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Endocrinology, doi:10.1210/en.2006-1244
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Endocrinology Vol. 148, No. 4 1561-1573
Copyright © 2007 by The Endocrine Society

Ablation of the Cholesterol Transporter Adenosine Triphosphate-Binding Cassette Transporter G1 Reduces Adipose Cell Size and Protects against Diet-Induced Obesity

Jana Buchmann, Christoph Meyer, Susanne Neschen, Robert Augustin, Katja Schmolz, Reinhart Kluge, Hadi Al-Hasani, Hella Jürgens, Karsten Eulenberg, Roland Wehr, Cord Dohrmann, Hans-Georg Joost and Annette Schürmann

Department of Pharmacology (J.B., S.N., R.A., K.S., R.K., H.A.-H., H.J., H.-G.J., A.S.), German Institute of Human Nutrition Potsdam-Rehbruecke, D-14558 Nuthetal, Germany; and DeveloGen AG (C.M., K.E., R.W., C.D.), D-37079 Goettingen, Germany

Address all correspondence and requests for reprints to: Dr. A. Schürmann, Department of Pharmacology, German Institute of Human Nutrition, Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114–116, D-14558 Nuthetal, Germany. E-mail: Schuermann{at}dife.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ATP-binding cassette transporter G1 (ABCG1) catalyzes export of cellular cholesterol from macrophages and hepatocytes. Here we identify an additional function of ABCG1 in the regulation of adiposity in screens of the Drosophila melanogaster and the New Zealand obese (NZO) mouse genomes. Insertion of modified transposable elements of the P-family upstream of CG17646, the Drosophila ortholog of Abcg1, generated lines of flies with increased triglyceride stores. In NZO mice, an Abcg1 variant was identified in a suggestive adiposity quantitative trait locus and was associated with higher expression of the gene in white adipose tissue. Targeted disruption of Abcg1 in mice resulted in reduced body weight gain (8.42 ± 0.6 g in Abcg1–/– vs. 13.07 ± 1.1 g in Abcg1+/+ mice) and adipose tissue mass gain (3.78 ± 1.3 g in Abcg1–/– vs. 9.39 ± 1.6 g in Abcg1+/+ mice) detected over a period of 12 wk. The reduction of adipose tissue mass in Abcg1–/– mice was associated with markedly decreased size of the adipocytes. In contrast to their wild-type littermates, male Abcg1–/– mice exhibited no high-fat diet-induced impairment of glucose tolerance and fatty liver. Furthermore, Abcg1–/– mice possess decreased food intake and elevated total energy expenditure (Abcg1–/– mice, 748.1 ± 5.4 kJ/kg metabolic body mass; Abcg1+/+ mice, 684.3 ± 5.0 kJ/kg metabolic body mass; P = 0.011), body temperature (Abcg1–/– mice, 37.82 ± 0.29 C; Abcg1+/+ mice, 36.83 ± 0.24 C; P < 0.05), and locomotor activity (Abcg1–/– mice, 3655 ± 189 counts/12 h during dark phase; Abcg1+/+ mice, 2445 ± 235 counts/12 h during dark phase; P < 0.01). Our data indicate a previously unrecognized role of ABCG1 in the regulation of energy balance and triglyceride storage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OBESITY REPRESENTS ONE of the most important contemporary health problems. It is well established that its pathogenesis has a polygenic basis, and considerable efforts are currently being made to identify genes and their variants involved in the regulation of energy balance and triglyceride storage (1). Toward this aim, we have employed a strategy that combined genome-wide screens performed in two different species. In a screen of the total triglyceride content of more than 10,000 mutant Drosophila melanogaster lines, we identified more than 200 genes with either significantly increased or decreased triglyceride levels (2). In outcross populations of the New Zealand obese (NZO) mouse with lean mouse strains, we identified chromosomal segments [quantitative trait loci (QTL)] associated with adiposity (3, 4, 5, 6). Several murine orthologs of the Drosophila genes associated with adiposity were located in one of the mouse adiposity QTL. These genes were considered strong candidates to be involved in the regulation of mammalian adiposity and therefore further investigated. One of them is the Drosophila CG17646 gene, which corresponds to the mammalian ATP-binding cassette (ABC) transporter ABCG1.

ABC transporters are a superfamily of conserved membrane proteins that transport a wide variety of substrates including ions, amino acids, peptides, sugars, lipids, and sterols across cell membranes (7). Several ABC transporters (ABCA1, ABCG1, ABCG5, and ABCG8) have been shown to modulate cholesterol and lipoprotein metabolism. Inactivation of ABCA1 via targeting of the Abca1 gene in mice resulted in a decrease in plasma cholesterol and phospholipids and in the virtual absence of plasma high-density lipoprotein (HDL) (8, 9, 10). ABCA1 was discovered as the cause of Tangier disease and familial HDL deficiency (11). The ABCG5 and ABCG8 transporters, defective in ß-sitosterolemia (12, 13), play a pivotal role in the regulation of intestinal cholesterol absorption (14). Interestingly, expression of ABCA1, ABCG1, ABCG5, and ABCG8 are highly activated by the nuclear liver X receptor (LXR) (15), an important regulator of cholesterol, lipid, and glucose homeostasis (16, 17).

