Stimulatory Effect of Cold Adaptation on Glucose Utilization by Brown Adipose Tissue RELATIONSHIP WITH CHANGES IN THE GLUCOSE TRANSPORTER SYSTEM*

The effect of cold adaptation (4 “C) on the in vivo glucose utilization and on the number and properties of the glucose transporters has been studied in brown adipose tissue of normal rats. Glucose utilization was assessed in vivo by the 2-deoxyglucose method. Glucose transporters in plasma and microsomal membranes were quantified by the [3H]cytochalasin B-binding assay. After cold adaptation the in vivo glucose utiliza- tion by brown adipose tissue increased 21-fold compared to controls (22 “C). The number of glucose trans- porters in plasma membranes of brown adipose tissue increased from 75 to 436 pmol/g tissue and that of total glucose transporters (plasma + microsomal membranes) from 438 to 754 pmol/g tissue. In addition, cold adaptation increased the Hill coefficient of the plasma membrane transporter for cytochalasin B from 0.90 to 2.03 and decreased the Kd from 100 to 54 nM. This study shows that cold adaptation promotes: (a) a translocation of glucose transporters from an intracellular pool to plasma membranes; (b) an increased number of plasma membrane glucose transporters unac- counted for by the translocation process (e.g. “de novo” synthesis); (c) an increase in the Hill coefficient for cytochalasin B that could also represent changes in the properties of the transporters vis-a-vis micro- membrane The enrichment in and microsomal membrane fractions estimated, respectively, measuring in the 5'-nucleotidase (11) and NADPH-cytochrome-c reductase (12). mitochondria determined cytochrome-c oxidase (13). Proteins by the Coomassie Brilliant Blue method using bovine y-globulin as standard (14).

In rodents adapted to a cold environment, brown adipose tissue (BAT)' is a major site of nonshivering thermogenesis (1). To fulfill this increased energy expenditure, the substrate requirement of BAT is augmented during cold exposure. Together with fatty acids, glucose may be an important fuel for this tissue (2, 3), either as a source of carbon for fatty acid synthesis or as a direct source of energy through its oxidation. Indeed, it has been observed that brown adipose tissue from cold-adapted rats utilizes more glucose when incubated in vitro than that of controls kept at normal temperature (4). In BAT, as in white adipose tissue, glucose transport appears to be the rate-limiting step for glucose utilization (5). Prelimi-* This work was supported by Grants 3.851.083 and 3.822.086 of the Swiss National Science Foundation (Berne, Switzerland) and by a grant-in-aid of Nest16 S. A. (Vevey, Switzerland). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed. The abbreviation used is: BAT, brown adipose tissue.
nary data have shown that cold adaptation produces an increase in the total number of glucose transporters in BAT plasma membranes of mice (24), although the underlying mechanism(s) of this effect was not investigated. The aim of the present study was therefore to measure the in viuo glucose utilization by BAT of control rats kept at 22 "C and of coldadapted (4 "C) ones and to attempt relating changes in glucose utilization to changes in the number or in the state of the glucose transporters of plasma and microsomal membranes. The data show that, in brown adipose tissue of cold-adapted rats, glucose utilization is markedly increased. This cold-induced metabolic effect is accompanied by a translocation of glucose transporters from an intracellular pool to the plasma membranes, by a further increase in the number of plasma membrane glucose transporters (suggestive of an increased "de IU)UO" synthesis), by an increase in the Hill coefficient of plasma membrane glucose transporter for cytochalasin B, possibly reflecting the occurrence of a positive cooperativity between plasma membrane transporters, and by a decrease in K d for cytochalasin B.

