Differentiation-dependent inhibition of proteolysis by norepinephrine in brown adipocytes

M. Desautels and S. Heal

Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The objective was to evaluate whether norepinephrine (NE) and other hormonal factors have direct effects on protein degradation in brown fat cells. NE inhibited proteolysis by 35-45% in mouse brown adipocytes differentiated in culture. Insulin also inhibited protein degradation but significantly less than NE, whereas glucagon and leptin had no effect. The inhibitory effect of NE was partially antagonized by propranolol but not by prazosin, and dose-response curves with BRL-37344 (a beta 3-agonist), isoproterenol (a beta 1/beta 2-agonist) and dobutamide (a beta 1-agonist) were consistent with the involvement of a beta 3-adrenergic receptor. Furthermore, forskolin mimicked the effects of NE, whereas additions of A-23187 or phorbol esters had no effect, alone or in combination with NE or forskolin. Thus inhibition of proteolysis by NE likely involves a beta 3-adrenergic receptor-mediated increase in cAMP. In contrast, NE, BRL-37344, and dobutamide had no effect on proteolysis in preadipocytes. Inhibition of proteolysis by NE was due at least in part to inhibition of autophagy. Thus inhibition of proteolysis by NE and insulin in mature brown adipocytes is likely an important process contributing to brown fat growth and atrophy under many physiological or pathological conditions.

brown fat; insulin; autophagy; beta 3-adrenergic receptor; lysosomes; protein turnover


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BROWN ADIPOSE TISSUE (BAT) is a sympathetically innervated heat-producing tissue involved in the control of energy homeostasis and body temperature (24, 25). Its capacity to produce heat is dependent on specialized mitochondria with a regulated uncoupling protein (UCP) pathway that dissipates as heat the energy normally conserved as ATP on oxidation of metabolic substrates (7, 25, 34). The capacity of BAT for thermogenesis varies with immediate or anticipated needs for energy dissipation. Growth and atrophy of the tissue is marked and rapid and occurs usually in synchrony with activation or suppression of sympathetic input to BAT (25). Indeed, norepinephrine not only stimulates metabolic activity but also promotes proliferation and differentiation of preadipocytes to the terminally differentiated phenotype (7).

Changes in BAT thermogenic capacity result from alterations in cell number as well as cell content of mitochondrial proteins and UCP-1 (7, 25, 34). The thermogenic contribution of two other mitochondrial UCP homologs, UCP-2 and UCP-3, expressed in brown fat and other tissues, is yet uncertain (4, 18, 40). Obviously, cellular hypertrophy/atrophy must result from changes in rates of protein synthesis and/or degradation. In mammalian cells, several proteolytic pathways are responsible for the degradation of intracellular proteins under basal conditions, with some becoming more active under certain physiological or pathological conditions. For instance, autophagy is an important route of delivery of cell constituents to lysosomes and is regulated by nutrients and hormones, allowing cells to match their degradative status to the environmental conditions (3, 16, 36). In contrast, nonlysosomal proteolysis as found in the cell cytosol or within certain organelles, such as mitochondria or endoplasmic reticulum, appears responsible for the breakdown of proteins with short half-lives, like some transcription factors, key enzymes in metabolic pathways, or proteins with structural damage from mutations or posttranslational modifications (20, 23, 27). In contrast to other organs such as liver or muscles, there is very little known about what controls proteolysis in brown fat cells.

Mice without food show marked BAT atrophy, characterized primarily by bulk loss of mitochondrial proteins without change in tissue cellularity (10, 14, 30). After some delay, loss of UCP-1 from the mitochondria is also observed (10, 30). At least two pathways are likely involved in the enhanced intracellular proteolysis occurring in the brown fat cells of fasting mice, an autophagic process and a change in proteolysis within mitochondria. BAT has a large complement of lysosomal enzymes, and the tissue atrophy can be reduced in vivo with blockers of lysosomal function (10). Proteolysis within mitochondria likely contributes also to fasting-induced tissue atrophy, because BAT mitochondria contain protease activity (11, 12) that may be involved in the selective loss of UCP-1 from the mitochondria (13) and the reduction in the apparent abundance of cristae (8). The neuroendocrine factors responsible for fasting-induced BAT atrophy are poorly defined. Food deprivation is associated with a suppression of sympathetic activity to BAT, reductions in circulating insulin and leptin levels, and an increase in circulating glucagon (24, 25, 29, 37). All of these are known to alter the thermogenic activity and/or capacity of the tissue by direct actions on brown adipocytes and/or modification of sympathetic activity to BAT (7, 21, 25, 35, 38). The observation that denervation of BAT mimics the tissue atrophy observed in fasting mice suggests that norepinephrine (NE) may exert an inhibitory influence on proteolysis (10, 14). However, there are currently no direct estimates of the effects of NE and other factors controlling the tissue thermogenic capacity on overall rates of protein degradation in brown fat cells.

