Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet
2008; Elsevier BV; Volume: 49; Issue: 5 Linguagem: Inglês
10.1194/jlr.m800042-jlr200
ISSN1539-7262
AutoresMary E. Rinella, Marc Elías, Robin R. Smolak, Tao Fu, Jayme Borensztajn, Richard M. Green,
Tópico(s)Folate and B Vitamins Research
ResumoThe methionine choline-deficient (MCD) diet results in liver injury similar to human nonalcoholic steatohepatitis (NASH). The aims of this study were to define mechanisms of MCD-induced steatosis in insulin-resistant db/db and insulin-sensitive db/m mice. MCD-fed db/db mice developed more hepatic steatosis and retained more insulin resistance than MCD-fed db/m mice. Both subcutaneous and gonadal fat were reduced by MCD feeding: gonadal fat decreased by 23% in db/db mice and by 90% in db/m mice. Weight loss was attenuated in the db/db mice, being only 13% compared with 35% in MCD-fed db/db and db/m mice, respectively. Both strains had upregulation of hepatic fatty acid transport proteins as well as increased hepatic uptake of [14C]oleic acid: 3-fold in db/m mice (P < 0.001) and 2-fold in db/db mice (P < 0.01) after 4 weeks of MCD feeding. In both murine strains, the MCD diet reduced triglyceride secretion and downregulated genes involved in triglyceride synthesis. Therefore, increased fatty acid uptake and decreased VLDL secretion represent two important mechanisms by which the MCD diet promotes intrahepatic lipid accumulation in this model. Feeding the MCD diet to diabetic rodents broadens the applicability of this model for the study of human NASH. The methionine choline-deficient (MCD) diet results in liver injury similar to human nonalcoholic steatohepatitis (NASH). The aims of this study were to define mechanisms of MCD-induced steatosis in insulin-resistant db/db and insulin-sensitive db/m mice. MCD-fed db/db mice developed more hepatic steatosis and retained more insulin resistance than MCD-fed db/m mice. Both subcutaneous and gonadal fat were reduced by MCD feeding: gonadal fat decreased by 23% in db/db mice and by 90% in db/m mice. Weight loss was attenuated in the db/db mice, being only 13% compared with 35% in MCD-fed db/db and db/m mice, respectively. Both strains had upregulation of hepatic fatty acid transport proteins as well as increased hepatic uptake of [14C]oleic acid: 3-fold in db/m mice (P < 0.001) and 2-fold in db/db mice (P < 0.01) after 4 weeks of MCD feeding. In both murine strains, the MCD diet reduced triglyceride secretion and downregulated genes involved in triglyceride synthesis. Therefore, increased fatty acid uptake and decreased VLDL secretion represent two important mechanisms by which the MCD diet promotes intrahepatic lipid accumulation in this model. Feeding the MCD diet to diabetic rodents broadens the applicability of this model for the study of human NASH. acyl-coenzyme A oxidase apolipoprotein B carnitine palmitoyltransferase fatty acid transport protein fast-protein liquid chromatography methionine choline-deficient nonalcoholic fatty liver disease nonalcoholic steatohepatitis peroxisome proliferator-activated receptor Quantitative Insulin Sensitivity Check Index real-time stearoyl-coenzyme A desaturase-1 sterol-regulatory element binding protein Nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal liver chemistry tests in the developed world and a major public health problem. Its progressive form, termed nonalcoholic steatohepatitis (NASH) (1.Skelly M.M. James P.D. Ryder S.D. Findings on liver biopsy to investigate abnormal liver function tests in the absence of diagnostic serology.J. Hepatol. 