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• W2169198250 abstract "ABCD2 (D2) is a peroxisomal transporter that is highly abundant in adipose tissue and promotes the oxidation of long-chain MUFA. Erucic acid (EA, 22:1ω9) reduces very long chain saturated fatty acids in patients with X-linked adrenoleukodystrophy but promotes dyslipidemia and dilated cardiomyopathy in rats. To determine the role of D2 in the metabolism of EA, we challenged wild-type and D2 deficient mice (D2 KO) with an enriched EA diet. In D2 KO mice, dietary EA resulted in the rapid expansion of adipose tissue, adipocyte hypertrophy, hepatic steatosis, and the loss of glycemic control. However, D2 had no impact on the development of obesity phenotypes in two models of diet-induced obesity. Although there was a significant increase in EA in liver of D2 KO mice, it constituted less than 2% of all fatty acids. Metabolites of EA (20:1, 18:1, and 16:1) were elevated, particularly 18:1, which accounted for 50% of all fatty acids. These data indicate that the failure to metabolize EA in adipose results in hepatic metabolism of EA, disruption of the fatty acid profile, and the development of obesity and reveal an essential role for D2 in the protection from dietary EA. ABCD2 (D2) is a peroxisomal transporter that is highly abundant in adipose tissue and promotes the oxidation of long-chain MUFA. Erucic acid (EA, 22:1ω9) reduces very long chain saturated fatty acids in patients with X-linked adrenoleukodystrophy but promotes dyslipidemia and dilated cardiomyopathy in rats. To determine the role of D2 in the metabolism of EA, we challenged wild-type and D2 deficient mice (D2 KO) with an enriched EA diet. In D2 KO mice, dietary EA resulted in the rapid expansion of adipose tissue, adipocyte hypertrophy, hepatic steatosis, and the loss of glycemic control. However, D2 had no impact on the development of obesity phenotypes in two models of diet-induced obesity. Although there was a significant increase in EA in liver of D2 KO mice, it constituted less than 2% of all fatty acids. Metabolites of EA (20:1, 18:1, and 16:1) were elevated, particularly 18:1, which accounted for 50% of all fatty acids. These data indicate that the failure to metabolize EA in adipose results in hepatic metabolism of EA, disruption of the fatty acid profile, and the development of obesity and reveal an essential role for D2 in the protection from dietary EA. Peroxisomes are essential for β-oxidation of very long chain fatty acids (VLCFAs) and α-oxidation of branched chain fatty acids (1.Wanders R.J. Waterham H.R. Biochemistry of mammalian peroxisomes revisited.Annu. Rev. Biochem. 2006; 75: 295-332Crossref PubMed Scopus (709) Google Scholar). However, peroxisomal fatty acid oxidation is incomplete, requiring chain-shortened fatty acyl-CoAs to be shuttled out of peroxisomes and into mitochondria for complete oxidation. Peroxisomes are also required for anabolic lipid metabolism, including the synthesis of ether lipids and some PUFAs. The endogenous synthesis of docosapentaenoic acid (22:5ω6) and docosahexaenoic acid (C22:6ω3, DHA) begins with the essential fatty acids linoleate (9,12-18:2) and linolenate (9,12,15-18:3), respectively (2.Sprecher H. Metabolism of highly unsaturated n-3 and n-6 fatty acids.Biochim. Biophys. Acta. 2000; 1486: 219-231Crossref PubMed Scopus (655) Google Scholar). Due to the limited types of desaturases in mammals (Δ5, Δ6, and Δ9), elongation and desaturation in the endoplasmic reticulum generates 6,9,12,15,18,21-24:6 and 6,9,12,15,18-24:5, both of which must be transported into peroxisomes and undergo one round of β-oxidation to yield 22 carbon PUFAs (1.Wanders R.J. Waterham H.R. Biochemistry of mammalian peroxisomes revisited.Annu. Rev. Biochem. 