Diurnal variations in activity of four pyridoxal enzymes in rat liver during metabolic transition from high carbohydrate to high protein diet.

The diurnal variations in enzyme activities including tyrosine aminotransferase (TAT), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT) and serine dehydratase (SDH) have been studied in rats trained to a 2 hour meal feeding schedule (″2+22″) during metabolic transition from 12.5 to 60% protein diets over a period of 21 days. Although the maximal TAT activity on the first day was slightly lower compared with other days, both TAT and ODC activities adapted rapidly to the increased dietary protein from the first day. The responses of TAT and ODC to the food were so rapid that the maximal value was observed only 4 hrs after the onset of feeding. After each feeding ODC activity decreased rapidly after 4 hours, while TAT activity declined only after 6 hours had elapsed. No clear diurnal rhythm was observed in either OAT or SDH, though OAT activity tended to decrease from the beginning of the dark period and to resume a slow adaptation after about four hours. In contrast to ODC and TAT both OAT and SDH required about 7 days to fully adapt to the high protein diet. The activities of the four enzymes were also compared after 4 groups of rats had been adapted to the ″2+22″ feeding of 12.5, 30 and 60% protein diets and to 60% diet, $\text{ad}$$\text{libitum}$, respectively. The enzyme activities were not directly proportional to the protein content of the diets although higher activity was observed on the high protein diets. The diurnal variations in both TAT and ODC were observed in all ″2+22″ groups although the timing of the peak values were slightly different from each other. The maximal activities of TAT were found at earlier times in 12.5 and 30% protein groups than in the 60% protein group. The peak time for ODC activity was found at a later time in the 12.5% protein group than in rats fed 30% and 60% protein. $\text{Ad}$$\text{libitum}$ rats fed 60% protein maintained relatively high levels of TAT activity compared to the rats on the schedule. However, the maximal activity of ODC on the 60% ″2+22″ protein diet $\text{ad}$$\text{libitum}$ was so low that a diurnal rhythm was not clearly evident.     

Fasting does not abolish the diurnal oscillation of ornithine decarboxylase activity in Morris hepatoma 5123-C.

The activities of ornithine decarboxylase (ODC) and tyrosine aminotransferase (TAT) were determined under conditions of feeding or fasting in the hepatomas and livers of rats bearing Morris hepatoma 5123-C. Prior to killing, the animals were entrained to a schedule of 12 hours of light followed by 12 hours of darkness with food (60% protein) available only during the first two hours of the dark period. With food available, ODC and TAT activities displayed diurnal oscillations in hepatomas and host livers, and in the livers of control (non-tumor bearing) animals, characterized by rapid increases in enzyme activity coincident with the onset of feeding followed by a decline to pre-feeding levels. When food was withheld the increase in ODC activity in host and control livers, and TAT activity in hepatoma, host and control livers was not evident. However, withholding food did not abolish the diurnal oscillation of ODC activity in hepatoma 5123-C.     

Induction of hepatic microsomal calcium uptake by glucocorticoids

Hepatic microsomes take up calcium in the presence of ATP and oxalate. In either fed or fasted adrenalectomized rats injections of dexamethasone 18 hours and then again 1 hour prior to sacrifice increased uptake of calcium by microsomes. Injections of estradiol had no similar effect indicating that the stimulation might be specific to glucocorticoids. Injection of Actinomycin D, an inhibitor of protein synthesis, 1 hour prior to dexamethasone administration resulted in a complete block of the stimulation. It is therefore likely that the increased calcium uptake is due to the induction of the microsomal calcium activated ATP-ase. The onset of this effect occurred later than the induction of tyrosine-amino transferase (TAT). The present data, in conjunction with the previous demonstration of glucagon stimulation and insulin inhibition of this system, indicates that microsomes might serve as a modifier of intracellular calcium distribution.     

Diurnal variation in the feeding pattern of rabbits

Laboratory rabbits maintained under controlled lighting and fed $\text{ad libitum}$ exhibit a weak but consistent diurnal fluctuation in feed intake. There are two major periods of eating; at the beginning and the end of the light period. This results in similar feed consumption for the light and dark periods. Water intake shows a similar diurnal variation to that of feed intake. If the normal lighting cycle is retarded by 6 hours, the animals adjust their diurnal rhythm of feeding behaviour to the new lighting cycle within 8 to 15 days. Comparative studies on rats are included.     

