Further studies of diabetes-prone BHE/Cdb rats: increased sensitivity to calcium ion suppression of oxidative phosphorylation

The responsiveness of hepatic mitochondria isolated from hyperthyroid and control Sprague-Dawley (SD) and diabetes-prone BHE/Cdb rats was studied. Hyperthyroidism was induced through the addition of thyroxine (T(4)) to the diet (2 mg/kg of diet). Oxidative phosphorylation (OXPHOS) with the addition of adenosine diphosphate (ADP) or an 8:1 mixture of adenosine monophosphate (AMP):ADP was studied. Dose response curves of state 3 and state 4 respiration, respiratory control (RC) ratio, and ADP:O ratio to calcium levels (0-7.5 microm) were generated. Mitochondria from BHE/Cdb rats were more sensitive to the addition of calcium than mitochondria from SD rats, as judged by losses in OXPHOS. T(4) treatment potentiated this strain difference and we conclude that the diabetes phenotype in the BHE/Cdb rat is probably related not only to the previously described mutation in the F(O)ATPase but also to a defect in the efflux of the calcium ion that, in turn, affected the regulation of OXPHOS.     

Nutrient-gene interactions in mitochondrial function: vitamin A needs are increased in BHE/Cdb rats

The BHE/Cdb rat has a maternally inherited mutation in the ATPase 6 mitochondrial gene that associates with impaired oxidative phosphorylation (OXPHOS) and glucose intolerance. A longevity study revealed that feeding an egg-rich (vitamin A-rich) diet delayed the onset of impaired glucose tolerance. Two experiments were conducted to test the hypothesis that BHE/Cdb rats require more dietary vitamin A than normal rats. Experiment 1 was a dose-response study examining OXPHOS in BHE/Cdb rats fed one of six levels of vitamin A. In experiment 2 BHE/Cdb and Sprague-Dawley rats were used. The rats were depleted of retinol stores, then repleted with 4 or 12 IU vitamin A/g diet. Vitamin A status was assessed in depleted, never depleted, and depleted/repleted rats. OXPHOS was optimized at 4 IU/g diet for the Sprague-Dawley rats and 12 IU/g diet for the BHE/Cdb rats. These results suggested that the criteria for vitamin intake adequacy in the BHE/Cdb rats is the optimization of mitochondrial OXPHOS. Using this criteria, we conclude that diabetes-prone BHE/Cdb rats require more dietary vitamin A than normal rats.     

Genetic control of circadian rhythms and aging

The review establishes a link between a group of genes which are conserved in evolution and form a molecular oscillator responsible for generation of circadian rhythms and genetic determinants of aging including associated pathways of intracellular signaling. An analysis of mechanisms of development of agedependent pathologies is conducted from the viewpoint of circadian genetics. Systematic data of circadian gene expression studies in animals demonstrating different rates of aging from accelerated to negligible are presented.     

Influence of menhaden oil on mitochondrial respiration in BHE rats

The effects of corn or menhaden oil and thyroxine treatment on hepatic mitochondrial respiration was studied. BHE rats were fed a 64% sucrose, 6% corn, or menhaden oil diet until they were 60-70 days of age. Succinate-supported mitochondrial respiration was studied at 3 degrees C intervals from 4 to 40 degrees C. Upper and lower activation energies and transition temperatures were determined through the calculation of Arrhenius plot. Menhaden oil plus daily thyroxine injection resulted in higher and lower activation energies than the other treatments. This combined treatment also resulted in lower state 3 and higher state 4 respiration rates and tighter coupling of respiration to ATP synthesis. These effects were thought to be due to the effect this treatment combination had on membrane fluidity.     

Effects of continuous infusion of vasopressin on circadian rhythms of food and water intake, urine output and electrolyte excretion in Brattleboro rats

Food intake (FI), water intake (WI), urine output (UO), Na+ and K+ excretions were investigated for 2 days at 4-h intervals during continuous infusion of saline or vasopressin (VP) 1.0 UI/day, in male Brattleboro vasopressin-deficient rats. Continuous VP infusion reduced significantly 24-h amounts of WI and UO, and increased Na+ excretion. A significant (3.5 h) phase advance of the circadian rhythm of WI was observed, while the group circadian rhythm of Na+ excretion was eliminated due to irregular phase shifts in the different rats. The results suggest that VP do not play a role in the generation of the circadian rhythms of water input and output, but it may participate in their internal synchronization.     

