Quantitative changes in the lysosomal vacuolar system of rat hepatocytes during short-term starvation

Summary Ultrastructural morphometric analysis was used to study time-dependent variations in macro and microautophagy in rat hepatocytes. Except during periods of shortterm starvation for up to 24 h, animals were kept under standardized conditions of food intake.In hepatocytes of meal-fed rats the volume fraction of macroautophagic vacuoles is significantly higher at 23:00 h, i.e., immediately before food intake, compared to 11:00 h, i.e., 12 h following feeding. During fasting, macroautophagy drops to a low level.Microautophagic vacuoles in hepatocytes of meal-fed rats, sacrificed at 11:00 or 23:00 h respectively, do not show any significant quantitative differences. However, during 12 h of starvation, the volume fraction of microautophagic vacuoles rises significantly, whereas the numerical density remains constant. Subsequently, during the second 12-h period of fasting, the volume fraction of microautophagic vacuoles remains unchanged, but the numerical density increases. Over a period of 24 h of starvation the volume fraction of the total lysosomal system does not change significantly, whereas the numerical density rises.The time-dependent changes of the macroautophagic vacuolar system correlate with the circadian, food-related variations in the protein content of individual hepatocytes from meal-fed animals. The increase in volume fraction and thereafter in number of microautophagic vacuoles, as observed during starvation, coincides with a large decrease in protein content of individual hepatocytes.     

Cytochemical determination of acid phosphatase activity in isolated rat hepatocytes during starvation-induced proteolysis

Summary Acid phosphatase activity in isolated rat liver parenchymal cells has been investigated with quantitative histochemical means during short-term starvation, which leads to a considerable loss in protein mass in the parenchyma. Animals trained to a meal-feeding regime in which food was available during 1 h only per 24 h (using an automatic food dispensing machine), were sacrified 6, 12, 18, 24 and 36 h after food had been withheld at the time point (23.00 h) of meal feeding. Acid phosphatase activity was analysed cytophotometrically in isolated hepatocytes incorporated into polyacrylamide gels before the enzyme reaction technique with the post-azo coupling was carried out. No indication could be found for any significant changes in the amount of acid phosphatase activity per individual hepatocyte during the entire period of fasting, as compared with two time points (11.00 and 23.00 h) before the theoretical onset of fasting. It is concluded that the considerable enhancement of protein degradation in the lysosomal apparatus during fasting is not reflected by changes in the cellular acid phosphatase activity.     

Circadian oscillations of thyroid hormones and insulin in the serum of fasting rats

Adult male Wistar rats adapted to a 12:12 h light:dark regimen, fed or after a 24- or 48-h fast, were decapitated at 3-h intervals during a single day. They were deprived of food at day-time intervals ensuring that on decapitation they had fasted for the same length of time, i.e. 24 or 48 h. Thyroid hormones, insulin and glucose concentrations were determined in their serum. Fasting did not significantly affect circadian thyroxine, triiodothyronine and reverse triiodothyronine rhythms compared with the findings in fed animals; 24, but not 48 hours' fasting led to a shift in the acrophase of circadian insulin and glucose oscillations compared with fed rats. The maintenance of original circadian thyroid hormones and insulin rhythm in rats which fasted for short lengths of time testifies to a dependence of the stimulus on the time of day.     

Effects of feeding time constraints on body mass regulation and energy expenditure in wintering dunlin (Calidris alpina)

