CCAAT/Enhancer-binding Protein β Deletion Reduces Adiposity, Hepatic Steatosis, and Diabetes in Lepr Mice
2007; Elsevier BV; Volume: 282; Issue: 21 Linguagem: Inglês
10.1074/jbc.m701329200
ISSN1083-351X
AutoresJill M. Schroeder‐Gloeckler, Shaikh Mizanoor Rahman, Rachel C. Janssen, Liping Qiao, Jianhua Shao, Michael Röper, Stephanie J. Fischer, Erin Lowe, David J. Orlicky, James L. McManaman, Carol A. Palmer, William L. Gitomer, Wan Huang, Robert M. O’Doherty, Thomas Becker, Dwight J. Klemm, Dalan R. Jensen, Leslie K. Pulawa, Robert H. Eckel, Jacob E. Friedman,
Tópico(s)Lipid metabolism and biosynthesis
ResumoCCAAT/enhancer-binding protein β (C/EBPβ) plays a key role in initiation of adipogenesis in adipose tissue and gluconeogenesis in liver; however, the role of C/EBPβ in hepatic lipogenesis remains undefined. Here we show that C/EBPβ inactivation in Leprdb/db mice attenuates obesity, fatty liver, and diabetes. In addition to impaired adipogenesis, livers from C/EBPβ-/- x Leprdb/db mice had dramatically decreased triglyceride content and reduced lipogenic enzyme activity. C/EBPβ deletion in Leprdb/db mice down-regulated peroxisome proliferator-activated receptor γ2 (PPARγ2) and stearoyl-CoA desaturase-1 and up-regulated PPARα independent of SREBP1c. Conversely, C/EBPβ overexpression in wild-type mice increased PPARγ2 and stearoyl-CoA desaturase-1 mRNA and hepatic triglyceride content. In FAO cells, overexpression of the liver inhibiting form of C/EBPβ or C/EBPβ RNA interference attenuated palmitate-induced triglyceride accumulation and reduced PPARγ2 and triglyceride levels in the liver in vivo. Leptin and the anti-diabetic drug metformin acutely down-regulated C/EBPβ expression in hepatocytes, whereas fatty acids up-regulate C/EBPβ expression. These data provide novel evidence linking C/EBPβ expression to lipogenesis and energy balance with important implications for the treatment of obesity and fatty liver disease. CCAAT/enhancer-binding protein β (C/EBPβ) plays a key role in initiation of adipogenesis in adipose tissue and gluconeogenesis in liver; however, the role of C/EBPβ in hepatic lipogenesis remains undefined. Here we show that C/EBPβ inactivation in Leprdb/db mice attenuates obesity, fatty liver, and diabetes. In addition to impaired adipogenesis, livers from C/EBPβ-/- x Leprdb/db mice had dramatically decreased triglyceride content and reduced lipogenic enzyme activity. C/EBPβ deletion in Leprdb/db mice down-regulated peroxisome proliferator-activated receptor γ2 (PPARγ2) and stearoyl-CoA desaturase-1 and up-regulated PPARα independent of SREBP1c. Conversely, C/EBPβ overexpression in wild-type mice increased PPARγ2 and stearoyl-CoA desaturase-1 mRNA and hepatic triglyceride content. In FAO cells, overexpression of the liver inhibiting form of C/EBPβ or C/EBPβ RNA interference attenuated palmitate-induced triglyceride accumulation and reduced PPARγ2 and triglyceride levels in the liver in vivo. Leptin and the anti-diabetic drug metformin acutely down-regulated C/EBPβ expression in hepatocytes, whereas fatty acids up-regulate C/EBPβ expression. These data provide novel evidence linking C/EBPβ expression to lipogenesis and energy balance with important implications for the treatment of obesity and fatty liver disease. Obesity is the most common nutritional disorder in Western societies. Today in the United States, more than 60% of people are either overweight (body mass index (BMI) > 25) or obese (BMI > 30) (1Mokdad A.H. Bowman B.A. Ford E.S. Vinicor F. Marks J.S. Koplan J.P. J. Am. Med. Assoc. 2001; 286: 1195-2000Crossref PubMed Scopus (2241) Google Scholar). Obesity is frequently associated with type II diabetes, hypertension, and hyperlipidemia, all known risk factors for cardiovascular disease (2Burton B.T. Foster W.R. Hirsch J. Van Itallie T.B. Int. J. Obes. 1985; 9: 155-170PubMed Google Scholar). Obesity is also a major risk factor for non-alcoholic fatty liver disease, one of the most common emerging liver diseases in Western countries coinciding with the worldwide obesity epidemic (3Clark J.M. J. Clin. 