Diurnal rhythms of hepatic carbohydrate metabolism during development of the rat

The ;8+16' feeding schedule (8h feeding and 16h without food in each 24h cycle) was applied to nursing mother rats to study enzyme development in neonatal rats in the absence of solid food. A ;16+8' suckling schedule (16h with the mother and 8h while the mother is fed in a separate cage) was used to show that the increases in pyruvate kinase, glucokinase and aldolase B activities that occur in the late suckling period of liver development do not require the intake of solid food at this time. Their activities may, however, be modulated by the composition of the diet at the time of weaning. Adaptation to the composition of the diet can occur within one feeding period, and to the periodicity of food provision in one or two feeding periods. In the early neonatal period, diurnal rhythms of tyrosine aminotransferase, liver glycogen and glucokinase are either greatly suppressed or absent, but develop rapidly after weaning. Food-dependent rhythms of glycogen and tyrosine aminotransferase were included in the late suckling period (day 14).     

Effects of cholecystokinin octapeptide (CCK-8) on food intake in adult and aged rats under different feeding conditions

The effects of CCK on food intake were investigated under fixed feeding conditions in comparison to a test meal taken after 16 h of food deprivation. The experiments were performed on young adult rats (8 weeks old) as well on aged rats (23 months old). Intraperitoneal CCK-8 (8 and 40 μg/kg) significantly reduced the size of a test meal following 16-h food deprivation. This effect was independent of the age of the rats. However, under fixed feeding conditions neither of the doses used in this study reduced food intake in the young adult rats, whereas the highest dose of 40 μg/kg did so in the aged rats. These results suggest that the hypophagic effect of exogenous CCK-8 depends on experimental conditions, food intake being reduced after a period of food deprivation but not under a fixed feeding regimen in adult animals. Furthermore, the data suggest that age is a factor contributing to the complex behavioral actions of CCK, because only old animals were more susceptible to an anorectic action of CCK under the fixed feeding schedule. An explanation may lie in an interaction of other known behavioral effects of CCK (e.g., anxiogenic, mnemonic action) with its effects under the different feeding schedules.     

Diurnal variations in inducibility of rat liver tyrosine aminotransferase activity.

The activity of tyrosine aminotransferase (TAT) was measured in the livers of rats which were entrained to eat for the first 2 hours of a daily 12 hour dark period (‘2+22’ schedule) and were treated with the synthetic glucocorticoid dexamethasone and with glucagon at several times of day. TAT activity in untreated animals varies diurnally with a maximum 4 to 6 hours after the beginning of feeding. In both fed and fasted rats there was a small diurnal variation in inducibility by dexamethasone: in fed rats induction was greatest near the beginning of the dark period, shortly after feeding; in fasted rats induction increased towards the end of the dark period. Glucagon induction showed a marked diurnal variation in fed rats with a decrease coincident with the decline in control TAT activity after its food-induced peak. This variation did not appear to be depemdent on food intake, however, since the decline in inducibility occurred in fasted rats at the same time as in fed rats. Co-treatment with dexamethasone did not affect the decrease in glucagon inducibility. The diurnal variation in TAT induction may reflect a diurnal rhythm in the components of the enzyme synthesizing system (e.g. in the availability of mRNA or in enzyme degradation).     

Rats Work for Food They then Reject: Support for the Information-primacy Approach to Learned Industriousness

