INTRODUCTION

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ALTHOUGH IT HAS BEEN KNOWN for a long time that brown adipose tissue (BAT) has relatively high levels of glycerokinase (GyK) activity, especially compared with levels in white adipose tissue (WAT), the physiological control of the enzyme has been studied little. Notwithstanding the possible role of GyK in the utilization of plasma glycerol by different tissues, in adipose tissue its activity has been usually associated with the recycling of free glycerol produced by hydrolysis of stored triacylglycerols (TAG). Hydrolysis of stored TAG to produce fatty acids (FA), which are both substrates and uncoupling messengers for BAT mitochondria, is an obligatory step in the process of activation of heat production in BAT in both diet-induced and nonshivering thermogenesis (11). Therefore, maintenance of adequate stores of TAG, through esterification, via glycerol-3-phosphate (G3P) of newly synthesized or preformed FA (taken up from the circulation or recycled after hydrolysis of endogenous TAG) seems to be essential for the normal functioning of BAT. There are three possible sources of G3P for acylation and TAG formation: 1) glucose, via dihydroxyacetone in the glycolytic pathway, and conversion to G3P by glycerophosphate dehydrogenase; 2) three carbon intermediates (such as lactate and pyruvate), via glyceroneogenesis, forming phosphoenolpyruvate via the dicarboxylic shuttle and subsequent formation of G3P by a partial reversion of glycolysis; and 3) glycerol, produced by hydrolysis of stored TAG or taken up by the tissue from the circulation, that is directly converted to G3P by GyK. We recently showed (3) that in BAT, similar to what has been found for WAT (7, 12, 17), glucose is a poor substrate for FA formation, being predominantly used for glyceride-glycerol synthesis. We also showed in the same work (3) that glyceroneogenesis is very active in BAT, glycerol-TAG synthesis from nonglucose sources representing ~80% of total glycerol production in rats fed a balanced diet. These findings clearly evidenced the importance of an active G3P production for the preservation of BAT normal metabolic activity and prompted us to investigate the contribution of GyK to this process. Previous findings in rats adapted to a high-protein, carbohydrate-free (HP) diet, a preparation that has been used in our laboratory to investigate the control of energy-linked metabolic processes, suggested that the sympathetic nervous system participated in the regulation of GyK activity. Thus we found that HP diet-adapted rats have a reduced BAT thermogenic capacity (5) that is accompanied by marked decreases in the activity of BAT GyK (2) and in the rate of norepinephrine (NE) turnover (4), which is mainly dependent on tissue sympathetic outflow. Also, an increased activity of the enzyme has been detected in BAT from rats submitted to a prolonged period (90 days) of exposure to cold (1), which is a well-known activator of tissue sympathetic activity (11). Therefore, the experiments of the present work were designed to investigate the participation of the sympathetic nervous system in the control of BAT GyK. The specific objective was to examine the effect of BAT denervation on the activity of GyK and on the response of the enzyme to two situations in which the sympathetic outflow to BAT is activated: 1) during cold exposure and 2) after replacement of the HP diet by a balanced diet, which we have shown to restore NE turnover rates to normal levels within 24 h (4).

	MATERIALS AND METHODS

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Male Wistar rats maintained at 25 ± 2°C on a 12:12-h light-dark cycle were used in all experiments. In addition to a commercial nutritionally balanced diet (% wt/wt, 19% protein, 50% carbohydrate, 5% lipid), two types of purified diets previously described in detail (5) were used in these studies. One of the diets, HP, contained (% wt/wt) 70% casein and no carbohydrate, whereas the other contained 17% casein and 66% carbohydrate. The two diets contained equal amounts of corn oil (8%), vitamins, and minerals, and they were approximately isocaloric. Rats initially weighing 90-110 g were adapted for 20 days to the purified diets. As in previous studies (13), after an initial period of adaptation of a few days, food ingestion and the rate of body weight increase were similar for the two groups of rats. The animals, including those fed the commercial diet, weighed 180-220 g when used for the experiments. In the cold exposure experiments, the animals were housed individually for a period of 6, 12, or 24 h in a cold room (4°C), and they were also maintained on a 12:12-h light-dark cycle. In the diet-reversion experiments, the HP diet was replaced by the control diet at 7:30 PM, and the metabolic parameters were determined within the next 24 h.

