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Deletion of Murine Arv1 Results in a Lean Phenotype with Increased Energy Expenditure

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Deletion of Murine Arv1 Results in a Lean Phenotype with Increased Energy Expenditure OPEN Citation: Nutrition & Diabetes (2015) 5, e181; doi:10.1038/nutd.2015.32 www.nature.com/nutd ORIGINAL ARTICLE Deletion of murine Arv1 results in a lean phenotype with increased energy expenditure WR Lagor1,5, F Tong2,5, KE Jarrett1, W Lin2, DM Conlon2, M Smith2, MY Wang2, BO Yenilmez2, MG McCoy2, DW Fields2,SMO’Neill2, R Gupta1, A Kumaravel2, V Redon2, RS Ahima3, SL Sturley4, JT Billheimer2 and DJ Rader2 BACKGROUND: ACAT-related enzyme 2 required for viability 1 (ARV1) is a putative lipid transporter of the endoplasmic reticulum that is conserved across eukaryotic species. The ARV1 protein contains a conserved N-terminal cytosolic zinc ribbon motif known as the ARV1 homology domain, followed by multiple transmembrane regions anchoring it in the ER. Deletion of ARV1 in yeast results in defective sterol trafficking, aberrant lipid synthesis, ER stress, membrane disorganization and hypersensitivity to fatty acids (FAs). We sought to investigate the role of Arv1 in mammalian lipid metabolism. METHODS: Homologous recombination was used to disrupt the Arv1 gene in mice. Animals were examined for alterations in lipid and lipoprotein levels, body weight, body composition, glucose tolerance and energy expenditure. RESULTS: Global loss of Arv1 significantly decreased total cholesterol and high-density lipoprotein cholesterol levels in the plasma. Arv1 knockout mice exhibited a dramatic lean phenotype, with major reductions in white adipose tissue (WAT) mass and body weight on a chow diet. This loss of WAT is accompanied by improved glucose tolerance, higher adiponectin levels, increased energy expenditure and greater rates of whole-body FA oxidation. CONCLUSIONS: This work identifies Arv1 as an important player in mammalian lipid metabolism and whole-body energy homeostasis. Nutrition & Diabetes (2015) 5, e181; doi:10.1038/nutd.2015.32; published online 19 October 2015 INTRODUCTION AHD is cytosolic. This is followed by a transmembrane region, a ACAT (Acyl-CoA cholesterol acyl transferase) related enzyme 2 large loop region located inside the ER lumen, and two more required for viability 1 (ARV1) is a transmembrane protein of the transmembrane domains with the extreme C-terminus of the 10 endoplasmic reticulum (ER) that is conserved between plants, yeast protein extending back into the ER lumen (Figure 1a). The and mammals.1,2 ARV1 was originally identified in a screen for genes murine Arv1 gene encodes a 266 amino acid (AA) protein. In required for viability in the absence of ARE1 and ARE2—the yeast contrast to its 321 AA yeast counterpart, murine Arv1 differs in that homologs of Acyl-CoA cholesterol acyl transferase 1 and 2.2 Yeast the AHD is preceded by a N-terminal 28 amino acid leader deficient in ARV1 accumulate sterols in the ER, and show reduced sequence (5–6). Expression of human ARV1 functionally comple- sterol content in the plasma membrane based on biochemical ments the loss of ARV1 in yeast, rescuing all of the known growth 2,7 membrane fractionation,2 as well as filipin staining.3–6 Concomi- and viability defects. Despite this functional conservation, very tant with ER lipid retention, yeast lacking ARV1 also harbor major little is known about the biochemical activity of Arv1 in any defects in phospholipid, sphingolipid7 and glycosylphosphatidy- organism. In the current study, we sought to test the involvement linositol synthesis,4 and are particularly susceptible to sterol- of Arv1 in mammalian lipid metabolism by generating mice with a binding antibiotics such as nystatin2,8 and amphotericin B.6 Lipid germline deletion of the gene. To our surprise, Arv1 knockout accumulation in the ER in ARV1-deficient yeast is accompanied by (Arv1 KO) mice have a striking metabolic phenotype including severe ER stress and activation of the unfolded protein response.5 major reductions in white adipose tissue (WAT) mass, improved Most recently, Ruggles et al. performed a genome-wide screen for glucose tolerance and increased energy expenditure. These palmitoleate-induced fatty acid (FA) toxicity in yeast.9 Yeast strains findings are consistent with an important role for Arv1 in FA deleted for ARV1 are viable but highly sensitive to unsaturated homeostasis, and identify it as a novel regulator of body FAs. In this same study, overexpression of human ARV1 increased composition and energy expenditure in mice. lipid synthesis in HEK293 cells and mouse liver, whereas knock- down of Arv1 in MIN6 pancreatic β-cells and HEK293 cells inhibited lipogenesis and caused lipoapoptosis.9 MATERIALS AND METHODS The ARV1 protein contains a conserved N-terminal zinc ribbon Generation of Arv1 knockout mice motif known as the ‘Arv1 homology domain,’ (AHD) which is A genomic library derived from female 129S6/SvEvTac (Taconic, Hudson, followed by several transmembrane regions. The predicted and