Effect of diet and starvation on the activity state of branched-chain 2-oxo-acid dehydrogenase complex in rat liver and heart

In rats fed a high-protein diet, the branched-chain 2-oxo-acid dehydrogenase complex in liver was essentially fully acitve and its activity state was unaffected by subsequent starvation for 48 h. Feeding with a low-protein diet led to a decrease in the activity state which was essentially reversed by 48 h of starvation. In heart, the enzyme was primarily inactive (activity state 18%) in rats fed a high-protein diet, with both low-protein diet and starvation leading to a further decrease in the activity state.     

Regulation of the branched-chain 2-oxo acid dehydrogenase complex in hepatocytes isolated from rats fed on a low-protein diet.

Hepatocytes isolated from rats fed on a chow diet or a low-protein (8%) diet were used to study the effects of various factors on flux through the branched-chain 2-oxo acid dehydrogenase complex. The activity of this complex was also determined in cell-free extracts of the hepatocytes. Hepatocytes isolated from chow-fed rats had greater flux rates (decarboxylation rates of 3-methyl-2-oxobutanoate and 4-methyl-2-oxopentanoate) than did hepatocytes isolated from rats fed on the low-protein diet. Oxidizable substrates tended to inhibit flux through the branched-chain 2-oxo acid dehydrogenase, but inhibition was greater with hepatocytes isolated from rats fed on the low-protein diet. 2-Chloro-4-methylpentanoate (inhibitor of branched-chain 2-oxo acid dehydrogenase kinase), dichloroacetate (inhibitor of both pyruvate dehydrogenase kinase and branched-chain 2-oxo acid dehydrogenase kinase) and dibutyryl cyclic AMP (inhibitor of glycolysis) were effective stimulators of branched-chain oxo acid decarboxylation with hepatocytes from rats fed on a low-protein diet, but had little effect with hepatocytes from rats fed on chow diet. Activity measurements indicated that the branched-chain 2-oxo acid dehydrogenase complex was mainly (96%) in the active (dephosphorylated) state in hepatocytes from chow-fed rats, but only partially (50%) in the active state in hepatocytes from rats fed on a low-protein diet. Oxidizable substrates markedly decreased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had much less effect in hepatocytes from chow-fed rats. 2-Chloro-4-methylpentanoate and dichloroacetate increased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had no effect on the activity state of the enzyme in hepatocytes from chow-fed rats. The results indicate that protein starvation greatly increases the sensitivity of the hepatic branched-chain 2-oxo acid dehydrogenase complex to regulation by covalent modification.     

The regulation of l-asparaginase activity in rats and mice. Effects of normal and malignant growth, of sex and of dietary changes

1. The activity of l-asparaginase was very low in the liver of newborn rats and mice, and increased within a few days of birth. 2. In rats, but not in mice, the enzyme activity was higher in females than in males, was enhanced by administration of oestradiol, and was decreased by gonadectomy. 3. The enzyme activity decreased in mice starved or fed on a low-protein diet; in rats it was enhanced by starvation, by feeding them on a high-protein diet, or by administration of l-asparagine. 4. The asparaginase activity was decreased in regenerating liver, and was almost absent in the Morris hepatoma 5123.     

Effects of diet and of alloxan-diabetes on the activity of branched-chain 2-oxo acid dehydrogenase complex and of activator protein in rat tissues.

The total activities (sum of active and inactive forms) of branched-chain 2-oxo acid dehydrogenase complex in tissues of normal rats fed on a standard diet were (unit/g wet wt.): liver, 0.82; kidney, 0.77; heart, 0.57; hindlimb skeletal muscles, 0.034. Total activity was decreased in liver by 9%- or 0%-casein diets and by 48 h starvation, but not by alloxan-diabetes. Total activities were unchanged in kidney and heart. The amount of active form of the complex (in unit/g wet wt. and as % of total) in tissues of normal rats fed on standard diet was: liver, 0.45, 55%; kidney, 0.55, 71%; heart, 0.03, 5%; skeletal muscle less than 0.007, less than 20% (below lower limit of assay). The concentration of the active form of the complex was decreased in liver and kidney, but not in heart, by low-protein diets, 48 h starvation and alloxan-diabetes. In heart muscle alloxan-diabetes increased the concentration of active complex. The concentration of activator protein (which activates phosphorylated complex without dephosphorylation) in liver and kidney was decreased by 70-90% by low-protein diets and 48 h starvation. Alloxan-diabetes decreased activator protein in liver, but not in kidney. Evidence is given that in tissues of rats fed on a normal diet approx. 70% of whole-body active branched chain complex is in the liver and that the major change in activity occasioned by low-protein diets is also in the liver.     

Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase

A radiochemical assay was developed for measuring branched-chain alpha-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain alpha-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids, established inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently starved, inhibition of branched-chain alpha-ketoacid dehydrogenase kinase by branched-chain alpha-ketoacids generated as a consequence of endogenous proteolysis most likely promotes the greater branched-chain alpha-ketoacid dehydrogenase activity state.     

Time course of urinary excretion of intraportal ammonia as uric acid and ammonia in chickens fed low- or high-protein diet

15N-ammonia was intraportally infused for 6 hr into chickens fed 5% or 20% protein diet to examine the time course of urinary excretion of intraportal ammonia and dietary effects on it. Urinary ammonia increased linearly for the first hour to the same extent in both dietary groups and thereafter further in the low-protein group. Urinary uric acid derived from the intraportal ammonia adaptively increased and reached a steady state level within 1.5 hr. This level was four times higher in the high-protein group. The infused ammonia was excreted into urine as both ammonia and uric acid, in relatively high proportions in the chickens fed the low-protein diet but was almost all excreted as uric acid in those fed the high-protein diet.     

The effects of nutritional status of rabbits and sheep on their resistance to the ticksRhipicephalus evertsi evertsi andR. appendiculatus

Rabbits and sheep were exposed to low-and high-protein diets and subsequently infested three times with adults ofRhipicephalus appendiculatus andRhipicephalus evertsi evertsi. The mean weight ofR.e. evertsi females which dropped from rabbits maintained on a high-protein diet decreased from 515.0±24.9 mg (naive) to 381.5±25.0 (second infestation) to 340.3±23.3 mg (third infestation) while the weight of ticks fed on animals which were exposed to a low-protein diet did not change significantly (2.7%). The mean weight of engorged females ofR. appendiculatus which completed their blood meal on rabbits (high protein) decreased from 520.9±31.8 (naive) to 369.3±39 mg (3rd infestation), a significant decrease of 29.1% compared to a 12.3% decrease in weight between the 1 st and 3rd infestation of females fed on animals on a low-protein diet.Rhipicephalus e. evertsi fed on sheep exhibited the same phenomenon. The mean decrease in weight of 4rd-infestation ticks which dropped from sheep fed lucerne was 26.2% compared to 16.6% for ticks from sheep which were fed on grass.Hosts maintained on a low-protein diet failed to acquire resistance to ticks, lost weight and developed anaemia while those on a high-protein diet developed resistance, maintained weight and did not develop anaemia.The nutritional stress of the hosts and its application in South Africa are discussed.     

The effect of starvation and refeeding on lipogenic enzymes in mammary glands and livers of lactating rats.

Lactating rats were starved for 48 h and refed a high-carbohydrate diet for a further 48 h. Starvation stops milk secretion, which resumes shortly after refeeding. Three lipogenic enzymes, fatty acid synthase, glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and 'malic' enzyme (EC 1.1.1.40) all decrease in the mammary gland during starvation and are restored to the pre-starvation levels 48 h after refeeding. The same enzymes in liver also decrease during starvation, but increase to values significantly higher than those for the normal fed rats after refeeding the high-carbohydrate diet. For the fatty acid synthase these values were four times the pre-starvation values. Serum insulin and prolactin concentrations also increased upon refeeding the high-carbohydrate diet.     

Changes in lung lysyl oxidase activity in streptozotocin-diabetes and in starvation

The effects of streptozotocin-induced diabetes and of starvation on the lysyl oxidase activity of rat lung were investigated. Enzyme activity was elevated 2–3 fold in the lungs of streptozotocin-diabetic rats. In contrast, starvation of rats produced a rapid loss of lung lysyl oxidase activity, with levels approximating 25% of control values after 48–72 h of starvation. Enzyme activity was essentially fully restored to control values upon refeeding the 48-h starved animals for 3 h. These studies demonstrate the responsiveness of lysyl oxidase to these physiological states and suggest a component, enzymatic basis of change in lung function known to occur in the diabetic state.     

Changes in lung lysyl oxidase activity in streptozotocin-diabetes and in starvation.

The effects of streptozotocin-induced diabetes and of starvation on the lysyl oxidase activity of rat lung were investigated. Enzyme activity was elevated 2--3 fold in the lungs of streptozotocin-diabetic rats. In contrast, starvation of rats produced a rapid loss of lung lysyl oxidase activity, with levels approximating 25% of control values after 48--72 h of starvation. Enzyme activity was essentially fully restored to control values upon refeeding the 48-h starved animals for 3 h. These studies demonstrate the responsiveness of lysyl oxidase to these physiological states and suggest a component, enzymatic basis of change in lung function known to occur in the diabetic state.     

