Biomedical subjects

R A Freedland

Publications and source records attributed to R A Freedland.

At least 19 recordsLinked to original sources

PubMed1996

Threonine is catabolized by L-threonine 3-dehydrogenase and threonine dehydratase in hepatocytes from domestic cats (Felis domestica).

Isolated hepatocytes were used to study threonine catabolism in kittens, and dietary threonine and crude protein were varied to study enzyme adaptation. Cells were isolated from 21-wk-old kittens which had been fed diets containing threonine at 4 or 8 g/kg of diet with either 200 or 500 g crude protein/kg of diet (2 x 2 factorial, n = 4/group). Production of CO2, glucose and various metabolites from [U-14C]threonine were measured. Inclusion of 10 mmol/L glycine, or glycine in combination with 10 mmol/L acetaldehyde +ethanol, in the incubation medium decreased formation of 14CO2 and [14C]glucose. At the same time, large amounts of [14C]glycine but no [14C]ethanol was formed. Inclusion of 10 mmol/L 2-ketobutyrate + 2-hydroxybutyrate decreased 14CO2 but not [14C]glucose production and resulted in the formation of [14C]2-hydroxybutyrate. Under all incubation conditions, 14CO2 and [14C]glucose production changed in response to alterations in dietary protein but not dietary threonine. It appears that threonine dehydratase and L-threonine 3-dehydrogenase, but not threonine aldolase, are active pathways for threonine metabolism in cats, and both enzymes are sensitive to levels of dietary protein.

Acetaldehyde↗

PubMed1993

Effects of estrogen on gluconeogenesis and related parameters in male rainbow trout.

To investigate the effects of estrogen, the hormone responsible for vitellogenesis, on gluconeogenesis, male rainbow trout were implanted with 17 beta-estradiol or given a sham procedure. Plasma glucose concentration in estrogenized fish was 50% of the control fish (6.4 mM). Glucose synthesis from physiological concentrations of alanine was 0.08 mumol.g cells-1 x h-1 compared with 0.20 mumol.g cells-1 x h-1 in control fish; synthesis from physiological concentrations of lactate was reduced by over 50% (0.88 vs. 0.36 mumol.g cells-1 x h-1) in implanted fish. Gluconeogenesis from 5 mM lactate was also significantly depressed in implanted fish. Oxidation of alanine, serine, and lactate was not significantly affected by estrogen implantation. The maximum clearance velocity of a key enzyme negatively regulating gluconeogenesis, pyruvate kinase, was 3.03 mumol.g cells-1 x h-1 in estrogen (E2) implanted fish compared with 7.83 mumol.g cells-1 x h-1 in control fish. No significant differences in plasma insulin or glucagon were found in the two groups. We conclude that estrogen depresses gluconeogenesis and that this reduction contributes to the lower plasma glucose concentration seen in vitellogenic trout.

Animals↗

PubMed1992

Bilirubin production and conjugation from newly formed heme in isolated rat hepatocytes.

1. Heme synthesis from delta-aminolevulinic acid (delta-ALA) in freshly isolated rat hepatocytes was maximal at 100 microM with a rate of approx. 7 nmol being synthesized per g wet weight cells. 2. Approximately 8% of synthesized heme was converted to bilirubin and 50% of the newly synthesized bilirubin was conjugated. 3. The ratio of di to monoconjugate was approx. 2.5. Incorporation of delta-ALA into bilirubin was increased by additional delta-ALA, heme and was also doubled in cells isolated from animals treated with CoCl2. 4. Bilirubin formation was inhibited approx. 90% by in vitro treatment with heme oxygenase inhibitors zinc and tin protoporphyrin.

Aminolevulinic Acid↗

PubMed1992

Bile pigments in gallbladder and freshly-secreted hepatic duct bile from fed and fasted rainbow trout, Oncorhynchus mykiss.

