Biochemical and ultrastructural hepatic changes during vitamin B12 deficiency in animals and man.

Vitamin B12 deficiency has been shown to result in an increase in content and activity of the hepatic cytosolic enzymes of fatty acid synthesis. The present study demonstrated that ATP citrate lyase, an enzyme whose activity has been positively correlated with rates of fatty acid biosynthesis, also increased in the livers of B12-deficient animals. Total and specific activity of hepatic citrate synthase, an enzyme whose activity is unaffected by a variety of dietary and hormonal changes, also was found to be increased in the B12-deprived state. By contrast, the activity of hepatic succinate-cytochrome c reductase, a portion of a multicomponent enzyme complex synthesized in part within the mitochondria, was unchanged in B12 deficiency. Vitamin B12 deprivation resulted in an increase in hepatic mitochondrial cristae membranes in both animals and man. Histochemical and chemical analysis demonstrated increased glycogen in the liver cells from B12-deficient animals and man. Thus, in the livers from vitamin B12-deficient animals there is an increased activity of the otherwise highly constant Krebs cycle enzyme citrate synthase, and in both animals and man there are increased mitochondrial cristae membranes.     

Effects of phenobarbital upon triacylglycerol metabolism in the rabbit.

1. The association between hepatic microsomal enzyme induction and triacylglycerol metabolism was examined in fasting male rabbits (2kg body wt.) injected intra-peritoneally with 50 mg of phenobarbital per kg for 10 days. 2. Occurrence of enzyme induction was established by a significant increase in hepatic aminopyrine N-demethylase activity and cytochrome P-450 content, as well as a doubling of microsomal protein per g of liver and a 54% increase in liver weight. Parallel increments in hepatic gamma-glutamyltransferase (EC 2.3.2.2) activity occurred; these were more pronounced in the whole homogenate than in the microsomes, which only accounted for 12.5% of the total enzyme activity in the controls and 17.0% in the animals given phenobarbital. Increased activity of gamma-glutamyltransferase activity was also observed in the blood serum of the test animals. 3. The rabbits given phenobarbital manifested increased hepatic triacylglycerol content and the triacylglycerol concentration of blood serum was also elevated. These changes were accompanied by a significantly enhanced ability of cell-free fractions of liver from the test animals (postmitochondrial supernatant and microsomal fractions) to synthesize glycerolipids in vitro from sn-[14C] glycerol 3-phosphate and fatty acids, when expressed per whole liver. Relative to the protein content of the fraction, glycerolipid synthesis in vitro was significantly decreased in the microsomes, presumably consequent upon the dramatic increase in their total protein content, whereas no change occurred in the postmitochondrial supernatant, possibly due to the protective effect of cytosolic factors present in this fraction and known to enhance glycerolipid synthesis. 4. Microsomal phosphatidate phosphohydrolase accounted for 85% of the total liver activity of this enzyme and its specific activity was 20-fold higher than that of the cytosolic phosphatidate phosphohydrolase (EC 3.1.3.4), when each was measured under optimal conditions. A significant increase in the activity of both enzymes per whole liver occurred in the rabbits given phenobarbital. A closer correlation between hepatic triacylglycerol content and and microsomal phosphatidate phosphohydrolase, as well as the above observation, suggest that this, rather than the cytosolic enzyme, may be rate-limiting for triacylglycerol synthesis in rabbit liver. 5. Significant correlations were observed between the various factors of hepatic microsomal-enzyme induction (aminopyrine N-demethylase and gamma-glutamyltransferase activity as well as cytochrome P-450 content) and hepatic triacylglycerol content, suggesting that that microsomal enzyme induction may promote hepatic triacylglycerol synthesis and consequently hypertriglyceridaemia in the rabbit.     

Comparative development of the pyruvate dehydrogenase complex and citrate synthase in rat brain mitochondria.