2006; 75: 295-332Crossref PubMed Scopus (709) Google Scholar, 3.Su H-M. Moser A.B. Moser H.W. Watkins P.A. Peroxisomal straight-chain Acyl-CoA oxidase and D-bifunctional protein are essential for the retroconversion step in docosahexaenoic acid synthesis.J. Biol. Chem. 2001; 276: 38115-38120Abstract Full Text Full Text PDF PubMed Google Scholar). A second round of peroxisomal oxidation of yields arachidonic acid (20:4ω6, AA) and eicosapentanoic acid (20:5ω3), but peroxisomal oxidation is not essential for the synthesis of these lipids. The entry of fatty acids into peroxisomes is thought to be dependent upon their esterification by a peroxisomal acyl-CoA synthetase followed by their transmembrane transport by members of the D-subfamily of ABC transporters (4.Coleman R.A. Lewin T.M. Van Horn C.G. Gonzalez-Baró M.R. Do long-chain Acyl-CoA synthetases regulate fatty acid entry into synthetic versus degradative pathways?.J. Nutr. 2002; 132: 2123-2126Crossref PubMed Scopus (248) Google Scholar, 5.Wanders R.J. Visser W.F. van Roermund C.W. Kemp S. Waterham H.R. The peroxisomal ABC transporter family.Pflugers Arch. 2007; 453: 719-734Crossref PubMed Scopus (80) Google Scholar). Among the four members of this subfamily, only ABCD1 (D1) has been associated with human disease, X-linked adrenoleukodystrophy (ALD). ALD is a pleiotropic disorder characterized by the accumulation of VLCFAs in plasma and tissues that presents with varying neurological, adrenocortical, and Leydig cell deficiencies (6.Cappa M. Bizzarri C. Vollono C. Petroni A. Banni S. Adrenoleukodystrophy.Endocr. Dev. 2011; 20: 149-160Crossref PubMed Scopus (18) Google Scholar, 7.Ferrer I. Aubourg P. Pujol A. General aspects and neuropathology of X-linked adrenoleukodystrophy.Brain Pathol. 2010; 20: 817-830Crossref PubMed Scopus (98) Google Scholar). The closest paralog of D1 is D2, which shares greater than 60% amino acid identity in humans and mice and at least some overlapping function in vitro and in vivo (8.Holzinger A. Kammerer S. Berger J. Roscher A.A. cDNA cloning and mRNA expression of the human adrenoleukodystrophy related protein (ALDRP), a peroxisomal ABC transporter.Biochem. Biophys. Res. Commun. 1997; 239: 261-264Crossref PubMed Scopus (73) Google Scholar–12.Fourcade S. Ruiz M. Camps C. Schluter A. Houten S.M. Mooyer P.A. Pampols T. Dacremont G. Wanders R.J. Giros M. et al.A key role for the peroxisomal ABCD2 transporter in fatty acid homeostasis.Am. J. Physiol. Endocrinol. Metab. 2009; 296: E211-E221Crossref PubMed Scopus (81) Google Scholar). Although direct evidence for transmembrane transport of fatty acyl-CoA substrates is lacking, human D1 and D2 transgenes have been shown to rescue growth in oleate-containing media and to promote the oxidation of distinct subsets of fatty acids in yeast lacking endogenous peroxisomal fatty acyl-CoA transporters (13.van Roermund C.W. Visser W.F. Ijlst L. van Cruchten A. Boek M. Kulik W. Waterham H.R. Wanders R.J. The human peroxisomal ABC half transporter ALDP functions as a homodimer and accepts acyl-CoA esters.FASEB J. 2008; 22: 4201-4208Crossref PubMed Scopus (181) Google Scholar, 14.van Roermund C.W.T. Visser W.F. Ijlst L. Waterham H.R. Wanders R.J.A. Differential substrate specificities of human ABCD1 and ABCD2 in peroxisomal fatty acid [beta]-oxidation.Biochim. Biophys. Acta. 2011; 1811: 148-152Crossref PubMed Scopus (99) Google Scholar). In this system, D2 was most active toward the 22-carbon saturated fatty acid and 22- and 24-carbon PUFAs (14.van Roermund C.W.T. Visser W.F. Ijlst L. Waterham H.R. Wanders R.J.A. Differential substrate specificities of human ABCD1 and ABCD2 in peroxisomal fatty acid [beta]-oxidation.Biochim. Biophys. Acta. 2011; 1811: 148-152Crossref PubMed Scopus (99) Google Scholar). Whereas D1 is constitutively expressed in many tissues, D2 is more restricted and dynamically regulated by transcription factors involved in lipogenesis, fatty acid oxidation, and cholesterol elimination. D2 mRNA and protein have been detected in brain, liver, lung, adrenal gland, and skeletal muscle but are most abundant in adipose tissue (9.Berger J. Albet S. Bentejac M. Netik A. Holzinger A. Roscher A.A. Bugaut M. Forss-Petter S. The four murine peroxisomal ABC-transporter genes differ in constitutive, inducible and developmental expression.Eur. J. Biochem. 1999; 265: 719-727Crossref PubMed Scopus (86) Google Scholar, 10.Pujol A. Ferrer I. Camps C. Metzger E. Hindelang C. Callizot N. Ruiz M. Pampols T. Giros M. Mandel J.L. Functional overlap between ABCD1 (ALD) and ABCD2 (ALDR) transporters: a therapeutic target for X-adrenoleukodystrophy.Hum. Mol. Genet. 2004; 13: 2997-3006Crossref PubMed Scopus (154) Google Scholar, 11.Lombard-Platet G. Savary S. Sarde C.O. Mandel J.L. Chimini G. A close relative of the adrenoleukodystrophy (ALD) gene codes for a peroxisomal protein with a specific expression pattern.Proc. Natl. Acad. Sci. USA. 1996; 93: 1265-1269Crossref PubMed Scopus (193) Google Scholar, 15.Liu J. Sabeva N.S. Bhatnagar S. Li X.A. Pujol A. Graf G.A. ABCD2 is abundant in adipose tissue and opposes the accumulation of dietary erucic acid (C22:1) in fat.J. Lipid Res. 2010; 51: 162-168Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Within adipose, D2 is restricted to adipocytes and is up-regulated during adipogenesis, presumably by the lipogenic transcription factor sterol receptor element binding protein 1 (SREBP1) (15.Liu J. Sabeva N.S. Bhatnagar S. Li X.A. Pujol A. Graf G.A. ABCD2 is abundant in adipose tissue and opposes the accumulation of dietary erucic acid (C22:1) in fat.J. Lipid Res. 2010; 51: 162-168Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 16.Weinhofer I. Kunze M. Rampler H. Bookout A.L. Forss-Petter S. Berger J. Liver X receptor alpha interferes with SREBP1c-mediated Abcd2 expression. Novel cross-talk in gene regulation.J. Biol. Chem. 2005; 280: 41243-41251Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Conversely, the expression of D2 is relatively low in liver, but is up-regulated by fasting and peroxisome proliferator activated receptor (PPAR) α agonists (9.Berger J. Albet S. Bentejac M. Netik A. Holzinger A. Roscher A.A. Bugaut M. Forss-Petter S. The four murine peroxisomal ABC-transporter genes differ in constitutive, inducible and developmental expression.Eur. J. Biochem. 1999; 265: 719-727Crossref PubMed Scopus (86) Google Scholar). Hepatic expression of D2 is suppressed by the liver X receptor (LXR), suggesting a role for D2 in cholesterol metabolism (16.Weinhofer I. Kunze M. Rampler H. Bookout A.L. Forss-Petter S. Berger J. Liver X receptor alpha interferes with SREBP1c-mediated Abcd2 expression. Novel cross-talk in gene regulation.J. Biol. Chem. 2005; 280: 41243-41251Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). However, the functional significance of this observation is unclear and has not been investigated. Despite its regulation by transcriptional factors essential for lipid homeostasis in liver and adipose, the physiological role for D2 in lipid metabolism remains largely unknown. The absence of D2 results in a modest accumulation of VLCFAs in liver after feeding with a diet enriched in saturated fat and after prolonged fasting, but these differences did not result in overt metabolic phenotypes, nor did they affect the expression of key metabolic enzymes (12.Fourcade S. Ruiz M. Camps C. Schluter A. Houten S.M. Mooyer P.A. Pampols T. Dacremont G. Wanders R.J. Giros M. et al.A key role for the peroxisomal ABCD2 transporter in fatty acid homeostasis.Am. J. Physiol. Endocrinol. Metab. 