UCP1 Induction during Recruitment of Brown Adipocytes in White Adipose Tissue Is Dependent on Cyclooxygenase Activity

Background

The uncoupling protein 1 (UCP1) is a hallmark of brown adipocytes and pivotal for cold- and diet-induced thermogenesis.

Methodology/Principal Findings

Here we report that cyclooxygenase (COX) activity and prostaglandin E₂ (PGE₂) are crucially involved in induction of UCP1 expression in inguinal white adipocytes, but not in classic interscapular brown adipocytes. Cold-induced expression of UCP1 in inguinal white adipocytes was repressed in COX2 knockout (KO) mice and by administration of the COX inhibitor indomethacin in wild-type mice. Indomethacin repressed β-adrenergic induction of UCP1 expression in primary inguinal adipocytes. The use of PGE₂ receptor antagonists implicated EP₄ as a main PGE₂ receptor, and injection of the stable PGE₂ analog (EP_3/4 agonist) 16,16 dm PGE₂ induced UCP1 expression in inguinal white adipose tissue. Inhibition of COX activity attenuated diet-induced UCP1 expression and increased energy efficiency and adipose tissue mass in obesity-resistant mice kept at thermoneutrality.

Conclusions/Significance

Our findings provide evidence that induction of UCP1 expression in white adipose tissue, but not in classic interscapular brown adipose tissue is dependent on cyclooxygenase activity. Our results indicate that cyclooxygenase-dependent induction of UCP1 expression in white adipose tissues is important for diet-induced thermogenesis providing support for a surprising role of COX activity in the control of energy balance and obesity development.     

Prostaglandin E2 Exerts Multiple Regulatory Actions on Human Obese Adipose Tissue Remodeling,Inflammation, Adaptive Thermogenesis and Lipolysis

Obesity induces white adipose tissue (WAT) dysfunction characterized by unremitting inflammation and fibrosis, impaired adaptive thermogenesis and increased lipolysis. Prostaglandins (PGs) are powerful lipid mediators that influence the homeostasis of several organs and tissues. The aim of the current study was to explore the regulatory actions of PGs in human omental WAT collected from obese patients undergoing laparoscopic bariatric surgery. In addition to adipocyte hypertrophy, obese WAT showed remarkable inflammation and total and pericellular fibrosis. In this tissue, a unique molecular signature characterized by altered expression of genes involved in inflammation, fibrosis and WAT browning was identified by microarray analysis. Targeted LC-MS/MS lipidomic analysis identified increased PGE₂ levels in obese fat in the context of a remarkable COX-2 induction and in the absence of changes in the expression of terminal prostaglandin E synthases (i.e. mPGES-1, mPGES-2 and cPGES). IPA analysis established PGE₂ as a common top regulator of the fibrogenic/inflammatory process present in this tissue. Exogenous addition of PGE₂ significantly reduced the expression of fibrogenic genes in human WAT explants and significantly down-regulated Col1α1, Col1α2 and αSMA in differentiated 3T3 adipocytes exposed to TGF-β. In addition, PGE₂ inhibited the expression of inflammatory genes (i.e. IL-6 and MCP-1) in WAT explants as well as in adipocytes challenged with LPS. PGE₂ anti-inflammatory actions were confirmed by microarray analysis of human pre-adipocytes incubated with this prostanoid. Moreover, PGE₂ induced expression of brown markers (UCP1 and PRDM16) in WAT and adipocytes, but not in pre-adipocytes, suggesting that PGE₂ might induce the trans-differentiation of adipocytes towards beige/brite cells. Finally, PGE₂ inhibited isoproterenol-induced adipocyte lipolysis. Taken together, these findings identify PGE₂ as a regulator of the complex network of interactions driving uncontrolled inflammation and fibrosis and impaired adaptive thermogenesis and lipolysis in human obese visceral WAT.     