The enzyme activity of the pyruvate dehydrogenase complex (PDHC) was measured in mitochondria prepared from developing rat brain, before and after steady-state dephosphorylation of the E1 alpha subunit. A marked increase in dephosphorylated (fully activated) PDHC activity occurred between days 10 and 15 post partum, which represented approx. 60% of the difference in fully activated PDHC activity measured in foetal and adult rat brain mitochondria. There was no detectable change in the active proportion of the enzyme during mitochondrial preparation nor any qualitative alteration in the detectable catalytic and regulatory components of the complex, which might account for developmental changes in PDHC activity. The PDHC protein content of developing rat brain mitochondria and homogenates was measured by an enzyme-linked immunoadsorbent assay. The development of PDHC protein in both fractions agreed closely with the development of the PDHC activity. The results suggest that the developmental increase in PDHC activity is due to increased synthesis of PDHC protein, which is partly a consequence of an increase in mitochondrial numbers. However, the marked increase in PDHC activity measured between days 10 and 15 post partum is mainly due to an increase in the amount of PDHC per mitochondrion. The development of citrate synthase enzyme activity and protein was measured in rat brain homogenates and mitochondria. As only a small increase in citrate synthase activity and protein was detected in mitochondria between days 10 and 15 post partum, the marked increase in PDHC protein and enzyme activity may represent specific PDHC synthesis. As several indicators of acquired neurological competence become apparent during this period, it is proposed that preferential synthesis of PDHC may be crucial to this process. The results are discussed with respect to the possible roles played by PDHC in changes of respiratory-substrate utilization and the acquisition of neurological competence occurring during the development of the brain of a non-precocial species such as the rat.     

Phosphopyruvate carboxylase induction by l-tryptophan. Effects on synthesis and degradation of the enzyme

1. The administration of l-tryptophan to fed rats produces a twofold increase in hepatic phosphopyruvate carboxylase activity that represents a comparable increase in enzyme protein. With specific antibody against the enzyme we have shown that the increase in phosphopyruvate carboxylase is partially mediated via an actinomycin D-sensitive increase in enzyme synthesis. 2. In starved animals tryptophan increases the enzyme activity without any change in the relative rate of phosphopyruvate carboxylase synthesis. In this condition degradation of the enzyme is retarded by tryptophan by a mechanism that is not prevented by cycloheximide.     

Dietary induction of hepatic malic enzyme activity: differentiation of the induction process

The activity of rat liver malic enzyme shows a marked increase when the animals are maintained on a restricted protein diet. Of the NADP-linked dehydrogenases tested (malic enzyme, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase), the response is confined only to malic enzyme. Dietary sucrose is not required for the increase in activity, but elevated dietary levels of this disaccharide increase hepatic malic enzyme regardless of dietary protein. Glucose-6-phosphate dehydrogenase activity is increased by dietary sucrose provided adequate dietary protein is supplied. The specificity of the response to lowered dietary protein shown by malic enzyme suggests that the control of the hepatic enzyme is mediated by processes different from those controlling the activity of glucose-6-phosphate dehydrogenase.     

RNA biosynthesis in mitochondria under conditions of prolonged inhibition of protein biosynthesis in rat liver cytoplasm]

When cytoplasmic protein synthesis is inhibited by cycloheximide (CHI) in vivo synthesis of water-soluble mitochondrial proteins and of mitochondrial RNA is decreased. These changes measured in isolated rat liver mitochondria are similar to those observed in vivo and correlate with the changes the synthesis of water-soluble proteins in mitochondria. When the cytoplasmic fraction (30,000 g-supernatant) had been added to the mitochondria showing decreased RNA synthesis, the RNA synthesis increased to the control level (the incubation conditions were favourable for the protein transport from microsomes to mitochondria). RNA synthesis in mitochondria was not stimulated by cytoplasmic fractions from the CHI-pretreated rats. After prolonged dialysis these fraction stimulated RNA synthesis even to a greater extent than cytoplasmic fractions from the untreated animals. Mitochondrial RNA polymerase activity (measured in mitochondrial extracts supplemented with exogenous DNA) was higher in extracts of mitochondria from livers of normal rats than in extracts of mitochondria from livers of animals injected with CHI.     