We examined the effects of time-restricted feeding on regulationof body mass and activity energy expenditure in captive winteringdunlin (Calidris alpina) held in outdoor aviaries at TomalesBay, California. In the first of two experiments, we comparedbirds under 24 h : 24 h (fasting : ad libitum feeding) foodrestriction with controls under continuous ad libitum feeding. In the second experiment, we compared birds under 24 h : 6 h: 12 h : 6 h (fasting : ad libitum : fasting : ad libitum)food restriction with birds under 24 h : 24 h food restriction.We estimated total energy expended on activities from dailymass balance using an additive model based on measures of grossenergy intake, thermoregulation, basal metabolism, and a sensitivity analysis of gross utilization efficiency and energy densityof reserve body tissue. Dunlin under 24 h : 24 h food restrictionovercompensated for body mass lost while fasting, increasingtheir body mass relative to controls fed ad libitum. Dunlinunder 24 h : 6 h : 12 h : 6 h food restriction were unable to recover body mass lost during the first fasting day. Whenallowed to feed, food-restricted birds reduced the amount ofenergy spent on being active and increased food intake andenergy storage relative to controls, but when forced to fast,they increased their activity energy expenditure. These patterns suggest winter body mass regulation consistent with the behaviorsof free-living dunlin in winter.     

Effects of withholding food for 0-72 h on mating, pregnancy rate and pituitary function in female rats

Food was withheld from female rats for 0-72 h at various stages of the oestrous cycle. Withholding food for periods of 24 h ending at 12:00 h on the day of pro-oestrus reduced the mating rate from 61 to 25% (P less than 0.05) but not the pregnancy rate of those rats that mated. Fasting for 24 h ending at 18:00 h on the day of pro-oestrus reduced the pregnancy rate from 82 to 18% (P less than 0.05) without affecting the mating rate and a 48-h fast starting at 12:00 h on the day of pro-oestrus reduced the pregnancy rate from 82 to 25% (P less than 0.05). Withholding food for 23 h ending at 17:00 h on the day of pro-oestrus prevented the LH and prolactin surges normally present at 17:00 h on this day. The treatments had no apparent effect on the ability of the adenohypophysis to release LH in response to injections of GnRH. When ovariectomized female rats fasted for 0-72 h and given 2 injections of oestradiol dibenzoate to test the ability of the hypothalamus to respond to an increasing plasma oestradiol concentration by stimulating the release of LH, a fast for 24 h reduced and a fast for 72 h completely prevented LH release.     

Food restricted schedules promote differential lipoperoxidative activity in rat hepatic subcellular fractions

Restricted access to food (from 12:00 to 14:00 h) produces a behavioral activation known as food anticipatory activity (FAA), which is a manifestation of the food entrained oscillator (FEO). Peripheral oscillators, especially in the liver, are thought to be part of the FEO. A variety of metabolic adaptations have been detected in the liver during the expression of this oscillator, including activation of mitochondrial respiration and changes in the cytoplasmic and mitochondrial redox states. Biological clocks are regulated by redox-sensitive factors. The present study explored the lipoperoxidative activity (LP) in the liver during the activity of the FEO. Conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS), with and without Fe2+-supplementation, were quantified in six subcellular fractions: whole homogenate, plasma membrane, mitochondria, microsomes, nucleus, and cytosol. The experimental protocol involved control groups of ad libitum fed and 24-h fasted rats, and groups under the restricted food schedule (RFS) which were sampled before FAA (08:00 h), during FAA (11:00 h) and after feeding (14:00 h). Clear differences in pro-oxidant activity was observed between ad libitum fed and 24-h fasted rats in almost all the subcellular fractions studied. RFS rats presented: CD levels more similar to the fasted rats, even at 14:00 h, after food presentation, and basal and Fe2+-supplemented TBARS levels tended to be lower than both controls, suggesting an increased antioxidant capacity associated with food restriction. In addition, a microarray analysis showed that several isoforms of peroxiredoxins, a family of antioxidant and hydrogen peroxide-catabolizing enzymes, were consistently up-regulated in each and every condition in which RFS was applied. Together, these data indicate a rheostatic adaptation of the liver in the handling of pro-oxidant reactions during the activity of the FEO.     

