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Endocrinology, doi:10.1210/en.2003-0857
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Endocrinology Vol. 145, No. 1 269-280
Copyright © 2004 by The Endocrine Society

Norepinephrine Increases Glucose Transport in Brown Adipocytes via ß3-Adrenoceptors through a cAMP, PKA, and PI3-Kinase-Dependent Pathway Stimulating Conventional and Novel PKCs

Ekaterina Chernogubova, Barbara Cannon and Tore Bengtsson

The Wenner-Gren Institute, 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
 Conclusions
 References
 
To identify the signaling pathways that mediate the adrenergic stimulation of glucose uptake in brown adipose tissue, we used mouse brown adipocytes in culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced 2-deoxy-D-glucose uptake 3-fold in a concentration-dependent manner (pEC50 ~6.5). The uptake was abolished by high doses of propranolol. The NE effect was mimicked by isoprenaline (pEC50 ~6.9), BRL 37344 (pEC50 ~8.6), CL 316243 (pEC50 ~9.7) and CGP 12177 (pEC50 ~7.3) and was thus mediated by ß3-adrenergic receptors. The NE-induced effect on 2-deoxy-D-glucose uptake was mediated by adenylyl cyclase and cAMP because responses were inhibited by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and the protein kinase A inhibitor 4-cyano-3-methylisoquinoline. Cholera toxin and 8-bromoadenosine cAMP were both able to increase 2-deoxy-D-glucose uptake. Involvement of other adrenergic signaling pathways ({alpha}1-and {alpha}2-adrenergic receptors) were excluded. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, abolished ß-adrenergic- or 8-bromoadenosine cAMP-stimulated 2-deoxy-D-glucose uptake, demonstrating that a cAMP-dependent PI3K-mediated pathway is positively connected to glucose uptake. Inhibition of the ß-adrenergically stimulated response with protein kinase C (PKC) inhibitors (Gö 6983, which inhibits ({alpha}, ß, {gamma}), ({delta}), and ({zeta}) isoforms and Ro-31–8220, which inhibits ({alpha}, ß1, ß2, {gamma}) and ({epsilon}) but not atypical isoforms) indicated that cAMP-mediated glucose uptake is stimulated via conventional and novel PKCs. These results demonstrate that adrenergic stimulation, through ß3-adrenergic receptors/cAMP/protein kinase A, recruits a PI3K pathway stimulating conventional and novel PKCs, which mediate glucose uptake in brown adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
IN RODENTS, BROWN ADIPOSE tissue (BAT) is an important organ for controlling energy dissipation by its ability to uncouple mitochondrial respiration, a process mediated by uncoupling protein-1 (UCP-1) (for a recent review, see Ref. 1). Furthermore, it may potentially play a significant role in the regulation of glucose homeostasis and insulin secretion in rodents (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 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. GLUT-1 is situated in the plasma membrane and provides a basal supply of glucose for most cells and is heavily expressed in BAT (8). GLUT-4 is exclusively found in adipose tissue (white and brown) and muscle, and its subcellular localization is controlled by insulin (7). This has also been demonstrated for brown adipocytes (9).

Thermogenesis in BAT is highly regulated by the sympathetic nervous system, and metabolic heat is produced in response to cold exposure or overfeeding. Glucose uptake in BAT is markedly stimulated by cold exposure (10, 11) and activation of the sympathetic nervous system (4, 5). In vitro glucose uptake is stimulated in brown adipocytes by norepinephrine and other adrenergic agents (3, 12, 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). We have previously shown that in muscle cells, glucose uptake is stimulated by ß2-adrenergic receptors via a process not directly related to increases in cAMP (19). In this study, we used brown adipocytes in primary culture that have previously been characterized to express intact adrenergic- and insulin-signaling systems (20, 21, 22, 23, 24). We have studied the norepinephrine-activated glucose uptake with focus on the adrenergic receptors and intracellular signaling pathways involved in this process.

We present a model for the signaling pathway and key molecules involved in the adrenergic receptor activation of glucose transport in brown adipocytes, indicating a ß3-adrenergic receptor/cAMP/protein kinase A (PKA) pathway which stimulates PI3K and protein kinase C (PKCs). The results indicate that this point represents the link between the opposing adrenergic and insulin pathways, at which the pathways converge and ultimately use the same signaling molecules downstream of PI3K.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
Cell isolation
The mice used in this study were 3-wk-old FVB mice (either sex) bred at the institute. Brown fat precursor cells were isolated in principle as previously described (15). The interscapular, axillary and cervical brown adipose tissue depots were dissected out under sterile conditions, minced, and transferred to the HEPES-buffered solution (pH 7.4) (detailed by Ref. 25), containing 0.2% (wt/vol) crude collagenase type II (Sigma, St. Louis, MO). 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 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.

Cell culture
The cell culture medium consisted of DMEM supplemented with 10% newborn calf serum (Life Technologies, Grand Island, NY), 2.4 nM insulin, 10 mM HEPES, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 25 µg/ml sodium ascorbate (25). 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-autozero B5061 incubator). On indicated days, the medium was discarded, cells washed with prewarmed DMEM (d 1), and fresh medium added (d 1, 3, and 5). 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 (26, 27, 28). In these cells, spontaneous induction of ß3-adrenergic receptor mRNA reaches a steady-state level at d 5 that coordinates with the ability of norepinephrine to induce the expression of the most specific brown adipocyte differentiation marker, the UCP1 (29).

Brown adipocytes in cell culture shows no significant change in cell number after d 7 (26). There is also very little difference in protein content between wells or series of cultures: the protein content was 9.86 ± 0.2 µg protein/cm2 (mean ± SE), with a variance of 0.64 µg protein/cm2 (n = 17 series in duplicate-quadruplicate).