Studies on hepatic injury and antioxidant enzyme activities in rat subcellular organelles following in vivo ischemia and reperfusion

The activities of rat hepatic subcellular antioxidant enzymes were studied during hepatic ischemia/reperfusion. Ischemia was induced for 30 min (reversible ischemia) or 60 min (irreversible ischemia). Ischemia was followed by 2 or 24 h of reperfusion. Hepatocyte peroxisomal catalase enzyme activity decreased during 60 min of ischemia and declined further during reperfusion. Peroxisomes of normal density (d = 1.225 gram/ml) were observed in control tissues. However, 60 min of ischemia also produced a second peak of catalase specific activity in subcellular fractions corresponding to newly formed low density immature peroxisomes (d = 1.12 gram/ml). The second peak was also detectable after 30 min of ischemia followed by reperfusion for 2 or 24 h. Mitochondrial and microsomal fractions responded differently. MnSOD activity in mitochondria and microsomal fractions increased significantly (p < 0.05) after 30 min of ischemia, but decreased below control values following 60 min of ischemia and remained lower during reperfusion at 2 and 24 h in both organelle fractions. Conversely, mitochondrial and microsomal glutathione peroxidase (GPx) activity increased significantly (p < 0.001) after 60 min of ischemia and was sustained during 24 h of reperfusion. In the cytosolic fraction, a significant increase in CuZnSOD activity was noted following reperfusion in animals subjected to 30 min of ischemia, but 60 min of ischemia and 24 h of reperfusion resulted in decreased CuZnSOD activity. These studies suggest that the antioxidant enzymes of various subcellular compartments respond to ischemia/reperfusion in an organelle or compartment specific manner and that the regulation of antioxidant enzyme activity in peroxisomes may differ from that in mitochondria and microsomes. The compartmentalized changes in hepatic antioxidant enzyme activity may be crucial determinant of cell survival and function during ischemia/reperfusion. Finally, a progressive decline in the level of hepatic reduced glutathione (GSH) and concomitant increase in serum glutamate pyruvate transaminase (SGPT) activity also suggest that greater tissue damage and impairment of intracellular antioxidant activity occur with longer ischemia periods, and during reperfusion.     

Studies on the biosynthesis of hepatic pyruvate kinase and its correlation with enhanced hepatic lipogenesis in meal-trained rats.

Metabolic and enzymic changes were measured in meal-trained rats fed on high-carbohydrate diet. Rates of hepatic fatty acid synthesis are probably greater than rates of gluconeogenesis throughout the 24 h day provided that animals are fed. The daily enhancement of fatty acid synthesis on meal feeding coincided with the maximum activation of hepatic pyruvate kinase. Maximum activation of this enzyme was reflected in increased total catalytic activity (Vmax.), increased activity at 0.5 MM-phosphoenolpyruvate (V0.5), decreased Vmax./V0.5 ratio and a decrease in co-operativity of phosphoenolpyruvate binding as measured by the Hill coefficient (h). The latter changes are consistent with a decrease in enzyme phosphorylation during activation of the enzyme. To estimate changes in enzyme protein, quantitative enzyme precipitation with rabbit antisera was used. Giving a high-carbohydrate diet to meal-trained animals induced enzyme synthesis within a few hours. Adaptations in diet that enhanced fatty acid synthesis (chow to high carbohydrate; starved to high carbohydrate) led to an increased steady-state concentration of pyruvate kinase protein. An approximate estimate of the half-life of hepatic pyruvate kinase was 56 h. Whenever pyruvate kinase specific activity was measured in liver tissue extracts it was always considerably less (20--100 mumol/min per mg of protein, depending on dietary status) than the specific activity of pure pyruvate kinase (200 mumol/min per mg of protein). Antigenically active, catalytically inactive protein was removed during enzyme purification from cytosol at the stage of (NH4)2SO4 fractionation. The fraction precipitated by 30--45%-satd. (NH4)2SO4 was enzymically active, antigenically reacting protein was identified in the remaining (NH4)2SO4 fractions (0--30%- and 45--85%-satd.) and this contained no enzyme activity. These may correspond to inactive proteolytic fragments of pyruvate kinase. The rate-determining step in adjusting enzyme concentration seems to be proteolysis.     