2009; 296: E211-E221Crossref PubMed Scopus (81) Google Scholar). Similarly, there is no apparent adipose dysfunction, and fatty acid profiles in adipose tissue were largely unaffected in D2 deficient mice (15.Liu J. Sabeva N.S. Bhatnagar S. Li X.A. Pujol A. Graf G.A. ABCD2 is abundant in adipose tissue and opposes the accumulation of dietary erucic acid (C22:1) in fat.J. Lipid Res. 2010; 51: 162-168Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). As in liver, adrenal, and neural tissues, there was a modest accumulation of 22- and 24-carbon saturated fatty acids (SFAs) and MUFAs. When acutely challenged with a diet enriched in erucic acid (EA) (22:1ω-9), there was a gene-dosage dependent increase in the levels in adipose and plasma (15.Liu J. Sabeva N.S. Bhatnagar S. Li X.A. Pujol A. Graf G.A. ABCD2 is abundant in adipose tissue and opposes the accumulation of dietary erucic acid (C22:1) in fat.J. Lipid Res. 2010; 51: 162-168Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). EA is abundant in native cultivars of rapeseed (Brassica napus) and is used clinically in combination with oleic acid to reduce plasma levels of VLCFA in patients with ALD due to its ability to inhibit the elongation of SFAs (6.Cappa M. Bizzarri C. Vollono C. Petroni A. Banni S. Adrenoleukodystrophy.Endocr. Dev. 2011; 20: 149-160Crossref PubMed Scopus (18) Google Scholar). However, it is also associated with a transient lipidosis, dyslipidemia, and the development of dilated cardiomyopathy in rats, effects not observed in mice and opposed by treatment with the PPARα agonist clofibrate (17.Bremer J. Norum K.R. Metabolism of very long-chain monounsaturated fatty acids (22:1) and the adaptation to their presence in the diet.J. Lipid Res. 1982; 23: 243-256Abstract Full Text PDF PubMed Google Scholar). We hypothesized that the absence of D2 would sensitize mice to disturbances in lipid metabolism by dietary EA. Our results indicate that in the absence of D2, dietary EA promotes obesity, insulin resistance, and steatosis. However, the absence of D2 had no impact on the development of obesity phenotypes when challenged with a high-fat (HF) diet or when crossed with leptin-deficient (ob/ob) mice. Although adipose tissue is greatly expanded after EA feeding, the lipid profile is only marginally affected with little accumulation of EA. Although there is a significant increase in EA in liver, it constitutes less than 2% of the total fatty acid pool. Conversely, hepatic SFA and MUFA content are elevated by 2- and 3-fold, respectively. These data suggest that in the absence of D2, peroxisomal metabolism of EA proceeds efficiently in the liver but that EA metabolites disrupt hepatic lipid metabolism, resulting in obesity, insulin resistance, and steatosis. Mice harboring the mutant Abcd2 allele are maintained on the C57BL/6J background as heterozygotes. Strain refreshing is conducted every five generations using C57BL6/J females obtained from The Jackson Laboratory (Bar Harbor, ME). Genotyping experiments to differentiate Abcd2-deficient (D2 KO) from heterozygous and wild-type mice were conducted as previously described (18.Ferrer I. Kapfhammer J. Hindelang C. Kemp S. Troffer-Charlier N. Broccoli V. Callyzot N. Mooyer P. Selhorst J. Vreken P. et al.Inactivation of the peroxisomal ABCD2 transporter in the mouse leads to late-onset ataxia involving mitochondria, Golgi and endoplasmatic reticulum damage.Hum. Mol. Genet. 