CCAAT/Enhancer-binding Protein β Deletion Reduces Adiposity, Hepatic Steatosis, and Diabetes in Lepr Mice

• DOI: 10.1074/jbc.m701329200
• Publication Date: 2007-03-27T01:15:40Z
• Authors: Jill M. Schroeder...; Shaikh Mizanoor R...; Rachel C. Janssen; Liping Qiao; Jianhua Shao; Michael Roper; Stephanie J. Fischer; Erin Lowe; David J. Orlicky; James L. McManaman; Carol Palmer; William L. Gitomer; Wan Huang; Robert M. O'Doherty; Thomas C. Becker; Dwight J. Klemm; Dalan R. Jensen; Leslie K. Pulawa; Robert H. Eckel; Jacob E. Friedman
• Keywords: Lipogenesis; Steatosis; Ccaat-enhancer-binding proteins

CCAAT/enhancer-binding protein β (C/EBPβ) plays a key role in initiation of adipogenesis adipose tissue and gluconeogenesis liver; however, the C/EBPβ hepatic lipogenesis remains undefined. Here we show that inactivation Leprdb/db mice attenuates obesity, fatty liver, diabetes. In addition to impaired adipogenesis, livers from C/EBPβ-/- x had dramatically decreased triglyceride content reduced lipogenic enzyme activity. deletion down-regulated peroxisome proliferator-activated receptor γ2 (PPARγ2) stearoyl-CoA desaturase-1 up-regulated PPARα independent SREBP1c. Conversely, overexpression wild-type increased PPARγ2 mRNA content. FAO cells, liver inhibiting form or RNA interference attenuated palmitate-induced accumulation levels vivo. Leptin anti-diabetic drug metformin acutely expression hepatocytes, whereas acids up-regulate expression. These data provide novel evidence linking energy balance with important implications for treatment obesity disease. Obesity is most common nutritional disorder Western societies. Today United States, more than 60% people are either overweight (body mass index (BMI) > 25) 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). frequently associated type II diabetes, hypertension, hyperlipidemia, all known risk factors cardiovascular disease (2Burton B.T. Foster W.R. Hirsch Van Itallie T.B. Int. Obes. 1985; 9: 155-170PubMed also major factor non-alcoholic disease, one emerging diseases countries coinciding worldwide epidemic (3Clark J.M. Clin. Gastroenterol. 2006; 40: S5-S10PubMed Scholar, 4Marchesini G. Babini M. Minerva Cardioangiol. 54: 229-239PubMed The underlying transcriptional events contribute its disorders not well understood. Some genes regulate body weight have been identified as additional neuropeptides, hormones, play regulation, particularly through β-adrenergic system (5Collins S. Surwit R.S. Recent Prog. Horm. Res. 56: 309-328Crossref (207) 6Schwartz M.W. Woods S.C. Porte Jr., D. Seeley R.J. Baskin D.G. Nature. 2000; 404: 661-671Crossref (4821) Discovery hormone leptin receptors, which suppress appetite reduce fat mass, has our understanding regulation (7Chen Koyama K. Yuan X. Lee Y. Zhou Y.T. O'Doherty R. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. A. 1996; 93: 14795-14799Crossref (285) 8Friedman Nutr. Rev. 1998; 56 (and S54-S75 (discussion)): S38-S46Crossref (230) More recently, study specific transcription their metabolism provided powerful new tools integrated mechanisms diabetes (9Beaven S.W. Tontonoz P. Annu. 57: 313-329Crossref (192) 10Finck B.N. Kelly D.P. Investig. 116: 615-622Crossref (1059) 11Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. 98: 1575-1584Crossref (695) This elegantly illustrated using tissue-specific gene knockouts models elucidate mechanism action PPAR 5The abbreviations used are: PPAR, receptor; ADPH, adipophilin; AMPK, AMP-activated kinase; C/EBP, protein; FAS, acid synthase; LAP, liver-enriched activating Leprdb/db, mutant mouse; LIP, inhibitory LXR, X NEFA, non-esterified free acids; PGC-1, peroxisomal proliferated-activated γ co-activator-1; Pref-1, preadipocyte factor-1; SCD-1, desaturase-1; SREBP, sterol response element-binding TG, triglycerides; WAT, white tissue; WT, wild type; RNAi, 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 protein. family nuclear receptors (12Chinetti-Gbaguidi Fruchart J.C. Staels B. Curr. Opin. Pharmacol. 