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Endocrinology, doi:10.1210/en.2006-0242
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Endocrinology Vol. 147, No. 12 5730-5739
Copyright © 2006 by The Endocrine Society

ß3-Adrenergic Receptors Stimulate Glucose Uptake in Brown Adipocytes by Two Mechanisms Independently of Glucose Transporter 4 Translocation

O. S. Dallner, E. Chernogubova, K. A. Brolinson and T. Bengtsson

The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden

Address all correspondence and requests for reprints to: Tore Bengtsson, The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden. E-mail: Tore.Bengtsson{at}zoofys.su.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To identify the mechanisms whereby norepinephrine induces glucose uptake in brown adipose tissue, we used mouse brown adipocytes in culture. Proliferating brown adipocytes had high levels of glucose transporter (GLUT) 1 mRNA and low levels of GLUT4 mRNA. The ratio of GLUT4/GLUT1 mRNA expression increased during differentiation, and mature brown adipocytes had high levels of GLUT4 mRNA. The endogenous adrenergic neurotransmitter norepinephrine induced a potent increase in GLUT1 mRNA and a decrease of GLUT4 mRNA in mature brown adipocytes. The norepinephrine effect was mimicked by isoprenaline and CL 316243 and was thus mediated by ß3-adrenergic receptors. The cAMP analog 8-bromoadenosine-cAMP partly mimicked the response on GLUT1 mRNA increase and fully mimicked the GLUT4 mRNA decrease. We found no involvement of {alpha}1 or {alpha}2-adrenergic receptors on GLUT1 or GLUT4 mRNA transcription. Norepinephrine treatment led to a large increase of GLUT1 protein amount in brown adipocytes as visualized with immunocytochemical staining and subcellular fractionation. A large part of the newly synthesized GLUT1 was found in the plasma membrane (PM). The potent transcriptional inhibitor actinomycin D fully abolished this increase of GLUT1 protein at all time points examined. Norepinephrine treatment shifted GLUT4 from the PM to an intracellular vesicular compartment. Norepinephrine increased 2-deoxy-D-glucose uptake 2-fold at an early time point (1 h) and 4-fold at later time point (5 h). Addition of actinomycin D did not block the early phase but blocked a large part of the later phase of 2-deoxy-D-glucose uptake. These results imply that adrenergic stimulation through ß3-adrenergic receptors induces glucose uptake in brown adipocytes via two mechanisms: 1) a mechanism not dependent on GLUT1 and GLUT4 translocation, 2) a mechanism that is dependent on de novo synthesis of GLUT1 protein and increase of GLUT1 protein at the PM.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BROWN ADIPOSE TISSUE (BAT) is an important organ for controlling energy dissipation in rodents. This is achieved by uncoupling mitochondrial respiration, via uncoupling protein-1 (UCP1) (for review see Ref. 1). Furthermore, it may potentially play a role in regulation of glucose homeostasis (2). Glucose uptake in BAT is markedly increased through two opposite metabolic pathways: during active anabolic processes, uptake is stimulated by insulin and during activation of thermogenesis, uptake is stimulated by norepinephrine (3, 4, 5). The insulin-signaling pathway has been extensively studied in muscle and white adipocytes and appears similar in brown adipocytes. Binding of insulin to the insulin receptor leads to the activation of the regulatory subunit of type 1A phosphatidylinositol 3-kinase (PI3K), which is one of the key intermediate targets in the insulin signaling pathway (for a recent review see Ref. 6). One of the downstream targets for insulin is translocation of intracellular vesicles containing glucose transporters (GLUTs) to the cell membrane (7). The GLUT isoforms differ in their tissue distribution profile, kinetic characteristics, and substrate specificity. GLUT1 is situated in the plasma membrane (PM) and provides basal supply of glucose for most cells and is heavily expressed in BAT (8). GLUT4 is exclusively found in adipose tissue (white and brown) and muscle, and its subcellular localization is controlled by insulin (7, 9), and this has also been demonstrated for brown adipocytes (10).

Cold exposure or overfeeding of rodents leads to stimulation of the sympathetic nervous system and activation of thermogenesis in BAT. A number of experiments show that glucose metabolism in rodents is affected and glucose uptake in BAT is markedly stimulated by cold exposure (11, 12) and activation of the sympathetic nervous system (4, 5). Interestingly, glucose uptake in BAT and skeletal muscle of cold-exposed fasting rats is increased in the same order of magnitude as in fed cold-exposed animals, suggesting that glucose uptake under these conditions is insulin independent (12, 13). Exposure to norepinephrine or other adrenergic agonists dramatically increases glucose uptake in brown adipocytes in both in vivo and in vitro models (5, 13, 14). BAT expresses several different adrenergic receptor subtypes (15, 16, 17) including the ß3-adrenergic receptor, which is a potential target for antiobesity and antidiabetic drug therapy (18). BAT has a very high uptake of glucose per gram of tissue, which means that even though the total amount of BAT in the body is not large, it can potentially be a significant glucose-clearing organ.