The inducibility of tyrosine aminotransferase in monolayer cultures of hepatocytes from neonatal rats

Hepatocytes from neo- and postnatal rat liver were isolated, purified from non-hepatocytes (erythropoietic cells), and cultured in sufficient quantity to investigate enzyme inducibility. Tyrosine aminotransferase (TAT) in neo- and postnatal hepatocytes was induced by maximally responsive doses of glucagon, dexamethasone, DB-cAMP, theophylline, and combinations thereof. In cultures from newborn parenchymal cells TAT enzyme-specific activity showed only a moderate inducibility; however, responsiveness to the combination was fully developed 10 days after birth and did not differ from values found in adult liver cells. The results also show the existence of the "permissive" effect of glucocorticoid during postnatal age, and indicate that the development of a possibly involved receptor complex for the induction of TAT is largely completed 5-10 days after birth.     

Cell-cycle regulation of insulin-stimulated tyrosine aminotransferase activity in rat hepatoma cells

Amongst the proteins that are subjected to variation during the cell division cycle few are under hormonal regulation. The variation in amount of tyrosine aminotransferase (TAT) in the hepatic tissue is under the control of glucagon, glucocorticoids and insulin. It has been reported that the inducibility of TAT activity by dexamethasone in rat hepatoma (HTC) is limited to the late G1 and the S portions of the cell cycle. Evidence is presented in this report that in the rat hepatoma Fao, insulin (which has the capability to promote both cell growth and hormonal effects via its own receptors) modulates the TAT activity during the cell cycle. The maximal insulin-stimulated induction of TAT activity was observed at the end of the G1 phase and then decreased as cells progressed through their mitotic cycle. The number of insulin binding sites per cell was decreased by only 30% during the same period of time. Furthermore, the extent of receptor autophosphorylation decreased in the same proportion, suggesting that insulin receptors remained functional through the whole cell cycle. In fact, another insulin-stimulated cellular function, neutral amino-acid transport, was not modified as cells progressed into the S phase. Hydroxyurea, which is known to prevent cell progression into the S phase, stabilized the insulin-induced TAT activity at its maximal level for several hours. Reciprocally, removal of hydroxyurea resulted in a concomitant decrease in TAT activity and reinitiation of DNA synthesis.     

Factors Affecting Developmental Increase of Tyrosine Aminotransferase in Rat Liver Immediately after Birth

Labeling with ³⁵S-methionine of dispersed hepatocytes prepared from neonatal rat liver and successive immunoprecipitation with antiserum against tyrosine aminotransferase (TAT) indicated that increase of TAT activity to a peak about 12 hours after birth and the decrease thereafter are mainly due to changes of TAT synthesis. Similar changes of TAT activity was also observed in the livers of premature neonates which were taken out by Caesarian section and nursed by foster mothers. This indicated that the appearance of TAT activity at this period is not an event programmed along with fetal development but is triggered by birth. The level of glucagon in neonatal plasma increased after birth. Administration of glucagon to neonates caused a great increase of TAT activity whereas the effect of dexamethasone was not so evident. These suggested that glucagon is an important factor affecting the abrupt appearance of TAT after birth.     

Circadian rhythm of rat hepatic cytochrome P-450 reductase.

The activity of cytochrome P-450 reductase was measured in liver microsomes prepared from adult male rats which had been surgically adrenalectomized, pinealectomized, pinealectomized-adrenalectomized, or sham adrenalectomized-pinealectomized and from intact controls. Rats of each class were killed at 1, 4, 6, 10 hours after the beginning of the light period and 1, 4, 6, 10 hours after the lights were turned off (dark period). The activity of cytochrome P-450 reductase shows a significant diurnal variation in the control group with minimum and maximum at 1 and 10 hours after dark, respectively. The rhythm was altered in the animals surgically treated and the average reductase activity was decreased.     