Northern blot
Cell cultures and experimental conditions were as detailed below (2-deoxy-D-[1-3H]glucose uptake) without the addition of the radioactive isotope. At the end of the experiment, cells were dissolved in 1 ml Ultraspec solution (Biotecx, Houston, TX), and the manufacturer’s procedure for RNA isolation was followed. Ten micrograms total RNA were separated by electrophoresis in an ethidium bromide-containing agarose-formaldehyde gel. The intensity of the 18S and 28S rRNA band under UV light was checked to verify that the samples were equally loaded and that no RNA degradation had occurred. mRNA levels were analyzed by Northern blotting as described earlier (29). The UCP-1 cDNA was as used earlier (15) and labeled with [{alpha}-32P] dCTP with Ready To Go DNA labeling beads (Amersham), according to the manufacturer’s instructions.

2-Deoxy-D-[1-3H]glucose uptake
Glucose uptake studies were performed as previously described (19, 30) with some modifications. All experiments were performed on d 7 of cell culture. On d 6, the cells were serum starved overnight in DMEM/Nutrient Mix F12 (1:1) with 4 mM L-glutamine, 0.5% BSA, 2.4 nM insulin, 10 mM HEPES, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 50 µg/ml sodium ascorbate. To reduce basal glucose uptake, the medium was changed to serum-free DMEM without insulin (containing 0.5% BSA, 0.125 mM of sodium ascorbate) for 30 min (longer periods did not further reduce basal uptake; data not shown) before the cells were challenged with drugs for a total of 2 h (time courses showed that 2 h stimulation gave the maximum response to insulin or adrenergic agonists; data not shown). Detailed protocols for the inhibitors used are found in the description of each experiment. After 1 h 50 min incubation with drugs, the medium was discarded, cells washed with prewarmed PBS buffer (10 mM phosphate buffer, 2.7 mM potassium chloride,137 mM sodium chloride, pH 7.4) before glucose-free DMEM (containing 0.5% BSA, 0.125 mM sodium ascorbate) was added and drugs readded with trace amounts of 2-deoxy-D-[1-3H]glucose (50 nM) (Amersham, specific activity 9.5–12 Ci/mmol) for 10 min. Reactions were terminated with washing in ice-cold PBS, cells lysed (500 µl of 0.2 M NaOH, 1 h at 55 C) and the incorporated radioactivity determined by liquid scintillation counting.

cAMP determinations
All experiments were performed on d 7 of cell culture. The cells were treated according to the first part of the protocol for the glucose uptake studies. After indicated times with drugs, the culture medium was aspirated, 0.8 ml of 95% ethanol added to each well, and the cells scraped off. The samples were routinely washed with 70% ethanol (0.4 ml) and the combined suspensions dried in a Speedvac centrifuge. The dried samples were dissolved in 150–500 µl of the buffer 1 provided with the cAMP (3H) assay system from Amersham (TRK 432), sonicated briefly and centrifuged at 14,000 rpm for 10 min. One 50-µl aliquot of the supernatant was analyzed for every sample according to the description in the assay system, and for every concentration of any agonist in each experiment, duplicate wells were used.

Analysis of results
For analysis of concentration-response curves, the curve-fitting option of the KaleidaGraph 3.08 program (Synergy Software, Reading, PA) was used. Monophasic concentration-response data were analyzed with the rearranged Michaelis-Menten equation VA = basal + Vmax/(1 + (EC50/[A])), where A is the concentration of adrenergic agent added, VA the response observed at that concentration, and Vmax the estimated maximal increase. In some calculations, basal was set as a constant to avoid a singular matrix that would make the fitting unsolvable.

Results are presented as the mean values ± SE t test (paired) was used to test for significance between the different treatments and/or controls. In studies in which inhibitors affected basal levels, differences between stimulated and inhibited effects were calculated as {delta} values above each corresponding control (i.e. control and inhibitor alone). Statistical significance in text means at least P <= 0.05 (statistical significance in figures: *, P <= 0.05 and **, P <= 0.01). In experiments in which antagonists were used, log(dr - 1) - log[antagonist], where dr = dose ratio (pKB) values were calculated according to the method of Furchgott (31) and values given as mean ± SE.

Chemicals
L-Norepinephrine bitartrate (Arterenol), (±)-isoproterenol, D,L-propranolol, collagenase (type II), BRL 37344, CGP-12177, cirazoline, prazosin, 8-bromoadenosine-cAMP (8-Br-cAMP), phorbol 12-myristate 13-acetate (TPA), LY294002, H89, and CL 316243 were obtained from Sigma, 4-cyano-3-methylisoquinoline (4CM), Ro-81–3220, and Gö 6983 from Calbiochem (San Diego, CA). ICI-89406 and ICI-118551 were from Zeneca (Wayne, PA). 2',5'-Dideoxyadenosine (DDA) was from ICN Biomedicals Inc. (Irvine, CA). Insulin (Actrapid) was from Novo Nordisk (Bagsvaerd, Denmark). 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclicmonophosphate sodium salt (8-CPT) was from Biolog (Germany). cAMP kit (TRK 432), 2-deoxy-D-[1-3H]glucose (specific activity 9.5–12 Ci/mmol) from Amersham. All cell culture media and supplements were from Life Technologies.

All adrenergic agents, 8-Br-cAMP, 8-CPT, and Ro 81–3220 were dissolved in water. Norepinephrine was dissolved in water with 0.125 mM sodium ascorbate. TPA, LY 294002, Gö 6983, DDA, and ICI 89406 were dissolved in dimethyl sulfoxide (final concentration was maximally of 0.1%).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Insulin-induced glucose uptake in cultured brown adipocytes
Insulin stimulates glucose uptake in brown adipocytes in vivo (32, 33, 34) and in freshly isolated cell systems (3, 12, 14, 35, 36, 37). In our system with spontaneously differentiated mouse brown adipocytes in culture, basal 2-deoxy-D-glucose uptake was 17.9 ± 1.6 pmol/min·mg (n = 8 series in duplicate) calculated with an average of 37.4 µg protein/well), comparable with other studies using rat brown adipocytes differentiated by high levels of insulin and a differentiation mix (8, 38). Brown adipocyte differentiation was confirmed by induction of UCP-1 gene expression. The addition of adrenergic agonists led to a large increase in UCP-1 mRNA in the mature cells (d 7) (Fig. 1Go), all in agreement with earlier observations (15, 39). To confirm the presence of a functional insulin signaling pathway and glucose transport system in our cells, we treated the cells with insulin for 2 h, as shown in Fig. 2AGo. Insulin increased 2-deoxy-D-glucose uptake in a concentration-dependent manner (Vmax 260 ± 3 4%; pEC50 8.8 ± 0.1), similar to other studies using differentiated rat brown adipocytes (24, 38, 40, 41). The results here show that the spontaneously differentiated mouse brown adipocytes in vitro are sensitive to insulin and have a functional insulin-signaling pathway that leads to increased glucose uptake.