It is evident that the mechanism underlying the insulin-stimulated glucose transport in adipocytes mainly depends on translocation of GLUT4 (9), but there is little evidence explaining the mechanism for norepinephrine stimulated glucose uptake in brown adipocytes. Glucose uptake can potentially be modulated by several different mechanisms. Stimulation can lead to translocation of intracellular vesicles containing GLUTs to the cell surface. Insulin stimulation of adipocytes leads to translocation of GLUT4-containing vesicles (9), resulting in an acute increase of glucose (7, 10). The intrinsic activity of the GLUTs could potentially be altered through interactions with other signaling molecules, a mechanism that has been proposed to alter GLUT1 transport activity in adipocytes (19, 20). Stimulation could, in addition, affect transcriptional or translational activity and/or degradation rate of mRNA and protein that has been previously indicated in adipocytes (21, 22, 23).

We have recently shown that, in muscle cells, glucose uptake is stimulated by ß2-adrenergic receptors through multiple signaling pathways including increase of cAMP and activation of PI3K (24, 25). Furthermore, we have also shown that ß3-adrenergic receptors can couple to multiple signaling pathways (26, 27) and in brown adipocytes glucose uptake is stimulated through ß3-adrenergic receptors through a cAMP/protein kinase A and PI3K pathway stimulating conventional and novel protein kinase Cs (28, 29). In this study, we used brown adipocytes in primary culture that have previously been characterized to express intact adrenergic- and insulin-signaling systems (29, 30, 31, 32) to elucidate the mechanism whereby norepinephrine stimulates glucose uptake in brown adipocytes. The results indicate that norepinephrine-induced glucose uptake depends on two separate mechanisms: a first mechanism, which cannot be explained by translocation or de novo synthesis of GLUT1 or GLUT4, and a second mechanism dependent on de novo synthesis of GLUT1 mRNA and increase of GLUT1 protein at the PM.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Drugs and reagents
L-Norepinephrine bitartrate (Arterenol), (-)-isoprenaline, collagenase (type II), cirazoline, 8-bromoadenosine-cAMP (8-Br-cAMP), and CL-316243 actinomycin D were obtained from Sigma Chemicals. Insulin (100 IU/ml, Actrapid) was from Novo Nordisk (Bagsvaerd, Denmark), 2-deoxy-D-[1-3H]-glucose (specific activity 9.5–12 Ci/mmol) from Amersham Biosciences (Arlington Heights, IL). All cell culture media were from HyClone (Logan, UT), plates, chamber slides and flasks from Corning (Corning, NY) or Nunc (Roskilde, Denmark) and supplements from Invitrogen (Carlsbad, CA). Complete mini protease inhibitor tablets were from Roche Diagnostics GmbH (Penzberg, Germany). Adrenergic agents and 8-Br-cAMP were dissolved in water, except norepinephrine that was dissolved in water with 0.125 mM sodium ascorbate.

Brown fat precursor cell isolation
The mice used in this study were 3-wk-old NMRI mice (BK Universal, Sollentuna, Sweden). Brown fat precursor cells were isolated in principle as previously described (15). The interscapular, axillary, and cervical BAT depots were dissected out under sterile conditions, minced, and transferred to the HEPES-buffered solution (pH 7.4) detailed in Ref. 33 , containing 0.2% (wt/vol) crude collagenase type II (Sigma). Routinely, pooled tissue from six mice was digested in 10 ml of the HEPES-buffered solution. The tissue was digested (30 min, 37 C) with vortexing every 5 min, and the digest filtered through a 250-µm filter into sterile tubes. The solution was placed on ice for 15 min to allow the mature brown fat cells and lipid droplets to float. The infranatant was filtered through a 25-µm filter, collected, and the precursor cells pelleted by centrifugation (10 min, 700 x g), resuspended in DMEM (4.5 g D-glucose/liter) and recentrifuged. The pellet was finally resuspended in a volume corresponding to 0.5 ml of cell culture medium for each mouse dissected. The experiments were conducted with ethical permission from the North Stockholm Animal Ethics Committee.

Primary cell culture of brown adipocytes
The cell culture medium consisted of DMEM supplemented with 10% newborn calf serum (Invitrogen), 2.4 nM insulin, 10 mM HEPES, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 25 µg/ml sodium ascorbate (33). Aliquots of 0.1 ml cell suspension were cultivated in 12-well culture dishes with 0.9 ml cell culture medium. Cultures were incubated at 37 C in a water-saturated atmosphere of 8% CO2 in air (Heraeus CO2-auto-zero B5061 incubator). On d 1, 3, and 5, the medium was discarded, cells washed with prewarmed DMEM, and fresh medium added. After 5 d in culture, the brown adipocyte precursor cells spontaneously convert from displaying fibroblast-like morphology to acquiring typical mature brown adipocyte features; this conversion occurs at the time of cellular confluence (29, 34, 35, 36). The primary culture is thus a good model system for investigating mature brown adipocytes.

GLUT1 and GLUT4 cDNA probes
GLUT1 cDNA was obtained from IMAGE clone 5320248. GLUT4 cDNA was obtained with RevertAid Minus First Strand cDNA Synthesis Kit from Fermentas (Lithuania) and after PCR from 6-wk-old Sprague Dawley rat white adipose tissue. Primers contained EcoRI and BamHI restriction sites and were 5'-CGC GAA TTC AAG GCA CCC TCA CTA CCC TT-3 (forward) and 5'-CGC GGA TCC CTC AAA GAA GGC CAC AAA GC-3 (reverse). Amplified fragment was cloned into pBluescript plasmid with EcoRI and BamHI. Both clones were fully sequence verified.

