Opposing effects of activation of central GABAA and GABAB receptors on brown fat thermogenesis in the rat

Baclofen (a GABAB agonist) stimulates body temperature, metabolic rate and brown adipose tissue (BAT) in the rat through a central action, but no effects of gamma-aminobutyric acid (GABA) itself on these parameters were observed. In the present study, it was found that the central effects of (+/-)baclofen (0.5-2.0 micrograms injection i.c.v.) on the temperature (1.2 degrees C increase) and metabolic rate (44-76% increase) of brown adipose tissue were inhibited by previous treatment with the GABAA agonist, muscimol (0.05 micrograms). Injection of GABA alone (12 micrograms) did not significantly affect these parameters, but in the presence of the GABAA antagonist bicuculline (2.5 micrograms), GABA significantly increased the temperature (0.3 degrees C) and oxygen consumption (22%) of brown fat. (-)Baclofen was found to be approximately 50-times more effective in stimulating the temperature of brown adipose tissue than (+/-)baclofen. The results indicate that activation of central GABAB receptors stimulates the activity and hence metabolic rate of brown adipose tissue. However, activation of the GABAA receptors opposes the effects of GABAB stimulation on the thermogenesis of brown fat.     

Activation of thermogenesis of brown fat in rats by baclofen

Injection of baclofen (0.5-5 micrograms) into the ventromedial hypothalamus (VMH) of anaesthetized rats produced marked increases in the temperature (over 2 degrees C) and thermogenic activity of brown adipose tissue (BAT). These effects were abolished by ganglionic or beta-adrenergic blockade, or denervation of the tissue, but unaffected by hypophysectomy, adrenalectomy or vagotomy. Injections into the hypothalamus close to, but outside the ventromedial hypothalamus did not affect brown adipose tissue. Intravenous administration of baclofen produced similar increases in the temperature of brown adipose tissue, but at larger doses (50-1000 micrograms), and a subcutaneous injection stimulated the metabolic rate in conscious rats. Chronic treatment with baclofen suppressed weight gain and stimulated activity of brown adipose tissue without affecting food intake. These effects of baclofen, which were not mimicked by injections of gamma-aminobutyric acid (GABA), indicate a novel action of baclofen in the ventromedial hypothalamus, leading to marked increases in metabolic rate and body temperature by stimulating sympathetic outflow to brown fat.     

Activation of brown fat thermogenesis in response to central injection of corticotropin releasing hormone in the rat

Injection of CRF-41 (2-5 nmol) into the third ventricle, or the paraventricular nucleus of anaesthetized rats caused a marked rise in the temperature of the interscapular brown adipose tissue (BAT) depot (peak rise 0.8-1 degree C) which was inhibited by prior intravenous injection of propranolol. There was also a significant increase in the mitochondrial proton conductance pathway of brown adipose tissue, assessed from the binding of guanosine diphosphate (GDP) to mitochondria isolated from the interscapular (89% above control) and perirenal and para-aortic depots (130%). Acute surgical sympathectomy of interscapular brown adipose tissue immediately prior to injection of CRF significantly attenuated the increase in mitochondrial GDP-binding. Hypophysectomized (HYPX) rats showed a large (180%) increase in GDP-binding of brown adipose tissue 7 days after surgery and this was almost completely prevented by denervation of the interscapular depot prior to hypophysectomy. Acute injection of morphine also reduced the GDP-binding in hypophysectomized, but not in control rats. These data demonstrate that central injection of CRF stimulates thermogenesis in brown adipose tissue, probably by modifying sympathetic outflow. The activation of brown adipose tissue following hypophysectomy was also dependent on the sympathetic innervation and could be due to an increase in release of CRF.     

Effects of the irreversible inhibition of GABA transaminase upon some morphine effects

The effects were investigated of two irreversible inhibitors of gamma-aminobutyric acid (GABA) transaminase on the analgesic response to morphine, on the intensity of tolerance produced by a slow release preparation of the opiate and on the degree of physical dependence to the analgesic. Both compounds, γ-acetylenic GABA and γ-vinyl GABA, produced an intensification of morphine analgesia, although large doses of γ-acetylenic GABA produced stimulant effects that masked a possible synergism. The intensification of analgesia was greater in tolerant mice as compared with the increment observed in naive animals, which may indicate a decrease in the intensity of tolerance, since the drugs per se were ineffective in the analgesic test. Physical dependence was attenuated by the two compounds assayed, except when γ-acetylenic GABA was administered in high doses. The results may indicate a relationship between some acute and chronic effects of morphine and GABA concentration in the nervous tissue. However an effect of the GABA transaminase inhibitors unrelated to GABA levels in the nervous system cannot be dismissed.     

