Juliana Bohnen Guimarães

Inhibition of tryptophan hydroxylase abolishes fatigue induced by central tryptophan in exercising rats

Fatigue during prolonged exercise is related to brain monoamines concentrations, but the mechanisms underlying this relationship have not been fully elucidated. We investigated the effects of increased central tryptophan (TRP) availability on physical performance and thermoregulation in running rats that were pretreated with parachlorophenylalanine (p‐CPA), an inhibitor of the conversion of TRP to serotonin. On the 3 days before the experiment, adult male Wistar rats were treated with intraperitoneal (ip) injections of saline or p‐CPA. On the day of the experiment, animals received intracerebroventricular (icv) injections of either saline or TRP (20.3 μM) and underwent a submaximal exercise test until fatigue. Icv TRP‐treated rats that received ip saline presented higher heat storage rate and a 69% reduction in time to fatigue compared with the control animals. Pretreatment with ip p‐CPA blocked the effects of TRP on thermoregulation and performance. Moreover, ip p‐CPA administration accelerated cutaneous heat dissipation when compared with saline‐pretreated rats. We conclude that an elevated availability of central TRP interferes with fatigue mechanisms of exercising rats. This response is modulated by serotonergic pathways, because TRP effects were blocked in the presence of p‐CPA. Our data also support that a depletion of brain serotonin facilitates heat loss mechanisms during exercise.

Fatigue is mediated by cholinoceptors within the ventromedial hypothalamus independent of changes in core temperature

We investigated brain mechanisms modulating fatigue during prolonged physical exercise in cold environments. In a first set of studies, each rat was subjected to three running trials in different ambient temperatures (Ta). At 8 °C and 15 °C, core body temperature (Tcore) decreased and increased, respectively, whereas at 12 °C, the Tcore did not change throughout the exercise. In another set of experiments, rats were randomly assigned to receive bilateral 0.2 μL injections of 2.5 × 10−2 M methylatropine or 0.15 M NaCl solution into the ventromedial hypothalamic nuclei (VMH). Immediately after the injections, treadmill exercise was started. Each animal was subjected to two experimental trials at one of the following Ta: 5 °C, 12 °C or 15 °C. Muscarinic blockade of the VMH reduced the time to fatigue (TF) in cold environments by 35–37%. In all Ta studied, methylatropine‐treated rats did not present alterations in Tcore and tail skin temperature compared with controls. These results indicate that, below the zone of thermoneutrality, muscarinic blockade of the VMH decreases the TF, independent of changes in Tcore. In conclusion, our data suggest that VMH muscarinic transmission modulates physical performance, even when the effects of thermoregulatory adjustments on fatigue are minimal.

Endurance training blocks uncoupling protein 1 up-regulation in brown adipose tissue while increasing uncoupling protein 3 in the muscle tissue of rats fed with a high-sugar diet

The mitochondrial uncoupling proteins (UCPs) of interscapular brown adipose tissue (iBAT) and of muscles play important roles in energy balance. For instance, the expression of UCP1 and UCP3 are modulated by free fatty acid gradients induced by high-sugar diets and acute exercise that is dependent on sympathetic stimulation. However, the effects of endurance training in animals fed with high-sugar diets are unknown. This study aims to evaluate the long-term effects of diet and exercise on UCP1 and UCP3 levels and energy balance efficiency. Rats fed with standard or high-sugar (HSD) diets were simultaneously subjected to running training over an 8-week period. After the training period, the rats were decapitated, and the iBAT and gastrocnemius muscle tissues were removed for evaluation of the β₃-receptor, Ucp1, and Ucp3 mRNA and protein expression, which were analyzed by quantitative reverse transcriptase polymerase chain reaction and Western blot, respectively. Groups fed with an HSD displayed a higher adiposity index and iBAT weight (P < .05), whereas exhibited an up-regulation of Ucp1 mRNA and protein levels (P < .05). Training increased β₃-receptor mRNA in iBAT and reduced the Ucp3 mRNA in muscle tissues. In association with an HSD, training restored the increasing β₃-receptor mRNA and greatly up-regulated the levels of Ucp3 mRNA. Therefore, training blocked the HSD-induced up-regulation of UCP1 expression in iBAT, whereas it up-regulated the expression of Ucp3 mRNA in muscle. These results suggest that training enhances the relationship between Ucp1/Ucp3 mRNA levels, which could result in higher energy efficiency, but not when HSD-induced elevated sympathetic activity is maintained.