Study on the role of endogenous polyamines in glucagon, isoproterenol or serum-mediated induction of tyrosine aminotransferase in cultured heart cells

In confluent and serum-starved embryonic heart cell cultures, the addition of serum (10%), glucagon (GLU, 0.1 microM) or isoproterenol (ISO, 10 microM), causes the onset of ornithine decarboxylase (ODC) activity, with a maximum after 5-6 hr. This is paralleled by polyamine accumulation and by the induction of TAT, which, in the case of GLU and ISO, exhibits maximal activity at 4-3 hr respectively, followed by a net decline. Cyclic AMP (cAMP) also accumulates after exposure to GLU or ISO. However, under different conditions of ODC inhibition, serum fails to induce TAT, thus supporting a relevant role of cellular polyamines in serum action. Conversely, cAMP and TAT responses to GLU or ISO are markedly improved under prevention of polyamine accumulation, which also leads to a longer lasting TAT inducibility. The suggestion is made that polyamines are not required in the cAMP-dependent mechanism of TAT induction, but rather in the restoration of the basal activity of the enzyme.     

Changes in the sensitivity of lipogenesis in rat hepatocytes to hormones and precursors over the diurnal cycle and during longer-term starvation of donor animals

In rat hepatocytes freshly isolated from donor rats at different times of the day, the rates of lipogenesis (de novo fatty acid synthesis) varied with a diurnal periodicity. The maximal rate occurred approximately 5 hr after the end of the normal 8-hr feeding period and at this time was four- to fivefold higher than the minimum rate which occurred midway through the feeding period. A similar diurnal pattern of change persisted even when the supply of lipogenic substrate, present in the medium as pyruvate, was not limiting. Although insulin stimulated the basal rates of lipogenesis to different relative extents in hepatocytes isolated at different times of the day, in absolute terms the hormone had little effect on the overall pattern of change during the diurnal cycle. The presence of pyruvate protected lipogenesis against inhibition by glucagon. The degree of protection varied over the diurnal cycle. During the early stages of starvation (up to 24 hr) there was a continuous decline in the rate of hepatocyte lipogenesis, irrespective of whether insulin and/or lipogenic substrate (pyruvate) were available or not. After this time the decline in the rate of lipogenesis was much less rapid. Seventeen hr after removal of food from donor rats, a point was reached beyond which pyruvate was incapable of supporting the maximum basal rate of lipogenesis which occurred during the normal diurnal cycle of fed rats. After this time lipogenesis in the presence of pyruvate was inhibited by glucagon to a much greater relative extent than that observed during feeding. The results suggest that variations in the rate of lipogenesis over the diurnal cycle and during the first 24 hr of starvation could not be accounted for entirely by fluctuations in substrate availability. In contrast, changes which occurred subsequent to this (up to 43 hr of starvation) could be eliminated when lipogenic substrate was made more abundant. Longer periods of starvation were marked by a relative increase in the ability of glucagon to prevent the substrate-induced stimulation of lipogenesis.     

Ornithine decarboxylase activity and DNA synthesis in Morris hepatomas 5123-C and 7800.

The activities of ornithine decarboxylase (ODC) and thymidine kinase (TK) and the rates of DNA synthesis were determined in hepatomas and livers of rats bearing Morris hepatoma 5123-C or 7800 and entrained to a schedule of 12 hours of light followed by 12 hours of darkness, with food (60% protein) available only during the first 2 hours of the dark period. ODC activity in hepatoma 5123-C displayed a diurnal oscillation, increasing 2-fold during the feeding period and then rapidly decaying to 20% of the peak level. The livers of rats bearing hepatoma 5123-C exhibited a similar oscillation of ODC activity, with peak values lower than in the hepatomas but higher than in the livers of control (non-tumor bearing) animals. TK activity and the rate of DNA synthesis in hepatoma 5123-C were low during most of the dark period but increased rapidly towards the end of the dark period. DNA synthesis reached a plateau at the dark-light interface and then rapidly declined, but TK activity remained high during the light period. Similar studies on hepatoma 7800 established that ODC activity in this hepatoma did not oscillate but remained at low levels throughout the day. Similarly, host livers of rats bearing hepatoma 7800 did not exhibit the diurnal oscillation of ODC activity characteristic of liver from control rats, but showed a slow increase in activity followed by a plateau and a slow decline to base-line levels. DNA synthesis in hepatoma 7800 was constant throughout the day, whereas TK activity may have increased during the dark period. In the livers of control rats and animals bearing hepatoma 5123-C or 7800, TK activity and rate of DNA synthesis were at low levels at all times studied and appeared not to oscillate.     