EXPERIMENTAL PROCEDURES
Normal male rats of the Zucker (FAFA) strain (253 k 5 g) were used throughout the experiments. They were initially all maintained at constant temperature (22 'C) with a fixed (12 h) light cycle. The animals were fed ad libitum with a standard laboratory chow (UAR, Epinary/Orge, France). For cold adaptation the rats were transferred for 12 days to a room refrigerated at 4 'C. At the time of the experiments the animals were 12-weeks-old.
BAT glucose utilization was studied in uiuo in conscious rats, using the 2-deoxyglucose technique described by Ferr6 et al. (6). This technique was as follows: at the age of 10 weeks, the rats were implanted, under anesthesia (Nembutal, 70 mg/kg body weight) with a cardiac catheter placed via the jugular vein and fixed on the skull with acrylic cement as described elsewhere (7, 8). Three to 4 days after the surgery the animals had regained their normal body weight and were placed at 4 'C or kept at room temperature. The catheters were rinsed every third day. On the day of the experiment, 40 pCi of 2-deoxy-~-[~H]glucose were injected through the catheter that was then rinsed with a 0.2-ml isotonic saline solution and used for blood sampling at 1, 3, 5, 10, 20, 40, and 60 min. Blood was collected in heparinized tubes and plasma used for measurements of immunoreactive insulin (9). Following plasma deproteinization in Ba(OH)*/ ZnSO,, supernatants were used for the determination of blood glucose levels (glucose oxidase kit) and for those of 2-deoxy-~-[~H]glucose (6). After the last blood sample, the rats were killed by the intravenous injection of 0.8 ml of Nembutal. Interscapular brown adipose tissue was removed and dissected from adjacent tissues. Ita content in 2deoxy-~-[~H]glucose 6-phosphate was determined as in (6). The rate of glucose utilization derived from the amount of 2-deoxy-~-[%] glucose 6-phosphate was calculated using a mathematical formula previously justified (6). It implied the determination of a correction factor (referred to as lumped constant) used for the possible discrimination of 2-deoxyglucose against glucose. Such a correction factor was determined in BAT of control and cold-adapted rats according to Ferre et al. (6) and was not modified by cold adaptation (0.70 f 0.08 for control and 0.81 f 0.04 for cold-adapted rats).
To obtain enriched plasma and microsomal membrane fractions, control and cold-adapted rats were anesthetized with Nembutal (70 mg/kg body weight). Their interscapular BAT was dissected out and transferred into a homogenization medium containing NaHC03 (1 mM), CaC12 (0.5 mM), and MgS04 (0.2 mM), pH 7.5. One gram of tissue (pooled from 4-5 animals) was placed in 10 volumes of medium and homogenized for 5 s in a Polytron (PTA 10-35 homogenizer, Kinematica GmbH, Switzerland). Plasma and microsomal membrane fractions were prepared following a method previously described for BAT (10) using 4 ml of 18.5% (w/v) metrizamide. After this, the respective membranes (plasma and microsomal) were immediately washed once in the homogenization buffer to remove metrizamide and centrifuged at 156,000 X g for 30 min. The pellets obtained were resuspended in a solution containing sucrose (250 mM), Tris-HCl(10 mM), and EDTA (1 mM) at pH 7.4, rapidly frozen in liquid nitrogen, and stored at -70 "C for subsequent use. Final plasma and microsomal membrane protein concentrations ranged between 4 and 6 mg/ ml.
The enrichment in plasma and microsomal membrane fractions was estimated, respectively, by measuring the changes in the specific activity of 5'-nucleotidase (11) and NADPH-cytochrome-c reductase (12). Contamination by mitochondria was determined by measuring cytochrome-c oxidase (13). Proteins were measured by the Coomassie Brilliant Blue method using bovine y-globulin as standard (14).
Glucose transporters of BAT were measured by the binding of [3H] cytochalasin B to plasma or microsomal membranes as described in Ref. 15, but with the following modifications: [3H]cytochalasin B in 100% ethanol was dried under nitrogen to remove 3H volatile contaminants and reconstituted in the binding assay buffer containing sucrose (255 mM), Tris-HC1 (10 mM), and EDTA (0.2 mM) at pH 7.4, immediately before use. A stock solution of cytochalasin E was prepared in dimethyl sulfoxide and stored at -20 "C until use. The final concentration of cytochalasin E in the assay was 2000 nM, that of dimethyl sulfoxide less than 0.2%. The concentrations of [3H] cytochalasin B were 40, 80, 160, 320, 640, and 1280 nM. These concentrations were used for both plasma and microsomal membranes of control rats (i.e. maintained at 22 "C) to ensure that the maximal binding of [3H]cytochalasin B had been reached. To measure glucose transporters following cold adaptation, the two highest concentration points just mentioned were replaced by the 420 nM concentration. The incubation of membranes with labeled cytochalasin B was carried out in the dark at 4 "C for 1 h. Sucrose was added when glucose was omitted to correct for viscosity. Similar data were obtained when sucrose was replaced with L-glucose (data not shown). Duplicate samples (50 pl) of each of the final membrane suspensions were transferred to small plastic tubes and deproteinized with 10 pl of 0.075 N barium hydroxide, immediately (less than 5 s) followed by 10 ~1 of 0.075 N zinc sulfate solution in order to precipitate all membrane protein. Five percent protein, at the most, remained in the supernatant when using either unlabeled glucose or sucrose. ["C] Urea was added for determining the trapped but unbound [3H] cytochalasin B. The samples were pelleted by centrifugation in a Beckman Microfuge at maximal speed for 10 min. Fifty pl of the supernatant were carefully removed for measurement of free [3H] cytochalasin B concentration, the remaining supernatant being aspirated and discarded. The pellet was obtained by cutting the lower end of the plastic tube with a lancet. This method of bound cytochalasin B precipitation differs from the technique previously reported (15) and shortens the time needed to separate bound from free [3H]cytochalasin B and gives comparable results (data not shown). All determinations were made in duplicates. Scintillation counting was carried out in polyethylene vials containing 3 ml of Aquasol-2 scintillator fluid, supernatants and pellets being counted for 10 and 30 min, respectively, in a Rackbeta liquid scintillation counter (LKB Instruments, Wallac Oy, Finland). The measured specific cytochalasin B-binding activities were expressed as pmol/mg of protein. The Hill equation (16) was used to analyze the data. Calculations were made on an Apple IIe microcomputer (Apple Corp., Cupertino, CA). Statistical analysis was carried out with Student's test for unpaired data. A computer program was developed (Dr. E. Perotto, CERN, Geneva, Switzerland) to find the best fit of the available data with the curve defined by the Hill equation (16). In this way, the values of R., K d , and Hill coefficient were found, and the binding curve was plotted (bound versus free) together with the averaged data points (+ S.E.). Plots were drawn on a Versatec V80F plotter, using an IBM 3081 computer running the VM/CMS operating system. (Program available on request.) Labeled compounds and Aquasol-2 scintillator were from New England Nuclear (Zurich, Switzerland), metrizamide from Nyeegard and Co. (Oslo, Norway), protein assay kit and bovine y-globulin were purchased from Bio-Rad Laboratories (Richmond, CA). Other reagents were obtained either from Sigma, Merck (Darmstadt, West Germany), or Boehringer Mannheim (Mannheim, West Germany).

