Carbohydrate-fed Rats Research Articles

Dietary regulation of glycolytic enzymes: VIII. Effect of diet and oral folate upon folate-metabolizing enzymes in rat jejum

1. 1. The effects of dietary fructose, glucose, casein and fasting and oral folate upon the activity of four enzymes involved in folate metabolism [glutamate formimonotransferase ( N- formimino- L- glutamate:tetrahydrofolate 5- formiminotransferase , EC 2.1.2.5), serine hydroxymethyltransferase ( L-serine: tetrahydrofolate 5,10-hydroxymethyltransferase, EC 2.1.2.1), methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate:NADP + oxidoreductase, EC 1.5.1.5) and formyltetra-hydrofolate synthetase (formate;tetrahydrofolate ligase (ADP), EC 6.3.4.3)] were studied in the jejenum of rats. 2. 2. Individual sugars (fructose and glucose) have specific carbohydrate effects on glutamate formiminotransferase activity, with the activity being highest in the fructose-fed rats. With the three other enzymes, no differential effect was noted with the two sugars. The activities of three of the enzymes (glutamate formiminotransferase, serine hydroxymethyltransferase and methylenetetrahydrofolate dehydrogenese) were highest on the carbohydrate diets, less on the casein diet and least in the fasting state. In contrast, the activities of formyltetrahydrofolate synthetase were lowest in the carbohydrate-fed rats and significantly higher in the fasted and caseinfed rats. 3. 3. Oral folate (two different dosages) produced significant increases in the activities of three of the folate-metabolizing enzymes: glutamate formmiminotransferase, serine hydroxymethyltransferase and methylenetetrahydrofolate dehydrogenase. The activity of glutamate formiminotransferase increased with increasing amounts of oral folate, whereas the activities of the other two enzymes were the same on the two levels of oral folate. Oral folate had no significant effect upon the activity of formyltetrahydrofolate synthetase.

Mitochondrial-bound hexokinase of the rat epididymal adipose tissue and its possible relation to the action of insulin

The amount of epididymal adipose tissue hexokinase activity found to be associated with the mitochondria varied with pH due to different stabilities of the binding. Highest stability was observed when using a homogenizing buffer adjusted to pH 8.0–8.5. The binding of hexokinase to mitochondria was stabilized by glucose in vitro. No such effect of insulin was observed. No difference in activity occured during release of bound hexokinase. This indicates that the adipose tissue mitochondria do not contain any readily releasable latent hexokinase. The percentage of the adipose tissue hexokinase associated with the mitochondrial fraction was increased in carbohydrate-fed rats as compared with fasted rats. This increase was specifically related to hexokinase II. The presence of insulin (in the absence of glucose) resulted in a rapid increase in mitochondrial-bound hexokinase activity of fat pads incubated in vitro. The results suggest an interrelationship between the binding of hexokinase to mitochondria and the dietary adaptation of the enzyme.

Activation in vitro of rat liver polyribosomes.

The increase in the incorporation of amino acids into protein in vitro by preparations obtained from protein-fed rats as compared with preparations obtained from carbohydrate-fed rats has been described previously. After molecular sieving through Sephadex G-25 of cell-free preparations, the difference in incorporating activity between the two types of rats was diminished in systems containing ATP, phosphoenolpyruvate, pyruvate kinase, GTP, and a mixture of amino acids. When, after molecular sieving, a mitochondrial (15,000 g) supernatant was incubated for 4 min at 35 degrees C the polysomal pattern of the preparations was unchanged. In the presence of ATP, phosphoenolpyruvate, and pyruvate kinase the polysomal incorporating activity was low and the polysomal pattern was only slightly changed. Addition of GTP increased the activity markedly, and a more pronounced activity was observed when a mixture of amino acids was added as well. As the amino acid incorporation ability increased, monosomes were formed from the polyribosomes. The activity of the polyribosomes was severalfold higher than that of non-Sephadex-treated preparations, indicating an activation of polysomal aggregates which under the usually applied conditions of incubation and prior to molecular sieving show little or insignificant activity. It was possible to activate polyribosomes from carbohydrate-fed and protein-fed rats to almost the same extent.

