Docosahexaenoic acid promotes neuronal differentiation by regulating basic helix-loop-helix transcription factors and cell cycle in neural stem cells.

Recent studies have suggested that docosahexaenoic acid (DHA) enhances neuronal differentiation of neural stem cells (NSCs) isolated from rat embryonic day 14.5. However the underlying mechanism remains largely unknown. One hypothesis supported by DHA controls the expression level of basic helix-loop-helix (bHLH) transcription factors, such as hairy and enhancer of split 1 (Hes1), Mash1, neurogenin1, and NeuroD; another is that previous studies in retinal progenitor cells DHA affects the cell cycle. In this study, we show that treatment with DHA under differentiation conditions without basic fibroblast growth factor, (1) increases Tuj-1 and MAP2 positive cells in NSCs, (2) that the expression level of Hes1 mRNA and protein decreased significantly from day 1 to day 4, on the other hand, the NeuroD mRNA expression level increased from day 1 to day 4 after treatment with DHA and (3) decreased the percentage of S-phase cells, which correlated with prolonged expression of cyclin-dependent kinase inhibitor p27(kip1), suggesting that DHA enhances neuronal differentiation of NSCs, in part, by controlling the bHLH transcription factors and promoting cell cycle exit. We therefore speculate that DHA is one of the essential key molecules for neuronal differentiation of NSCs.

Docosahexaenoic acid promotes neurogenesis in vitro and in vivo.

Docosahexaenoic acid (22:6n-3), one of the main structural lipids in the mammalian brain, plays crucial roles in the development and function of brain neurons. We examined the effect of docosahexaenoic acid on neuronal differentiation of neural stem cells in vitro and in vivo. Neural stem cells obtained from 15.5-day-old rat embryos were propagated as neurospheres and cultured under differential conditions with or without docosahexaenoic acid for 4 and 7 days. Docosahexaenoic acid significantly increased the number of Tuj1-positive neurons compared with the control on both culture days, and the newborn neurons in the docosahexaenoic acid group were morphologically more mature than in the control. Docosahexaenoic acid significantly decreased the incorporation ratio of 5-bromo-2'-deoxyuridine, the mitotic division marker, during the first 24 h period; it also significantly decreased the number of pyknotic cells on day 7. Thus, docosahexaenoic acid promotes the differentiation of neural stem cells into neurons by promoting cell cycle exit and suppressing cell death. Furthermore, dietary administration of docosahexaenoic acid significantly increased the number of 5-bromo-2'-deoxyuridine(+)/NeuN(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. These results demonstrate that docosahexaenoic acid effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis.

Relationship between age-related increases in rat liver lipid peroxidation and bile canalicular plasma membrane fluidity.

Aging is associated with increased cellular levels of lipid peroxides and reactive oxygen species. This increase in the radical species may affect membrane fluidity and consequently membrane functions. We attempted to determine whether age-related increases in lipid peroxide and reactive oxygen species levels affect the annular fluidity of the bile canalicular plasma membrane in young (2-3 weeks old), adult (20 weeks old) and old (100 weeks old) rats. Hepatic levels of lipid peroxides measured with thiobarbituric acid reactive substances (TBARS), reactive oxygen species and reduced glutathione were significantly higher in the old rats than in the young and adult rats. Arachidonic acid (AA) levels increased and docosahexaenoic acid (DHA) levels decreased in the isolated canalicular plasma membrane of the old rats, and the ratio of DHA to AA thus decreased significantly with rat aging. Hepatic TBARS levels correlated negatively with molar ratios of DHA to AA. The annular fluidity of the bile canalicular plasma membrane decreased significantly in the old rats compared with that in the young and adult rats. These results suggest that the age-related decrease in DHA content of the bile canalicular plasma membrane and the molar ratio of DHA to AA may be associated with age-related deterioration of membrane annular fluidity.

Anticipatory fall in core temperature in rats acclimated to heat given for various hours at a fixed daily time.

Rats were subjected to daily heat exposure limited to a fixed time for 10 consecutive days. An ambient temperature (T(a)) inside an animal room initially set at 24.0 degrees C was raised to 32.0 degrees C, starting at the middle of the dark phase. The high T(a) was maintained for a subsequent 1.5, 3, or 5 h and was then returned to 24.0 degrees C. After the heat exposure schedule, their core temperature significantly fell for about 30 min before and during the period when they had previously been exposed to heat without thermal stimuli. In rats, a time memory for heat exposure could be formed even when the duration of daily heat exposure was short.

