Vol. 185, No. 3, 1992 June 30, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1078-1082
COLD EXPOSURE INCREASES GLUCOSE UTILIZATION AND GLUCOSE TRANSPORTER EXPRESSION IN BROWN ADIPOSE TISSUE Hideki Nikamis, Yasutake Shimizus, Daiji EndohS, Hideki Yanoll and Masayuki Saito$ Departments of §Biochemistry and SRadiation Biology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan, and llSecond Division, Department of Internal Medicine, Kyoto University School of Medicine, Kyoto 606, Japan
Received
May 18,
1992
SUMMARY. When rats were exposed to a cold environment (4°C) for 10 days, tissue glucose utilization was increased in brown adipose tissue (BAT), a tissue specified for nonshivering thermogenesis, but not in skeletal muscle. Cold exposure also caused an increase in the amount of GLUT4, an isoform of glucose transporters expressed in insulin-sensitive tissues, in parallel with an increased cellular level of GLUT4 mRNA. In contrast to BAT, no significant effect of cold exposure was found in skeletal muscle. The results suggest the coldinduced increase in glucose utilization by BAT is attributable, at least in part, to the increased expression of GLUT4. 0 1992 Academic Press, Inc.
Brown adipose tissue (BAT) is a tissue specified for sympathetically controlled heat production during cold acclimation, spontaneous hyperphagia and recovery from hypothermia (l-3). The major substrate for BAT thermogenesis is considered to be fatty acids derived from triglyceride in this tissue and from blood lipoproteins. Together with fatty acids, glucose may be an important fuel in BAT, not only as a carbon source for fatty acid synthesis but also as a precursor for sufficient supply of oxaloacetate which is essential for rapid oxidation of fatty acids. In fact, it has been demonstrated that glucose utilization by BAT is increased in parallel with heat production after cold exposure (4-6) and sympathetic nerve stimulation (6). However, there is few information about the cellular mechanisms of the increased glucose utilization, besides those through the P-adrenergic action of norepinephrine. In BAT, as in white adipose tissue, glucose transport across the plasma membrane appears to be the rate-limiting step for intracellular glucose utilization. Among five isoforms of glucose transporters (GLUT) so far described, GLUT1 and GLUT4 are expressed in BAT (7,8). GLUT1 is the isoform widely present in most tissues, whereas GLUT4 is expressed at highest levels in the insulin-sensitive tissues, such as muscle and adipose tissues (9,lO). In the present 0006-291X/92
Copyright All rights
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
1078
Vol.
185,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
study, to elucidate the regulatory mechanism for glucose utilization
COMMUNICATIONS
in BAT, we have
investigated the expression of GLUT4 in BAT of rats exposed to a cold environment. The results were compared with those of skeletal muscle. Our results confirm a parallel increase in the expression of GLUT4 and glucose utilization specifically in BAT after cold exposure. MATERIALS
AND
METHODS
Animals: Female Wister rats weighting 160-220 g were housed in plastic cages in rooms at either 24 or 4 “C with a 12-h light-dark cycle (lights on at 07:00-19:00 h) and given free accessto laboratory chow and water. Ten days later, they were starved for 20-24 h and used for the measurement of 2-deoxy-D-glucose (2-DG) uptake or Western and Northern blot analyses. 2-DC uptake: Tissue glucose utilization was assessedfrom the tissue uptake of 2-DG in viva according to the procedure described by Horn et al. (11) with slight modifications (6). Briefly, rats were injected with 2-deoxy-D-PHI glucose (2-[3H]DG), and blood and tissue samples (interscapular BAT, gastrocnemius) were taken 11-13 min later. The rate constant of the tissue uptake of 2-DG was calculated from the intracellular concentration and plasma disappearance rate of 2-[JH]DG. The glucose metabolic index was calculated as a product of the rate constant and plasma glucose concentration, and expressed as ng glucose/mg tissue/min (12). Western blotting: GLUT4 protein was measured by Western blot analysis using rabbit antiserum raised against a synthesized peptide corresponding to the C-terminal region of GLUT4 (residues 498-509, TELEYLGPDEND). The