l-Dopa and brown fat hemorrhage in the rat pup

Female Sprague-Dawley rats were given l-dopa during gestation. Several hours after birth, hemorrhages appeared in the interscapular brown adipose tissue of some of the offspring of l-dopa treated rats. Histological examination of the brown fat of these pups showed large hemorrhages and vasodilation. Administration of l-dopa during the first 5 days of gestation and dopamine for the first 7 days of gestation caused a dose-related occurrence of hemorrhage in the offspring. Cross-foster experiments demonstrated that pups exposed to l-dopa in utero had a significantly increased chance of hemorrhage, while pups nursed by l-dopa-treated rats did not. During Days 1–21 of gestation, haloperidol (1 mg/kg/day) partially blocked the effect of l-dopa (100 mg/kg/day) while propranolol (5 mg/kg/day) and phenoxybenzamine (10 mg/kg/day) did not. When given with l-dopa, the decarboxylase inhibitors MK 486 and RO 4–4602 reduced the frequency of hemorrhages. The combination of l-dopa (100 mg/kg), pyrogallol (100 mg/kg), and tranylcypromine (1.5 mg/kg) produced 20.8% hemorrhage in the pups, a significant increase compared to l-dopa (100 mg/kg) alone (7.9%). Among eight different amino acids and amines tested, dopamine was the most hemorrhagic. The dopamine-β-hydroxylase inhibitor, dimethyldithiocarbamate (125 mg/kg), given daily for the first 7 days of gestation, led to brown fat hemorrhage in 17.6% of the offspring, suggesting that dopamine may be implicated in this effect.     

GABA and behavioral inhibition in the neonatal rat pup

The effects of the GABA agonist muscimol and GABA antagonist picrotoxin were examined in 3–4-day-old deprived rat pups under conditions of low (absence of milk) and high (milk presence) activity baselines. A low dose of muscimol was observed to have activating effects under low baseline conditions, whereas higher doses of muscimol were observed to depress milk-induced activity and mouthing. Picrotoxin treatment was observed to increase activation of these neonates under both baseline conditions. Taken together, these results provide evidence for a functional GABAergic inhibition of behavior in the neonate.This research was supported in part by National Institute of Mental Health grant R01MH35761     

Synergism between caffeine and dl-phenylpropanolamine on brown adipose tissue thermogenesis in the adult rat

Caffeine produces enhanced oxygen consumption, an effect that may reflect an action of caffeine on brown adipose thermogenesis. In Experiment 1, adult male rats were anesthetized with 1.2 g/kg urethane and treated (IP) with either 0.9% saline or 10, 20 or 40 mg/kg caffeine (n = 4 each group). Interscapular BAT (IBAT) and rectal temperatures were recorded every minute for 10 minutes prior to and 30 minutes following drug injection. Stable IBAT and rectal temperatures were observed prior to and after saline injection whereas rats treated with 20 and 40 mg/kg caffeine exhibited moderate increases in IBAT, but not rectal, temperature. In Experiment 2, adult male rats were treated with either 0.9% saline or 10 mg/kg caffeine, anesthetized with urethane (1.2 g/kg) and treated (30 minutes after pretreatment injections) with either 0.9% saline or 10 mg/kg dl-phenylpropanolamine (dl-PPA). A combination of caffeine and dl-PPA produced significantly greater BAT thermogenesis than just dl-PPA alone. The implications of these data for the inclusion of caffeine in over-the-counter diet-pills are discussed.     

