Reduction of Hepatosteatosis and Lipid Levels by an Adipose Differentiation-Related Protein Antisense Oligonucleotide
2007; Elsevier BV; Volume: 132; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2007.02.046
ISSN1528-0012
AutoresYumi Imai, Gladys M. Varela, Malaka B. Jackson, Mark J. Graham, Rosanne M. Crooke, Rexford S. Ahima,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoBackground & Aims: Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive triglyceride accumulation in hepatocytes. Expression of the lipid droplet protein adipose differentiation-related protein (ADRP) is increased in NAFLD, but whether this is causally linked to hepatic lipid metabolism is unclear. We postulated that a reduction in ADRP would ameliorate hepatic steatosis and improve insulin action. Methods: Leptin deficient Lepob/ob and diet-induced obese (DIO) mice were treated with antisense oligonucleotide (ASO) against ADRP, and effects on hepatic and serum lipids and glucose homeostasis were examined. Results: ADRP ASO specifically decreased ADRP mRNA and protein levels in the livers of Lepob/ob and DIO mice, without altering the levels of other lipid droplet proteins, that is, S3-12 and TIP47. ADRP ASO suppressed expression of lipogenic genes, reduced liver triglyceride content without affecting cholesterol, attenuated triglyceride secretion, and decreased serum triglyceride and alanine aminotransaminase levels. The reduction in hepatic steatosis by ADRP ASO was associated with improvement in glucose homeostasis in both Lepob/ob and DIO mice. Conclusions: This study demonstrates a crucial role for the lipid droplet protein ADRP in regulation of lipid metabolism. Reduction in hepatic ADRP level using an antisense oligonucleotide reverses hepatic steatosis, hypertriglyceridemia, and insulin resistance in obese mice, suggesting that ADRP may be targeted for the treatment of NAFLD and associated lipid and glucose abnormalities. Background & Aims: Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive triglyceride accumulation in hepatocytes. Expression of the lipid droplet protein adipose differentiation-related protein (ADRP) is increased in NAFLD, but whether this is causally linked to hepatic lipid metabolism is unclear. We postulated that a reduction in ADRP would ameliorate hepatic steatosis and improve insulin action. Methods: Leptin deficient Lepob/ob and diet-induced obese (DIO) mice were treated with antisense oligonucleotide (ASO) against ADRP, and effects on hepatic and serum lipids and glucose homeostasis were examined. Results: ADRP ASO specifically decreased ADRP mRNA and protein levels in the livers of Lepob/ob and DIO mice, without altering the levels of other lipid droplet proteins, that is, S3-12 and TIP47. ADRP ASO suppressed expression of lipogenic genes, reduced liver triglyceride content without affecting cholesterol, attenuated triglyceride secretion, and decreased serum triglyceride and alanine aminotransaminase levels. The reduction in hepatic steatosis by ADRP ASO was associated with improvement in glucose homeostasis in both Lepob/ob and DIO mice. Conclusions: This study demonstrates a crucial role for the lipid droplet protein ADRP in regulation of lipid metabolism. Reduction in hepatic ADRP level using an antisense oligonucleotide reverses hepatic steatosis, hypertriglyceridemia, and insulin resistance in obese mice, suggesting that ADRP may be targeted for the treatment of NAFLD and associated lipid and glucose abnormalities. Nonalcoholic fatty liver disease (NAFLD) includes a spectrum of abnormalities, ranging from excessive lipid accumulation (steatosis) to inflammation, cirrhosis, and hepatic failure.1Angulo P. Nonalcoholic fatty liver disease.N Engl J Med. 2002; 346: 1221-1231Google Scholar NAFLD is increasing at an alarming rate, and has become the leading cause of abnormal liver function tests in the United States.2Clark J.M. Brancati F.L. Diehl A.M. The prevalence and etiology of elevated aminotransferase levels in the United States.Am J Gastroenterol. 2003; 98: 960-967Google Scholar Although NAFLD is closely linked with obesity, dyslipidemia, and insulin resistance, the pathogenesis is unclear.1Angulo P. Nonalcoholic fatty liver disease.N Engl J Med. 2002; 346: 1221-1231Google Scholar Lipid droplets are composed of a core of neutral lipids surrounded by proteins, for example, perilipin, adipose differentiation-related protein (ADRP), S3-12, and TIP47.3Londos C. Sztalryd C. Tansey J.T. Kimmel A.R. Role of PAT proteins in lipid metabolism.Biochimie. 