2001; 35: 195-199Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), is a significant predisposing factor for the development of cryptogenic cirrhosis and hepatic failure and an increasingly common indication for liver transplantation (2.Caldwell S.H. Oelsner D.H. Iezzoni J.C. Hespenheide E.E. Battle E.H. Driscoll C.J. Cryptogenic cirrhosis: clinical characterization and risk factors for underlying disease.Hepatology. 1999; 29: 664-669Crossref PubMed Scopus (976) Google Scholar, 3.Sanjeevi A. Lyden E. Sunderman B. Weseman R. Ashwathnarayan R. Mukherjee S. Outcomes of liver transplantation for cryptogenic cirrhosis: a single-center study of 71 patients.Transplant. Proc. 2003; 35: 2977-2980Crossref PubMed Scopus (45) Google Scholar). Insulin resistance is a very significant contributor to the development of NASH that is then exacerbated by hepatic steatosis (4.Sanyal A.J. Mechanisms of disease. Pathogenesis of nonalcoholic fatty liver disease.Nat. Clin. Pract. Gastroenterol. Hepatol. 2005; 2: 46-53Crossref PubMed Scopus (184) Google Scholar, 5.Sanyal A.J. Campbell-Sargent C. Mirshahi F. Rizzo W.B. Contos M.J. Sterling R.K. Luketic V.A. Shiffman M.L. Clore J.N. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.Gastroenterology. 2001; 120: 1183-1192Abstract Full Text Full Text PDF PubMed Scopus (1773) Google Scholar, 6.Samuel V.T. Liu Z.X. Qu X. Elder B.D. Bilz S. Befroy D. Romanelli A.J. Shulman G.I. Mechanism of hepatic insulin resistance in non-alcoholic fatty liver disease.J. Biol. Chem. 2004; 279: 32345-32353Abstract Full Text Full Text PDF PubMed Scopus (1044) Google Scholar). Furthermore, strong evidence exists demonstrating that in NAFLD patients, insulin does not suppress adipose tissue lipolysis to the same extent that it does in healthy individuals (5.Sanyal A.J. Campbell-Sargent C. Mirshahi F. Rizzo W.B. Contos M.J. Sterling R.K. Luketic V.A. Shiffman M.L. Clore J.N. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.Gastroenterology. 2001; 120: 1183-1192Abstract Full Text Full Text PDF PubMed Scopus (1773) Google Scholar). Human data have shown that most hepatic triglyceride in patients with NASH originates from increased delivery and subsequent uptake of free fatty acids to the liver (7.Donnelly K.L. Smith C.I. Schwarzenberg S.J. Jessurun J. Boldt M.D. Parks E.J. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.J. Clin. Invest. 2005; 115: 1343-1351Crossref PubMed Scopus (2470) Google Scholar). NASH is closely associated with obesity and the metabolic syndrome; thus, obese, diabetic murine models such as leptin-deficient ob/ob mice have been used to study experimental steatohepatitis. Despite the presence of profound fatty liver, obesity, and diabetes, ob/ob mice do not develop significant liver injury because of the essential role of leptin in hepatic injury and fibrosis (8.Leclercq I.A. Farrell G.C. Schriemer R. Robertson G.R. Leptin is essential for the hepatic fibrogenic response to chronic liver injury.J. Hepatol. 2002; 37: 206-213Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 9.Aleffi S. Petrai I. Bertolani C. Parola M. Colombatto S. Novo E. Vizzutti F. Anania F.A. Milani S. Rombouts K. et al.Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.Hepatology. 2005; 42: 1339-1348Crossref PubMed Scopus (299) Google Scholar). In contrast to ob/ob mice, leptin-resistant db/db mice are hyperinsulinemic, hyperleptinemic, and hyperlipidemic. They retain some leptin signaling, likely through the short-form leptin receptor, and develop hepatic fibrosis when fed a methionine choline-deficient (MCD) diet (10.Sahai A. Malladi P. Melin-Aldana H. Green R.M. Whitington P.F. Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model.Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 287: G264-G273Crossref PubMed Scopus (163) Google Scholar, 11.Sahai A. Malladi P. Pan X. Paul R. Melin-Aldana H. Green R.M. Whitington P.F. Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin.Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 287: G1035-G1043Crossref PubMed Scopus (245) Google Scholar). Feeding mice a MCD diet is a frequently used nutritional model of NASH that induces aminotransferase elevation and hepatic histological changes characterized by steatosis, focal inflammation, hepatocyte necrosis, and fibrosis (12.Rinella M.E. Green R.M. The methionine-choline deficient dietary model of steatohepatitis does not exhibit insulin resistance.J. Hepatol. 2004; 40: 47-51Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 13.Ip E. Farrell G. Hall P. Robertson G. Leclercq I. Administration of the potent PPARalpha agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice.Hepatology. 2004; 39: 1286-1296Crossref PubMed Scopus (307) Google Scholar, 14.Yamaguchi K. Yang L. McCall S. Huang J. Yu X.X. Pandey S.K. Bhanot S. Monia B.P. Li Y.X. Diehl A.M. Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.Hepatology. 2007; 45: 1366-1374Crossref PubMed Scopus (763) Google Scholar). These histological changes occur rapidly and are morphologically similar to those observed in human NASH. db/db mice fed the MCD diet have greater hepatic injury, steatohepatitis, and hepatic fibrosis than db/m mice fed the MCD diet. In addition, they develop a more proinflammatory cytokine profile than db/m mice fed the MCD diet or db/db mice on a control diet (11.Sahai A. Malladi P. Pan X. Paul R. Melin-Aldana H. Green R.M. Whitington P.F. Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin.Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 287: G1035-G1043Crossref PubMed Scopus (245) Google Scholar, 14.Yamaguchi K. Yang L. McCall S. Huang J. Yu X.X. Pandey S.K. Bhanot S. Monia B.P. Li Y.X. Diehl A.M. Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.Hepatology. 2007; 45: 1366-1374Crossref PubMed Scopus (763) Google Scholar). The mechanisms responsible for the development of hepatic steatosis in the MCD dietary model have not been fully delineated; however, previous rat studies using a choline-deficient (methionine-containing) diet suggest that the pathogenesis of hepatic steatosis may be attributable, at least in part, to impaired hepatic VLDL secretion (15.Yao Z.M. Vance D.E. The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes.J. Biol. Chem. 1988; 263: 2998-3004Abstract Full Text PDF PubMed Google Scholar). This assumption is further supported by the fact that methionine and choline are precursors to phosphatidylcholine, the main phospholipid coating VLDL particles (16.Vance J.E. Vance D.E. The role of phosphatidylcholine biosynthesis in the secretion of lipoproteins from hepatocytes.Can. J. Biochem. Cell Biol. 1985; 63: 870-881Crossref PubMed Scopus (85) Google Scholar). In wild-type mice, others have found that the MCD diet results in downregulation of stearoyl-coenzyme A desaturase-1 (SCD-1), a key enzyme in triglyceride synthesis, with minimal upregulation of β-oxidation genes (17.Rizki G. Arnaboldi L. Gabrielli B. Yan J. Lee G.S. Ng R.K. Turner S.M. Badger T.M. Pitas R.E. Maher J.J. Mice fed a lipogenic methionine-choline-deficient diet develop hypermetabolism coincident with hepatic suppression of SCD-1.J. Lipid Res. 2006; 47: 2280-2290Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Thus, impaired VLDL secretion may play a role in MCD diet-induced intrahepatic lipid accumulation in mice. The aims of this study were to examine the effect