cAMP-dependent Signaling Regulates the Adipogenic Effect of n-6 Polyunsaturated Fatty Acids
2007; Elsevier BV; Volume: 283; Issue: 11 Linguagem: Inglês
10.1074/jbc.m707775200
ISSN1083-351X
AutoresLise Madsen, Lone Møller Pedersen, Bjørn Liaset, Tao Ma, Rasmus K. Petersen, Sjoerd A.A. van den Berg, Jie Pan, Karin Müller‐Decker, Erik Dülsner, Robert Kleemann, Teake Kooistra, Stein Ove Døskeland, Karsten Kristiansen,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoThe effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial. Using in vitro cell culture models, we show that n-6 PUFAs was pro-adipogenic under conditions with base-line levels of cAMP, but anti-adipogenic when the levels of cAMP were elevated. The anti-adipogenic action of n-6 PUFAs was dependent on a cAMP-dependent protein kinase-mediated induction of cyclooxygenase expression and activity. We show that n-6 PUFAs were pro-adipogenic when combined with a high carbohydrate diet, but non-adipogenic when combined with a high protein diet in mice. The high protein diet increased the glucagon/insulin ratio, leading to elevated cAMP-dependent signaling and induction of cyclooxygenase-mediated prostaglandin synthesis. Mice fed the high protein diet had a markedly lower feed efficiency than mice fed the high carbohydrate diet. Yet, oxygen consumption and apparent heat production were similar. Mice on a high protein diet had increased hepatic expression of PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) and genes involved in energy-demanding processes like urea synthesis and gluconeogenesis. We conclude that cAMP signaling is pivotal in regulating the adipogenic effect of n-6 PUFAs and that diet-induced differences in cAMP levels may explain the ability of n-6 PUFAs to either enhance or counteract adipogenesis and obesity. The effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial. Using in vitro cell culture models, we show that n-6 PUFAs was pro-adipogenic under conditions with base-line levels of cAMP, but anti-adipogenic when the levels of cAMP were elevated. The anti-adipogenic action of n-6 PUFAs was dependent on a cAMP-dependent protein kinase-mediated induction of cyclooxygenase expression and activity. We show that n-6 PUFAs were pro-adipogenic when combined with a high carbohydrate diet, but non-adipogenic when combined with a high protein diet in mice. The high protein diet increased the glucagon/insulin ratio, leading to elevated cAMP-dependent signaling and induction of cyclooxygenase-mediated prostaglandin synthesis. Mice fed the high protein diet had a markedly lower feed efficiency than mice fed the high carbohydrate diet. Yet, oxygen consumption and apparent heat production were similar. Mice on a high protein diet had increased hepatic expression of PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) and genes involved in energy-demanding processes like urea synthesis and gluconeogenesis. We conclude that cAMP signaling is pivotal in regulating the adipogenic effect of n-6 PUFAs and that diet-induced differences in cAMP levels may explain the ability of n-6 PUFAs to either enhance or counteract adipogenesis and obesity. The effect of dietary fat on human health is not solely a matter of quantity but depends also on the nature of the fatty acids. The current recommendation is to replace saturated fat by polyunsaturated fatty acids (PUFAs). 5The abbreviations used are:PUFApolyunsaturated fatty acidCOXcyclooxygenasePGE2 and PGF2αprostaglandin E2 and F2α, respectivelyRTreverse transcriptionqPCRquantitative PCRGCgas chromatographyMIXmethylisobutylxanthineCREcAMP-response elementCREBCRE-binding protein6-MB-cAMPN6-monobutyryl-cAMP.5The abbreviations used are:PUFApolyunsaturated fatty acidCOXcyclooxygenasePGE2 and PGF2αprostaglandin E2 and F2α, respectivelyRTreverse transcriptionqPCRquantitative PCRGCgas chromatographyMIXmethylisobutylxanthineCREcAMP-response elementCREBCRE-binding protein6-MB-cAMPN6-monobutyryl-cAMP. Today, more than 85% of the total dietary PUFA intake in Western diets is n-6 PUFAs, mainly linoleic acid, a precursor of arachidonic acid, whereas the consumption of n-3 PUFAs has declined (1Simopoulos A.P. Biomed. Pharmacother. 