Unilateral sympathetic denervation of BAT. While the rats were under ether anesthesia, after a careful dissection of interscapular BAT from the surrounding muscle and WAT, five branches of the right intercostal nerve bundles were isolated and a section of ~5 mm was removed from these nerves. Surgical hemidenervation was performed 6 days before the utilization of the animals for the experiments. After this period, the NE content of the denervated side, measured as described (8), was reduced to <2% of values in the control, innervated side.

NE infusion and BAT temperature. NE was infused (0.26 or 2.2 µg · min¹ · 100 g body wt¹ for 45 min) in nonanesthetized rats through a catheter previously inserted into the jugular vein. After this procedure, the animals were killed by cervical dislocation, and the BAT was removed for GyK activity measurement. The response of BAT temperature to NE infusion (2 µg/min for 3 min) was measured under pentobarbital sodium anesthesia (40 mg/kg ip) by placing a small thermistor between the two lobes of the interscapular tissue as previously described (5).

Measurement of BAT GyK activity. Tissue was homogenized in ice-cold 1% KCl in l mM EDTA. After centrifugation of the homogenate for 10 min at 2,000 g at 4°C, and removal of the top fat layer, the activity of GyK was measured following the recommendations of Newsholme et al. (15). Aliquots of the 2,000 g supernatant were incubated for 30 min in an assay mixture containing: 0.1 M Tris, pH 7.5, 6 mM ATP, 4 mM MgCl₂, 1 mM EDTA, 25 mM NaF, 20 mM 2--mercaptoethanol, 10 mM phosphocreatine, 26 U/ml creatine kinase, 1 mM glycerol, 10 µCi/ml U-¹⁴C-glycerol, and 1% albumin. After the incubation period, the reaction was stopped by the addition of 100 µl of 97% ethyl alcohol, and the ¹⁴C-glycerol phosphate formed was isolated by ascending chromatography in ethanol:NH₃:H₂O (80:4:16), using Whatman n°1 paper (6). The labeled glycerol phosphate remained in the origin of the chromatogram, which was cut out and placed in toluene-PPO for counting. Protein content of homogenates was determined by the method of Lowry et al. (14). Preliminary experiments showed that in BAT from rats kept at ambient temperature or exposed to 4°C for 24 h, the activity of the enzyme in mitochondria was <5% of that in cytosol, thus ensuring that the measurements were not significantly affected by mitochondria retained in the floating fat layer after homogenate centrifugation.

BAT NE turnover rates and NE content. BAT NE turnover rates were assessed from the decline of tissue NE levels after inhibition of catecholamine synthesis with DL--methyl-p-tyrosine methyl ester (-MT; Sigma). -MT (300 mg/kg body wt) was injected intraperitoneally, and the animals were killed by cervical dislocation 0, 6, and 12 h after injection. The interscapular BAT was rapidly removed, weighed, frozen on dry ice, and stored at 70°C until NE assay could be performed, usually within 2 wk. The procedure used for determination of NE concentration has been described in detail (8). Briefly, tissues were homogenized in 0.2 N perchloric acid with EDTA and sodium metabisulfite as antioxidants. Dihydroxybenzylamine was used as internal standard. After protein removal by centrifugation, catecholamines were adsorbed in alumina, eluted with 0.1 N perchloric acid, and isolated using HPLC (LC-7A, Shimadzu Instruments) with a Spherisorb ODS-2 (5 µm) (Sigma-Aldrich) reversed-phase column. NE and internal standard were quantitated with an electrochemical detector (LC-ECD-6A, Shimadzu).

Other methods of chemical analysis. The plasma concentration of insulin was measured by radioimmunoassay using a commercial kit from Amersham (Little Chalfont, UK). Plasma levels of glucose were determined with glucose oxidase in a Beckman glucose analyzer.

Statistical methods. Except for turnover rate measurements, results are expressed as means ± SE, and differences between means were analyzed by one- or two-way ANOVA, as appropriate, with P < 0.05 as the criterion of significance. For comparison of turnover rates, 95% confidence intervals were determined as described by Taubin et al. (18).

