Circadian rhythms in ingestive behavior and responsiveness to glucoprivic and osmotic challenges in rats with rostral zona incerta lesions

Albino rats with lesions in the rostral zona incerta (ZI) displayed an exaggerated diurnal rhythm of ingestive behavior, consuming nearly all of their daily food and water during the dark portion of a 12-hr light/12-hr dark cycle. When tested during the light phase of the cycle, rats with ZI lesions ate little or nothing in response to 2-deoxy-D-glucose (2DG) while their response to insulin was only slightly impaired. When these tests were repeated during the dark phase of the cycle, the response to insulin was entirely normal but the response to 2DG was even more depressed than during daytime testing. During the light phase of the cycle, rats with ZI lesions also drank little or nothing in response to hypertonic saline. This impairment was ameliorated in some but not all experimental rats when these tests were repeated in the dark. A correlational analysis failed to establish a significant relation between changes in the diurnal distribution of ad lib food and water intake and feeding responses to 2DG or insulin or drinking responses to hypertonic saline in the light or dark phases of the cycle.     

Central but not peripheral glucoprivation is impaired in monosodium glutamate-treated rats

In the present study, newborn male Wistar rats were injected, subcutaneously, five times, every other day, with monosodium glutamate (MSG, 4 g/kg bw) or saline (as control, C), during the neonatal period. MSG animals developed destruction of the arcuate nuclei (ARC) with absence of NPY-immunoreactive cell bodies, which impaired both the food intake (baseline) and the 2-deoxy-D-glucose (2DG) glucoprivic feeding response. Increases in the immunoreactivity of corticotropin-releasing hormone-cell bodies in the paraventricular nuclei might have developed to compensate for the atrophy of the pituitary in MSG-treated rats. After systemic 2DG injection, neither the C nor the MSG rats increased their food intake, but they showed similar hyperglycemic responses, whereas plasma free fatty acids (FFA) increased only in the C group. In other groups, 2DG, norepinephrine (NE), neostigmine (NEO) and saline were intracerebroventricularly (i.c.v.) administered. In this condition, impairment of the hyperglycemic and food intake responses, associated to a lower increase in plasma FFA levels, were observed. As opposed to this, the MSG treatment gives support to NE effects, enhancing food intake, as well as plasma glucose and FFA levels. After NEO, plasma glucose increased only in the MSG group, while plasma FFA levels were elevated in the C rats. Taken together, the results obtained after MSG treatment point to a separate neural control of the hyperglycemic response and of the lipid mobilization when stimulated by central 2DG, NE or NEO administration. It seems likely that the excitatory neural pathway that controls lipid metabolism and is present in C rats was destroyed by the MSG treatment.     

2-deoxy-D-glucose inhibits food intake in rats after ventromedial hypothalamic lesions

Changes in food induced by 2 deoxy-D-glucose (2 DG) have been investigated in newly lesioned rats during the two parts of the diurnal cycle. In a first experiment rats were injected with saline or 2 DG (250, 500, 750 mg/kg) at the beginning of the light or the dark period. In a second experiment rats were injected with saline or 2 DG (250 mg/kg) in the middle of the two periods after 0, 2, 4 or 6 hr of fasting. Results show that 2 DG exerts an inhibitory effect on food intake at night, as it does in intact rats, under ad lib conditions or after a short deprivation time. The stimulating effects of 2 DG on diurnal food intake observed in intact rats is not replicated in VMH rats. On the contrary an inhibition of intake follows a short food deprivation. The data suggest that 2 DG stimulates food intake only under metabolic and feeding conditions characteristic of the diurnal phase in intact rats. Since lipogenesis and hyperphagia are observed 24 hr a day in VMH animals, only inhibition could follow 2 DG.     

Feeding behavior responses of Zucker rats to naloxone

The effects of naloxone on feeding patterns were studied in both obese and lean Zucker rats during both light and dark phases of the diurnal cycle. Eight female obese (471±9 g) and lean (225±6 g) Zucker rats were trained to bar press for food. They were administered 0, 0.5, 1.0 or 2.0 mg/kg naloxone at the initiation of the light or dark phase of the diurnal cycle and feeding behavior was recorded for the subsequent 12 hr using an automated real-time data collection system. First meal size and duration were decreased and first postmeal interval was increased by naloxone and responses did not vary with phenotype or phase of the diurnal cycle. Naloxone decreased food intake during the 12-hr period by decreasing average meal size but meal frequency was not affected. Overall, the feeding behavior responses of obese rats to naloxone were greater than those of lean rats, supporting the hypothesis of an association between opioid peptides and obesity. Opioid involvement in diurnal control of food intake is also supported by the greater responses generally demonstrated in the light compared with dark phases.     