Mammalian Abcg1 mRNA was detected in macrophages, spleen, lung, thymus, and brain, and lower expression was detected in kidney, liver, and heart (18, 19). Abcg1 expression is regulated by cholesterol uptake, and inhibition of its expression by an antisense approach resulted in reduced efflux of cholesterol and choline-phospholipids (20). Recently, Vaughan and Oram (21) demonstrated that ABCG1 traffics to the plasma membrane and redistributes cholesterol to a cell-surface pool that is accessible to enzymatic oxidation and to removal by HDL particles (21). While our study was in progress, it was described that targeted disruption of the Abcg1 gene in mice fed a high-fat and high-cholesterol diet caused accumulation of cholesterol and phospholipids in hepatocytes and macrophages (22). Conversely, tissues of mice overexpressing ABCG1 were protected from cholesterol accumulation (22). In addition, it was shown recently that ABCG1 is involved in the regulation of pulmonary homeostasis. Lungs of chow-fed Abcg1–/– mice exhibited a progressive subpleural cell proliferation and an accumulation of phospholipids and cholesterol in pneumocytes and macrophages when mice were older than 6 months (23).

Here we report data from a Drosophila screen and from a polygenic obese model, the NZO mouse, suggesting that ABCG1 plays a previously unrecognized role in lipid metabolism and the function of adipose tissue. To corroborate this notion, we generated an Abcg1 knockout mouse and characterized its phenotype with regard to high-fat diet-induced adiposity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Genetics and generation of a modified transposable element of the P-family (EP-element) insertion collection
EP-lines of Drosophila melanogaster were used that contained single insertions bearing two different EP-elements as described in Staudt et al. (24). EPg was modified to function in the female germline and contains the white+ gene as a selectable marker in white mutant individuals. The second EP-element, P{Mae-UAS.6.11}, contained the yellow gene as a corresponding marker. Chromosomes bearing EP-element integrations were kept either as homozygous lines or in trans to a corresponding balancer chromosome. Male F1 offspring of EP-element-bearing fly lines were screened for altered triglyceride content. Depending on the integration site into a gene locus, e.g. exon, promoter, or transcription control elements, the EP-element often reduces gene activity but is also able to enhance gene expression.

Molecular analysis and screening for genes that interfere with whole-body triglyceride content
The whole-body triglyceride content of two to three batches of 10 flies of each EP-element integration line was determined (triglyceride INT kit; Sigma, Munich, Germany) and normalized per protein content (Bio-Rad DC protein assay; Bio-Rad, Hercules, CA). The determination of triglyceride content was repeated at least one time for each EP-line. The EPg and the pMae elements have been described previously (2). An inverse PCR approach (Berkeley Drosophila Genome Project, http://www.fruitfly.org/) with EP-element-specific primers was used for identification of the genomic integration site of an individual EP-element. Fragments were amplified for the 5'-end of P{EP,y+}. Primers used for the pMae were 5'-CAG CTG CGC TTG TTT ATT TGC-3' (forward) and 5'-TGG GAA TTC GTT AAC AGA TCC AC-3' (reverse) and for the EPg were Pw new up (5'-CAG CCG AAT TAA TTC TAG TTC CAG TGA A-3') and Pw new low (5'-ACT TCG GCA CGT GAA TTA ATT TTA CTC C-3'). The amplified DNA was sequenced and used to determine the insertion site.

Sequencing
All cDNA clones and PCR products were sequenced in both directions by the method of Sanger using the ABI PRISM 3100-Avant Genetic Analyzer in combination with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).

RNA preparation and first strand cDNA synthesis
Total RNA from epididymal mouse fat was extracted with the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the guidelines of the manufacturer. Extraction of total RNA from other tissues was performed with peqGOLD RNA Pure (PeqLab Biotechnologie GmbH, Erlangen, Germany). cDNA was generated from 2 µg total RNA with Superscript III and random hexamers as primers (Invitrogen, Carlsbad, CA). Quality of cDNA was controlled by PCR with murine GAPDH primers (forward, 5'-ACC ACA GTC CAT GCC ATC AC-3'; reverse, 5'-TCC CAC CAC CCT GTT GCT GTA-3').

Quantitative real-time PCR
Quantitative real-time PCR analysis was performed with the Applied Biosystems 7300 Real-Time PCR System. The PCR mix (25 µl) was composed of TaqMan Universal PCR Master Mix, No AmpErase UNG, a cDNA amount corresponding to 25 ng RNA used for cDNA synthesis (each sample in a triplicate), the TaqMan Gene Expression Assay (Mm01348250_m1) for the Abcg1 mRNA. The assay amplifies the region between exons 3 and 4, which is deleted in Abcg1–/– mice. For the determination of fat-specific genes, the following TaqMan gene expression assays were used: adiponectin (Mm00456425_m1), GLUT4 (Mm00436615_m1), LXR{alpha} (Mm00443454_m1), PPAR{gamma} (Mm00440945_m1), ATGL (Mm00503040_m1), and HSL (Mm00495359_m1). Analysis of FAS expression was performed with primer probe pairs (forward, 5'-TTG CTG GCA CTA CAG AAT GC-3'; reverse, 5'-AAC AGC CTC AGA GCG ACA AT-3'). Data were normalized referring to Livak and Schmittgen (25), whereas a ß-actin expression assay (Mm00607939_si; Applied Biosystems) and primers for 18S RNA (forward, 5'-TGA GGC CAT GAT TAA GAG GG-3'; reverse, 5'-TTC TTG GCA AAT GCT TTC G-3'), respectively, were used as endogenous control.