The template (25–50 ng cDNA fragment) was denatured by boiling for 5 min and then added to a DNA labeling bead from (Amersham Biosciences) with 5 µl [{alpha}-32P] d-CTP (3000 Ci/mmol). After incubation for 1 h in 37 C the probe was isolated with a NICK column (Amersham Biosciences). Approximately 1 x 106 cpm was added per ml hybridization solution and 10–15 ml of hybridization solution were used for incubation with each membrane.

RNA isolation and analysis of mRNA levels
Approximately 10 mg of each tissue or cells from a 9.5-cm2 cell culture well were homogenized in 1 ml Ultraspec RNA reagent from Biotexc Labs (Houston, TX), and total RNA was isolated according to manufacturer’s instructions. RNA concentration and possible contaminations was measured in a DU-50 Beckman (Fullerton, CA) spectrophotometer at 260 and 280 nm. The 260/280 ratios for all samples were higher than 1.8, indicating low amounts of contaminations.

Ten micrograms of total RNA from each sample were separated by electrophoresis in an ethidium bromide-containing agarose-formaldehyde gel and blotted to a Hybond-XL membrane (Amersham Biosciences) as described earlier (31).

The intensity of the 28S rRNA band was analyzed in a PhosphoImager (Fujifilm FLA-3000) (Fujifilm, Tokyo, Japan) and quantified in Image Gauge (Fujifilm) to verify that no RNA degradation had occurred. Results were analyzed with the same procedure and normalized to 28S rRNA values.

2-Deoxy-D-[1-3H]-glucose uptake in primary brown adipocytes
Glucose uptake studies were performed in principle as previously described (24, 28). All experiments were performed on d 7 of cell culture. The culture media was changed at d 1, 3, and 6. On d 7, the cells were challenged with drugs for totally 0–16 h. Ten minutes before 2-deoxy-D-[1-3H]-glucose uptake measurement, the medium was discarded, cells washed with prewarmed PBS [10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl (pH 7.4)] buffer. Glucose-free DMEM (containing 0.5% BSA, 0.25 mM sodium ascorbate) was added and drugs readded with trace amounts of 2-deoxy-D-[1-3H]-glucose (50 nM) (specific activity 9.5–12 Ci/mmol) for 10 min. Reactions were terminated by washing in ice-cold PBS, cells lysed (500 µl of 0.2 M NaOH, 1 h at 60 C) and the incorporated radioactivity determined by liquid scintillation counting.

Subcellular fractionation and total cell lysate
Subcellular fractionation was in principle performed as described in Ref. 37 . Differentiated primary brown adipocytes (~30 million cells) were stimulated for 0–5 h, and then washed with prewarmed Ca2+/Mg2+-free PBS. Cells were then collected with a rubber policeman in prewarmed Ca2+/Mg2+-free PBS containing 2 mM EDTA. Cells were collected by centrifugation at 4 C for 10 min at 600 x g. The pellet was resuspended in sonication buffer [Tris-Hcl, 50 mM (pH 7.4); NaCl, 150 mM; EDTA, 2 mM; EGTA, and one complete mini protease inhibitor tablet/10 ml], and homogenized 30 times in a Dounce glass homogenizer. Samples were centrifuged at 4 C for 5 min at 1100 x g to remove cell debris, and supernatant was then centrifuged at 4 C for 60 min at 100,000 x g. Supernatant was collected as low-density microsomal (LDM) fraction and 1% Triton X was added, and the pellet (PM fraction) was resuspended in sonication buffer with 1% Triton X. The resuspended pellet was vortexed, sonicated for 2 x 5 sec, and vortexed again. The samples were then centrifuged at 4 C for 15 min at 21,000 x g, and supernatant was collected as PM fraction.

Total cell lysates was prepared by direct addition of 65 C lysis buffer [62.5 mmol/liter Tris (pH 6.8), 2% (vol/vol) sodium dodecyl sulfate, 10% (vol/vol) glycerol, 50 mmol/liter dithiothreitol, and 1% (vol/vol) bromophenol blue]. The samples were sonicated briefly and boiled for 5 min.

Western blot
Protein concentrations were determined (38) and 10 µg of protein (PM or LDM fractions) or 10 µl sample (total cell lysate) from each sample was loaded onto a 10% NuPAGE Bis-Tris Gel (Invitrogen) and run with a 2-(N-morpholino)ethanesulfonic acid sodium dodecyl sulfate running buffer according to the manufacturer’s instructions. Samples were loaded next to SeeBlue Plus2 marker (Invitrogen) and separated for 35 min at 200 V. Gels were blotted onto Hybond-P polyvinylidene difluoride membranes (pore size, 0.45 µm; Amersham Biosciences) for 1 h at 30 V. Primary and secondary antibodies hybridizations were performed as previously described (39). The primary antibodies used were rabbit polyclonal GLUT1 (AbCam, Cambridge, UK), rabbit polyclonal GLUT4 (AbCam), goat ß3-Adrenergic receptor (M-20; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit ERK 1/2 (Cell Signaling Technology, Boston, MA). All primary antibodies were detected using a secondary antibody (horseradish peroxidase-linked goat antirabbit IgG for GLUT1 and GLUT4 from Cell Signaling Technology; horseradish peroxidase-linked donkey antigoat IgG from Santa Cruz Biotechnology for ß3-adrenergic receptor) diluted 1:2000 and enhanced chemiluminescence (ECL, Amersham Biosciences).