NY, USA) mice was screened with a cDNA probe corresponding to the Arv1 experimentally defined topology of the yeast Arv1p indicated the gene to identify positive BAC clones. Two BAC clones were used to 1Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA; 2Division of Translational Medicine and Human Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; 3Division of Endocrinology, Diabetes and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA and 4Department of Pediatrics, Columbia University Medical Center, New York, NY, USA. Correspondence: Dr DJ Rader, Division of Translational Medicine and Human Genetics, Perelman School of Medicine, University of Pennsylvania, 11–125 Smilow Translational Research Center, 3400 Civic Center Boulevard, Philadelphia, PA 19104, USA. E-mail [email protected] 5These authors contributed equally to this work. Received 7 June 2015; accepted 28 June 2015 A role for Arv1 in mammalian energy homeostasis WR Lagor et al 2 Probe A A A A A Wild Type 1 2 3 4 5 6 7.2 kb A A A A A Targeted 1 2 3 Neo 4 5 6 8.4 kb FLP Conditional 1 2 3 4 5 6 CRE Null 1 4 5 6 Arv1 mRNA +/+ Targeted * 1.5 * 8.4 kb - 1.0 -Actin 7.2 kb - β 1 / 0.5 Relative Qty Arv 0.0 ES Cells WT Het KO Figure 1. Generation of Arv1 knockout mice. (a) Predicted structure of the Arv1 protein showing the N-terminal Zn2+ binding domain, Arv1 homology domain (AHD), transmembrane domains (barrels) and luminal C-terminus. (b) Arv1 messenger RNA (mRNA) expression in tissues from male C57BL6/J mice 12–14 weeks of age (n = 4), fold changes are shown relative to spleen, the lowest expression tissue. (c) Diagram of the murine Arv1 locus showing the locations of the PGK-neomycin resistance cassette (‘Neo’), LoxP sites (triangles), FRT sites (diamonds), Exons (closed boxes), AseI restriction sites (‘A’), and Southern blotting probe (probe). (d) Southern blot of wild-type (lane 1) and targeted (lane 2) embryonic stem cells. Digestion of the wild-type Arv1 allele with AseI produces a 7.2 kb fragment, while inclusion of the Neo cassette removes this AseI site and introduces a new site closer to exon 3, generating an 8.4 kb fragment. (e) Arv1 mRNA levels measured by real-time reverse transcriptase PCR (RT-PCR) of liver RNA isolated from wild-type (WT), heterozygous (Het) and homozygous null mice (KO) generated using CMV-Cre to delete Arv1 in the germline. Values are expressed as means ± s.d. where the asterisk (*) indicates Po0.05 relative to the wild-type mice. CMV, cytomegalovirus. generate the targeting vector illustrated in Figure 1. Positive embryonic 350 bp.11 Primers from Taconic for mouse chromosome 11 were included stem cell clones (R1 cells, SCRC-1011, American Type Culture Collection, as a positive control (1260_1: 5′-GAGACTCTGGCTACTCATCC-3′, 1260_2: Manassas, VA, USA) were selected with G418, and genomic integration was 5′-CCTTCAGCAAGAGCTGGGGAC-3′; 585 bp). The WT Arv1 allele was confirmed by Southern blotting with an AseI digest. Mycoplasma testing detected using the primers WTArv1For: 5′-CTTTGTTATGTTGTACAGG was not performed on embryonic stem cells before injection. Correctly GTTAC-3′, WTArv1Rev: 5′-CCCGTGGACATGGACATGGGAAC-3′; 218 bp, targeted embryonic stem cells were injected into blastocysts from C57Bl/6J where the forward primer binding site is unique to the WT allele in Intron 1. mice. Male chimeras were bred to C57BL6/J mice to obtain F1 progeny. The floxed Arv1 allele was detected using the primers FlxFor: These mice were then crossed with FLP recombinase transgenic mice 5′-GCAGCCCAATTCCGATCATATTC-3′, FlxRev:5′-CAGGAACAGAAATCACAGG (C57BL6/J) to remove the neomycin resistance gene, creating a conditional CAG-3′, to amplify a 162 bp fragment of intron 1 where the forward primer Arv1 allele with exons 2 and 3 flanked by loxP sites. The mice harboring the binds to the loxP site. In the reactions to detect the WT and floxed alleles, conditional Arv1 allele were then crossed with CMV-Cre transgenic mice on primers that detect the murine IL2 genomic sequence from JAX were a C57BL6/J background from Jackson Labs to delete Arv1 in the germline included as a positive control (JAX-oIMR7338:5′-CTAGGCCACAGAATTGAAA (B6.C-Tg(CMV-cre)1Cgn/J, Jax stock number 006054), and backcrossed an ′ ′ ′ additional two to four times before experiments. GATCT-3 , JAX-oIMR7339:5-GTAGGTGGAAATTCTAGCATCATCC-3 ; 324 bp). The deleted (null) Arv1 allele was detected with the primers: GENO3W- TARV1F:5′-CATTTGAGAGGGTAGCACAACTAC-3′, GENO-WTARV1-Rev:5′-GAC Genotyping TTCCTCTCAGACTCCTCTACCGTGAG-3′ and GENO3DELARV1R:5′-CCTGGCTT The Cre transgene was detected by PCR using published primers—Cre-For: AAGGCAAACGCTTCTGAAG-3′. The first two primers flank the loxP site in 5′-ACGACCAAGTGACAGCAATG-3′, Cre-Rev: 5′-CTCGACCAGTTTAGTTACCC-3′; intron 1, while the third primer binds to intron 3, which is brought close Nutrition & Diabetes (2015) 1 – 7 A role for Arv1 in mammalian energy homeostasis WR Lagor et al 3 enough for amplification when the allele is deleted (Floxed 409 bp, WT Metabolic phenotyping and activity measurements 391 bp, null 182 bp).
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