Effects of docosahexaenoic acid on annular lipid fluidity of the rat bile canalicular plasma membrane.

The role of docosahexaenoic acid (DHA) in the fluidity of the annular lipid regions and their associated membrane-bound proteins is still not as well understood as that in the global (bulk) lipid regions. We therefore studied the effects of dietary DHA on the relationship between annular and global lipid fluidity and membrane-bound enzymes such as 5'-nucleotidase and Mg(2)+-ATPase in the rat bile canalicular membrane. Dietary DHA caused significant increases in 5'-nucleotidase and Mg(2)+-ATPase activity and in global and annular lipid fluidity, a higher increase in fluidity in the annular lipids than the global lipids, and a decrease in the cholesterol-to-phospholipid molar ratio in the canalicular membrane. Plasma total cholesterol and LDL cholesterol decreased, and fecal cholesterol increased in the DHA-fed rats. No changes were observed in oxidative markers, but glutathione peroxidase increased in the liver with DHA feeding. Annular lipid fluidity, but not global lipid fluidity, correlated remarkably well with DHA, synchronously with the activities of 5'-nucleotidase and Mg(2)+-ATPase. The data indicate that the DHA-induced increase in annular lipid fluidity is responsible for the increases observed in the enzyme activity. We therefore concluded that the increased activity of membrane-bound enzymes and transporters induced by DHA and the concomitant increase in annular lipid fluidity comprise one of the mechanisms involved in DHA-induced clearance of plasma cholesterol.

Nicorandil--induced ATP release in endothelial cells of rat caudal artery is associated with increase in intracellular Ca(2+).

The effect of nicorandil, an ATP-sensitive K(+) channel opener, on the level of intracellular Ca(2+) ([Ca(2+)](i)) and on ATP release in endothelial cells of the rat caudal artery was examined using a fluorescent confocal microscopic imaging system and high-performance liquid chromatography (HPLC) with fluorescent detection, respectively. Nicorandil significantly increased [Ca(2+)](i) and the overflow of ATP and its metabolites. The former reaction was abolished in the absence of extracellular Ca(2+), but it did not change in the presence of thapsigargin or cyclopiazonic acid. The increase in the overflow of ATP and [Ca(2+)](i) induced by nicorandil was markedly suppressed by glibenclamide, an ATP-sensitive K(+) channel blocker. The increase of [Ca(2+)](i) induced by nicorandil was significantly and inversely correlated with the level of intracellular ATP in the endothelial cells, suggesting that activation of ATP-sensitive K(+) channels by nicorandil increases Ca(2+) influx in endothelial cells. The increase of [Ca(2+)](i) might be associated with ATP release.

Ambient temperatures preferred by humans acclimated to heat given at a fixed daily time.

We investigated preferred ambient temperatures (T(pref)) of heat-acclimated humans to assess their behavioral thermoregulation. Seven male volunteers were exposed to an ambient temperature (T(a)) of 42 degrees C and relative humidity (RH) of 40% for 4 h (14:00-18:00 h)/day for 9-10 consecutive days. Rectal temperature (T(re)) was measured, and T(pref) was determined at two distinct times of day, 09:00-11:00 h (AM test) and 14:00-16:00 h (PM test), in both heat- and nonheat-acclimated (control) conditions. Heat acclimation significantly decreased T(re) only in the PM test. There was no difference in the T(pref) between the two tests in the control condition. However, T(pref) in the PM test was significantly lower than that of the AM test in the heat-acclimated condition. The findings suggest that repeated heat exposure in humans for 4 h at a fixed time daily alters the core temperature level and behavioral thermoregulatory function, particularly during the period when the subjects had previously been exposed to heat.

Autonomic and behavioural thermoregulation in starved rats.