Two different mechanisms for modulation of bronchoconstriction in guinea-pigs by cyclooxygenase metabolites

Leukotriene D₄ (LTD₄)-induced bronchoconstriction in guinea-pig airways has a cyclooxygenase (COX)-dependent component. The main objective of this study was to establish if prostaglandin (PG) D₂-induced bronchoconstriction also was modulated by COX products. The effects of non-selective and selective COX-1 and COX-2 inhibitors on bronchoconstriction induced by LTD₄ and PGD₂ were investigated in the perfused and ventilated guinea-pig lung (IPL). Both LTD₄-induced bronchoconstriction and thromboxane (TX) A₂ release was suppressed by COX inhibitors or by TX synthesis inhibition. The release of additional COX products following CysLT₁ receptor activation by LTD₄ was established by measurements of immunoreactive 6-keto PGF_1α (a stable metabolite of PGI₂) and PGE₂. In contrast, TP receptor-mediated bronchoconstriction by PGD₂ was somewhat enhanced by COX inhibitors, and there was no measurable release of COX products after TP receptor activation with U-46619. PGE₂ was bronchoprotective in IPL as it inhibited the histamine-induced bronchoconstriction. In the isolated guinea-pig trachea, neither PGD₂ nor U-46619 actively released PGE₂, but continuous production of PGE₂ and PGI₂ was established, and the response to PGD₂ was enhanced also in the trachea by COX inhibition. The study documented that bronchoconstriction induced by LTD₄ and PGD₂ in IPL was modulated differently by COX products. Whereas bronchoconstriction induced by LTD₄ was amplified predominantly by secondarily released TXA₂, that induced by PGD₂ was attenuated by bronchoprotective PGE₂ and PGI₂, presumably tonically produced in the airways.     

Thrombin induces cyclooxygenase‐2 expression and prostaglandin E2 release via PAR1 activation and ERK1/2‐ and p38 MAPK‐dependent pathway in murine macrophages

Thrombin levels increase at sites of vascular injury and during acute coronary syndromes. It is also increased several fold by sepsis with a reciprocal decrease in the anti‐thrombin III levels. In this study we investigate the effects of thrombin on the induction of cyclooxygenase‐2 (COX‐2) and prostaglandin (PG) production in macrophages. Thrombin‐induced COX‐2 protein and mRNA expression in RAW264.7 and primary cultured peritoneal macrophages. A serine proteinase, trypsin, also exerted a similar effect. The inducing effect by thrombin in macrophages was not affected by a lipopolysaccharide (LPS)‐binding antibiotic, polymyxin B, excluding the possibility of LPS contamination. The increase of COX‐2 expression by thrombin was functionally linked to release of PGE₂ and PGI₂ but not thromboxane A₂ into macrophage culture medium. Thrombin‐induced COX‐2 expression and PGE₂ production were significantly attenuated by PD98059 and SB202190 but not by SP600125, suggesting that ERK1/2 and p38 MAPK activation were involved in this process. This was supported by the observation that thrombin could directly activate ERK1/2 and p38 MAPK in macrophages. A further analysis indicated that the proteinase‐activated receptor 1 (PAR1)‐activating agonist induced effects similar to those induced by thrombin in macrophages and the PAR1 antagonist‐SCH79797 could attenuate thrombin‐induced COX‐2 expression and PGE₂ release. Taken together, we provided evidence demonstrating that thrombin can induce COX‐2 mRNA and protein expression and PGE₂ production in macrophages through PAR1 activation and ERK1/2 and p38 MAPK‐dependent pathway. The results presented here may explain, at least in part, the possible contribution of thrombin and macrophages in these pathological conditions. J. Cell. Biochem. 108: 1143–1152, 2009. © 2009 Wiley‐Liss, Inc.     