Hypothalamic Temperature of Rats Subjected to Treadmill Running in a Cold Environment

Different strategies for cooling the body prior to or during physical exercise have been shown to improve prolonged performance. Because of ethical and methodological issues, no studies conducted in humans have evaluated the changes in brain temperature promoted by cooling strategies. Therefore, our first aim sought to measure the hypothalamic temperature (Thyp) of rats subjected to treadmill running in a cold environment. Moreover, evidence suggests that Thyp and abdominal temperature (Tabd) are regulated by different physiological mechanisms. Thus, this study also investigated the dynamics of exercise-induced changes in Thyp and Tabd at two ambient temperatures: 25°C (temperate environment) and 12°C (cold). Adult male Wistar rats were used in these experiments. The rats were implanted with a guide cannula in the hypothalamus and a temperature sensor in the abdominal cavity. After recovery from this surgery, the rats were familiarized with running on a treadmill and were then subjected to the two experimental trials: constant-speed running (20 m/min) at 12°C and 25°C. Both Thyp and Tabd increased during exercise at 25°C. In contrast, Thyp and Tabd remained unchanged during fatiguing exercise at 12°C. The temperature differential (i.e., Thyp - Tabd) increased during the initial min of running at 25°C and thereafter decreased toward pre-exercise values. Interestingly, external cooling prevented this early increase in the temperature differential from the 2nd to the 8th min of running. In addition, the time until volitional fatigue was higher during the constant exercise at 12°C compared with 25°C. Together, our results indicate that Thyp and Tabd are regulated by different mechanisms in running rats and that external cooling affected the relationship between both temperature indexes observed during exercise without environmental thermal stress. Our data also suggest that attenuated hypothalamic hyperthermia may contribute to improved performance in cold environments.

Muscarinic receptors within the ventromedial hypothalamic nuclei modulate metabolic rate during physical exercise.

The involvement of muscarinic cholinoceptors within the ventromedial hypothalamic nuclei (VMH) on the exercise-induced increase in oxygen consumption (VO(2)) was investigated. Rats were fitted with bilateral cannulae into the VMH for local delivery of drugs. On the day of the experiments, the animals were submitted to running exercise (20 m/min; 5% grade) until the point of fatigue. VO(2) was continuously measured after bilateral injections of either 0.2 μL of 5 × 10(-9)mol methylatropine or 0.15M NaCl solution into the VMH. Control experiments were conducted in freely moving rats on the treadmill. Muscarinic blockade within the VMH reduced time to fatigue by 32% and enhanced the increase in VO(2) from the 8th until the 17th min of exercise when compared to the control trial. In fact, time to fatigue was negatively correlated to the rate of increase in VO(2) (r(2)=0.747; P<0.001). However, bilateral injections of methylatropine in freely moving rats did not change VO(2) in comparison to saline injections. In conclusion, muscarinic cholinoceptors within the VMH are activated during exercise to modulate the increase in metabolic rate. Furthermore, blocking muscarinic transmission leads to a faster increase in VO(2) that is associated with the early interruption of exercise.

Thermoregulatory responses in exercising rats: methodological aspects and relevance to human physiology

Rats are used worldwide in experiments that aim to investigate the physiological responses induced by a physical exercise session. Changes in body temperature regulation, which may affect both the performance and the health of exercising rats, are evident among these physiological responses. Despite the universal use of rats in biomedical research involving exercise, investigators often overlook important methodological issues that hamper the accurate measurement of clear thermoregulatory responses. Moreover, much debate exists regarding whether the outcome of rat experiments can be extrapolated to human physiology, including thermal physiology. Herein, we described the impact of different exercise intensities, durations and protocols and environmental conditions on running-induced thermoregulatory changes. We focused on treadmill running because this type of exercise allows for precise control of the exercise intensity and the measurement of autonomic thermoeffectors associated with heat production and loss. Some methodological issues regarding rat experiments, such as the sites for body temperature measurements and the time of day at which experiments are performed, were also discussed. In addition, we analyzed the influence of a high body surface area-to-mass ratio and limited evaporative cooling on the exercise-induced thermoregulatory responses of running rats and then compared these responses in rats to those observed in humans. Collectively, the data presented in this review represent a reference source for investigators interested in studying exercise thermoregulation in rats. In addition, the present data indicate that the thermoregulatory responses of exercising rats can be extrapolated, with some important limitations, to human thermal physiology.

Increased brain L-arginine availability facilitates cutaneous heat loss induced by running exercise.