Effects of Feeding a Histidine-excess Diet and Subsequent Starvation on Liver and Muscle Glycogen,and on Serum Glucose

The effects of feeding with a histidine-excess diet and subsequent starvation on liver and muscle glycogen, and on serum glucose were investigated in young and adult rats.

Feeding with a histidine-excess diet resulted in the accumulation of liver glycogen in both young and adult rats. The hepatic glycogen continued to decrease during starvation, and the liver became almost totally depleted of glycogen after starvation for 48 hr. Glycogen in the liver of young rats starved for 24 hr after previous feeding with a histidine-excess diet was significantly higher than that of young rats starved for 24 hr after previous feeding with a basal diet.

Muscle glycogen after feeding and subsequent starvation was not affected by the types of diets fed previously, muscle glycogen during starvation showing a slight decrease in young rats and a slight increase in adult rats.

Feeding with a histidine-excess diet caused a significant decrease of serum glucose in young rats, but not in adult rats. Serum glucose in young rats was markedly reduced by starvation after previous feeding with a basal diet, but not after previous feeding with a histidine-excess diet. In adult rats, there were no changes in serum glucose between rats starved after feeding with either a basal diet or a histidine-excess diet, and serum glucose was decreased slightly by starvation after feeding with the test diets.

The overall results indicate that the maintenance of serum glucose in young rate even during starvation after previous feeding with a histidine-excess diet might be partially concerned with the export of glucose from the accumulated glycogen in the liver due to the diet.     

Sensitivity of inhibition of rat liver mitochondrial outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and temperature-induced changes in membrane fluidity.

In the fed state, hyperthyroidism increased glucose utilization indices (GUIs) of skeletal muscles containing a lower proportion of oxidative fibres. Glycogen concentrations were unchanged, but active pyruvate dehydrogenase (PDHa) activities were decreased. Hyperthyroidism attenuated the effects of 48 h of starvation to decrease muscle GUI. Glycogen concentrations and PDHa activities after 48 h of starvation were low and similar in euthyroid and hyperthyroid rats. The increase in glucose uptake and phosphorylation relative to oxidation and storage in skeletal muscle induced by hyperthyroidism may contribute to increased glucose re-cycling in the fed hyperthyroid state and to glucose turnover in the starved hyperthyroid state.     

Inter-organ relationships between glucose, lactate and amino acids in rats fed on high-carbohydrate or high-protein diets

1. Inter-organ relationships between glucose, lactate and amino acids were studied by determination of plasma concentrations in different blood vessels of anaesthetized rats fed on either a high-carbohydrate diet [13% (w/w) casein, 79% (w/w) starch] or a high-protein diet [50% (w/w) casein, 42% (w/w) starch]. The period of food intake was limited (09:00-17:00h), and blood was collected 4h after the start of this period (13:00h). 2. Glucose absorption was considerable only in rats fed on a high-carbohydrate diet. Portal-vein-artery differences in plasma lactate concentration were higher in rats fed on this diet, but not proportional to glucose absorption. Aspartate, glutamate and glutamine were apparently converted into alanine, but when dietary protein intake was high, a net absorption of glutamine occurred. 3. The liver removed glucose from the blood in rats fed on a high-carbohydrate diet, but glucose was released into the blood in rats fed on the high-protein diet, probably as a result of gluconeogenesis. Lactate uptake was very low when amino acid availability was high. 4. In rats on a high-protein diet, increased uptake of amino acids, except for ornithine, was associated with a rise in portal-vein plasma concentrations, and in many cases with a decrease in hepatic concentrations. 5. Hepatic concentrations of pyruvate and 2-oxo-glutarate decreased without a concomitant change in the concentrations of lactate and malate in rats fed on the high-protein diet, in spite of an increased supply of pyruvate precursors (e.g. alanine, serine, glycine), suggesting increased pyruvate transport into mitochondria. 6. High postprandial concentrations of plasma glucose and lactate resulted in high uptakes of these metabolites in peripheral tissues of rats on both diets. Glutamine was released peripherally in both cases, whereas alanine was taken up in rats fed on a high-carbohydrate diet, but released when the amino acid supply increased. 7. It is concluded that: the small intestine is the main site of lactate production, and the peripheral tissues are the main site for lactate utilization; during increased ureogenesis in fed rats, lactate is poorly utilized by the liver; the gut is the main site of alanine production in rats fed on a high-carbohydrate diet and the liver utilizes most of the alanine introduced into the portal-vein plasma in both cases.     