The objective of this work was to evaluate whether NE and other hormonal factors directly influence proteolysis in brown fat cells differentiated in culture.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of preadipocytes and cell cultures. The isolation, culture, and differentiation of brown preadipocytes were carried out as described previously (31, 32). Newborn mice (CD1 strain, 2-4 days old) were killed by decapitation, and the interscapular BAT was removed under sterile conditions and cut into small pieces with a scalpel blade. The tissue was digested for 30 min at 37°C under a 95% O2-5% CO2 gas phase (vortex shaking every 2.5 min) with 1 mg/ml collagenase in DMEM (GIBCO) supplemented with 33 µM biotin, 17 µM pantothenic acid, 100 µM ascorbic acid, 100 µg/ml streptomycin, 6 µg/ml penicillin G, and 4% (wt/vol) bovine serum albumin. The digestion mixture was filtered through Nitex mesh (Thompson, 243 µM) into a sterile 50-ml centrifugation tube, rinsed with 30 ml of media, and allowed to stand at room temperature for 20 min. After centrifugation (800 g, 5 min), the top layer and supernatant phase containing mature brown fat cells were discarded, and the pellet was resuspended in 40-50 ml of culture media.

Cells were inoculated in 35-mm Falcon culture dishes and incubated in a CO2 incubator (5% CO2-95% air) at 37°C and 100% relative humidity. After 1-2 h, the media were aspirated off, and fresh culture media were added. The standard culture media consisted of DMEM with antibiotics, biotin, pantothenic acid, ascorbic acid, and 10% (vol/vol) fetal bovine serum (ICN, CELLect-Silver) and were replaced every 2nd to 3rd day. Near or at confluence (day 5), 3,5,3'-triiodothyronine (T3; 1 nM) and insulin (50 nM) were added to the culture media, and cells were allowed to differentiate until day 10. Differentiation to brown adipocytes was followed by the appearance of fat vacuoles by light microscopy and of UCP-1 by immunofluorescence microscopy and Western blots (12).

Cell protein labeling and measurements of protein degradation. Standard culture media were replaced by culture media with reduced methionine content made of 10% (vol/vol) fetal bovine serum, 10% (vol/vol) DMEM, and 80% (vol/vol) methionine-free DMEM. Supplementation with vitamins and antibiotics was as before. [35S]methionine was added at 10 µCi/ml. After 3 days, cell monolayers were washed 3 times with 2 ml of DMEM + 2 mM unlabeled methionine and were incubated with 2.5 ml of DMEM + 2 mM unlabeled methionine and other additions, as will be detailed in captions to Figs. 1-6. At time 0 and various times after addition, 0.25 ml of culture media was collected into microcentrifuge tubes and mixed with 0.035 ml trichloroacetic acid (TCA, 100% wt/vol) and 0.015 ml BSA (5% wt/vol). After >= 1 h at 4°C, samples were centrifuged, and 0.15 ml of acid-soluble supernatant was added to 4 ml of Amersham PCS scintillator fluid and counted. Protein degradation was measured as the release of acid-soluble radioactivity from the cells into the cell culture media per unit time after subtraction of residual acid-soluble radioactivity at time 0. Specific activity of labeled cell proteins was estimated at time 0 after cell collection and disruption by sonication into 1 ml TCA (10% wt/vol) + 5 mM methionine (TCA+Met). An aliquot (50 µl) was collected by filtration (Whatmann GF/C glass fiber filters), washed 5 times with 5 ml TCA+Met, and counted, while another aliquot was centrifuged, solubilized in 0.5 N NaOH, and used for protein estimation by the method of Bradford (5).