Immunocytochemistry
Brown adipocytes were isolated as described above and seeded onto BD Falcon culture chamber slides (BD Biosciences, Franklin Lakes, NJ). After norepinephrine stimulation, the cells were washed with warm PBS and fixed for 15 min with 4% formaldehyde in PBS. Cells were washed with PBS and formaldehyde, quenched with 50 mM glycine in PBS, and washed three times for 5 min each with PBS. Cells were blocked for 1 h at room temperature with 8% BSA in PBS, and washed three times for 5 min each with PBS. Primary antibody solution (2 µg/ml antibody, 1.5% BSA in PBS) was added and slides were incubated for 1 h at room temperature and washed three times for 5 min each with PBS. Slides were then incubated with secondary antibody solution (3 µg/ml antibody, 3% BSA in PBS) and washed three times for 5 min each with PBS. Slides were mounted with mounting media [8% 1,4-diazabicyclooctane, 75% glycerol in PBS] and sealed. Antibodies used were GLUT1 (AbCam), GLUT4 (N-20; Santa Cruz Biotechnology), secondary alexa488-conjugated goat antirabbit IgG from Molecular Probes (Eugene, OR)/Invitrogen and Texas red-conjugated donkey antigoat IgG (Santa Cruz Biotechnology).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of GLUT1/4 expression by ß-ARs
Norepinephrine and adrenergic agents induce glucose uptake in brown adipocytes in vivo and in vitro in the absence of insulin (5, 13, 14, 28, 29). To determine whether activation of adrenergic receptors induce glucose uptake through stimulation of GLUT1 and GLUT4 gene expression, we stimulated mature primary brown adipocytes with 0.1 µM norepinephrine for 2 h. Norepinephrine stimulation leads to a potent increase of GLUT1 mRNA, and a surprising decrease of GLUT4 mRNA (Fig. 1Go).


Figure 1
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  		FIG. 1. mRNA expression of GLUT4 and GLUT1 in brown adipocytes stimulated with norepinephrine. Isolated brown adipocytes were cultured for 7 d, and stimulated for 2 h with 0.1 µM norepinephrine (NE). A total of 10 µg of each sample were analyzed by Northern blot analysis and hybridization with GLUT4 or GLUT1 probes as described in Materials and Methods. C, Control.

 
To evaluate the expression levels of GLUT1 and GLUT4 mRNA during differentiation, isolated brown adipocytes were cultured for 3–8 d and analyzed. GLUT1 gene expression was high during the proliferative stage (d 3), and decreased by 60% on d 4, and remained unchanged until d 8 (Fig. 2AGo). During the proliferative stage (d 3) GLUT4 mRNA levels were barely detectable but dramatically increased 10-fold to d 8 (Fig. 2BGo). The brown adipocytes proliferated at d 3, differentiated, and gradually matured and were fully confluent at d 6. This morphology was displayed at d 7 by aggregation of multilocular fat droplets, and as we have previously reported, by the characteristic induction of UCP1 mRNA expression by norepinephrine (not shown) (28). The ratio of GLUT4/GLUT1 mRNA expression (Fig. 2CGo) increases during differentiation and reaches maximum levels at d 6, indicating that the brown adipocytes are fully mature at that stage. We performed subsequent experiments on d 7 when the brown adipocytes were fully differentiated and displayed a high GLUT4/GLUT1 ratio.


Figure 2
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  		FIG. 2. mRNA expression of GLUT1 and GLUT4 during differentiation of brown adipocytes in culture. Isolated brown adipocytes were cultured for 8 d, and total RNA was isolated and 10 µg of each sample were analyzed by Northern blot analysis as described in Materials and Methods. Levels of GLUT1 (A) and GLUT4 (B) were analyzed at d 3–8 of differentiation. Ratio of GLUT4/GLUT1 was also calculated (C). Each point in A and B represents mean ± SEM of three experiments performed in duplicate.

 
To identify how rapidly norepinephrine treatment regulates GLUT1 and GLUT4 gene expression, we stimulated brown adipocyte primary cultures with 0.1 µM norepinephrine and analyzed mRNA levels of GLUT1 and GLUT4 at 0.5, 2, 5, 8, and 16 h of stimulation. Norepinephrine induced a dramatic increase of GLUT1 gene expression after 2 h, followed by 2- to 3-fold increase after 8 and 16 h (Fig. 3AGo). Stimulation with 0.1 µM norepinephrine decreased GLUT4 gene expression 50% of control after 2 and 5 h and returned back to control level after 16 h (Fig. 3BGo).


Figure 3
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  		FIG. 3. mRNA expression of GLUT4 and GLUT1 during 16 h of norepinephrine stimulation. Isolated brown adipocytes were cultured for 7 d in culture, and stimulated with 0.1 µM norepinephrine. Total RNA was isolated and 10 µg of each sample was analyzed by Northern blot analysis as described in Materials and Methods. Levels of GLUT1 (A) and GLUT4 (B) were analyzed after 0.5, 2, 5, 8, and 16 h of stimulation. Each point represents mean ± SEM of three experiments performed in duplicate.