Generation of Abcg1 knockout mice
For generation of Abcg1 knockout mice, the targeting construct subcloned into the vector pBS-K+ was linearized with NotI, and embryonic stem cells from 129 SvJ were transfected by electroporation. Cells were subsequently cultured in the presence of 400 µg G418/ml for 7–9 d. Neomycin-resistant clones were genotyped by PCR (forward-neo-primer 5'-GTG GGG GTG GGG TGG GAT TAG ATA-3' and add reverse-gene-primer 5'-TGT GCT GTT GTG TAG GGG ACA TTC-3'). One ES cell clone that had incorporated the targeting vector by homologous recombination was used for a morula aggregation. Blastocysts were then transferred into a pseudopregnant (d 2.5) female mouse. Male chimeric mice were mated with C57BL/6 females. Offspring carrying the transgene were backcrossed on to C57BL/6 three times and subsequently intercrossed. In each experiment, littermates (wild-type, heterozygous, and knockout) of the offspring of heterozygous crossings were compared to characterize mice with comparable genetic background. Genotyping was performed by PCR (for wild-type mice, forward primer 5'-GAG TGG AGT GGG GTA GTT TCT TGG-3' and reverse primer 5'-ATG GCA ACA GGA TGG AGC AGA GGG AC-3'; for knockout mice, forward-neo-primer 5'-GTG GGG GTG GGG TGG GAT TAG ATA-3' and add reverse-gene-primer 5'-TGT GCT GTT GTG TAG GGG ACA TTC-3'). The animals were housed in air-conditioned rooms (temperature, 20 ± 2 C; relative moisture, 50–60%) under a 12-h light, 12-h dark cycle. They were kept in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals, and all experiments were approved by the ethics committee of the Ministry of Agriculture, Nutrition, and Forestry (State of Brandenburg, Germany).

Immunochemical detection of ABCG1 and perilipin
For immunohistochemical detection of ABCG1, brains and lungs from Abcg1+/+ and Abcg1–/– mice were homogenized and centrifuged for 1 h at 200,000 x g at 4 C. Membrane proteins (20 µg) were separated by SDS-10% PAGE and transferred onto nitrocellulose. For immunochemical detection, an anti-ABCG1 antibody (ABR-Affinity BioReagents, Golden, CO) was used in a dilution of 1:1000, and bound Ig was detected with goat antirabbit IgG (whole molecule) peroxidase-conjugated antibody (1:20,000; Dianova, Hamburg, Germany). For detection of perilipin, total membranes of white adipose tissue (WAT) and brown adipose tissue (BAT) from Abcg1+/+ and Abcg1–/– mice (10 µg) were detected by Western blots with an anti-perilipin antibody (PROGEN Biotechnik GmbH, Heidelberg, Germany) in a dilution of 1:1000. Bound Ig was detected with rabbit anti-guinea pig IgG (whole molecule) peroxidase-conjugated antibody (1:50,000; Sigma Chemical Co., St. Louis, MO).

Diets
From the age of 3 wk, mice were fed either a normal maintenance diet for rats and mice (item no. 1320; Altromin, Lage, Germany) containing 11,825 kJ/kg digestible energy with 19% (wt/wt) protein, 4% fat, and 50.5% carbohydrates or a high-fat diet (C1057; Altromin) containing 17% (wt/wt) protein, 15% fat, and 52% carbohydrates.

Body composition
Body fat and lean mass were determined with a nuclear magnetic resonance spectrometer (Bruker-Mini-Spec-NMR-Analyzer mq10; Bruker Optics, Houston, TX). In addition, body weights were measured with an electronic scale.

Serum parameters
Blood glucose levels were determined with a glucometer elite (Bayer, Leverkusen, Germany). Cholesterol and HDL cholesterol were measured with the analyzing system VITROS DT60 II (Johnson & Johnson, Neckargemünd, Germany). Nonesterified fatty acids (NEFA) (Wako NEFA C kit; Wako Chemicals, Neuss, Germany), insulin (insulin mouse ultrasensitive ELISA; DRG Instruments GmbH, Germany), leptin (Mouse Leptin Quantikine ELISA Kit; R & D Systems GmbH, Wiesbaden-Nordenstadt, Germany), triglycerides (triyglyceride reagent, Sigma, Munich, Germany), and glycerol (free glycerol reagent; Sigma) were analyzed by ELISA.

Tissue triglyceride and cholesterol contents
The tissue lipid extraction procedure was adapted from methods described previously (26, 27). Triglyceride and cholesterol concentrations were measured in triplicate after evaporation of the organic solvent using an enzymatic method (Randox Laboratories, Crumlin, UK, and Wako Chemicals, respectively).

Feeding behavior
Food intake was recorded with an automated drinking and feeding monitor system (TSE, Bad Homburg, Germany), consisting of macrolon type III cages equipped with baskets connected to weight sensors. The baskets contained high-fat diet pellets and were freely accessible to the mice. Mice were habituated to the test cages for 2 d before trials, and the measurement period lasted 5–6 d. Recorded data were analyzed as metabolic body mass (MBM)-specific food intake [food intake/(body weight)0.75].

Indirect calorimetry
Total energy expenditure (TEE) was measured at 22 C for 23 h with an open circuitry calorimetry system (Hartmann & Braun GmbH and Co. KG, Frankfurt/Main, Germany; VO2 analyzer Magnos 16; VCO2 analyzer Uras 14) as described previously (28). Before recording the rate of oxygen consumption (VO2) and the rate of carbon dioxide production (VCO2), mice were allowed to adapt to the new cage and to the system for 48 h. Air-tight respiratory cages with a flow rate of about 30 liters/h were placed in climate chambers (Vötsch Industrietechnik GmbH, Reiskirchen-Lindenstruth, Germany) to achieve the desired temperatures of 22 C. VO2 and VCO2 were recorded in 6-min intervals for each animal, and TEE was calculated with the equation (29) TEE = 16.17VO2 + 5.03VCO2 + 5.98N, where TEE is in kilojoules per day and VO2 and VCO2 are in liters per day. N is excreted nitrogen and was assumed to be 0.1 g/d. MBM-specific TEE was calculated by dividing TEE (kJ/d) by the MBM of the animal and expressed as kilojoules per kilogram MBM per day.

Rectal body temperature
Rectal body temperature in wild-type and Abcg1–/– mice was measured with a signal conditioner (ML312 T-type Pod) in combination with a rectal probe for mice (MLT1404; AD Instruments GmbH, Spechbach, Germany) when mice were resting. Analysis of the data was performed with the program Chart version 3.4.8.