Cold-induced changes in gene expression in brown adipose tissue: implications for the activation of thermogenesis

Brown adipose tissue (BAT) is the site of heat production (thermogenesis). This unique function is performed by uncoupling protein 1 (UCP1) specifically expressed in mitochondria of BAT. UCP1 dissipates the driving force of ATP synthesis, and thus causes heat production followed by energy expenditure. The thermogenic function of BAT has the role of maintaining body temperature under cold conditions. When animals are exposed to cold, the expression of UCP1 gene is increased to activate thermogenesis. To date, functional analysis of BAT has been focused on UCP1, because it plays an indispensable role in thermogenesis. However, the gene expression of not only UCP1 but also that of other genes in BAT is expected to be regulated to achieve effective thermogenesis. Our previous investigations showed increased expression of genes that encode several energy metabolic enzymes in the BAT of rats kept in the cold. These changes in gene expression imply that the enhancement of energy metabolism is needed to activate thermogenesis. Furthermore, various reports from studies focused on genes whose expression is changed in response to cold stimulation have provided new insights into the function of BAT. In this review, to understand the thermogenic function of BAT systematically, we have provided an overview of previous findings on changes in the expression of genes thought to be related to the activation of thermogenesis in BAT.     

Thiopental inhibition of γ-aminobutyrate transaminase in rat brain synaptosomes

The kinetic parameters for thiopental inhibition of γ-aminobutyrate: 2-oxoglutarate transaminase solubilized from rat brain synaptosomes were studied, V_max is 0.76 μmole/hr/mg of protein. K_m for GABA (γ-aminobutyric acid) is 16.0 mM and K_m for 2-OG (2-oxoglutarate: α-ketoglutarate) is 0.35 mM. Thiopental inhibition is noncompetitive and K_i 1.1 mM. is similar when either GABA or 2-OG is the limiting substrate. The implication of these findings in clinical anesthesia is discussed.     

Glutamate receptors in the raphe pallidus mediate brown adipose tissue thermogenesis evoked by activation of dorsomedial hypothalamic neurons

Disinhibition of DMH neurons with the GABAA receptor antagonist, bicuculline, increases heart rate (HR) and augments both brown adipose tissue sympathetic nerve activity (BAT SNA) and renal SNA (RSNA) contributing to the evoked increases in BAT thermogenesis and arterial pressure (AP). We determined the role of glutamate receptor activation in the rostral raphe pallidus (RPa) in mediating the sympathoexcitatory responses in HR, BAT SNA and RSNA following disinhibition of DMH neurons in urethane/chloralose anesthetized, artificially ventilated rats. Microinjections of either the selective NMDA receptor agonist, NMDA, or the selective non-NMDA receptor agonist, kainic acid (KA), into the RPa produced increases in BAT SNA (peak: + 502% and + 408% of control, respectively) and BAT temperature (peak: + 0.6 degrees C and + 1.0 degrees C) accompanied by rises in HR (peak: + 38 and + 63 bpm), RSNA (peak: + 57% and + 58% of control) and MAP (peak: + 12 and 15 mmHg). These responses were reversed by subsequent microinjection into RPa of the respective selective glutamate receptor antagonists, AP5 and CNQX. Microinjections of the non-selective glutamate receptor antagonist, kynurenic acid (Kyn), the NMDA receptor antagonist, AP5, or the non-NMDA receptor antagonist, CNQX, were effective in reversing the increases in BAT SNA (for Kyn, from peak of + 419% of control to + 9% of control) and BAT temperature, but not those in HR, MAP or RSNA (for Kyn, from peak of + 143% of control to + 124% of control) evoked by unilateral microinjection of bicuculline into the DMH. These results indicate that both NMDA and non-NMDA glutamate receptors in the RPa play a significant role in mediating the excitatory synaptic transmission producing the activation of BAT thermogenesis following disinhibition of DMH neurons. Glutamate receptors in the RPa may not be important for transmitting cardiovascular responses induced by activation of the DMH neurons.     

Chronic inhibition of GABA synthesis in the bed nucleus of the stria terminalis elicits anxiety-like behavior

The current study tested the hypothesis that chronic loss of inhibitory GABAergic tone in the bed nucleus of the stria terminalis (BNST), a region implicated in anxiety behavior, results in generalized anxiety disorder-like behaviors without panic-like responses (i.e., tachycardia, hypertension and tachypnea) following panicogenic stimuli (e.g., sodium lactate infusions). To test this hypothesis, the GABA synthesis inhibitor L-allylglycine (L-AG) or its inactive isomer D-AG was chronically infused into the BNST of male rats via osmotic mini-pumps. L-AG, but not D-AG, treated rats had increased anxiety-like behavior as measured by social interaction (SI) and elevated-plus maze paradigms. Restoring GABAergic tone, with 100pmoles/100nl of muscimol (a GABA(A) receptor agonist), in the BNST of L-AG treated rats attenuated L-AG-induced anxiety-like behavior in the SI test. To assess panic-like states, L-AG treated rats were intravenously infused with 0.5 M sodium lactate, a panicogenic agent, prior to assessing SI and cardiorespiratory responses. L-AG decreased SI duration again; however, sodium lactate did not induce panic-like cardiorespiratory responses. These findings demonstrate that GABA inhibition in the BNST elicits anxiety-like behavior without increasing sensitivity to lactate, thus suggesting a behavioral profile similar to that of generalized anxiety-like behavior rather than that of panic.     