1. Chromatographic analyses of bile pigments in rainbow trout reveal the presence of primarily unconjugated biliverdin (BV) and bilirubin (BR) glycosyl conjugates. Only trace amounts of unconjugated BR are present in hepatic duct (HD) bile: no beta-glucuronidase activity is detectable. 2. The per cent of BV and BR in HD and gallbladder biles is similar in fasted trout; however, the per cent of BV is significantly increased in HD bile from fed fish. 3. Fasting decreases the rate of choleresis but does not alter the excretory rate of endogenous BV or BR. 4. Erythrocyte life span is estimated to be approximately 500 days.

Animals↗

PubMed1992

Effects of estrogen on whole animal and tissue glucose use in female and male rainbow trout.

Reports of changes in carbohydrate metabolism during vitellogenesis in fish prompted an investigation of the effects of estrogen on glucose utilization in rainbow trout. Estrogen pellets were implanted in both female and male fish, and a third group of male fish was given a sham operation. After cannulation of the dorsal aorta, D-[1-3H]glucose and 2-deoxy-D-[U-14C]glucose were injected into the fish to observe whole animal and tissue glucose use. We found that estrogen does not affect glucose turnover rate or transit time but causes a decrease in plasma glucose concentration and size of the glucose mixing pool. Adipose tissue in female fish utilized glucose at a higher rate than sham fish. Ovarian tissue used more glucose per kilogram of body weight than the testes of the male fish. Regardless of treatment, brain had the highest rate of glucose consumption per gram of tissue, followed by gonads and red blood cells. Muscle and adipose tissue utilized only small amounts (< 1 nmol.g tissue-1.min-1) of glucose. We conclude that an increase in the rate of whole body glucose use is not responsible for the fall in plasma glucose caused by estrogen and seen during vitellogenesis.

Animals↗

PubMed1992

Fasting hyperbilirubinemia in normal squirrel monkeys.

The plasma of Bolivian squirrel monkeys, unlike that of Brazilian squirrel monkeys, is markedly yellow due to unconjugated hyperbilirubinemia after an overnight fast. The fasting hyperbilirubinemia in Bolivian squirrel monkeys is likely due to two mechanisms. First, a twofold increase in the bilirubin turnover/production rate occurs during a 24-hour fast. A second mechanism is the decreased hepatic conjugation potential for bilirubin due to the presence of a higher bilirubin UDP-glucuronosyltransferase UDPGAKm and a lower Vm; this results in higher steady-state plasma and hepatic bilirubin levels during a fast when hepatic UDP-glucuronic acid levels are low. The Bolivian squirrel monkey provides an excellent animal model for human Gilbert's syndrome type I in which to study rate-limiting mechanisms in the movement of bilirubin from plasma to bile.

Animals↗

PubMed1991

Kinetic properties of bilirubin UDP-glucuronyltransferase in squirrel monkeys exhibiting fasting hyperbilirubinemia.

1. Bolivian squirrel monkeys (BoSM), unlike Brazilian squirrel monkeys (BrSM), exhibit a marked fasting hyperbilirubinemia (FH) and serve as animal models for Gilbert's syndrome type I. 2. Compared to BrSM, BoSM possess a higher apparent UDPGAKm (0.51 vs 0.29 mM) and lower Vm (0.36 vs 0.48 nmol BR conjugated/min per mg microsomal protein) for hepatic bilirubin (BR) UDP-glucuronyl-transferase (BR UDPG-T). 3. Lineweaver-Burk plots are linear and obey Michaelis-Menten kinetics when UDP-acetylglucosamine is used as activator and UDPGA substrate concentrations are within the physiologic range present in the liver during the fed and fasted state (0.10-0.71 mM); above these concentrations, there is a discontinuity of kinetic plots as noted in other species. 4. There is no effect of fasting on the Km of BR conjugation (i.e. sum of mono- and diglucuronides) in either monkey; however, fasting is associated with lower Vm values (15-20%) in each subspecies. 5. By calculating the potential BR flux (nmol BR conjugated/min per kg) using known hepatic UDPGA concentrations, liver weights and in vitro Km and Vm, a markedly lower BR flux is observed in BoSM (58.4 nmol/min per kg) than in BrSM (91.6 nmol/min per kg). 6. Significantly higher apparent UDPGAKm and lower Vm of BR UDPG-T for conjugation of BR to BR monoglucuronide appears responsible in part for the four- to five-fold elevations in unconjugated BR in the liver and plasma in the fasted BoSM.