Regulation of the rate of amino acid incorporation into protein by rat liver cell sap components

Cell sap from 8 g of liver of non-starved rats was lyophilized, resuspended in a smaller volume and applied to a column of Sephadex G-50. The collected fractions were lyophilized and resuspended in 0.2 ml of distilled water. Part of each fraction was used for thinlayer chromatography (TLC) and the chromatograms were tested for ultraviolet (UV) absorption and for ninhydrin staining. The effect of each fraction on amino acid incorporating activity in the absence and presence of GTP was studied in systems containing ATP, an ATP regenerating system, ribosomes and cell sap from normal rats. The cell sap had been passed through a column of Sephadex G-25 to remove small molecular weight components. Cell sap of normal and carbohydrate-fed rats was separated on the Sephadex G-50 columns into fractions of inhibitory and stimulatory activity. Subfractions of cell sap from stress-induced and protein-fed rats had a low or non-inhibitory activity but a very pronounced stimulatory activity. Fractions of stimulatory activity were characterized further by means of thinlayer chromatography. They contained amino acids and nucleoside triphosphates, primarily ATP and GTP. The fractions of inhibitory activity obtained after column chromatography on Sephadex G-50 contained RNA, protein and lipoid components. The inhibitory activity was retained after thinlayer chromatography in organic solvent. The eluate containing the inhibitory activity showed a decrease in protein content.

Effects of Insulin and Fatty Acids on Gluconeogenesis in the Rat

The present study was undertaken to examine the mechanism of insulin action on gluconeogenesis in the rat. Conversion of pyruvate to glucose was estimated by measuring the radioactivity of the blood glucose and liver glycogen at short intervals, usually between 5 min and 1 hour, after injection of pyruvate-3-14C. In alloxan-diabetic rats insulin at high dosage markedly lowered incorporation of pyruvate-3-14C into blood glucose, but also stimulated its incorporation into liver glycogen. However, total incorporation into blood glucose and liver glycogen was decreased by insulin. When insulin was withdrawn from insulin-treated, alloxan-diabetic rats, the incorporation rose gradually over 3 days to the untreated diabetic level. Insulin lowered the incorporation of pyruvate-3-14C into blood glucose of fasted or fat-fed rats, and also lowered incorporation in liver glycogen. Octanoate injected intraperitoneally into normal, carbohydrate-fed rats markedly enhanced pyruvate-3-14C incorporation into blood glucose. This effect was accompanied by lowered liver glycogen and increased blood glucose levels, but was without effect on the incorporation of pyruvate-3-14C into liver glycogen. On the basis of present knowledge of insulin action, taken in conjunction with known effects of long chain acyl coenzyme A esters and acetyl-CoA on intermediary metabolic reactions in liver, these results suggest that the high hepatic gluconeogenesis of fasting and diabetes is promoted by high hepatic acyl-CoA levels, and that the prompt and marked suppression of hepatic gluconeogenesis by insulin is due to its antilipolytic action on adipose tissue.

Comparison of oxidative metabolism in starved, fat-fed and carbohydrate-fed rats.

1. Rats were starved for 48hr. or fed for 1 week on a high-fat or a high-carbohydrate diet. The effects of these dietary alterations on the rate of production of (14)CO(2) from trace amounts of [U-(14)C]glucose, [1-(14)C]palmitate or [1-(14)C]acetate administered intravenously were studied. 2. The oxidation of [(14)C]glucose was most rapid in the carbohydrate-fed condition and was decreased significantly and to the same extent after starvation and after feeding with fat. 3. Under all dietary regimes studied the maximum rate of elimination of (14)CO(2) from [(14)C]palmitate occurred within a few minutes after injection, but considerably more was oxidized after starvation and feeding with fat than after feeding with carbohydrate. 4. Alterations in diet had no effect on the oxidation and high recovery of administered [(14)C]acetate as (14)CO(2). 5. Graphical analysis showed the presence of several exponential components in the (14)CO(2)-elimination curves. 6. In all studies a marked similarity in oxidative pattern was noted between the starved and the fat-fed rat.