Circadian rhythms of food intake in gastroduodenally-ulcerated rats: effects of three anti-ulcer drugs

The effects of three anti-ulcers drugs on the temporal distribution of food intake and of the two parameters, meal size and meal frequency, were studied in ulcerated and non-ulcerated rats exposed to light-dark (LD 12:12) cycles. Experimental ulceration with indomethacin reduces the amplitude of meal frequency and brings the acrophase forward, compared with non-ulcerated animals. These effects were reversed by the oral administration of either ranitidine, sucralfate or pirenzepine along with the food. However, the administration of either pirenzepine or sucralfate alone to non-ulcerated rats is accompanied by significant (P less than 0.05) changes in the circadian patterns of meal size and meal frequency without the total daily food intake being affected in any way (pirenzepine treatment caused large intake of food during the light period while sucralfate treatment resulted in marked food intake during the dark period). The results indicate that circadian modification of meal patterns in the ulcerated rats are attributable to indomethacin-induced gastrointestinal mucosal injury and anti-ulcer medications.     

Cryptochrome restores dampened circadian rhythms and promotes healthspan in aging Drosophila

Circadian clocks generate daily rhythms in molecular, cellular, and physiological functions providing temporal dimension to organismal homeostasis. Recent evidence suggests two‐way relationship between circadian clocks and aging. While disruption of the circadian clock leads to premature aging in animals, there is also age‐related dampening of output rhythms such as sleep/wake cycles and hormonal fluctuations. Decay in the oscillations of several clock genes was recently reported in aged fruit flies, but mechanisms underlying these age‐related changes are not understood. We report that the circadian light–sensitive protein CRYPTOCHROME (CRY) is significantly reduced at both mRNA and protein levels in heads of old Drosophila melanogaster. Restoration of CRY using the binary GAL4/UAS system in old flies significantly enhanced the mRNA oscillatory amplitude of several genes involved in the clock mechanism. Flies with CRY overexpressed in all clock cells maintained strong rest/activity rhythms in constant darkness late in life when rhythms were disrupted in most control flies. We also observed a remarkable extension of healthspan in flies with elevated CRY. Conversely, CRY‐deficient mutants showed accelerated functional decline and accumulated greater oxidative damage. Interestingly, overexpression of CRY in central clock neurons alone was not sufficient to restore rest/activity rhythms or extend healthspan. Together, these data suggest novel anti‐aging functions of CRY and indicate that peripheral clocks play an active role in delaying behavioral and physiological aging.     

Hypoxic depression of circadian rhythms in adult rats.

Because the circadian rhythms of oxygen consumption (VO(2)) and body temperature (T(b)) could be contributed to by differences in thermogenesis and because hypoxia depresses thermogenesis in its various forms, we tested the hypothesis that hypoxia blunts the normal daily oscillations in VO(2) and T(b). Adult rats were instrumented for measurements of T(b) and activity by telemetry; VO(2) was measured by an open-flow method. Animals were exposed to normoxia (21% O(2)), hypoxia (10.5% O(2)), and normoxia again, each 1 wk in duration, in either a 12:12-h light-dark cycle ("synchronized") or constant light ("free running"). In this latter case, the period of the cycle was approximately 25 h. In synchronized conditions, hypoxia almost eliminated the T(b) circadian oscillation, because of the blunting of the T(b) rise during the dark phase. On return to normoxia, T(b) rapidly increased toward the maximum normoxic values, and the normal cycle was then reestablished. In hypoxia, the amplitude of the activity and VO(2) oscillations averaged, respectively, 37 and 56% of normoxia. In free-running conditions, on return to normoxia the rhythm was reestablished at the expected phase of the cycle. Hence, the action of hypoxia was not on the clock itself but probably at the hypothalamic centers of thermoregulation. Hyperoxia (40% O(2)) or hypercapnia (3% CO(2)) had no significant effects on circadian oscillations, indicating that the effects of hypoxia did not reflect an undifferentiated response to changes in environmental gases. Modifications of the metabolism and T(b) rhythms during hypoxia could be at the origin of sleep disturbances in cardiorespiratory patients and at high altitude.     

Evidence for time-of-day dependent effect of neurotoxic dorsomedial hypothalamic lesions on food anticipatory circadian rhythms in rats