of the MCD diet within the physiologic milieu of the metabolic syndrome that occurs in diabetic db/db mice. Furthermore, we examined the mechanisms by which the MCD diet promotes hepatic steatosis in both insulin-sensitive db/m mice and insulin-resistant db/db mice. Our data demonstrate that db/db mice fed the MCD diet retain many aspects of the metabolic syndrome and have relatively preserved visceral fat stores compared with db/m mice. Increased hepatic fatty acid uptake and reduced VLDL secretion are two important mechanisms by which the MCD diet promotes hepatic steatosis. Female BKS·Cg-m+/+ Leprdb/J (db/db) mice and insulin-sensitive heterozygote db/m mice, 8–10 weeks of age, were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice received humane care in compliance with institutional guidelines. Animals had free access to chow and water until the first day of the study, when they received their corresponding diet. The mice were fed one of two diets: a lipogenic diet deficient in methionine and choline (MCD diet) or the MCD diet supplemented with methionine and choline (control diet) (ICN Biomedicals, Inc., Costa Mesa, CA) for 4 weeks. After a 4 h fast, animals were euthanized using CO2 narcosis. Whole blood was obtained from the right atrium by cardiac puncture, and the livers were excised and weighed. Livers were flash-frozen in liquid nitrogen and stored at −70°C. All animal experiments were approved by the Animal Care and Use Committee of Northwestern University Feinberg School of Medicine. The determination of serum alanine aminotransferase was performed using a spectrophotometric assay kit (Biotron, Hemet, CA), and values are reported in Sigma-Frankel units/ml. Triglyceride (TG) and cholesterol were measured enzymatically (Thermo Electron, Louisville, KY) on serum and hepatic homogenate. Fasting blood glucose was measured by the glucose oxidase method using a reflectance glucometer (One Touch II; LifeScan, Milpitas, CA), and serum insulin and leptin levels were determined via radioimmunoassay (Linco, St. Charles, MO). Quantitative Insulin Sensitivity Check Index (QUICKI) was calculated from fasting insulin and glucose levels. Serum β-hydroxybutyrate levels were determined using the β-hydroxybutyrate Liquicolor method (Sanbio, Boerne, TX). After CO2 narcosis and removal of the liver, representative depots of both gonadal (parametrial and paraovarian), representative of visceral fat, and subcutaneous adipose tissue were obtained from all mice in each group as described previously (18.Reed D.R. McDaniel A.H. Li X. Tordoff M.G. Bachmanov A.A. Quantitative trait loci for individual adipose depot weights in C57BL/6ByJ × 129P3/J F2 mice.Mamm. Genome. 2006; 17: 1065-1077Crossref PubMed Scopus (26) Google Scholar). Subcutaneous fat was removed in a standardized manner from the skin overlying the abdominal wall bordered inferiorly by the perineum, medially by the abdominal midline (defined by the most medial aspect of the rectus abdominus), laterally by the midaxillary line, superiorly by the xyphoid process, and posteriorly by the spine. Four cohorts of five mice each were fasted overnight, anesthetized with 70 mg/kg ketamine and 7 mg/kg xylazine ip, and then injected with 0.2 ml of a 10% (v/v) solution of Triton WR-1339 in saline. Blood samples were collected from the tail vein at 0, 3, and 6 h. During the entire period, the animals had free access to water. Triglyceride and cholesterol assays were performed as described above. Assuming that total blood volume in the mouse represents 3.5% of its body weight (19.Vanpatten S. Karkanias G.B. Rossetti L. Cohen D.E. Intracerebroventricular leptin