Statistical analysis. Differences between groups were examined by one-way and two-way ANOVA and Duncan's multiple range test for comparisons between means. NCSS statistical software (Kaysville, UT) was used for computation. The results were considered statistically significant when P < 0.05. Results are presented as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Norepinephrine and insulin inhibit protein degradation in brown adipocytes differentiated in culture. Our cell cultures had the pattern of differentiation typically observed by others (31, 32). Preadipocytes grew rapidly with a fibroblast-like appearance until confluence at about day 5 and thereafter started to round up and fill with lipid droplets. By day 10, almost all cells had the appearance of mature brown adipocytes. These cells expressed UCP-1, as visualized by immunofluorescence microscopy with an antiserum specific to UCP-1 (data not shown). Western blots showed no expression of UCP-1 on day 5, some expression on day 7, and maximal expression on day 10 (see Fig. 2A). Thus our brown fat cells did acquire the terminal differentiation phenotype typical of mature brown adipocytes.

A pulse-chase approach was then used to measure rates of proteolysis. Cells were labeled with [35S]methionine over 72 h to label all cell proteins uniformly. Typically, the specific activity of labeled proteins was 30,000-40,000 counts · min-1 · µg protein-1, with 290 ± 11 µg proteins/culture dish. The protein content per culture dish was similar within and between experiments. Labeling conditions (reduced methionine content of the culture media) between days 7 and 10 did not affect the apparent differentiation of the cells. After three washes to remove the labeled amino acid, the residual radioactivity in the supernatant was <0.02% of acid-precipitable radioactivity at time 0. Release of acid-soluble radioactivity from the cells into the culture media over time in the presence of 2 mM unlabeled methionine (to minimize reincorporation of labeled precursors) was then used to estimate rates of protein degradation. In DMEM without serum or hormonal additions, release of acid-soluble radioactivity was linear over a 6-h period (r2 = 0.64-0.75) and averaged 5.9 ± 1.6 µg proteins degraded · h-1 · culture dish-1 (Fig. 1). The extent of proteolysis represents ~2% of the labeled cell proteins turning over per hour. Addition of 1 µM NE reduced protein degradation significantly (Fig. 1). However, inhibition of proteolysis by NE was observed only in fully differentiated brown fat cells (day 10 cultures, Fig. 2B), with no effect of NE in preadipocytes (day 3 and day 5 cultures). There was some inhibition of proteolysis by NE in day 7 cultures, in keeping with some differentiation of the cells (low expression of UCP-1; Fig. 2A) that was not significant. Addition of insulin also inhibited protein degradation but was less effective than NE (Fig. 1). The combination of insulin and NE did not reduce protein degradation any more than NE alone. Both NE and insulin were effective inhibitors of proteolysis, with maximal inhibition observed at concentrations of 50 nM for insulin and 100 nM for NE (Fig. 3). Two other hormones, glucagon and leptin, had no effect on proteolysis (Fig. 3).


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Fig. 1.   Insulin and norepinephrine (NE) inhibit protein degradation. Brown adipocytes were labeled with [35S]methionine for 72 h (days 7-10), washed 3× in DMEM + 2 mM unlabeled methionine, and then incubated without addition (open circle ), with 50 nM insulin (), with 1 µM NE (), or with both insulin and NE (). Protein degradation was measured as described in MATERIALS AND METHODS, and results are expressed as µg of labeled proteins released from cells as acid-soluble products over 0-6 h. Results are means ± SE, with n = 3 separate cell cultures. Analysis of variance indicates significant accumulation of labeled material in the culture medium over time [F(2,72) = 57; P < 0.0001] and a significant effect of insulin [F(1,72) = 5; P < 0.04] and NE [F(1,72) = 29; P < 0.0001].