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  		FIG. 1. Northern blot confirming adrenergic induction of UCP-1 gene expression in spontaneously differentiated brown adipocytes in culture. Brown adipocytes (d 7 in culture) were cultured under conditions identical to those used for 2-deoxy-D-glucose uptake as described in Materials and Methods. Cells were treated for 2 h with the indicated concentrations of drugs. Control (C), 1 µM BRL 37344 (BRL), 1 µM isoprenaline (ISO), and 1 µM norepinephrine (NE). Total RNA was isolated as described in Materials and Methods, and 10 µg/sample were analyzed by Northern blot analysis by hybridization with the UCP-1 probe.

 


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  		FIG. 2. Concentration-response curves for insulin (A), norepinephrine and isoprenaline (B), norepinephrine plus 1 µM prazosin and cirazoline (C), and norepinephrine plus 10 µM yohimbine (D) on 2-deoxy-D-glucose (2-DG) uptake in cultured mouse brown adipocytes. Confluent cultures of brown adipocytes (d 7 in culture) were treated for 2 h with the indicated concentrations of drugs and measured for 2-deoxy-D-glucose uptake as described in Materials and Methods. The values are means ± SE A, Insulin ({blacksquare}-{blacksquare}) (n = 5 series in duplicate). B, Norepinephrine ({diamondsuit}-{diamondsuit}) (n = 7 series in duplicate), and isoprenaline ({bullet}-{bullet}) (n = 7 series in duplicate). C, Norepinephrine ({diamondsuit}-{diamondsuit}) in absence and presence of prazosin (1 µM) ({triangledown}-{triangledown}) (n = 3 series in quadruplicate) and cirazoline in parallel ({blacktriangledown}-{blacktriangledown}) (n = 3 series in duplicate). D, Norepinephrine ({diamondsuit}-{diamondsuit}) in absence and presence of yohimbine (10 µM) ({dotsquare}-{dotsquare}) (n = 1 series in duplicate). All antagonists were added before addition of agonist. Vehicle-treated wells were set to 100% for each cell culture series and the other values given relative to this. The results were fitted with the general curve-fitting procedure for Michaelis-Menten kinetics.

 
Norepinephrine-induced glucose uptake in cultured brown adipocytes
Norepinephrine and adrenergic agents induce glucose uptake in brown adipocytes in vivo (32, 42, 43) and in vitro (3, 12, 14, 38, 42) in the absence of insulin. To determine which adrenergic receptor subtype(s) mediate the norepinephrine effect, we stimulated brown adipocytes with different selective adrenergic agonists. The spontaneously differentiated mouse brown adipocytes in culture responded to norepinephrine in a concentration-dependent manner (Vmax 330 ± 28%; pEC50 6.5 ± 0.1) (Fig. 1BGo). The unspecific ß-adrenergic agonist isoprenaline gave similar increases in 2-deoxy-D-glucose uptake (Vmax 272 ± 48%; pEC50 6.9 ± 0.1) as did norepinephrine (Fig. 2BGo). There was no statistically significant difference between the norepinephrine and isoprenaline responses, indicating that ß-adrenergic receptors appear to mediate the norepinephrine response. To investigate whether {alpha}-adrenergic receptors are involved in the norepinephrine response, we used a selective {alpha}1-adrenergic receptor antagonist prazosin and a selective {alpha}2-adrenergic receptor antagonist yohimbine. The results showed no statistically significant difference between the norepinephrine effect in the presence or absence of prazosin (1 µM) (Fig. 2CGo) or yohimbine (1 µM, not shown; or 10 µM, Fig. 2DGo). Intriguingly, the {alpha}1-adrenergic agonist cirazoline by itself gave a very small but significant increase in 2-deoxy-D-glucose uptake (Vmax 130 ± 7%; pEC50 8.3 ± 0.2) (Fig. 2CGo).

Norepinephrine stimulated glucose uptake through ß3-adrenergic receptors
BAT expresses all three ß-adrenergic receptors (17, 44, 45), but it is likely that most of the ß2-adrenergic receptor expression is in the vascular system. To elucidate through which ß-adrenergic receptor norepinephrine stimulates glucose uptake, we used selective ß-adrenergic agonists and antagonists. Neither the selective ß1-adrenergic receptor antagonist ICI 89406 (Fig. 3AGo) nor the ß2-adrenergic receptor antagonist ICI 118551 (Fig. 3BGo) displaced the norepinephrine concentration-response curve. The nonselective ß-adrenergic receptor antagonist propranolol (which has a much lower affinity for ß3-adrenergic receptors than for ß1- and ß2-adrenergic receptors) shifted the norepinephrine curve significantly to the right at 10 µM, resulting in a pKB of 6.1 ± 0.1 (Fig. 3DGo) but not at 1 µM (Fig. 3CGo). This is in accordance with the pA2 values (5.5–6.1) found for ß3-adrenergic receptor-stimulated respiration in mouse, rat, and hamster brown adipocytes (46, 47, 48, 49). ß3-Adrenergic receptors are characterized by a very low affinity for classical ß-adrenergic receptor antagonists such as propranolol, compared with a pA2 of about 9 on ß1-/ß2-adrenergic receptors (50). This indicated that neither ß1- nor ß2-adrenergic receptors are involved in the norepinephrine-induced increase in glucose transport in brown adipocytes in culture.



