Liver growth, biosynthesis of cytidine nucleotides and level of cytochrome P-450 in rat liver after administration of alpha-hexachlorocyclohexane.

The biosynthesis of cytidine nucleotides and the level of microsomal cytochrome P-450 in intact and regenerating rat liver after repeated administration of alpha-hexachlorocyclohexane (alpha-HCH) were compared. In alpha-HCH treated animals the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides is suppressed. In 24-h regenerating liver the incorporation of labelled orotic acid into cytidine nucleotides is markedly activated; the degree of activation is lower in regenerating livers of alpha-HCH treated animals. The changes in the level of cytochrome P-450 vary inversely with the changes in the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides. The activity of cytidine triphosphate synthetase of liver cytosol increases shortly after the administration of alpha-HCH; uridine-cytidine kinase is enhanced in the later stages of the drug action. Within 15-45 min after the administration of alpha-HCH the uptake of [U-14 C] cytidine into the liver and its incorporation into RNA cytosine are increased. After the administration of the drug the uptake of [2-14 C] uridine and its incorporation into RNA uracil is also enhanced whereas its utilization for the synthesis of cytidine nucleotides of the acid-soluble extract as well as for the RNA cytosine are suppressed.     

Biosynthesis of cytidine nucleotides in rat liver after administration of D-galactosamine

Following the administration of D-galactosamine the utilization of [2-¹⁴C]orotic acid for the synthesis of the cytidine components of the acidsoluble extract and liver RNA cytosine is markedly decreased. The depression of the specific activity of the cytidine components takes place after application of low doses of the drug which do not interfere with the specific activity of the uridine components of the acid-soluble extract or of liver RNA uracil. Simultaneously the administration of [U-¹⁴C]cytidine paralleled by its enhanced liver uptake. The total amount of uridine as well as cytidine components of the acid-soluble extract following the administration of D-galactosamine increases; however, the molar ratio of both pyrimidines does not change. The alterations of the cytidine metabolism after the administration of the drug are accompanied by the increased level of microsomal cytochrome P-450.     

Circadian variations in pyrimidine nucleotide synthesis in rat liver

The biosynthesis of pyrimidine components in rat liver varies with the time of the day. The concentrations of both the cytidine and the uridine components of the acid-soluble extract are lowest in the morning hours and highest around midnight. The utilization of [2-¹⁴C]orotic acid for the synthesis of the pyrimidine components of the acid-soluble extract, RNA, and DNA has a similar character. Analogous changes also are seen in the uptake of [U-¹⁴C]cytidine and its utilization for the synthesis of RNA cytosine.     

Cytidine nucleotide biosynthesis and the level of cytochrome P-450 in rat liver microsomes after administration of colchicine.

Colchicine displays a biphasic effect on the biosynthesis of cytidine nucleotides in rat liver; an initial depression is followed by activation of synthesis. Depending on the duration of exposure to colchicine, the changes of the values of the ratio of specific radioactivity of cytosine to uracil in the acid-soluble pool and 3′-CMP to 3′-UMP of cytoplasmic ribosomal RNA are inversely proportional to changes of the levels of cytochrome P-450 in liver microsomes. The utilization of [2-¹⁴C]orotic acid for the biosynthesis of DNA cytosine as affected by the exposure to colchicine reflects the changes in the specific radioactivity of the cytidine components of the acid-soluble pool. However, the maximal radioactivity in DNA thymine is reached under these conditions only at longer time intervals.     

Glucose homeostasis during the early stages of liver regeneration in fasted rats

Kinetic studies with [2-3H]glucose in vivo and gluconeogenic activity measurements in vivo and in vitro were performed in 70% hepatectomized rats submitted to fasting, which represents an extra burden for glucose synthesis but does not impair liver regeneration. Rates of glucose replacement, under steady-state conditions, 14 and 24 h postoperatively, did not differ in partially hepatectomized fasted rats and sham-operated controls. Phosphoenolpyruvate carboxykinase activities increased more rapidly during fasting in remnant livers than in intact livers from controls. Rates of incorporation of 14C from alanine into circulating glucose in hepatectomized rats were already maximal 14 h after surgery, whereas in controls they continued to augment. The maximal rates after partial hepatectomy could not be surpassed by performing the operation in diabetic animals. It is concluded that the relatively high blood sugar levels during fasting in hepatectomized rats do not depend on a reduced peripheral utilization of glucose, but only on a rapid increase in the gluconeogenic activity. The data suggest that hepatocytes in remnant liver can proliferate under conditions of maximal gluconeogenic and low glycolytic activities.     