	RESULTS

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The data in Fig. 1 show that adaptation to the HP diet resulted in a reduction of ~50% in the activity of BAT GyK. Figure 1 also shows that denervation induced a further reduction in the activity of the enzyme, both in control and HP-adapted rats. As a result, levels of BAT GyK activity were ~50% lower in denervated pads from HP-adapted rats than in denervated pads from animals fed the control diet. Finally, the results in Fig. 1 show that replacement of the HP diet by the control diet for 24 h did not significantly affect the activity of GyK in both intact and denervated BAT pads from HP-adapted rats. On the other hand, as shown by the data in Table 1, BAT NE fractional turnover rate and calculated turnover rate, which were significantly reduced in rats fed the HP diet, increased after replacement of the diet by the control diet for 24 h, attaining levels that did not differ significantly from those in rats fed the control diet. BAT NE content was not significantly affected by diet substitution, remaining lower than in control rats (Table 1). The data in Table 1 also show that diet reversion resulted in a marked increase in plasma levels of glucose and insulin of HP-adapted rats, which attained concentrations higher than those of normally fed animals. The effect was specially pronounced in plasma insulin levels, which were lower in HP rats (before reversion) than in controls.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effect of denervation on the activity of glycerokinase in brown adipose tissue from rats adapted to the high-protein (HP) or control (N) diet and from HP-adapted rats 24 h after replacement of their diet by the N diet (HP-N 24). Data are means ± SE from 5 to 7 animals. * P < 0.05 vs. innervated and # P < 0.05 vs. N diet.

Cold acclimation at 4°C for 10 days induced an increase in the activity of BAT GyK that, when expressed per milligram protein, was more marked in HP-adapted rats, in which the activity of the enzyme was already very low at ambient temperature (Fig. 2). Because the hypertrophic effect of cold exposure was greater on control rats, when expressed per whole tissue, the activity of the enzyme did not differ significantly in cold-acclimated rats of the two experimental groups (controls: 0.74 ± 0.05 µmol/min; HP adapted: 0.65 ± 0.07 µmol/min).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Effect of cold acclimation (10 days at 4°C) on the activity of glycerokinase in brown adipose tissue from rats adapted to the HP or N diet. Data are means ± SE from 5 to 7 animals. * P < 0.05 vs. 25°C and # P < 0.05 vs. N diet.

The results of experiments with shorter periods of cold (4°C) exposure (6, 12, and 24 h), using rats fed the commercial diet, are shown in Figs. 3 and 4. The data in Fig. 3 show that cold exposure for 6 h did not affect BAT GyK activity, but increased activities of the enzyme were observed at the 12- and 24-h time intervals. As shown in Fig. 4, denervation induced a marked reduction in GyK activity in BAT from rats kept at ambient temperature and blocked the cold-induced increase in enzyme activity, which in these experiments was observed only after 24 h of cold exposure.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Effect of cold exposure (4°C) for 6, 12, or 24 h on the activity of glycerokinase in brown adipose tissue from normally fed rats. Data are means ± SE from 6 to 8 animals. * P < 0.05 vs. 25°C.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of denervation on the activity of glycerokinase in brown adipose tissue from rats exposed to cold (4°C) for 12 or 24 h. Data are means ± SE from 6 to 8 animals. * P < 0.05 vs. innervated and # P < 0.05 vs. 25°C.

Intravenous infusion of NE for 45 min (for a total dose of either 12 or 100 µg) did not significantly affect the activity of BAT GyK (Fig. 5). On the other hand, 3 min of NE infusion (2 µg/min) was sufficient to induce a significant increase of BAT temperature (0.90 ± 0.07°C, 5 animals).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effect of a 45-min intravenous infusion of norepinephrine (NE; 0.26 or 2.2 µg · min1 · 100 g body wt1) or saline on the activity of glycerokinase in rat brown adipose tissue. Data are means ± SE from 6 to 8 animals.