Effect of Cold Adaptation on Glucose Utilization in Brown
Adipose Tissue-Glucose utilization by BAT of control (i.e. kept at 22 "C) or cold-adapted (4 "C) rats was measured in conscious animals using the 2-deoxyglucose method described under "Experimental Procedures." As can be seen by Table I, this process was increased by 21-fold in BAT of cold-adapted rats compared to controls, without any change in either plasma glucose or insulin levels.
Characteristics of BAT P h m a and Microsomal Membranes-In another group of control (22 "C) and cold-adapted (4 "C) rats, plasma and microsomal membranes were prepared. The preparation procedure was carried out at the same time from the same homogenate. As shown by Table 11, the enrichment of the membranes was assessed by the specific activity of 5'-nucleotidase and NADPH-cytochrome-c reductase used, respectively, as markers of plasma and microsomal membranes. A 14-22-fold enrichment was obtained for plasma membranes, a 7-9 one for microsomal membranes. Contamination by mitochondria (as measured with cytochrome-c oxidase) was negligible in all the preparations (data not shown). The percent recovery of the plasma and microsomal membrane enzyme markers was slightly higher in cold-adapted animals and the protein content was doubled after cold adaptation (Table 11). The slight difference in the percent recovery of either 5'-nucleotidase or NADPH-cytochrome-c reductase was taken into account for the calculation of the total number of glucose transporters (see Table 111).
Glucose transporters were measured in plasma membranes by studying the D-glucose displaceable [3H]cytochalasin B, in the presence of increasing concentration of [3H]cytochalasin B. The binding assays were carried out in the absence or presence of 500 mM D-glucose first by using 12 different concentrations of cytochalasin B, as depicted by Fig. 1. Similar measurements were performed in plasma and microsomal membranes from BAT of control and cold-adapted rats, using now five to six standard concentrations of the ligand, as represented in Figs. 2 and 3, panels A and C. Subtraction of the curves obtained in the presence of D-glucose from the respective curves obtained in the absence of D-glucose represents the specific cytochalasin B-binding. These values (plotted as described under the "Experimental Procedures") are TABLE I Glucose utilization by interscapular BAT in rats kept at normal temperature or cold-adapted at 4 "C 12-week-old normal male rats of the Zucker (FA/FA) strain. Cold adaptation was of 12-day duration at 4 "C. Glucose utilization was assessed 60 min after 2-deoxy-~-[~H]glucose administration via a chronic cardiac catheter to conscious rats (see "Experimental Procedures"). Results are the mean f S.E. of three individual determinations.