1. We investigated the mechanism of starvation-induced hypothermia in rats. 2. Threshold core temperatures (Tcor) for tail skin vasodilatation and cold-induced thermogenesis were determined after a 3 day starvation using a chronically implanted intravenous thermode. Food deprivation significantly lowered the threshold Tcor for heat production, but did not affect the heat loss threshold. 3. Thermogenic response to a fall in Tcor below its threshold was enhanced by starvation. 4. Preferred ambient temperatures (Tpref) and Tcor were measured before and during a 3 day starvation in a thermal gradient. The 3 day starvation significantly lowered Tcor only in the light phase of the day. The level of hypothermia was the same throughout the fasting period, while Tpref gradually increased during the 3 days of starvation. 5. When rats were starved at a constant ambient temperature of 25 C (no thermal gradient), their Tcor levels were comparable with those of the rats kept in the thermal gradient. 6. The results suggest that, in rats, hypothermia caused by starvation was not due to a decrement in thermogenic capability, but was due to a decrease in the threshold for the activation of thermogenesis.

Changes in ambient temperature at the onset of thermoregulatory responses in exercise-trained rats.

Spontaneous running in a wheel has emerged as a useful method of exercise in rodents. We investigated how exercise training with a running wheel affects ambient temperatures (T(a)) at the onset of thermoregulatory responses in rats. Female rats were allowed to run freely in the wheel for 6 months. Sedentary control rats did not exercise during the same period. After the exercise training period, they were loosely restrained and T(a) values at the onset of tail skin vasodilation and cold-induced thermogenesis were determined by raising or lowering T(a). Resting levels of core temperature and heat production of the exercise-trained rats were significantly higher than those of the controls. T(a) values at the onset of tail skin vasodilation and cold-induced thermogenesis of the exercise-trained rats were higher than those of the controls. The results suggest that, in rats, exercise training with a running wheel elevates ambient temperatures for heat loss and heat production, which may then contribute to maintaining the core temperature at a high level.

Effects of food deprivation on daily changes in body temperature and behavioral thermoregulation in rats.

Homeothermic animals regulate body temperature (T(b)) by using both autonomic and behavioral mechanisms. In the latter process, animals seek out cooler or warmer places when they are exposed to excessively hot or cold environments. Thermoregulation is affected by the state of energy reserves in the body. In the present study, we examine the effects of 4-day food deprivation on circadian changes in T(b) and on cold-escape and heat-escape behaviors in rats. Continuous measurement of T(b) during food deprivation indicated that the peak T(b) amplitude was not different from baseline values, but the trough amplitude continuously decreased after the onset of food deprivation. Cold-escape behavior was facilitated by food deprivation, whereas heat-escape behavior was unchanged. After the termination of food deprivation, the lowered T(b) returned to normal on the first day. However, cold-escape behavior was still facilitated on the third day after food reintroduction. Autonomic and behavioral thermoregulatory effectors are modulated in the face of food shortage so as to maintain optimal performance during the active period, whereas increasing energy conservation occurs during the quiescent phase.

Selected ambient temperatures of rats acclimated to heat given on various schedules.

We investigated the behavioral thermoregulation of heat-acclimated rats by measuring their selected ambient temperatures (Ts). Rats kept in a light:dark cycle of 12:12 h were subjected to one of four different heat exposure regimes for 10 consecutive days; a constant ambient temperature (Ta) of 32 degrees C (CH), a Ta of 32 degrees C for 5 h daily in the latter half of the dark phase (IHF), a Ta of 32 degrees C for 5 h daily at a random time of day (IHR), or a constant Ta of 24 degrees C (control). After the heat exposure schedule, the rats were placed in a thermal gradient and their intra-abdominal temperature (Tab), Ts and spontaneous activity were measured for 3 days. There were clear day-night variations of Tab and Ts in all groups. The levels of Tab and Ts of the CH rats were significantly higher than those of the IHF, IHR and control rats. The Tab and activity levels of the IHF rats were significantly lower than those of the IHR and control rats only in the latter half of the dark phase. The Ts values of the IHF rats did not differ from those of the IHR and control rats. These results suggest that, after rats were constantly subjected to heat, heat-seeking behavior was induced so that the core temperature was maintained at a high level. However, when rats were acclimated to heat given for several hours at a fixed time daily, core temperature was lowered during the same time period of previous heat exposure in association with a depression of thermogenic behavior.

Thermoregulatory responses to acute heat loads in rats following spontaneous running.