Animals↗

PubMed1991

Time course changes in glycogen accretion, 6-phosphogluconate, fructose-2,6-bisphosphate, and lipogenesis upon refeeding a high sucrose diet to starved rats.

1. Starved rats refed 60% sucrose diets were used to determine in vivo lipogenesis and levels of hepatic metabolites. 2. Fatty acid synthesis increased 11-fold 4 hr after refeeding. 3. Glycogen rose from 3 to 100 mg/g liver after 8 hr. 4. Fructose-2,6-bisphosphate rose to 6 nmol/g at 1 hr and remained constant. 5. 6-Phosphogluconate increased from 10 to 45 nmol/g liver after 2 hr and remained constant.

Animals↗

PubMed1991

Effects of dietary fibers on nonfasting plasma lipoprotein and apolipoprotein levels in rats.

This study was undertaken to determine whether selected dietary fibers had an effect on plasma lipoproteins, apolipoproteins and enzymes involved in cholesterol metabolism in rats. Each experimental diet contained 8% dietary fiber by weight; all animals were killed in a nonfasted state. After 4 wk, final body weight and liver cholesterol were similar in fiber-free controls and in rats fed diets containing cellulose, pectin, oat bran or wheat bran. Pectin-fed animals has significantly lower plasma cholesterol, HDL-cholesterol and apolipoprotein A-I levels, and exhibited significantly higher hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity than did the fiber-free control group. In addition, plasma triglyceride concentrations were lowest in pectin-fed animals. These multiple effects on lipid metabolism were not observed when oat bran, containing one-third soluble fiber, was used. Although total plasma cholesterol levels in wheat bran-fed animals were not different from those in the fiber-free controls or the cellulose-oat bran-fed animals, the LDL cholesterol level was significantly higher than in fiber-free controls or pectin-fed animals. This study demonstrate that dietary fibers included in the diet of rats are able to alter nonfasting lipoprotein cholesterol and apolipoproteins and that pectin, a soluble fiber, was most effective in lowering plasma cholesterol levels.

Animals↗

PubMed1991

The effect of pyruvate or dihydroxyacetone on parenterally induced liver lipid accumulation in the rat.

Orally fed pyruvate (pyr) and dihydroxyacetone (DHA) have been shown to decrease liver lipid accumulation in animal models. These compounds lessen the degree of fatty liver in ethanol-fed rats and in a genetic strain of hens predisposed to fatty liver. Total parenteral nutrition can result in liver dysfunction, including fatty infiltration of the liver. In this study, rats were assigned to either control, pyr, or DHA groups. All rats were fitted with jugular vein catheters, and following a 3-day recovery, were infused continuously for 7 days. The infusate provided adequate nutrition (including 7% kcal as fat) with 5% pyr or 5% DHA (g/liter) substituted for dextrose in the experimental groups. Plasma triglycerides were lower in the pyr groups relative to controls: 62.2 +/- 34.7 (SE) vs 96.8 +/- 44.3 mg/dl, though this was significant only at P less than 0.10. Neither pyr nor DHA decreased liver lipids. Pyr and DHA were administered intravenously in this study, and therefore passed through the heart and to peripheral tissues first. These compounds may need to be fed orally, passing via the portal system, to produce the liver lipid-lowering effects seen in other studies.

Animals↗

PubMed1990

Effect of 6-aminonicotinamide on pentose cycle activity in isolated hepatocytes.