2005; 5: 177-183Crossref (80) Scholar).The (C/EBP) includes five factors, C/EBP α, β, γ, δ, ϵ, encoded by separate 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 (626) 14Williams Cantwell C.A. 1991; 1553-1567Crossref (438) Collectively, C/EBPs expressed across variety cell types, large exists patterns, promoters they regulate, signals modulate and/or activity (15Poli V. Biol. Chem. 273: 29279-29282Abstract Full Text PDF (550) 16Ramji Foka Biochem. 2002; 365: 561-575Crossref great majority this information was obtained vitro. Members can homo- heterodimers within genes, often making it difficult distinguish between unique redundant functions However, recent years generated null mutations each allowing identification physiologically relevant functions.Although C/EBPα shares similar distribution C/EBPβ, differentially regulated during development changes nutrition hormonal status. knock-out born without lipid glycogen reserves die neonatal hypoglycemia several hours after birth due part absence phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, synthase (17Wang N.D. Finegold M.J. Bradley Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref (828) There no significant increase -δ these mice, suggesting may be developmental program aimed at preparing fetus early prenatal period (18Darlington Wang N. Hanson R.W. Genet. 565-570Crossref (115) Adult lacking fail fasting deposition, resulting (NEFA) act systemically insulin sensitivity (19Liu Croniger C. Arizmendi Harada-Shiba Ren Poli Friedman J.E. 1999; 103: 207-213Crossref (79) 20Wang L. Shao Muhlenkamp Liu Klepcyk 275: 14173-14181Abstract (41) leads lower blood glucose carboxykinase induction (21Arizmendi 274: 13033-13040Abstract (70) Scholar), indicating deleting anti-obesity effects. Using replacement strategy where locus, Chen et al. (22Chen S.S. J.F. Muppala Y.H. Mol. Cell. 20: 7292-7299Crossref (87) Scholar) showed rescued but (WAT), emphasizing gluconeogenesis.Although gluconeogenesis, ability relatively unexplored. Recently, Matsusue (23Matsusue Gavrilova O. Lambert Brewer H.B. Ward Inoue LeRoith Gonzalez F.J. Endocrinol. 2004; 18: 2751-2764Crossref (60) demonstrated liver-specific prevented accelerated Leprob/ob mice. disruption normal adult appears cause an exacerbation hyperglycemia (24Inoue Yim S.H. 279: 44740-44748Abstract (64) age-dependent hepatosteatosis 25Yang C.M. Lekstrom-Himes Zhang Fenyus Tenen 280: 38689-38699Abstract (66) decrease encoding lipoprotein transport oxidation. reason(s) differences C/EBPα-/- unclear highlight regard metabolic phenotype.To address potential pathogenesis related disorders, background. affected critical (TG) protection steatosis, element binding 1c (SREBP1c). Moreover, forced induced TG along PPARγ (SCD-1) vivo, vivo cells vitro block accumulation, coincident reduction PPARγ. Together results demonstrate regulating necessary sufficient SREBP1c.EXPERIMENTAL PROCEDURESAnimal Crossing GenotypingThe generation genotyping procedures described previously (26Nizielski S.E. Shteyngarts A.R. Farrell C.J. Physiol. 270: R1005-R1012PubMed 27Screpanti Romani Musiani Modesti Fattori E. Lazzaro Sellitto Scarpa Bellavia Lattanzio Bistoni Frati Cortese Gulino Ciliberto Costantini EMBO 14: 1932-1941Crossref (370) Mice deficient were backcrossed up eight generations C57Bl/6J Jackson Laboratories (Bar Harbor, ME) intercrosses heterozygous derived littermates. Double offspring then intercrossed produce following genotypes: (WT) (+/+ db locus), Lepr+/+, C/EBPβ+/+ double All kept 12-h light/dark cycle fed standard mouse chow ad libitum. Experiments sample collection took place afternoon 6-h daytime food withdrawal steady state measurements. University Colorado Denver Health Sciences Center Animal Care Use Committee approved procedures.Determination Serum Insulin, Adiponectin, Free Fatty Acids, TGsBlood collected tail vein times indicated. Plasma measured automatic monitor (Roche Diagnostics). adiponectin (Linco Research, St. Charles, MO Alpco Diagnostics, Windham, NH), (Waco Wako, TX), TGs (Sigma-Aldrich) commercial kits, according manufacturers' recommended protocols.Determination Body Fat ContentTo assess composition (percentage fat), whole-body measurements intact performed dual-energy x-ray absorptiometry (DEXA, PIXImus; GE-Lunar Corp., Madison, WI) 16 weeks age (28Barbour L.A. Qiao Pulawa L.K. Jensen Bartke Garrity Draznin Obstet. Gynecol. 