interscapular BAT was homogenized in a solution containing 1 mM NaHCO 3, 0.5 mM CaC12 and 0.2 mM MgSO 4 (pH 7.5), and skeletal muscle (gastrocnemius) in a solution containg 250 mM sucrose, 1 mM EDTA and 10 mM Tris-HCl (pH 7.4), using a Polytron. After centrifugation at 1,500 xg for 10 min, the fat cakes were discarded, and the infranants were used for Western blot analysis. Protein was measured by the method of Lowry et al. (13) using bovine serum albumin (BSA) as a standard. Samples (20-30 pg protein) were solubilized, subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis as described by Laemmli (14), and transferred to a nitrocellulose filter. After blocking the filter with 6 % BSA, it was incubated first with the antiserum diluted at x200 and then with [12q] protein-A. The dried blots were autoradiographed and their radioactivity was counted in a gamma counter. Northern blotting: Total cellular RNA was extracted from 0.5-l g of pooled tissue as described by Birnboim (15), and was applied on an oligo-(dT) cellulose column. Poly(A)+ RNA (4yg) thus obtained was denatured at 70 “C, separated on a 1 % agarose/formaldehyde gel, and transferred to and fixed on a nylon membrane. Rat GLUT4 cDNA probe corresponding to nucleotides l-1330 of the published cDNA sequence (9) was labeled with a-t32p] dCTP (16). The blots were hybridized to the labeled cDNA probe at 42 “C for 20 h in the presence of 200 pg / ml salmon sperm DNA. The autoradiographs of the blots were analyzed by a densitometer to determine the relative amount of GLUT4 mRNA. RESULTS Effects of cold exposure on glucose utilization by BAT and skeletal muscle were examined by measuring tissue 2-DG uptake in vivu in rats kept at 24 (control) or 4 “C (cold-exposed) for 10 days. As shown in Table 1, glucose utilization in BAT was about 4.5 times higher in coldexposed rats than control rats, while glucose utilization in skeletal muscle was almost equal in the two groups. These results were essentially the same as our previous results (6), though the increment by cold exposure seemed rather less. 1079
Vol.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 1. In vivo glucoseutilization in brown adiposetissueand skeletalmuscleof rats kept at 24 or 4°C for 10 days
Brown adiposetissue Rate constantof 2-DG uptake (min-’ x 10w3) Glucosemetabolicindex (ng/mg/min) Skeletalmuscle Rateconstantof 2-DG uptake (min-’ x 10-3) Glucosemetabolicindex (ng/mg/min)
24°C (n=6)
4°C (n=7)
14.1 r 1.1
63.4 + 10.2*
22.2 t 2.0
97.9 It 17.1*
16.1 -c 1.9
15.0 + 1.3
27.0 -c 4.2
23.5 * 3.6
Valuesare means+ SE. * ~~0.05 comparedwith the value at 24°C.
In order to estimate the protein level of GLUT4 by Western blotting, we used a rabbit antiserum raised against a synthesized 12-amino acid peptide corresponding to the C-terminal region of GLUT4. The specificity of the antiserum was confirmed by its cross-reactivity to the homogenates of BAT, white adipose tissue, skeletal muscle and heart, but not to those of brain, liver and kidney (data not shown). This tissue distribution was identical to the previous reports (7,9,10) and also to the distribution of GLUT4 mRNA as determined by Northern blot analysis with a cDNA probe for GLUT4 (data not shown). Fig. 1 shows typical examples of Western and Northern blot analyses of BAT and skeletal muscle in control and cold-exposed rats. It seems obvious that both GLUT4 protein and GLUT4 mRNA increase much in BAT of cold-exposed rats, while they do not change in skeletal muscle. To assessthe effects of cold exposure quantitatively, the amounts of GLUT4 protein and mRNA of cold-exposed rats were estimated as those relative to the respective 24°C controls (Fig. 2). In BAT, the amount of GLUT4 protein and GLUT4 mRNA were 3 and 2 times higher, respectively, in cold-exposed rats than control rats. In skeletal muscle, however, no significant difference was found between the two groups of rats. Thus, cold exposure increases GLUT4 expression specifically in BAT but not in skeletal muscle. DISCUSSION In this study, we demonstrated a parallel increase of glucose utilization and the expression of GLUT4, an isoform of glucose transporters, in BAT after cold exposure. 1080
Vol.
BIOCHEMICAL
185, No. 3, 1992
A. Protein
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
B. mRNA BAT
BAT
Muscle
Muscle .I. ..,: .I.