Role of endogenous regucalcin in nuclear regulation of regenerating rat liver: suppression of the enhanced ribonucleic acid synthesis activity

The role of endogenous regucalcin in the regulation of ribonucleic acid (RNA) synthesis activity in the nucleus of normal and regenerating rat livers was investigated. Nuclear RNA synthesis was measured by the incorporation of [(3)H]-uridine 5'-triphosphate into the nuclear RNA in vitro. The presence of regucalcin (0.25 or 0.5 microM) in the reaction mixture caused a significant decrease in nuclear RNA synthesis of normal rat liver. alpha-Amanitin (10(-8)-10(-6) M), an inhibitor of RNA polymerase II and III, decreased significantly nuclear RNA synthesis activity. The effect of regucalcin (0.25 microM) in decreasing nuclear RNA synthesis activity was not seen in the presence of alpha-amanitin (10(-6) M). The calcium chloride (10 microM)-increased nuclear RNA synthesis activity was significantly suppressed by the addition of regucalcin (0.25 microM). RNA synthesis activity was significantly enhanced in the nuclei of regenating rat liver obtained at 24, 48, or 72 h after partial hepatectomy. This enhancement was significantly inhibited in the presence of PD98059 (10(-5) M), staurosporine (10(-6) M), or vanadate (10(-3) M). Western analysis of the nuclei of regenerating liver obtained at 24, 48, or 72 h after partial hepatectomy showed a significant increase in regucalcin protein as compared with that of sham-operated rats. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the reaction mixture caused a significant increase in nuclear RNA synthesis activity of normal rat liver. This increase was completely blocked by the addition of regucalcin (1.0 microM). The effect of anti-regucalcin monoclonal antibody (50 ng/ml) in increasing nuclear RNA synthesis activity was significantly enhanced in the nuclei of regenerating liver obtained at 24, 48, or 72 h after partial hepatectomy. This enhancement was significantly suppressed by the addition of alpha-amanitin (10(-6) M), PD98059 (10(-5) M), staurosporine (10(-6) M), or vanadate (10(-3) M) in the reaction mixture. The present study demonstrates that endogenous regucalcin has a suppressive effect on the enhancement of RNA synthesis activity in the nucleus of regenerating rat liver with proliferative cells.     

Exercise endurance and fuel utilization: a reevaluation of the effects of fasting

The effect of fasting on energy utilization during running or swimming was studied in adult male Wistar rats. Compared with fed rats, fasted animals displayed a decreased contribution of carbohydrates in energy supply, with decreased liver and muscle glycogen contents and decreased rate of glycogen breakdown. This was compensated by an enhanced rate of beta-oxidation. In addition, fasting induced an exaggerated sympathoadrenal response during exercise, reflected by a greater epinephrine plasma level and a higher norepinephrine turnover rate in both liver and soleus. Nevertheless, endurance capacity was similar in fasted and fed animals. These results contrast with the impairment of endurance observed in fasting humans but also with the improvement of endurance in rats previously reported by Dohm et al. (J. Appl. Physiol. 55: 830-833, 1983). These data suggest that the metabolic responses to exercise subsequent to food deprivation depend not only on the considered species but also, in the same species (rat), on the age of the animals and the duration of the fast. These factors probably determine the hormonal secretion and substrate utilization during prolonged exercise in fasting conditions.     

Selective recognition in erythrophagocytosis by mouse peritoneal macrophages. Isolation and purification of a possible self-marker from mouse erythrocyte membranes

The percental participation of exogenous cytidine in liver RNA synthesis was determined after application of ³H-cytidine to rats. The amount of exogenous cytidine was varied by a factor of 5 × 10⁵, between 0.000 02 and 10.0 μg/g rat. With the ³H-cytidine doses and specific activities most frequently reported in the literature, the percental participation of the exogenous precursor is only about 0.1%, with 99.9% of the cytidylic acid incorporated into RNA under these conditions being of endogenous origin.The results show that the upper limit of the tracer dose of exogenous cytidine is about 1.0 μg/g rat. Within this tracer region 1.8% of ³H-activity—and therefore 1.8% of the amount of exogenous cytidine—is incorporated into liver RNA. The dependence of the percental participation on the duration of the experiments is examined.It is shown that autoradiographic grain density and specific activity of RNA can only be regarded as direct measures for the rate of RNA synthesis in different cells and animals if the percental participation of exogenous cytidine in RNA synthesis is generally of equal value.Comparable situations exist in the incorporation of ³H-thymidine into DNA as shown by earlier experimental work.     