 
We show that norepinephrine mediates an increase in glucose uptake through ß3-adrenergic receptor (Table 1Go), which previously has been shown to be through signaling with cAMP and PI3K in brown adipocytes (28). To determine which adrenergic receptor subtypes that mediate the induction of GLUT1 mRNA and reduction of GLUT4 mRNA, cultured brown adipocytes were stimulated for 2 h with 0.1 µM norepinephrine, ß-AR agonist (1 µM isoprenaline), a ß3-AR agonist (1 µM CL-316243), or a cAMP analog (1 mM 8-Br-cAMP). Stimulation with norepinephrine induced a marked 8-fold increase in GLUT1 mRNA expression (Fig. 4AGo). This effect was partially mimicked by stimulation with the cAMP analog 8-Br-cAMP. Stimulation with adrenergic receptor agonists, isoprenaline and CL-316243, increased GLUT1 mRNA levels compared with that found with norepinephrine. On the contrary, GLUT4 mRNA levels were repressed to approximately 50% of control by 8-Br-cAMP, isoprenaline, CL-316243, and norepinephrine (Fig. 4BGo). Brown adipocytes were also stimulated for 2 h with 1 µM cirazoline ({alpha}1-AR agonist) and 1 µM clonidine ({alpha}2-AR agonist) without any effect on GLUT1 or GLUT4 mRNA expression (data not shown).


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  		TABLE 1. Glucose uptake in brown adipocytes stimulated with agonists

 

Figure 4
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  		FIG. 4. mRNA expression of GLUT4 and GLUT1 in brown adipocytes stimulated with adrenergic agonists. Isolated brown adipocytes were cultured for 7 d, and stimulated for 2 h with 1 mM 8-Br-cAMP, 1 µM -(-)isoproterenol (ISO), 1 µM CL-316243 (CL), or 0.1 µM norepinephrine (NE). Total RNA was isolated and 10 µg of each sample was analyzed for mRNA levels of GLUT1 (A) and GLUT4 (B) by Northern blot analysis as described in Materials and Methods. Each column represents mean ± SEM of three experiments performed in duplicate. Asterisks represent values that are significantly different (*, P < 0.05; **, P < 0.01) from control, as analyzed by paired two-tailed Student’s t test.

 
Regulation of subcellular distribution of GLUT1/4 by norepinephrine
Because norepinephrine stimulation induces a marked increase in GLUT1 gene expression, we examined the content and localization of GLUT1 and GLUT4 protein with immunocytochemistry. Brown adipocytes were stimulated for 0, 2, or 5 h with norepinephrine and treated as described in Materials and Methods. Compared with 0 and 2 h, 5 h of norepinephrine stimulation induces a marked increase of GLUT1 protein in a perinuclear compartment. The GLUT1 protein and GLUT4 protein was located mainly in two different compartments as visualized with the merged GLUT1/GLUT4 image in Fig 5Go. An increase of GLUT4 in the intracellular compartment could be seen after 5 h of norepinephrine treatment. Control experiment with insulin was performed to ascertain that GLUT4 translocation could be detected by our assay. To further investigate whether this corresponds to protein levels in the PM, we made total cell lysate to investigate whole cell GLUT content and in addition isolated the LDM and PM contents of GLUT1 and GLUT4 protein, after norepinephrine stimulation (Fig. 6Go). Fractions were prepared from brown adipocyte primary cultures after 0, 2, and 5 h stimulation with 0.1 µM norepinephrine, and the protein levels were analyzed. GLUT1 protein content in the PM was approximately 2-fold after 5 h of norepinephrine stimulation compared with unstimulated cells (0 h) and was accompanied by an increase in the LDM fraction at the same time point, consistent with a de novo synthesis of GLUT1. GLUT4 protein was present in PM in the 0 h (control) and then shifted to the LDM fraction after norepinephrine stimulation. Virtually no GLUT4 was present in PM fraction after 5 h of stimulation. A part of the GLUT4 protein was located in the PM (culture conditions with a low concentration of insulin was used to keep the brown adipocytes viable), but most GLUT4 was located in intracellular vesicles, and during the experiment we saw no indication of GLUT4 translocation to the PM; instead, the amount of GLUT4 protein decreased in the PM and increased in intracellular compartment after norepinephrine treatment. ß3-AR protein was exclusively detected in PM fraction and ERK could only be found in LDM fraction indicating high integrity of the two fractions.


Figure 5
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  		FIG. 5. Immunostaining of transcriptional influence on GLUT4 and GLUT1 PM content of norepinephrine-stimulated brown adipocytes. Isolated brown adipocytes were seeded in culture chamber slides, cultured for 7 d, and stimulated with 0.1 µM norepinephrine for 0, 2, or 5 h (A), or insulin (INS) for 2 h (B). NC, Negative control. Cells were then fixed, subjected to GLUT1 or GLUT4 antibodies, and Texas red- or alexa488-labeled secondary antibodies, as described in Materials and Methods. A, Representative of three separate experiments; B, performed as one control experiment. Pictures were taken with x40 magnification.