regulates hepatic cholesterol metabolism.Biochem. J. 2004; 379: 229-233Crossref PubMed Scopus (0) Google Scholar), the rate of triglyceride accumulation in mg/kg/h was determined after inhibitor (Triton WR-1339) injection. Plasma apolipoprotein B-100 (apoB-100) and apoB-48 were detected using Western immunoblotting. Aliquots of the plasma before and after Triton WR-1339 injection were diluted 1:2 (v/v) with denaturing sample buffer, heated at 70°C for 10 min, and run on a 3–8% Tris acetate gel using the NuPAGE electrophoresis system (Invitrogen, Carlsbad, CA). The aliquots were then transferred to a nitrocellulose membrane using the wet electrophoretic transfer. Immunoblotting was performed on a nitrocellulose membrane with rabbit polyclonal antibodies (Biodesign International, Saco, ME) against mouse apoB-48/apoB-100 as described previously (20.Fu T. Mukhopadhyay D. Davidson N.O. Borensztajn J. The peroxisome proliferator-activated receptor alpha (PPARalpha) agonist ciprofibrate inhibits apolipoprotein B mRNA editing in low density lipoprotein receptor-deficient mice: effects on plasma lipoproteins and the development of atherosclerotic lesions.J. Biol. Chem. 2004; 279: 28662-28669Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Serum lipoproteins were fractionated with an ÄKTA fast-protein liquid chromatography (FPLC) system using equal volumes of pooled plasma from mouse cohorts on tandem Superose 6 FPLC columns (Amersham Biosciences, Piscataway, NJ). The columns were then eluted with 200 mmol/l sodium phosphate (pH 7.4), 50 mmol/l NaCl, 0.03% (w/v) EDTA, and 0.02% (w/v) sodium azide at a flow rate of 0.4 ml/min. The contents of TG and cholesterol in the eluted fractions and plasma were measured with a microplate assay technique using enzymatic assay reagent kits (Sigma, St. Louis, MO). Hepatic [14C]oleate uptake was measured as described previously by Doege et al. (21.Doege H. Baillie R.A. Ortegon A.M. Tsang B. Wu Q. Punreddy S. Hirsch D. Watson N. Gimeno R.E. Stahl A. Targeted deletion of FATP5 reveals multiple functions in liver metabolism: alterations in hepatic lipid homeostasis.Gastroenterology. 2006; 130: 1245-1258Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Briefly, in a separate experiment, four cohorts of five female db/m and db/db mice were treated for 4 weeks on their respective diets and then fasted overnight with free access to water. Each mouse then received an intragastric 200 μl olive oil bolus containing 2 μCi of [14C]oleic acid (0.1 mCi/ml; ARC 0297-50μCi; American Radiolabeled Chemicals, Inc., St. Louis, MO). Because the exact oleic acid content of the olive oil was unknown, results were analyzed as relative uptake in disintegrations per minute in the four groups rather than as absolute uptake. Before (time 0) and at 30, 60, 120, and 240 min after administration, blood samples were collected via tail bleed. The liver was resected at the 240 min time point, and 250 mg of liver was homogenized in 1 ml of Dulbecco's phosphate-buffered saline. Liver homogenate (0.8 ml) was added to a mixture of 1 ml of chloroform and 2 ml of methanol. The sample was vortexed for 1 min, and 1 ml of Millipore water and an additional 1 ml of chloroform were then added. The sample was vortexed again and then spun at 2,000 rpm for 5 min. The resultant lipid phase was dried, and 5 ml of ScintiVerse (Fisher Scientific, Hampton, NH) was added, in small scintillation vials, vortexed, and then counted for 2 min in a Beckman LS6500 scintillation counter whose efficiency was calculated to be 95%. Total RNA was extracted from liver by homogenizing snap-frozen liver tissue samples in TRIzol