Effect of hepatic vagotomy and/or coeliac ganglionectomy on the delayed eating response to insulin and 2DG injection in rats

The eating response that occurs following recovery from the effects of insulin or 2-deoxy-D-glucose (2DG) injection was examined in rats with hepatic vagotomy and/or coeliac ganglionectomy. Rats were deprived of food and injected with either saline (1 ml/kg), regular insulin (3 U/kg) or 2DG (200 mg/kg). Plasma glucose was measured periodically over the next 6 hr and then food was returned and intakes were measured over the next 2 hr. Rats increased food intake 6–8 hr after insulin or 2DG injection compared to the saline (control) condition. Nerve section did not affect the plasma glucose or food intake responses to insulin or 2DG injection. The results indicate that the innervation of the liver via the vagus nerve or coeliac ganglion is not involved in the delayed eating response to insulin and 2DG injection.     

Dual 24-hour feeding response to 2DG in rats: daytime increase and nighttime decrease

Thirty-six rats were injected IP with 2DG (0, 250, or 500 mg/kg) at 7-day intervals, once at light onset (7 a.m.) and once at dark onset (7 p.m.), and postinjection food intake was monitored for 24 hours. Five hundred mg/kg 2DG caused food intake to rise above control levels during the first 6 hours of daylight, regardless of whether the injection had occurred that morning or the previous evening, whereas intake during the first 6 hours of darkness was consistently below control levels. In a second study, 24 rats were injected first at 7 a.m. (500 mg/kg 2DG or saline), and 7 days later at 7 p.m. (opposite drug), and food was withheld 12 hours until the light:dark period had changed. For 12 hours after food was returned, 2DG again decreased nighttime food intake (Injection 1) and increased daytime intake (Injection 2). 2DG's dual long-term effects cannot be accounted for either by malaise or by an initial action that later is compensated by its opposite. Rather, 2DG (500 mg/kg) appears to exert two independent, opposite alimentary effects which persist 18-24 hours and which change direction with phase changes in the light:dark cycle.     

Physiological and behavioral responses to glucoprivation in the golden hamster

Golden hamsters failed to increase their food intake following food deprivation alone or in combination with insulin or 2-deoxy-D-glucose (2DG) treatment. 2DG also failed to induce feeding in hamsters tested at night. In this latter experiment, there was no effect of 2DG on wheel running or general alertness. Insulin administration significantly decreased plasma levels of glucose and free fatty acids (FFA). 2DG treatment produced a dose-related hyperglycemia associated with increased ketone levels. These data are discussed in terms of cerebral energy status and its relation to food intake and physiological responses.     

Entrainment of anticipatory activity to various durations of food access

Rats with lesions of the suprachiasmatic nuclei (SCN) and intact rats were maintained on restricted feeding with the duration of food access ranging from 4 to 12 hr. All rats with SCN lesions displayed at least some anticipatory activity (AA) at all food access durations. The amount of AA diminished when food access was extended to 12 hr and was lowest in a group that was exposed only to a 10-hr access period. The onset of AA (phase angle of entrainment) appeared to be more sensitive to the time of food availability than to its termination. Most intact rats maintained in constant light displayed some AA at food access durations between 4 and 10 hr. In most cases the period of the free running rhythm increased as it crossed food access and the free running rhythm became increasingly disrupted as the experiment progressed in all rats. In some cases the free running rhythm appeared to force AA out of entrainment. These results demonstrate that AA occurs in conditions that impose only minor deficits on the energy balance of rats. Furthermore, they provide additional evidence of interactions between two separate circadian pacemaking systems.     

Influences of feeding and drinking on circadian rhythms of opioid peptides in plasma, hypothalamus and pituitary gland in rats