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This is most elegantly illustrated using tissue-specific gene knockouts and overexpression models to elucidate the mechanism of action of the PPAR 5The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; ADPH, adipophilin; AMPK, AMP-activated protein kinase; C/EBP, CCAAT/enhancer-binding protein; FAS, fatty acid synthase; LAP, liver-enriched activating protein; Leprdb/db, leptin receptor mutant mouse; LIP, liver-enriched inhibitory protein; LXR, liver X receptor; NEFA, non-esterified free fatty acids; PGC-1, peroxisomal proliferated-activated receptor γ co-activator-1; Pref-1, preadipocyte factor-1; SCD-1, stearoyl-CoA desaturase-1; SREBP, sterol response element-binding protein; TG, triglycerides; WAT, white adipose tissue; WT, wild type; RNAi, RNA interference; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDL, low density lipoprotein; shRNA, short hairpin RNA; pfu, plaque-forming units; Adv, adenovirus; GFP, green fluorescent protein.5The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; ADPH, adipophilin; AMPK, AMP-activated protein kinase; C/EBP, CCAAT/enhancer-binding protein; FAS, fatty acid synthase; LAP, liver-enriched activating protein; Leprdb/db, leptin receptor mutant mouse; LIP, liver-enriched inhibitory protein; LXR, liver X receptor; NEFA, non-esterified free fatty acids; PGC-1, peroxisomal proliferated-activated receptor γ co-activator-1; Pref-1, preadipocyte factor-1; SCD-1, stearoyl-CoA desaturase-1; SREBP, sterol response element-binding protein; TG, triglycerides; WAT, white adipose tissue; WT, wild type; RNAi, RNA interference; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDL, low density lipoprotein; shRNA, short hairpin RNA; pfu, plaque-forming units; Adv, adenovirus; GFP, green fluorescent protein. family of nuclear hormone receptors (12Chinetti-Gbaguidi G. Fruchart J.C. Staels B. Curr. Opin. Pharmacol. 2005; 5: 177-183Crossref PubMed Scopus (80) Google Scholar).The CCAAT/enhancer-binding protein (C/EBP) family includes five nuclear transcription factors, C/EBP α, β, γ, δ, and ϵ, encoded by separate genes located on different chromosomes (13Landschulz W.H. Johnson P.F. Adashi E.Y. Graves B.J. McKnight S.L. Genes Dev. 1988; 2: 786-800Crossref PubMed Scopus (626) Google Scholar, 14Williams S.C. Cantwell C.A. Johnson P.F. Genes Dev. 1991; 5: 1553-1567Crossref PubMed Scopus (438) Google Scholar). Collectively, C/EBPs are expressed across a variety of cell types, and a large body of data exists on their expression patterns, the promoters they regulate, and the signals that modulate expression and/or activity (15Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar, 16Ramji D.P. Foka P. Biochem. J. 2002; 365: 561-575Crossref PubMed Google Scholar). The great majority of this information was obtained in vitro. Members of the C/EBP family can form homo- and heterodimers within the promoters of genes, often making it difficult to distinguish between unique and redundant functions of the transcription factors on gene expression. However, in recent years mice have been generated with null mutations for each of the C/EBP genes, allowing the identification of unique and physiologically relevant functions.Although C/EBPα shares a similar tissue distribution with C/EBPβ, they are differentially regulated during development and in response to changes in nutrition and hormonal status. C/EBPα knock-out mice are born without lipid or glycogen reserves and die of neonatal hypoglycemia several hours after birth due in part to the absence of phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and glycogen synthase (17Wang N.D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (828) Google Scholar). There was no significant increase in either C/EBPβ or -δ mRNA in the livers of these mice, suggesting C/EBPα may be part of a developmental program aimed at preparing the fetus for metabolism during the early prenatal period (18Darlington G.J. Wang N. Hanson R.W. Curr. Opin. Genet. Dev. 1995; 5: 565-570Crossref PubMed Scopus (115) Google Scholar). Adult mice lacking C/EBPβ fail to increase gluconeogenesis during fasting and have reduced fat deposition, resulting in hypoglycemia and reduced non-esterified free fatty acids (NEFA) that act systemically to increase insulin sensitivity (19Liu S. Croniger C. Arizmendi C. Harada-Shiba M. Ren J. Poli V. Hanson R.W. Friedman J.E. J. Clin. Investig. 