28s ) 45kD * 18Sb
g E E 9 F .!I! 2
400
200
0
0
CL
1
Control
BAT
(24-C)
(473
2 0 (B) blot analyses of GLUT4. Rats were kept at 24°C
CL
CL
CL
Ml&de (4’C)
F&.& Western (A) and Northern (C) or 4°C (L) for 10 days. Protein samples from BAT (3Opg) and skeletal muscle (2Op.g) were used for Western blot analysis. For Northern blot analysis, 4 pg poly(A)+RNA was used. Fj& Effects of cold exposure on glucose utilization and GLUT4 expression in BAT and skeletal muscle. The values are means * SE for 5 (GLUT4 protein) and 4 (GLUT’4 mRNA) experiments, and expressed as those relative to the respective control at 24 “C. Glucose utilization (rate constant of 2-DG uptake) was calculated from the data in Table 1. * ~~0.05 compared with the value at 24°C.
We observed using a specific antiserum against GLUT4 that the amount of GLUT4 protein in BAT was several times higher in cold-exposed rats than control rats. This finding seems compatible with those of Greco-Perotto et al. (4) who quantified the total number of glucose transporters by the cytochalasin B binding method to find a 2-fold increase in BAT of coldexposed animals. In BAT, there is an another isoform of glucose transporter, GLUT?, in addition to GLUT4. However, since GLUT4 has been recognized as the predominant isoform in adipose tissues in adult rats (17) it seems rational to consider that the changes in cytochalasin B binding and glucose transport itself are attributable largely to the change in GLUT4. In fact, in a preliminary study, we found no change in the amount of immunoreactive GLUT1 after cold exposure. Northern
blot analysis
demonstrated
that the cellular
level of GLUT4 mRNA was also
increased remarkably in BAT of cold-exposed rats, suggesting that the increased GLUT4 protein
is due to an enhanced
de novo synthesis
of this protein.
It is now established
that
GLUT4 is an insulin-regulated glucose transporter isoform, and that its expression is activated by insulin
(18). Camps etal. suggested recently
possible that insulin takes part dominantly
this is also the case in BAT (19). It seems thus
in the cold-induced
activation
of GLUT4
expression.
However, there are reports indicating that glucose utilization in BAT can be activated directly, independently of the action of insulin, by sympathetic nerve via the f3-adrenergic pathway (620). Moreover, cold exposure results in a decreased plasma insulin level (6) and an increased sympathetic nerve activity in BAT (21). It is therefore more likely that the increased GLUT4 1081
Vol.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
expression in BAT seen after cold exposure is mediated through the p-adrenergic mechanism rather than insulin. The finding that GLUT4 expression in skeletal muscle is not influenced by cold exposure also supports the idea of a minor contribution of humoral factors such as insulin. Similar stimulatory effects of sympathetic nerves and / or adrenergic agonists on gene expression in BAT has been documented with mitochondrial uncoupling protein (22) and lipoprotein lipase (23) which play critical roles for BAT thermogenesis. ACKNOWLEDGMENTS We are very grateful to Dr. G. I. Bell (The University of Chicago, Chicago) for providing the cDNA clone of GLUT4 and Dr. J. Utsumi (Toray Basic Research Laboratory, Kamakura) for synthesizing the C-terminal peptide of GLUTC
REFERENCES
1) Himms-Hagen, J. (1986) in Brown Adipose Tissue (Trayhurn,P., & Nicholls,D.G., eds.) pp. 214-268, Edward Arnold, London. & 2) Rothwell, N.J., & Stock, M.J. (1986) in Brown Adipose Tissue (Trayhurt& Nicholls,D.G., eds.) pp. 269-298, Edward Arnold, London. 3) Shimizu, Y., & Saito, M. (1991) Am. J. Physiol. 261, R301-R304. 4) Greco-Perotto, R., Zaninetti, D., Assimacopoulos-Jeannet, F., Bobbioni, E., & Jeanrenaud, B. (1987) J. Biol. Chem. 262, 7732-7736. 5) Shibata, H., Perusse, F., Vallerand, A., & Bukowiecki, L.J. (1989) Am. J. Physiol. 257, R96-RlOl. 6) Shimizu, Y., Nikami, H., & Saito, M. (1991) J. Biochem. 110, 688-692. James, D.E., Strube, M., & Mueckler, M. (1989) Nature 338, 83-87. :; Pessin, J.E., & Bell, G.I. (1992) Annu. Rev. Physiol. 54, 911-930. 