Regulation of cytoplasmic acetyl-CoA and acetoacetyl-CoA synthetases by dexamethasone phosphate in rat liver and adipose tissue.

The activity of cytoplasmic acetyl-CoA synthetase and acetoacetyl-CoA synthetase in both liver and adipose tissue was measured in five groups of rats : fed, fasted, fasted and re-fed, fasted, re-fed and injected with dexamethasone during the re-feeding period, fed and injected with dexamethasone. Fasting was found to have a strong inhibitory effect on the activity of the two enzymes in both adipose tissue and liver, which could be abolished by re-feeding. In adipose tissue, dexamethasone prevented this re-establishment of normal enzyme activity but was without effect in the liver. It can therefore be concluded that dexamethasone inhibits the synthesis of acetyl-CoA and acetoacetyl-CoA synthetases in adipose tissue.     

Diurnal rhythm of rat liver mRNAs encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase. Correlation of functional and total mRNA levels with enzyme activity and protein

Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase exhibits a diurnal rhythm of activity which coincides with a diurnal rhythm of reductase protein and reductase mRNA levels. This diurnal rhythm of reductase activity, polypeptide mass, and mRNA exists in rats fed a normal diet (unsupplemented rat chow) and in rats fed a diet supplemented with cholestyramine plus or minus mevinolin. Levels of reductase protein were determined by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Reductase mRNA was measured by in vitro translation or blot hybridization of liver RNA. Functional reductase mRNA levels in rats fed a normal diet were approximately 10-fold higher during the middle of the dark cycle than during the middle of the light cycle. Maximum induction of functional reductase mRNA was observed in rats fed cholestyramine and mevinolin. This latter level was 157-fold higher than the level measured at the diurnal low point in rats fed a normal diet. Blot hybridization of liver RNA showed two predominant mRNAs of 4.6 and 4.2 kilobase pairs and a minor species at 6.9 kilobase pairs. These mRNAs exhibited a diurnal rhythm for rats on all three diets and reached peak levels during the 12-h dark period. These data indicate that the diurnal rhythm of reductase mass and activity is closely paralleled by the level of its mRNA.     

Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.     

Circadian rhythms of urinary electrolyte excretion in freely moving rats

In 8 freely moving rats the circadian variation in the eletrolyte excretion was studied. Food was available during either the dark or the light period. The lights were on from 0800–2000 hr. Potassium, phosphate and magnesium showed peak excretion values during the dark period under both feeding conditions, although the maxima occurred 2.5 hr earlier when the rats were fed during the light period; minimum excretion was recorded just prior to feeding. Sodium excretion followed a different pattern; for animals fed during the night, maximum excretion occurred almost at the end of the dark period and minimum excretion at the start of the feeding period. For day-fed animals these values were recorded 5 and 4 hours earlier, respectively. Calcium excretion reached a maximum after the feeding period and a minimum shortly after the onset of feeding. From this study it can be concluded that the peak excretions of potassium, phosphate and magnesium are only slightly influenced by the feeding regimen, indicating that they depend mainly on an endogenous rhythm. In contrast, the minimum excretion of these ions is determined by feeding. For calcium maximum as well as minimum excretion is correlated with the feeding regimen. The excretion pattern of sodium differs from that of calcium, as well as potassium, phosphate and magnesium, indicating that it is controlled by a different mechanism.     