1999; 103: 207-213Crossref PubMed Scopus (79) Google Scholar, 20Wang L. Shao J. Muhlenkamp P. Liu S. Klepcyk P. Ren J. Friedman J.E. J. Biol. Chem. 2000; 275: 14173-14181Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The absence of C/EBPβ leads to lower blood glucose and reduced phosphoenolpyruvate carboxykinase gene induction in diabetes (21Arizmendi C. Liu S. Croniger C. Poli V. Friedman J.E. J. Biol. Chem. 1999; 274: 13033-13040Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), indicating that deleting C/EBPβ may have anti-diabetic as well as anti-obesity effects. Using a gene replacement strategy where C/EBPβ was expressed from the C/EBPα gene locus, Chen et al. (22Chen S.S. Chen J.F. Johnson P.F. Muppala V. Lee Y.H. Mol. Cell. Biol. 2000; 20: 7292-7299Crossref PubMed Scopus (87) Google Scholar) showed that C/EBPβ rescued the role of C/EBPα in liver but not in white adipose tissue (WAT), emphasizing the unique role of C/EBPα in adipogenesis and C/EBPβ in gluconeogenesis.Although C/EBPs regulate adipogenesis and gluconeogenesis, their ability to regulate hepatic lipid metabolism remains relatively unexplored. Recently, Matsusue et al. (23Matsusue K. Gavrilova O. Lambert G. Brewer Jr., H.B. Ward J.M. Inoue Y. LeRoith D. Gonzalez F.J. Mol. Endocrinol. 2004; 18: 2751-2764Crossref PubMed Scopus (60) Google Scholar) demonstrated that liver-specific deletion of C/EBPα prevented accelerated lipogenesis in Leprob/ob mice. However, disruption of hepatic C/EBPα in normal adult mice appears to cause an exacerbation of hyperglycemia (24Inoue Y. Inoue J. Lambert G. Yim S.H. Gonzalez F.J. J. Biol. Chem. 2004; 279: 44740-44748Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and age-dependent increase in hepatosteatosis (24Inoue Y. Inoue J. Lambert G. Yim S.H. Gonzalez F.J. J. Biol. Chem. 2004; 279: 44740-44748Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 25Yang J. Croniger C.M. Lekstrom-Himes J. Zhang P. Fenyus M. Tenen D.G. Darlington G.J. Hanson R.W. J. Biol. Chem. 2005; 280: 38689-38699Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), due in part to a decrease in genes encoding lipoprotein transport and fatty acid oxidation. The reason(s) for these differences in C/EBPα-/- mice is unclear but highlight important differences between C/EBPα and C/EBPβ with regard to their regulation and the metabolic phenotype.To address the potential role of C/EBPβ in the pathogenesis of obesity and related disorders, we generated C/EBPβ-/- mice on a Leprdb/db null background. Here we show that C/EBPβ inactivation in Leprdb/db mice attenuates adipogenesis, obesity, fatty liver, and diabetes. C/EBPβ deletion affected critical genes that regulate hepatic lipogenesis and triglyceride (TG) metabolism resulting in protection from liver steatosis, independent of sterol response element binding protein 1c (SREBP1c). Moreover, forced overexpression of C/EBPβ induced TG accumulation along with PPARγ and stearoyl-CoA desaturase-1 (SCD-1) in vivo, whereas liver-specific inactivation in vivo and in liver cells in vitro can block TG accumulation, coincident with a reduction in PPARγ. Together these results demonstrate that in addition to its role in regulating adipogenesis and gluconeogenesis, C/EBPβ is necessary and sufficient to regulate hepatic lipogenesis independent of SREBP1c.EXPERIMENTAL PROCEDURESAnimal Crossing and GenotypingThe generation and genotyping procedures for C/EBPβ-/- and Leprdb/db mice have been described previously (26Nizielski S.E. Arizmendi C. Shteyngarts A.R. Farrell C.J. Friedman J.E. Am. J. Physiol. 