Adipocyte-derived lipoprotein lipase induces macrophage activation and monocyte adhesion: role of fatty acids

Objective: We evaluated the effect of adipocyte‐derived lipoprotein lipase (LPL) on macrophage activation and monocyte adhesion and the role of fatty acids in these effects. Research Methods and Procedures: 3T3‐L1 adipocytes were incubated with heparin or insulin to induce LPL secretion; then adipocyte conditioned media (CM) were added to cultured J774 macrophages or human aortic endothelial cells (HAECs). Macrophage cytokine production and monocyte adhesion to HAECs were determined. Results: Incubation of macrophages with heparin‐ or insulin‐treated adipocyte CM increased tumor necrosis factor α, interleukin‐6, and nitric oxide production by these cells. LPL neutralization and heparinase treatment prevented these effects. Addition of active LPL or palmitate to cultured macrophages replicated these effects. Blockade of leptin also reduced the effect of insulin‐treated adipocyte CM on macrophage inflammatory changes. Induction of macrophage cytokine secretion by leptin was prevented by LPL immunoneutralization. Finally, addition of CM of heparin‐ or insulin‐treated adipocytes to HAECs stimulated monocyte adhesion to these cells, an effect that was abrogated by an anti‐LPL antibody. This effect was reproduced by treating HAECs with active LPL or palmitate. Discussion: These results point to an effect of LPL‐mediated lipolysis in macrophage activation and monocyte adhesion.     

Prostaglandin E3 attenuates macrophage-associated inflammation and prostate tumour growth by modulating polarization

Alternative polarization of macrophages regulates multiple biological processes. While M1-polarized macrophages generally mediate rapid immune responses, M2-polarized macrophages induce chronic and mild immune responses. In either case, polyunsaturated fatty acid (PUFA)-derived lipid mediators act as both products and regulators of macrophages. Prostaglandin E₃ (PGE₃) is an eicosanoid derived from eicosapentaenoic acid, which is converted by cyclooxygenase, followed by prostaglandin E synthase successively. We found that PGE₃ played an anti-inflammatory role by inhibiting LPS and interferon-γ-induced M1 polarization and promoting interleukin-4-mediated M2 polarization (M2a). Further, we found that although PGE₃ had no direct effect on the growth of prostate cancer cells in vitro, PGE₃ could inhibit prostate cancer in vivo in a nude mouse model of neoplasia. Notably, we found that PGE₃ significantly inhibited prostate cancer cell growth in a cancer cell-macrophage co-culture system. Experimental results showed that PGE₃ inhibited the polarization of tumour-associated M2 macrophages (TAM), consequently producing indirect anti-tumour activity. Mechanistically, we identified that PGE₃ regulated the expression and activation of protein kinase A, which is critical for macrophage polarization. In summary, this study indicates that PGE₃ can selectively promote M2a polarization, while inhibiting M1 and TAM polarization, thus exerting an anti-inflammatory effect and anti-tumour effect in prostate cancer.     

Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages

Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A₂ group IVA (cPLA₂α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA₂α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA₂α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E₂ production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA₂α inhibitor blocked HA-induced AA release and PGE₂ production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA₂α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE₂ production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE₂ production and COX2 expression were blocked in Tlr4^−/− and Myd88^−/− mice, but not in Cd44^−/− mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA₂α/COX2^high and COX1/ALOX15/ALOX5/LTA4H^low gene and PGE₂/PGD₂/15-HETE^high and LXA₄^low eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.     

α-Adrenergic stimulants, prostaglandins and catecholamine release from the adrenal gland in vitro

Prostaglandin D₂ was found to be a potent inhibitor of platelet aggregation. Aggregation of human platelets by ADP, collagen and prostaglandin G₂ was inhibited more strongly by PGD₂ than by PGE₁. Although ADP-induced aggregation of rabbit platelets was inhibited more strongly by PGE₁ than by PGD₂ the latter prostaglandin gave a more long-lasting inhibitory effect on platelet aggregation following intravenous or oral administration. These results coupled with the finding that PGD₂ has less hypotensive effects on the cardiovascular system than PGE₁ suggest the possible use of PGD₂ as an antithrombotic agent.     

Prostaglandin D2 as a potential antithrombotic agent

Prostaglandin D₂ was found to be a potent inhibitor of platelet aggregation. Aggregation of human platelets by ADP, collagen and prostaglandin G₂ was inhibited more strongly by PGD₂ than by PGE₁. Although ADP-induced aggregation of rabbit platelets was inhibited more strongly by PGE₁ than by PGD₂ the latter prostaglandin gave a more long-lasting inhibitory effect on platelet aggregation following intravenous or oral administration. These results coupled with the finding that PGD₂ has less hypotensive effects on the cardiovascular system than PGE₁ suggest the possible use of PGD₂ as an antithrombotic agent.     