Telemetric measurement of spontaneous locomotor activity
Transponders (22 x 8 mm; Mini Mitter Co. Inc., Bend, OR) were implanted into the abdominal cavity under ketamine (100 mg/kg body weight) and xylazine hydrochloride (10 mg/kg body weight) anesthesia. The abdominal cavity was sutured with absorbable surgery thread (PGA Resorba; Resorba, Nürnberg, Germany), and skin was closed with metal clips (Becton Dickinson, Sparks, MD) that were removed after a 1-wk recovery period.

After an adaptation period of 48 h, spontaneous locomotor activity data were collected continuously with the VitalView Data Acquisition System (Mini Mitter) in parallel with indirect calorimetry measurement as described previously (28). Values were recorded in 6-min intervals for each animal for 24 h at 22 C.

Histological analysis
Organs (WAT, BAT, lung, and liver) of wild-type and Abcg1–/– animals at the age of 12 and 30 wk were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin. Cryosections of livers of Abcg1+/+ and Abcg1–/– animals at the age of 12 and 37 wk were incubated with 0.3% oil-red O for 10 min, washed with 60% isopropanol, and counterstained with hematoxylin.

Glucose tolerance test
Animals received the high-fat diet for 19 wk and were fasted for 16 h before the experiment. Each animal received a single ip injection of glucose (20% solution, 1.5 g/kg body weight). Samples for determination of blood glucose concentrations were obtained at 0, 15, 30, 60, and 120 min from the tail tip. To minimize distress, mice were kept in their accustomed cage and had free access to water. For sampling, a box-shaped restraining device was used.

Statistical analysis
Values are reported as mean ± SE. Statistical significance (P value < 0.05) was determined by unpaired Student’s t test (Statview; Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of ABCG1 as a candidate gene for obesity
By random insertion of EP-elements into the D. melanogaster genome, loss- as well as gain-of-function mutants were generated, and phenotypes were analyzed by determination of their triglyceride and glycogen content. Among the mutants with increased triglyceride stores, we identified two integrations of the EP-elements [HD-EP(2)0388 and EP(2)2482] in the 5'-flanking region of the CG1764 gene (Fig. 1AGo). As is illustrated in Fig. 1BGo, male flies with homozygous integration at position 1,737,342 bp or 1,737,369 bp of chromosome 2L exhibited a 1.5- or 1.7-fold increase in triglyceride content, respectively. Therefore, independent integration events, both between 100 and 130 bp upstream of the CG17646 transcription start, confirmed the metabolic phenotype.


Figure 1
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  		FIG. 1. Identification of CG17646 as obesity gene. A, Position of the insertion of the EP-elements HD-EP(2 )0388 and EP(2 )2482 into the D. melanogaster genome. B, triglyceride content of control flies (EP control) and flies with homozygous insertion of the EP-elements. Bars represent means ± SE of n > 12 flies. *, P < 0.03.

 
The mammalian ortholog of CG17646 is the ABC transporter ABCG1, which exhibits 37% identity to the gene product of CG17646 (alignment is shown in supplemental Fig. 1Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). The murine Abcg1 gene is located on chromosome 17 close to the microsatellite marker D17Mit175. In an outcross of the NZO mouse with the lean Swiss/Jim Lambert (SJL) mouse strain (3, 5), this marker located a suggestive obesity QTL. Mice carrying both NZO alleles at D17Mit175 exhibited a 2.9-g higher body weight than the controls (P = 0.0184) at wk 12. Furthermore, a comparison of the single-nucleotide polymorphism maps of NZO and SJL indicated that the gene was located in a polymorphic region. Sequencing of the Abcg1 gene revealed identical deduced amino acid sequences of NZO, SJL, and C57BL/6. However, NZO exhibited a 210-bp insertion in intron 4 between the exons 2 and 3 (see supplemental Fig. 2Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). This intron has been described to contain multiple LXR-responsive elements that are necessary and sufficient to activate expression of ABCG1 in response to LXR ligands (30).


Figure 2
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  		FIG. 2. Expression of Abcg1 is elevated in WAT of the polygenic obese NZO mouse. Relative expression of ABCG1 was determined in WAT (A) and liver (B) of C57BL/6 (B6), SJL, NZB, NZO, and ob/ob mice at the age of 8 wk by quantitative RT-PCR. Body weight (C) and fat mass (D) were measured at the age of 8 wk. Data represent means ± SE of five animals. E, Time course of Abcg1 expression during differentiation of 3T3-L1 cells. F, Quantification of Abcg1 mRNA expression of 3T3-L1 fibroblasts and adipocytes. Bars represent means ± SE of three different batches of cells. *, P < 0.05; **, P < 0.01.

 
Because we assumed that the 210-bp insertion in the NZO Abcg1 gene might influence its expression, quantitative real-time PCR was performed in tissues from several mouse strains, NZO, NZB (which is the closest relative of NZO), SJL, C57BL/6, and ob/ob mice, which are a model for monogenic obesity. As is shown in Fig. 2Go (left), mRNA levels of Abcg1 in WAT were significantly higher in NZO than in the other mouse strains. We also observed a significantly (P = 0.0014) increased expression in BAT of NZO mice (relative expression in NZO was 1.49 ± 0.03 in comparison with C57BL/6, which was 1.01 ± 0.07). In contrast, no significant differences in Abcg1 expression between the NZO, NZB, SJL, and C57BL/6 were detected in other tissues, e.g. in liver. In liver, only the ob/ob mice exhibited a higher expression compared with the other strains (Fig. 2Go, right). To support the conclusion that Abcg1 expression in fat cells is relevant for the function of WAT, we determined its expression in 3T3-L1 cells during the process of their differentiation to adipocytes. As shown in Fig. 2EGo, mRNA of Abcg1 was nearly absent in 3T3-L1 fibroblasts (d 0). When 3T3-L1 cells were treated with the differentiation cocktail, expression appeared at d 4 and increased until d 10 when differentiation of cells was completed. There was an approximately 140-fold increase in Abcg1 expression from fibroblasts (relative expression 1.01 ± 0.08) to differentiated adipocytes (139.18 ± 10.49). These data suggest that ABCG1 plays a specific role in the function of the fat cell.