1. 6-aminonicotinamide (6AN), a purported inhibitor of 6-phosphogluconate (6PG) dehydrogenase, has been regarded as an inhibitor of the pentose cycle. 2. Incubation of isolated hepatocytes with 6AN caused a time- and concentration-dependent accumulation of 6PG. 3. At 5 mM, 6AN increased the 6PG level 1000-times to values comparable to those observed in the livers of rats injected with this niacin antagonist. 4. Despite the accumulation of 6PG, neither the total rate of lipogenesis, nor the incorporation of radioactivity from [3-3H]glucose, used to estimate the activity of the pentose cycle, were impaired to a large extent. 5. The evidence presented suggests that the accumulation of 6PG is not a sufficient criterion to establish blockade of the pentose cycle.

6-Aminonicotinamide↗

PubMed1990

Effect of diabetes and time after in vivo insulin administration on ketogenesis and gluconeogenesis in isolated rat hepatocytes.

1. Diabetic rats had elevated rates of gluconeogenesis and ketone body production. 2. Four hours after insulin administration the rate of gluconeogenesis returned to normal and the rate of ketogenesis decreased with certain substrates, but did not return to normal. 3. Eight hours after insulin treatment ketogenesis from all substrates was significantly reduced, but still significantly higher than normal.

3-Hydroxybutyric Acid↗

PubMed1990

Contribution of several amino acids and lactate to gluconeogenesis in hepatocytes isolated from rats fed various diets.

The contribution under various nutritional regimens of several amino acids and lactate to gluconeogenesis was estimated by measuring the glucose formation from 14C-labeled substrates. Isolated rat hepatocytes were incubated for 60 min in a Krebs-Ringer bicarbonate buffer pH 7.4 containing lactate, pyruvate and all the amino acids at concentrations similar to their physiological levels found in rat plasma, with one precursor labeled in each flask. In all conditions, lactate was the major glucose precursor, providing over 60% of the glucose formed. Glutamine and alanine were the major amino acid precursors of glucose, contributing 9.8% and 10.6% of the glucose formed, respectively, in hepatocytes isolated from starved rats. Serine, glycine and threonine also contributed to gluconeogenesis in the starved liver cells at 2.6, 2.1 and 3.8%, respectively, of the glucose formed. The rate of glucose formation from the isolated hepatocytes of the starved rats and those fed either high protein or high fat was higher than that from rats fed a nonpurified diet.

Amino Acids↗

PubMed1990

Effects of propionate on lipid biosynthesis in isolated rat hepatocytes.

The effects of propionate, a product of intestinal fiber fermentation, on fatty acid and sterol synthesis were studied in isolated rat hepatocytes. Fatty acid synthesis, as measured by tritium incorporation from 3H2O, was inhibited in the presence of 1 mmol/L propionate with no substrate additions or additions of acetate, butyrate, lactate or oleate. Incorporation of [1-14C]acetate into fatty acids was also inhibited in the presence of propionate. Although propionate markedly depressed [1-14C]acetate incorporation into sterols in hepatocyte preparations, tritium incorporation from 3H2O into sterols was not inhibited, indicating that overall sterol synthesis was not affected. Thus, in vitro, the effect of propionate on lipid metabolism is apparently limited to inhibition of de novo fatty acid synthesis.

Acetates↗

PubMed1990

The effects of dietary fiber feeding on cholesterol metabolism in rats.

The flux through the sterol biosynthetic pathway was studied in hepatocytes isolated from male Sprague-Dawley rats fed diets containing one of four fiber sources: cellulose, pectin, oat bran and wheat bran. Sterol synthesis measured by the incorporation of tritiated water or [2-14C]mevalonic acid was not inhibited in hepatocytes isolated from animals fed diets containing cellulose, pectin, oat bran or wheat bran when compared to animals fed a fiber-free diet. Based on these results, it is concluded that the intake of fiber has no inhibitory effect on endogenous sterol synthesis. In fact, in comparison to that in fiber-free controls, sterol synthesis was markedly elevated in pectin- and wheat bran-fed animals. In the case of the pectin-treated animals, the higher synthetic rate corresponded to an increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

Animals↗

PubMed1989

Effect of glycerol and dihydroxyacetone on hepatic lipogenesis.