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  		FIG. 3. Concentration-response curves for norepinephrine plus 1 µM ICI89406 (A), norepinephrine plus 1 µM ICI11854 (B), norepinephrine plus 1 µM propranolol (C), and norepinephrine plus 10 µM propranolol (D) on 2-deoxy-D-glucose (2-DG) uptake in cultured mouse brown adipocytes. Experiments were performed as described in the legend to Fig. 2Go. The values are means ± SE A, Norepinephrine ({diamondsuit}-{diamondsuit}) in absence and presence of 1 µM ICI89406 ({circ}-{circ}) (n = 3 series in duplicate). B, Norepinephrine ({diamondsuit}-{diamondsuit}) in absence and presence of 1 µM ICI118551 ({diamond}-{diamond}) (n = 3 series in duplicate). C, Norepinephrine ({diamondsuit}-{diamondsuit}) in absence and presence of 1 µM propranolol ({triangleup}-{triangleup}) (n = 3 series in duplicate and quadruplicate). D, Norepinephrine ({diamondsuit}-{diamondsuit}) in absence and presence of 10 µM propranolol ({triangleup}-{triangleup}) (n = 3 series in duplicate). Vehicle-treated wells were set to 100% for each cell culture series and the other values given relative to this. The results were fitted with the general curve-fitting procedure for Michaelis-Menten kinetics.

 
Isoprenaline stimulates glucose uptake through ß3-adrenergic receptors
To further confirm which ß-adrenergic receptor mediated increases in glucose uptake in mouse brown adipocytes, we used the nonselective ß-adrenergic receptor agonist isoprenaline in the presence or absence of selective ß-adrenergic receptor antagonists. The ß1-adrenergic receptor antagonist ICI 89406 or the ß2-adrenergic receptor antagonist ICI 118551 did not significantly shift the isoprenaline concentration-response curves (Figs. 3BGo and 4AGo). However, propranolol significantly shifted the isoprenaline concentration-response curve to the right at both 1 and 10 µM, giving pKB values of 7.1 ± 0.1 and 7.0 ± 0.2, respectively (Fig. 4Go, C and D). That 1 µM propranolol failed to shift the norepinephrine concentration-response curve (Fig. 3CGo) could perhaps indicate the presence of a small {alpha}1-adrenergic receptor component in the norepinephrine response. However, the pKB values nonetheless indicated that isoprenaline activation of glucose uptake is probably mediated through ß3-adrenergic receptors in mature mouse brown adipocytes in culture.



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  		FIG. 4. Concentration-response curve for isoprenaline plus 1 µM ICI-89406(A), isoprenaline plus 1 µM ICI 118551 (B), isoprenaline plus 1 µM propranolol (C), and isoprenaline plus 10 µM propranolol (D) on 2-deoxy-D-glucose uptake (2-DG) in cultured mouse brown adipocytes. Experiments were performed as described in legend to Fig. 2Go. The values are means ± SE. A, Isoprenaline ({bullet}-{bullet}) in absence and presence of 1 µM ICI-89406 ({circ}-{circ}) (n = 4 series in duplicate). B, Isoprenaline ({bullet}-{bullet}) in absence and presence of 1 µM ICI 118551 ({diamond}-{diamond}) (n = 3 series in duplicate). C, Isoprenaline ({bullet}-{bullet}) in absence and presence of 1 µM propranolol ({triangleup}-{triangleup}) (n = 3 series in duplicate). D, Isoprenaline ({bullet}-{bullet}) in absence and presence of 10 µM propranolol ({triangleup}-{triangleup}) (n = 4 series in duplicate). Vehicle-treated wells were set to 100% for each cell culture series and the other values given relative to this. The results were fitted with the general curve-fitting procedure for Michaelis-Menten kinetics.

 
ß3-adrenergic receptor agonists
The selective ß3-adrenergic receptor agonist BRL 37344 produced a significant increase in glucose uptake (Vmax 176 ± 16%; pEC50 8.6 ± 0.0) (Fig. 5AGo), as did the selective ß3-adrenergic receptor agonist, CL 316 243 (Vmax 176 ± 11%; pEC50 9.7 ± 0.1) (Fig. 5BGo). The specific ß3-agonist CGP 12177 [also a ß1- and ß2-adrenergic receptor antagonist (51)] increased glucose uptake (Vmax 196 ± 0%; pEC50 7.3 ± 0.1) (Fig. 5CGo). The ß3-adrenergic receptor agonists mimicked the norepinephrine response to a large extent.



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  		FIG. 5. Concentration-response curve for BRL 37344 (A), CL 316243 (B), and CGP 12177 (C) on 2-deoxy-D-glucose (2-DG) uptake in cultured mouse brown adipocytes. Experiments were performed as described in legend to Fig. 2Go. The values are means ± SE. A, BRL 37344 ({blacksquare}-{blacksquare}) (n = 3 series in duplicate). B, CL 316243 ({blacksquare}-{blacksquare}) (n = 4 series in duplicate). C, CGP 12177 ({blacksquare}-{blacksquare}) (n = 5 series in duplicate). Vehicle-treated wells were set to 100% for each cell culture series and the other values given relative to this. The results were fitted with the general curve-fitting procedure for Michaelis-Menten kinetics.

 
cAMP is the mediator of ß-adrenergic receptor stimulated glucose uptake
Our results showed that a major component of the norepinephrine-increased glucose transport is mediated through ß3-adrenergic receptors in mouse brown adipose cells. ß3-Adrenergic receptor stimulation leads to activation of Gs and subsequent activation of adenylyl cyclase to increase intracellular cAMP levels and PKA activity. Cholera toxin (2 µg/ml) (Gs protein activator) significantly increased 2- deoxy-glucose (338 ± 37%) uptake to a similar extent as insulin (306 ± 21%), and the cAMP analog 8-Br-cAMP (1 mM) increased 2-deoxy-glucose uptake more than 2-fold (228 ± 7%) (n = 3 series in duplicate), demonstrating that elevation of cAMP levels mimics adrenergic stimulation. Pertussis toxin pretreatment (0.2 µg/ml, 16 h) of cells did not significantly affect the norepinephrine-induced glucose uptake (n = 3 series in duplicate), indicating that Gi plays no role in activating glucose uptake in brown adipocytes.

