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.     

Hormonal triggering of the diurnal variation of sterol carrier protein

Rat liver sterol carrier protein (SCP) is a major intracellular protein regulating lipid metabolism and transport. During a dark-light cycle, SCP undergoes a dramatic diurnal variation in synthesis and level, reflecting translational events. Several hormones participate in the control of SCP synthesis. Insulin was implicated when the circadian rhythm of SCP was lost in both diabetes and fasting, states where insulin is low. After a 12-h fast the amplitude of the diurnal rhythm is diminished; after a 48-h fast it disappears, although SCP synthesis and level remain high. When endogenous insulin secretion is increased in fasted rats by glucose administration, SCP increases 2-fold in less than 30 min. When food intake is manipulated, but the dark-light cycle is unchanged, the circadian rhythm of SCP corresponds to feeding patterns and not light cycling. During feeding, increases in SCP are triggered following the expected increase in serum insulin. However, SCP is rapidly and significantly elevated in response to insulin only when glucocorticoids are normally high or increased by injection of the synthetic glucocorticoid, dexamethasone. Hepatocyte SCP levels are also induced by a combination of insulin and dexamethasone (2.3-fold) or insulin alone (1.3-fold). Dexamethasone alone causes a striking depression of SCP (2.4-fold). Thus, insulin is a major regulator of the diurnal variation of SCP synthesis. Glucocorticoids and other hormones (e.g. triiodothyronine) are also essential for maximum induction of SCP but play permissive roles.     

Circadian rhythm of blood glucose and tissue glycogen in fed and fasted rats

Blood glucose and tissue glucogen circadian rhythms were determined in male Wistar rats adapted 3 weeks to an artificial lighting regimen of 12 hours' light and 12 hours' darkness. Over a period of 24 hours we examined at 3-hour intervals the blood glucose concentration and the glycogen content of the liver, heart, skeletal muscle (quadriceps femoris) and white (epididymal) and brown (interscapular) adipose tissue of fed rats and rats fasted for 24 hours. The experiments were carried out in the autumn and the results were evaluated statistically by an analysis of variance and the cosinor test. The blood glucose level and the glycogen concentration in all the given tissues, in both fed and starved rats, displayed rhythmic oscillations with a 24- or 12-hour period in the course of the day, with the exception of glycogen in the white adipose tissue of fed rats, in which cosinor analysis failed to demonstrate any rhythm. One day's fasting did not affect the character of circadian rhythm.     

Effect of thyroid hormones on the activity of hepatic glucose-6-phosphatase in fed and fasted rats

The action of thyroid hormones on hepatic glucose-6-phosphatase was studied in rats. Fed and 24-h fasted rats received T3 (10 micrograms/day) or T4 (25 micrograms/day) 1 h, 1 or 3 days before sacrificing. In addition a group of fed rats was treated with T4 for 7 and 14 days. The glucose-6-phosphatase activity was measured in the isolated microsomes prepared from the liver. The intactness of the microsomal preparation was checked using 2 mM mannose-6-phosphate as a substrate. In fed rats a single injection of T3 or T4 augmented the activities of the translocase and hydrolase components of glucose-6-phosphatase provided that the rats were killed 24 h after the administration of hormone. This effect was more pronounced in animals treated for 3-14 days. As expected, fasting per se increased the activities of both components of the enzyme. Moreover, in fasted rats treatment with T3 and T4 for 3 days further augmented the activities of the translocase and the hydrolase components of glucose-6-phosphatase. In fed animals T3 and T4 increased the latency of the enzyme whereas in fasted animals thyroid hormones increased the activities of the translocase and hydrolase components in parallel, maintaining the level of latency of the enzyme system. Administration of T3 and T4 increased blood glucose level in fasted rats after one day, while in fed rats a significant hyperglycaemia appeared after 7-14 days of treatment. In conclusion, T3 and T4 increase the activities of the translocase and hydrolase components of hepatic glucose-6-phosphatase in fed and fasted rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian patterns of plasma leptin,insulin and glucose concentration in the toad-headed lizard Phrynocephalus versicolor