2005; 87: 45-49Google Scholar Perilipin expression is confined to steroidogenic cells and adipocytes, and increases triacylglyceride storage in adipocytes by decreasing lipolysis.3Londos C. Sztalryd C. Tansey J.T. Kimmel A.R. Role of PAT proteins in lipid metabolism.Biochimie. 2005; 87: 45-49Google Scholar In agreement, mice lacking perilipin exhibit an increased basal lipolytic rate and resistance to obesity.4Tansey J.T. Sztalryd C. Gruia-Gray J. Roush D.L. Zee J.V. Gavrilova O. Reitman M.L. Deng C.X. Li C. Kimmel A.R. Londos C. Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity.Proc Natl Acad Sci U S A. 2001; 98: 6494-6499Google Scholar ADRP was named for its high expression in the early stages of adipogenesis; however, later studies revealed that ADRP was only transiently associated with perilipin on the surface of lipid droplets, and very low levels persisted in mature adipocytes.5Jiang H.P. Serrero G. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.Proc Natl Acad Sci U S A. 1992; 89: 7856-7860Google Scholar, 6Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein.J Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF Google Scholar However, ADRP is abundantly expressed in a variety of cells, including hepatocytes, macrophages, breast epithelium, and kidneys, in which ADRP coats lipid droplets.6Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein.J Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF Google Scholar, 7Heid H.W. Moll R. Schwetlick I. Rackwitz H.R. Keenan T.W. Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases.Cell Tissue Res. 1998; 294: 309-321Google Scholar Overexpression of ADRP stimulates fatty acid uptake and triglyceride formation, whereas inhibition of ADRP expression decreases lipid accumulation.8Gao J. Serrero G. Adipose differentiation related protein (ADRP) expressed in transfected COS-7 cells selectively stimulates long chain fatty acid uptake.J Biol Chem. 1999; 274: 16825-16830Google Scholar, 9Imamura M. Inoguchi T. Ikuyama S. Taniguchi S. Kobayashi K. Nakashima N. Nawata H. ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts.Am J Physiol Endocrinol Metab. 2002; 283: E775-E783Google Scholar, 10Larigauderie G. Furman C. Jaye M. Lasselin C. Copin C. Fruchart J.C. Castro G. Rouis M. Adipophilin enhances lipid accumulation and prevents lipid efflux from THP-1 macrophages: potential role in atherogenesis.Arterioscler Thromb Vasc Biol. 2004; 24: 504-510Google Scholar, 11Schadinger S.E. Bucher N.L. Schreiber B.M. Farmer S.R. PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes.Am J Physiol Endocrinol Metab. 2005; 288: E1195-E1205Google Scholar Studies suggest that ADRP is actively involved in hepatic lipid metabolism. ADRP is increased in fatty liver in humans and rodents.11Schadinger S.E. Bucher N.L. Schreiber B.M. Farmer S.R. PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes.Am J Physiol Endocrinol Metab. 2005; 288: E1195-E1205Google Scholar, 12Dalen K.T. Ulven S.M. Arntsen B.M. Solaas K. Nebb H.I. PPARalpha activators and fasting induce the expression of adipose differentiation-related protein in liver.J Lipid Res. 2006; 47: 931-943Google Scholar, 13Motomura W. Inoue M. Ohtake T. Takahashi N. Nagamine M. Tanno S. Kohgo Y. Okumura T. Up-regulation of ADRP in fatty liver in human and liver steatosis in mice fed with high fat diet.Biochem Biophys Res Commun. 2006; 340: 1111-1118Google Scholar, 14Steiner S. Wahl D. Mangold B.L. Robison R. Raymackers J. Meheus L. Anderson N.L. Cordier A. Induction of the adipose differentiation-related protein in liver of etomoxir-treated rats.Biochem Biophys Res Commun. 1996; 218: 777-782Google Scholar An increase in ADRP levels in hepatocytes stimulates triglyceride deposition, while suppression of ADRP prevents lipid accumulation in hepatocytes.11Schadinger S.E. Bucher N.L. Schreiber B.M. Farmer S.R. PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes.Am J Physiol Endocrinol Metab. 