reagent (Invitrogen). cDNA was synthesized from 2 μg of total RNA using the SuperScript First Strand System for real-time (RT)-PCR (Invitrogen) and random hexamer primers. The resulting cDNA was subsequently used as a template for quantitative RT-PCR. RT-PCR was performed using 4 μl of the total cDNA in a 50 μl reaction containing QuantiTect SYBR Green PCR Master Mix (Qiagen, Valencia, CA) with specific RT-PCR primers (Table 1). GAPDH (Integrated DNA Technologies, Coralville, IA) was used as a control. RT-PCR was performed using the Applied Biosystems Prism 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). Results are reported as relative differences in gene expression.TABLE 1.Primer sequences for real-time quantitative PCRPrimerSequenceAOX-fwd5′-CTT GTT CGC GCA AGT GAG G-3′AOX-rev5′-CAG GAT CCG ACT GTT TAC C-3′CPT2-fwd5′-GCC CAG CTT CCA TCT TTA CT-3′CPT2-rev5′-CAG GAT GTT GTG GTT TAT CCG C-3′FAS-fwd5′-TGC TCC CAG CTG CAG GC-3′FAS-rev5′-GCC CGG TAG CTC TGG GTG TA-3′FATP1-fwd5′-CGC TTT CTG CGT ATC GTC TG-3′FATP1-rev5′-GAT GCA CGG GAT CGT GTC T-3′FATP2-fwd5′-GGT ATG GGA CAG GCC TTG CT-3′FATP2-rev5′-GGG CAT TGT GGT ATA GAT GAC ATC-3′FATP3-fwd5′-AGT GCC AGG GAT TCT ACC ATC-3′FATP3-rev5′-GAA CTT GGG TTT CAG CAC CAC-3′FATP4-fwd5′-GAT GGC CTC AGC TAT CTG TGA-3′FATP4-rev5′-GGT GCC CGA TGT GTA GAT GTA-3′FATP5-fwd5′-CTA CGC TGG CTG CAT ATA GAT G-3′FATP5-rev5′-CCA CAA AGG TCT CTG GAG GAT-3′GAPDH-fwd (1)5′-ACC ACC ATG GAG AAG GCC GG-3′GAPDH-rev (1)5′-CTC AGT GTA GCC CAA GAT GC-3′GAPDH-fwd (2)5′-GTC GTG GAT CTG ACG TGC C-3′GAPDH-rev (2)5′-TGC CTG CTT CAC CAC CTT C-3′LCAD-fwd5′-AAG GAT TTA TTA AGG GCA AGA AGC-3′LCAD-rev5′-GGA AGC GGA GGC GGA GTC-3′L-FABP-fwd5′-GTG GTC CGC AAT GAG TTC AC-3′L-FABP-rev5′-GTA TTG GTG ATT GTG TCT CC-3′SCD-1-fwd5′-TGG GTT GGC TGC TTG TG-3′SCD-1-rev5′-GCG TGG GCA GGA TGA AG-3′SREBP-1a-fwd5′-TAG TCC GAA GCC GGG TGG GCG CCG GCG CCA T-3′SREBP-1a-rev5′-GAT GTC GTT CAA AAC CGC TGT GTG TCC AGT TC-3′SREBP-1C-fwd5′-ATC GGC GCG GAA GCT GTC GGG GTA GCG TC-3′SREBP-1C-rev5′-ACT GTC TTG GTT GTT GAT GAG CTG GAG CAT-3′AOX, acyl-coenzyme A oxidase; CPT, carnitine palmitoyltransferase; FATP, fatty acid transport protein; LCAD, long-chain acyl-coenzyme A dehydrogenase; L-FABP, fatty acid binding protein; SCD-1, stearoyl-coenzyme A desaturase-1; SREBP, sterol-regulatory element binding protein. Open table in a new tab AOX, acyl-coenzyme A oxidase; CPT, carnitine palmitoyltransferase; FATP, fatty acid transport protein; LCAD, long-chain acyl-coenzyme A dehydrogenase; L-FABP, fatty acid binding protein; SCD-1, stearoyl-coenzyme A desaturase-1; SREBP, sterol-regulatory element binding protein. ANOVA was used for multiple group comparisons. When ANOVA resulted in a nonparametric distribution of the data, a log transformation was performed. When two groups were compared, unpaired t-tests were used for data analysis. The MCD diet has been shown previously to alter glucose metabolism in other mouse strains (12.Rinella M.E. Green R.M. The methionine-choline deficient dietary model of steatohepatitis does not exhibit insulin resistance.J. Hepatol. 2004; 40: 47-51Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Thus, we compared the effects of the MCD diet on these metabolic parameters in db/m and db/db mice. Table 2 summarizes the metabolic effects of the MCD diet on db/db and db/m mice. db/db mice fed either the control diet or the MCD diet remained hyperinsulinemic relative to db/m mice fed either diet (3.7 ± 0.8 and 1.6 ± 0.5 ng/ml, compared with 0.6 ± 0.1 and 0.7 ± 0.2 ng/ml, in db/m mice fed the control or MCD diet, respectively; P < 0.05). Similarly, db/db mice remained more insulin-resistant (lower