There is good evidence that in many situations, animals prefer to work for food even when identical food is freely available. This phenomenon is called ‘learned industriousness’ or ‘contrafreeloading’ and has been found in several species. This study shows that wild rats will also work hard for contaminated food that they associate with sickness and subsequently reject, even when wholesome food is continuously available at no extra cost. Seven wild rats (Rattus novegicus) lived for 4 wk in cages containing two operant levers and associated pellet dispensers. The rats earned all their food by lever pressing for 45-mg food pellets on a variable-interval 10 s (VI10s) schedule in each dispenser. After 1 wk, pellets containing a sub-lethal dose of an acute rodenticide were available for 12 h (the test period) from the preferred dispenser, before being replaced by normal food pellets. This procedure was repeated 1 wk and 4 wk later. The number of lever presses, pellets delivered, and pellets consumed at each dispenser were recorded for 12 h prior to the test period, during the 12-h test period, and 12 h after the test period. The rats quickly learned to shift their feeding preference during the test period away from the dispenser that provided the rodenticide pellets. However, during the second half of the test period, lever pressing at this dispenser increased, even though the pellets thereby obtained were still rejected. This divergence between the lever pressing rate and the pellet consumption rate increased over the three trials. This ‘learned-industriousness’ behaviour is not easily explained by the self-reinforcing hypothesis, by the obligate species-specific response hypothesis, or as some artefact of the operant situation. It is suggested that the lever pressing towards the end of the trial, for pellets that were rejected, enabled the animals to gather information about a rare, but very important, event, namely, the presence of dangerous food at a previously preferred and normally safe feeding site. The data lend support to the information-primacy theory of learned industriousness.     

Apolipoprotein-E-gene expression in rat liver during development in relation to insulin and glucagon

An apolipoprotein-E (apo-E) cDNA probe, cloned by immunoscreening of a lambda GT11 rat liver cDNA library, was used to further characterize the expression of the apo-E gene in rat liver during development, in relation to plasma insulin and glucagon levels. The apo-E mRNA level was low in fetus liver, then abruptly increased at birth and rose further during the suckling period. It returned to the level at birth in 10-week-old adults. These variations were paralleled with dramatic changes in plasma glucagon, which rose at birth and remained high during suckling. At the same time, the insulin/glucagon molar ratio fell. Administration of N6,O2-dibutyryl cAMP to 5-day-old rats resulted in a significant induction of liver apo-E mRNA. Moreover, liver apo-E mRNA rose in 10-h-fasted suckling rats as compared to controls, while plasma glucagon increased and the insulin/glucagon ratio decreased. Conversely, glucose feeding of suckling rats did not induce any increase in liver apo-E mRNA, the insulin/glucagon ratio was 10-fold higher than in fasted animals. Our results are consistent with liver apo-E gene expression being under the control of plasma glucagon and of the glucagon/insulin balance.     

The influence of diurnal rhythms of carbohydrate metabolism in adult rat liver on the metabolic characteristics of isolated liver parenchymal cells.

Rats trained to the "8 + 16" controlled feeding cycle where food is only available for the first 8 h of the 12 h dark period exhibit a pronounced diurnal rhythm of hepatic glycogen metabolism. Glycogen is stored within the liver parenchymal cells during the dark period and subsequently mobilized for energy production during the light period. Hepatocytes, isolated by collagenase perfusion, from livers of such animals have differing capacities for glycogen synthesis when incubated with glucose. Cells prepared at the end of the 16 h period without food have very little capacity for synthesis compared with much higher rates obtained in cells obtained during the feeding period. Cells obtained from liver containing a large glycogen concentration produce a net breakdown of glycogen during incubations with glucose, however experiments using radioactively labelled glucose indicate that synthesis does occur in these cells. The changes in the capacity of the cells for glycogen synthesis appear to be due, in part, to changes in the percentage of the cell population involved in synthesis and in the activity of glycogen synthetase a. Attempts of influence the rate of glycogen synthesis at any time of day with insulin or dexamethasone were unsuccessful.     

Ontogeny of glutamine transport by rat liver plasma membrane vesicles.