Effect of social isolation on 24-h pattern of stress hormones and leptin in rats

This work analyzes the effect of social isolation of growing male rats on 24-h changes of plasma prolactin, growth hormone, ACTH and leptin, and on plasma and adrenal corticosterone concentrations. At 35 days of life, rats were either individually caged or kept in groups (6-8 animals per cage) under a 12:12 h light/dark schedule (lights on at 08:00 h). A significant arrest of body weight gain regardless of unchanged daily food intake was found in isolated rats after 2 weeks of isolation. On the 4th week, rats were killed at 6 time intervals during a 24-h cycle, beginning at 09:00 h. In isolated rats the 24-h pattern of all parameters tested became distorted, as assessed by Cosinor analysis. When analyzed as a main factor in a factorial analysis of variance, isolation decreased plasma prolactin and growth hormone, increased plasma leptin and corticosterone while decreased adrenal corticosterone. Plasma corticosterone levels correlated significantly with plasma ACTH and with adrenal corticosterone levels in group-caged rats only. These changes can be attributed to an effect of mild stress on the endogenous clock that modulates the circadian hormone release.     

Effects of starvation,re-feeding and timing of food supply on daily rhythm features of gut melatonin in carp (Catla catla)

Influences of starvation, re-feeding and time of food supply on daily rhythm features of melatonin (5-methoxy-N-acetyltryptamine) and its key regulator AANAT (arylalkylamine N-acetyltransferase) protein in the gut tissues were separately evaluated in carp Catla catla. The first experiment was aimed at demonstration of duration dependent effects of starvation and re-feeding after starvation on the daily profiles and rhythm features of gut melatonin and AANAT. Accordingly, juvenile carp were randomly distributed in three groups, which were (a) provided with balanced diet daily at a fixed time, that is, 10:00 clock hour or zeitgeber time (ZT) 4 (control), or (b) starved (for 2-, 4-, 6- or 8 days), or (c) initially starved for 8 days and then re-fed (for 2-, 4-, 6-, 8-, 12- or 16 days) daily with the same food and at the time (ZT4) used for control fish. The carp in each group were sampled for collection of gut tissues at six different time points at a regular interval of 4?h in a daily cycle. In another experiment, the influences of timing of food supply were separately examined in four fish groups, which were provided with a fixed amount of food once daily either at 06:00 or 12:00 or 18:00 or 24:00 clock hour corresponding to ZT0 or ZT6 or ZT12 or ZT18, respectively, for 7 days before sampling at 12 different time points with a regular interval of 2?h in a 24-h cycle. The study revealed a gradual increase in the mesor and amplitude values of melatonin and AANAT in gut with the progress of starvation till their values reached maximum at day-6 and remained steady thereafter. In contrast, re-feeding of 8-day starved fish resulted in a sharp decrease in their mesor and amplitude values after 2 days and then followed by a steady-state increase till re-attainment of their values close to control fish at the end of 16 days. The acrophase of these gut variables in each control, starved and re-fed fish was noted mostly at midday or ZT6. However, the results of another experiment demonstrated that a shift of food supply time led to a shift in their acrophase. The amount of residual food in the gut lumen in each, but not starved, fish by showing a significant positive correlation independently with the gut levels of melatonin and AANAT also indicated possible role of food as the synchronizer for their daily rhythms. Collectively, it appears reasonable to argue that daily profiles of gut melatonin and AANAT are strongly influenced by the availability of food, while their daily rhythm features seem to be dependent mostly on the time of food supply in carp.     

The role of non-parenchymal cells in liver growth

The main non-parenchymal cells of the liver, Kupffer cells, sinusoidal endothelial cells and stellate cells, participate in liver growth with respect to both their own proliferation, and effects on hepatocyte proliferation. In the well-characterised paradigm of 70% partial hepatectomy, they undergo DNA synthesis and cell division 20-24h later than the hepatocyte population. They exert both positive and negative influences on hepatocyte proliferation, including provision of an extracellular matrix-bound reservoir of hepatocyte growth factor that is activated after damage; priming of hepatocytes for DNA synthesis through rapid generation of TNF-alpha and IL-6; and generation of factors at later time points that curb hepatocyte DNA synthesis (IL-1, TGF-beta) and initiate reconstruction and reformation of matrix proteins.     