Earlier studies showed that spontaneous exercise training in rodents shifted their core temperature and thermoeffector thresholds to high levels. The present study investigated heat loss and heat production responses to acute heat loads of exercise-trained rats. The exercise-trained rats were allowed to run in a running wheel freely for 6 months, while the sedentary controls were denied access to the wheel during the same period. Then, they were loosely restrained and put in a direct calorimeter. After thermal equilibrium had been attained, they were warmed for 30 min with an intraperitoneal electric heater (internal heating). At least 2 h later, the rats were externally warmed for 90 min by raising the ambient temperature from 24 to 38C (external warming). Hypothalamic temperature (Thy), evaporative and nonevaporative heat loss (R+C+K) and heat production were measured. Internal and external heating significantly increased Thy. During internal heating, the magnitude of the increase in Thy was significantly smaller and the amount of increase in (R+C+K) was significantly greater in the exercise-trained rats than in the controls. The slope showing the relationship between Thy and (R+C+K) in the trained rats was significantly steeper than that in the controls. During external warming, the magnitude of increase in Thy of the exercise-trained rats was significantly greater than that of the controls. The slope showing the relationship between Thy and (R+C+K) in the trained rats was not different from that in the controls. Changes in evaporative heat loss and heat production during the two types of heat load did not differ between the two groups. The results suggest that, in rats, exercise training with voluntary running improves heat tolerance through enhancing nonevaporative heat loss response. However, this may be the case only when the rats are subjected to a direct internal heat load.

Core temperature and sweating onset in humans acclimated to heat given at a fixed daily time.

The thermoregulatory functions of rats acclimated to heat given daily at a fixed time are altered, especially during the period in which they were previously exposed to heat. In this study, we investigated the existence of similar phenomena in humans. Volunteers were exposed to an ambient temperature (Ta) of 46 degrees C and a relative humidity of 20% for 4 h (1400-1800) for 9-10 consecutive days. In the first experiment, the rectal temperatures (Tre) of six subjects were measured over 24 h at a Ta of 27 degrees C with and without heat acclimation. Heat acclimation significantly lowered Tre only between 1400 and 1800. In the second experiment, six subjects rested in a chair at a Ta of 28 degrees C and a relative humidity of 40% with both legs immersed in warm water (42 degrees C) for 30 min. The Tre and sweating rates at the forearm and chest were measured. Measurements were made in the morning (0900-1100) and afternoon (1500-1700) on the same day before and after heat acclimation. Heat acclimation shortened the sweating latency and decreased the threshold Tre for sweating. However, these changes were significant only in the afternoon. The results suggest that repeated heat exposure in humans, limited to a fixed time daily, alters the core temperature level and thermoregulatory function, especially during the period in which the subjects had previously been exposed to heat.

Thermoregulatory responses to acute heat loads in the FOK rat.

FOK is an inbred rat strain with a genotypic adaptation to hot environments. The present study investigated the mechanism of the high heat tolerance of the FOK rat. Male FOK and WKAH rats were used. They were loosely restrained and placed individually in a direct calorimeter with an ambient temperature of 24 degrees C. Their hypothalamic temperature, evaporative and non-evaporative heat loss and heat production were measured. After thermal equilibrium had been attained, the rats were warmed for 30 min with a chronically implanted intraperitoneal electric heater (internal heating). At least 90 min after the heating, the jacket water temperature surrounding the calorimeter chamber was gradually raised from 24 degrees C to 36 degrees C in 80 min (external warming). During the internal heating, changes in the thermoregulatory parameters did not differ between the groups. During the external warming, the evaporative heat loss of the FOK rat was significantly greater than that of the WKAH rat, while changes in nonevaporative heat loss and heat production did not differ between the groups. The results suggest that in the FOK rat, the improved heat tolerance is attributable to an enhanced evaporative heat loss response, but not to a facilitation of nonevaporative heat loss or of metabolic depression.

Changes in core temperature and thermoeffector thresholds in exercise-trained rats.

Spontaneous running in a running wheel has emerged as an alternative method of exercise in small animals. The present study investigated how exercise training with a running wheel affects core temperature level and thermoeffector thresholds in rats. Female rats were allowed to run freely in the wheel for 6 months. Sedentary controls did not exercise during the same period. After the exercise training period, they were loosely restrained and their threshold core temperatures for tail skin vasodilation and cold-induced thermogenesis were determined by warming or cooling the animals by use of a chronically implanted intravenous thermode. Resting and threshold core temperatures of the exercise-trained rats were higher than those of the sedentary controls. The results suggest that in rat, exercise training with a running wheel shifts threshold temperatures for heat loss and heat production to high levels, which may result in a rise in core temperature level.

