The obesogenic effects of polyunsaturated fatty acids are dependent ...

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Cyclooxygenases and UCP1 Although it has been estimated that 50 g of brown adipocytes would be sufficient to burn 20% of the daily energy intake [14], BAT has traditionally been considered to be virtually absent and of no physiological relevance in adult humans. This view has recently changed dramatically with the demonstration of functional BAT in adult humans [15–19] adding to the observation of brown-like multilocular adipocytes expressing UCP1 interspersed within human WAT [20–22]. Actually, UCP1 mRNA has been detected in all adipose tissues in adult humans, and it has been estimated that 1 in 100–200 adipocytes in human intraperitoneal adipose tissue expresses UCP1 [23]. Classic interscapular brown adipocytes and brown-like adipocytes found in WAT depots appear to originate from distinct lineages. Brown pre-adipocytes derived from the interscapular region (iBAT) demonstrate myogenic gene expression [24] and classic brown adipocytes arise from Myf5 expressing progenitors [25]. In contrast, brown-like adipocytes appearing in white adipose tissue by b-adrenergic stimulation (‘‘brite adipocytes’’) appear to originate from another lineage, much closer to white adipocytes [26–29] and display different molecular markers [30]. Several lines of evidence suggest that the number of brown-like adipocytes in WAT depots might influence whole body energy balance. Increased occurrence of brown-like adipocytes within WAT depots is a feature of mouse strains resistant to dietary obesity, such as the A/J strain [31;32], and reduced adiposity associated with aP2-transgenic expression of UCP1 is linked to increased energy dissipation in white, but not interscapular brown, adipose tissue [33]. Human obesity is associated with a reduced expression of UCP1 and other thermogenesis related genes in WAT depots [34;35]. Thus, identification of factors controlling induction of UCP1 expression and an increase in the number of brown-like adipocytes in white depots obviously deserves further attention. It is intriguing that the cold-induced occurrence of brown-like adipocytes and UCP1 requires the presence of the b 3 -adrenoceptor in previously white adipose tissue, but not in interscapular brown adipose tissue [36]. Furthermore, the presence of the b 3 - adrenoceptor is required for full stimulation of energy expenditure and oxygen consumption in white adipose tissue [37]. Adipocytes from lean rats have higher isoprenalin-stimulated prostaglandin E 2 (PGE 2 ) synthesis, than adipocytes from obese Zucker rats [38]. We therefore hypothesized that prostaglandins or related products synthesized by cyclooxygenases (COXs) might be involved in the recruitment of brown adipocytes in white depots. The COXs have previously been implicated in adipogenesis [39–41], but no specific role has been assigned. Here, we demonstrate that COX activity is crucially involved in the induction of UCP1 expression in WAT providing further evidence for a role of COXs in the control of energy balance and obesity development. In view of the worldwide epidemic of obesity and associated metabolic disorders it is obviously of importance to identify pathways that can be manipulated genetically or pharmacologically and regulate the induction of UCP1 expression and recruitment of brown-like adipocytes in white adipose tissues. Results COX1 and COX2 protein expression is upregulated in iWAT during cold treatment When mice are kept at 28uC, close to thermoneutrality, the majority of the adipocytes in the inguinal white adipose depot (iWAT) – the major subcutaneous depot in the mouse – appear as UCP1-negative, spherical unilocular adipocytes [42]. In iWAT from warm-acclimated mice, only endothelial cells and macrophages stained positive for COX1 and COX2, respectively (Figure 1A and B). However, when mice were transferred to a cold environment, the emerging multilocular adipocytes stained positive for COX1 and COX2. In particular, cells that appeared to be in a transition state between uni- and multilocular cells stained strongly (Figure 1A and B). Western blotting demonstrated increased expression of COX1 and COX2 in iWAT, and also in iBAT, after cold exposure (Figure 1C). Real time qPCR analysis verified that genes preferentially expressed in brown vs. white adipose tissue, such as UCP1, peroxisome proliferator activated receptor gamma coactivator 1a (PGC1a), type II thyroxine deiodinase (Dio2), cytochrome C oxidase subunit 8b (Cox8b), epithelial V like antigen 1 (Eva1) and Cidea were all highly induced in iWAT upon cold exposure, whereas expression of 4E- BP1 and of nuclear receptor interacting protein 140 (Nrip1/ RIP140) was reduced (Figure 1D). Immunohistochemical staining of iBAT from cold-treated mice demonstrated that adipocytes stained positive for COX2, whereas only endothelial cells stained positive for COX1 (Figure S1A). The lack of COX1 and COX2 expression in adipocytes from iBAT in warm-acclimated mice was verified by analysis of protein and RNA isolated from fractionated adipose tissue, in which COX1 and COX2 was detected solely in the stromal vascular fraction (Figure S1B). Inhibition of COX activity represses induction of UCP1 expression Differentiated mouse embryo fibroblasts (MEFs) lacking the retinoblastioma (Rb) gene, resemble brown or brown-like adipocytes in demonstrating b-adrenergic induction of UCP1 expression [43]. To achieve a robust induction of UCP1 expression, differentiated Rb 2/2 MEFs were treated with a combination of isoproterenol and 9-cis retinoic acid [44]. Just as cold exposure increased COX1 and COX2 mRNA and protein levels in brown-like adipocytes (Figure 1), isoproterenol/9-cis retinoic acid treatment increased COX1 and COX2 mRNA and protein expression (Figure 2A and B) in this model system. Upregulation of COX1 and COX2 expression in Rb 