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.     

A method to evaluate dynamics and periodicity of hormone secretion

Spontaneous hormone secretory dynamics include tonic and pulsatile components and a number of periodic processes. Circadian variations are usually found for melatonin, TSH and GH, with peak secretions at night, and in cortisol secretion, which peaks in the morning. Free thyroxine (FT4) and insulin-like growth factor (IGF)1 levels do not always change with circadian rhythmicity or show only minor fluctuations. Fractional variations explore the dynamics of secretion related to time intervals, and the rate of change in serum levels represents a signal for the receptorial system and the target organ. We evaluated time-related variations and change dynamics for melatonin, cortisol, TSH, FT4, GH and IGF1 levels in blood samples obtained every 4 h for 24 h from eleven healthy males, ages 35-53 years (mean ? SE 43.6 ± 1.7). Nyctohemeral (i.e., day-night) patterns of hormone secretion levels and the fractional rate of variation between consecutive 4-hourly time-qualified hormone serum levels (calculated as percent change from time 1 to time 2) were evaluated for circadian periodicity using a 24 and 12-h cosine model. A circadian rhythm was validated for serum level changes in cortisol with peaks of the 24-h cosine model at 07:48 h, and melatonin, TSH and GH, with phases at 01:35 h, 23:32 h, and 00:00 h, respectively. A weak, but significant, 12-h periodicity was found for FT4 serum levels, with minor peaks in the morning (10:00 h) and evening (22:00 h), and for IGF1, with minor peaks in the morning (07:40 h) and evening (19:40 h). Circadian rhythmicity was found in the 4-hourly fractional variations with phases of increase or surge at 02:00 h for cortisol, 22:29 h for melatonin, 05:14 h for FT4, and 21:19 h for GH. A significant 12-h periodicity was found for the 4-hourly fractional variations of TSH with two peaks in the morning (decrease or drop at 04:42 h) and afternoon (surge at 16:28 h), whereas IGF1 fractional variation changes did not show a significant rhythmic pattern. In conclusion, the calculation of the time-qualified fractional rate of variation allows evaluation of the dynamics of secretion and the specification of the timepoint(s) of maximal change of secretion, not only for hormones whose secretion is characterized by a circadian pattern of variation, but also for hormones that show no circadian or only weak ultradian (12 h) variations (i.e., FT4).     

Time-dependent effects of leptin on food intake and locomotor activity in goldfish

The present study investigates the possible circadian dependence of leptin effects on food intake, locomotor activity, glycemia and plasma cortisol levels in goldfish (Carassius auratus). Fish were maintained under 12L:12D photoperiod and subjected to two different feeding schedules, one group fed during photophase (10:00) and the other one during scotophase (22:00). Leptin or saline were intraperitoneally injected at two different times (10:00 or 22:00), coincident or not with the meal time. To eliminate the entraining effect of the light/dark cycle, goldfish maintained under 24 h light (LL) were fed and leptin-injected at 10:00. A reduction in food intake and locomotor activity and an increase in glycemia were found in goldfish fed and leptin-injected at 10:00. No significant changes in circulating cortisol were observed. Those effects were not observed when leptin was administered during the scotophase, regardless the feeding schedule; neither in fish maintained under LL, suggesting that a day/night cycle would be necessary to observe the actions of leptin administered during the photophase. Changes in locomotor activity and glycemia were only observed in goldfish when leptin was injected at daytime, coincident with the feeding schedule, suggesting that these leptin actions could be dependent on the feeding time as zeitgeber. In view of these results it appears that the circadian dependence of leptin actions in goldfish can be determined by the combination of both zeitgebers, light/dark cycle and food. Our results point out the relevance of the administration time when investigating regulatory functions of hormones.     