9) Birnbaum, M.J. (1989) Cell 57, 305-315. 10) Charron, M.J., Brosius III, EC., Alper, S.L., & Lodish H.E (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2535-2539. 11) Horn, F.G., Goodner, C.J., & Berrie, M.A. (1984) Diabetes 33,141-152. 12) Kraegen, E.W., James, D.E., Jenkins, A.B., & Chisholm, D.J. (1985) Am. J. Physiol. 248, E353-E362. 13) Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 14) Laemmli, U.K. (1970) Nature 227, 680-685. 15) Birnboim, H.C. (1988) Nucleic Acids Res. 16, 1487-1497. 16) Feinberg, A.F!, & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 17) Zorzano,A., Wilkinson, W., Kotliar, N., Thoidis, G., Wadzinkski, B.E., Ruoho, A.E., & Pilch, PF. (1989) J. Biol. Chem. 264, 12358-12363. 18) Kahn, B.B., Charron, M.J., Lodish, HF., Cushman, S.W., & Flier, J.S. (1989) J. Clin. Invest. 84, 404-411. 19) Camps, M., Castello, A., Munoz, P., Monfar, M., Testar, X., Palacin, M., & Zorzano. A. (1992) B&hem. J. 282, 765-772. 20) Marette, A., & Bukowiecki, L.J. (1989) Am. J. Physiol. 257, C714-C721. 21) Young, J.B., Saville, E., Rothwell, N.J., Stock, M.J., & Landsberg, L. (1982) J. Clin. Invest. 69, 1061-1071. 22) Bouillaud, F., Ricquier, D., Mory, G., & Thibault, J. (1984) J. Biol. Chem. 259, 1158311586. 23) Mitchell, J.R., Carneheim, C.M.H., Jacobsson, A., Kirchgessner, T, Schotz, M.C., Alexson, S.E.H., Cannon, B., & Neclergaard, J. (1989) in Obesity in Europe 88 (Bjorntorp, I?, & Rossner, S. eds.) pp. 235-239, Libbey, London. 1082
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1078-1082
COLD EXPOSURE INCREASES GLUCOSE UTILIZATION AND GLUCOSE TRANSPORTER EXPRESSION IN BROWN ADIPOSE TISSUE Hideki Nikamis, Yasutake Shimizus, Daiji EndohS, Hideki Yanoll and Masayuki Saito$ Departments of §Biochemistry and SRadiation Biology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan, and llSecond Division, Department of Internal Medicine, Kyoto University School of Medicine, Kyoto 606, Japan
Received
May 18,
1992
SUMMARY. When rats were exposed to a cold environment (4°C) for 10 days, tissue glucose utilization was increased in brown adipose tissue (BAT), a tissue specified for nonshivering thermogenesis, but not in skeletal muscle. Cold exposure also caused an increase in the amount of GLUT4, an isoform of glucose transporters expressed in insulin-sensitive tissues, in parallel with an increased cellular level of GLUT4 mRNA. In contrast to BAT, no significant effect of cold exposure was found in skeletal muscle. The results suggest the coldinduced increase in glucose utilization by BAT is attributable, at least in part, to the increased expression of GLUT4. 0 1992 Academic Press, Inc.
Brown adipose tissue (BAT) is a tissue specified for sympathetically controlled heat production during cold acclimation, spontaneous hyperphagia and recovery from hypothermia (l-3). The major substrate for BAT thermogenesis is considered to be fatty acids derived from triglyceride in this tissue and from blood lipoproteins. Together with fatty acids, glucose may be an important fuel in BAT, not only as a carbon source for fatty acid synthesis but also as a precursor for sufficient supply of oxaloacetate which is essential for rapid oxidation of fatty acids. In fact, it has been demonstrated that glucose utilization by BAT is increased in parallel with heat production after cold exposure (4-6) and sympathetic nerve stimulation (6). However, there is few information about the cellular mechanisms of the increased glucose utilization, besides those through the P-adrenergic action of norepinephrine. In BAT, as in white adipose tissue, glucose transport across the plasma membrane appears to be the rate-limiting step for intracellular glucose utilization. Among five isoforms of glucose transporters (GLUT) so far described, GLUT1 and GLUT4 are expressed in BAT (7,8). GLUT1 is the isoform widely present in most tissues, whereas GLUT4 is expressed at highest levels in the insulin-sensitive tissues, such as muscle and adipose tissues (9,lO). In the present 0006-291X/92
Copyright All rights
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
1078
Vol.