Targeted disruption of the Abcg1 gene in mice
To further test the conclusion that the mammalian ABCG1 is involved in the regulation of triglyceride storage, we generated Abcg1 knockout mice by exchange of exons 3–5 for a neomycin-resistance cassette. The targeting construct contained 1045 and 5975 kb derived from the 5'- and 3'-side flanking regions of the Abcg1 gene, respectively (Fig. 3AGo). Transfection of embryonic stem cells with the targeting construct generated one clone with a homologous recombination as ascertained by Southern blot analysis with a specific probe (Fig. 3BGo, left). This stem cell clone was fused with morula of mice. Male chimeric mice were mated with C57BL/6 females, and F1 progeny carrying the transgene were backcrossed three times on to the C57BL/6 background. In the subsequent F2 progeny, which was used for characterization of the phenotype, we obtained the Abcg1+/+, Abcg1+/–, and Abcg–/– mice (Fig. 3BGo, right) in the expected ratio (data not shown). To verify the knockout, cDNA was generated from wild-type and Abcg1–/– mice, amplified by PCR, and sequenced. Sequence analysis confirmed the absence of the region between bp 6803 (within exon 2) and bp 46,210 (in exon 6) in the genomic sequence. As shown in Fig. 3CGo, mRNA of Abcg1 was absent in all investigated tissues, whereas tissues of heterozygous mice expressed approximately 50% of Abcg1 (Fig. 3CGo). In addition, Western blot analysis of total membranes from brain and lung of Abcg1+/+ and Abcg1–/– mice demonstrated the absence of the ABCG1 protein in knockout mice (Fig. 3DGo).


Figure 3
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  		FIG. 3. Targeted disruption of the Abcg1 gene by homologous recombination. A, Abcg1 gene, targeting construct, and organization of the disrupted Abcg1 allele. B, Southern blot of control ES cells and the ES cell clone used for generation of the Abcg1 knockout mouse (left panel). Genomic DNA was digested with XbaI or ScaI and analyzed with the 5'-probe. Southern blot analysis of genomic DNA from the F2 progeny (right panel). The DNA was digested with XbaI and hybridized with the 5' probe. C, Abcg1 mRNA levels of 30-wk-old high-fat diet-fed Abcg1+/+, Abcg1+/–, and Abcg1–/– littermates in brown adipose tissue (BAT), brain, hypothalamus, liver, lung, pancreas, and WAT were detected by quantitative RT-PCR as described in Materials and Methods. D, Western blot analysis of total membrane fractions from brains and lungs of Abcg1+/+ and Abcg1–/– littermates was performed with an anti-ABCG1 antibody as described in Materials and Methods. An anti-GAPDH antibody was used as a loading control.

 
Abcg1–/– mice grew normally, were in an apparently good health, showed normal grooming and exploratory behavior, and exhibited no noticeable coat problems. In addition, we did not observe a reduced life span of Abcg1–/– mice in comparison with wild-type littermates. In a pilot experiment, we monitored the survival rate of 17 wild-type and 28 Abcg1–/– mice over a period of 82 wk, and 29% of wild-type and 46% of Abcg1–/– mice survived until wk 82 of life.

Deletion of Abcg1 reduces body weight and adipose tissue depots
On a high-fat diet containing 35% of total calories from fat, male Abcg1–/– mice exhibited a significantly lower body weight gain over a period of 12 wk than their wild-type littermates (Fig. 4Go, A and B). Heterozygous mice show an intermediate effect on body weight gain. The reduced weight gain of Abcg1–/– was entirely due to reduced fat accumulation; knockout mice gained only 3.8 ± 1.3 g of body fat under the high-fat diet, whereas heterozygous gained 6.9 ± 1.7 g and wild-type mice 9.4 ± 1.59 g over a time period of 12 wk (Fig. 4CGo, left). Under a standard diet (15% of total calories from fat), there was a much smaller difference in fat gain, which did not reach statistical significance (Abcg1–/–, 2.2 ± 1.01 g; Abcg1+/–, 1.9 ± 1.1 g; wild-type, 3.93 ± 0.94 g). In contrast to the body fat content, no significant differences in the lean mass were observed (Fig. 4CGo, right). In the Abcg1–/– females, the effects on body weight and body fat content were weaker and started later, approximately at the age of 14 wk (data not shown).


Figure 4
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  		FIG. 4. Reduced body weight and body fat content in Abcg1–/– mice. A, Photographs showing reduced body fat pads of 30-wk-old Abcg1–/– mice in comparison to wild-type mice, both fed a high-fat diet. B, Body weight development of male Abcg1+/+ and Abcg1–/– mice fed a high-fat diet (left panel). Body weight gain determined over a period of 12 wk (between wk 14 and 26 for mice fed standard diet; between wk 8 and 20 for mice fed high-fat diet; right panel). C, Body composition (fat mass, left panel; lean mass, right panel) of male Abcg1+/+, Abcg1+/–, and Abcg1–/– mice fed standard diet or high-fat diet. Data represent mean of 9 Abcg1+/+, 25 Abcg1+/–, and 19 Abcg1–/– mice ± SE (*, P < 0.03).