The dorsomedial hypothalamus (DMH) is a site of circadian clock gene and immediate early gene expression inducible by daytime restricted feeding schedules that entrain food anticipatory circadian rhythms in rats and mice. The role of the DMH in the expression of anticipatory rhythms has been evaluated using different lesion methods. Partial lesions created with the neurotoxin ibotenic acid (IBO) have been reported to attenuate food anticipatory rhythms, while complete lesions made with radiofrequency current leave anticipatory rhythms largely intact. We tested a hypothesis that the DMH and fibers of passage spared by IBO lesions play a time-of-day dependent role in the expression of food anticipatory rhythms. Rats received intra-DMH microinjections of IBO and activity and body temperature (T(b)) rhythms were recorded by telemetry during ad-lib food access, total food deprivation and scheduled feeding, with food provided for 4-h/day for 20 days in the middle of the light period and then for 20 days late in the dark period. During ad-lib food access, rats with DMH lesions exhibited a lower amplitude and mean level of light-dark entrained activity and T(b) rhythms. During the daytime feeding schedule, all rats exhibited food anticipatory activity and T(b) rhythms that persisted during 2 days without food in constant dark. In some rats with partial or total DMH ablation, the magnitude of the anticipatory rhythm was weak relative to most intact rats. When mealtime was shifted to the late night, the magnitude of the food anticipatory activity rhythms in these cases was restored to levels characteristic of intact rats. These results confirm that rats can anticipate scheduled daytime or nighttime meals without the DMH. Improved anticipation at night suggests a modulatory role for the DMH in the expression of food anticipatory activity rhythms during the daily light period, when nocturnal rodents normally sleep.     

Effect of starvation and refeeding on the circadian rhythms of hematological and clinico-biochemical values, and water intake of rats

We investigated the effect of starvation for 24 hr and subsequent refeeding for 12 hr on the circadian rhythms of 39 hematological and clinico-biochemical parameters, and water intake of F344 rats. The rats scarcely drank any water during the starvation period, but subsequently their intake of water were normal, even in the light period. During starvation, 12 parameters such as serum levels of alkaline phosphatase activity and PaCO2 decreased with time-related and time-related increases of 8 parameters such as the erythrocyte count and cholinesterase activity. During refeeding for 12 hr, almost all these biochemical parameters were normalized, but none of the hematological values except the leukocyte count returned to normal levels. Starvation and refeeding had little affect on the circadian rhythms of others.     

N-Phthaloyl gamma-aminobutyric acid affects biochemical circadian rhythms in Wistar rats

N-Phthaloyl gamma-aminobutyric acid (P-GABA) was administered to Wistar rats and 24 hr rhythms of glucose, cholesterol, total protein and lactic acid levels in blood were studied under semi-natural light dark conditions. P-GABA administration caused desynchronisation of the rhythms; while glucose and lactic acid rhythms were advanced, cholesterol and total protein rhythms were delayed. Since GABA is being involved in conveying dark information to the clock, exogenous administration of P-GABA may reduce the photic information received by the clock. The results could be explained by slightly less than 1 hr daily delays (or) advances respectively which would bring the peak times to the points 21 days after the start of administration.     

Changes in circadian rhythms of thermoregulation and motor activity in rats as a function of aging: effects of d-amphetamine and alpha-MSH

Thermoregulatory and motor activity circadian cycles are age-dependent. While the level of thermoregulation and motor activity remained almost at the same level during the first 1-15 months during the light portion of the 24-hr cycle, a significant decrease in the level of both rhythms was observed during the dark period. Therefore, older rats exhibited reversed cycles compared with younger rats. Treatments with d-amphetamine resulted in the enhancement of reversal of the cycles. Rats treated with alpha-MSH failed to exhibit a reversal of the cycles. While the effects of d-amphetamine are mediated by the brain DA mesolimbic pathway, it seems that alpha-MSH acts on the dopaminergic system at different sites of action.     

Disturbance of cardiovascular circadian rhythms by pertussis vaccine in freely-moving rats

Vaccination of young children with diphtheria, tetanus, poliomyelitis and pertussis (DTPoP) vaccine is effective in preventing outbreaks of whooping cough but adverse events sometimes occur. This pilot study shows that in freely-moving rats, multiple treatment with DTPoP (at day 0 and day 5, 6 ml/kg i.v.) increased heart rate (HR) for 5 days after the first treatment and decreased diastolic blood pressure (DBP) for at least 26 days after the first treatment and inhibited the circadian rhythm of HR and DBP for at least 10 days. DTPo vaccine, containing no pertussis vaccine, was free of such effects. Thus, in rats, the pertussis component of DTPoP acts on the cardiovascular system and disturbs its circadian rhythm. The contribution of these findings to clinical adverse effects is as yet unknown and needs further research.     

Litorin suppresses food intake in rats

Litorin (LIT), a bombesin-like nonapeptide, decreased food intake in rats in a dose-related manner after parenteral injection. LIT decreased deprivation-induced water intake only at a dose much higher than required to suppress feeding. LIT administration did not significantly alter the frequency of observed feeding-associated behaviors, nor did it result in subsequent aversion to an associated novel solution. Litorin shares with bombesin structural features and pharmacological actions that include the suppression of food intake in a manner that mimics natural satiation.