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Fig. 2.   Effect of differentiation on inhibition of proteolysis by NE. A: expression of uncoupling protein-1 (UCP-1) as visualized by Western blots after 5, 7, and 10 days in culture. Ten micrograms of cell proteins were separated by SDS-PAGE, followed by immunoblotting with a polyclonal antibody against rat UCP-1 and detection by enhanced chemoluminescence (13). B: proteolysis was measured in cells in culture for 3, 5, 7, and 10 days incubated with (solid bar) or without (open bar) 1 µM NE. Results are expressed as µg labeled proteins degraded to acid-soluble products in 4 h (left) and as % of control [(proteolysis + NE/proteolysis - NE) × 100; right] for each time point. A significant effect of NE [F(3,11) = 14, * P < 0.002; n = 3] was observed on day 10 only.



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Fig. 3.   Effects of increasing concentrations of NE, insulin, glucagon, and leptin on protein degradation. Protein degradation in brown adipocytes (day 10 cultures) without addition was 54.5 ± 6.4 µg labeled proteins released as acid-soluble products in 4 h (control, 100%). Significant reductions in proteolysis were observed with increasing concentrations of NE [triangle ; F(6,21) = 3, P < 0.04] and insulin [; F(9,30) = 11, P < 0.001] but not with glucagon [; F(4,9) = 0.1, P < 1.0] and leptin [open circle ; F(4,9) = 0.1, P < 1.0]. Results are means ± SE, with n = 2-5 separate cell cultures.

A beta 3-adrenergic receptor-mediated increase in cAMP inhibits proteolysis. In day 10 cultures, additions of BRL-37344 (a selective beta 3-agonist), isoproterenol (a beta 1/beta 2-agonist), and dobutamide (a selective beta 1-agonist) inhibited proteolysis with a relative potency of BRL-37344 > isoproterenol > dobutamide (Fig. 4A). The apparent EC50 values were, for BRL-37344, 3.9 nM, for isoproterenol, 6.4 nM, and for dobutamide, 100 nM. Addition of propranolol, a beta -adrenergic antagonist, significantly prevented the reduction in protein degradation caused by 0.1 µM NE (Fig. 4B). However, even at a concentration 100 times that of NE, propranolol reduced the effect of NE by only ~60%. Addition of prazosin, a alpha 1-adrenergic receptor antagonist, had no effect on NE-dependent inhibition of proteolysis (Fig. 4B). In keeping with the lack of effect of NE in preadipocytes (Fig. 3), neither BRL-37344 nor dobutamide had any effect in day 4 cultures (data not shown). Thus the inhibitory effect of NE is predominantly a beta 3-adrenergic-mediated process, known to be linked to activation of adenylate cyclase (43). Accordingly, addition of forskolin, which activates adenylate cyclase, inhibited protein degradation to the same extent as NE (Fig. 5). In contrast, additions of A-23187, a calcium ionophore, or phorbol 12-myristate-13-acetate (PMA), a phorbol ester known to activate kinase C, had no effect. When A-23187 or PMA was added, together with either NE or forskolin, there were no additive effects, suggesting that increased cAMP levels by NE or forskolin are sufficient to inhibit proteolysis. Concentrations of forskolin, A-23187, and phorbol esters in this study were similar to or slightly higher than those used by others in brown adipocytes (39, 43).


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Fig. 4.   Effects of adrenergic agonists (A) and antagonists (B) on NE-dependent inhibition of protein degradation. A: brown adipocytes (day 10 cultures) were incubated with increasing concentrations of BRL-37344 (), isoproterenol (), or dobutamide (open circle ) for up to 4 h, and rates of proteolysis were measured as described in MATERIALS AND METHODS. Proteolysis in the absence of drugs was 65.4 ± 3.1 µg labeled proteins released as acid-soluble products in 4 h (Control = 100%). Results are means ± SE, with n = 3 separate cell cultures. There were significant differences among drugs [F(3,215) = 4; P < 0.01] and among concentrations [F(12,215) = 6; P < 0.0001]. B: brown adipocytes were incubated for 4 h with 0.1 µM NE in the presence of increasing concentrations of propranolol () or prazosin (open circle ). Proteolysis was 56.3 ± 4.2 µg labeled proteins degraded to acid-soluble products in 4 h in the absence of NE (control, 100%) and 33.8 ± 2.4 with 0.1 µM NE. Results are means ± SE, with n = 7 separate cell cultures. There was a significant effect of propranolol at concentrations of 5, 10, and 25 µM (P < 0.05) but not with prazosin.