 

Figure 6
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  		FIG. 6. Subcellular distribution and total cell content of GLUT4 and GLUT1 with norepinephrine (NE) stimulation. Isolated brown adipocytes were cultured for 7 d and stimulated with 0.1 µM norepinephrine for 0, 2, or 5 h [with or without actinomycin D (act); D–F]. Cell fractionation and preparation of total cell lysate was performed as described in Materials and Methods. A, 10 µg of protein from PM or LDM fractions, or 10 µl total cell lysate (D) was subjected to SDS-PAGE and immunoblotting with GLUT4, GLUT1, ß3-AR, or ERK 1/2 antibodies (A and D). GLUT1 and GLUT4 were analyzed and columns represent mean ± SEM of three experiments.

 
Effect of actinomycin D on norepinephrine-stimulated increase of GLUT1 protein and glucose uptake
To further examine the transcriptional control of the GLUT1 gene by norepinephrine, we performed immunocytochemistry experiments on brown adipocytes stimulated for 0, 2, or 5 h with/without 2 µg/ml of the transcriptional inhibitor actinomycin D. Actinomycin D has been shown to block transcription in brown adipocytes at 1–5 µg/ml without being detrimental to the cells (32, 40). Stimulation with norepinephrine resulted in a marked increase of GLUT1 protein, and this effect was completely abolished with actinomycin D treatment (Fig. 7Go).


Figure 7
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  		FIG. 7. Immunostaining of transcriptional influence on PM content of GLUT1 protein in norepinephrine-stimulated brown adipocytes. Isolated brown adipocytes were seeded in culture chamber slides, cultured for 7 d, and stimulated with 0.1 µM norepinephrine for 0, 2, or 5 h with 2 µg/ml of actinomycin D (act). Cells were then fixed and subjected to GLUT1 antibodies, and an alexa448-labeled secondary antibody, as described in Materials and Methods. Pictures were taken with x20 magnification. The picture is representative of three separate experiments.

 
A transcriptional activation of the GLUT1 gene, leading to GLUT1 protein accumulation and exocytosis, could have a physiological significance in altering the glucose transport over the PM. We investigated whether norepinephrine could induce glucose uptake in differentiated brown adipocytes due to transcriptional activation, and performed glucose uptake measurements at different time points of norepinephrine stimulation, in the absence or presence of actinomycin D in mature brown adipocytes. Norepinephrine induced an increase in glucose uptake at all time points measured with more than 350% increase at 5 h (Fig. 8AGo). Insulin-stimulated glucose uptake after 12 h treatment with 2 µg/ml actinomycin D was 182.8% ± 0.75% above control (n = 2 in duplicate) and in the same magnitude as treatment without actinomycin D (199 ± 5%), indicating that addition of actinomycin D was not detrimental to the cells or affected the insulin translocation system. Addition of 2 µg/ml of actinomycin D inhibited norepinephrine-induced glucose uptake at 2 h to some extent and heavily inhibited at the 5- and 8-h points. Plotting {delta} values when transcription is inhibited indicate two mechanisms. The first mechanism is not dependent on transcription and reaches a maximum after 2 h. This mechanism contributes solely to norepinephrine-induced glucose uptake at 1 h and contributes significantly up to 5 h. The second mechanism is dependent on transcription and starts to contribute to glucose uptake after 1 h and reaches its maximum at 5 h (Fig. 8BGo).


Figure 8
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  		FIG. 8. Transcriptional influence on norepinephrine-stimulated glucose uptake. A, Brown adipocytes were grown in culture and stimulated on d 7 with 0.1 µM norepinephrine with or without 2 µg/ml of actinomycin D for indicated time and glucose uptake was measured as described in Materials and Methods. Each point in A represents mean ± SE of three experiments performed in triplicate, and control 0.5 h is set to 100%, {delta} values of (NE)-(NE+Act) and (Ne+Act)-(basal) were also calculated from panel A (B), displaying mechanism 1 (M1) not dependent on transcription and mechanism 2 (M2) dependent on transcription.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
BAT has a very high uptake of glucose per gram of tissue (5, 13) and it could therefore potentially be an important glucose-clearing organ when activated by norepinephrine. In this study, we have investigated norepinephrine-induced glucose uptake and glucose transporter expression in mouse brown adipocytes grown in primary culture. We used primary cultures of mouse brown adipocytes that differentiate spontaneously in the presence of newborn calf serum (NSC) and physiological levels of insulin and at d 6 display characteristics associated with mature adipocytes, such as UCP1 expression, ß3-adrenergic receptor expression, multilocular fat droplets and insulin sensitivity (15, 28, 31, 35). In this study, we confirmed the spontaneous induction of the above mentioned parameters in primary mouse brown adipocytes (not shown). This is in contrast to primary cultures of rat brown adipocytes that do not differentiate spontaneously. Primary culture of rat brown adipocytes are normally supplemented with fetal calf serum (which is more proliferative and presumably has higher levels of growth factors than newborn calf serum) and a differentiation mix (including D-pantothenic acid, D-biotin, ascorbic acid, octanoic acid, triiodothyrine, and high levels of insulin). When rat cultured brown adipocytes cells reach confluence, they further need to be treated with dexamethasone to induce glucose transport in response to either insulin or norepinephrine (41). Because this mix artificially up-regulates GLUT4 gene expression in brown adipocytes (42, 43), we believe that spontaneously differentiated primary mouse brown adipocytes are a better suited model system for investigating norepinephrine effects on GLUT gene expression in brown adipocytes.