To further investigate the role of cAMP formation in activation of glucose uptake, we blocked cAMP formation with DDA, which blocks adenylyl cyclase (52). DDA significantly inhibited ß-adrenergic receptor-activated cAMP formation to a very large extent. BRL 37344- and isoprenaline-activated cAMP formation was inhibited approximately 88 and 80%, respectively (Fig. 6AGo). Insulin did not cause any significant increase in cAMP levels (Fig. 6AGo). Parallel cultures were analyzed for 2-deoxy-D-glucose uptake. DDA significantly inhibited BRL 37344- and isoprenaline-mediated 2-deoxy-D-glucose uptake (approximately 64 and 70% inhibition) (Fig. 6BGo). This indicated that a large component (73–88% in these experiments) of the ß-adrenergic receptor-stimulated increase in glucose uptake in brown adipocytes is mediated through cAMP. Treatment with DDA did not affect insulin-stimulated 2-deoxy-D-glucose uptake.



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  		FIG. 6. Effects of adenylyl cyclase inhibitor DDA on cAMP formation on 2-deoxy-D-glucose (2-DG) uptake in cultured mouse brown adipocytes. The values are means ± SE for two series performed in quadruplicate in which half were analyzed for 2-deoxy-D-glucose (2-DG) uptake and the other half for cAMP levels. A, DDA (50 µM) was added 30 min before addition of agonist, BRL 37344 (1 µM), isoprenaline (1 µM), and insulin (1 µM). Vehicle-treated wells were set to 100% for each cell culture series and the other values given relative to this. B, The cultured cells were fixed with ethanol 10 min after agonist addition and the cAMP levels obtained as described in Material and Methods. The values are given as picomoles cAMP per 3.8 cm2 well (insulin, 9.6 cm2/well). *, P <= 0.05 and **, P <= 0.01 indicate differences compared with agonist.

 
PKA mediates the ß-adrenergic receptor signal to glucose uptake
The downstream mediator of cAMP is PKA. We inhibited PKA with a highly specific PKA inhibitor (4CM). Treatment with the PKA inhibitor clearly reduced isoprenaline- (approximately 52% inhibition) and 8-Br cAMP-mediated (approximately 68% inhibition) glucose uptake (Fig. 7AGo). Insulin-induced glucose uptake was not significantly reduced by PKA inhibition (Fig. 7AGo). This indicated that a large fraction of the ß-adrenergic receptor signal is indeed mediated through PKA activation. We tested another PKA inhibitor (H89), but this drug inhibited basal glucose uptake and had a detrimental effect on the cells (50 µM). We further examined the possibility that cAMP could activate glucose uptake through a pathway not involving PKA. In other tissues, there is evidence for at least one additional cAMP-activated pathway: the GTP-exchange protein Epac (53). Although 8-Br cAMP increased glucose uptake in a concentration-dependent manner, the Epac-specific activator 8-CPT (53) did not increase glucose uptake (Fig. 7BGo). We thus found no evidence that Epac could stimulate glucose uptake in these cells.



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  		FIG. 7. Effects of PKA inhibition on insulin- and isoprenaline- induced 2-deoxy-D-glucose (2-DG) uptake in cultured mouse brown adipocytes. The values are means ± SE for four series performed in duplicate and quadruplicate. A, 4CM (10 µM) was added 5 min before addition of agonist; insulin (1 µM), 8-Br-cAMP (1 mM), and isoprenaline (1 µM). Vehicle-treated wells were set to 100% for each cell culture series and the other values given relative to this. *, P <= 0.05 and **, P <= 0.01 indicate differences compared with agonist. B, Concentration-response curve for 8-Br-cAMP ({blacktriangleup}-{blacktriangleup}) and 8-CPT-2Me-cAMP ({bullet}-{bullet}). Vehicle-treated wells were set to 100% for each cell culture series and the other values given relative to this (n = 3 series in duplicate).

 
PI3K is necessary for ß-adrenergic receptor activation of glucose uptake
Insulin-mediated glucose uptake involves PI3K. We used a specific PI3K inhibitor (LY294002) to elucidate whether ß-adrenergic receptor signaling increases glucose uptake through this kinase. Basal glucose uptake levels were to some extent inhibited by PI3K inhibition (approximately 22% inhibition), indicating that PI3K could be constitutively active and regulate glucose uptake even under basal conditions. The ß-adrenergic receptor-mediated increase in glucose uptake were largely inhibited (approximately 65% inhibition) by LY294002. Furthermore, LY294002 blocked 8-Br cAMP-mediated glucose uptake to approximately the same extent (approximately 66% inhibition). Insulin-stimulated glucose uptake was inhibited by the PI3K inhibitor to a higher degree (approximately 80% inhibition) (Fig. 8AGo). To examine whether PI3K inhibition could influence cAMP formation, we measured cAMP levels. There was no inhibition of ß- adrenergic receptor-stimulated cAMP formation in the presence of LY294002 (Fig. 8BGo).



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  		FIG. 8. Effects of PI3K inhibition on insulin-, isoprenaline-, and 8-Br-cAMP-induced 2-deoxy-D-glucose (2-DG) uptake in cultured mouse brown adipocytes. The values are means ± SE for three to four series performed in duplicate-quadruplicate. LY294002 (10 µM) was added 5 min before agonist addition; insulin (1 µM), isoprenaline (1 µM), 8-Br-cAMP (1 mM). Vehicle-treated wells were set to 100% for each cell culture series and the other values given relative to this. *, P <= 0.05 and **, P <= 0.01 indicate differences compared with agonist.

