy balance. Even though PFDA-treated rats had a lower body weight than their pair-fed counterparts at each dose level, they were found to have a greater carcass fat content. Thus, at maintenance (i.e., zero change of body weight) PFDA-treated rats require a higher caloric intake associated with a greater body fat content than vehicle-treated animals.

Changes in the cyclic AMP phosphodiesterase of the thymus were observed during leukemogenesis in the AKR mouse. Upon maturation, activity at 1.0 muM and 100 muM cyclic AMP concentrations decreased in both the AKR mouse and normal Swiss mice. The thymus of the leukemic mouse had sharply higher phosphodiesterase activity. Much of the phosphodiesterase activity of normal and leukemic mice was associated with particulate fractions, and, as indicated by marker enzymes for particulate components, the high affinity phosphodiesterase was enriched in a fraction containing plasma membranes.

Perfluorodecanoic acid (PFDA) administration to adult male rats increased both the activity of hepatic malic enzyme and liver weight in a dose-dependent manner. Hepatomegaly and augmented activity of malic enzyme in liver were apparent within one day following PFDA administration and reached a plateau by three days posttreatment. Malic enzyme quantity per liver in PFDA-treated rats was elevated within one day following dosing and increased continually throughout five days posttreatment. Administration of PFDA to rats in the fed state also led to an increase in the specific activity of hepatic malic enzyme that peaked at three days following dosing. When compared to the fed condition, rats fasted for 48 hours had a decrease in both relative liver weight and the quantity of supernatant protein per liver. The total activity (U/liver) and specific activity of malic enzyme in the liver were also reduced in the fasted state. During the 24 hours after treatment in rats fasted for 48 hours, the body weight as well as the absolute and relative liver weight of animals receiving vehicle declined continuously in the absence of feed. Following the administration of PFDA to fasted rats, body weight was maintained until eight hours posttreatment but then declined at a rate similar to that found with the vehicle-treated group. Absolute and relative liver weight in PFDA-treated rats were increased significantly at eight hours posttreatment when compared to those receiving vehicle, and this increment was maintained throughout the rest of the 24 hours following dosing. While the activity and enzyme content of hepatic malic enzyme decreased in the vehicle-treated group, administration of PFDA to rats fasted for 48 hours prevented their decline. The specific activity of hepatic malic enzyme in 48 hours fasted rats receiving PFDA was also elevated significantly at 16 hours posttreatment. Thus, the administration of PFDA to the adult male rat in both the fed and fasted nutritional states was found to regulate hepatic malic enzyme by not only increasing enzyme quantity but also by augmenting the specific activity, (ie, catalytic state) of the enzyme.