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Fig. 5.   NE inhibition of protein degradation is mediated by a cAMP-dependent process. Brown adipocytes were incubated for 4 h in the presence of forskolin (10 µM), A-23187 (5 µM), and phorbol esters [phorbol 12-myristate-13-acetate (PMA), 5 µM] alone or in combination with NE (1 µM) or forskolin (10 µM). Results (µg labeled proteins degraded to acid-soluble products in 4 h) are shown as means ± SE, with n = 3-6 separate cell cultures. * Significant difference (P < 0.05) relative to cell cultures without addition.

Inhibition of proteolysis by NE is mediated at least in part by inhibition of autophagy. The effect of NE was mimicked by the addition of 3-methyladenine (3-MA), a widely used inhibitor of autophagy (3, 15, 22, 36), at concentrations of 1-5 mM (Table 1). Because recent work in hepatocytes suggests that 3-MA exerts its action from inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) (2, 3), we tested also two structurally unrelated inhibitors of PI 3-kinase, wortmannin and LY-294002 (Table 1). They inhibited proteolysis in brown fat cells, as did the inhibition observed with 3-MA. The doses of wortmannin and LY-294002 were the lowest doses causing maximal inhibition of proteolysis (dose-response curves not shown) and are consistent with doses required to inhibit PI 3-kinase (2). Addition of chloroquine, an acidotropic agent that raises the pH of acidic compartments such as lysosomes (14, 16, 22), and leupeptin, a thiol protease inhibitor that inactivates cathepsins B, H, and L also present in BAT (14), inhibited proteolysis markedly, chloroquine more effectively than leupeptin at the concentrations used (Table 1). Addition of NE, together with inhibitors of autophagy and lysosomal function, did reduce proteolysis somewhat further, but the differences did not reach statistical significance (Table 1).

                              
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Table 1.   Effects of inhibitors of autophagy and lysosomal function on protein degradation in brown adipocytes

In a second set of experiments, we made use of the observations that proteins with short half-lives are not broken down primarily by autophagy (20, 23), and thus the inhibitory effects of NE should be sensitive to the length of the labeling period in a pulse-chase experiment. In mature brown adipocytes under basal conditions, reducing the period of labeling to 1 h is expected to increase the relative proportion of the total labeled protein pool made of proteins with short half-lives. Such proteins are degraded mainly by proteolytic systems present in the cytosol or within organelles (20, 23). After 72 h of labeling, all proteins are uniformly labeled, and proteins with short half-lives should constitute a relatively smaller fraction of the total labeled protein pool. Thus, if NE inhibits some aspect of autophagy, one would expect a greater inhibition after 72 h of labeling than after 1 h, which is exactly what was observed (Fig. 6).


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Fig. 6.   Effects of reducing labeling period on subsequent inhibition of protein degradation by NE. Brown adipocytes were incubated with [35S]methionine in methionine-free DMEM for 1 h. After removal of labeled precursor, measurement of protein degradation was carried out with (open circle ) or without () NE (1 µM) as described in MATERIALS AND METHODS. [35S]methionine incorporation after 1 h was 2,448 ± 305 counts · min-1 · µg protein-1 with n = 3 separate cell cultures. There was significant release of acid-soluble radioactivity over 4 h [F(3,72) = 115, P < 0.0001] that was reduced by NE [F(3,72) = 7.5, P < 0.001]. Inset, %inhibition of protein degradation by NE over 4 h in cells previously labeled with radioactive methionine for 1 or 72 h [F(1,15) = 6.4, P < 0.03].