2002; 56: 365-379Crossref PubMed Scopus (2558) Google Scholar). Since the high intake of n-6 has been associated with childhood obesity, concerns regarding this matter have been raised (2Ailhaud G. Massiera F. Weill P. Legrand P. Alessandri J.M. Guesnet P. Prog. Lipid Res. 2006; 45: 203-236Crossref PubMed Scopus (367) Google Scholar). However, animal studies have yielded conflicting results, with some studies demonstrating that a diet enriched in n-6 PUFAs decreases adipose tissue mass (3Matsuo T. Takeuchi H. Suzuki H. Suzuki M. Asia Pac. J. Clin. Nutr. 2002; 11: 302-308Crossref PubMed Scopus (40) Google Scholar, 4Okuno M. Kajiwara K. Imai S. Kobayashi T. Honma N. Maki T. Suruga K. Goda T. Takase S. Muto Y. Moriwaki H. J. Nutr. 1997; 127: 1752-1757Crossref PubMed Scopus (119) Google Scholar), whereas others have associated intake of n-6 PUFAs with an increased propensity for obesity (5Cleary M. Phillips F. Morton R. Proc. Soc. Exp. Biol. Med. 1999; 220: 153-161Crossref PubMed Google Scholar, 6Massiera F. Saint-Marc P. Seydoux J. Murata T. Kobayashi T. Narumiya S. Guesnet P. Amri E.Z. Negrel R. Ailhaud G. J. Lipid Res. 2003; 44: 271-279Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 7Prentice A.M. Obes. Res. 2001; 9: 234S-238SCrossref PubMed Scopus (53) Google Scholar).Adipose tissue increases in size by hypertrophy of preexisting adipocytes and recruitment and differentiation of new adipocytes from a preadipocyte population (8Faust I.M. Johnson P.R. Stern J.S. Hirsch J. Am. J. Physiol. 1978; 235: E279-E286Crossref PubMed Google Scholar). The dichotomy of action of n-6 PUFAs in feeding experiments is mirrored by the dichotomy of the effects of arachidonic acid on fat cell differentiation in vitro. On one hand, arachidonic acid has been identified as one of the adipogenic components of serum and is required for induction of differentiation of 3T3-F442A cells and Ob1771 preadipose cells (9Gaillard D. Negrel R. Lagarde M. Ailhaud G. Biochem. J. 1989; 257: 389-397Crossref PubMed Scopus (162) Google Scholar). On the other hand, arachidonic acid and its metabolites generated by cyclooxygenases (COXs) inhibit differentiation of primary preadipocytes (10Serrero G. Lepak N.M. Goodrich S.P. Biochem. Biophys. Res. Commun. 1992; 183: 438-442Crossref PubMed Scopus (42) Google Scholar), 1246 cells (11Serrero G. Lepak N.M. Goodrich S.P. Endocrinology. 1992; 131: 2545-2551Crossref PubMed Google Scholar), and 3T3-L1 cells (12Miller C.W. Casimir D.A. Ntambi J.M. Endocrinology. 1996; 137: 5641-5650Crossref PubMed Scopus (0) Google Scholar).In the present study, we present data that reconcile and explain the disparate effects of n-6 PUFAs on adipocyte differentiation in vitro and in vivo. We demonstrate that cAMP signaling plays a pivotal role controlling the production of antiadipogenic prostaglandins. In vivo, the obesigenic action of n-6 PUFAs is determined by the balance between dietary carbohydrates and protein. A high carbohydrate/protein ratio translated into a high plasma insulin/glucagon ratio, and in this setting, dietary n-6 PUFAs promoted strongly adipose tissue expansion. Conversely, a high protein/carbohydrate ratio translated into a high plasma glucagon/insulin ratio and enhanced cAMP-dependent signaling. In this setting, COX-mediated prostaglandin synthesis was enhanced, and dietary n-6 PUFAs decreased white adipose tissue mass. The decreased obesigenic action of n-6 PUFAs in mice fed a protein-rich diet did not result from increased dissipation of energy by uncoupled respiration but rather reflected increased energy expenditure in relation to gluconeogenesis and urea formation.EXPERIMENTAL PROCEDURESCell Culture and Differentiation—3T3-L1 cells were cultured and induced to differentiate by 0.5 mm methylisobutylxanthine, 1 μm dexamethasone, 1 μg/ml insulin (MDI) as previously described (13Hansen J.B. Zhang H. Rasmussen T.H. Petersen R.K. Flindt E.N. Kristiansen K. J. Biol. Chem. 2001; 276: 3175-3182Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Fatty acids, prostaglandins, and inhibitors were dissolved in Me2SO and added when differentiation was induced unless otherwise stated in the figure legends. Cells not treated received similar volumes of vehicle. Staining of lipid by Oil Red-O was