186: 512-517Abstract (143) 29Haugen B.R. Sharma Hays Krezel W. Chambon Eckel Endocrinology. 145: 3679-3685Crossref (42) Total fat, lean, mineral evaluated, excluding head tail. term precision error <2%. Results presented total weight.Indirect CalorimetryMetabolic open circuit indirect calorimetry (30Smith S.J. Cases H.C. Sande Tow Sanan D.A. Raber Farese R.V. Nat. 25: 87-90Crossref (725) calibrated against gas mixture measure O2 consumed CO2 produced (ml/kg/h). Oxygen consumption assessed individually After 24-h adaptation chamber, VO2 5-min intervals 72-h period. access water night oxygen represents mean samples experiment. Measurements intake expenditure corrected lean weight.TG AssaysLiver homogenized microcentrifuge tubes Kontes disposable pestles, muscle Duall® homogenizers (Kimble/Kontes, Vineland, NJ). tissues initially 8 volumes deionized facilitate disruption; subsequently, 1 volume 5 m NaCl added enhance partitioning lipids into organic phase. analyzed directly, homogenates extracted modification method Bligh Dyer (31Bligh E.G. W.J. Can. 1959; 37: 911-917Crossref (42174) Briefly, 200-μl aliquot 10% homogenate prepared 0.5 mixed 500 μl methanol, vortexed, 250 chloroform vortexed again 2 min. single-phase broken two phases followed mixing addition. centrifugation, lower, phase shell vials. Complete extraction any residual ensured reextracting μlof chloroform:methanol (9:1). separated centrifugation combined first extraction. Samples dried N-Evap (Organomation Associates, Berlin, MA) under flowing nitrogen. re-dissolved solution 90% isopropanol, Triton X-100 (2%) disperse assay. colorimetrically (TR0100, Sigma-Aldrich). For (RNAi) experiments, rat tert-butanol (32Perdomo Commerford S.R. Richard A.T. Adams Corkey B.E. R.M. N.F. 27177-27186Abstract (132) Scholar).Histology ImmunofluorescencePortions gonadal removed anesthetized fixed overnight 4% paraformaldehyde, embedded paraffin, sectioned 4 μm thickness. Sections stained hematoxylin eosin photographed ×50 magnification. immunohistochemistry, sections deparafinized xylenes rehydrated, antigen retrieval heated citrate buffer. blocked 1.5% goat serum saline h room temperature incubated primary antibody diluted °C. Primary antibodies targets were: (sc-61, Santa Cruz Biotechnology, Cruz, CA), (2492, Cell Signaling Technology, Danvers, MA), factor-1 (Pref-1) (PREF-12A, Alpha Diagnostic, San Antonio, proliferating (M0879, DakoCytomation, Glostrup, Denmark), Akt1 (Upstate, Chicago, IL). rinsed 3× PBS Texas Red-conjugated anti-rabbit IgG (Vector Laboratories, Burlingame, CA) PBS. three coverslips mounted Vectamount 4′,6-diamidino-2-phenylindole (fluorescent stain, Vector Laboratories). Images fluorescent-labeled taken SPOT RT camera connected Nikon Eclipse TE2000U microscope captured PowerMac G4 computer. Phase contrast images Insight attached same microscope. processed Adobe Photoshop CS software. Fluorescent terminal dUTP nick-end labeling staining apoptotic ApoAlert DNA fragmentation assay kit Clontech (Mountain View, manufacturer's protocol.Western Blot AnalysisTo SREBP1c levels, extracts frozen samples. Liver (50-70 mg) 300-500 hypotonic buffer (10 mm HEPES, 10 KCl, 0.1 EDTA, EGTA, dithiothreitol, phenylmethylsulfonyl fluoride, μg/ml aprotinin leupeptin, mg/ml benzamidine). supernatants (cytoplasmic extracts) saved. pellets re-suspended 40 high salt (20 400 aprotinin, benzamidine) 30 min ice occasional vortexing. 15,000 rpm min, (nuclear concentration Bradford (Bio-Rad) bovine albumin standard. Lysates subjected SDS/PAGE gradient gels. Proteins transferred immobilized Immobilon-P membrane. membranes immunoblotted respective secondary antibodies, bands visualized enhanced chemiluminescence quantified densitometry. SCD-1 (sc-1475, Cruz), (sc-9000), (sc-22020), (sc-150), CD36 (ABM5526, Cascade Biosciences), phospho-AMP-activated kinase (AMPK; Thr-172, 2531, Signaling), (sc-366), adipophilin (ADPH; RDI-PROGP40, Fitzgerald Industries, Concord, GAPDH (sc-20358). LDL supplied kind Dr. Jay (UT-Southwestern). 