The Hepatic Genes for Immunoproteasome Are Upregulated by Refeeding after Fasting in the Rat

Considering that animals maintain energy homeostasis in response to nutrient levels, experiments were done to elucidate the temporal effects of refeeding after fasting on gene expression profiles in the rat liver. Using DNA microarray technology, we first compared gene expression profiles in the livers of rats allowed to feed for 6 h after fasting for 18 h and those in 24-h fasting rats, and found that the expression levels of energy metabolism-related genes in the two groups were different. In addition, refeeding induced upregulation of the genes encoding immunoproteasome components. Finally, immunoblot analysis confirmed changes in protein levels, suggesting that refeeding after fasting enhanced immune function.     

On lysosomal fragility and induction of liver hexose-monophosphate dehydrogenases in the fasted-refed rat.

Young adult male rats were fasted for 3 days, then fed a glucose-rich diet, ad libitum. At the end of the fasting period, the specific activity of liver glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was decreased to 60% of control (nonfasted) levels. After 24 to 72 h of refeeding, the specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase increased seven- and twofold, respectively. During the fasting period, the liver lysosome fragility increased, as judged by increased release of bound acid phosphatase and β-N-acetylglucosammidase activity during standard homogenization. Three hours after feeding a carbohydrate-rich diet, a further increase in liver lysosomal fragility was observed that returned to control values prior to the induction of the dehydrogenases. Similarly, the susceptibility of liver lysosomes from fasted rats to increased fragility by the intraperitoneal injection of glucose or galactose was also observed. Prior starvation was not a requisite for labilization of lysosomal membranes by injected glucose, but induction of the pentose phosphate shunt dehydrogenase was not observed.In a group of 6-week old male rats fed a commercial pellet diet throughout, the injection of insulin caused no change in liver lysosomal fragility, though hypoglycemia resulted. Similar animals made diabetic by treatment with Streptozotocin and diabetic rats given insulin, showed no change in liver lysosmal fragility based on the percentage of free to total activities of β-N-acetylglucosaminidase, β-glucuronidase, β-galactosidase, and Cathespin D. However, when adult female rats were fasted for 24 h, then injected with sufficient insulin to produce hypoglycemia, liver lysosomal fragility, based on the release of β-N-acetylglucosaminidase during homogenization, increased nearly threefold. These studies demonstrate that stimulated lysosomal fragility can be initiated by refeeding fasted animals a carbohydrate-rich diet, by intraperitoneal injections of fasted rats with glucose or galactose, or by administering insulin alone to fasted rats. However, hyperglycemia induced by diabetogenic doses of Streptozotocin, or hypoglycemia induced in well-fed animals by insulin injection failed to elicit an enhanced liver lysosomal fragility. Whether induction of the enzymes of lipogenesis by rat liver is dependent upon a prior lysosomal membrane labilization remains to be determined.     

Physiological regulation of acetyl-CoA carboxylase gene expression: effects of diet, diabetes, and lactation on acetyl-CoA carboxylase mRNA

We measured acetyl-CoA carboxylase mRNA levels in various tissues of the rat under different nutritional and hormonal states using a cDNA probe. We surveyed physiological conditions which are known to alter carboxylase activity, and thus fatty acid synthesis, to determine whether changes in the levels of carboxylase mRNA are involved. The present studies include the effects of fasting and refeeding, diabetes and insulin, and lactation on carboxylase mRNA levels. Northern blot analysis of liver RNA revealed that fasting followed by refeeding animals a fat-free (high carbohydrate) diet dramatically increased the amount of carboxylase mRNA compared to the fasted condition. These changes in the level of mRNA correspond to changes in the activity and amount of acetyl-CoA carboxylase. Acetyl-CoA carboxylase mRNA levels in epididymal fat tissue decreased upon fasting and increased to virtually normal levels after 72 h of refeeding, closely resembling the liver response. The amount of acetyl-CoA carboxylase mRNA decreased markedly in epididymal fat tissue of diabetic rats as compared to nondiabetic animals. However, 6 h after injection of insulin the mRNA level returned to that of the nondiabetic animals. Gestation and lactation also affected the levels of carboxylase mRNA in both liver and mammary gland. Maximum induction in both tissues occurred 5 days postpartum. These studies suggest that these diverse physiological conditions affect fatty acid synthesis in part by altering acetyl-CoA carboxylase gene expression.     