L-tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo

The administration of l-tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of l-tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in vivo. When l-tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-Hydroxy-dl-tryptophan and indole administration in amounts equimolar to l-tryptophan also result in similar increases in activity whereas α-methyl-dl-tryptophan produces little or no increase.Utilizing pulse-labeling in vivo with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.     

Triacylglycerol metabolism in the phenobarbital-treated rat

1. Various aspects of triacylglycerol metabolism were compared in rats given phenobarbital at a dose of 100mg/kg body wt. per day by intraperitoneal injection; controls were injected with an equal volume of 0.15m-NaCl by the same route. Animals were killed after 5 days of treatment. 2. Rats injected with phenobarbital demonstrated increased liver weight, and increased microsomal protein per g of liver. Other evidence of microsomal enzyme induction was provided by increased activity of aminopyrine N-demethylase and cytochrome P-450 content. Increased hepatic activity of γ-glutamyltransferase (EC 2.3.2.2) occurred in male rats, but not in females, and was not accompanied by any detectable change in the activity of this enzyme in serum. 3. Phenobarbital treatment increased the hepatic content of triacylglycerol after 5 days in starved male and female rats, as well as in non-starved male rats; non-starved females were not tested in this regard. At 5 days after withdrawal of the drug, there was no difference in hepatic triacylglycerol content or in hepatic functions of microsomal enzyme induction between the treated and control rats. 4. After 5 days, phenobarbital increased the synthesis in vitro of glycerolipids in cell-free liver fractions fortified with optimal concentrations of substrates and co-substrates when results were expressed per whole liver. The drug caused a significant increment in the activity of hepatic diacylglycerol acyltransferase (EC 2.3.1.20), but did not affect the activity per liver of phosphatidate phosphohydrolase (EC 3.1.3.4) in cytosolic or washed microsomal fractions. A remarkable sex-dependent difference was observed for this latter enzyme. In female rats, the activity of the microsomal enzyme per liver was 10-fold greater than that of the cytosolic enzyme, whereas in males, the activities of phosphohydrolases per liver from both subcellular fractions were similar. 5. The phenobarbital-mediated increase in hepatic triacylglycerol content could not be explained by a decrease in the hepatic triacylglycerol secretion rate as measured by the Triton WR1339 technique. Since the hepatic triacylglycerol showed significant correlation with microsomal enzyme induction functions, with hepatic glycerolipid synthesis in vitro and with diacylglycerol acyltransferase activity, it is likely to be due to enhanced triacylglycerol synthesis consequent on hepatic microsomal enzyme induction. 6. In contrast with rabbits and guinea pigs, rats injected with phenobarbital showed a decrease in serum triacylglycerol concentration in the starved state; this decrease persisted for up to 5 days after drug administration stopped, and did not occur in non-starved animals. It seems to be independent of the microsomal enzyme-inducing properties of the drug, and may be due to the action of phenobarbital at an extrahepatic site.     

Effects of anabolic steroids on acute phase responses in intra-abdominal sepsis

The acute phase response is an important adaptive response to sepsis and injury. As anabolic steroids increase protein synthesis we postulated that these agents might also increase hepatic acute phase protein synthesis. Male Wistar rats were pretreated with testosterone or danazol for 48 h prior to caecal ligation and puncture (CLP). Thirty-six h following surgery the animals were killed and blood taken for full blood count, total protein, albumin, alpha, beta and gamma globulin fractions on serum electrophoresis, complement C(3) and transferrin levels. Danazol increased the alpha1, alpha2 and beta1 globulin serum protein fractions in comparison with no surgery and CLP alone groups. These results indicate that danazol increases plasma acute phase proteins, as measured by electrophoresis, in this model of intra-abdominal sepsis.     