 
ß-Adrenergic receptors activate glucose uptake in brown adipocytes through conventional and novel PKCs
There is evidence that implicates the involvement of PKCs in insulin-stimulated glucose uptake in skeletal muscle and fat (6). We investigated whether PKCs are involved in ß-adrenergic receptor-activated glucose uptake in brown adipocytes. We used Gö 6983, which inhibits conventional ({alpha}, ß, {gamma}), novel ({delta}), and atypical PKC ({zeta}) isoforms, and Ro-31-8220, which inhibits conventional ({alpha}, ß1, ß2, {gamma}) and novel ({epsilon}) but not atypical PKC isoforms (54, 55). The phorbol ester TPA stimulates conventional and novel PKCs. TPA activated glucose uptake in brown adipocytes to a large extent (394 ± 54%; 1 µM, data not shown). TPA-induced glucose uptake was largely inhibited by inhibition of conventional, novel, and atypical PKCs with Gö 6983 (73% inhibition) (data not shown). The insulin-mediated increase in glucose uptake was only partially inhibited by Gö 6983 (approximately 37% inhibition) (Fig. 9AGo). Interestingly, the ß-adrenergic receptor-mediated increase in glucose uptake was fully inhibited by Gö 6983 (approximately 93% inhibition), as was the increase in glucose uptake mediated by 8-Br-cAMP. This indicates that PKCs are very significant in ß-adrenergic receptor-induced glucose uptake and that there could be a difference in which PKCs are activated in insulin- and ß- adrenergic receptor-mediated glucose uptake. TPA-induced glucose uptake was fully inhibited by Ro-31-8220 in brown adipocytes (98% inhibition) (data not shown), confirming that TPA acts via conventional and novel PKCs. Surprisingly, insulin-stimulated glucose uptake was also inhibited (63% inhibition) (Fig. 9BGo) by blocking conventional and novel PKCs with Ro-31-8220, showing that insulin signaling in brown adipocytes may be through PKCs other than atypical PKCs ({zeta}). Isoprenaline- and 8-Br cAMP- induced glucose uptake were almost fully inhibited, to approximately 80–91% by Ro-31-8220. This indicates that conventional and novel PKCs are important in ß-adrenergic receptor-induced glucose uptake.



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  		FIG. 9. Effects of PKC inhibition on insulin-, isoprenaline-, and 8-Br-cAMP-induced 2-deoxy-D-glucose (2-DG) uptake in cultured mouse brown adipocytes. The values are means ± SE for three to four series performed in duplicate. A, Ro 31–8220 (5 µM) was added 5 min before agonist addition; insulin (1 µM), isoprenaline (1 µM), 8-Br-cAMP (1 mM). B, Gö 6983 (5 µM) was added 5 min before agonist addition; insulin (1 µM), isoprenaline (1 µM), 8-Br-cAMP (1 mM). Vehicle-treated wells were set to 100% for each cell culture series and the other values given relative to this. *, P <= 0.05 and **, P <= 0.01 indicate differences compared with agonist.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
In the present study, we investigated norepinephrine- induced activation of glucose uptake in brown adipocytes grown in culture because these cells exhibit a highly differentiated and well-characterized phenotype (15, 21, 26, 29). We found that norepinephrine elevates glucose transport mainly through ß3-adrenergic receptor activation. Thus, there could be a significant difference between adrenergic receptor signaling in brown fat and white fat because {alpha}1-adrenergic receptors play a significant role in stimulating glucose uptake in white adipocytes (56, 57). Our results indicate that {alpha}1-adrenergic receptors play only a very small role in increasing glucose uptake in brown adipocytes with endogenously expressed adrenergic receptors. With the use of PTX, we did not find any evidence for an {alpha}2- or ß-adrenergic receptor coupling to Gi and glucose uptake. The ß3-adrenergic receptor pathway was mediated via an increase in cAMP levels and activation of PKA. Furthermore, PI3K and PKCs were involved in the signaling event leading to increase in glucose uptake (Fig. 10Go).



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  		FIG. 10. Suggested model for the norepinephrine induced 2-deoxy-D-glucose uptake in brown adipocytes. For explanation, see text. NE, Norepinephrine; AC, adenylyl cyclase; {alpha}1, {alpha}1-adrenergic receptor; {alpha}2, {alpha}2-adrenergic receptor; ß3, ß3-adrenergic receptor.

 
ß3-Adrenoceptors activate glucose uptake
In this study, we were able to mimic the maximal norepinephrine-induced glucose uptake with a ß-receptor agonist, indicating that most of the norepinephrine signal is mediated by ß-adrenergic receptors. ß1- or ß2-adrenergic receptor antagonists did not block norepinephrine- or ß- agonist-induced glucose uptake, showing that ß1- or ß2-adrenergic receptors are not involved in norepinephrine- or isoprenaline-induced glucose uptake. This is in accordance with our earlier studies in which ß1-adrenergic receptors, even though they are expressed, are coupled to cAMP formation only in brown preadipocytes and not in mature brown adipocytes in culture (29). Even though BAT expresses ß2- adrenergic receptors (probably mainly on blood vessels), there is no ß2-adrenergic receptor gene expression in nonstimulated mature brown adipocytes in culture (29). In addition, we showed that three different ß3-adrenergic receptor agonists activated glucose uptake to approximately the same extent as did a nonselective ß-adrenergic receptor agonist. It is therefore most likely that ß3-adrenergic receptors mediate most, if not all, of the norepinephrine signal.

ß3-Adrenoceptor atypical signaling?
It is plausible that ß3-adrenergic receptors can activate glucose transport through at least three pathways: 1) classical Gs activation; 2) interaction with other G proteins, e.g. Gi (the release of Gß{gamma}) or Gq; or 3) a direct receptor interaction with an atypical signaling molecule. It has been appreciated that ß-adrenergic receptors (ß2- and ß3-adrenergic receptors) may couple not only to Gs but also to Gi (23, 58, 59, 60, 61). Concerning the ß2-adrenergic receptor, a switch in ß2-adrenergic receptor coupling from Gs to Gi is induced by PKA- and G-protein coupled kinase-dependent phosphorylation of the third intracellular loop and C-terminal tail region after agonist stimulation (58). This mechanism may not be responsible for the ability of the ß3-adrenergic receptor to couple to Gi because ß3-adrenergic receptors lack many of the sites in the intracellular domains for phosphorylation by PKA and G-protein coupled kinase. 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 (see references in Ref. 62).