185,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
study, to elucidate the regulatory mechanism for glucose utilization
COMMUNICATIONS
in BAT, we have
investigated the expression of GLUT4 in BAT of rats exposed to a cold environment. The results were compared with those of skeletal muscle. Our results confirm a parallel increase in the expression of GLUT4 and glucose utilization specifically in BAT after cold exposure. MATERIALS
AND
METHODS
Animals: Female Wister rats weighting 160-220 g were housed in plastic cages in rooms at either 24 or 4 “C with a 12-h light-dark cycle (lights on at 07:00-19:00 h) and given free accessto laboratory chow and water. Ten days later, they were starved for 20-24 h and used for the measurement of 2-deoxy-D-glucose (2-DG) uptake or Western and Northern blot analyses. 2-DC uptake: Tissue glucose utilization was assessedfrom the tissue uptake of 2-DG in viva according to the procedure described by Horn et al. (11) with slight modifications (6). Briefly, rats were injected with 2-deoxy-D-PHI glucose (2-[3H]DG), and blood and tissue samples (interscapular BAT, gastrocnemius) were taken 11-13 min later. The rate constant of the tissue uptake of 2-DG was calculated from the intracellular concentration and plasma disappearance rate of 2-[JH]DG. The glucose metabolic index was calculated as a product of the rate constant and plasma glucose concentration, and expressed as ng glucose/mg tissue/min (12). Western blotting: GLUT4 protein was measured by Western blot analysis using rabbit antiserum raised against a synthesized peptide corresponding to the C-terminal region of GLUT4 (residues 498-509, TELEYLGPDEND). The interscapular BAT was homogenized in a solution containing 1 mM NaHCO 3, 0.5 mM CaC12 and 0.2 mM MgSO 4 (pH 7.5), and skeletal muscle (gastrocnemius) in a solution containg 250 mM sucrose, 1 mM EDTA and 10 mM Tris-HCl (pH 7.4), using a Polytron. After centrifugation at 1,500 xg for 10 min, the fat cakes were discarded, and the infranants were used for Western blot analysis. Protein was measured by the method of Lowry et al. (13) using bovine serum albumin (BSA) as a standard. Samples (20-30 pg protein) were solubilized, subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis as described by Laemmli (14), and transferred to a nitrocellulose filter. After blocking the filter with 6 % BSA, it was incubated first with the antiserum diluted at x200 and then with [12q] protein-A. The dried blots were autoradiographed and their radioactivity was counted in a gamma counter. Northern blotting: Total cellular RNA was extracted from 0.5-l g of pooled tissue as described by Birnboim (15), and was applied on an oligo-(dT) cellulose column. Poly(A)+ RNA (4yg) thus obtained was denatured at 70 “C, separated on a 1 % agarose/formaldehyde gel, and transferred to and fixed on a nylon membrane. Rat GLUT4 cDNA probe corresponding to nucleotides l-1330 of the published cDNA sequence (9) was labeled with a-t32p] dCTP (16). The blots were hybridized to the labeled cDNA probe at 42 “C for 20 h in the presence of 200 pg / ml salmon sperm DNA. The autoradiographs of the blots were analyzed by a densitometer to determine the relative amount of GLUT4 mRNA. RESULTS Effects of cold exposure on glucose utilization by BAT and skeletal muscle were examined by measuring tissue 2-DG uptake in vivu in rats kept at 24 (control) or 4 “C (cold-exposed) for 10 days. As shown in Table 1, glucose utilization in BAT was about 4.5 times higher in coldexposed rats than control rats, while glucose utilization in skeletal muscle was almost equal in the two groups. These results were essentially the same as our previous results (6), though the increment by cold exposure seemed rather less. 1079
Vol.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 1. In vivo glucoseutilization in brown adiposetissueand skeletalmuscleof rats kept at 24 or 4°C for 10 days
Brown adiposetissue Rate constantof 2-DG uptake (min-’ x 10w3) Glucosemetabolicindex (ng/mg/min) Skeletalmuscle Rateconstantof 2-DG uptake (min-’ x 10-3) Glucosemetabolicindex (ng/mg/min)
24°C (n=6)
4°C (n=7)
14.1 r 1.1
63.4 + 10.2*
22.2 t 2.0
97.9 It 17.1*
16.1 -c 1.9
15.0 + 1.3
27.0 -c 4.2
23.5 * 3.6
Valuesare means+ SE. * ~~0.05 comparedwith the value at 24°C.