Age-related changes of protein- and RNA-synthetic processes in experimental hyper- and hypothyroidism

The rate of liver and plasma protein synthesis and the activity of liver RNA polymerases 1 and 2 were investigated in rats of various age under experimental hyper- and hypothyroidism. The rate of plasma protein synthesis decreased with age more dramatically than that of liver proteins. Hyper- and hypothyroidism exerted opposite effects on protein synthesis in rats: stimulation and inhibition, respectively. The manifestation of these effects was age related. The thyroid status of animals also influenced the balance of protein synthesis. Thyroxin administration caused preferential incorporation of a label into blood plasma proteins. Changes of thyroid status of old animals insignificantly affected the absolute values of the label incorporation into proteins and the ratio of the label incorporation into local and secreted liver proteins. Age-related decrease of total hepatic nuclear RNA-polymerase activity was due to reduction of the template-bound functionally active forms of RNA-polymerases 1 and 2. Administration of thyroxin caused initial redistribution of the enzyme activity between template-bound and free fractions accompanied by the increase of template bound RNA-polymerases. Prolonged hormonal stimulus also caused an increase of free RNA-polymerases, which reflects the increased synthesis of these enzymes. Mecrazolyl administration reduced the activity of RNA-polymerase 1 and 2. All age groups were characterized by preferential reduction of the bound form. RNA-polymerase 2 activity decreased to a greater extent than that of RNA-polymerase 1. The data suggest age-determined reactions of the body to altered thyroid status.     

The regulation of ornithine aminotransferase synthesis by glucagon in the rat.

Ornithine aminotransferase (OAT) from rat liver mitochondria was purified to homogeneity. A monospecific antiserum against the enzyme protein was prepared in rabbits. Immunotitrations were performed on OAT present in crude mitochondrial extracts obtained from the livers and kidneys of rats in several hormonal and dietary states. No evidence was found for the existence of an immunologically reactive but enzymatically inactive form of OAT. The relative rate of enzyme synthesis in vivo was studied by pulselabeling rats with [4, 5-³H]leucine, isolating the enzyme protein by immunoprecipitation, and dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels. Nine hours after a single subcutaneous injection of a glucagon oil emulsion, a 3-fold increase in OAT activity and a 12-fold increase in the synthetic rate of the enzyme were observed. Serine dehydratase activity increased on a time-course very similar to that of OAT following glucagon injection. These increases occurred only on low (0–12.5%) protein diets. At higher levels of dietary protein (30% and up), no further stimulation of OAT synthesis by glucagon was observed. Administration of actinomycin D within the first 2 h after glucagon injection resulted in an inhibition of OAT induction. When the administration of the antibiotic was delayed until 4 h after glucagon, no inhibition of OAT induction was observed. Glucose repression of the glucagon induction of the enzyme in hepatic mitochondria was demonstrated to be the result of a rapid inhibition of OAT synthesis.     

Binding of specific ATP citrate lyase and fatty acid synthetase antibodies to heavy populations of rat liver polysomes.

The polysome fractions involved in the synthesis of the rat-liver inducible lipogenic enzymes, ATP citrate lyase and fatty acid synthetase, were identified by their binding of radioiodinated specific antibodies to enzyme. Both of these populations of specific polysomes were shown to be markedly heavier than specific polysomes involved in albumin synthesis. The quanity of antibody bound to the lipogenic enzyme-related polysomes was markedly affected by the dietary status of the animal. A dietary regimen which induced ipogenesis resulted in a tenfold increase in the hepatic activities of these enzymes found in normally fed animals. The radioactivity bound to hepatic polysomes of induced rats was likewise greater than tenfole higher, presumably reflecting an increase in the number of polysomes active in enzyme synthesis. The fasting state resulted in lower hepatic enzyme activity than normal and correspondingly less binding of ATP citrate lyase and fatty acid synthetase antibodies to the heavy polysomes of the sucrose gradient.     

Mitochondrial 3 beta-hydroxysteroid dehydrogenase (HSD) is essential for the synthesis of progesterone by corpora lutea: An hypothesis