Glucose uptake stimulated by ß2-adrenergic receptors may signal through the Gß/{gamma} subunit because it has been shown that the ß/{gamma}-subunit can activate PI3K{gamma} under certain conditions (63) and Gq and Gß{gamma} (dissociated from Gi) are involved in stimulated glucose transport in adipocytes (64, 65, 66). ß3-Adrenergic receptor signaling 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 (67). Splicing of the first intron results in a ß3-adrenergic receptor variant, termed the ß3B-adrenergic receptor, which has a unique C-terminal tail that differs from the known ß3A-adrenergic receptor. In BAT, the ß3A-adrenergic receptor variant is the most abundant (67). Our earlier results show that in transfected CHO-K1 cells, the ß3A- adrenergic receptor couples solely to Gs to increase cAMP levels, whereas the ß3B-adrenergic receptor couples to both Gs and Gi to stimulate and inhibit cAMP, respectively (61). Both splice variants increase Erk1/2 phosphorylation via a mechanism independent of their effect on Gi, Gs, or cAMP levels (61). This indicates that the ß3-adrenergic receptor may mediate cellular responses via different signaling pathways in different cell systems [coupling of the ß3-adrenergic receptor to Erk1/2 differs in different cell backgrounds (23, 59, 60)] but also possibly through other undefined signaling pathways. Functionally, the ß3-adrenergic receptor has been demonstrated to couple to Gi in immortalized cell systems (59, 60) but not in brown fat primary cultures (23). This is consistent with our study, in which ß3-adrenergic receptor-mediated activation of glucose uptake was not affected by PTX treatment.

ß3-Adrenoceptors stimulate glucose uptake through cAMP
We mimicked the norepinephrine-mediated glucose uptake response with cholera toxin (an established Gs activator). The cell-permeable cAMP analog 8-Br cAMP was also able to mimic the norepinephrine effect. It is therefore plausible that norepinephrine stimulates glucose uptake through the classical Gs{alpha} pathway.

Addition of 8-Br cAMP to the cells raised cAMP levels (8-Br cAMP) to higher values than isoprenaline. However, 8-Br cAMP and isoprenaline gave the same response in glucose uptake in the parallel experiments (Fig. 8AGo). This could indicate that 8-Br cAMP and cAMP have different affinity for their target in the assay system or that endogenous local concentrations of cAMP regulate targets in specific microdomains throughout the cell (68). It is also possible that the cAMP levels reached by isoprenaline saturate the system.

It is not fully established which of the 10 known adenylyl cyclase isoforms mediates the ß-adrenergic receptor signal in BAT (69, 70), but it is possible to inhibit all known isoforms of mammalian adenylyl cyclases by 3'-nucleotides in a domain referred to as the P-site (52). By blocking this site with DDA, we concluded that cAMP formation is very important and necessary in ß3-adrenergic receptor-activated glucose uptake in brown adipocytes. The observation that we could not totally block cAMP formation with DDA could reflect the fact that different adenylyl cyclase isoforms are more difficult to inhibit than others (52) or that the concentration used was too low. Because most of the glucose uptake and the cAMP response was inhibited to the same degree, we believe that if an additional pathway(s) exists, it is probably of minor importance in stimulating glucose uptake in these cells. Our results show the first evidence that inhibition of cAMP elevation blocks ß-adrenergic receptor-stimulated glucose transport in brown adipocytes. This is very interesting because cAMP elevation has been shown to inhibit glucose transport in other cell types (71, 72) but clearly not in these cells.

The ß3-adrenoceptor-induced glucose uptake is mediated via PKA
ß3-Adrenergic receptor-mediated increases in glucose uptake were mediated via PKA activation, as evidenced by the ability of the PKA inhibitor 4CM to inhibit this increase. Because we did not inhibit the response totally (even though we used a rather high concentration of 4CM), this could be because a part of the signal is through another pathway not involving PKA or not all of the PKA activity was inhibited. In other tissues, there is evidence for cAMP/PKA-independent pathways: the GTP-exchange protein Epac and cyclic-nucleotide-gated cation channels (73, 74). We could not find any evidence for an Epac pathway to activate glucose uptake using an Epac-specific cAMP analog (53), and there was no effect of the Ca2+ ionophore A23187 (data not shown) on glucose uptake, making it unlikely that cation channels have a stimulatory effect on glucose uptake. Although it has been discussed whether or not pathways other than PKA are activated by ß3-adrenergic receptor stimulation in brown adipocytes (23, 75, 76), we find it most likely that PKA activation is not only a possible but also an essential step in ß-adrenergic receptor-mediated glucose uptake in brown adipocytes.

ß3-Adrenoceptor and insulin pathways converge at PI3K
Because PI3K is crucial in the insulin-signaling pathway, we examined whether this kinase was involved in norepinephrine-induced glucose transport, using the established PI3K inhibitor LY294002 (77, 78). ß3-Adrenergic receptor-mediated increases in glucose uptake were almost fully inhibited by this specific PI3K inhibition. This result is not in agreement with earlier studies performed in rat brown adipocytes, in which it was indicated that PI3K plays a critical role in insulin-induced translocation of GLUT4 but not in norepinephrine-increased glucose transport (41). The discrepancy could be due to the selectivity of different PI3K blockers for other protein kinases (79) or that other mechanisms in addition to PI3K-mediated glucose uptake occur in other systems. The mechanism by which G protein-coupled adrenergic receptors activate PI3K is unknown, but it has earlier been postulated to involve the activation of distinct isoforms of this kinase by Gß{gamma}-subunits from Gi proteins. Glucose uptake stimulated by ß2-adrenergic receptors in other systems may signal through the Gß/{gamma}-subunit because it has been shown that the Gß/{gamma}-subunit can activate the PI3K {gamma}-isoform under certain conditions (63). Because our results showed that ß3-adrenergic receptor-mediated increase in glucose uptake is mediated through cAMP, we examined whether PI3K inhibition could block a cAMP analog-induced glucose uptake. Surprisingly, glucose uptake mediated by a cAMP analog (8-Br-cAMP) was blocked with PI3K inhibition (LY 294002). It is therefore plausible to suggest that most (or all) of the ß3-adrenergic receptor signal stimulating glucose uptake is through cAMP/PKA and activation of PI3K and is independent of Gß/{gamma}-subunits. There was no evidence indicating a cAMP/PKA-independent pathway for glucose uptake.