Local proliferation initiates macrophage accumulation in adipose tissue during obesity

Obesity-associated chronic inflammation is characterized by an accumulation of adipose tissue macrophages (ATMs). It is generally believed that those macrophages are derived from peripheral blood monocytes. However, recent studies suggest that local proliferation of macrophages is responsible for ATM accumulation. In the present study, we revealed that both migration and proliferation contribute to ATM accumulation during obesity development. We show that there is a significant increase in ATMs at the early stage of obesity, which is largely due to an enhanced in situ macrophage proliferation. This result was obtained by employing fat-shielded irradiation and bone marrow reconstitution. Additionally, the production of CCL2, a pivotal chemoattractant of monocytes, was not found to be increased at this stage, corroborating with a critical role of proliferation. Nonetheless, as obesity proceeds, the role of monocyte migration into adipose tissue becomes more significant and those new immigrants further proliferate locally. These proliferating ATMs mainly reside in crown-like structures formed by macrophages surrounding dead adipocytes. We further showed that IL-4/STAT6 is a driving force for ATM proliferation. Therefore, we demonstrated that local proliferation of resident macrophages contributes to ATM accumulation during obesity development and has a key role in obesity-associated inflammation.The accumulation of adipose tissue macrophages (ATMs) is a significant characteristic of obesity-associated chronic inflammation. It is also critical in regulating obesity development. In lean animals, there is a low cellularity of resident ATMs interspersing among adipocytes, which are considered as M2 macrophages. During obesity, significantly increased macrophages accumulate in adipose tissue and form the so-called ‘crown-like structures'' (CLSs) around the dead adipocytes.^{1, 2} Those macrophages exhibit M1 phenotype and produce various types of inflammatory cytokines, such as TNF-α, resulting in the propagation of obesity-related inflammation and the development of metabolic disorders, such as insulin resistance.^{3, 4, 5}Traditionally, the accumulated ATMs are considered as a consequence of peripheral monocyte migration under inflammatory conditions. Recently, increasing evidences have shown that the maintenance of tissue macrophages is probably independent of the replenishment of circulating monocytes and even independent of precursors from bone marrow.⁶ Indeed, several kinds of tissue macrophages are capable of self-renewal and proliferate locally in naive state, such as microglia,^{7, 8} Kupffer cells,⁹ and Langerhans cells.¹⁰In acute inflammation status, for instance, during parasitic infection, local proliferation of macrophages is boosted and these macrophages exhibit phenotypes of alternatively activated macrophages, a process driven by Th2 cytokines.¹¹ In chronic inflammation conditions, such as atherosclerosis, local proliferation of macrophages also occurs and contributes to macrophage accumulation in arterial walls.¹² Most recently, it has been reported that local proliferation of macrophages could contribute to the ATM accumulation in obesity.^{13, 14}Given the potential contributions of monocyte migration and macrophage proliferation to ATM accumulation, an important question about the respective role of each event in ATM accumulation during obesity is raising. To address it, we first focus on the initiation of ATM accumulation in obesity. We found that, although there is no significant change in the level of chemokine (C-C motif) ligand 2 (CCL2) either in adipose tissue or in circulation, the cellularity of ATMs is dramatically elevated at the early stage of obesity. Interestingly, the increase of ATMs was accompanied with vigorous ATM proliferation. By inducing obesity in chimeric mice that were generated by fat-shielded irradiation and bone marrow transplantation, we demonstrated that in situ proliferation of resident macrophages dominates the initiation of ATM accumulation at early stage of obesity, and the recruited monocytes make contribution to ATM accumulation at a relatively late stage of obesity. This study sheds light on the dynamic process of ATM accumulation and provides insight on the initiation of obesity-associated inflammation.     