Changes of plasma, hypothalamic and pituitary immunoreactive beta-endorphin (IR-beta-end), methionine-enkephalin (IR-Met-enk) and ACTH (IR-ACTH) were studied under various conditions of feeding and watering in rats. When rats were fed from 17:00 to 09:00 hr and water was given ad lib, plasma IR-beta-end and IR-ACTH had parallel circadian rhythms with a peak before feeding and drinking. In the hypothalamus, IR-beta-end and IR-Met-enk showed parallel circadian rhythms with a decrease before these behaviors. When rats were fed from 09:00 to 17:00 hr, the peaks of plasma IR-beta-end and IR-ACTH shifted to one hour before the onset of feeding and drinking. When feeding and watering were restricted to 17:00-09:00 hr and 09:00-12:00 hr respectively, plasma IR-beta-end and IR-ACTH exhibited parallel circadian rhythms with two separate peaks at one hour before drinking and feeding, respectively. In the hypothalamus, IR-beta-end, IR-Met-enk and IR-ACTH showed parallel circadian rhythms with a decrease before feeding but not before drinking. When rats were fed from 17:00 to 20:00 hr, plasma IR-beta-end increased and neurohypophysial IR-beta-end and IR-Met-enk decreased at 16:00 hr, one hour before feeding. It was observed that locomotor activities increased at the time of transition from light to dark and at one hour before the onset of feeding and drinking. The present results suggest that endogenous opioid peptides may have some physiological roles in feeding and drinking behaviors.     

Effects of estrogen upon feeding inhibition produced by intragastric glucose loads

Two studies were performed to determine if estrogen could potentiate the inhibitory effects of glucose on feeding behavior. In the first experiment, ovariectomized rats were injected with either 6 μg of estradiol benzoate (EB) or with sesame oil, and two days later were given 5 ml intragastric loads of either 40% glucose or 13.5% urea by gavage. Gavage was performed after a 12-hr fast, during the light period. Food intake (FI) was measured at hourly intervals, starting 2 hr after gavage and reaccess to food as well as daily throughout the experiment. In a second experiment, rats received a similar treatment, except that gavage was performed in an unfasted condition, during the dark period. It was found that (1) a 12-hr fast abolishes anorexic effects of EB, but leaves the inhibitory effects of glucose intact, (2) EB does not potentiate short-term effects of glucose, and (3) EB enhances delayed (22 hr after gavage) effects of glucose upon feeding. These findings are consistent with the hypothesis that EB modified the long-term control of feeding behavior, probably at the level of the ventromedial hypothalamus.     

Meal patterns of pygmy goats fed hay and concentrate ad lib

The meal patterns of pygmy goats fed hay and pelleted concentrate ad lib were recorded and analyzed. The pygmy goats consumed 8 hay meals [6 during the light phase (= light)/2 during the dark phase (= dark)] and 10 concentrate meals (7 during light/3 during dark) during 24 hr (12 hr light/12 hr dark). Sixty-two percent of hay and 74% of concentrate intake occurred during light. Total 24-hr hay (280 g) and concentrate (264 g) intakes were similar, but concentrate was preferred during dark. Concentrate meals were smaller during light than during dark. The mean feeding rate (g/min) within meals for both hay and concentrate was higher during dark than during light. Meal size and duration of postmeal interval were positively correlated for concentrate but not for hay. No significant positive correlation was found between meal size and duration of premeal interval. Separate analysis of diurnal and nocturnal meals indicated that the postmeal correlation for concentrate was evident primarily during dark. The results demonstrate that food intake in pygmy goats shows distinct diurnal variations and suggest that food intake in ruminants is regulated from meal to meal, with different factors prevailing during light and dark.     

Feeding in response to insulin but not to 2-deoxy-D-glucose in the hamster

Adult hamsters did not increase their food intake in response to subcutaneous injection of 2-deoxy-D-glucose (2DG) at doses of 50, 200, 350, 500, 750, 1,000, or 1,500 mg/kg. The failure to feed in response to 2DG was apparent regardless of time of testing and length of test. Hamsters did display marked hyperglycemia after 2DG treatment. Therefore, hamsters do detect and form neural responses to 2DG. In contrast to 2DG, injection of regular insulin, 2, 4, or 8 U/animal, caused hamsters to significantly increase their food intake over a 5-h test period. Furthermore, injection of 4 U of protamine zinc insulin twice daily also caused pronounced increases in food intake and weight gain by hamsters. Because hamsters do not seem to posses a feeding response to 2DG-induced glucoprivation, it seems unlikely that insulin elicits feeding via the glucoprivic control in this species. The results suggest that 1) hamsters lack the glucoprivic control of feeding behavior as exercised by 2DG, 2) 2DG and insulin treatments are not equivalent ingestive challenges, and 3) insulin-induced feeding may result in part from activation of a nonglucoprivic control of food intake.     