Glutamine metabolism in the liver is essential for gluconeogenesis and ureagenesis. During the suckling period there is high hepatic protein accretion and the portal vein glutamine concentration is twice that in the adult, whereas hepatic vein glutamine concentration is similar between adult and suckling rats. Therefore, we hypothesized that glutamine uptake by the liver could be greater in the suckling period compared to the adult period. The present studies were, therefore, designed to investigate the transport of glutamine by plasma membranes of rat liver during maturation (suckling--2-week old, weanling--3-week old and adult--12-week old). Glutamine uptake by the plasma membranes of the liver represented transport into an osmotically sensitive space in all age groups. Inwardly directed Na+ gradient resulted in an "overshoot" phenomenon compared to K+ gradient. The magnitude of the overshoot was greater in suckling rats plasma membranes compared to adult membranes. Glutamine uptake under Na+ gradient was electrogenic and maximal at pH 7.5, whereas uptake under K+ gradient was electroneutral. Glutamine uptake with various concentrations of glutamine under Na+ gradient was saturable in all age groups with a Vmax of 1.5 +/- 0.1, 0.7 +/- 0.1 and 0.5 +/- 0.06 nmoles/mg protein/10 seconds in suckling, weanling and adult rats, respectively (P < 0.01). Km values were 0.6 +/- 0.1, 0.5 +/- 0.1 and 0.5 +/- 0.1 mM respectively. Vmax for Na(+)-independent glutamine uptake were 0.6 +/- 0.1, 0.55 +/- 0.07 and 0.54 +/- 0.06 nmoles/mg protein with Km values of 0.54 +/- 0.2, 0. +/- 0.1 and 0.5 +/- 0.2 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian organization of running-wheel activity, food intake and drinking of old male rats

Circadian rhythms of running-wheel activity, food intake and drinking were monitored in old male rats of Long-Evans strain over 22 months of age in both entrained (light:dark 12:12, LD) and free running condition (continuous illumination, LL) and were compared with those of young adult male rats of 3.5 to 6.5 months of age. Twenty-four hour distribution of running activity, feeding events and licking events of young rats as well as old rats showed bi- or tri-modal patterns during the 12 hr dark period of the LD schedule. In the light period, 2 out of 8 old rats, 6 out of 10 old rats and 1 out of 6 old rats had 1 or 2 medium or high peaks in running activity, feeding events and licking events, respectively, leading to equal distribution between the dark and light period. In the LD schedule, old rats showed a decrease in running-wheel activity, its patterns and power spectra, a decrease in feeding events and its power spectra in 6 rats which lost circadian rhythms and increase in feeding events and its power spectra in 4 rats which still showed circadian rhythms and increase in licking events. LL suppressed running-wheel activity, its patterns and power spectra, licking events and its power spectra and feeding events in young rats. However, LL could suppress only feeding events of 4 rats which still showed circadian rhythms and licking events and its spectral level in old rats. The possible causes of decreased response to LL in old rats and its implication are discussed.     

Modification of the template capacity of liver chromatin for form-B ribonucleic acid polymerase by food intake in rats under controlled feeding schedules.

Nuclei from liver of rats accustomed to eating during the first 8h of a daily 12h dark period demonstrate an increased capacity to synthesize RNA 6H after the beginning of the feeding period. 2. This increase is accompanied by a higher yield of extractable form-B DNA-dependent RNA polymerase activity. 3. The endogenous RNA polymerase activity associated with nuclear chromatin is also stimulated by food intake. Both purified and chromatin-associated form-B enzyme activities exhibit different ionic strength requirements after food intake. 4. The sensitivity of exogenous (added) form-B-enzyme to changes in ionic strength changes after feeding when chromatin is used as template. 5. Chromatin extracted from the liver of fed rats is a better template for form-B-enzyme than chromatin extracted from starved rats.     

Effects of cortisone and thyroxine on suckling rat liver N-acetyl-ß-glucosaminidase isozymes: Comparison with adult reactivity

Suckling rat liver N-acetyl-β-glucosaminidase (hexosaminidase) activity undergoes considerable fluctuation during the first two weeks of life. As two major forms of hexosaminidase (A, heat-labile, and B, heat-stable) are known to exist in both human and adult rat liver, we choose to examine the effect of the maturative hormones, thyroxine and cortisone, upon these isozymes during the suckling period. Between days 7 and 15, the observed developmental change is attributable solely to an increase in the ‘A-like’ (heat-labile) form of the enzyme; an enhanced response is seen in thyroxine-injected 11–15-day old animals. The response may be considered ‘age-independent’, as adult animals react in the same manner. In contrast, cortisone-injected sucklings show a decrease in both A and B isozymes, while in adults no changes in total activity or isozyme distribution are evoked. The ratio of hexosaminidase A to hexosaminidase B in suckling rat liver appears to shift in favor of the labile (A) isozyme early in development.     