Metabolism of chyle triglycerides in the liver: I. Studies on the mechanisms for liver uptake of intravenously injected, glycerol- and fatty acid-labeled chyle in the carbohydrate-fed rat

1. 1. Blood and liver radioactivity was studied after the intravenous injection of chyle, containing triglycerides labeled with [1- 14C]glycerol and [9, 10- 3H 2] palmitic acid or [9,10- 3H 2]oleic acid into carbohydrate-fed male rats. 2. 2. More than 25% of the fatty acid radioactivity in the livers 10 min after the injection of chyle could be washed out by an immediate perfusion in situ with saline. A major part of this radioactive material consisted of chyle triglycerides. 3. 3. Labeled palmitic acid liberated by the hydrolysis of chyle triglycerides was distributed in approximately equal amounts in liver endogenous non-phospholipids and phospholipids. Labeled oleic acid was found to a higher extent in the non-phospholipids. 4. 4. It is concluded that, after the intravenous injection of labeled chyle into carbohydrate-fed rats, chyle particles are removed by the liver intact and deposited mainly in liver extracellular spaces. The chyle triglycerides are then hydrolysed and the fatty acids and, to a smaller extent, the glycerol are used for the synthesis of liver lipids. Labeled fatty acids recirculated via the blood after hydrolysis of chyle triglycerides at peripheral capillary beds contributed less than 20% to the labeled endogenous liver lipids.

Radioactivity in blood and liver partial glycerides, and liver phospholipids after intravenous administration to carbohydrate-fed rats of chyle containing double-labeled triglycerides

A chromatographic method permitting fast and complete separation of all nonphospholipid fractions in rat blood and liver except for 1,2-diglycerides from cholesterol, and 1- from 2-monoglycerides is described. The level of radioactivity and the ratio 14C/ 3H were determined in partial glycerides and free fatty acids from blood and liver and in liver phospholipids at short time intervals after the intravenous injection into carbohydrate-fed rats of chyle containing triglycerides labeled with [ 14C]glycerol and [ 3H]palmitic acid. The ratios 14C/ 3H in blood partial glycerides showed that they were not formed by breakdown of chyle triglycerides in the circulating blood, but were probably original constituents of the chyle. The sum of 3H radioactivity in partial glycerides and free fatty acids in the liver never exceeded one tenth of the total 3H radioactivity in this organ. The ratios 14C/ 3H in the labeled liver partial glycerides indicated that these were mainly formed during the resynthesis of triglycerides and phospholipids. There was a progressive rise of fatty acid radioactivity in the liver phospholipids. Only a small amount of 14C radioactivity was found in these—slightly relatively more in the phosphatidyl ethanolamine than in the phosphatidyl choline fraction.

The metabolism of palmitic acid-1-C14 in functionally hepatectomized rats

Normal carbohydrate-fed rats and rats on which a functional hepatectomy had been performed by ligating the vessels of the porta hepatis were injected with C14-labeled palmitic acid bound to albumin, and the distribution of activity studied. The concentration of free fatty acids (FFA) in plasma rapidly rises after the hepatectomy. The half life of the injected activity, however, does not seem to change significantly in these animals. In the normal rat a considerable fraction of the injected FFA is recirculated in the blood in glycerides and to a lesser extent in phospholipids. This recirculation is almost completely abolished after hepatectomy, indicating that the liver is the chief organ for plasma lipoprotein synthesis. The peripheral tissues are capable of esterifying the FFA but do not release any esterified fatty acids into plasma, with the possible exception of adipose tissue. There is less activity going into the adipose tissue after hepatectomy. The uptake of fatty acids into adipose tissue is discussed.

Effects of intravenous heparin on oxidation of fat

The intravenous injection of heparin to carbohydrate-fed rats results in an elevation of plasma free fatty acid levels and in a lowering of the respiratory quotient from 1.0 to 0.9. The injection of heparin simultaneously with palmitic acid-C14-labeled chylomicrons results in an elevation of the specific radioactivity of the expired CO2. These observations suggest that heparin causes an obligatory oxidation of fat, presumably through the release of free fatty acid from triglyceride.

The liberation of lipoprotein lipase by heparin from adipose tissue incubated in vitro

Epididymal fat tissues of rats, incubated in vitro in the presence of heparin, have been found to release lipoprotein lipase into the medium. This release occurs rapidly and shows little dependence upon the composition of the medium. The tissues of carbohydrate-fed rats are more active with respect to lipoprotein lipase liberation than those of fasted animals.