Norepinephrine stimulates GLUT1 but decreases GLUT4 gene expression
We show here that brown adipocytes had high levels of GLUT1 mRNA in the proliferative stage but decreased rapidly during differentiation and reached low levels in confluent mature brown adipocytes. In contrast, GLUT4 mRNA levels were lower in proliferative cells but increased rapidly in spontaneously differentiating cells and reached a maximum in the mature brown adipocytes. The ratio of GLUT4/GLUT1 mRNA expression increased during differentiation from proliferating to mature brown adipocytes. This switch coincides with a shift in ß-adrenergic receptor expression. ß1-Adrenergic receptor mRNA follows the same pattern as GLUT1 mRNA, and ß3-adrenergic receptors mRNA follow the same pattern as GLUT4 mRNA levels during differentiation (31). The induction of ß3-adrenergic receptors and GLUT4 is an innate part of the differentiation program and does not require any additional exogenous stimulation. We believe this reflects the change from proliferating cells, which needs a high basal glucose uptake for processes contributing to cellular division, to mature cells sensitive to insulin and ß3-adrenergic receptor stimulation leading to storage or dissipation of energy through UCP1. Surprisingly, when mature brown adipocytes are challenged with norepinephrine that couples through ß3-adrenergic receptors, they react with a large increase in GLUT1 mRNA and a decrease in GLUT4 mRNA, which is sustained for at least 8 h. Norepinephrine stimulation also leads to similar effects on ß3-adrenergic receptors (full cessation of expression) and ß1-adrenergic receptors (markedly enhanced expression) (32, 44). Norepinephrine activates many metabolic processes in brown adipocytes and increases the overall energy consumption of the tissue mainly through ß3-adrenergic receptors and activation of UCP1 (1), but glucose probably does not play a large role as a thermogenic substrate for UCP1 (45). It is possible that the large increase in GLUT1 and ß1-adrenergic receptor and decrease in GLUT4 and ß3-adrenergic receptor expression reflect an activated state where the cells increase in overall energy consumption is dependent on ß1-adrenergic receptors and GLUT1, which we are currently investigating.

ß-Adrenergic receptor signaling involved in regulation of GLUT gene expression
Significant uncertainty exists regarding the physiological importance and the relative role of ß3-adrenergic receptors vs. ß1- and ß2-adrenergic receptors in mediating signal transduction in BAT. It has been discussed that ß-adrenergic receptors (ß2-adrenergic receptors and ß3-adrenergic receptors) may not only couple to Gs, but also to Gi (26, 39, 46, 47, 48). Furthermore, the G proteins to which ß-adrenergic receptors are coupled, are heterotrimeric proteins with {alpha}-, ß-, and {gamma}-subunits, and the ß/{gamma} subunit from Gi could function as a signal to several downstream targets such as PI3K involved in glucose uptake (see references in Ref. 49). ß3-Adrenergic receptor signaling in mouse cells could be more complicated because the mouse ß3-adrenergic receptor gene contains two exons, both of which undergo alternative splicing and produce expressed splice variants of the ß3-adrenergic receptor (50).

We have previously reported that ß3-adrenergic receptors may couple to multiple signaling pathways (26, 27, 28), and it is possible that other signaling pathways except the classical second messenger cAMP could influence GLUT gene expression.

It is evident from our results in this study that norepinephrine regulates GLUT1 and GLUT4 gene expression through ß3-adrenergic receptors, but we cannot with these data fully conclude whether ß3-adrenergic receptor-induced GLUT1 mRNA synthesis is fully governed by cAMP or other signaling pathways. We did not achieve the same GLUT1 mRNA increase with 8-Br-cAMP as with a ß3-adrenergic agonist but interestingly the GLUT4 mRNA decrease is fully governed by cAMP. This could reflect different sensitivity for the GLUT1/GLUT4 gene promoters to cAMP or the inability of 8-Br-cAMP to mimic local concentrations in specialized compartments in the cell, or possible that ß3-adrenergic signaling such as activation of PI3K or other signaling molecules, together with cAMP, is governing GLUT1 gene expression. PI3K is important in ß3-adrenergic receptor-induced glucose uptake in mature wild-type brown adipocytes (28), and therefore investigations of PI3K involvement on GLUT1 promoter and gene expression could be envisaged, but this was not within the scope of this investigation.

Norepinephrine stimulates glucose uptake through two mechanisms
Norepinephrine increases glucose uptake mainly through ß3-adrenergic receptors in mature brown adipocytes (28, 29). Our results here indicate that norepinephrine induces glucose uptake through two separate mechanisms, a mechanism that is actinomycin D insensitive and a second mechanism that is relatively slower and actinomycin D sensitive. Actinomycin D is fast-acting general transcription inhibitor, and we have previously shown that actinomycin D rapidly stops transcription of multiple genes in brown adipocytes (32, 44). We therefore find it unlikely that the first mechanism would reflect a specific slow inhibition of GLUT transcription. Furthermore, we see a complete inhibition of de novo synthesis of GLUT1 with actinomycin D with immunocytochemistry. We found no inhibitory effects on insulin-stimulated glucose uptake in actinomycin D-treated cells leading to the conclusion that norepinephrine-induced glucose uptake is different from that of insulin in brown adipocytes and that actinomycin D does not inhibit translocation per se.