The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats.

The response of muscle and liver protein metabolism to either a single or three successive daily injections of an endotoxin (Escherichia coli lipopolysaccharide, serotype 0127 B8; 1 mg/ml, 0.3 mg/100 g body wt.) was studied in vivo in the fed rat, and at 24 and 30 h after endotoxin treatment during fasting. In the fed rats there was a catabolic response in muscle, owing to a 60-100% increase in muscle protein degradation rate, and a 52% fall in the synthesis rate. Although there was a 20% decrease in food intake, the decrease in protein synthesis was to some extent independent of this, since rats treated with endotoxin and fasted also showed a lower rate of muscle protein synthesis, which was in excess of the decrease caused by fasting alone. The mechanism of this decreased protein synthesis involved decreased translational activity, since in both fed and fasted rats there was a decreased rate of synthesis per unit of RNA. This occurred despite the fact that insulin concentrations were either maintained or increased, in the fasted rats, to those observed in fed rats. In the liver total protein mass was increased in the fed rats by 16% at 24 h, and the fractional synthesis rate at that time was increased by 35%. In rats fasted after endotoxin treatment the liver protein mass was not decreased as it was in the control fasted rats, and the fractional synthesis rate was increased by 22%. In both cases the increased synthesis rate reflected an elevated hepatic RNA concentration. The extent of this increase in hepatic protein synthesis was sufficient at one point to compensate for the fall in estimated muscle protein synthesis, so that the sum total in the two tissues was maintained.     

FLOW OF GLUCOSE CARBON INTO BRAIN LIPIDS IN ADULT RATS FOLLOWING FOOD DEPRIVATION

Abstract— Rates of flow of glucose carbon in vivo into brain cholesterol, phospholipids, cerebrosides and gangliosides and concentrations of these lipids in the brain, were determined in adult rats after various periods of food deprivation. The rates were calculated from two measurements, the curve representing the decrease of plasma [¹⁴C]glucose specific activity with time and the specific activity of the brain lipid 180 min after intravenous injection of a tracer dose of d -[U-¹⁴C]glucose. Specific activities of brain lipids in rats deprived of food for 72h were significantly higher than in postabsorptive rats which were treated with the same dose of [¹⁴C]glucose. These higher specific activities were interpreted as a result of more labelled glucose available to lipid synthesis in the brain of fasted rats due to the substantial decrease in the rate of irreversible disposal of glucose by the whole body, commonly observed in fasted animals. The possibility that the higher specific activity values resulted from enhanced synthesis of brain lipids from glucose was ruled out since no changes were observed in the rate of flow of glucose carbon into brain lipids after food deprivation. The rate of flow of glucose carbon into gangliosides (15.4 ng C/min/mg C) was more than twice as fast as into either phospholipids or cerebrosides and about 4 times as fast as into cholesterol. The rates of carbon flow were used to calculate half lives of glucose carbon in the different classes of brain lipids. These half life values were 31 days for gangliosides, 72 days for phospholipids, 82 days for cerebrosides and 133 days for cholesterol. The results suggest that the synthesis of brain lipids from glucose is not affected by prolonged starvation in the adult rat.     

Protein turnover in adipose tissue from fasted or diabetic rats

Protein synthesis and degradation in vitro were compared in epididymal fat pads from animals deprived of food for 48 h or treated 6 or 12 days prior with streptozotocin to induce diabetes. Although both fasting and diabetes led to depressed (-24% to -57%) protein synthesis, the diminution in protein degradation (-63% to -72%) was even greater, so that net in vitro protein balance improved dramatically. Insulin failed to inhibit protein degradation in fat pads of these rats as it does for fed animals. Although insulin stimulated protein synthesis in fat pads of fasted and 12 day diabetic rats, the absolute change was much smaller than that seen in the fed state. The inhibition of protein degradation by leucine also seems to be less in fasted animals, probably because leucine catabolism is slower in fasting. These results show that fasting and diabetes may improve protein balance in adipose tissue but diminish the regulatory effects of insulin.     