In order to estimate the protein level of GLUT4 by Western blotting, we used a rabbit antiserum raised against a synthesized 12-amino acid peptide corresponding to the C-terminal region of GLUT4. The specificity of the antiserum was confirmed by its cross-reactivity to the homogenates of BAT, white adipose tissue, skeletal muscle and heart, but not to those of brain, liver and kidney (data not shown). This tissue distribution was identical to the previous reports (7,9,10) and also to the distribution of GLUT4 mRNA as determined by Northern blot analysis with a cDNA probe for GLUT4 (data not shown). Fig. 1 shows typical examples of Western and Northern blot analyses of BAT and skeletal muscle in control and cold-exposed rats. It seems obvious that both GLUT4 protein and GLUT4 mRNA increase much in BAT of cold-exposed rats, while they do not change in skeletal muscle. To assessthe effects of cold exposure quantitatively, the amounts of GLUT4 protein and mRNA of cold-exposed rats were estimated as those relative to the respective 24°C controls (Fig. 2). In BAT, the amount of GLUT4 protein and GLUT4 mRNA were 3 and 2 times higher, respectively, in cold-exposed rats than control rats. In skeletal muscle, however, no significant difference was found between the two groups of rats. Thus, cold exposure increases GLUT4 expression specifically in BAT but not in skeletal muscle. DISCUSSION In this study, we demonstrated a parallel increase of glucose utilization and the expression of GLUT4, an isoform of glucose transporters, in BAT after cold exposure. 1080
Vol.
BIOCHEMICAL
185, No. 3, 1992
A. Protein
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
B. mRNA BAT
BAT
Muscle
Muscle .I. ..,: .I.
28s ) 45kD * 18Sb
g E E 9 F .!I! 2
400
200
0
0
CL
1
Control
BAT
(24-C)
(473
2 0 (B) blot analyses of GLUT4. Rats were kept at 24°C
CL
CL
CL
Ml&de (4’C)
F&.& Western (A) and Northern (C) or 4°C (L) for 10 days. Protein samples from BAT (3Opg) and skeletal muscle (2Op.g) were used for Western blot analysis. For Northern blot analysis, 4 pg poly(A)+RNA was used. Fj& Effects of cold exposure on glucose utilization and GLUT4 expression in BAT and skeletal muscle. The values are means * SE for 5 (GLUT4 protein) and 4 (GLUT’4 mRNA) experiments, and expressed as those relative to the respective control at 24 “C. Glucose utilization (rate constant of 2-DG uptake) was calculated from the data in Table 1. * ~~0.05 compared with the value at 24°C.
We observed using a specific antiserum against GLUT4 that the amount of GLUT4 protein in BAT was several times higher in cold-exposed rats than control rats. This finding seems compatible with those of Greco-Perotto et al. (4) who quantified the total number of glucose transporters by the cytochalasin B binding method to find a 2-fold increase in BAT of coldexposed animals. In BAT, there is an another isoform of glucose transporter, GLUT?, in addition to GLUT4. However, since GLUT4 has been recognized as the predominant isoform in adipose tissues in adult rats (17) it seems rational to consider that the changes in cytochalasin B binding and glucose transport itself are attributable largely to the change in GLUT4. In fact, in a preliminary study, we found no change in the amount of immunoreactive GLUT1 after cold exposure. Northern
blot analysis
demonstrated
that the cellular
level of GLUT4 mRNA was also
increased remarkably in BAT of cold-exposed rats, suggesting that the increased GLUT4 protein
is due to an enhanced
de novo synthesis
of this protein.
It is now established
that
GLUT4 is an insulin-regulated glucose transporter isoform, and that its expression is activated by insulin
(18). Camps etal. suggested recently
possible that insulin takes part dominantly
this is also the case in BAT (19). It seems thus
in the cold-induced
activation
of GLUT4
expression.
However, there are reports indicating that glucose utilization in BAT can be activated directly, independently of the action of insulin, by sympathetic nerve via the f3-adrenergic pathway (620). Moreover, cold exposure results in a decreased plasma insulin level (6) and an increased sympathetic nerve activity in BAT (21). It is therefore more likely that the increased GLUT4 1081
Vol.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
expression in BAT seen after cold exposure is mediated through the p-adrenergic mechanism rather than insulin. The finding that GLUT4 expression in skeletal muscle is not influenced by cold exposure also supports the idea of a minor contribution of humoral factors such as insulin. Similar stimulatory effects of sympathetic nerves and / or adrenergic agonists on gene expression in BAT has been documented with mitochondrial uncoupling protein (22) and lipoprotein lipase (23) which play critical roles for BAT thermogenesis. ACKNOWLEDGMENTS We are very grateful to Dr. G. I. Bell (The University of Chicago, Chicago) for providing the cDNA clone of GLUT4 and Dr. J. Utsumi (Toray Basic Research Laboratory, Kamakura) for synthesizing the C-terminal peptide of GLUTC
REFERENCES
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