performed as described previously (13Hansen J.B. Zhang H. Rasmussen T.H. Petersen R.K. Flindt E.N. Kristiansen K. J. Biol. Chem. 2001; 276: 3175-3182Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar).Plasmids—Wild type and CRE site-mutated COX-2 promoter luciferase reporter constructs were kindly provided by Dr. H. R. Hershman (14Wadleigh D.J. Reddy S.T. Kopp E. Ghosh S. Herschman H.R. J. Biol. Chem. 2000; 275: 6259-6266Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). β-Galactosidase expression vector pCMVβ is from Clontech. Retroviral vectors pLXSN-hygro and pBabe-puro were kindly obtained from Dr. O. A. MacDougald. pLXSN-COX-1 was nondirectionally cloned as a HindIII fragment from pSVL-COX-1 into HindIII-digested pLXSN-hygro. pBabe-COX-2 was made by directional cloning of the BamHI/XbaI fragment from pcDNA3-COX-2 into BamHI/XbaI-digested pBabe-puro.Transient Transfection—Preconfluent 3T3-L1 cells were transfected at 50-75% confluence with 0.95 μg of wild type or CRE site-mutated COX-2 promoter luciferase reporter constructs (14Wadleigh D.J. Reddy S.T. Kopp E. Ghosh S. Herschman H.R. J. Biol. Chem. 2000; 275: 6259-6266Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) and 0.05 μg of β-galactosidase expression vector for normalization (pCMVβ; Clontech) per well (6-well plates) using METAFECTENE™ (Biotex). Six hours after transfection, the medium was changed, and cells treated with vehicle (0.1% Me2SO), 0.5 mm methylisobutylxanthine, and/or 100 μm arachidonic acid. Twenty-four hours after transfection cells were harvested, and luciferase and β-galactosidase activities were measured as described (13Hansen J.B. Zhang H. Rasmussen T.H. Petersen R.K. Flindt E.N. Kristiansen K. J. Biol. Chem. 2001; 276: 3175-3182Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Retrovirus production and transduction was performed as described earlier (13Hansen J.B. Zhang H. Rasmussen T.H. Petersen R.K. Flindt E.N. Kristiansen K. J. Biol. Chem. 2001; 276: 3175-3182Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar).Prostaglandin Levels in Culture Supernatants—PGE2 and PGF2α were determined as described previously (15Muller-Decker K. Furstenberger G. Marks F. Toxicol. Appl. Pharmacol. 1994; 127: 99-108Crossref PubMed Scopus (63) Google Scholar) using 1 ml of medium and following the instructions of the manufacturer of the PGE2- and PGF2α-specific enzyme immunoassays (Cayman).Animals—Male C57BL/6JBomTac mice ∼6 weeks of age were obtained from Taconic Europe (Ejby, Denmark) and were divided into groups (n = 6). The mice were kept at a 12-h light/dark cycle at 22 °C. After acclimatization, the animals were fed ad libitum or pair-fed experimental diets. The compositions of the diets are presented in supplemental Table 1. Corn oil was chosen as the n-6 fatty acid source, since this oil is particularly enriched in linoleic acid, and analysis of the diet confirmed that more than 50% of the fatty acids in the diets were linoleic acid (supplemental Table 2). The diet did not contain arachidonic acid, but analysis of the fatty acid composition of red blood cells confirmed conversion of ingested n-6 PUFAs to arachidonic acid (supplemental Table 2). Body weight was recorded twice a week. Mice were killed by cardiac puncture under anesthesia (Dormitor (1 mg/kg body weight) and Ketalar (75 mg/kg body weight)), and serum was prepared from blood. Tissues were dissected out, freeze-clamped, and frozen at -80 °C.Analyses in Computerized Metabolic Cages—Male C57BL/6J mice 6 weeks of age were purchased from Charles River Laboratories (Maastricht, The Netherlands). Animals received a standard chow diet (AIN93G/95). After 1 week of acclimatization in the experimental facility, the animals were fed a corn oil diet supplemented with sucrose (n = 8) or protein (n = 8) for a period of 5 weeks before the start of the metabolic cage experiments. Mice were acclimatized to the metabolic cage environment for 1 day prior to starting of the monitoring period. During the metabolic cage experiment, oxygen consumption, CO2 production, food intake, and activity (x-y-z-axis) were measured as described elsewhere (16den Hoek van A.M. Heijboer A.C. Voshol P.J. Havekes L.M. Romijn J.A. Corssmit E.P.M. Pijl H. Am. J. Physiol. 