2/2 adipocytes was accompanied by increased production of PGE 2 , the primary prostaglandin produced by mature adipocytes [45;46], but not of PGF 2a and 6-keto-PGF 1a (Figure 2C). This indicates that Rb 2/2 adipocytes resemble mature adipocytes in producing PGE 2 as the major prostaglandin species. To investigate the importance of COX activity for induction of UCP1 expression, we treated differentiated Rb 2/2 adipocytes with isoproterenol/9-cis retinoic acid in the absence or presence of the general COX inhibitor indomethacin. Indomethacin prevented induction of UCP1 mRNA and protein expression (Figure 2D and E), thus suggesting the intriguing possibility that COX activity is required for induction of UCP1. To examine if COX activity was required also in primary adipocytes, we induced cells from the stromal vascular fraction of iBAT and iWAT to differentiate and then treated the mature adipocytes with the b-adrenergic agonist isoproterenol in the absence and presence of indomethacin. Interestingly, indomethacin inhibited isoproterenol-induced UCP1 expression in cells derived from iWAT but not from iBAT (Figure 3A), indicating that COX activity is required for b-adrenergic induction of UCP1 expression in adipocytes from iWAT, but not in iBAT adipocytes. In keeping with this notion, indomethacin only marginally attenuated induction of UCP1 expression in the WT-1 cell model representing interscapular brown adipocytes (Text S1, Figure S2) [47]. To investigate the role of COX activity during induction of UCP1 expression in iWAT and iBAT in vivo, we treated warm- PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11391
Cyclooxygenases and UCP1 Figure 1. Cold exposure induces COX1 and COX2 expression in iWAT and iBAT. Sv129 mice were warm-acclimated at 28–30uC for 6 days and then transferred to 4–6uC. Samples for cryosections, RNA and protein extractions were prepared from iBAT and iWAT after 2, 4 and 6 days at 4– 6uC. A–B. Representative COX1 (A) and COX2 (B) immunoreactivity in iWAT from mice kept at 28–30uC for 6 days and after 6 days of cold exposure. C. Proteins were isolated from warm-acclimated mice (lane 1) and after 2, 4 and 6 days of cold exposure. COX1 and COX2 expression were determined by Western blotting. D. RNA was harvested from iBAT and iWAT from individual mice (n = 4 in each group) that were warm-acclimated or coldexposed for 6 days. Expressions of genes were measured by RT-qPCR in duplicates and normalized to TBP (TATA box binding protein). The bars represent mean 6 standard error. * indicates statistical difference (p,0.05) compared to expression in warm acclimated mice. doi:10.1371/journal.pone.0011391.g001 acclimated mice with the COX inhibitor indomethacin and transferred the mice to 4uC. Measurements of rectal temperature revealed that mice treated with indomethacin had slightly, but significantly lower body temperature (Figure 3B). As expected, UCP1 expression was induced in both iBAT and iWAT in vehicle-treated mice (Figure 3C and D). While indomethacin treatment only slightly attenuated cold-induced UCP1 expression in iBAT, it almost completely prevented the induction of UCP1 expression in iWAT (Figure 3C and D). Thus, COX activity appeared to be necessary for cold-induced UCP1 expression in iWAT, but not in iBAT. In addition, indomethacin treatment attenuated cold-induced enhancement of PGC1a, Dio2,Cox8b, Eva1 and Cidea expression in iWAT, while preventing coldinduced repression of RIP140 and 4E-BP1 expression in iWAT (Figure 3D). Forced expression of COX2 induces UCP1 expression in Rb 2/2 adipocytes Since indomethacin attenuated b-adrenergically stimulated UCP1 expression in Rb 2/2 adipocytes and primary inguinal PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11391
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The obesogenic effects of polyunsat
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Table of Contents Acknowledgement .
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Abstract in Danish Polyumættede n-
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Polyunsaturated fatty acids in the
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was more than 3 times higher in the
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gluconeogenesis and ureagenesis (Fi
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high dietary PUFAs, the modulation
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In a pair of population studies, an
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18. Massiera, F. et al. Arachidonic
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54. Xu, J. et al. Polyunsaturated f
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Annexes 1. Ma T, Liaset B, Hao Q, P
Page 23 and 24: Fish Oil, Sucrose and Obesity Obesi
Page 25 and 26: Fish Oil, Sucrose and Obesity Figur
Page 27 and 28: Fish Oil, Sucrose and Obesity Table
Page 33 and 34: Fish Oil, Sucrose and Obesity 6. Ma
Page 35 and 36: Carbohydrate source and insulin sec
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Page 51 and 52: 350 351 FIGURE LEGENDS 352 353 354
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Page 57 and 58: 484 485 486 TABLE 1. Macronutrient
Page 59 and 60: Energy (kJ/g) 17.16 25.12 25.12 25.
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Page 80 and 81: Cyclooxygenases and UCP1 PLoS ONE |
Page 82 and 83: Cyclooxygenases and UCP1 E synthase
Page 84 and 85: Cyclooxygenases and UCP1 29. Granne
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Page 88 and 89: hydrolysate (SPH), would be protect
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Page 92 and 93: In order to demonstrate that bile a
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Page 100 and 101: FOOTNOTES This work was carried out
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Page 108 and 109: Figure 7 A Plasma ALAT B Liver Ucp2
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