Changes in body weight, feed intake, hepatic cellularity, and intermediary metabolism were assessed in the mature male (450 g) rat following 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administration. All animals were schedule-fed (8-hr feeding period/24 hr) and treated with a single oral dose of either TCDD (75 micrograms/kg) or vehicle. Blood and tissues were sampled 16 to 18 hr following the end of the feeding period on 2, 4, 6, and 8 days post-treatment. Mature rats treated with TCDD exhibited a slight but progressive reduction in both body weight and feed intake throughout the 8-day experimental period. An increase in liver mass that was apparent at 2 days and plateaued by 4 days after TCDD treatment was associated with a decrease in the concentration of DNA per gram of wet liver. However, the total liver content of DNA in TCDD-treated rats remained similar to pair-fed animals. Thus, TCDD treatment produced liver enlargement in the mature rat that was the result of hepatocellular hypertrophy and not an increase in cell number. Hepatic glycogen content in TCDD-treated rats was threefold higher than their pair-fed counterparts at 2 to 6 days post-treatment, and this augmentation would account, in part, for the hypertrophy of the liver cell found after administration of TCDD. Plasma glucose and lactate concentrations were similar in TCDD-treated and pair-fed rats, suggesting that the Cori cycle remained unaltered following TCDD administration. Likewise, heart and gastrocnemius glycogen concentrations were similar in all experimental groups. Urinary excretion of urea, ammonia, and creatinine was comparable in TCDD-treated rats and their pair-fed counterparts, indicative of a nitrogen balance that was not disturbed by TCDD. Plasma glutamine concentrations in TCDD-treated rats tended to be reduced and were significantly lower at Day 6 post-treatment when compared to those of pair-fed counterparts, suggestive that amino acid release from muscle was not enhanced in TCDD-treated rats. Likewise, plasma concentrations of branched-chain amino acids, which are metabolized to a large extent in muscle, tended to be lower on Day 6 following TCDD treatment. Yet at Day 6 post-treatment, the circulating concentrations of amino acids that are metabolized by the liver were elevated in TCDD-treated animals. TCDD administration also resulted in an increase in total hepatic protein concentration which was evident at 4 days and increased progressively at 6 and 8 days post-treatment. Liver content of phospholipids also increased gradually following administration of TCDD.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of nicotinic acid on regional myocardial blood flow, percentage of segment shortening and myocardial uptake of free-fatty acids during a 15-min occlusion of the left anterior descending coronary artery and 3-hr reperfusion period was compared to a saline-treated control group. Nicotinic acid (2.4 mumol/kg/min i.v.) was infused 30 min before and throughout the occlusion period. Heart rate, arterial blood pressure and left ventricular systolic and end diastolic pressures were not different during occlusion and reperfusion in the nicotinic acid or saline-treated groups. However, left ventricular dP/dt, an index of global myocardial function and percentage of segment shortening in the ischemic region were greater during occlusion and reperfusion after nicotinic acid. Even though myocardial blood flow was unaltered in the normal or ischemic region during nicotinic acid infusion, subendocardial blood flow during reperfusion was enhanced significantly when compared to the control group. Nicotinic acid also decreased free-fatty acid uptake by the heart during occlusion which returned gradually to the pretreatment control during 3 hr of reperfusion. Thus, the improvement in percentage of segment shortening, dP/dt and subendocardial blood flow during reperfusion may be related to the ability of nicotinic acid to reduce free-fatty acid uptake by the heart during coronary occlusion.

Hypertrophy of liver cells, as measured by decreased mg DNA per g wet liver, from obese mice (C57BL/6J ob/ob) was associated with a marked increase in triacylglycerol content per g wet liver when compared with control lean mice (C57BL/6J ?/+). The difference between the rates of net glucose production from lactate in isolated perfused livers from obese and lean mice was small when expressed per g wet liver. However, calculation of these rates either on a DNA or body weight basis indicated that the rate of gluconeogenesis in livers from obese mice was twice that of lean mice. Using perfused livers from lean mice, the ratio of net uptake of C3-substrate to gluconeogenesis indicated that a major proportion of lactate disappearing from the perfusion medium could not be accounted for in the appearance of glucose; but using the perfused livers of obese mice, almost all of the net lactate disappearance could be accounted for by a net increase in glucose production.

Chronic treatment of rats with dithiobiuret (DTB) produces a delayed onset muscle weakness and, as suggested by a preliminary study, a distal axonopathy. An inhibition of glycolysis resulting in an energy deficit has been suggested as a possible mechanism of neurotoxin-induced distal axonopathies. To determine whether chronic DTB administration (1 mg/kg/day X 7 days; sacrificed on Day 7) might be associated with a perturbation of glucose metabolism in neuronal tissues, lactate production was measured in brain and spinal cord homogenates prepared from rats which exhibited hindlimb skeletal muscle weakness. Kinetic and statistical analysis showed that glucose-dependent lactate production in these homogenates was not different from that of controls. Lactate production was also determined in brain homogenates prepared from rats intoxicated with acrylamide (50 mg/kg/day X 9 days; sacrificed on Day 9), p-bromophenylacetylurea (200 mg/kg/day X 2 days; sacrificed on Day 14) or 2,5-hexanedione (400 mg/kg/day X 21 days; sacrificed on Day 21). The results indicated that whereas 2,5-hexanedione produced a slight decrease in Vmax, acrylamide and p-bromophenylacetylurea did not cause changes in glucose-dependent lactate production. These findings do not support the suggestion that an inhibition of glycolysis represents a common biochemical lesion associated with neurotoxins which cause delayed onset muscle weakness and distal axonopathy.