2007; 292: E238-E245Google Scholar).Serum Analysis—Glucose was determined enzymatically with reagents from Dialab; insulin was determined with the mouse insulin ELISA kit (EIA 3439) from DRG Diagnostics; glucagon was determined with a radioimmune assay kit (catalog number GL-32K; Linco); and prostaglandins were determined with the PGF2α immunoassay (catalog number DE1150) and PGE2 immunoassay (catalog number DE0100) from R&D Systems.Real Time RT-qPCR—Total RNA was purified from mouse tissue or cells using Trizol, and cDNA was synthesized and analyzed by real time qPCR using the ABI PRISM 7700 sequence detection system (Applied Biosystems) as described earlier (17Madsen L. Petersen R.K. Sørensen M.B. Jørgensen C. Hallenborg P. Pridal L. Fleckner J. Amri E.-Z. Krieg P. Furstenberger G. Berge R.K. Kristiansen K. Biochem. J. 2003; 375: 539-549Crossref PubMed Scopus (114) Google Scholar). Primers for real time PCR (supplemental Table 3) were designed using Primer Express 2.0 (Applied Biosystems).Western Blotting—Preparation of extracts from mouse tissue or whole cell dishes, electrophoresis, blotting, visualization, and stripping of membranes was performed as described (13Hansen J.B. Zhang H. Rasmussen T.H. Petersen R.K. Flindt E.N. Kristiansen K. J. Biol. Chem. 2001; 276: 3175-3182Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Primary antibodies used were goat anti-COX-1, goat anti-COX-2, rabbit anti-PGC-1α, rabbit anti-TFIIB (all from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA)), mouse anti-phospho-CREB (Upstate Biotechnology, Inc.), and mouse anti-Vimentin (DAKO). Secondary antibodies were horseradish peroxidase-conjugated anti-mouse, anti-goat, or anti-rabbit antibodies obtained from DAKO.Fatty Acid Composition—The fatty acids in diets and isolated red blood cells were extracted with chloroform/methanol (2:1) (v/v) and saponified and methylated using 12% BF3 in methanol. The fatty acid composition of total lipids was analyzed on a gas chromatograph-mass spectrometer as previously described (18Arslan G. Brunborg L.A. Frøyland L. Brun J.G. Valen M. Berstad A. Lipids. 2002; 37: 935-940Crossref PubMed Scopus (33) Google Scholar).Urea and Amino Acid Levels—Liver samples were homogenized and deproteinated in 10% sulfosalicylic acid. Free amino acids and urea were analyzed on an amino acid analyzer (Biochrom 20 plus, Cambridge, UK).Energy in Feces and Diets—The energy content was determined in a bomb calorimeter following the manufacturer's instruction (Parr Instruments, Moline, IL).Statistics—Data represent mean ± S.E. Analysis of variance was performed by post hoc pairwise comparison: Student's t test (RT-PCR analysis), Tukey HSD test (relative eWAT weight and absolute liver weight), and Newman-Keuls test due to nonhomogenous variances (rest of data). Data were considered statistically significant when p was <0.05.RESULTSExpression of Cyclooxygenases and Production of Antiadipogenic Prostaglandins Are Regulated by cAMP in 3T3-L1 Cells—n-6 PUFAs, including arachidonic acid, have been reported to act both pro- and antiadipogenically in vitro and in vivo (5Cleary M. Phillips F. Morton R. Proc. Soc. Exp. Biol. Med. 1999; 220: 153-161Crossref PubMed Google Scholar, 6Massiera F. Saint-Marc P. Seydoux J. Murata T. Kobayashi T. Narumiya S. Guesnet P. Amri E.Z. Negrel R. Ailhaud G. J. Lipid Res. 2003; 44: 271-279Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 7Prentice A.M. Obes. Res. 2001; 9: 234S-238SCrossref PubMed Scopus (53) Google Scholar). These disparate findings may be explained by our previous finding that the effect of n-6 PUFAs depends on the intracellular level of cAMP (19Petersen R.K. Jorgensen C. Rustan A.C. Froyland L. Muller-Decker K. Furstenberger G. Berge R.K. Kristiansen K. Madsen L. J. Lipid Res. 2003; 44: 2320-2330Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Accordingly, in the presence of the cAMP-elevating agent methylisobutylxanthine (MIX), arachidonic acid inhibited adipogenesis (Fig. 1A). Inhibition of PKA activity by (Rp)-cAMPS/(Rp)-8-Br-cAMPS or of cyclooxygenase activity by indomethacin restored adipocyte differentiation in the presence of arachidonic acid and MIX (Fig. 1A). By contrast, in the