1996; 270: R1005-R1012PubMed Google Scholar, 27Screpanti I. Romani L. Musiani P. Modesti A. Fattori E. Lazzaro D. Sellitto C. Scarpa S. Bellavia D. Lattanzio G. Bistoni F. Frati L. Cortese R. Gulino A. Ciliberto G. Costantini F. Poli V. EMBO J. 1995; 14: 1932-1941Crossref PubMed Scopus (370) Google Scholar). Mice deficient in C/EBPβ were backcrossed for up to eight generations with C57Bl/6J mice from The Jackson Laboratories (Bar Harbor, ME) and intercrosses between heterozygous mice derived from these littermates. Double heterozygous offspring were then intercrossed to produce offspring with the following genotypes: wild-type (WT) mice (+/+ at the C/EBPβ and db locus), C/EBPβ-/- x Lepr+/+, C/EBPβ+/+ x Leprdb/db, and double knock-out C/EBPβ-/- x Leprdb/db mice. All mice were kept on a 12-h light/dark cycle and were fed a standard mouse chow ad libitum. Experiments and sample collection took place in the early afternoon after a 6-h daytime food withdrawal for steady state measurements. The University of Colorado at Denver and Health Sciences Center Animal Care and Use Committee approved all procedures.Determination of Serum Insulin, Adiponectin, Free Fatty Acids, and TGsBlood was collected from the tail vein at the times indicated. Plasma glucose levels were measured using an automatic glucose monitor (Roche Diagnostics). Serum insulin and adiponectin levels (Linco Research, St. Charles, MO and Alpco Diagnostics, Windham, NH), free fatty acids (Waco Diagnostics, Wako, TX), and TGs (Sigma-Aldrich) were measured using commercial kits, according to the manufacturers' recommended protocols.Determination of Body Fat ContentTo assess body composition (percentage of fat), whole-body measurements of intact mice were performed using dual-energy x-ray absorptiometry (DEXA, PIXImus; GE-Lunar Corp., Madison, WI) as described previously in mice at 16 weeks of age (28Barbour L.A. Shao J. Qiao L. Pulawa L.K. Jensen D.R. Bartke A. Garrity M. Draznin B. Friedman J.E. Am. J. Obstet. Gynecol. 2002; 186: 512-517Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 29Haugen B.R. Jensen D.R. Sharma V. Pulawa L.K. Hays W.R. Krezel W. Chambon P. Eckel R.H. Endocrinology. 2004; 145: 3679-3685Crossref PubMed Scopus (42) Google Scholar). Total fat, lean, and mineral mass were evaluated, excluding the head and tail. The short term precision error for whole-body measurements was <2%. Results are presented as fat content and total body weight.Indirect CalorimetryMetabolic measurements were obtained using an open circuit indirect calorimetry system as described previously (30Smith S.J. Cases S. Jensen D.R. Chen H.C. Sande E. Tow B. Sanan D.A. Raber J. Eckel R.H. Farese Jr., R.V. Nat. Genet. 2000; 25: 87-90Crossref PubMed Scopus (725) Google Scholar). The system was calibrated against a standard gas mixture to measure O2 consumed and CO2 produced (ml/kg/h). Oxygen consumption was assessed individually in mice. After a 24-h period for adaptation to the metabolic chamber, VO2 and CO2 produced were assessed at 5-min intervals for a 72-h period. Mice had free access to water and food during the 12-h night period. Total oxygen consumption represents the mean of all samples collected during the experiment. Measurements of energy intake and energy expenditure were corrected for lean body weight.TG AssaysLiver samples were homogenized in microcentrifuge tubes using Kontes disposable pestles, whereas muscle was homogenized in Kontes Duall® homogenizers (Kimble/Kontes, Vineland, NJ). All tissues were initially homogenized in 8 volumes of deionized water to facilitate cell disruption; subsequently, 1 volume of 5 m NaCl was added to enhance partitioning of lipids into the organic phase. Plasma samples were analyzed directly, whereas tissue sample homogenates were extracted using a modification of the method described by Bligh and Dyer (31Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42174) Google Scholar). Briefly, a 200-μl aliquot of 10% homogenate prepared in 0.5 m NaCl was mixed with 500 μl of methanol, vortexed, then mixed with 250 