1:300-1:500 5% nonfat dry milk. Secondary anti-mouse IgG-horseradish peroxidase conjugates 1:10000 milk.Hepatic Enzyme ActivityLivers 16-week-old fasted 6 h. Animals 150 avertin (2.5%) rapid removal freezing stored -80 °C until analysis. Tissue (100 s 2.0 ml 0.25 sucrose, Tris acetate, pH 7.4, Tissumizer (Polytron) homogenizer. centrifuged 130 68,000 × g. From supernatant gel-filtered NAP columns (GE Healthcare). lyase derivation equilibrated eluted 20 Tris/HCl, 7.5, assays potassium phosphate buffer, 7.0. Activity ATP determined spectrophotometrically (33Corrigan A.P. Rider C.C. 1983; 214: 299-307Crossref (16) duplicate linear least 15 CoA. (FAS) activated before analysis (34Nepokroeff Lakshmanan M.R. Porter J.W. Methods Enzymol. 1975; 35: 37-44Crossref (228) incubating speed 37 FAS malonyl CoA acetyl CoA.Adenovirus PurificationAdenovirus propagated HEK293 cells. Cells harvested when cytopathic effects visible Adenovirus released freeze/thawing. purified via cesium chloride gradients dialyzed virion dialysis Tris-HCl, 8.0, 135 NaCl, MgCl2, 50% glycerol). Titer A260.RNAi ConstructionShort (shRNA) designed targeted 3′-untranslated region (5′-CCGGGCCCTGAGTAATCAC-3′). Sense antisense oligonucleotides cloned pSUPER (OligoEngine, Seattle, WA) (35Brummelkamp T.R. Bernards Agami 296: 550-553Crossref (3945) Cloning adenoviral shuttle vector EH006, virus propagation (36Bain J.R. Schisler Takeuchi Becker T.C. Diabetes. 53: 2190-2194Crossref (69) Scholar).Adenovirus TransductionAnimals—Adenovirus (1 1010-1 1011 pfu/ml adenovirus PBS) expressing isoforms, LAP (Adv-LAP) LIP (Adv-LIP), GFP (Adv-GFP) alone (control) injected sacrificed days post-injection. removed, blotting preserved RNAlater (Qiagen, Valencia, CA).Cells—FAO transduced 24 plating Adv-GFP, Adv-LAP, Adv-LIP treated 200 palmitate (molar ratio palmitate:3 albumin) harvesting. RNAi adenovirus-delivered shRNA targeting (Adv-C/EBPβ-shRNA) 48 harvesting.Treatment MetforminFAO cultured Dulbecco's modified Eagle's medium supplemented fetal calf antibiotics (penicillin 100 IU/ml, streptomycin μg/ml) CO2. subsequently serum-starved (20, 50, 200, μm). Separate cytoplasmic Protein (50 μg) electrophoresed, blotted, probed antibodies.Liver Perfusion StudiesIsolated perfused preparation (37Huang Dedousis Bhatt 21695-21700Abstract (63) Livers re-circulating oxygenated Krebs-Henseleit (9-fold above basal) 90 presence phosphatidylinositol 3-kinase inhibitors wortmannin nm) LY294002 blot analysis.Quantitative Real-time PCRTotal RNeasy (Qiagen). cDNA reverse 250-1000 ng Superscript III random hexamers (Invitrogen). cDNAs amplified Platinum quantitative PCR SuperMix-UDG (Invitrogen) TaqMan Gene Expression (ABI, City, custom Primer Express (ABI) software Opticon ABI 7700 real-time system. normalized 18 S RNA, unaffected transduction animal genotype. ID number follows: acyl-coenzyme A oxidase 1, Mm00443579_m1; Mm00662319_m1; PPARα, Mm00440939_m1; PPARγ, Mm00440945_m1; Mm00772290_m1; 4308313; 4308329. (forward primer, 5′-AAGAGCCGCGACAAGGC-3′; 5′-GTCAGCTCCAGCACCTTGTG-3′, probe 5′-AAGATGCGCAACCTGGAGACGCA-3′).Statistical AnalysisStatistical comparisons groups made Student's t test variance appropriate. values reported plus minus S.E., considered statistically p less 0.05.RESULTSC/EBPβ Deletion Decreases Weight Gain Mass Mice—To determine whether deficiency protect genetic syndromes, C/EBPβ+/- Leprdb/+ both (C/EBPβ-/- Leprdb/db). frequency obtaining progeny expected partial embryonic lethality (38Croniger Trus Lysek-Stupp Cohen Reshef 1997; 272: 26306-26312Abstract (93) At weaning (4 age) indistinguishable (Fig. 1A). 12 overtly obese, weighed significantly (p < 0.05). By 13.21 ± 0.76 g less, difference 25% 0.01). accompanied 10.7 0.56 DEXA scanning 1B). 3.1 0.15 g, could perhaps intake. To investiga