Glycerol is a dietary component which is metabolized primarily by the liver and kidney where it is used mainly for glucose synthesis. The metabolism of glycerol is very similar to that of dihydroxyacetone which can be considered its more oxidized counterpart. The effects of these substrates on hepatic lipogenesis and gluconeogenesis were examined. In isolated hepatocytes, 10 mM dihydroxyacetone caused a large increase in glucose output and stimulated lipogenesis without affecting the lactate/pyruvate ratio or the total ATP content of the cells. (As compared to dihydroxyacetone, 10 mM glycerol was less effective as a gluconeogenic substrate, increased the lactate/pyruvate ratio, caused a slight decrease in the total ATP content, and inhibited lipogenesis by at least 40% depending on the type of diet fed to the rats.) The fall in ATP levels was very small and did not correlate with the changes in fatty acid synthesis. The immediate cause of the inhibition of lipogenesis, brought about by glycerol in hepatocytes from sucrose fed rats, seemed to be a large decrease in pyruvate levels. This did not result from impairment of glycolysis but from a rise in the cytosolic NADH/NAD ratio.

3-Hydroxybutyric Acid↗

PubMed1989

A new approach of the estimation of Km of carbamyl phosphate synthetase for ammonia in isolated rat hepatocytes.

1. The Km for ammonia of carbamyl phosphate synthetase was determined by preincubating isolated liver cells for 30 min in the absence of ammonia and bicarbonate and in the presence of ornithine, chloroquine, which blocks lysosomal proteolysis, and aminoxy acetic acid, which inhibits transaminases. 2. The reaction was started with the addition of varying concentrations of ammonia and 10 mM bicarbonate. 3. The rate of citrulline formation was measured as related to ammonia concentration. 4. The pre-incubation with ornithine permits an accumulation of intracellular and mitochondrial ornithine concentrations which in turn allow rapid citrulline formation in the carbamyl phosphate form. 5. This prevents any feedback inhibition on a carbamyl phosphate synthetase or decreases in activity due to accumulation of carbamyl phosphate and/or absence of ornithine. 6. Using these methods in combination with [14C]bicarbonate permitted an estimation of exogenous ammonia for carbamyl phosphate synthesis. 7. The Km for ammonia was 1.5 mM, using a pK of 8.88 the Km for free NH3 was 48 microM.

Animals↗

PubMed1989

Comparison among the lipogenic potential of various substrates in rat hepatocytes: the differential effects of fructose-containing diets on hepatic lipogenesis.

Fructose feeding has been reported to cause hypertriglyceridemia in rats. Apparently this is due to increased hepatic fatty acid synthesis. In hepatocytes from female rats fed a 60% sucrose or fructose diet, the rate of lipogenesis was two times higher than in cells from rats fed a 60% glucose diet and three times higher than in cells from rats fed a commercial nonpurified diet. In hepatocytes from rats fed the fructose-containing diets, lactate was a better substrate than either butyrate or acetate, whereas in cells from rats fed either the glucose diet or the non-purified diet, butyrate was the best lipogenic substrate, and the lipogenic potential of lactate and acetate was similar. In all cases, 1 mM fructose caused a 30-40% stimulation of lipogenesis, while 10 mM glucose did not enhance fat synthesis above the endogenous rates. These results suggest that the differential effect of fructose-containing diets on hepatic lipogenesis results from activation of pyruvate dehydrogenase, thereby increasing the efficiency by which lactate is used as a carbon source for fatty acid synthesis. The differences in lipogenic potential of the various substrates tested is discussed.

Animals↗