Effects of ultradian lighting and feeding schedules on the circadian rhythm of body temperature in monkeys

In estimating, by use of cosinor-test, the 12- and 24-h component parameters of body temperature circadian rhythm in monkeys under ultradian schedules of lighting and feeding (LD 6:6; DL 6:6) we have shown that an intensive 12-h component is registered in both cases. The presence of a 24-h component of circadian rhythm depends on the zeitgeber phase. This component is present in LD 6:6 (lighting hours 07:00-13:00 and 19:00-01:00) and is absent in DL 6:6 (01:00-07:00 and 13:00-19:00). We hold that the most satisfactory explanation of the phenomena observed is that 12-h component is the result of a masking effect induced by the 12-h schedule (exogenous component) whereas the 24-h component reflects the intrinsic pacemaker work (endogenous component). It should be noted that in our case the masking effect in body temperature rhythm is circadian phase-dependent.     

Differences in proton leak kinetics, but not in UCP3 protein content, in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from fed and fasted rats

We have investigated the effect of 24-h fasting on basal proton leak and uncoupling protein (UCP) 3 expression at the protein level in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. In fed rats, the two mitochondrial populations displayed different proton leak, but the same protein content of UCP3. In addition, 24-h fasting, both at 24 and 29 degrees C, induced an increase in proton leak only in subsarcolemmal mitochondria, while UCP3 content increased in both the populations. From the present data, it appears that UCP3 does not control the basal proton leak of skeletal muscle mitochondria.     

Behaviour of dairy cows when fed concentrates with an automatic feeding system

On Dutch commercial farms automatic systems for feeding concentrates in a feeding station are being used increasingly. Various systems have been developed, which differ in the timing and amount of concentrates made available to the cows over a 24-h period. Three types of a fixed-time system and one variable-time system are compared with a traditional system. The fixed-time systems provided a ration of concentrates every 12 h. The starting time of these 12-h cycles varied, hence the fixed-7-time system started at 07:00 and 19:00 h; the fixed-11-time system cycles started at 11:00 and 23:00 h; and the fixed-3-time system cycles started at 15:00 and 03:00 h. The variable-time system continuously added small amounts of concentrates to the available ration per cow throughout a 24-h cycle. The traditional system provided concentrates mixed with maize silage at the feeding rack. The aim of the investigation was to describe the pattern of intake of concentrates, and the possible consequences of the various feeding systems on the cows' general activities such as eating, standing and lying down.

Three experiments were carried out, each with a group of 20 lactating dairy cows kept in a cubicle house with a single concentrates feeding station. In each of the experiments, feeding systems were tested during 3-week experimental periods. Behaviour was observed during three 24-h periods in each experimental period.

It was found that each of the five feeding systems tested evoked a typical pattern of visits to the feeding station. The number, the duration and the timing of rewarded and unrewarded visits differed between the various systems. The cows adapted to each feeding system, eating all their rations of concentrates as soon as they were made available.

Adaptation to the feeding system affected several of the general activities, in particular the time spent at the feeding rack and the time spent lying in the cubicles. With the fixed-11-time system, new feeding cycles started at times when many cows were lying down. Under these conditions the lowranking cows sometimes had to wait a long time before they could enter the station, which resulted in a reduced lying time for them.     