Differential effects of GABA transaminase inhibitors on sexual behavior, locomotor activity, and motor execution in the male rat

The GABA transaminase inhibitors gamma-acetylen GABA (GAG) and sodium valproate were administered intraperitoneally and their effects on locomotor activity, motor execution and sexual behavior were analyzed. It was found that sodium valproate, administered 15 min before observation, reduced locomotor activity only at a dose of 200 mg/kg. Doses of 100 and 400 mg/kg had no effect. Motor execution was impaired in a dose-dependent way, the lowest effective dose being 200 mg/kg. Sexual behavior was also dose-dependently reduced. Sodium valproate, administered 60 min before observation, inhibited all behaviors. The lowest effective dose was 200 mg/kg for locomotor activity and 400 mg/kg for motor execution and sexual behavior. GAG also inhibited all behavior, in doses ranging from 25 mg/kg (locomotor activity) to 100 mg/kg (motor execution and sexual behavior). The data showed that there is no relation between effects on locomotor activity and the effects on sexual behavior, whereas sexual behavior is inhibited whenever motor execution is impaired. Moreover, there is no correlation between effects on locomotor activity and motor execution. It is suggested that GABA transaminase inhibitors effect sexual behavior only indirectly, via an impairment of motor execution. Therefore it is doubtful whether GABAergic mechanisms play any role in the normal regulation of sexual behavior.     

Chronic alcohol intake induces the oxidative capacity of brown adipose tissue in the rat

The present study was carried out to elucidate the effect of long-term alcohol intake on the oxidative capacity of brown adipose tissue in the rat. Rats housed at room temperature were given water containing 10% ethanol for six months, while controls received water alone. Fully cold-acclimated rats (exposed to +4 degrees C for 6 weeks) served as the second control group. Alcohol did not alter the food intake of the rats compared with the controls kept at room temperature, but it did cause a mean decrease of 8 ml in fluid consumption. There was no difference in the increase in body weight between the groups housed at room temperature. Body weight of the rats exposed to cold did not change during cold acclimation. No morphological liver changes were observed in alcohol-fed rats, but some changes related to long-term alcohol consumption were found in the myocardium. Chronic alcohol intake increased the quantity of brown adipose tissue and its protein content but changes were not as great as in the cold-acclimated rats nor did alcohol increase protein content per unit of the adipose tissue as did cold. On the other hand, the specific activity of mitochondrial cytochrome oxidase increased by 90% and that of succinate dehydrogenase by 130% in alcohol-fed rats, whereas specific activities of these enzymes displayed little or no change in the cold-acclimated rats. Results suggest that chronic alcohol ingestion induces the oxidative capacity of the interscapular brown adipose tissue in the rat, increasing the mass of BAT and specific activities of mitochondrial enzymes.     

Calcium-independent inhibition of the spontaneous release of GABA by baclofen in the rat hippocampus]

The mechanism of calcium-independent spontaneous GABA release in a long-living (3-10 weeks) rat hippocampal neuron culture was studied. It was found that Cd2+ (100 microM), a Ca2+ channel blocker, reversibly decreased the frequency and amplitude of GABAergic spontaneous miniature inhibitory postsynaptic currents (smIPSCs). Besides, Cd2+ decreased the currents evoked by muscimol application onto the neurons, which is evidence of an additional postsynaptic effect. The GABAB receptor agonist baclofen (0.1-50 microns) produced a concentration-dependent decrease in the smIPSC frequency, while not affecting the current amplitude. The baclofen effect was blocked by the pertussis toxin. The baclofen efficacy both in the presence of Cd2+ (presumably compete blocking of Ca2+ channels) and in the absence of this agent were similar and could be completely accounted for by the loss of an equivalent smIPSC fraction. Thus, the presynaptic baclofen-induced inhibition of the spontaneous GABA release can be entirely independent of the calcium channel modulation (the latter playing a decisive role in a mediator release induced by the action potential). Therefore, the smIPSC measurements (widely used in the past decade for the study of presynaptic drug activity) may inadequately reflect the drug effect in the intact brain.     