μl of chloroform and vortexed again for 2 min. The single-phase mixture was broken into two phases by the addition of 250 μl of water followed by 250 μl of chloroform with mixing between each addition. After centrifugation, the lower, organic phase was collected in shell vials. Complete extraction of any residual lipids was ensured by reextracting with 250 μlof chloroform:methanol (9:1). The organic phase was separated by centrifugation and combined with the first extraction. Samples were dried with an N-Evap (Organomation Associates, Berlin, MA) under flowing nitrogen. The lipids were re-dissolved in a solution of 90% isopropanol, 10% Triton X-100 (2%) to disperse the TGs for assay. TGs were measured colorimetrically (TR0100, Sigma-Aldrich). For RNA interference (RNAi) experiments, TGs were extracted from FAO rat liver cells after the tert-butanol extraction method described previously (32Perdomo G. Commerford S.R. Richard A.T. Adams S.H. Corkey B.E. O'Doherty R.M. Brown N.F. J. Biol. Chem. 2004; 279: 27177-27186Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar).Histology and ImmunofluorescencePortions of gonadal adipose tissue were removed from anesthetized mice, fixed overnight in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm thickness. Sections were stained with hematoxylin and eosin and photographed at ×50 magnification. For immunohistochemistry, sections were deparafinized in xylenes and rehydrated, and antigen retrieval was performed in heated citrate buffer. Sections were blocked in 1.5% goat serum in phosphate-buffered saline for 1 h at room temperature and then incubated overnight with primary antibody diluted in the PBS, 1.5% goat serum overnight at 4 °C. Primary antibodies to the following targets were: C/EBPα (sc-61, Santa Cruz Biotechnology, Santa Cruz, CA), PPARγ2 (2492, Cell Signaling Technology, Danvers, MA), preadipocyte factor-1 (Pref-1) (PREF-12A, Alpha Diagnostic, San Antonio, TX), and proliferating cell nuclear antigen (M0879, DakoCytomation, Glostrup, Denmark), Akt1 (Upstate, Chicago, IL). Sections were then rinsed 3× in PBS and incubated with Texas Red-conjugated anti-rabbit IgG (Vector Laboratories, Burlingame, CA) for 1 h in PBS. Sections were rinsed three times in PBS, and coverslips were mounted with Vectamount with 4′,6-diamidino-2-phenylindole (fluorescent nuclear stain, Vector Laboratories). Images of fluorescent-labeled sections were taken with a SPOT RT camera connected to a Nikon Eclipse TE2000U microscope and captured to a PowerMac G4 computer. Phase contrast images were taken with a SPOT Insight camera attached to the same microscope. Images were processed using Adobe Photoshop CS software. Fluorescent terminal dUTP nick-end labeling staining for apoptotic cells was performed using the ApoAlert DNA fragmentation assay kit from Clontech (Mountain View, CA) according to the manufacturer's protocol.Western Blot AnalysisTo measure nuclear C/EBPβ and SREBP1c protein levels, liver nuclear extracts were prepared from frozen liver samples. Liver tissue (50-70 mg) was homogenized in 300-500 μlof hypotonic buffer (10 mm HEPES, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml each of aprotinin and leupeptin, and 0.5 mg/ml benzamidine). The supernatants (cytoplasmic extracts) were saved. The pellets were re-suspended in 40 μl of high salt buffer (20 mm HEPES, 400 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 0.5 mg/ml benzamidine) for 30 min on ice with occasional vortexing. After centrifugation at 15,000 rpm for 30 min, the supernatants (nuclear extracts) were saved. The protein concentration was measured with the Bradford assay (Bio-Rad) using bovine serum albumin as standard. Lysates were subjected to SDS/PAGE on 10% gradient gels. Proteins were transferred and immobilized on Immobilon-P membrane. The membranes were immunoblotted with respective primary and secondary antibodies, and bands were visualized by using enhanced chemiluminescence and quantified by densitometry. Primary antibodies used were SCD-1 (sc-1475, Santa