2005; 214: 3565-3577Crossref Scopus (85) Google Scholar). Animals were housed in individually ventilated cages in a temperature-controlled room with 14:10 light:dark cycle and provided with enrichment in the form of acrylic huts and nesting material. All mice were maintained on standard rodent chow (Harlan Teklad 2014S) until initiation of experiments. All animal procedures conformed to PHS policies for humane care and use of laboratory animals and were approved by the institutional animal care and use committee at the University of Kentucky. The EA-enriched diet was custom formulated to contain levels of EA found in native rapeseed oil and contained 21.6% total energy from fat (Research Diets, New Brunswick, NJ) (Supplementary Tables I and II). EA accounted for 55.6% of total fatty acids as determined by GC-MS. The diet was stored at 4°C, provided ad libitum, and replaced twice weekly to limit oxidation. Diets were initiated at 8 weeks of age and continued until termination of the experiment after 8 weeks of feeding (n = 7). A second cohort of wild-type and D2 KO littermates were analyzed for the development of obesity phenotypes after low fat (LF) (10% kCal, Research Diets #D12450B, n = 10) or HF (45% kCal, Research Diets #D12451, n = 13 wt, 8 D2 KO) diets. Diets were initiated at 8 weeks of age. Mice were monitored weekly for weight gain and monthly for body composition. Mice were euthanized at 24 weeks (16 weeks on diet). D2 KO mice were also crossed to a leptin-deficient strain (Lepob, Strain #000632; The Jackson Laboratory), maintained on standard rodent chow, and analyzed at 8, 12, and 16 weeks of age (n = 11 ob/ob; n = 8 ob/ob D2 KO). Body composition was determined by EchoMRI at the initiation of the feeding period and the day before the termination of the study. During week 7, fasting blood glucose levels were measured using a standard glucometer from a drop of blood obtained by tail-vein prick after a 4 h fast beginning at lights-on. To determine glucose tolerance, mice were injected with sterilized 20% glucose solution (10 μl/g of body weight, i.p.). Blood glucose levels were measured before and 30, 60, 90 and 120 min after glucose injection. At termination of the experiments, mice were euthanized by exsanguination under ketamine/xylazine anesthesia after a 4 h fast beginning shortly after lights-on. Blood was collected from the right ventricle with a 1 ml syringe fitted with a 20 gauge hypodermic needle. Serum was separated by centrifugation and stored at −20°C. Tissues were excised, rinsed with PBS to remove blood, and snap frozen in liquid nitrogen. Liver, heart, and individual fat pats were dissected and weighed. Tissue samples were stored at −80°C. Additional tissue samples were immediately embedded in OCT, frozen on dry ice, and stored at −20°C or formalin fixed overnight and stored in 70% ethanol at 4°C until processing and histological analysis. Tissue was processed in a dehydrating ethanol gradient followed by xylene incubation and paraffin embedding. Paraffin blocks of tissue were cut into sections of 1 to 3 μm thickness and stained for hematoxylin and eosin (H&E). Frozen sections were cut at 10 μm thickness and subjected to Oil-Red-O staining. Total lipids from liver and adipose were analyzed as previously described (15.Liu J. Sabeva N.S. Bhatnagar S. Li X.A. Pujol A. Graf G.A. ABCD2 is abundant in adipose tissue and opposes the accumulation of dietary erucic acid (C22:1) in fat.J. Lipid Res. 2010; 51: 162-168Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Hepatic cholesterol and triglycerides were determined using enzymatic, colorometric assays. For GC/MS, total lipid extracts were saponified and esterified with 10% boron trifluoride in methanol (BF3-methanol 10%; Supelco, Bellefonte, PA). BF3 and methanol were removed, and samples were injected into a gas