absence of MIX, arachidonic acid promoted adipogenesis, which, however, was inhibited by selective activation of PKA by N6-monobutyryl-cAMP (6-MB-cAMP). Inclusion of indomethacin rescued differentiation in the presence of 6-MB-cAMP (Fig. 1B).COX-2 protein expression is transiently induced when adipocyte differentiation is induced by MDI treatment (19Petersen R.K. Jorgensen C. Rustan A.C. Froyland L. Muller-Decker K. Furstenberger G. Berge R.K. Kristiansen K. Madsen L. J. Lipid Res. 2003; 44: 2320-2330Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). A cAMP-responsive element (CRE) has been identified in the COX-2 promoter (20Xie W. Fletcher B. Andersen R. Herschman H.R. Mol. Cell. Biol. 1994; 14: 6531-6539Crossref PubMed Scopus (189) Google Scholar), and hence it is likely that COX-2 expression is regulated via cAMP-dependent signaling in 3T3-L1 preadipocytes. Treatment with the cAMP-elevating agent MIX induced the expression of a luciferase reporter gene driven by the wild-type COX-2 promoter transiently transfected into 3T3-L1 preadipocytes, whereas no induction was observed when the regulatory CRE element in the COX-2 promoter was mutated. Of note, combined treatment with MIX and arachidonic acid resulted in a greater activation than treatment with MIX alone (Fig. 1C).To further analyze the interplay between cAMP and arachidonic acid in regulation of COX expression, 3T3-L1 cells were induced to differentiate in the absence or presence of MIX with or without arachidonic acid, and RNA was harvested at different time points. A combined treatment with arachidonic acid and MIX led to a strong and sustained expression of both COX-1 and COX-2 during initiation of differentiation, whereas treatment with arachidonic acid or MIX alone led to a weak induction of COX-1 and COX-2 expression (Fig. 1D). As anticipated, omitting MIX from the induction mixture abolished the transient induction of COX-2 (and COX-1). Together, these results suggest the existence of a regulatory circuit by which cAMP/PKA-dependent induction of COX expression sensitizes the cell to an inhibitory action of arachidonic acid, which depends on COX activity, and further that the synthesized prostaglandins feed back, securing sustained expression of COX-1 and COX-2.To corroborate the existence of such a regulatory circuit in mediating an inhibitory effect of arachidonic acid on adipocyte differentiation, COX-1 and COX-2 were retrovirally expressed, alone or in combination in 3T3-L1 preadipocytes. Retroviral expression was confirmed by Western blotting (Fig. 1E). Since forced expression of COX-1 induced COX-2 expression, a selective COX-2 inhibitor (NS398) was added to the COX-1-expressing cells. Similarly, cells with forced expression of COX-2 were treated with a selective COX-1 inhibitor (SC560). Twenty-four hours after the induction of differentiation, media were collected and analyzed for the main prostaglandins produced by 3T3-L1 cells (21Hyman B.T. Stoll L.L. Spector A.A. Biochim. Biophys. Acta. 1982; 713: 375-385Crossref PubMed Scopus (66) Google Scholar). Forced expression of COX-1 or COX-2 alone did not per se enhance prostaglandin synthesis, but when COX-1 and COX-2 were simultaneously expressed, the production of PGE2 and PGF2α was increased. However, exogenous arachidonic acid was required to boost the synthesis of prostaglandins (Fig. 1F). In accordance with this, forced expression of the COXs was not able to inhibit MDI-induced differentiation per se (Fig. 1G) but sensitized 3T3-L1 cells for arachidonic acid-mediated inhibition of differentiation, as indicated by the lack of Oil Red-O staining of cells expressing COX-1 and COX-2 treated with 30 μm arachidonic acid (Fig. 1G).If the role of cAMP/PKA signaling in mediating arachidonic acid-dependent inhibition of adipocyte differentiation is solely linked to induction of COX expression, forced expression of the COXs should alleviate the requirement for elevated cAMP levels in arachidonic acid-mediated inhibition of adipocyte differentiation. In keeping with this prediction, arachidonic acid completely prevented adipocyte differentiation of 3T3-L1 cells with forced expression of COX-1 and COX-2 also in the absence of MIX (Fig. 1G). Finally, both