Cruz), PPARα (sc-9000), PPARγ2 (sc-22020), C/EBPβ (sc-150), CD36 (ABM5526, Cascade Biosciences), phospho-AMP-activated protein kinase (AMPK; Thr-172, 2531, Cell Signaling), SREBP1c (sc-366), adipophilin (ADPH; RDI-PROGP40, Fitzgerald Industries, Concord, MA), and GAPDH (sc-20358). The antibody to the LDL receptor was supplied in kind from Dr. Jay Horton (UT-Southwestern). Primary antibodies were diluted 1:300-1:500 in 5% nonfat dry milk. Secondary antibodies were goat anti-mouse and goat anti-rabbit IgG-horseradish peroxidase conjugates (Bio-Rad) and were diluted 1:10000 in 5% nonfat dry milk.Hepatic Enzyme ActivityLivers were from 16-week-old mice fasted for 6 h. Animals were anesthetized with 150 μl of avertin (2.5%) followed by rapid removal and freezing on dry ice within 1 min. The samples were stored at -80 °C until analysis. Tissue (100 mg) was homogenized for 30 s in 2.0 ml of 0.25 m sucrose, 10 mm Tris acetate, 1 mm EDTA, 1 mm dithiothreitol, pH 7.4, using a Tissumizer (Polytron) homogenizer. The homogenate was centrifuged at 4 °C for 130 min at 68,000 × g. From the supernatant 0.5 ml was gel-filtered using NAP 5 columns (GE Healthcare). For citrate lyase derivation the columns were equilibrated and eluted with 20 mm Tris/HCl, 1 mm EDTA, 1 mm dithiothreitol, pH 7.5, whereas for fatty acid synthase assays the columns were equilibrated and eluted with 0.5 m potassium phosphate buffer, 5 mm dithiothreitol, pH 7.0. Activity of ATP citrate lyase was determined spectrophotometrically as described previously (33Corrigan A.P. Rider C.C. Biochem. J. 1983; 214: 299-307Crossref PubMed Scopus (16) Google Scholar). All assays were performed in duplicate and were linear for at least 15 min after the addition of CoA. Fatty acid synthase (FAS) was activated before analysis (34Nepokroeff C.M. Lakshmanan M.R. Porter J.W. Methods Enzymol. 1975; 35: 37-44Crossref PubMed Scopus (228) Google Scholar) by incubating the gel-filtered high speed supernatant at 37 °C for 15 min before assay. Activity of FAS was then determined spectrophotometrically (34Nepokroeff C.M. Lakshmanan M.R. Porter J.W. Methods Enzymol. 1975; 35: 37-44Crossref PubMed Scopus (228) Google Scholar). All assays were performed in duplicate and were linear for at least 5 min after the addition of malonyl CoA and acetyl CoA.Adenovirus PurificationAdenovirus was propagated in HEK293 cells. Cells were harvested when cytopathic effects were visible in more than 90% of the cells. Adenovirus was released from cells through rapid freeze/thawing. Adenovirus was purified via cesium chloride gradients and dialyzed into virion dialysis buffer (10 mm Tris-HCl, pH 8.0, 135 mm NaCl, 1 mm MgCl2, and 50% glycerol). Titer was measured using A260.RNAi Adenovirus ConstructionShort hairpin RNA (shRNA) designed for C/EBPβ targeted the 3′-untranslated region (5′-CCGGGCCCTGAGTAATCAC-3′). Sense and antisense oligonucleotides were designed and cloned into pSUPER (OligoEngine, Seattle, WA) as described previously (35Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3945) Google Scholar). Cloning into the adenoviral shuttle vector EH006, and virus propagation was described previously (36Bain J.R. Schisler J.C. Takeuchi K. Newgard C.B. Becker T.C. Diabetes. 2004; 53: 2190-2194Crossref PubMed Scopus (69) Google Scholar).Adenovirus TransductionAnimals—Adenovirus (1 × 1010-1 × 1011 pfu/ml adenovirus in 150 μl of PBS) expressing the C/EBPβ isoforms, LAP (Adv-LAP) or LIP (Adv-LIP), or GFP (Adv-GFP) alone (control) were injected via tail vein into mice. Animals were sacrificed 4 days post-injection. Liver was removed, and nuclear homogenates were prepared for Western blotting or preserved in RNAlater (Qiagen, Valencia, CA).Cells—FAO cells were transduced 24 h after plating with Adv-GFP, Adv-LAP, or Adv-LIP and treated with or without 200 μm palmitate (molar ratio of 1 palmitate:3 albumin) for 24 h before harvesting. For RNAi experiments, FAO cells were transduced with or without adenovirus-delivered shRNA targeting C/EBPβ (Adv-C/EBPβ-shRNA) 24 h and