chromatography system (6890 GC G2579A system; Agilent, Palo Alto, CA) equipped with a split injection system with parallel OMEGAWAXTM 250 capillary columns (Supelco) and either an FID detector (quantitative) or Agilent 5973 network mass selective detector (target identity). The GC program was: injector: 1 µl at 10:1 spilit, 250°C; detector: FID, 260°C; oven: 160°C (5 min) to 220°C at 4°C/min; carrier: helium, 1.2 ml/min. Hepatic levels of diacylglycerol and ceramide were determined as previously described (19.Sumanasekera, C., Kelemen, O., Beullens, M., Aubol, B. E., Adams, J. A., Sunkara, M., Morris, A., Bollen, M., Andreadis, A., Stamm, S. 2011. C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1. Nucleic Acids Res. In press.Google Scholar, 20.Ren H. Federico L. Huang H. Sunkara M. Drennan T. Frohman M.A. Smyth S.S. Morris A.J. A phosphatidic acid binding/nuclear localization motif determines lipin1 function in lipid metabolism and adipogenesis.Mol. Biol. Cell. 2010; 21: 3171-3181Crossref PubMed Scopus (54) Google Scholar). The acyl-carnitine extraction method is a modification of the method previously described (21.van Vlies N. Tian L. Overmars H. Bootsma A.H. Kulik W. Wanders R.J. Wood P.A. Vaz F.M. Characterization of carnitine and fatty acid metabolism in the long-chain acyl-CoA dehydrogenase-deficient mouse.Biochem. J. 2005; 387: 185-193Crossref PubMed Scopus (73) Google Scholar). Tissues were weighed and homogenized in water and extracted with 80% acetonitrile with a series of vortexing, sonication, and centrifugation at 4000 rpm for 10 min. The supernatant was transferred into a vial, dried under nitrogen, and reconstituted with 100 μl of methanol. D3-C2, D3-C4, and D3-C16 acylcarnitines were used as internal standards for short- and long-chain acyl carnitines, respectively. Analyses of acylcarnitines was carried out using a Shimadzu UFLC coupled with an AB Sciex 4000-Qtrap hybrid linear ion trap triple quadrupole mass spectrometer in multiple reaction monitoring mode. Acyl carnitines were analyzed using an XTerra MS C8 3.5 μm, 3.0 × 100 mm column. The mobile phase consisted of 75/25 methanol/water, 0.5% formic acid + 0.1% ammonium formate as solvent A and 80% of (99/1 methanol/water, 0.5% formic acid + 0.1% ammonium formate): 20% chloroform as solvent B. For the analyses of acyl carnitines, the separation was achieved using a gradient of 0 to 100% solvent B in 10 min and maintaining at 100% B for the next 8 min. The column equilibrated back to the initial conditions in 3 min. The flow rate was 0.5 ml/min with a column temperature of 30°C. The sample injection volume was 10 uL. The mass spectrometer was operated in the positive electrospray ionization mode with optimal ion source settings determined by synthetic standards of acylcarnitines with a declustering potential of 86 V, entrance potential of 10 V, collision energy of 53 V, collision cell exit potential of 6 V, curtain gas of 20 psi, ion spray voltage of 5500 V, ion source gas1/gas2 of 40 psi, and temperature of 550°C. Multiple species of acyl carnitines wewre quantitated by monitoring species specific precursor product ion pairs. Fatty acid elongation was determined as described previously (22.Moon Y-A. Hammer R.E. Horton J.D. Deletion of ELOVL5 leads to fatty liver through activation of SREBP-1c in mice.J. Lipid Res. 2009; 50: 412-423Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Briefly, microsomes (30 μg total protein) were incubated in a reaction mixture containing 150 μM [14C] malonyl-CoA (American Radiolabeled Chemicals, Inc., St. Louis, MO) and 20 μM of the indicated fatty acids in the presence of 100 μM CoA, 1 mM ATP, and 1 mM NADPH at 37°C for 10 min. Fatty acids were extracted from the