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There are very few studies that examine the control of proteolysis in adipocytes in general and brown fat cells in particular. This is a first survey of what controls proteolysis in brown adipocytes. Brown fat cells differentiated in culture had a high rate of protein turnover (~2%/h; Fig. 1), consistent with the net protein loss observed by others when brown fat cells were incubated with cycloheximide to block protein synthesis (31). This proteolysis has both an autophagic and a nonautophagic component, as shown by partial inhibition with blockers of autophagy and lysosomal function (Table 1). For instance, 3-MA is an effective autophagy inhibitor in all mammalian cell types (3, 16, 22, 36) and reduced proteolysis by 40-45% in brown fat cells (Table 1). However, because 3-MA was shown recently to inhibit PI 3-kinase, likely underlying its mechanism of action as an inhibitor of autophagy (2, 3), it may have numerous other effects. In hepatocytes, classical inhibitors of PI 3-kinase, wortmannin and LY-294002, inhibit the sequestration step in the formation of autophagic vacuoles without the cytotoxic effects associated with high concentrations of 3-MA (2). Like 3-MA, wortmannin and LY-294002 were also effective inhibitors of proteolysis in brown adipocytes (Table 1), suggesting that, as in hepatocytes (2), some inositol phosphate metabolites are required for proteolysis.

Proteolysis was markedly inhibited by NE (Figs. 1, 2, 3, 5; Table 1). NE appears to inhibit autophagy, as shown by a comparable inhibition by 3-MA, wortmannin, and LY-294002, without net additive effects when added together with NE, suggesting that they are affecting the same pathway (Table 1). Furthermore, reducing the labeling period from 72 to 1 h, which biases the measurements of proteolysis toward proteins with short half-lives [not degraded primarily by autophagy (20, 23)], reduced the inhibitory effect of NE (Fig. 6). This is not to say that reduction of autophagy is the only way by which NE inhibits proteolysis. When NE was added together with inhibitors of autophagy or lysosomal function, proteolysis was always slightly less than from additions of inhibitors alone (Table 1). This slightly greater effect of NE on proteolysis did not reach statistical significance but is suggestive that NE may be affecting other proteolytic pathways in addition to autophagy. This nonautophagic component is likely cytosolic and/or mitochondrial, as both compartments have proteolytic systems capable of breaking down proteins to amino acids (20, 23, 27). There are extensive changes in mitochondrial structure and protein composition (e.g., loss of UCP-1) under conditions of brown adipose tissue atrophy, and it is possible that NE may affect the expression or activity of proteases located within mitochondria.

A beta 3-adrenergic receptor-mediated increase in intracellular cAMP is likely responsible for the inhibitory action of NE. First, propranolol, a beta -adrenergic antagonist, partially antagonized the effect of NE, whereas prazosin, an alpha 1-adrenergic antagonist, had no effect (Fig. 4B), indicating a role for beta -adrenergic receptors. The concentration of propranolol required to inhibit even partially the effect of 0.1 µM NE on proteolysis was high (10-50 µM). This result points to a beta 3-receptor-mediated effect, which is known to be more resistant to propranolol than beta 1- or beta 2-adrenergic receptor-mediated events (43). Second, the order of potency of adrenergic agonists in reducing proteolysis (BRL-37344 > isoproterenol > dobutamide; Fig. 4A) is consistent with similar results by others showing an important role of the beta 3-adrenoreceptor in mediating the lipolytic and thermogenic effects of NE (1, 43). Third, forskolin, an activator of adenylate cyclase, inhibited proteolysis to the same extent as NE, likely underlying its mechanism of action (Fig. 5). The lack of effect of A-23187 and phorbol esters on proteolysis with or without NE (Fig. 5) is also consistent with the inability of prazosin to inhibit NE's effects on proteolysis (Fig. 4B). Altogether, these results point to a beta 3-receptor-mediated increase in cAMP as responsible for the inhibitory effects of NE on proteolysis.

The physiologically important features of beta 3-adrenergic receptors are low affinity for NE and resistance to desensitization during prolonged agonist exposure (1). The concentration of NE that maximally inhibited proteolysis (100 nM; Fig. 3) is in keeping with the concentration suggested to occur in the synaptic cleft (1). Even plasma concentrations of NE of 1-25 nM [depending on the environmental conditions (1)] would inhibit proteolysis to some extent (Fig. 3). Thus there should be chronic inhibition of proteolysis with prolonged sympathetic stimulation, as during cold exposure, and increased intracellular proteolysis when sympathetic activity is suppressed, as during deacclimation, food deprivation, lactation, and drug-induced diabetes (25). This interpretation is consistent with in vivo data showing marked protein loss from brown fat on surgical denervation or sympathectomy (10, 13, 30).