Endoplasmic reticulum stress in adipose tissue augments lipolysis

The endoplasmic reticulum (ER) is an organelle important for protein synthesis and folding, lipid synthesis and Ca²⁺ homoeostasis. Consequently, ER stress or dysfunction affects numerous cellular processes and has been implicated as a contributing factor in several pathophysiological conditions. Tunicamycin induces ER stress in various cell types in vitro as well as in vivo. In mice, a hallmark of tunicamycin administration is the development of fatty livers within 24–48 hrs accompanied by hepatic ER stress. We hypothesized that tunicamycin would induce ER stress in adipose tissue that would lead to increased lipolysis and subsequently to fatty infiltration of the liver and hepatomegaly. Our results show that intraperitoneal administration of tunicamycin rapidly induced an ER stress response in adipose tissue that correlated with increased circulating free fatty acids (FFAs) and glycerol along with decreased adipose tissue mass and lipid droplet size. Furthermore, we found that in addition to fatty infiltration of the liver as well as hepatomegaly, lipid accumulation was also present in the heart, skeletal muscle and kidney. To corroborate our findings to a clinical setting, we examined adipose tissue from burned patients where increases in lipolysis and the development of fatty livers have been well documented. We found that burned patients displayed significant ER stress within adipose tissue and that ER stress augments lipolysis in cultured human adipocytes. Our results indicate a possible role for ER stress induced lipolysis in adipose tissue as an underlying mechanism contributing to increases in circulating FFAs and fatty infiltration into other organs.     

Adipocyte-derived Th2 cytokines and myeloid PPARdelta regulate macrophage polarization and insulin sensitivity

The polarization of adipose tissue-resident macrophages toward the alternatively activated, anti-inflammatory M2 phenotype is believed to improve insulin sensitivity. However, the mechanisms controlling tissue macrophage activation remain unclear. Here we show that adipocytes are a source of Th2 cytokines, including IL-13 and to a lesser extent IL-4, which induce macrophage PPARdelta/beta (Ppard/b) expression through a STAT6 binding site on its promoter to activate alternative activation. Coculture studies indicate that Ppard ablation renders macrophages incapable of transition to the M2 phenotype, which in turns causes inflammation and metabolic derangement in adipocytes. Remarkably, a similar regulatory mechanism by hepatocyte-derived Th2 cytokines and macrophage PPARdelta is found to control hepatic lipid metabolism. The physiological relevance of this paracrine pathway is demonstrated in myeloid-specific PPARdelta(-/-) mice, which develop insulin resistance and show increased adipocyte lipolysis and severe hepatosteatosis. These findings provide a molecular basis to modulate tissue-resident macrophage activation and insulin sensitivity.     

Effects of non-steroidal anti-inflammatory drugs on prostaglandin E2 production by cyclooxygenase-2 from endogenous and exogenous arachidonic acid in rat peritoneal macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) stimulated prostaglandin E₂ (PGE₂) formation and induction of cyclooxygenase-2 (COX-2) expression without changing the levels of COX-1 protein in rat peritoneal macrophages. Non-steroidal anti-inflammatory drugs (NSAIDs) (nimesulide, indomethacin and ibuprofen) strongly inhibited LPS-stimulated PGE₂ production without any effect on COX-2 protein expression, suggesting that NSAIDs are active in inhibiting the ability of COX-2 to convert arachidonic acid (AA) endogenously released in response to LPS stimulation. Exogenous AA can be converted to PGE₂ by both COX isoforms even in LPS-stimulated macrophages. NSAIDs inhibited PGE₂ production from exogenous AA mediated by both COX-1 and COX-2. However, the two isoforms interacted differentially with different NSAIDs. Furthermore, NSAIDs were distinctly more active in inhibiting PGE₂ production from endogenous AA than that from exogenous AA. These data suggest that PGE₂ production through COX-2 from exogenous AA may not be subject to the same regulatory processes as that from endogenous AA and the two metabolic processes may be differentially sensitive to different NSAIDs.     