QUICKI values) than db/m mice fed either diet. MCD-fed db/db mice had a QUICKI of 0.44 ± 0.01, compared with 0.63 ± 0.05 in db/m mice fed the MCD diet (P < 0.05). db/db mice remained hyperleptinemic on the MCD diet relative to db/m mice fed either diet: 133 ± 23 ng/ml compared with 19 ± 2 and 21 ± 3 ng/ml in db/m mice fed the MCD and control diet, respectively (P < 0.001). Table 2 also demonstrates that db/db mice fed the MCD diet had significantly higher serum cholesterol levels than db/m mice fed either diet. In addition, there was an upward trend in serum triglyceride levels for db/db mice fed the MCD diet compared with db/m mice on this diet (NS; P = 0.09). Thus, the db/db mice fed a MCD diet retained many of the features of the metabolic syndrome.TABLE 2.Metabolic parameters of fasting db/db and db/m mice fed the MCD or control diet for 4 weeksdb/db Micedb/m MiceParameterControl DietMCD DietControl DietMCD DietInsulin (ng/ml)3.7 ± 0.8adb/db versus db/m mice on the control diet.1.6 ± 0.5bdb/db versus db/m mice on the MCD diet.0.6 ± 0.10.7 ± 0.2Glucose (mg/dl)426 ± 74124 ± 16195 ± 2476 ± 8Quantitative Insulin Sensitivity Check Index0.32 ± 0.02adb/db versus db/m mice on the control diet.0.44 ± 0.01bdb/db versus db/m mice on the MCD diet.0.52 ± 0.030.63 ± 0.05Leptin (ng/ml)123 ± 40adb/db versus db/m mice on the control diet.133 ± 23bdb/db versus db/m mice on the MCD diet.21 ± 319 ± 2Alanine aminotransferase (SFU/ml)107 ± 7adb/db versus db/m mice on the control diet.,cdb/db mice on the control versus the MCD diet.383 ± 18bdb/db versus db/m mice on the MCD diet.29 ± 10ddb/m mice on the control versus the MCD diet.101 ± 3Liver triglyceride (mg/g liver)65 ± 12adb/db versus db/m mice on the control diet.,cdb/db mice on the control versus the MCD diet.106 ± 522 ± 3ddb/m mice on the control versus the MCD diet.89 ± 13Serum triglyceride (mg/dl)79 ± 3cdb/db mice on the control versus the MCD diet.56 ± 370 ± 4ddb/m mice on the control versus the MCD diet.45 ± 5Serum cholesterol (mg/dl)216 ± 7adb/db versus db/m mice on the control diet.,cdb/db mice on the control versus the MCD diet.148 ± 11bdb/db versus db/m mice on the MCD diet.97 ± 3ddb/m mice on the control versus the MCD diet.69 ± 5MCD, methionine choline-deficient; SFU, sigma-Frankel units. Data were analyzed using ANOVA (n = 4–7 for each group). Values represent means ± SEM. For all, P < 0.05.a db/db versus db/m mice on the control diet.b db/db versus db/m mice on the MCD diet.c db/db mice on the control versus the MCD diet.d db/m mice on the control versus the MCD diet. Open table in a new tab MCD, methionine choline-deficient; SFU, sigma-Frankel units. Data were analyzed using ANOVA (n = 4–7 for each group). Values represent means ± SEM. For all, P < 0.05. To determine whether these metabolic differences were associated with changes in body weight or food consumption, we treated separate cohorts of mice with the MCD diet for 4 weeks. Table 3 shows the effect of the MCD diet on body weight and food consumption. All mice lost weight on the MCD diet, as expected; however, the weight loss was attenuated in the db/db mice compared with db/m mice: 13% versus 35%, respectively. The db/db MCD diet group remained significantly more obese than db/m mice on either diet. At the end of the dietary treatment, the db/db MCD group weighed 34.6 ± 2.2 g, compared with 21.3 ± 0.5 and 14.2 ± 0.3 g in db/m mice fed the control and MCD diet, respectively (P < 0.05). Considering the possibility that weight loss could have been associated with alterations in food consumption, we measured food intake over a 4 week period (Table 3). The MCD diet eliminated baseline