In mouse ovaries, the enzyme 3 beta-hydroxysteroid dehydrogenase (HSD) is distributed between microsomes and mitochondria. Throughout the follicular phase of the estrous cycle, the HSD activity in microsomes is predominant; whereas, after LH stimulation, HSD activity during the luteal phase is highest in the mitochondria. The current study examined whether or not LH stimulation always results in an increase in mitochondrial HSD activity. This was accomplished by measuring the HSD activity in microsomal and mitochondrial fractions from ovaries of pregnant mice. These animals have two peaks of LH during gestation, and one peak of LH after parturition. It was found that mitochondrial HSD activity was highest after each peak of LH. It is proposed that mitochondrial HSD is essential for the synthesis of high levels of progesterone. The increase in HSD activity in mitochondria after LH stimulation occurs because: 1) LH initiates the simultaneous synthesis of HSD and the cholesterol side-chain cleavage enzyme (P450scc); and, 2) HSD and P450scc bind together to form a complex, which becomes inserted into the inner membrane of the mitochondria. High levels of progesterone are synthesized by mitochondrial HSD because: 1) the requisite NAD+ cofactor for progesterone synthesis is provided directly by the mitochondria, rather than indirectly via the rate limiting malate-aspartate shuttle; and, 2) the end-product inhibition of P450scc by pregnenolone is eliminated because pregnenolone is converted to progesterone.     

Regulation of hepatic fatty acid synthetase in the obese-hyperglycemic mutant mouse.

Regulation of fatty acid synthetase has been studied in the obese-hyperglycemic mouse and compared with regulation in non obese, littermate control animals. The mechanisms underlying the regulatory changes were defined by immunochemical techniques. Several major conclusions are justified from the data obtained: (1) Although the hepatic specific activity of fatty acid synthetase is higher in obese than in non obese animals pair-fed chow, no difference in hepatic activities is apparent in animals pair-fed the fat-free diet; (2) The higher enzymatic activity in obese animals fed chow is related to a higher content of enzyme, and this higher content is associated with a higher rate of enzyme synthesis; (3) The decrease in hepatic synthetase activity with starvation is distinctly more striking in non obese than in obese animals, and the changes in activity reflect changes in content of enzyme; (4) With starvation there is a decrease in synthesis of enzyme in obese and non obese animals, but only in non obese animals is there also a marked increase in the rate of synthetase degradation (t1/2 = 24 h during starvation, t1/2 = 76 h during normalfeeding); (5) Refeeding starved mice a fat-free diet results in a more striking increase in hepatic synthetase activity in non obese than in obese animals; (6) Administration of triiodothyronine causes a more marked increase in hepatic synthetase activity in non obese than in obese animals. The data thus define a variety of differences in regulation of hepatic fatty acid synthetase in mutant and normal animals. The roles of enzyme synthesis and degradation in the etiology of these differences are defined, and possible mechanisms underlying regulation of synthetase synthesis and degradation in normal mammalian liver are suggested by the observations.     

Effects of 4-hydroxynonenal on mitochondrial 3-hydroxy-3-methylglutaryl (HMG-CoA) synthase

Chronic ethanol consumption causes increased production of reactive oxygen species in hepatic mitochondria accompanied by elevations in products of lipid peroxidation such as 4-hydroxynonenal (4-HNE). In the current study we investigated the effects of chronic ethanol consumption on a prominent protein-4-HNE adduct in liver mitochondria. Male Sprague-Dawley rats were fed a liquid diet for 31 days in which ethanol constituted 36% of total calories. Immunoblot analyses of liver mitochondria from ethanol-fed and control animals, using an antibody to a 4-HNE-protein adduct, demonstrated elevated 4-HNE binding (+50%) to a mitochondrial protein of approximately 55 kDa due to chronic ethanol consumption. Analysis of this protein using AspN digestion and tandem mass spectrometry identified it as the mitochondrial form of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. Activity of the activated form of this enzyme was unchanged in livers from ethanol-fed animals, but the protein level was elevated by 36%, which suggests a compensatory mechanism to maintain constant levels of synthase activity in the mitochondrion in the face of continuous inactivation by 4-HNE. Treatment of isolated mitochondria with 4-HNE demonstrated that the enzyme activity decreased as a function of 4-HNE concentration and with time of exposure. This study demonstrates that ethanol consumption increases the formation of a 4-HNE adduct with mitochondrial HMG-CoA synthase, which has the potential to inactivate the enzyme in situ.     