It was surprising that NE did not inhibit proteolysis in preadipocytes, as it did in mature cells (Fig. 2). NE stimulates proliferation of preadipocytes by a beta 1-adrenergic receptor process, linked to an increase in cAMP (6). One possible explanation is the appearance late in differentiation of a protein, perhaps a protein or lipid kinase, to couple beta 3-adrenergic receptor function and proteolysis. Changes in protein and lipid phosphorylation are involved in the control of autophagy (3). Another possibility is a link between NE-dependent uncoupling of metabolism and proteolysis, because expression of UCP-1 is also a late event in differentiation (Fig. 2A). Autophagy in hepatocytes is sensitive to reductions in intracellular ATP (3, 16). We did not find a significant change in ATP content of rat brown adipocytes exposed to NE (41), but others have reported a fall of 15-20% in the ATP content of hamster brown fat cells (28). Our current work suggests it is unlikely that uncoupling of oxidative phosphorylation is the major mechanism of action of NE, because addition of fatty acids that are effective activators of UCP-1 (25) has little effect on proteolysis (unpublished observations).

Of the other three hormones tested, only insulin was able to inhibit proteolysis (Fig. 1), at concentrations in the upper end of the physiological range (Fig. 3). This is likely a function of the 4 h of incubation used to measure proteolysis, during which some insulin may be degraded or inactivated. In vivo, changes in insulin status are known to alter sympathetic nervous system activity to BAT (24, 25), the expression of the beta 3-adrenergic receptor, and the responsiveness of brown adipocytes to NE (17). It is likely that these effects may be quantitatively more significant to BAT growth and atrophy than insulin's direct action on proteolysis, which was only about one-half that seen with NE (Fig. 1). The mechanism of action of insulin, like that of NE, is uncertain. They are likely distinct.

Leptin and glucagon had no effect on proteolysis (Fig. 3). Leptin is a hormone produced by white adipocytes and important in the control of energy balance (19, 42). Leptin infusions increase the thermogenic and lipolytic capacity of BAT in vivo (35), presumably because of increased sympathetic activity (21, 29), but also from direct actions (8, 38). Leptin receptor mRNA is present in brown fat cell cultures, and leptin activates the Jak/Stat signal transduction pathway and transcription of many genes involved in lipid metabolism (38). This pathway is obviously not involved in the acute control (<= 4 h) of protein degradation, but longer-term effects (e.g., modulation of the cell proteolytic capacity) are possible. Glucagon increases lipolysis and thermogenesis in BAT, presumably via increases in cAMP, and in pharmacological doses induces tissue growth (24). It may be that glucagon receptors are not expressed in brown fat cells in culture, or that our incubation conditions are not appropriate for detecting its effects. In intact liver, glucagon increases protein degradation, whereas in isolated hepatocytes, glucagon increases it, decreases it, or has no effect depending on the incubation conditions (26). Recent work also questions whether the thermogenic actions of injected glucagon are due to a direct action on brown adipocytes (15).

In summary, there is an important capacity for proteolysis in brown fat cells kept under negative control by NE and insulin, which likely contributes significantly to BAT growth and atrophy. For instance, during food deprivation, reductions in sympathetic tone to BAT and in circulating insulin levels would have the effect of both reducing protein synthesis and enhancing protein degradation, thus contributing to the rapid and marked atrophy of the tissue. The reverse would be true during refeeding or cold exposure.


    ACKNOWLEDGEMENTS

We are grateful to R. Hutchinson for artwork and photography.


    FOOTNOTES

This work was supported by grants from the Medical Research Council of Canada and Saskatchewan Health. S. Heal was a medical student supported by the summer student research fund of the College of Medicine of the University of Saskatchewan.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Desautels, Dept. of Physiology, College of Medicine, University of Saskatchewan, Health Sciences Bldg., 107 Wiggins St., Saskatoon, SK, Canada S7N 5E5 (E-mail: desautel{at}duke.usask.ca).

Received 14 September 1998; accepted in final form 22 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 277(2):E215-E222
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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