The influence of diurnal rhythms of carbohydrate metabolism in adult rat liver on the metabolic characteristics of isolated liver parenchymal cells

Rats trained to the “8 + 16” controlled feeding cycle where food is only available for the first 8 h of the 12 h dark period exhibit a pronounced diurnal rhythm of hepatic glycogen metabolism. Glycogen is stored within the liver parenchymal cells during the dark period and subsequently mobilized for energy production during the light period. Hepatocytes, isolated by collagenase perfusion, from livers of such animals have differing capacities for glycogen synthesis when incubated with glucose. Cells prepared at the end of the 16 h period without food have very little capacity for synthesis compared with much higher rates obtained in cells obtained during the feeding period. Cells obtained from livers containing a large glycogen concentration produce a net breakdown of glycogen during incubations with glucose, however experiments using radioactively labelled glucose indicate that synthesis does occur in these cells. The changes in the capacity of the cells for glycogen synthesis appear to be due, in part, to changes in the percentage of the cell population involved in synthesis and in the activity of glycogen synthetase $\text{a}$. Attempts to influence the rate of glycogen synthesis at any time of day with insulin or dexamethasone were unsuccessful.     

Glycolytic isoenzymes and glycogen metabolism in regenerating liver from rats on controlled feeding schedules

Intact rats trained on a controlled feeding and lighting schedule designated ;8+16' exhibited diurnal oscillations in liver weight, glucokinase activity and liver glycogen content. Glucokinase activity expressed as units/g of liver decreased to 30% of that from unoperated controls during the first 48h after partial hepatectomy and returned to near normal values in 2 weeks. When the glucokinase activity was expressed as units/liver per 100g body wt., a decrease to 50% of control activity was observed between 24 and 48h after the operation. A similar pattern was found for pyruvate kinase type I. In contrast, pyruvate kinase type III activity increased after partial hepatectomy. It is suggested that the newly divided cells after partial hepatectomy do not synthesize glucokinase and pyruvate kinase I but do synthesize pyruvate kinase III. Glycogen was found to accumulate as early as 24h after partial hepatectomy, and normal concentrations were reached after 48h if the operation was performed at times other than during the feeding periods.     

Fatty acid oxidation in isolated rat liver mitochondria. Developmental changes and their relation to hepatic levels of carnitine and glycogen and to carnitine acyltransferase activity

Prompted by an apparent relationship between ketosis and fatty acid utilization, we studied the capacities for fatty acid oxidation through β-oxidation and Krebs cycle in liver mitochondria isolated from fetal and suckling rats. Rates of state 3 oxidation, as measured by oxygen consumption, were low for both palmitylcarnitine and palmityl CoA plus carnitine at 2 days before term and at birth, but increased at least ninefold during the first 8 days of life and at least sixfold during the remaining suckling period. Despite these sharp increases, oxygen consumption in suckling rats did not exceed the value for fed adult rats. Also, the rates of state 3 oxidation of succinate were low in suckling rats. Respiratory control indices, determined with each of the three substrates, were lower in suckling rats than fed adults. By contrast, ratios of fatty acyl ester to succinate oxidation, a relative measure of the oxidation of palmitylcarnitine and palmityl CoA, were 21–66% and 27–77% higher in suckling than in fed adult rats. The increased ratios indicate that the capacity for fatty acid oxidation is higher during postnatal development than in the fetal stage or adulthood. The oxidation capacity was inversely related to glycogen content in the liver. Although hepatic carnitine concentration and carnitine palmityltransferase activity increased during suckling period, they are not rate limiting for fatty acid oxidation. Studies of the partitioning of fatty acids showed that about two-thirds of the fatty acid oxidized through β-oxidation did not enter Krebs cycle for further oxidation. These results support our working hypothesis that ketosis of suckling rats stems from rapid oxidation of fatty acids and increased partitioning of acetyl CoA into ketogenesis.     