hyperphagia in db/db mice; however, daily food consumption was not reduced compared with db/m mice on either diet.TABLE 3.Body weight and food consumption after 4 weeks of feeding with the MCD or the MCD control dietdb/db Micedb/m MiceParameterControl DietMCD DietControl DietMCD DietLiver weight (g)2.78 ± 0.13adb/db mice versus db/m mice on the control diet.,cdb/db mice on the control versus the MCD diet.1.80 ± 0.22bdb/db mice on the versus db/m mice MCD diet.1.00 ± 0.07ddb/m mice on the control versus the MCD diet.0.74 ± 0.03Baseline body weight (g)35.2 ± 1.5adb/db mice versus db/m mice on the control diet.39.5 ± 2.4bdb/db mice on the versus db/m mice MCD diet.19.6 ± 0.321.7 ± 0.4Final body weight (g)49.8 ± 1.1adb/db mice versus db/m mice on the control diet.34.6 ± 2.2bdb/db mice on the versus db/m mice MCD diet.21.3 ± 0.514.2 ± 0.3Change in body weight (g) (%)14.5 ± 0.6 (42%)adb/db mice versus db/m mice on the control diet.,cdb/db mice on the control versus the MCD diet.−4.9 ± 0.6 (−13%)bdb/db mice on the versus db/m mice MCD diet.1.8 ± 0.5 (9%)ddb/m mice on the control versus the MCD diet.−7.5 ± 0.2 (−35%)Daily food consumption (g)6.1 ± 0.4adb/db mice versus db/m mice on the control diet.,cdb/db mice on the control versus the MCD diet.3.4 ± 0.52.9 ± 0.63.5 ± 1.3Data were analyzed using ANOVA (n = 5 for each group). Values represent means ± SEM. For all, P < 0.001.a db/db mice versus db/m mice on the control diet.b db/db mice on the versus db/m mice MCD diet.c db/db mice on the control versus the MCD diet.d db/m mice on the control versus the MCD diet. Open table in a new tab Data were analyzed using ANOVA (n = 5 for each group). Values represent means ± SEM. For all, P < 0.001. Both subcutaneous and visceral fat compartments were differentially reduced by MCD feeding in db/m and db/db mice (Fig. 1). Subcutaneous fat was diminished significantly in both strains by the MCD diet: from 0.32 ± 0.04 to 0.13 ± 0.01 g in db/m mice (P < 0.01) and from 4.5 ± 0.15 to 3.0 ± 0.21 g in db/db mice (P < 0.001), corresponding to 60% and 34% reductions in db/m and db/db mice, respectively. In db/m mice, gonadal fat decreased from 0.52 ± 0.05 g in the control diet group to 0.05 ± 0.01 g in the MCD diet group, a 90% decrease (P < 0.001). In contrast, the decrease in gonadal fat in db/db mice receiving the MCD diet was less marked: from 4.1 ± 0.17 g for db/db mice fed a control diet to 3.1 ± 0.3 g in the MCD diet group (P < 0.05), a 23% reduction. Therefore, gonadal fat mass in db/db mice remained 60-fold higher than in db/m mice on the MCD diet. To understand the potential mechanisms by which the MCD diet induces hepatic steatosis, we examined the effects of the MCD diet on hepatic genes involved in fatty acid synthesis, oxidation, uptake, and secretion. In db/db mice, gene expression of sterol-regulatory binding element binding protein 1c (SREBP-1c) was unaffected by MCD diet feeding; however, the SREBP-1c downstream targets SCD-1 and FAS were decreased significantly, from 0.9 ± 0.1 to 0.2 ± 0.1 and from 1.2 ± 0.2 to 0.5 ± 0.1, respectively, after 4 weeks of MCD feeding (P < 0.05). In addition, gene expression of SREBP-1a was also decreased by the MCD diet in db/db mice (1.1 ± 0.2 and 0.6 ± 0.1; P < 0.01). The MCD diet significantly downregulated SREBP-1c and its downstream targets in db/m mice: 1.0 ± 0.1 and 0.6 ± 0.1 (P = 0.02), for control and MCD diet-fed mice, respectively (Table 4).TABLE 4.Real-time quantitative PCR of hepatic lipid genes in db/db and db/m mice treated with the MCD or the control diet for 4 weeksdb/db Micedb/m MiceGeneControl DietMCD DietC
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