Patterns and mechanisms of growth of fifth-instar Manduca sexta caterpillars following exposure to low- or high-protein food during early instars.

For many insect herbivores, variation in protein availability is a pervasive part of the environment. I explore how variable protein availability affects growth rates of fifth-instar Manduca sexta caterpillars and how growth is related to behavior and physiology. Groups of larvae were reared on low- or high-protein artificial diets (5.9% and 17.7% casein by dry weight, respectively) and then transferred in the fifth instar to the same or opposite diet. During or after the 24-h period following transfer, I measured growth rate, consumption rate, growth efficiency, midgut proteolytic activity, and masses of midgut contents and tissues. Fifth-instar caterpillars reared in earlier instars on high-protein diet grew about 20% more rapidly over 24 h than did caterpillars reared on low-protein diet. This growth pattern appears to be caused by differences in consumption and growth efficiency: caterpillars reared on high protein consumed more food, and used it more efficiently, than did caterpillars reared on low-protein diet. Over the short term (24 h), in contrast, fifth instars that received low-protein diet grew as rapidly as caterpillars that received high-protein diet. Increased (compensatory) consumption appears to be the primary mechanism by which caterpillars consuming low-protein food maintained growth rates.     

Nutritional regulation of milk protein messenger RNA concentrations in mammary acini isolated from lactating rats

The effect of starvation, starvation and refeeding and feeding a low-protein diet on concentrations of the mRNAs for the alpha-, beta- and gamma-caseins, alpha-lactalbumin and whey acidic protein has been determined using dot-blot hybridization analyses with total RNA extracted from mammary acini isolated from lactating rats. Starvation for 48 h decreased the concentrations of all RNA species to between 5 and 20% of those for rats feeding ad libitum when expressed on a DNA basis. Refeeding for 24h restored the concentrations to control values. Consumption of a low protein diet reduced the concentrations of each mRNA by about 50%. Only minor changes were detected in the mRNA concentrations for alpha-casein, alpha-lactalbumin and whey acidic protein in samples prepared at six hourly intervals from rats receiving a restricted intake (40% ad libitum) of the control diet.     

Quantification of liver and kidney phosphofructokinase by radioimmunoassay in fed, starved and alloxan-diabetic rats.

A newly developed specific radioimmunoassay was used to quantify phosphofructokinase protein directly and independently of assayable activity in liver and kidney cytosol of normal fed, starved and alloxan-diabetic rats. In the fed state, liver phosphofructokinase concentration was 0.096 microM and the kidney enzyme was 0.086 microM (mumol/kg of tissue). In the starved state (24h), liver and kidney phosphofructokinase concentrations decreased by 30%. Prolonged starvation up to 72h did not further decrease enzyme concentration. In liver, total enzyme content during starvation declined by more than 50%, secondary also to a decrease in liver weight. In the alloxan-diabetic rats, there was a 22% decrease in enzyme protein concentration in liver and kidney. Total enzyme content per liver actually decreased much more (46%), because diabetes also resulted in a decrease in liver size. In conjunction with assayable activity measurements, the results of the radioimmunoassay allowed us to calculate the apparent specific activity of the enzyme. The specific activity of the kidney enzyme was 2-3 times that of the liver. Little or no change in specific activity of the liver or kidney enzyme occurred as a result of starvation or chemically induced diabetes. Tissue enzyme concentrations of phosphofructokinase unequivocally reconcile the ultimate results of changing rates of synthesis and degradation and are useful data in the design of spectrophotometric, kinetic, aggregation-disaggregation and other studies.     

High-protein nutrition during pregnancy and lactation programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner

Maternal low-protein diet during pregnancy is a risk factor for cardiovascular disease of the offspring in later life. The impact of high-protein diet during pregnancy on the cardiovascular phenotype of the offspring, however, is still unknown. We examined the influence of a high-protein diet during pregnancy and lactation on the renal, hemodynamic, and metabolic phenotype of the F1 generation. Female Wistar rats were either fed a normal protein diet (20% protein: NP) or an isocaloric high-protein diet (40% protein: HP) throughout pregnancy and lactation. At weaning, the offspring were fed with standard diet, and they were allocated according to sex and maternal diet to four groups: normal-protein male (NPm, n = 25), normal-protein female (NPf, n = 19), high-protein male (HPm, n = 24), high-protein female (HPf, n = 29). During the experiment (22 wk), the animals were characterized by repeated measurement of body weight, food intake, blood pressure, glucose tolerance, energy expenditure, and kidney function. At the end of the study period histomorphological analyses of the kidneys and weight measurement of reproductive fat pads were conducted. There were no differences in birth weight between the study groups. No influence of maternal diet on energy expenditure, glucose tolerance, and plasma lipid levels was detected. Blood pressure and glomerulosclerosis were elevated in male offspring only, whereas female offspring were characterized by an increased food efficiency, higher body weight, and increased fat pads. Our study demonstrates that a high-protein diet during pregnancy and lactation in rats programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner.     