The effects of increased brain availability of l‐arginine (l‐arg), a precursor for nitric oxide synthesis, on core body temperature (Tcore) and cutaneous heat loss were evaluated in running rats. One week prior to the experiments, adult male Wistar rats received the following implants: a chronic guide cannula in the lateral cerebral ventricle and a temperature sensor in the abdominal cavity. On the day of the experiments, the rats were assigned to receive a 2‐μL intracerebroventricular injection of either NaCl (0.15 mol/L) or l‐arg solution (0.825, 1.65 or 3.30 mol/L); Tcore and tail skin temperature were measured while the rats ran at a speed of 18 m/min until they were fatigued. l‐arginine induced a dose‐dependent reduction in the threshold Tcore required for cutaneous heat loss (38.09 ± 0.20°C for 3.30‐mol/L l‐arg vs 38.61 ± 0.10°C for saline; P < 0.05), which attenuated the exercise‐induced hyperthermia. Although the rats treated with l‐arg presented a lower Tcore at the end of exercise (~0.7°C lower after treatment with the highest dose), no changes in the time to fatigue were observed relative to the control trial. These results suggest that brain l‐arg controls heat loss during exercise, most likely by modulating the sympathetic vasoconstrictor tonus to skin vessels. Furthermore, despite facilitating cutaneous heat loss mechanisms, increased brain l‐arg availability did not enhance physical performance.

Endurance Training Increases Leptin Expression in the Retroperitoneal Adipose Tissue of Rats Fed with a High-Sugar Diet

The presence of leptin receptors in white adipose tissue (WAT) suggests a type of peripheral control during the development of obesity and other metabolic disorders. Both diet composition and exercise influence serum leptin; however, the effect of their combination on long-term WAT leptin metabolism is unknown. In this study, rats fed with standard or high-sugar diets (HSD) were simultaneously subjected to running training for 4- and 8-week periods, and the retroperitoneal WAT (rWAT) was evaluated for adipocyte cell size, lipid and catecholamine content, Lep, OB-Rb and Ucp2 mRNA transcription levels, and circulating leptin and non-esterified fatty acids (NEFA). The HSD groups displayed a higher adiposity index and rWAT weight, Lep mRNA and protein upregulation, and a period-dependent effect on OB-Rb mRNA expression. Exercise decreased serum leptin and upregulated the OB-Rb mRNA levels. However, in rats fed with an HSD, the increase in OB-Rb mRNA and reduction in catecholamine levels resulted in a high level of adiposity and hyperleptinemia. The combination of training and an HSD decreases the NEFA levels and upregulating the Ucp2 mRNA expression in the 4-week period, while downregulating the Ucp2 mRNA expression in the 8-week period without changing the NEFA levels. Our results suggest that an HSD induces an increase in leptin expression in rWAT, while reducing adipocytes via leptin-mediated lipolysis after an 8-week period. In exercised rats fed an HSD, TAG synthesis and storage overlaps with lipolysis, promoting fat store development and Lep mRNA and plasma protein upregulation in adult rats.

The central administration of C75, a fatty acid synthase inhibitor, activates sympathetic outflow and thermogenesis in interscapular brown adipose tissue

The present work investigated the participation of interscapular brown adipose tissue (IBAT), which is an important site for thermogenesis, in the anti-obesity effects of C75, a synthetic inhibitor of fatty acid synthase (FAS). We report that a single intracerebroventricular (i.c.v.) injection of C75 induced hypophagia and weight loss in fasted male Wistar rats. Furthermore, C75 induced a rapid increase in core body temperature and an increase in heat dissipation. In parallel, C75 stimulated IBAT thermogenesis, which was evidenced by a marked increase in the IBAT temperature that preceded the rise in the core body temperature and an increase in the mRNA levels of uncoupling protein-1. As with C75, an i.c.v. injection of cerulenin, a natural FAS inhibitor, increased the core body and IBAT temperatures. The sympathetic IBAT denervation attenuated all of the thermoregulatory effects of FAS inhibitors as well as the C75 effect on weight loss and hypophagia. C75 induced the expression of Fos in the paraventricular nucleus, preoptic area, dorsomedial nucleus, ventromedial nucleus, and raphé pallidus, all of which support a central role of FAS in regulating IBAT thermogenesis. These data indicate a role for IBAT in the increase in body temperature and hypophagia that is induced by FAS inhibitors and suggest new mechanisms explaining the weight loss induced by these compounds.