PGF2α and PGE2 were able to inhibit differentiation in the absence or presence of MIX (Fig. 1, H and I), providing further evidence for the importance of the cAMP-PKA-COX-prostaglandin axis in regulating the effect of arachidonic acid on adipocyte differentiation.The Effect of Corn Oil on Body Weight and Adipose Tissue Mass Is Regulated by the Balance between Carbohydrate and Protein in the Feed—As for in vitro studies, fundamentally opposite effects of n-6 fatty acids on adipose tissue development in vivo have been reported. Some studies have demonstrated that a diet enriched in n-6 PUFAs decreases adipose tissue growth (3Matsuo T. Takeuchi H. Suzuki H. Suzuki M. Asia Pac. J. Clin. Nutr. 2002; 11: 302-308Crossref PubMed Scopus (40) Google Scholar, 4Okuno M. Kajiwara K. Imai S. Kobayashi T. Honma N. Maki T. Suruga K. Goda T. Takase S. Muto Y. Moriwaki H. J. Nutr. 1997; 127: 1752-1757Crossref PubMed Scopus (119) Google Scholar), whereas other studies have associated dietary n-6 PUFAs with an increased propensity to obesity (5Cleary M. Phillips F. Morton R. Proc. Soc. Exp. Biol. Med. 1999; 220: 153-161Crossref PubMed Google Scholar, 6Massiera F. Saint-Marc P. Seydoux J. Murata T. Kobayashi T. Narumiya S. Guesnet P. Amri E.Z. Negrel R. Ailhaud G. J. Lipid Res. 2003; 44: 271-279Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 7Prentice A.M. Obes. Res. 2001; 9: 234S-238SCrossref PubMed Scopus (53) Google Scholar). Since the adipogenic potential of n-6 PUFAs is dependent on the cAMP status in vitro, we hypothesize that the hormonal status, such as the glucagon/insulin ratio in particular, might be of importance in regulating the effect of n-6 PUFAs on adipose tissues also in vivo. Since the glucagon/insulin ratio is altered in response to intake of carbohydrates versus protein, we predicted that the adipogenic effect of n-6 PUFAs might be determined by the ratio between carbohydrates and protein in the feed.To test this hypothesis, obesity-prone C57BL/6J mice were fed an energy-dense high fat diet enriched in n-6 fatty acids (corn oil), supplemented with either protein or sucrose (supplemental Tables 1 and 2) for 53 days. The C57BL/6J mice were chosen in order to limit adaptive thermogenesis that occurs in most mice strains when fed an energy-dense diet (22Watson P.M. Commins S.P. Beiler R.J. Hatcher H.C. Gettys T.W. Am. J. Physiol. 2000; 279: E356-E365Crossref PubMed Google Scholar). Corn oil was chosen as an n-6 fatty acid source, since this oil is enriched in linoleic acid, the predominant PUFA in Western diets (23Zhou L. Nilsson A. J. Lipid Res. 2001; 42: 1521-1542Abstract Full Text Full Text PDF PubMed Google Scholar). Analysis of the diet confirmed that more than 50% of the fatty acids in the diets were linoleic acid (supplemental Table 2). The diet did not contain arachidonic acid, but analysis of the fatty acid composition of red blood cells confirmed conversion of the dietary n-6 PUFAs to arachidonic acid (supplemental Table 2). The corn oil-enriched diets were isocaloric and contained a total of 24.3 ± 0.3 and 24.9 ± 0.1 weight % fat, respectively. It should be noted that the sucrose-enriched diet contained 20 weight % protein and hence was not protein-deficient.Mice fed the sucrose-supplemented corn oil diet ad libitum gained considerably more weight than mice fed the high protein-supplemented corn oil diet (Fig. 2A). The higher total body weight gain in mice fed corn oil in combination with sucrose was to a large extent due to an increase in white adipose tissue mass (Fig. 2A).FIGURE 2A high corn oil diet supplemented with sucrose, but not protein, induces obesity in C57BL/6J mice. C57BL/6J mice (n = 6) were fed a high corn oil diet supplemented with protein or sucrose ad libitum. After 53 days, the experiment was terminated, and the mice and adipose tissue were weighted (A). B-H, C57BL/6J mice (n = 6) were pair-fed a high corn oil-supplemented diet with protein or sucrose, whereas a third group received a normal chow diet. The weight was recorded twice per week (B). After 56 days, the experiment was terminated, the mice were photographed, and adipose tissues (interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (iWAT), and epididymal white adipose tissue (eWAT)) were dissected out and weighed (C). D, glucose, insulin, and glucagon levels in serum. Expression of the cAMP-responsive element modulator (CREM) and cAMP-specific phosphodiesterase 4b (PDE4b) was measured by RT-qPCR and normalized to TATA-box-binding protein (TBP) in adipose tissue (E). Phosphorylation of CREB was determined by Western blotting. Expression of vimentin verified equal loading of protein (F). Expression of COX-1 and COX-2 in adipose tissues was measured by RT-qPCR. The expression in each animal was normalized to TBP (G). H, serum PGE2 and PGF2α.