TABLE I1
Enzyme markers of plasma and microsomal membranes obtained from brown adipose tissue of control and coldadapted normal rats. Plasma and microsomal membranes were obtained as described under "Experimental Procedures." Enrichment of each membranes fraction is assessed by the changes in specific activities of respective marker enzymes compared to the activities of the initial homogenate. Recovery is expressed as percent values found in the initial homogenate.
Results are the mean * S.E. of six individual preparations for each group of rats.

TABLE I11
Hill coefficient, Kd and number of D-glucose inhibitable cytochalasin B-binding sites in plasma and microsomal membranes of brown adipose tissue from control and cold-adapted normal rats Values obtained after correction for cross-contamination. Plasma and microsomal membranes were obtained as described under "Experimental Procedures." Results are the mean f S.E. of four (control) and three (cold-adapted) independent preparations. * = specific cytochalasin B-binding activities have been adjusted to those which would have been observed, had the membrane fractions been free of cross-contamination. Adjustments were based on the enzyme marker specific activities and on the assumption that only 5"nucleotidase activity is localized specifically to the plasma membrane and that NADPH-cytochrome-c reductase is a specific marker for microsomal membranes.

Control rats
Cold-adapted rata ( further shown by Figs. 2 and 3 (panels B and D ) . As can be seen, binding curves obtained in the BAT membranes fit a rectangular hyperbola in control (22 "C) rats (Fig. 2, panels B and D ) , whereas a sigmoidal shape was observed in BAT membranes of cold-adapted rats (Fig. 3, panels B and D ) .

. [SH]Cytochalasin B-binding sites in brown adipose tissue plasma membranes from control and cold-adapted rats.
[3H]Cytochalasin B-binding to plasma membranes from control (panel A ) and cold-adapted (panel B) rats has been measured at 12 standard concentrations of the ligand, in the absence (4-) or presence (-O-) of 500 mM D-glucose as described under "Experimental Procedures." Panel A : R., 6.2 pmol/mg of protein; Hill coefficient, 1.13. Panel B: R,, 12.7 pmol/mg of protein; Hill coefficient, 1.93.
individual experiments as described under "Experimental Procedures," and are presented on Table 111. In BAT plasma membranes of control rats, the Hill coefficient was close to 1 and Kd was 99 nM. Cold adaptation resulted in a doubling of the Hill coefficient and a significant decrease in the K d of the plasma membranes. The properties of the glucose transporter in the microsomal membranes of control animals were very similar to those of the plasma membranes. Cold adaptation promoted slight changes in the Hill coefficient of microsomal membranes and a decrease in the Kd toward cytochalasin B. [3H]Cytochalasin B binding to plasma and microsomal membranes from control rats has been measured at six standard concentrations in the absence (*-) or presence (e) of 500 mM Dglucose (panels A and C), as described under "Experimental Procedures." The differences of the values obtained in the presence of Dglucose from their respective values obtained in the absence of Dglucose were analyzed according to the Hill equation (16) in order to obtain the number of cytochalasin B-binding sites (Ro, in picomoles per milligram of membrane protein), the K d (dissociation constant, in nanomolar), and the Hill coefficient (Hc). Using the values of R,, Kd, and Hill coefficient thus found, the binding curve was plotted (panels B and D ) . Kd and Hill coefficient were calculated from the averaged curves shown by the figure. Results are the means f S.E. of four independent preparations in duplicate determinations. Table I11 also summarizes the results of the cytochalasin B-binding sites in the plasma and microsomal membranes from control (22 "C) and cold-(4 "C) adapted rats. The measured specific cytochalasin B-binding activities have been adjusted to those which would have been observed, had the membrane fractions been free of cross-contamination. Adjustments were based on the enzyme marker specific activities and on the assumptions that 5'-nucleotidase activity is localized specifically to the plasma membrane and NADPH-cytochrome-c reductase is a specific marker for microsomal membranes. These corrections did not modify the Hill coefficient or the Kd. As can be seen by Table 111, cold adaptation produced a significant increase (by 5-6-fold) in the number of glucose transporters associated to the plasma membranes and a decrease (by 40%) in those present in the microsomal fraction, when expressed per milligram of protein. Furthermore, the total number of glucose transporters (plasma + microsome membranes) was increased by %fold by cold adaptation.