To understand the circadian rhythms of the glucose metabolism and related hormones in desert reptiles, we studied the daily changes in plasma leptin, insulin, and glucose in the toad-headed lizard, Phrynocephalus versicolor, in the semi-desert of the Alashan Plateau. Blood samples were obtained from groups of 20 lizards (about equal sex ratio for each group) at 4-h intervals throughout a 24-h period. Leptin, insulin, and glucose levels were determined using ELISA, RIA, and the glucose oxidase method, respectively. Ambient temperature and humidity were also measured when the blood samples were taken. We found that: (1) there was a significant difference in plasma leptin concentrations at different times of day; (2) no significant circadian rhythm could be measured for plasma insulin and glucose; (3) ambient temperature, humidity, and body condition had no significant effect on plasma leptin, insulin, and glucose levels; and (4) there were significant correlations between plasma glucose concentrations and leptin concentrations 8 h later, and between insulin concentrations and leptin concentration 8–12 h later. These results suggested that the secretions of leptin and insulin played an important role in glucose metabolism in reptiles in an arid environment.     

The stimulation of lipogenesis in white adipose tissue from fed rats by corticosterone

The direct effects of a physiological concentration of corticosterone (50 ng ml-1) in presence of insulin (200 microU ml-1) on lipid synthesis and CO2 production from glucose and glycerol release were evaluated in vitro in white adipose tissue after pre-incubation with the hormones. Lipid synthesis was 27% higher after 24 h and 66% higher after 48h pre-incubation with corticosterone and insulin compared with insulin alone. Basal and adrenaline-stimulated glycerol release and CO2 production were unchanged after pre-incubation with both hormones compared with insulin alone. We propose that corticosterone acts as a pro-lipogenic hormone on adipose tissue in the fed rat, in contrast to its glucose sparing effects in the fasted animal.     

Metabolic and hormonal response to short term fasting after endurance training in the rat

The metabolic and hormonal response to short term fasting was studied after endurance exercise training. Rats were kept running on a motor driven rodent treadmill 5 days/wk for periods up to 1 h/day for 6 wk. Trained and untrained rats were then fasted for 24 h and 48 h. Liver and muscle glycogen, blood glucose, lactate, beta OH butyrate, glycerol, plasma insulin, testosterone and corticosterone were measured in fed and fasted trained and untrained rats. 48 h fasted trained rats show a lower level of blood lactate (1.08 +/- 0.05 vs 1.33 +/- 0.08 mmol/l-1 of blood glycerol (1 +/- 0.11 vs 0.84 +/- 0.08 mmol/l-1), and of muscle glycogen. There is a significant increase in plasma corticosterone in 48 h fasted trained rats from fed values. Plasma testosterone decreases during fasting, the values are higher in trained rats. Plasma insulin decreases during fasting without any difference between the two groups. These results show higher lipolysis, and decreased glycogenolysis in trained animals during 48 h fasting. The difference between the groups in steroid hormone response could reduce neoglucogenesis and muscle proteolysis in trained animals.     

Circadian rhythms of food intake, plasma glucose and insulin levels in fed and fasted rabbits.

The rhythm of circadian variations of plasma insulin level was similar in 48 h fasted and in fed rabbits; however, the amplitude of variations was less important in fasted animals. Plasma glucose level did not change during circadian cycle. In conclusion, we showed in rabbit a circadian rhythm of insulin with two maxima: one diurnal and the other nocturnal.     

Metabolic control of food intake and estrous cycles in syrian hamsters. I. Plasma insulin and leptin

The "adipostat hypothesis" refers to the idea that circulating hormone concentrations reflect levels of body adiposity and act as signals to control food intake and reproduction. Implicit in the adipostatic hypothesis are the following two assumptions: 1) plasma levels of adipostatic hormones accurately reflect body fat content and 2) decreased plasma concentrations of adipostatic hormones necessarily result in increased food intake and inhibited reproductive processes. The present experiments are designed to test these assumptions. Fat and lean Syrian hamsters were either fasted for 12, 24, 36, or 48 h or allowed ad libitum access to food. Contrary to the first assumption, plasma leptin and insulin levels in fat hamsters dropped dramatically by 12 h after the start of a fast, with no significant change in body fat content and no postfast hyperphagia. Lean hamsters showed anestrus after a 48-h fast but not after a 24-h fast. Contrary to the second assumption of the lipostatic hypothesis, lean hamsters fasted for 24 h and then refed for the next 24 h had leptin levels that were not significantly elevated compared with those of 48-h-fasted hamsters. Thus, in adult female Syrian hamsters, plasma leptin concentrations do not accurately reflect body fat content under all conditions; normal estrous cyclicity does not necessarily require plasma leptin concentrations higher than those of fasted hamsters; and decreased plasma leptin levels do not result in increased food intake.     