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To evaluate whether the different effect of the diets could be explained by altered energy expenditure and/or voluntary activity of the animals, the mice were individually housed in metabolic cages. No difference in oxygen consumption was found between the two groups, but the respiratory exchange rate was lower in mice fed corn oil in combination with protein than with sucrose (Fig. 3), indicating that relatively more fat and possibly protein were used as substrates for oxidation. However, no increase in expression of key enzymes involved in fatty acid oxidation in muscle or liver was observed (Fig. 4). In fact, a lower heat production, indicating lower total energy expenditure, was observed in mice fed the protein-supplemented diet (Fig. 3). This was not an effect of reduced animal activity but could partly be explained by the observed lower food intake (Fig. 3).FIGURE 3Metabolic parameters of mice fed corn oil in combination with protein or sucrose. Obesityprone C57BL/6J mice (n = 8) were fed a high corn oil diet supplemented with protein or sucrose. After 5 weeks, respiratory exchange ratio, heat production, oxygen consumption, total X activity, accumulated feeding, and accumulated drinking were measured during a 50-h period.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4The expression of genes involved in fatty acid oxidation is similar in liver and muscle of mice fed corn oil in combination with protein or sucrose. Obesity-prone C57BL/6J mice (n = 8) were fed ad libitum a high corn oil diet supplemented with protein or sucrose. After 5 weeks, liver and muscle (gastrocnemius/plantaris) were collected. RNA was isolated, and cDNA was synthesized from each individual animal. Expression of CPT1, acyl-CoA oxidase (ACO), UCP1, UCP2, and UCP3 was measured by RT-qPCR.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A high protein intake is known to increase satiety and thereby to reduce energy intake (24Halton T.L. Hu F.B. J. Am. Coll. Nutr. 2004; 23: 373-385Crossref PubMed Scopus (572) Google Scholar, 25Westerterp-Plantenga M.S. Lejeune M.P.G.M. Appetite. 2005; 45: 187-190Crossref PubMed Scopus (55) Google Scholar). Thus, in order to exclude the possibility that reduced adipose tissue mass in mice fed corn oil and protein ad libitum was simply due to reduced caloric intake, a third set of mice was pair-fed the same diets for 56 days. The mice fed corn oil in combination with sucrose gained an average of 11.3 g of body weight and became visibly obese (Fig. 2, B and C, and Table 1). The mice fed corn oil in combination with protein gained on average less than 1.8 g of body weight during the 56 days of feeding and had small amounts of white adipose tissue (Table 2 and Fig. 2, B and C). In fact, the weight gain and amount of body fat in mice fed a high corn oil diet supplemented with protein was comparable with the body weight gain and adipose tissue mass in mice fed an energy-restricted low fat chow diet (Fig. 2, B and C, and Table 1).TABLE 1Feed intake, weight gain, and energy efficiency in pair-fed miceChowHigh sucrose plus corn oilHigh protein plus corn oilFeed intake (g)Total142.4 ± 0.7140.9 ± 0.3137.1 ± 2.4Daily2.66 ± 0.012.63 ± 0.062.56 ± 0.04Energy intake (kcal)Total634 ± 3aDifferent letters indicate significant differences (p < 0.05).767 ± 16bDifferent letters indicate significant differences (p < 0.05).842 ± 14cDifferent letters indicate significant differences (p < 0.05).Daily12.0 ± 0.1aDifferent letters indicate significant differences (p < 0.05).14.5 ± 0.3bDiff
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