Interestingly, the first mechanism of glucose uptake is not due to translocation or de novo synthesis of GLUT1/4 in brown adipocytes. Norepinephrine treatment still gives a substantial glucose uptake at early time points when transcription has been blocked and we find no translocation of GLUT1 or GLUT4. Thus, neither gene expression nor translocation of transporters from intracellular pools, the paradigm for insulin action, appear to be involved in acute regulation of glucose transport in brown adipocytes. It is possible that this mechanism reflects a change in the intrinsic activity of GLUTs, which has been proposed before in several cell system (8, 19, 51, 52, 53, 54). In these instances, transporter activation has been ascribed to an increase in rate of glucose transport despite little, if any, increase in the content of GLUT in the PM. Although the molecular mechanism underlying such activation have not yet been identified, it is possible that modifications of GLUT or GLUT-protein interactions (55) play a role in activating GLUT1 or translocation/effects on other GLUTs that we have not investigated here. In most cases, the enhanced uptake of glucose is associated with an increase in the Vmax for transport, with little or no change in the apparent affinity (Km) for the substrate. It has been postulated that AMP-activated protein kinase (AMPK) is important in activation of GLUT1 in rat liver epithelial cell line Clone 9 (56). We have shown that ß-adrenergic receptors, but not {alpha}-adrenergic receptors, stimulate AMPK in brown adipocytes. The activation of AMPK is partially responsible for the norepinephrine-induced glucose uptake (57). We are currently investigating a possible link between ß-adrenergic receptors, AMPK and GLUT1 activation and/or effects on GLUT gene expression relative to glucose uptake in brown adipocytes.

The second phase of norepinephrine-induced glucose uptake consists of the first and second mechanisms. The second mechanism depends on cAMP-induced GLUT1 de novo synthesis and elevated GLUT1 amount at the PM, which leads to a large increase in glucose uptake that can be blocked by actinomycin D. At 5 h, the second mechanism dominates largely over the first mechanism. We found that GLUT1 was concentrated to a subcellular compartment outside the nucleus and this compartment was different from the compartment containing GLUT4. Norepinephrine treatment leads to a large increase of GLUT1 and a concentration of GLUT4 in these two respective compartments within 5 h. We cannot at this point fully conclude which type of subcellular compartments GLUT1 and GLUT4 resides in, but the morphological data suggest that GLUT1 is found in the endoplasmatic reticulum and GLUT4 in vesicles apart from the endoplasmatic reticulum. Norepinephrine treatment heavily increases the numbers of GLUT1 in the PM and in the LDM, suggesting de novo synthesis of GLUT1 and translocation of the newly synthesized GLUT1 to the PM. Interestingly, at all times activation of the pathways downstream of the adrenergic receptors does not seem to cause GLUT4 translocation, but actually shifts this transporter isoform from the PM to an intracellular vesicular compartment. This surprising finding is in accordance with data observed in adipocytes (53, 58), making it plausible that even though GLUT4 is heavily expressed in brown adipocytes they do not contribute to norepinephrine-induced glucose uptake. Furthermore, we found that norepinephrine decreases GLUT4 mRNA, making it unlikely that GLUT4 de novo synthesis is involved in norepinephrine-induced glucose uptake in brown adipocytes.

Conclusions
We put forward the hypothesis that norepinephrine through ß3-adrenergic receptor induces glucose uptake via two separate mechanisms. The response is dependent on a first mechanism that is not due to GLUT4 translocation and a relatively slower mechanism dependent on an increase of GLUT1 mRNA and de novo synthesis of GLUT1 that is translocated to the PM. At present, it remains unknown which isoform(s) of GLUT is responsible for the first mechanism of norepinephrine-induced increase in glucose uptake. We are currently investigating whether the first response is due to activation of GLUT1 or translocation/effects on other GLUTs, but it is unlikely that this mechanism involves GLUT4 because this transporter is shifted from the PM to intracellular vesicles. Both mechanisms are probably important during activation of BAT. The second mechanism dominates largely over the first mechanism at later time points, and it is therefore vital to investigate and separate these two mechanisms when elucidating norepinephrine signaling and effects on glucose uptake in brown adipocytes and possible in other cell types.


   Acknowledgments
 
We thank Professor Barbara Cannon, Professor Jan Nedergaard, Dr. Dana Hutchinson, and Dr. Julia Nevzorova for valuable discussions.


   Footnotes
 
This investigation was supported by research grants from the Swedish Science Research Council, Novonordiskfonden and Stiftelsen Svenska Diabetesförbundets Forskningsfond.

Disclosure summary: The authors have nothing to disclose.

First Published Online September 7, 2006

Abbreviations: AMPK, AMP-activated protein kinase; 8-Br-cAMP, 8-bromoadenosine-cAMP; BAT, brown adipose tissue; GLUTs, glucose transporters; LDM, low-density microsomal; PI3K, phosphatidylinositol 3-kinase; PM, plasma membrane; UCP1, uncoupling protein-1.

Received February 22, 2006.

Accepted for publication August 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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