Restricted feeding schedules alter the circadian rhythms of serum insulin and gastric inhibitory polypeptide

Insulin and gastric inhibitory polypeptide (GIP) have a circadian rhythm of secretion that is altered by various feeding schedules. We acclimated rats over 3 weeks to one of 6 different feeding schedules. They were then killed at intervals over one feeding cycle. Blood was collected, and their stomachs were weighed. Hormones in the serum were measured by radioimmunoassay. When highest and lowest measured concentrations were compared in ad libitum fed rats, insulin more than doubled (445 +/- 50 to 993 +/- 180 pg/ml) and GIP more than tripled (682 +/- 108 to 1964 +/- 145 pg/ml) during a 24-h period. With restricted schedules, concentrations correlated with the feeding schedule, not the light-dark cycle. Hormone levels rose higher during feeding and fell lower with fasting than in ad lib fed rats. For example, GIP in one study fluctuated from 468 +/- 22 to 6433 +/- 432 pg/ml. In another example, insulin ranged from 30 +/- 5 to 2259 +/- 406 pg/ml during a 24-h period. However, insulin did not always correlate well with stomach weight. Circadian rhythms occurred for insulin with all feeding schedules and for GIP with all schedules except fasted rats. This finding implies an endogenous insulin rhythm, whereas food intake controls GIP secretion. Thus, disruption of normal circadian cycles of feeding may yield misleading information about gut hormone secretion.     

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.     

Influence of fasting on glycogen depletion in rats during exercise

We recently observed that a 24-h fasted group of rats could run longer than an ad libitum fed control group before becoming exhausted. Because of the demonstrated importance of glycogen levels and free fatty acid availability during endurance exercise, we have investigated several parameters of carbohydrate and lipid metabolism in exercised and nonexercised rats that were either fed ad libitum or fasted for 24 h. A 24-h fast depleted liver glycogen, lowered plasma glucose concentration, decreased muscle glycogen levels, and increased free fatty acid and beta-hydroxybutyrate concentrations in plasma. During exercise the fasted group had lower plasma glucose concentration, higher plasma concentration of free fatty acids and beta-hydroxybutyrate, and a lower muscle glycogen depletion rate than did the ad libitum fed group. Since fasted rats were able to continue running even when plasma glucose had dropped to levels lower than those of fed-exhausted rats, it seems unlikely that blood glucose level, per se, is a factor in causing exhaustion. These results suggest that fasting increases fatty acid utilization during exercise and the resulting "glycogen sparing" effect may result in increased endurance.     

Long-term infusion of nutrients (total parenteral nutrition) suppresses circulating ghrelin in food-deprived rats