	DISCUSSION

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The data of the present work clearly show that an adequate sympathetic flow to BAT is required: 1) for the maintenance of normal levels of BAT GyK activity, as evidenced by the reduction in enzyme activity produced by denervation and also by the lower levels of GyK activity in BAT from rats adapted to the HP diet (Fig. 1), in which BAT sympathetic activity (NE turnover rate) is decreased (Table 1), and 2) for the increase in the activity of the enzyme that occurs in situations in which the sympathetic flow to the tissue is increased, as shown by the suppression of the cold-induced increase in GyK activity in denervated tissue (Fig. 4). On the other hand, the magnitude of the effect of the different treatments in the experiments of Fig. 1 can only be reasonably explained if after suppression of total or partial BAT sympathetic flux, the activity of GyK does not fall rapidly to a new level but decreases in a gradual, progressive and, therefore, time-dependent manner. Under this assumption, the finding that practically the same reduction (~50%) occurs in denervated pads of normally fed rats, with total suppression of sympathetic input, and in intact pads from animals adapted to the HP diet, in which sympathetic activity is reduced but not suppressed, could be explained by the shorter period of denervation (6 days), compared with the period of adaptation to the diet (20 days). Also, the finding that the activity of GyK in denervated pads from HP-fed rats was about one-half of that in denervated pads from normally fed controls, despite a total suppression of sympathetic flux in both groups, could be explained by the already reduced levels of the enzyme in the animals fed the HP diet. This interpretation implies that lower levels of BAT GyK activity can be obtained by extending the period of tissue denervation. In agreement with the experiments with sympathetic flux suppression, evidence of a gradual, time-dependent response of BAT GyK was also obtained in situations in which the tissue sympathetic activity increased. Thus, an increase in BAT GyK could be detected only in the experiments in which the animals were exposed to cold (4°C) for 12 h (Fig. 3). In contrast, the spontaneous activity of the efferent sympathetic nerves reaching the BAT of normal rats has been shown to be immediately increased in response to an acute cold stimulus, the increase failing to occur in nerves of ventromedial hypothalamus-lesioned rats (16). In previous studies, we found that BAT NE turnover was already increased after 8 h of cold exposure (4). Also, no change in the activity of BAT GyK was observed 24 h after replacement of the diet of HP-adapted rats by the control diet (Fig. 1), despite the increase in BAT sympathetic activity, which returned to control values after the same period of diet substitution (Table 1). The failure of diet reversion of HP rats to change BAT GyK activity also suggests that glucose and/or insulin has no effect on the enzyme, as the plasma concentration of both compounds increased markedly to levels above control values after diet replacement (Table 1). Preliminary results from our laboratory also show that intravenous infusion of glucose in normally fed rats does not affect BAT GyK activity.

The activity of GyK in adipose tissue is usually associated with the reesterification of free glycerol produced by hydrolysis of stored TAG. It is well established (11) that the hydrolysis of TAG is under direct control of the sympathetic nervous system, which has a pivotal role in the initiation of the events leading to a rapid activation of hormone-sensitive lipase, TAG breakdown, increased FA oxidation, and heat production in BAT. Therefore, a close correlation between sympathetic input, BAT thermogenesis (TAG hydrolysis), and GyK activity was somewhat to be expected. In fact, in the present study, the activity of BAT GyK changed parallel with the changes in tissue sympathetic flux in the different experimental conditions. However, BAT GyK responses to the modifications of tissue sympathetic flux were very slow, compared with the known rapid responses of TAG hydrolysis. In agreement with these observations, the activity of BAT GyK was not affected by intravenous infusion of NE (12 or 100 µg in 45 min), in contrast to the rapid increase in thermogenesis, evidenced by the rise in tissue BAT temperature 3 min after NE infusion (see RESULTS). In view of the present data, longer periods of administration of NE are needed to verify possible effects of the hormone on the enzyme activity.

The response, albeit delayed, of GyK activity to changes in the sympathetic flux to BAT is in agreement with the idea that the activity of the enzyme is associated with the recycling of the glycerol produced by hydrolysis of stored TAG. However, the possibility cannot be excluded of BAT GyK also having a role in the esterification of glycerol taken up by the tissue from the circulation. This additional source of glycerol phosphate could be important, for instance, during acclimation to cold, when, in addition to an activation of BAT thermogenesis, there is an activation of WAT lipolysis (10) and consequent increased release of glycerol to plasma. Recent studies (9) showed that even in tissues considered as lacking significant levels of GyK, such as skeletal muscle, plasma glycerol is an important precursor of glycerol phosphate for TAG synthesis, thus confirming the presence of functionally important amounts of GyK in this tissue. Attempts to induce changes in the activity of BAT GyK by infusing glycerol intravenously were unsuccessful (data not shown).

The present data do not allow any conclusion about the biochemical mechanism through which the activity of BAT GyK is affected by the sympathetic nervous system. The delayed response suggests that the possible biochemical agents behave rather as inducers of the enzyme expression than as allosteric effectors.

Perspectives