A new protocol for studying the early events during liver regeneration

Hepatic amino acid transport and ornithine decarboxylase (ODC) activity were measured as early events during liver regeneration in rats adapted to a meal-feeding protocol in which food was presented during the first 2 hr of a daily 12-hr dark period. Surgeries were performed during the early hours of the light period, and food was withheld on the day following surgery to control the effect of feeding on the parameters measured. Initial experiments indicated that rats maintained on this schedule were capable of surviving partial hepatectomy. The survival rate was greater and the fat pads larger among 56-day-old than among 42-day-old rats; this indicated that animals with greater metabolic reserves were better suited for these experiments. The hepatic α-aminoisobutyric acid (α-AIB) distribution ratio and ODC activity increased above control values by 2 hr following partial hepatectomy. The α-AIB distribution ratio peaked at 10 hr after surgery and remained above control values for an additional 16 hr. In contrast, ODC activity peaked by 4 hr after surgery, followed by two smaller peaks at 10 and 20 hr. In sham-hepatectomized rats both the α-AIB distribution ratio and the ODC activity increased above control values by 3 hr after surgery, but fell to control values by 15 hr. These findings show that increases in amino acid transport and ODC activity following partial hepatectomy occur independently of feeding. The identical time course for the initiation of both of these events suggests that they result from a common effector.     

Eine technische Modifikation im Aufbau von Respirationssystemen nach dem HALDANE-Prinzip

ABSTRACT

The objectives of the present study were to quantify the deposition of carotenoids and tocopherols in the tissues of suckling lamb and to use the levels of those compounds to trace the maternal feeding. Twenty suckling lambs were raised with their dams in vegetative-stage pastures, and 19 suckling lambs were stalled indoors with dams that received hay ad libitum, until the lambs reached 10–12 kg. The lambs’ weekly intake of carotenoids and tocopherols was estimated from the milk production of the ewes and the carotenoid and tocopherol content of the milk. Samples of the subcutaneous and perirenal fat, longissimus thoracis muscle, and liver of the suckling lambs were collected at 24 h after slaughter. The pasture-raised lambs had greater intake of lutein than their indoor counterparts throughout the suckling period (p < 0.05), more retinol during the second and third weeks of the suckling period (p < 0.05), and more α-tocopherol during the first three weeks of the suckling period (p < 0.05), being similar thereafter. The pasture-raised lambs, when compared to the lambs raised indoors, had greater lutein content in the muscle and liver (p < 0.001), retinol and α-tocopherol content in all tissues (p < 0.001) and muscle and liver γ-tocopherol content (p < 0.05). The maternal feeding could be accurately predicted from the carotenoid and tocopherol content of whole lamb carcasses and muscle tissue but not from those of ewes’ milk, lamb liver tissue or lamb fat deposits.     

The dominance boundary method of determining motivational state

A model is outlined in which physiological state affects behaviour through command variables. Theoretically command space can be divided into regions in which different behaviours are dominant. These regions are bounded by switching lines which should be experimentally observable. It is shown that, in the case of the Barbary dove, it is possible by use of a suitable reward schedule (the ‘double interruption’ schedule) to hold all causal factors for feeding and drinking other than physiological deficit constant (or to mask their variation). When this is done either, feeding or drinking occurs according to whether the deficit for food or water is greater. This is equivalent to saying that there exists a straight switching line (estimated by the dominance boundary) in a deficit space with axes food deficit and water deficit, both measured in grams. The position of this line in deficit space does not vary systematically over a period of time, and is not affected either by initial deprivation or by radical alterations in the consequences of feeding and drinking. It is possible to estimate the motivational state of the animal from a knowledge of its position in deficit space and the position of the dominance boundary. Analogous experiments with pigs give less clear-cut results.     

Effect of supplementary food on suckling patterns and ovarian activity during lactation.