 
Increased TEE, locomotor activity, and body temperature in Abcg1–/– mice
To study the main parameter of energy balance, we determined daily food intake, TEE, locomotor activity, and body temperature before onset of obesity at the age of 8–9 wk in wild-type and Abcg1–/– mice fed a high-fat diet. Under these conditions, cumulative food intake was slightly less in Abcg1–/– mice compared with that of wild-type litter controls (Fig. 5AGo). However, this small difference in food intake failed to reach statistical significance. To assess energy balance directly, we monitored 8- to 9-wk-old mice in open-circuit indirect calorimetry. In comparison with wild-type mice Abcg1–/– mice showed an increased O2 consumption (wild-type, 31.289 ± 0.24, and Abcg1–/– mice, 34.163 ± 0.190, detected as liters/d·kg MBM; P = 0.08), increased CO2 production (wild-type, 30.59 ± 0.28, and Abcg1–/– mice, 33.10 ± 0.30, detected as liters/d·kg MBM; P = 0.03), whereas respiratory quotient was not altered (wild-type, 0.914 ± 0.003, and Abcg1–/– mice, 0.903 ± 0.0.003). In addition, Abcg1–/– mice exhibited significantly (P < 0.02) higher TEE (748.1 ± 5.4 kJ/d·kg MBM of Abcg1–/– mice vs. 684.3 ± 5.0 kJ/d·kg MBM of Abcg1+/+ mice; P = 0.011) in the average of 24 h (Fig. 5BGo) and significantly elevated locomotor activity (3655 ± 189 counts of Abcg1–/– mice vs. 2445 ± 235 counts during dark phase of Abcg1+/+ mice; P < 0.01; Fig. 5CGo). In addition, rectal body temperature was significantly (P < 0.05) elevated in Abcg1–/– mice (37.82 ± 0.29 C) compared with wild-type mice (36.83 ± 0.24 C; Fig. 5DGo). This result was confirmed by recording core temperature with the telemetry system; wild-type mice had a mean temperature of 37.51 ± 0.58 C, whereas the mean temperature of Abcg1–/– mice was higher (38.35 ± 0.64; n = 4). These data suggest that slightly reduced food intake together with increased TEE, locomotor activity, and body temperature resulted in lower body weight of the Abcg1–/– mice.


Figure 5
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  		FIG. 5. Abcg1–/– mice have increased energy expenditure, locomoter activity, and body temperature. After weaning male Abcg1+/+ and Abcg1–/– littermates were fed a high-fat diet and (A) cumulative food intake, (B) total energy expenditure as kJ per MBM, (C) locomotor activity, and (D) rectal body temperature was measured at the age of 8 wk as described in Materials and Methods. Data represent mean of 8–9 mice ± SE (*, P < 0.05).

 
Improved glucose homeostasis in Abcg1–/– mice
At the age of 22 wk, fasting blood glucose levels of Abcg1–/– males fed a high-fat diet were significantly lower (83.00 ± 7.23 mg/dl) than that of wild-type males (120.25 ± 21.18 mg/dl). Because this finding suggested that ablation of Abcg1 corrects diet-induced insulin resistance, ip glucose tolerance tests were performed. Indeed, high-fat diet-fed Abcg1–/– mice exhibited a normal glucose tolerance, whereas wild-type mice showed a sustained hyperglycemia after the glucose load (Fig. 6Go).


Figure 6
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  		FIG. 6. Glucose tolerance in Abcg1+/+ and Abcg1–/– mice at the age of 22 wk. Intraperitoneal glucose tolerance tests were performed in four wild-type and five Abcg1–/– males as described in Materials and Methods. Data represent mean ± SE (*, P < 0.05).

 
Ablation of Abcg1 reduces the volume of white and brown adipocytes and prevents diet-induced lipid accumulation in the liver
The histological analysis of WAT from mice fed a high-fat diet (Fig. 7AGo) indicated that the size of adipocytes of Abcg1–/– mice was markedly lower before (12 wk) and after (30 wk) the differences in body weight and body fat content were visible. White adipocytes of wild-type mice were univacuolar cells containing a big lipid droplet. In contrast, adipose tissue from Abcg1–/– mice contained numerous multivacuolar cells, and fat cells appeared to be considerably smaller. The same effect was observed in BAT (Fig. 7BGo). To quantify the fat cell size, morphometric analysis of WAT sections were performed on four wild-type and four knockout mice. In a blinded study, we measured the maximal diameter of approximately 200 cells per animal and detected significant differences (P = 0.038) between wild-type (40.48 ± 2.57 µm) and Abcg1–/– mice (24.59 ± 3.27 µm).


Figure 7
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  		FIG. 7. Reduced adiposity of Abcg1–/– mice is associated with decreased size of adipocytes. A, Photomicrographs of gonadal WAT after staining with hematoxylin and eosin of 12-wk-old male Abcg1+/+ and Abcg1–/– littermates fed a high-fat diet (upper panel) and 30-wk-old mice fed a standard diet (lower panel). B, hematoxylin and eosin staining of brown adipose tissue of 12-wk-old male Abcg1+/+ and Abcg1–/– mice fed a high-fat diet. Bars, 100 µm.

 
To exclude a lipid storage defect in Abcg1–/– mice that would lead to hepatic triglyceride accumulation, we performed liver histology in 12- and 37-wk-old mice (Fig. 8Go, A and B). No difference was observed in livers from 12-wk-old mice (Fig. 8AGo). After 34 wk on a high-fat diet (age 37 wk), livers of wild-type mice exhibited extensive intracellular vacuolization (Fig. 8BGo, middle left) and a significant lipid accumulation (Fig. 8BGo, lower left). In contrast, only scattered small lipid droplets were detected in livers from Abcg1–/– mice (Fig. 8BGo, lower right). For further analysis, liver lipids were extracted and concentrations of triglyceride and cholesterol levels were evaluated. As expected from the histological analysis, we detected a reduction in liver triglyceride content (15.56 ± 4.0 vs. 21.24 ± 3.4 µg/mg liver of Abcg1–/– and Abcg1+/+ mice, respectively; n = 6–8 mice) and nearly no difference in cholesterol levels of Abcg1–/– mice (2.48 ± 0.15 µg/mg liver) in comparison with Abcg1+/+ mice (2.10 ± 0.1 µg/mg liver).