BACKGROUND: Ghrelin derives from endocrine cells (A-like cells) in the stomach (mainly the oxyntic mucosa). Its concentration in the circulation increases during fasting and decreases upon re-feeding. This has fostered the notion that the absence of food in the upper gastrointestinal (GI) tract stimulates the secretion of ghrelin. The purpose of the present study was to determine the concentration of ghrelin in serum and oxyntic mucosa after replacing food with intravenous (iv) infusion of nutrients for 8 days using the technique known as total parenteral nutrition (TPN) MATERIALS AND METHODS: Male Sprague-Dawley rats (200-250 g) were given nutrients (lipids, glucose, amino acids, minerals and vitamins) by iv infusion for 8 days during which time they were deprived of food and water; another group was deprived of food for 24-48 h (fasted controls), while fed controls had free access to food and water. Serum ghrelin, gastrin and pancreastatin concentrations were measured together with the ghrelin content of the oxyntic mucosa. Plasma insulin and glucose as well as serum lipid concentrations were also determined. RESULTS: Fasted rats had higher serum ghrelin than TPN rats and fed controls. The oxyntic mucosal ghrelin concentration (and content) was lower in TPN rats than in fasted rats or fed controls. The serum gastrin and pancreastatin concentrations were lower in TPN rats and fasted rats than in fed controls. The plasma insulin concentration was 87 pmol/l+/-8 (SEM) in TPN rats compared to 101+/-16 pmol/l in fed controls; it was 26+/-14 pmol/l in fasted rats. The basal plasma glucose level was 11+/-0.6 mmol/l in TPN rats and 12+/-0.8 mmol/l in fed controls; it was 7+/-0.3 mmol/l in fasted rats. In TPN rats, the serum concentrations of free fatty acids, triglycerides and cholesterol were increased by 100%, 50% and 25%, respectively, compared to fed controls. Fasted rats had higher circulating concentrations of free fatty acids (20%) and lower concentrations of triglycerides (-40%) than fed controls; fasted rats did not differ from fed controls with respect to serum cholesterol. CONCLUSION: The circulating ghrelin concentration is high in situations of nutritional deficiency (starvation) and low in situations of nutritional plenty (free access to food or TPN). The actual presence or absence of food in the GI tract seems irrelevant. Circulating insulin and glucose concentrations did not differ much between TPN rats and fed controls; serum lipids, however, were elevated in the TPN rats. We suggest that elevated blood lipid levels contribute to the suppression of circulating ghrelin in rats subjected to TPN for 8 days.     

Circadian oscillations of thyroid hormones, insulin and glucagon in the blood of laboratory rats in the course of the year

The concentration of thyroid hormones and insulin in the serum that of glucagon in the plasma and glucose in the blood was determined at 3-hour intervals, in the course of one day, at different times of the year, in adult male rats (Wistar strain) of a conventional breed kept under standard conditions with a 12:12 h light:dark regimen. The lowest thyroid hormone and glucagon concentrations and the highest insulin and blood glucose levels were found in the winter. In various seasons, circadian oscillations of the thyroid hormones culminated in the dark part of the day and that of glucagon in the light part, with the exception of the autumn. Circadian oscillation of insulin levels culminated at different times of day during the year. The pronounced changes found in the examined hormones in the laboratory rat at various times of the year are evidently the outcome of adaptation to changes in external environmental conditions during phylogenesis. In the polarity of changes in these indicators between the winter and the summer or the spring, the laboratory rat bears the closest resemblance to wild mammals.     

Circadian rhythms of glucose, triiodothyronine and insulin in relation to composition of diet and meal-timing in rats

A possible role of nutrition as a synchronizer has been recently emphasized, particularly the effect of controlled diet composition of circadian variations of many functions of the organism. The aim of the study was to determine whether diet composition (low or high fat diet) could be a synchronizer of circadian rhythms of glucose, insulin and triiodothyronine. The effect of diet composition on diurnal changes in glucose tolerance was also tested. After 24 h starvation period the biochemical parameters in the serum were measured every 4 hours i.e. 600, 1000, 1400, 1800, 2200, 200. Glucose tolerance was tested between 500-700, 1100-1300, 1700-1900 and 2300-100. The circadian variations of glucose and insulin levels were observed in animals fed both diets. An increase of glucose level was noted during reduced activity of the animals and the acrophase was recorded at 1659 h (low fat diet) and 1514 h (high fat diet). The acrophase of insulin level was observed at 526 h (low fat diet) and 352 hrs (high fat diet) in the period of activity of the animals. Circadian changes of triiodothyronine level were noted in animals fed the low fat diet only, the acrophase appeared at 1447. Simultaneously, no variations occurred in animals fed the high fat diet. A consequence of the high fat diet was also a disappearance of diurnal variations in glucose tolerance test at 60, 90 and 120 min.     