Circadian feeding rhythm after hypothalamic knife-cut isolating suprachiasmatic nucleus

Bilateral parasagittal knife-cut between the suprachiasmatic nucleus (SCN) and lateral hypothalamic area (LH) or coronal knife-cut between the SCN and ventromedial hypothalamic nucleus (VMH) resulted in a partial loss of the circadian feeding rhythm in rats; after either operation the rats consumed about 30% of their total daily food intake during the light period. However, after the parasagittal and coronal knife-cuts were made in combination, the circadian feeding rhythm was completely lost (50% food intake during the light period). Rats which lost the circadian feeding rhythm partially or completely showed neither obesity nor anorexia. These findings suggest that there are dual informational pathways from the SCN, possibly between the SCN and LH and between the SCN and VMH, through which circadian time signals generated in the SCN are transmitted to the LH and VMH to drive the circadian feeding rhythm.     

Effects of forced exercise on the food intake of rats maintained on a 23-hr deprivation schedule

Adult male rats of the Sprague-Dawley strain were given daily forced exercise of progressively higher levels on motor-driven treadmills while maintained on a 23-hr food deprivation schedule. Food intake measures were taken for 68 days, spanning a range of 2–8 hr exercise-sessions per day (1–4 hr actual running time) at 1.46 km/hr. The food intake of both exercising and sedentary food-restricted animals was lower as compared to that of ad lib feeding animals. Exercise did not further suppress food intake. Shifts to higher exercise levels did not result in any transitory or permanent depression in food intake. It is concluded that high levels of exercise in trained male rats maintained on a 1-hr/day feeding schedule has no suppressive effects on food intake.     

Decreased food intake of rats kept under adiurnal feeding cycles: effect of suprachiasmatic lesions

The circadian feeding rhythm and food intake under restricted feeding conditions, including adiurnal feeding cycles, were examined in rats with bilateral lesions of the suprachiasmatic nucleus (SCN). Although rats with SCN-lesions ate nearly as much food per day as those with control-lesions, their feeding pattern did not show circadian rhythmicity. When rats with control-lesions were fed for 5 hr once every 20 hr or for 7 hr once every 28 hr, they ate less than when they were fed for 6 hr once every 24 hr, probably due to some effect of desynchronization between the feeding cycle and an endogenous circadian oscillator. Decreased food intake under adiurnal feeding cycles was also observed in rats with SCN-lesions as in those with control-lesions. It is suggested that the circadian rhythm entrained by food is related to an endogenous time-keeping system that does not include the SCN.     

Enhancement of amphetamine-induced locomotor response in rats on different regimens of diet restriction and 2-deoxy-D-glucose treatment

Diet restriction (DR) in rodents increases lifespan, reduces age-related disease and pathology, increases stress responses, and maintains better function later into life compared with conventional ad libitum (AL) feeding. We have been investigating different DR regimens and also DR mimetics that stimulate stress response pathways that are activated by DR. By inhibiting glycolysis, feeding or injection of 2-deoxy-D-glucose (2DG) has been proposed as a DR mimetic and has been shown to provide neuroprotection. In the current study, we examined whether 2DG treatment produces behavioral changes similar to those observed in DR rats following stimulation of the dopaminergic (DA) system by D-amphetamine (AMPH). Male Fischer 344 rats were maintained on different dietary regimens: 40% daily DR (40% DR); every-other-day feeding (EOD); or AL with some groups provided food containing 0.4% 2DG or injected i.p. with 2DG. In addition, we examined the persistence of effects of DR or 2DG feeding after switching rats to AL. When locomotor activity was assessed at different time points following initiation of dietary treatments, we noted that the enhancement of AMPH-induced locomotor responses emerged earlier in DR rats than observed in 2DG fed rats, but 40% DR and EOD rats responded in a similar manner. Enhanced locomotor responses persisted in 2DG fed rats even when returned to normal diet for 1 month and in the case of DR rats even after 2 months of AL feeding. Three weeks of 2DG injections also enhanced AMPH response, but this effect was transient. The most important finding was that 2DG did not affect body weight or diet intake yet had effects similar to DR. Thus, 2DG appears to activate DA pathways in the same direction as DR does but without the necessity of reducing caloric intake.     

Feeding and macronutrient selection patterns in rats: adrenalectomy and chronic corticosterone replacement

To analyze further the role of corticosterone (CORT) in the control of feeding behavior, we examined the impact of adrenalectomy (ADX) and chronic CORT implants on the food intake and macronutrient self-selection patterns of adult male rats at different periods of the diurnal cycle. Consistent with a separate study of acute CORT injection in ADX rats (Kumar and Leibowitz, 1988), the present findings indicate that ADX significantly attenuated the rats' daily (24 hr) ingestion of all three macronutrients, namely, protein, carbohydrate and fat. However, food intake in the dark cycle, specifically during the first few hours after dark onset, was significantly more affected (-70%) than feeding in the later dark and light periods (-25%). Moreover, during this early dark time when circulating CORT level normally peaks, ADX appeared to have its strongest suppressive effect on carbohydrate ingestion. Chronic subcutaneous CORT implants in the ADX animals reversed these effects of surgery and generally restored the rats' eating patterns to that of the cholesterol-implanted SHAM animals. These findings suggest that CORT exerts a decisive influence on caloric intake, on the diurnal pattern of feeding, and on appetite for specific macronutrients. The impact of CORT on carbohydrate intake is apparent specifically during the active eating period, particularly at dark onset when endogenous CORT levels normally peak and carbohydrate is exhibited as the preferred macronutrient.     