DISCUSSION
The present data show for the first time that, when the experiments are performed at 4 "C, glucose utilization by BAT is markedly increased (21-fold) by cold adaptation (Table I). This is in keeping with other data that suggested indirectly an increased glucose utilization by this tissue, used in partic- [3H]Cytochalasin B binding to plasma and microsomal membranes from cold-adapted rats has been measured at five standard concentrations in the absence (+-) or presence (e-) of 500 mM D-glucose (panels A and C), as described under "Experimental Procedures." Data are calculated and expressed as in Fig. 1. Results are the mean k S.E. of three independent preparations in duplicate determinations. Hc, Hill coefficient. ular for heat dissipation and fatty acid synthesis during cold adaptation (2-4). This study also shows the basic characteristics of the glucose transporters of brown adipose tissue of rats (kept at 22 "C) and underscores the major changes which are occurring in the glucose transporters after cold adaptation. As depicted in Table 111, exposure to cold results in a 5-6fold increase (on a per milligram of membrane protein or on a per gram of tissue basis) in the number of the glucose transporters in the plasma membranes, together with a 40% decrease in the number of the glucose transporters in microsomal membranes (when expressed on a per milligram of protein basis but not per gram of tissue).
In addition, cold adaptation brings about other changes in the BAT glucose transporters. Thus, when data are analyzed according to the Hill equation (16) to obtain a precise evaluation of the properties of glucose transporters, as well as the Hill coefficient, it is observed that this latter value is close to 1 in both plasma and microsomal membranes of control rats maintained at 22 "C. In marked contrast, cold adaptation increases the Hill coefficient of plasma membrane transporters (to a value of 2.0) and, to a lesser extent (1.3), that of the microsomal ones. This suggests the existence of a positive cooperativity amongst the glucose transporters that would be induced by cold adaptation. Functionally, this could mean that the binding of each molecule of cytochalasin B increases the affinity for the next one and that cold adaptation would thus change the properties of the glucose transporters by making them more efficient. Cold adaptation also increases the affinity of the glucose transporter for cytochalasin B by decreasing the Kd. Hill coefficient and K d of the glucose transporters are measured toward cytochalasin B. They could reflect changes in the affinity and cooperativity of the transporter toward glucose and thereby contribute, in addition to the well-characterized translocation process, to the increased glucose uptake. Indeed, additional mechanisms involving "activation" of transporters have been postulated (20,21) to explain the stimulatory effect of insulin on glucose transport in white adipocytes or the inhibitory effect of hormones counterregulating insulin-stimulated glucose transport (22,23).
It can be speculated that the marked increase in glucose utilization produced by cold adaptation could be due to an increased glucose uptake. This view is based on the finding that cold adaptation produced a 5-6-fold increase of the plasma membrane glucose transporter number when expressed per gram of tissue, together with a doubling of the plasma and microsomal membrane Hill coefficients toward cytochalasin B indicating the appearance of positive cooperativity. A marked increase in the Hill coefficient and a decrease in Kd toward cytochalasin B are also observed in BAT plasma membranes after insulin,* a condition known to markedly increase glucose utilization (3).
These data together are compatible with the view that cold adaptation produces a dual effect on brown adipose tissue: ( a ) a marked increase in the total number of the glucose transporters (plasma + microsomal membranes), possibly due to a stimulation of their de mu0 synthesis (6) a stimulation of the translocation of these transporters from the intracellular pool to the plasma membrane in a way that may be similar to that observed after the addition of insulin to isolated white adipose tissue (15,17) or to isolated diaphragm (18, 19); and (c) changes in the properties of the transporter uis-u-uis cytochalasin B that may possibly reflect changes in the properties of the system for glucose as proposed by others (20, 21). All these modifications may participate in the marked increase in glucose utilization measured in brown adipose tissue after cold adaptation.