Induction of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes. Requirement for insulin and dexamethasone

To determine the relative contributions of glucose, insulin, dexamethasone, and triiodothyronine to the induction of hepatic glucose-6-phosphate dehydrogenase, hepatocytes isolated from normal or adrenalectomized rats, either fasted or fed, were examined in culture. Addition of insulin (42 milliunits/ml, 0.9 microM) and dexamethasone (1 microM) to hepatocytes obtained from 3-day-fasted rats and cultured for 48 h in serum-free Dulbecco's medium resulted in a 7- to 11-fold increase in Glc-6-P dehydrogenase specific activity compared with a 2- to 3-fold increase in activity in control cultures incubated without added hormones. The effects of insulin and dexamethasone were independent of DNA synthesis, dose-dependent, and additive; each contributing about one-half of the total response. Medium glucose was neither sufficient nor necessary for the insulin- or dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Addition of triiodothyronine (10 microM) preferentially blocked the dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Insulin failed to stimulate the induction of Glc-6-P dehydrogenase in hepatocytes obtained from normal fed rats or from fasted and fed adrenalectomized rats. However, insulin caused a significant increase in the Glc-6-P dehydrogenase specific activity of these cells when dexamethasone was concurrently added to the culture medium.     

Regulation of hepatic malic enzyme by perfluorodecanoic acid

Perfluorodecanoic acid (PFDA) administration to adult male rats increased both the activity of hepatic malic enzyme and liver weight in a dose-dependent manner. Hepatomegaly and augmented activity of malic enzyme in liver were apparent within one day following PFDA administration and reached a plateau by three days posttreatment. Malic enzyme quantity per liver in PFDA-treated rats was elevated within one day following dosing and increased continually throughout five days posttreatment. Administration of PFDA to rats in the fed state also led to an increase in the specific activity of hepatic malic enzyme that peaked at three days following dosing. When compared to the fed condition, rats fasted for 48 hours had a decrease in both relative liver weight and the quantity of supernatant protein per liver. The total activity (U/liver) and specific activity of malic enzyme in the liver were also reduced in the fasted state. During the 24 hours after treatment in rats fasted for 48 hours, the body weight as well as the absolute and relative liver weight of animals receiving vehicle declined continuously in the absence of feed. Following the administration of PFDA to fasted rats, body weight was maintained until eight hours posttreatment but then declined at a rate similar to that found with the vehicle-treated group. Absolute and relative liver weight in PFDA-treated rats were increased significantly at eight hours posttreatment when compared to those receiving vehicle, and this increment was maintained throughout the rest of the 24 hours following dosing. While the activity and enzyme content of hepatic malic enzyme decreased in the vehicle-treated group, administration of PFDA to rats fasted for 48 hours prevented their decline. The specific activity of hepatic malic enzyme in 48 hours fasted rats receiving PFDA was also elevated significantly at 16 hours posttreatment. Thus, the administration of PFDA to the adult male rat in both the fed and fasted nutritional states was found to regulate hepatic malic enzyme by not only increasing enzyme quantity but also by augmenting the specific activity, (ie, catalytic state) of the enzyme.     

Beta-(2-furyl)-acryloyl phosphate hydrolase activity in insect cell surface membranes

In primary cultures of adult rat hepatocytes, dexamethasone (10^?5M) induced tyrosine aminotransferase (TAT) 24 h after its addition. Glucagon (10^?7M) alone had no effect, but strongly enhanced the induction by dexamethasone. Glucagon could be replaced by butyryl cyclic-AMP (10^?4M), which caused about 20-fold increase in activity. In contrast to many previous reports that insulin induced TAT activity $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$, it inhibited the inductions of TAT by dexamethasone and dexamethasone plus glucagon 24 h after its addition. However, insulin significantly induced TAT activity in the early pahse, 4 h after its addition. Dose-response curves of the effect of insulin on TAT activity showed reverse relations to activity in early and late phase. These results show that TAT activity is regulated by insulin in a two phase fashion.     

Diurnal rhythmicity of mammalian DNA-dependent RNA polymerase activities I and II: dependence on food intake

Diurnal variations of DNA-dependent RNA polymerase activities I and II have been found in rats maintained under controlled feeding schedules. RNA polymerase I has two peaks of activity in a 24-hours cycle: one 6 hours after the onset of dark period and a second one in the middle of the light period. Polymerase II shows only one peak coinciding with the first one of polymerase I. These diurnal fluctuations are not present in the liver of rats denied food on the day of the experiment. Both polymerases do not exibit different optima for divalent metal ions and ionic strength in the different feeding conditions studied.