Figure 8
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  		FIG. 8. Ablation of Abcg1 prevents diet-induced lipid accumulation in liver. A, Oil-red stainings of liver sections of 12-wk-old male Abcg1+/+ and Abcg1–/– littermates fed a high-fat diet. B, Macroscopic view of representative livers (upper panel) and hematoxylin and eosin (HE) (middle panel) and oil-red stained (lower panel) sections of livers from 37-wk-old male Abcg1+/+ and Abcg1–/– mice fed a high-fat diet. Bars, 100 µm.

 
Disruption of Abcg1 results in decreased plasma lipid levels after fasting
The reduced lipid volume in adipocytes of WAT and BAT of Abcg1–/– mice might indicate that Abcg1–/– mice catabolize lipids at a higher rate. To assess the rate of lipid metabolism in Abcg1–/– mice, we measured plasma levels of NEFA, triglycerides, and glycerol in fed and fasted mice. Abcg1–/– mice and wild-type mice had similar levels of NEFA and triglycerides when fed, but levels of NEFA were about 30% lower in Abcg1–/– mice (P < 0.05) after prolonged fasting. In addition, triglyceride and glycerol levels were slightly reduced compared with wild-type mice after 24 h fasting (Table 1Go). According to the increased body temperature and locomotor activity of Abcg1–/– mice, this observation might indicate that Abcg1–/– mice use their lipids for heat production and physical activity.


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  		TABLE 1. Metabolic parameters of male Abcg1+/+ and Abcg1–/– mice (n = 4) at the age 30 wk fed a high-fat diet

 
Expression analysis of adipose tissue of Abcg1–/– mice
The reduced fat cell size of the Abcg1–/– mice might be due to an increased lipid mobilization or decreased lipogenesis. As is illustrated in Fig. 9AGo, 12-wk-old Abcg1–/– males exhibited increased mRNA levels of expression of hormone-sensitive lipase (HSL) and of adipose triglyceride lipase, which catalyze the hydrolysis of triacylglycerol stored in the adipocytes, whereas mRNA levels of fatty acid synthase, was not altered in comparison with wild-type littermates. Stimulation of hormone-sensitive lipase activity is known to be associated with its translocation to the surface of lipid storage droplets (31). This translocation has recently been shown to be controlled by perilipin (32). Western blot analysis of WAT and BAT performed with an anti-perilipin antibody detected several bands due to the two splicing products perilipin-A (57 kDa) and perilipin-B (46 kDa) (Fig. 9BGo). In addition, both isoforms show an upward shift as result of their phosphorylation (33). As shown in Fig. 9BGo, expression of perilipin-A and the phosphorylated form are elevated in WAT and BAT of Abcg1–/– compared with control mice, suggesting that lipolysis might be enhanced in adipose tissue of these mice. In addition, expression levels of other fat-cell-specific genes such as the transcription factor peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), the glucose transporter GLUT4, and the adipokine adiponectin were increased in WAT of Abcg1–/– mice (Fig. 9AGo).


Figure 9
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  		FIG. 9. Expression levels of adipocyte specific genes in adipose tissue of male wild-type and Abcg1–/– mice. A, Quantitative real-time PCR in gonadal WAT of male mice at the age of 12 wk fed a high-fat diet (*, P < 0.05). B, Expression of perilipin in WAT and BAT of 30-wk-old Abcg1+/+ and Abcg1–/– mice fed a high-fat diet was analyzed by Western blotting as described in the Materials and Methods section. The antibody detects perilipin-A (57 kDa), perilipin-B (46 kDa), and the phosphorylated isoforms. An anti-ß-actin antibody was used as loading control. ATGL, Adipose triglyceride lipase.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ABCG1 is a transporter that mediates the export of cholesterol from cells to HDL (20, 30). It was recently shown to play a critical role for lipid homeostasis by controlling tissue lipid levels and efflux of cellular cholesterol (1, 22). In addition, the data presented here demonstrate for the first time that the transporter is also involved in the control of adipose tissue stores. We identified ABCG1 as a potential regulator of adiposity in two species, D. melanogaster and Mus musculus. First, insertion of an EP-element upstream of the CG17646 gene, the Drosophila ortholog of Abcg1, altered the triglyceride content of the fly (Fig. 1Go). Second, insertion of 210 bp in a regulatory intron of Abcg1 was associated with increased mRNA levels in WAT and BAT (Fig. 2Go) and higher body weight in the NZO mouse. Third, targeted deletion of the Abcg1 gene produced resistance to the effects of a high-fat diet on body weight (Fig. 4Go), lipid metabolism (Table 1Go), glucose tolerance (Fig. 6Go), and liver lipid accumulation (Fig. 8Go) by increased energy expenditure due to enhanced locomotor activity and thermogenesis (Fig. 5Go). These data strongly suggest that ABCG1, presumably by altering intracellular cholesterol levels, is involved in the regulation of energy balance and lipid storage.