treated with or without 200 μm palmitate for 48 h before harvesting.Treatment of FAO Cells with MetforminFAO cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (penicillin 100 IU/ml, streptomycin 100 μg/ml) in 5% CO2. Cells were subsequently serum-starved overnight then treated for 4 h with metformin (20, 50, 200, 500 μm). Separate cytoplasmic and nuclear extracts were prepared as described previously (21Arizmendi C. Liu S. Croniger C. Poli V. Friedman J.E. J. Biol. Chem. 1999; 274: 13033-13040Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Protein (50 μg) was electrophoresed, blotted, and probed with specific antibodies.Liver Perfusion StudiesIsolated perfused rat liver preparation has been described previously (37Huang W. Dedousis N. Bhatt B.A. O'Doherty R.M. J. Biol. Chem. 2004; 279: 21695-21700Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Livers were perfused with 400 ml of re-circulating oxygenated Krebs-Henseleit buffer with or without leptin (9-fold above basal) for 90 min in the presence or absence of phosphatidylinositol 3-kinase inhibitors wortmannin (100 nm) or LY294002 (10 μm). Livers were removed and kept frozen at -80 °C until analysis of C/EBPβ protein by Western blot analysis.Quantitative Real-time PCRTotal RNA was extracted from homogenized mouse liver or FAO cells using the RNeasy kit (Qiagen). cDNA was prepared by reverse transcription of 250-1000 ng of total RNA using the Superscript III enzyme and random hexamers (Invitrogen). cDNAs were amplified using Platinum quantitative PCR SuperMix-UDG (Invitrogen) and TaqMan Gene Expression assays (ABI, Foster City, CA), or custom assays were designed using Primer Express (ABI) software on an Opticon 2 (Bio-Rad) or ABI 7700 real-time PCR system. RNA expression data were normalized to levels of 18 S RNA, which was unaffected by adenoviral transduction or animal genotype. The TaqMan ID number for genes analyzed are as follows: acyl-coenzyme A oxidase 1, Mm00443579_m1; FAS, Mm00662319_m1; PPARα, Mm00440939_m1; PPARγ, Mm00440945_m1; SCD-1, Mm00772290_m1; GAPDH, 4308313; 18 S RNA, 4308329. For C/EBPβ, a custom designed assay from ABI was used (forward primer, 5′-AAGAGCCGCGACAAGGC-3′; reverse primer, 5′-GTCAGCTCCAGCACCTTGTG-3′, probe 5′-AAGATGCGCAACCTGGAGACGCA-3′).Statistical AnalysisStatistical comparisons between groups were made using Student's t test or analysis of variance where appropriate. All values are reported as the mean plus or minus S.E., and differences were considered to be statistically significant at p values less than 0.05.RESULTSC/EBPβ Deletion Decreases Weight Gain and Fat Mass in Leprdb/db Mice—To determine whether C/EBPβ deficiency can protect against a genetic form of obesity and its related syndromes, we intercrossed C/EBPβ+/- and Leprdb/+ mice and obtained mice deficient in both C/EBPβ and leptin receptors (C/EBPβ-/- x Leprdb/db). The frequency for obtaining C/EBPβ-/- x Leprdb/db progeny was lower than expected due to partial embryonic lethality of C/EBPβ-/- mice (38Croniger C. Trus M. Lysek-Stupp K. Cohen H. Liu Y. Darlington G.J. Poli V. Hanson R.W. Reshef L. J. Biol. Chem. 1997; 272: 26306-26312Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). At weaning (4 weeks of age) the body weight differences between Leprdb/db and C/EBPβ-/- x Leprdb/db mice were indistinguishable (Fig. 1A). However, by 12 weeks of age Leprdb/db mice were overtly obese, whereas C/EBPβ-/- x Leprdb/db mice weighed significantly less (p < 0.05). By 16 weeks of age C/EBPβ-/- x Leprdb/db mice weighed 13.21 ± 0.76 g less, a difference of 25% (p < 0.01). The lower body weight in C/EBPβ-/- x Leprdb/db mice was accompanied by a reduction in total fat mass of 10.7 ± 0.56 g as determined by whole-body DEXA scanning (Fig. 1B). However, C/EBPβ-/- x Leprdb/db mice also had significantly less lean body mass by 3.1 ± 0.15 g, suggesting the decrease in body weight could be associated with increased energy expenditure or perhaps reduced energy intake. To investiga
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