Irreversible inhibitors of GABA transaminase induce antinociceptive effects and potentiate morphine

The irreversible inhibitors of GABA-transaminase, γ-acetylenic GABA and γ-vinyl GABA, were examined for antinociceptive actions in rodents. An antinociceptive effect unaccompanied by ataxia was demonstrated in mice on the 52°C hot-plate and in rats in a tail-stimulation procedure and was maximal at 4–6 hr after drug administration, which correlates temporally with reported maximal increases of brain GABA. The antinociceptive effect was antagonized by a subconvulsive dose of bicuculine (0.5 mg/kg, i.p.) in rats, but could not be prevented by naloxone (1 mg/kg, i.p.) in mice. Only the (+)-stereoisomer γ-vinyl GABA, active as a GABA-transaminase inhibitor, was antinociceptive. The effects appear to be causally related to elevated cerebral GABA levels. The profile of these agents in the tail stimulation test in rats suggests a specific antinociceptive effect since vocalization and vocalization after-discharge were similarly affected. The analgesic actions of morphine in mice on the 56°C hot-plate were enhanced 5 hr after the administration of γ-acetylenic GABA or γ-vinyl GABA. The compounds did not alter the naloxone-precipitated morphine withdrawal syndrome in the rat.It is concluded that γ-acetylenic GABA and γ-vinyl GABA can produce distinct antinociceptive effects in rats and mice related to increased brain GABA levels, but which are not opioid-like in nature.     

Ambient temperature modulation of fenfluramine-induced thermogenesis in the rat

The anti-obesity drug fenfluramine, promotes loss of weight by reducing food intake; however, there is controversy as to whether the drug can also elevate expenditure of energy. Resting consumption of oxygen (VO2) was measured in conscious rats to determine whether the injection of fenfluramine increased metabolic rate and whether prior fasting, or ambient temperature altered the response. Regardless of whether the rats were fed or had been fasted for 22 hr, in a thermoneutral environment (28 degrees C), the intraperitoneal injection of dl-fenfluramine (20 mg/kg) caused a raised oxygen consumption. This elevation was sustained to the end of the 60-min period of measurement after the injection, at which point the colonic temperature was found to be increased. This metabolic response to fenfluramine was largely attenuated when the drug was administered at 23 degrees C, and the colonic temperature of the rats was decreased by 60 min after the injection. At 4 degrees C, the injection of fenfluramine inhibited thermogenesis against cold, the oxygen consumption fell and the rats exhibited hypothermia. It was concluded that fenfluramine can increase the metabolic rate, but that this effect is not conditional on associated food intake, as has been reported. Rather, the ambient temperature governs whether stimulation or inhibition of thermogenesis will be evoked. These metabolic effects of fenfluramine explain, in part, its divergent effects on body temperature, reported previously.     

Halothane: inhibition and activation of rat hepatic glutathione S-transferases

Multiple halothane anesthesias (1.25 MAC for 1 hr on 3 alternate days) of male Long-Evans rats initially decreased by up to 30% and subsequently increased to up to 185% liver cytosolic glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene, 3,4-dichloro-1-nitrobenzene and trans-4-phenyl-3-buten-2-one and glutathione peroxidase activity. Halothane rapidly and reversibly activated hepatic cytosolic glutathione S-transferases and purified isoenzyme 1-2 but not isoenzymes 1-1 and 3-3. At high concentrations of halothane (ca. 22 mM), maximal activation was ca. 25%. Halothane, enflurane, isoflurane and methoxyflurane, but not the halothane metabolite 1-chloro-2,2-difluoroethylene, inhibited a mixture of liver cytosolic glutathione S-transferases with time (ca. 30% inhibition/15 min). The inhibition exhibited pseudo-first order kinetics (kobs = 0.13 min-1) and an I50 for halothane of greater than or equal to 15 mM. Halothane inhibited glutathione S-transferases 3-3, 3-4, and 4-4 by 50-60%, but did not affect isoenzymes 1-1 and 1-2. The ability of halothane to diminish hepatic glutathione S-transferase activity in vivo may in part reflect the time-dependent inhibition of glutathione S-transferase isoenzymes containing the 3- and 4-subunits.