TRH decreases food intake and increases water intake and body temperature in rats

Thyrotropin-releasing hormone (TRH) is a key regulator of the hypothalamus-pituitary-thyroid axis, which plays an important role in energy homeostasis and is involved in the regulation of feeding behavior. In the present study, we investigated the effects of acute and chronic TRH treatment on water intake, body temperature and feeding behavior in rats. TRH (0, 4, 16 and 64 mg/kg) was injected subcutaneously twice a day (06:00 and 18:00 h) in rats fed ad libitum. TRH decreased food and water intake in the first few hours (P < .05). There was a small reduction in food intake over the 24-h period, but body weight was not affected (P < .05). When TRH was injected at a dose of 32 mg/kg twice a day (06:00 and 18:00 h) for 5 days, T(3) and T(4) concentrations were increased (P < .05). TRH increased body temperature for 2 h after injection. Water intake was markedly increased (P < .05), but there was no effect on food intake or body weight. These results show that whereas a single injection of TRH decreases short-term food and water intake in rats, repeated daily treatments stimulate water intake but not food intake, and, thus, the increase in water consumption is mediated independently of food intake under these conditions.     

Dependence of memory of meal time upon circadian biological clock in rats

Water intake was measured in rats under a free feeding schedule and under a meal feeding schedule (food was presented for 3 hours each day from 14:00 to 17:00 hr), both under LD condition. Both schedules allowed animals free access to water. Under the meal feeding schedule, drinking occurred mainly during the food presentation time, but it was also observed during the dark period, when food was not present. After 3 weeks of meal feeding, the rats were transferred to the free feeding schedule. They showed a habit of drinking at the meal time of the previous schedule (meal-feeding-induced drinking) up to 7 days of free feeding. When the rats were made blind in the transition from the meal feeding to the 2nd free feeding, the meal-feeding induced drinking occurred with a progressively increasing delay relative to the clock time, in the same way as seen with the nocturnal drinking. Rats with bilateral lesions of the suprachiasmatic nucleus did not show nocturnal drinking during the meal feeding schedule, and no signs of the meal-feeding-induced drinking were observed after the 2nd free feeding schedule was started. It was concluded that the persistence of the memory of the time of meal feeding in the following free feeding period could be traced with water-drinking as an index and such memory seems to be formed depending on the biological clock which is ascribed to activity of the suprachiasmatic nucleus under LD condition.     

Circadian variation in the effects of nitric oxide synthase inhibitors on body temperature, feeding and activity in rats

We have investigated whether there is circadian variation in the effects of nitric oxide synthase inhibitors on body temperature, physical activity and feeding. We used nocturnally active Sprague-Dawley rats, housed at approximately 24 degrees C with a 12:12 h light:dark cycle (lights on 07:00 hours) and provided with food and water ad libitum. Nitric oxide synthesis was inhibited by intraperitoneal injection of the unspecific nitric oxide synthase inhibitor N-nitro- L-arginine methyl ester ( L-NAME, 100, 50, 25, 10 mg/kg), or the relatively selective inducible nitric oxide synthase inhibitor aminoguanidine (100, 50 mg/kg), during the day ( approximately 09:00 hours) or night ( approximately 21:00 hours). Body temperature and physical activity were measured using radiotelemetry, while food intake was calculated by weighing each animal's food before as well as 12 and 24 h after each injection. We found that daytime injection of L-NAME and aminoguanidine had no effect on daytime body temperature. However, daytime injection of both drugs did decrease nocturnal food intake ( P<0.05) and activity ( P<0.05). When injected at night, L-NAME reduced night-time body temperature ( P<0.01), activity ( P<0.05) and food intake ( P<0.05) in a dose-dependent manner, but night-time injection of aminoguanidine inhibited only night-time activity ( P<0.05). The effects of nitric oxide synthase inhibition on body temperature, feeding and activity therefore are primarily a consequence of inhibiting constitutively expressed nitric oxide synthase, and are subject to circadian variation.