Short-term effect of a high-protein/low-carbohydrate diet on aminopeptidase in adult rat jejunoileum. Site of aminopeptidase response.

The short-term effects of high-protein/low-carbohydrate diet on aminopeptidase N activity were studied in the brush-border membranes of proximal jejunum and proximal ileum of adult rats. The animals were starved overnight and re-fed for 15 h either with a standard diet (20% protein, 55% carbohydrate, in terms of energy content) or with a high-protein/low-carbohydrate diet of equal energy content (70% protein, 5% carbohydrate). All rats consumed similar amounts of diet, and measurements were made 15 h after initiation of re-feeding. In the proximal jejunum a slight increase in aminopeptidase activity was observed after the high-protein intake. In contrast, considerable stimulation (52%) of the enzyme specific activity was obtained in the proximal ileum. This increase in ileal aminopeptidase activity was more prominent in the mature cells of the upper villus. To determine if the increase of aminopeptidase activity was due to an increased amount of enzyme protein, rocket immunoelectrophoresis was performed with detergent-solubilized brush-border protein from ileum on agarose gels containing anti-(rat brush-border) antiserum. When the same amount of enzyme activity was loaded on the gels, the peaks of immunoprecipitate for aminopeptidase were similar for animals fed on a standard or a high-protein diet. When the same amount of protein was loaded, the peak of immunoprecipitate for aminopeptidase was higher (81%) after a high-protein diet. These results showed that the high protein intake evoked an increase in aminopeptidase activity, with a concomitant increase in the amount of immunoreactive protein.     

Distribution of pyruvate kinase type L and M2 in microdissected periportal and perivenous rat liver tissue with different dietary states

Pyruvate kinase type L and M2 activities were measured in microdissected periportal and perivenous liver tissue from rats in different dietary states. A specific antibody against pyruvate kinase type L was used to distinguish the two isoenzymes. Using separated cells it was found that the L-isoenzyme was essentially restricted to the parenchymal and the M2-isoenzyme to the non-parenchymal cells. Pyruvate kinase type L activity in the perivenous zone was about twice as high as in the periportal zone in both male and female fed rats. Starvation for 48 h led to a decrease of the overall activity and to a lower perivenous-periportal gradient. After refeeding for 48 h the overall activity and the gradient were increased to above the normal level. Pyruvate kinase type M2 was homogenously distributed within the liver acinus. After 48 h starvation no change in the overall activity nor in the zonal distribution was observed. Refed rats exhibited a slightly reduced overall activity. Since the hepatocytes contain the total regulatory L- but no M2-pyruvate kinase the heterogeneous distribution of the L-isoenzyme under different dietary states supports the model of metabolic zonation of liver parenchyma with glycolysis predominantly in the perivenous zone.     

Hepatic mitochondrial inner membrane properties and carnitine palmitoyltransferase A and B. Effect of diabetes and starvation.

Intact mitochondria and inverted submitochondrial vesicles were prepared from the liver of fed, starved (48 h) and streptozotocin-diabetic rats in order to characterize carnitine palmitoyltransferase kinetics and malonyl-CoA sensitivity in situ. In intact mitochondria, both starved and diabetic rats exhibited increased Vmax., increased Km for palmitoyl-CoA, and decreased sensitivity to malonyl-CoA inhibition. Inverted submitochondrial vesicles also showed increased Vmax. with starvation and diabetes, with no change in Km for either palmitoyl-CoA or carnitine. Inverted vesicles were uniformly less sensitive to malonyl-CoA regardless of treatment, and diabetes resulted in a further decrease in sensitivity. In part, differences in the response of carnitine palmitoyltransferase to starvation and diabetes may reside in differences in the membrane environment, as observed with Arrhenius plots, and the relation of enzyme activity and membrane fluidity. In all cases, whether rats were fed, starved or diabetic, and whether intact or inverted vesicles were examined, increasing membrane fluidity was associated with increasing activity. Malonyl-CoA was found to produce a decrease in intact mitochondrial membrane fluidity in the fed state, particularly at pH 7.0 or less. No effect was observed in intact mitochondria from starved or diabetic rats, or in inverted vesicles from any of the treatment groups. Through its effect on membrane fluidity, malonyl-CoA could regulate carnitine palmitoyltransferase activity on both surfaces of the inner membrane through an interaction with only the outer surface.