Circadian Variation in Alloxan Sensitivity of Mice as Indicated by Mortality and Blood Glucose Alteration

The objective of this study was to determine in mice if a time-dependent pancreatic β-cell susceptibility to alloxan could be correlated to daily changes in blood glucose levels and to monitor the pattern of blood glucose at various times of day as mice became diabetic. Food was removed from mice standardized to a 12-h light:dark cycle (lights on at 0600 h CST, during the month of June) at 12 h before subcutaneous injection with 0.27 mg/g of alloxan. Six groups of 30 fasted mice were injected at 4-h intervals. Blood glucose levels were measured from each group immediately prior to injection, and at 2, 4, 8, 12, 24, 48, and 216 h after treatment. Animals receiving alloxan during the early- to middark period had an increase in blood glucose after 2 h, followed by a decline to hypoglycemic levels between 4 and 8 h, and recovery to hyperglycemic levels 48 h after alloxan exposure. Three and 30% of these animals were dead at 8 and 48 h, respectively. Mice treated during the midlight span had decreased blood glucose levels 2 h after alloxan treatment followed by an increase to diabetic hyperglycemia within 48 h. Twenty-three and 70% of the animals treated at 1430 h were dead at 8 and 48 h, respectively. At 216 h, total mortality was 45.6% and 81 of the 98 surviving mice were hyperglycemic. These data suggest a greater sensitivity to alloxan during the midlight resting period of the mice. This may be the result of increased sensitivity to the insulin released from the β cells when alloxan was given during the light span.     

Author index to volume 140

The effects of varied durations of food deprivation on the rates and kinetics of glucose phosphorylation by isolated rat hepatocytes have been examined. Glucokinase activity was measured concurrently in extracts from these cells prepared from livers of rats which had fasted for 0, 24, 48 and 72 h. Significant levels of hepatocyte glucose phosphorylation were noted even when glucokinase levels were extrapolated to zero. The K_0.5-glucose value of 33 mM in cells from fed rats increased to 48 mM after a 72-h fast. It is concluded that a high K_0.5 glucose-phosphorylating enzyme or enzymes compensatory to insulin-dependent glucokinase is/are involved in rat liver glucose phosphorylation.     

Effect of cold-acclimation on oxygen uptake and glucose production of perfused duckling liver

The control of hepatic metabolism by substrates and hormones was assessed in perfused liver from young Muscovy ducklings. Studies were performed in fed or 24-h fasted 5-week-old thermoneutral (25 degrees C; TN) or cold-acclimated ducklings (4 degrees C; CA) and results were compared with those obtained in rats. Basal oxygen uptake of perfused liver (LVO2) was higher after cold acclimation both in fed (+65%) and 24-h fasted (+29%) ducklings and in 24-h fasted rats (+34%). Lactate (2 mM), the main gluconeogenic substrate in birds, similarly increased LVO2 in both TN and CA ducklings and the effect was larger after fasting. Both glucagon and norepinephrine dose-dependently increased LVO2 in ducklings and rats, but cold acclimation did not improve liver response and liver sensitivity to norepinephrine in ducklings was even reduced in CA animals. Liver contribution to glucagon-induced thermogenesis in vivo was estimated to be 22% in TN and 12% in CA ducklings. Glucagon stimulated gluconeogenesis from lactate in duckling liver and the stimulation was 2.2-fold higher in CA than in TN fasted birds. These results indicate a stimulated hepatic oxidative metabolism in CA ducklings but hepatic glucagon-induced thermogenesis (as measured by LVO2) was not improved. A role of the liver is suggested in duckling metabolic acclimation to cold through an enhanced hepatic gluconeogenesis under glucagon control.     

Effect of starvation on nutrition and insulin secretion in pregnant rats and their fetuses

Pregnancy is thought to create a metabolic condition of accelerated starvation. To clarify this idea, the effect of fasting on pregnant rats (day 21 of gestation) and their fetuses was examined. Although pregnancy significantly increased plasma insulin, plasma ketone body concentrations in fed pregnant rats were higher than those of age-matched fed virgin rats. After 48 hr fasting (i.e., fasting during days 19-21 of gestation), plasma insulin was markedly decreased in virgin rats compared with term pregnant rats, while ketone bodies were significantly higher in pregnant rats than in virgin rats. Body weight was lower in fetuses from fasted mothers than those from fed mothers. Starvation also markedly diminished the insulin response to glucose in isolated, perfused pancreases in both virgin and pregnant rats. The amount of insulin released during glucose stimulation was greater in pregnancy, and the inhibitory effect of 48 hr fasting on insulin release was greater in virgin rats than in pregnant rats. It is possible, therefore, that in term pregnant rats a decrease in insulin release caused by fasting may cause more profound catabolism than in nongravid rats.