Intron 4 of Abcg1 has been described to contain multiple LXR-responsive elements that are necessary and sufficient to activate expression of the gene (30). The finding that NZO mice exhibit a higher Abcg1 expression only in WAT and BAT suggests that the NZO-specific 210-bp insertion carries a binding site for an adipocyte-specific transcription factor. Database analysis of the inserted sequence identified several putative binding sites, including a DR2 site (see supplemental Fig. 2Go). This site is described to bind several transcription factors such as PPAR{gamma}, retinoid X receptor, and Rev-erb{alpha}, which play important roles for the fat cell function (34). The latter is a negative regulator of transcription binding to the same responsive element as receptor-related orphan receptor {alpha}. Rev-erb{alpha} is, like Abcg1, differentially expressed in 3T3-L1 cells and is induced by PPAR{gamma} activation in 3T3-L1 cells in vitro and in rat adipose tissue in vivo (35).

Several other genes were identified as obesity-resistant genes that fall into different functional categories. Deletion or overexpression of genes such as Pparg disrupt adipocyte differentiation and lipid storage (36), affect lipid synthesis like Scd1 (37), or lipid hydrolysis such as perilipin (38), increase fatty acid oxidase like Ppard (39), or induce respiratory uncoupling like Ucp1 (40, 41). These alterations produce a common phenotype characterized by increased energy expenditure and enhanced glucose tolerance and insulin sensitivity, a phenotype we also observed in Abcg1–/– mouse. Thus, the present data suggest a link between cholesterol transport and the regulation of lipid storage. However, it remains to be determined whether the effect is primarily due to alterations in adipocyte metabolism, BAT function, liver metabolism, or the function of the central orexigenic network.

It has been suggested previously that cholesterol levels and/or metabolism in adipocytes might serve as an intracellular signal regulating triglyceride storage and the size of adipocytes (42). Overexpression of ABCG1 in HEK cells redistributes cellular cholesterol to cell-surface domains accessible to treatment with cholesterol oxidase (21). Conversely, we assume that cholesterol content in the adipocytes might increase as a consequence of Abcg1 ablation and, therefore, influence triglyceride storage and/or activate cholesterol-regulated transcription factors such as LXR{alpha} (43) in the fat cell. In addition, smaller fat cells are associated with improved insulin response, whereas adipocyte hypertrophy leads to ectopic fat storage and insulin resistance (44). The present data are consistent with such a scenario in that they show that fat cells from Abcg1–/– mice are considerably smaller than those from wild-type mice and that diet-induced impaired glucose tolerance is fully prevented in Abcg1–/– mice. However, the smaller adipocytes may also be the result of the increased systemic energy expenditure.

Two alternative scenarios for the significantly smaller fat cells in Abcg1–/– mice have to be considered. First, reduced lipid stores in Abcg1–/– mice may be due to enhanced locomotor activity, higher body temperature, and reduced food intake. A similar phenotype has been reported for mice lacking Cidea, a protein interacting with and inhibiting uncoupling protein 1. Cidea null mutants, which are lean and resistant to diet-induced obesity, have higher metabolic rate, lipolysis, and body temperature in addition to decreased NEFA levels after fasting (45). Second, an increased differentiation of preadipocytes, possibly stimulated by intracellular cholesterol, to small, metabolically active fat cells might have prevented the high-fat diet-induced insulin resistance and fatty liver in Abcg1–/– mice. This pattern is similar to the effects of an activation of PPAR{gamma} with a thiazolidinedione (46, 47).

It was described recently that targeted disruption of the Abcg1 gene in mice causes accumulation of macrophages in lung tissue (1, 22) and of cholesterol, triglycerides, and phospholipids in hepatocytes (22). Our knockout mice exhibited a similar macrophage accumulation in lung tissue (data not shown) but the opposite phenotype with regard to lipid accumulation in liver. A potential explanation for this discrepancy is the different diets employed in the two studies. In our study, mice were challenged with a high-fat diet that was devoid of cholesterol, whereas Kennedy et al. (22) employed a high-cholesterol diet. Likewise, Kalaany et al. (48) demonstrated an impact of cholesterol for obesity-resistance genes. LXR–/– mice were resistant to high-fat diet-induced obesity only when the diet contained cholesterol (48). Cholesterol activates LXR, and this factor stimulates triglyceride synthesis via sterol regulatory element-binding protein-1c (SREBP-1c) (43). We assume that this pathway is activated in Abcg1–/– mice fed a high-fat, cholesterol-enriched diet but not in mice kept in the absence of cholesterol. Indeed, Kennedy et al. (22) detected an up-regulation of SREBP-1c expression in both wild-type and Abcg1–/– mice fed a high-fat/high-cholesterol diet in comparison with chow diet-fed mice, whereas no change of either LXRß or SREBP-1c expression was detected with the cholesterol-free diet used in our experiments (data not shown). Thus, we assume that dietary cholesterol can modify the response of Abcg1–/– mice to the high-fat diet.


   Acknowledgments
 
The skillful technical assistance of Michaela Fiebich, Elisabeth Meyer, Susanne Neubert, and Carola Plaue is gratefully acknowledged.


   Footnotes
 
This work was supported by grants from the German Ministry of Education, Research, and Technology (0313128B and NGFN2: 01GS0487) and in part by the European Community’s FP6 EUGENE2 (LSHM-CT-2004-512013).

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 28, 2006

Abbreviations: ABC, ATP-binding cassette; BAT, brown adipose tissue; EP-elements, modified transposable elements of the P-family; HDL, high-density lipoprotein; HSL, hormone-sensitive lipase; LXR, liver X receptor; MBM, metabolic body mass; NEFA, nonesterified fatty acids; PPAR{gamma}, peroxisome proliferator-activated receptor; QTL, quantitative trait loci; SREBP-1c, sterol regulatory element-binding protein-1c; TEE, total energy expenditure; VCO2, rate of CO2 production; VO2, rate of O2 consumption; WAT, white adipose tissue.

Received September 12, 2006.

Accepted for publication December 19, 2006.


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 Discussion
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