In vivo studies on pathways for the biosynthesis of lecithin in the rat

The in vivo biosynthesis of lecithin in rats has been studied with the precursors choline-1,2-(14)C, ethanolamine-1,2-(14)C and methionine-CH(3)-(14)C or -CH(3)-(3)H. Lecithin synthesis from choline is rapid in all organs. No sex difference was observed in this pathway. The biosynthesis of lecithin by methylation of phosphatidyl ethanolamine is of quantitative significance in the liver, but not in extrahepatic tissues. More lecithin is synthesized by this pathway in female rats. In liver the lecithin synthesized via both pathways enters a common pool which is in rapid equilibrium with lecithin of blood plasma. A sex difference in the utilization of radioactive ethanolamine for the formation of phosphatidyl ethanolamine was observed (greater utilization in the female). Incorporation of ethanolamine into phospholipids of extrahepatic tissues was slow in both sexes. With labeled methionine as precursor the liver cytidine diphosphate (CDP) choline had a specific activity identical with that of liver lecithin after 20 min, while the specific activity of phosphoryl choline remained low. With labeled choline as precursor the phosphoryl choline reached a specific activity 50 times that of lecithin after 20 min, while the specific activity of CDP choline was only four times that of lecithin. These findings indicate that the reaction: CDP choline + diglyceride right harpoon over left harpoon phosphatidyl choline + CMP is freely reversible in vivo.     

Rates of rat liver RNA degradation in vivo as determined from cytidine release during brief cyclic perfusion in situ.

The breakdown of RNA and of long-lived proteins in rat liver is believed to occur largely within the lysosomal-vacuolar system. Both processes are induced by amino acid lack and suppressed by insulin, and in all circumstances a consistent lag of 15-20 min was observed between the introduction of a physiological regulator and onset of the degradative response. This lag has allowed us to determine rates of liver RNA degradation in vivo during brief cyclic perfusions, as was done previously for long-lived-protein breakdown [Hutson & Mortimore (1982) J. Biol. Chem. 257, 9548-9554]. Degradation was measured from the release of [14C]cytidine in livers of rats previously labelled in vivo with [6-14C]orotic acid. Release was linear and unaffected by physiological regulators between 2 and 12 min of perfusion. In contrast with protein breakdown, no short-lived component was observed. In animals trained to feed between 16:00 and 20:00 h, the content of liver RNA fell at an average rate of 0.26 mg/h per 100 g initial body wt. between 07:00 and 16:00 h, a loss that was within 9% of that predicted from the net release (total release minus reutilization) of cytidine in vivo. In addition, the total rate of RNA degradation determined at the end of the meal was only 12% of that at the start of the post-absorptive period 14 h later (2.1 versus 17.1%/day). This finding is fully consistent with a lysosomal mechanism for RNA degradation, since autophagy is strongly suppressed by food intake. This approach provides a comparatively simple means of approximating moment-to-moment rates of RNA degradation in the rat liver in vivo.     

Glutamine synthetase in cultured whole retinas from the embryonic chick. Role of protein and RNA syntheses in 4 degrees C storage enhancement

Glutamine synthetase (GS) activity is enhanced in cultured whole retinas when a 72 h incubation at 37 degrees C is preceded by storage at 4 degrees C for 2-24 h. This enhancement occurs even in the absence of glucocorticoids and is maximal in retinas from 11 to 14 d embryos. In comparison, cortisol-induced increases in retinal GS activity at 37 degrees C are optimal in retinas from 8 to 12 d embryos. This study, using cycloheximide (an inhibitor of protein synthesis) and cordycepin (an inhibitor of RNA synthesis), indicates that both protein and RNA synthesis are required for the 4 degrees C storage enhancement of GS activity. The necessary RNA synthesis occurs within the first 48 h following transfer to 37 degrees C and does not require concomitant protein synthesis. Uridine uptake, but not incorporation into trichloroacetic acid-precipitable material, is increased by initial 4 degrees C storage when compared with whole retina controls incubated at 37 degrees C for the total time. In contrast, both uptake and incorporation of amino acids are increased in 4 degrees C-stored retinas for as long as 72 h subsequent to transfer from 4 to 37 degrees C. This suggests that enhancement GS activity may arise from a combination of elevated general protein synthesis and specific messenger-RNA synthesis following 4 degrees C storage.