reaction mixture, and the total incorporation of [14C] malonyl-CoA to fatty acid substrates was measured by scintillation counting. Stearoyl CoA desaturase (SCD) activity was determined by measuring the amount of [1-14C] oleic acid generated from [1-14C] stearic acid as previously described (23.Garg M.L. Thomson A.B. Clandinin M.T. Effect of dietary cholesterol and/or omega 3 fatty acids on lipid composition and delta 5-desaturase activity of rat liver microsomes.J. Nutr. 1988; 118: 661-668Crossref PubMed Scopus (104) Google Scholar, 24.Garg M.L. Wierzbicki A.A. Thomson A.B. Clandinin M.T. Dietary cholesterol and/or n-3 fatty acid modulate delta 9-desaturase activity in rat liver microsomes.Biochim. Biophys. Acta. 1988; 962: 330-336Crossref PubMed Scopus (61) Google Scholar). The reaction medium contained 4 mM ATP, 100 μM CoA, 1.25 mM NADH, 500 μM nicotinamide, 5 mM MgCl2, 64 mM NaF, 1.5 mM glutathione, 64 mM potassium phosphate (pH 7.2), and 100 μM [1-14C] stearic acid (6650 dpm/nmol) as a BSA emulsion with a total reaction volume of 500 μl. The reaction was initiated by adding 0.3 mg mouse hepatic microsomes into the reaction medium. After incubation at 37°C in a water bath for 10 min, the reaction was stopped by adding 0.5 ml of 5 M potassium hydroxide in 10% methanol. Lipids were saponified, extracted with Folch reagent (chloroform/methanol 2:1 + 0.01% BHT (v/v/w), and dried under streaming nitrogen gas. Lipids were methylated in 1 ml of 14% BF3/methanol overnight at 55°C. Lipid esters were reextracted in 2 ml of chloroform, washed with 1 ml of water, and dried under nitrogen. Samples were loaded onto 10% argentinated TLC plates (#310496 56; Whatman, Piscataway, NJ). Plates were developed in hexane/diethyl ether (17:3, v/v), and radioactivity was measured by phosporimaging on a Typhoon FLA9000 Imaging System. SCD activity was expressed as nmol of oleate formed from stearate C18:0 per mg microsomal protein per hour. Membrane proteins were prepared and analyzed by SDS-PAGE and immunoblot analysis as previously described (25.Sabeva N.S. Rouse E.J. Graf G.A. Defects in the leptin axis reduce abundance of the ABCG5-ABCG8 sterol transporter in liver.J. Biol. Chem. 2007; 282: 22397-22405Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The isolation of total RNA and the determination of relative transcript abundance by quantitative real-time PCR for both tissues and cells was conducted as previously described (25.Sabeva N.S. Rouse E.J. Graf G.A. Defects in the leptin axis reduce abundance of the ABCG5-ABCG8 sterol transporter in liver.J. Biol. Chem. 2007; 282: 22397-22405Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Body composition data were analyzed by two-way repeated measures ANOVA. Bonferroni posts tests were used where indicated. Comparisons between genotypes for other measures were conducted with two-tailed t-tests. All statistical analyses were conducted using GraphPad Prism statistical analysis software. To determine the role of D2 in the metabolism of dietary EA, mice were challenged with an EA-enriched diet for 8 weeks beginning at 8 weeks of age. As previously reported, differences in body weight, body composition, and other measures of obesity and lipid metabolism were not observed at the initiation of the study (Fig. 1, Reference 15). After EA feeding, we observed a modest but significant increase in body weight in D2 KO mice compared with wild-type controls. Analysis of body composition by MRI indicated a significant increase in fat mass in D2 KO mice after 8 weeks on diet. The accumulation of excess adipose tissue occurred primarily in epididymal and retroperitoneal depots (Fig. 1B). Whereas lean mass increased in wild-type mice during EA feeding, it