Reversal of Inflammation‐Induced Impairment of Glucose Uptake in Adipocytes by Direct Effect of CB1 Antagonism on Adipose Tissue Macrophages

Macrophage infiltration into adipose tissue (AT‐MP) is thought to induce insulin resistance and diabetes in obesity. Here, we investigated the effect of the antiobesity drug SR141716 (a CB1 antagonist) on macrophage‐mediated inhibition of insulin signaling in adipocytes. THP1 macrophages (THP1) were stimulated in vitro with lipopolysaccharide (LPS) and SR141716 or vehicle. The resulting conditioned medium (CM) was analyzed and incubated on human adipocytes. CM from LPS‐stimulated THP1 inhibited insulin‐induced AKT phosphorylation in adipocytes, in contrast to CM from nonactivated THP1. Moreover, it contained higher concentrations of tumor necrosis factor‐α (TNFα) and lower levels of the anti‐inflammatory cytokine IL‐10. SR141716 reduced TNFα production and increased IL‐10 secretion, resulting in a rescue of insulin signaling in adipocytes. To confirm these findings in vivo, AT‐MP CM from cafeteria diet‐fed or Zucker diabetic fatty (ZDF) rats that had received SR141716 for 3 weeks were isolated, analyzed, and incubated with adipocytes. Cafeteria diet induced macrophage‐mediated inhibition of insulin signaling in adipocytes. Interestingly, SR141716 rescued insulin‐induced glucose uptake in adipocytes. Finally, AT‐MP CM from obese ZDF rats inhibited insulin‐stimulated glucose uptake in adipocytes in contrast to AT‐MP CM from lean ZDF rats. After treatment with SR141716, AT‐MP CM rescued insulin‐induced glucose uptake in adipocytes. In summary, our data indicate that CB1 receptor antagonism in macrophages modified their cytokine production and improved the insulin responsiveness of adipocytes that had been incubated with macrophage CM. Thus, SR141716 ameliorated adipose tissue insulin resistance by direct action on AT‐MP demonstrating a novel peripheral mode of action of CB1 antagonism.     

Metabolism and effect of prostaglandin H2 in adipose tissue

Prostaglandin H₂ (PGH₂) inhibited noradrenaline induced cyclic AMP accumulation in isolated rat fat cells in a dose-dependent manner. IC₅₀ was 10 – 25 ng/ml both in the absence and in the presence of theophylline. The degree of inhibition produced by PGH₂ increased with time of incubation. A stable PGH₂ analog did not inhibit cyclic AMP accumulation. PGH₂ was rapidly converted by isolated fat cells to PGD₂, PGE₂ and PGH_2α, but no formation of thromboxane B₂ was found either or . PGE₂ was a more potent inhibitor than PGH₂ of noradrenaline induced cyclic AMP accumulation. PGD₂ enhanced cyclic AMP accumulation in a limited concentration interval, while PGF_2α was essentially uneffective.Our results suggest that PGH₂ is an inhibitor of cyclic AMP formation in isolated rat fat cells only after conversion to PGE₂. A physiological role for PGH₂ as a modulator of lipolysis is considered unlikely.     

Prostaglandin E2 increases the calcium concentration in rat brown adipocytes and their consumption of oxygen

Effects of prostaglandin E₂ (PGE₁) were examined on the oxygen consumption and intracellular calcium concentration of rat brown adipose tissue (BAT). PGE₂ 0.1 nM-1 μM increased oxygen consumption of the tissue blocks of BAT, with a maximum 2–13 min after PGE2 administration. PGE₂ was most effective at 1 and 10 nM, and the oxygen consumption was elevated for over 40 min. Pretreatment of BAT with indomethacin, a prostaglandin synthesis inhibitor, did not affect the increase in oxygen consumption induced by noradrenaline. PGE₂ at 1–10 nM gradually increased the intracellular calcium concentration of freshly dispersed single brown adipocytes by 3–4 times in 30 min. PGE₂ also increased the intracellular calcium concentration of brown adipocytes in calcium-free medium. These results raise the possibility that PGE₂ and noradrenaline affect heat genesis and metabolism of BAT independently.