Patterns of infant feeding, basal prolactin concentrations, and ovarian activity were studied longitudinally in 27 breast-feeding mothers from delivery until first ovulation. Suckling frequency (6.1 feeds/day) and suckling duration (122 mins/day) reached peak values four weeks post partum and remained relatively constant until the introduction of supplementary food at a mean of 16 weeks post partum. There were subsequently sharp declines in both the frequency and duration of suckling, both of which correlated closely with basal prolactin concentrations. None of the 27 mothers ovulated during unsupplemented breast-feeding, but within 16 weeks of introducing supplements ovarian follicular development had returned in 20 and ovulation in 14 mothers. The mothers who ovulated within 16 weeks of giving supplements had reduced frequency and duration of suckling more quickly and weaned more abruptly than those who continued to suppress ovulation. These data suggest that the introduction of supplementary food may exert an important and hitherto unrecognised effect on the timing of first ovulation by reducing the frequency and duration of suckling episodes.     

Scheduled feeding versus the light-dark cycle on the rhythms of circulating tryptophan, serotonin and N-acetylserotonin in rats

Both the environmental light-dark cycle and scheduled feeding can act as entrainers of biological rhythms. The present study investigated the relative potency of these two environmental cues in entraining the rhythms of circulating tryptophan (TRP), serotonin (5HT) and N-acetylserotonin (NAS). Four groups of rats were subjected for one month to an identical light-dark cycle of 14 h light and 10 h dark with food availability restricted to the 3 h period beginning 2 h after onset of light or onset of darkness. Two groups of animals were food deprived on the day of experiment. The 24 h rhythms of serum TRP, 5HT and NAS were determined. Serum TRP showed a sharp increase after food presentation and declined gradually to a trough just before feeding. Withholding food on the day of experiment abolished this increase. The trough of serum 5HT occurred just before feeding, increased gradually after feeding and peaked 10-13 h afterwards. Serum NAS levels, however, demonstrated an anticipatory rise before feeding, which peaked during feeding and declined to a trough 8 h afterwards. Unlike TRP, withholding food had no effect on either the 5HT or the NAS rhythm. These results indicated that feeding schedule was the common and stronger entrainer for the rhythms of serum TRP, 5HT and NAS. However, each indole had a different rhythm pattern in relation to the feeding schedule which could not be explained by a simple precursor-product relationship.     

Rats made cirrhotic by chronic CCl4 treatment still exhibit anticipatory activity to a restricted feeding schedule

Food entrainment of clock genes in the liver suggests that this organ may underlie a food entrained oscillator (FEO), which manifests under restricted feeding schedule (RFS). In order to test the importance of a functional liver for the expression of FEO, chronic CCl4-treated cirrhotic rats and oil-treated controls were entrained to RFS and drinking behavior was continuously monitored. After 20 d of free-running conditions, food access was restricted to 2 h, followed by a refeeding-fasting protocol to test persistence of anticipatory drinking. Present data show no differences between groups for the onset and intensity of anticipation during RFS. After RFS, however, cirrhotic rats exhibited a significantly longer free-running period and a delay and lower intensity of the persistence of activity under fasting conditions. Histology confirmed injury of the liver chronically treated with CCl4. Present data indicate that a dysfunctional liver due to chronic CCl4 does not prevent animals from exhibiting anticipatory activity but may promote metabolic derangement of the clock mechanisms of the suprachiasmatic nucleus and the FEO.     

Glycogen metabolism in adult rat liver parenchymal cell primary cultures.

Parenchymal cells were isolated from adult rat liver with an enzyme perfusion technique. The single-cell suspension, representing 40-50% of the liver's hepatocytes was suspended in medium and maintained in primary culture for up to four days. The cells were found to carry out glycogen synthesis for the first eight hours in culture after which time the accumulated glycogen was gradually degraded. The ability of the liver cell cultures to accumulate glycogen was found to be dependent upon the metabolic state of the animal prior to cell isolation. Cells prepared during the feeding period from animals on the 8+16 feeding schedule had markedly different capacities for glycogen accumulation. Changes in glycogen metabolism were found to be due, in part, to changes in the fraction of cells involved in metabolism at any given time. High concentrations of glucose stimulated the cells to deposit glycogen but the response was reduced the longer the cells were in culture over a 3-day period. This loss of glycogen synthesizing capacity appears to be due to a decrease in glycogen synthetase activity. The activities of pyruvate kinase, hexokinase and aldolase also decrease during the culture period.