Conventional and novel PKCs are involved in the ß3-adrenoceptor-mediated increase in glucose uptake
We hypothesized that it is likely that after activation of PI3K, the ß3-adrenergic receptor pathway uses the same kinases as the insulin pathway. Both insulin and phorbol ester TPA stimulate conventional and novel PKCs. However, atypical PKC{zeta} is stimulated by insulin but not by TPA (80). The atypical PKC isoforms, PKC{zeta} and PKC{lambda}, are involved in insulin-stimulated glucose uptake in adipocytes (81, 82, 83, 84), whereas conventional PKCs ({alpha}, ß1, ß2, and {gamma},) and novel PKCs ({delta}, {epsilon}, {eta},and {theta}) do not appear to be required for insulin-stimulated glucose transport in white adipocytes (81, 83). Phorbol esters induce glucose uptake mainly through conventional PKCs (84). TPA is a very potent activator of glucose transport in brown adipocytes (data not shown). This activation can be largely blocked with Gö 698, which inhibits conventional, novel, and atypical PKCs. Insulin-mediated glucose uptake was only partially inhibited by Gö 6983. These results indicate that PKCs could be involved in the insulin-signaling pathway.

That the insulin-stimulated uptake was only partially inhibited could be explained if the small GTP-binding protein TC10, which is also involved in glucose transporter translocation by insulin, were active. The TC10 pathway functions in parallel with the PI3K pathway to stimulate glucose transporter translocation but probably does not activate the same kinases (85). Isoprenaline- or 8-Br cAMP-stimulated glucose uptake was fully inhibited by inhibition of PKCs (Gö 6983). Furthermore, the TPA-induced glucose transport was fully inhibited by Ro-31–8220, which is a potent inhibitor of conventional and novel PKCs but inhibits PKC{zeta} only at relatively high concentrations (81). These results confirm that TPA activates glucose uptake mainly through conventional and novel PKCs in brown adipocytes. The insulin response was only partially inhibited with Ro-31–8220, indicating that conventional or novel PKCs could to some extent be involved in insulin stimulation of glucose uptake in brown adipocytes. However, the Ro-31–8220 concentration used (5 µM) slightly inhibit insulin-induced glucose uptake in rat white adipocytes through inhibition of PKC{zeta}, which would indicate that this could be the case also in brown adipocytes (81). Interestingly, the isoprenaline-induced glucose uptake was almost fully inhibited and the 8-Br cAMP-stimulated uptake was completely inhibited by Ro-31–8220, indicating that conventional and novel PKCs are very important in ß3-adrenergic receptor-induced glucose uptake. Further studies would be needed to delineate whether these conventional and novel PKCs can be activated directly by ß3-adrenergic receptors. However, it is generally accepted that PKCs are downstream effectors of PI3K (81, 82, 83, 86). It is therefore likely that these conventional and novel PKCs are downstream of PI3K and not directly activated by elevated cAMP levels.

The mechanism for adrenergic- and insulin-stimulated glucose uptake
The mechanism for insulin-induced glucose uptake in brown adipocytes is similar to that in other tissues such as skeletal muscle. Insulin induces the translocation of GLUT4 from intracellular stores to the plasma membrane (9, 37, 87). There is some translocation of GLUT1 in response to insulin in isolated mature brown adipocytes (37), but it is unclear how significant this is to the total insulin-induced glucose transport. Earlier studies have indicated that the mechanism by which norepinephrine stimulates glucose transport in brown adipocytes is through a different mechanism from that of insulin (38). It was proposed that norepinephrine stimulates glucose transport in brown adipocytes by enhancing the functional activity of GLUT1 by a cAMP-dependent mechanism (8). Our results indicate that most of the norepinephrine signal is through the classical insulin pathway from PI3K, and thus translocation of GLUT4 would be most probable. However, it is possible that, in addition, norepinephrine and cAMP also affect GLUT1 and thus reflect an additional pathway in norepinephrine-stimulated glucose transport. Investigating the GLUT isoforms needed for insulin- and norepinephrine-activated glucose uptake is interesting, and further studies in this area are needed.


   Conclusions
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
We put forward the hypothesis that norepinephrine- induced glucose uptake in brown adipocytes is mainly through ß3-adrenergic receptors. The ß3-adrenergic receptor activation of glucose uptake is through activation of Gs, activation of adenylyl cyclase, the classical second messenger cAMP and activation of PKA. Activated PKA, through a mechanism not yet clarified and perhaps via additional signaling molecules, although not Gi, stimulates PI3K. This activation leads to stimulation of PKCs and subsequently activation of glucose transport (Fig. 10Go). In brown adipocytes, both conventional and unconventional PKCs play a major role in ß3-adrenergic receptor-activated glucose transport. It is likely that that ß3-adrenergic receptor activation downstream of PI3K leads to the same signal transduction pathway stimulated by insulin in elevating glucose transport.


   Acknowledgments
 
We thank Dr. Dana Hutchinson for valuable discussions and helpful comments on the manuscript and Valeria Golozoubova for the large series of protein measurements on brown adipocytes in culture.


   Footnotes
 
This work was supported by research grants from the Swedish Science Research Council, Magn. Bergvalls stiftelse, Jeanssons stiftelser, and Tore Nilssons stiftelse.

Abbreviations: BAT, Brown adipose tissue; 8-Br-cAMP, 8-bromoadenosine-cAMP; 4CM, 4-cyano-3-methylisoquinoline; 8-CPT, 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclicmonophosphate sodium salt; DDA, 2',5'-dideoxyadenosine; 2-DG, 2-deoxy-D-glucose; GLUT, glucose transporter; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; pKB, log(dr - 1) - log[antagonist], where dr = dose ratio; PKC, protein kinase C; TPA, phorbol 12-myristate 13-acetate; UCP-1, uncoupling protein-1.

Received July 10, 2003.

Accepted for publication September 25, 2003.


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