C 14O 2 excretion after the intravenous administration of labeled chylomicrons in the rat

Fasting and carbohydrate-fed rats were injected with chylomicrons labeled with palmitic acid-1-C 14. In the following 90 min. the former expired 45% of the injected radioactivity and the latter less than 5%. There was a significant difference in the amount of unexpired radioactivity found in the fat depots in the two groups.

Carbohydrate sparing of fatty acid oxidation. I. The relation of fatty acid chain length to the degree of sparing. II. The mechanism by which carbohydrate spares the oxidation of palmitic acid

1. I. Carboxyl-labeled fatty acids of the even series (C 8C 16) and the triglycerides of carboxyl-labeled palmitic and capric acids were injected into fasted and carbohydrate-fed rats. The expired CO 2 was collected at various intervals for 24 hr., and the cumulative C 14O 2 and the specific activity-time curves were derived for each rat. 1.1. 1. The conversion of the C 14 of the injected fatty acids to CO 2 was lower in the fed than in the fasted rat. This sparing action of carbohydrate was more pronounced in the case of the longer-chain fatty acids. Administered carbohydrate failed, under the conditions employed, to spare the oxidation of acetate to CO 2. 1.2. 2. The sparing action of carbohydrate lasted several hours, and was followed by a secondary rise in the specific activity of the expired CO 2. This secondary rise, suggestive of an increased breakdown of labeled fatty acid, was more pronounced in the case of the longer-chain fatty acids. 2. II. To study the mechanism of the action of carbohydrate in sparing the oxidation of palmitic acid to CO 2, the triglyceride and the nonlipide fractions were isolated from carbohydrate-fed and fasted rats 5 hr. after the injection of palmitic acid-1-C 14. The C 14 contents of the carboxyl carbon and decarboxylated moieties of the triglyceride 16-` and 18-carbon fatty acids and the C 14 contents of the nonlipides were determined. 2.1. 1. The results obtained indicate that the extent of utilization of acetyl-S-CoA, derived from palmitic acid breakdown, for fatty acid synthesis is far greater in the carbohydrate-fed than in the fasted rat. But diversion of the acetyl-S-CoA from an oxidative to a synthetic fate can account for only a small part of the difference in the C 14O 2 recoveries observed in the fed and fasted rats. 2.2. 2. The results obtained for the C 14 nonlipides in the carbohydrate-fed and fasted rats indicate that carbohydrate feeding did not bring about a diversion of C 14 into this fraction. 2.3. 3. The conclusion was drawn that the principal action of carbohydrate in sparing the oxidation of palmitic acid is in restricting its breakdown. 2.4. 4. In both the carbohydrate-fed and fasted rats, the injected palmitic acid was recovered after 5 hr. chiefly as 16-carbon fatty acids. It was found, however, that a much greater proportion of the unoxidized palmitic acid was converted to 18-carbon fatty acids in the carbohydrate-fed than in the fasted rats. 2.5. 5. It is suggested that the degree of sparing, by carbohydrate, of the oxidation of fatty acids of chain lengths less than 16 carbons is related to the capacity of the organism to convert a particular fatty acid directly to 16- and 18-carbon fatty acids.

EFFECT OF DIET ON GLUCOSE TOLERANCE AND LIVER AND MUSCLE GLYCOGEN OF HYPOPHYSECTOMIZED AND NORMAL RATS1,2

THE HYPOPHYSECTOMIZED RAT on an ad libitum or force-fed diet has been shown to exhibit an abnormal glucose tolerance curve (1, 2, 3). During the period of rapid glycogen formation the rate of removal of glucose from the blood is less than normal, but during the later period when glycogenolysis maintains the bloodsugar level in normal animals, the blood-sugar level of the hypophysectomized rat continues to fall. This appears to be the result of the rapid removal of glucose from the blood stream by the peripheral tissues as demonstrated by Greeley in the hypophysectomized rabbit (4). In balance studies on rats fed high carbohydrate and high fat diets by stomach tube for 10 to 17 days after hypophysectomy, it was found that while the carbohydrate-fed rats showed a low metabolic rate and lower carbohydrate stores than control animals on the same diet, the fat-fed animals still had a normal metabolic rate and a normal amount of carbohydrate in their bodies (5).