Circadian Rhythms of Renal Hemodynamics in Unanesthetized, Unrestrained Rats

Catheters were placed in the jugular vein and femoral artery of male Sprague-Dawley rats and connected to a specially designed perfusor for continuous constant infusion of 0.9% NaCl and a syringe to perform simultaneous and intermittent blood collections. This permitted continuous 24-h study of renal hemodynamics, estimated by inulin (C_in) and p-amino-hippuric acid (C_PAH) clearances; C_in represents glomerular filtration rate and C_PAH renal plasma flow. Animals were individually housed in metabolism cages in a controlled environment with light/dark 12:12 h. Urine was collected every 4 h (12:00, 16:00, 20:00, 24:00, 04:00, and 08:00) and blood sampled at the midpoint of urine collection periods. Urine and plasma sodium, potassium, inulin, and PAH were spectrophotometrically assessed. During continuous infusion of isotonic saline, C_in exhibited circadian changes with large decrease between 12:00 and 20:00 h (0.9 ± 0.2 ml/min) and acrophase at 00:30 h. Rhythmicity in C_PAH was similar with the minimum between 16:00 and 20:00 h (2.5 ± 0.3 ml/min) and peak between 00:00 and 04:00 h (acrophase at 00:25 h). Water and electrolyte excretion were also circadian rhythmic with a similar nighttime enhancement and daytime minimum. Such circadian changes persisted during continuous 0.9% NaCl infusion for several consecutive days. The unanesthetized, unrestrained rat model enables investigations in renal chronopharmacology and chronotoxicology.     

Cadmium chronotoxicity at pituitary level: effects on plasma ACTH,GH, and TSH daily pattern

Cadmium is an endocrine disruptor that has been shown to induce chronotoxic effects. The present study was designed to evaluate the possible cadmium effects on the daily secretory pattern of adrenocorticotropin hormone (ACTH), growth hormone (GH), and thyroid-stimulating hormone (TSH) in adult male Sprague-Dawley rats. For this purpose, animals were treated with cadmium at two different doses [25 and 50 mg/l cadmium chloride (CdCl₂)] in the drinking water for 30 days. Control age-matched rats received cadmium-free water. After the treatment, rats were killed at six different time intervals throughout a 24-h cycle. Cadmium exposure modified the 24-h pattern of plasma ACTH and GH levels, as the peak of ACTH content between 12:00 and 16:00 h in controls appeared at 12:00 h in the group treated with the lowest dose used, while it appeared between 16:00 and 20:00 h in rats exposed to 50 mg/l CdCl₂. In addition, the peak of GH content found at 04:00 h in controls moved to 16:00 h in rats exposed to 25 mg/l CdCl₂, and the highest dose used abolished 24-h changes of GH secretion. The metal treatment did not modify ACTH secretory pattern. Exposure to cadmium also increased ACTH and TSH medium levels around the clock with both doses used. These results suggest that cadmium modifies ACTH and TSH medium levels around the clock, as well as disrupted ACTH and GH secretory pattern, thus confirming the metal chronotoxicity at pituitary level.     

Diurnal rhythms of retinal phospholipid synthetic enzymes are retained but their activities are decreased in rats under alpha-linolenic acid deficiency

Rats fed a safflower oil (alpha-linolenic acid (ALNA)-deficient) diet over the course of two generations had significantly decreased docosahexaenoic acid (22:6n-3) and increased docosapentaenoic acid (22:5n-6) contents in the major retinal phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) when compared with those fed a perilla oil (ALNA-sufficient) diet, but the compositions of phosphatidylinositol acyl chains were relatively unaffected. The contents of individual phospholipids in the retina were essentially the same for the two dietary groups. The activities of the rate-limiting enzymes in the de novo synthesis of PC and PE, CTP:phosphocholine cytidylyltransferase (CT), and CTP:phosphoethanolamine cytidylyltransferase (ET), respectively, were measured in the retinas excised at 5:00, 9:00, 13:00, and 17:00 h from rats adapted to a 24-h cycle with lights on from 7:00 to 19:00 h. Both enzymes exhibited significant diurnal rhythms with the lowest activities at 5:00 h and gradually increasing activities following exposure of the rats to light; the maximum activities were at 13:00 h for CT and 17:00 h for ET. The diurnal rhythms were not significantly affected by the above-mentioned diets. However, both enzyme activities at each collection time point were significantly lower in the safflower oil group than in the perilla oil group. These results suggest that retinal phospholipid turnover associated with shedding, phagocytosis, and resynthesis of the rod outer segments is limited by ALNA deficiency.