Calcitonin increases pyruvate carboxylase activity in hepatic mitochondria of rats

The effect of calcitonin (CT) on calcium content and enzyme activity in the hepatic mitochondria of intact rats was investigated. A single subcutaneous administration of CT (80 MRC mU/100 g BW) produced a significant increase in the content of calcium, the activity of pyruvate carboxylase, succinate dehydrogenase and ATPase 15 min after the hormone treatment. The significant increases in calcium content and pyruvate carboxylase activity were also observed 30 min after CT administration, while succinate dehydrogenase and ATPase activity began to decrease. A physiological dose of CT (20 MRC mU/100 g BW) caused a marked increase in calcium content and pyruvate carboxylase activity but not succinate dehydrogenase of ATPase-activity. The removal of calcium by 10 mM EGTA washing of the mitochondria produced a remarkable reduction in pyruvate carboxylase activity increased by CT administration. The addition of calcium ion of 2.5 x 10(-2) - 2.5 x 10(1) nmoles Ca2+ per mg mitochondrial protein produced a marked increase in pyruvate carboxylase activity. The present results suggest that calcium taken up by the hepatic mitochondria after CT administration activates pyruvate carboxylase.     

Multiple effects of tumor necrosis factor on lipoprotein lipase in vivo

A single dose of recombinant murine tumor necrosis factor (TNF) suppressed lipoprotein lipase activity in adipose tissue of fed rats, mice, and guinea pigs for 48 h, even though TNF itself is rapidly metabolized in vivo. Immunoprecipitation of [35S]lipoprotein lipase from fat pads pulse-labeled with [35S]methionine showed a decrease in relative synthesis of the enzyme, which correlated to the decrease in activity. There was no decrease in general protein synthesis and no change in distribution of the enzyme between adipocytes and extracellular locations in the tissue. This is in contrast to fasting in which case there is redistribution of the enzyme within the tissue, decrease in general protein synthesis, but no change in relative synthesis of lipoprotein lipase. TNF did not decrease lipoprotein lipase activity in any tissue other than the adipose but increased the activity in several cases, most markedly in the liver. No [35S]methionine was incorporated into lipoprotein lipase by liver slices from normal or TNF-treated animals. Thus, the increased activity can not be ascribed to enhanced hepatic synthesis of the enzyme. There was an increase in lipoprotein lipase activity in plasma, which correlated to the increase in liver. Thus, TNF suppresses lipoprotein lipase synthesis in adipocytes, but not in other tissues, and has some as yet undefined effect on lipoprotein lipase turnover in extrahepatic tissues, which results in increased transport of active lipase through plasma to the liver.     

Changes in the rates of synthesis and messenger RNA levels of hepatic glucose-6-phosphate and 6-phosphogluconate dehydrogenases following induction by diet or thyroid hormone

The lipogenic capacity of rat liver is increased in animals fed a high carbohydrate, fat-free diet or by the administration of 2,2',5'-triiodo-L-thyronine. Underlying this change is a generalized induction of the enzymes involved in lipogenesis, including glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme, which together serve to generate the additional NADPH required for increased fatty acid synthesis. This report presents evidence indicating that induction of the hexose-shunt dehydrogenases involves increased enzyme synthesis secondary to elevated enzyme specific mRNA levels, as has previously been shown for malic enzyme. Activities of specific mRNAs, estimated by cell-free translation of hepatic poly(A)-containing RNA in the mRNA dependent rabbit reticulocyte lysate, were compared with enzyme specific activities and relative rates of specific enzyme synthesis. The 2-fold increase in glucose-6-phosphate dehydrogenase specific activity in hyperthyroid rats and the 13-fold increase in rats fed a high carbohydrate, fat-free diet, relative to euthyroid, chow-fed controls were paralleled by comparable increases in the synthetic rates and mRNA levels of this enzyme. Similarly, consonant changes in the rate of enzyme synthesis and concentration of 6-phosphogluconate dehydrogenase mRNA accompanied the 2.5- and 3-fold increases in specific activity of this enzyme observed in response to hormonal and dietary induction, respectively. Thus, both thyroid hormone and carbohydrate feeding appear to induce glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase primarily by increasing the effective cellular concentrations of their respective mRNAs and, consequently, their rates of synthesis.