Can our thermoregulatory system anticipate temperature exposure?

After daily heat exposure for approximately 5 h/day during a fixed time for more than 5 consecutive days, body core temperature of rats decreases during the period when they were previously exposed to heat. The fall in body temperature persists for a few days after terminating the timed daily heat exposure. Additionally, various thermoregulatory changes to resist heat are brought about, especially during the specific heat-exposure period. It is therefore hypothesized that the thermoregulatory system can memorize a time for heat exposure and that, in accordance with the memory, thermoregulatory responses for neutralizing heat stress are induced, even without actual heat exposure, around the period corresponding to that of the previous heat exposure.

Thermoeffector thresholds and preferred ambient temperatures of the FOK rat.

The FOK is an inbred rat strain with a genotypic adaptation to hot environments. The present study compared the thermoeffector thresholds and preferred ambient temperatures (Tpref) of the FOK rat with those of other rat strains. Male FOK, WKAH, and Donryu rats were used. First, they were loosely restrained and placed individually in a metabolic chamber with an ambient temperature of 26.0 degrees C. Their hypothalamic temperature (T(hy)), tail skin temperature (Tsk), and heat production (M) were measured. After thermal equilibrium had been attained, the rats were gradually warmed and then cooled using an intravenous thermode. The threshold T(hy) values for tail skin vasodilation and cold-induced thermogenesis were defined as the points at which sharp increases in Tsk and M occurred, respectively. The two thresholds of the FOK rat were lower than those of the WKAH and Donryu rats. In a second set of experiments, the FOK and WKAH rats were placed individually in a thermocline. Their intra-abdominal temperatures (T(ab)) were measured by a biotelemetry system, and the rats' Tpref values were estimated with the thermal gradient. Mean T(ab) and Tpref over a 24-h period for the FOK rat were significantly lower than those of the WKAH rat. The results suggest that in the FOK rat the control ranges of autonomic and behavioral thermoregulation are lower than those of the other rat strains examined. This contributes to the maintenance of core temperature at low levels.

Body core temperature of rats subjected to daily exercise limited to a fixed time.

Several timed daily environmental cues alter the pattern of nycthemeral variations in body core temperature in rodents. The present study investigated the effect of timed exercise on variations of daily body core temperature. Male rats were housed in cages with a running wheel at an ambient temperature of 24 degrees C with a 12:12 h light/dark cycle. Timed daily exercise rats (TEX) were allowed access to the wheel for 6 h in the last half of the dark phase, freely exercising rats (FEX) could run at any time, and sedentary rats (NEX) were not allowed to run. After a 3-week exercise period, all animals were denied access to the wheel. The intraabdominal temperatures (Tab) and spontaneous activities of rats were measured for 6 days after the exercise period. The Tab values of the TEX rats were significantly higher than those of the other two groups only in the last half of the dark phase, while Tab in the FEX and NEX rats showed no significant difference. The specific Tab changes in the TEX rats lasted for 2 days after the exercise period. Spontaneous activity levels were higher in the TEX rats than the FEX and NEX rats in the last half of the dark phase for 1 day after the exercise period. The results suggest that daily exercised limited to a fixed time per day modifies nycthemeral variations of body core temperature in rats so that the temperature increases during the period when the animals had previously exercised. Such a rise in body core temperature is partly attributed to an increase in the spontaneous activity level.

Lipopolysaccharide-induced fever in rats prolonged by a needle prick.

We studied the effect of an abdominal prick with a needle on LPS-induced fever in freely-moving rats. LPS was injected intraperitoneally by the following 3 methods: 1) through a hypodermic needle pricked into the abdominal cavity, 2) through a catheter chronically indwelt in the abdominal cavity, and 3) through a catheter chronically indwelt in the abdominal cavity immediately after an abdominal prick was made. In the second method, core body temperature (Tb) began to rise about 1 h after the injection, reaching a maximal level at around 2.5 h and decreasing gradually thereafter. In the first and third methods, Tb rose again to make a second peak after making the first peak of fever. This was the same when LPS was injected through a hypodermic needle pricked into the abdominal cavity under restrained condition. These results suggest that the abdominal prick with a needle is responsible for the development of the second peak (or prolongation) of LPS-fever in rats.