remained relatively constant in D2 KO and was significantly lower than in wild-type controls at the termination of the study (Fig. 1A). The expansion of adipose mass in the absence of an increase in lean mass resulted in a substantial increase in adiposity in D2 KO mice (Fig. 1C). Histological analysis of epididymal fat indicates that the expansion of adipose tissue was associated with the enlargement of adipocytes (Fig. 1D). Fasting glucose levels were increased in D2 KO mice compared with wild-type controls (Fig. 1E). A glucose tolerance test conducted after 7 weeks on diet revealed a decrease in glucose disposal consistent with the development of insulin resistance (Fig. 1F, G). Whereas fasting levels of triglycerides were unaffected, serum cholesterol was significantly elevated (Table 1).TABLE 1Serum lipid and adipokine levels in wild-type and D2 KO mice after an EA-enriched dietWild-typeD2 KOTriglycerides (mg/dl)24.5 ± 2.823.8 ± 3.0Cholesterol (mg/dl)67.05 ± 6.036125.7 ± 6.5 *Leptin (ng/ml)2.70 ± 1.1820.24 ± 6.53 *IL-6 (pg/ml)7.99 ± 0.8910.30 ± 2.31Resistin (pg/ml)2991 ± 1872,570 ± 245TNF-α (pg/ml)3,104 ± 2762,806 ± 348PAI-1 (pg/ml)3,104 ± 2762,806 ± 348Insulin (ng/ml)0.30 ± 0.060.299 ± 0.13 Open table in a new tab The expansion of adipose and the loss of glycemic control are generally associated with adipose tissue inflammation. Consistent with the expansion of adipose tissue, leptin levels were elevated in D2 KO mice (Table 1). However, we failed to detect any differences in other adipocytokines typically associated with inflammation. We also stained adipose tissue sections for CD68 and did not observe an accumulation of macrophages or the presence of crown-like structures (data not shown). These data suggest the absence of inflammation in D2 KO adipose tissue after EA feeding despite the increase in adipose mass and adipocyte hypertrophy. D2 is up-regulated in liver of obese and insulin-resistant mice, suggesting that D2 might modulate the development of obesity phenotypes in multiple mouse models (26.Kozawa S. Honda A. Kajiwara N. Takemoto Y. Nagase T. Nikami H. Okano Y. Nakashima S. Shimozawa N. Induction of peroxisomal lipid metabolism in mice fed a high-fat diet.Mol. Med. Report. 2011; 4: 1157-1162PubMed Google Scholar) (Supplementary Figure I). First, mice were challenged with a HF (45% kCal) diet for 12 weeks beginning at 8 weeks of age (Fig. 2). Although there was a modest increase in fat mass, we observed no differences in body weight, adiposity, fasting glucose, or glucose tolerance. There were also no differences in food intake or blood lipids (Supplementary Table III). We next crossed D2 deficient mice with the leptin-deficient (ob/ob) strain, maintained them on standard rodent chow, and evaluated them at 8, 12, and 16 weeks of age. As in HF feeding, there was a tendency toward increased fat mass at 12 weeks, but there were no differences in other measures of obesity phenotypes (Supplementary Figure II, Supplementary Table III). GC-MS analysis of the fatty acid profile in epididymal adipose showed that the relative abundance of EA was similar in wild-type and D2 KO mice and comprised less than 7% of the total fatty acids within adipose tissue (Fig. 3). The relative abundance of C18 and C20 MUFA was lower in D2 deficient mice and offset by an increase in C14 and C16 SFAs. The reduction in 20:1 and 18:1 in D2 KO mice is consistent with a reduction in β-oxidation and shortening of dietary EA before storage in adipose tissue triglycerides (TGs). However, given the increase in adipose tissue mass, the total amount of all fatty acids within adipose is greater in D2 KO than in wild-type mic" @default.
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