2005; 288: E1195-E1205Google Scholar, 15Magnusson B. Asp L. Bostrom P. Ruiz M. Stillemark-Billton P. Linden D. Boren J. Olofsson S.O. Adipocyte differentiation-related protein promotes fatty acid storage in cytosolic triglycerides and inhibits secretion of very low-density lipoproteins.Arterioscler Thromb Vasc Biol. 2006; 26: 1566-1571Google Scholar Although ablation of the adrp gene in mice decreased hepatic triglyceride content, the effect was very mild.16Chang B.H. Li L. Paul A. Taniguchi S. Nannegari V. Heird W.C. Chan L. Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein.Mol Cell Biol. 2006; 26: 1063-1076Google Scholar Unexpectedly, ADRP deficiency in this model did not affect lipid synthesis, secretion, or oxidation.16Chang B.H. Li L. Paul A. Taniguchi S. Nannegari V. Heird W.C. Chan L. Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein.Mol Cell Biol. 2006; 26: 1063-1076Google Scholar Furthermore, the reduction in hepatic lipid content in ADRP knockout mice did not affect plasma glucose and lipid levels.16Chang B.H. Li L. Paul A. Taniguchi S. Nannegari V. Heird W.C. Chan L. Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein.Mol Cell Biol. 2006; 26: 1063-1076Google Scholar Genetic ablation of ADRP did not affect the expression of other lipid droplet proteins in the liver, but a recent study has shown that the TIP47 level is increased in adipocytes.17Sztalryd C. Bell M. Lu X. Mertz P. Hickenbottom S. Chang B.H. Chan L. Kimmel A.R. Londos C. Functional compensation for adipose differentiation-related protein (ADFP) by TIP47 in an adfp null embryonic cell line.J Biol Chem. 2006; 281: 34341-34348Google Scholar Thus, we postulated that these discrepancies may be attributable to developmental changes in other lipid droplet proteins. To clarify the role of ADRP in lipid and glucose metabolism, we administered an antisense oligonucleotide (ASO) to adult Lepob/ob and diet-induced obese (DIO) mice, which are well known to develop steatosis, insulin resistance, and elevated plasma lipid and glucose levels.18Takahashi N. Patel H.R. Qi Y. Dushay J. Ahima R.S. Divergent effects of leptin in mice susceptible or resistant to obesity.Horm Metab Res. 2002; 34: 691-697Google Scholar, 19Takahashi N. Qi Y. Patel H.R. Ahima R.S. A novel aminosterol reverses diabetes and fatty liver disease in obese mice.J Hepatol. 2004; 41: 391-398Abstract Full Text Full Text PDF Scopus (34) Google Scholar, 20Yu X.X. Murray S.F. Pandey S.K. Booten S.L. Bao D. Song X.Z. Kelly S. Chen S. McKay R. Monia B.P. Bhanot S. Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.Hepatology. 2005; 42: 362-371Google Scholar ASO treatment has been used successfully to investigate the roles of various enzymes in lipid and glucose metabolism.20Yu X.X. Murray S.F. Pandey S.K. Booten S.L. Bao D. Song X.Z. Kelly S. Chen S. McKay R. Monia B.P. Bhanot S. Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.Hepatology. 2005; 42: 362-371Google Scholar, 21Jiang G. Li Z. Liu F. Ellsworth K. Dallas-Yang Q. Wu M. Ronan J. Esau C. Murphy C. Szalkowski D. Bergeron R. Doebber T. Zhang B.B. Prevention of obesity in mice by antisense oligonucleotide inhibitors of stearoyl-CoA desaturase-1.J Clin Invest. 2005; 115: 1030-1038Google Scholar, 22Savage D.B. Choi C.S. Samuel V.T. Liu Z.X. Zhang D. Wang A. Zhang X.M. Cline G.W. Yu X.X. Geisler J.G. Bhanot S. Monia B.P. Shulman G.I. Reversal of diet-induced hepatic steatosis and hepatic insulin resistance by antisense oligonucleotide inhibitors of acetyl-CoA carboxylases 1 and 2.J Clin Invest. 2006; 116: 817-824Google Scholar Our results demonstrate that ADRP ASO treatment specifically decreases ADRP mRNA and protein levels in the livers of Lepob/ob and DIO mice, resulting in suppression of lipogenic genes, reduction in triglyceride secretion, reversal of hepatic steatosis and hypertriglyceridemia, and enhancement of insulin sensitivity. Chimeric second-generation antisense oligonucleotides were synthesized by Isis Pharmaceuticals (Carlsbad, CA) and formulated in phosphate-buffered saline. ADRP ASO, ISIS 384423 (5′GGTCATCTGGCCAGCAACAT3′), is a 20-mer phosphorothioate oligonucleotide complementary to the mRNA for mouse ADRP. The mouse ADRP ASO is composed of 3 2′-O-methoxyethyl-modified ribonucleosides (2′-MOE) at the 3′- and 5′-ends with 2′-deoxynucleosides in between. A control oligo, ISIS 141923 (5′CCTTCCCTGAAGGTTCCTCC-3′) contains similar chemical modifications, with no complementarity to known genes including the ADRP mRNA. The cytosines in all of these molecules are methylated. The efficacy of ADRP ASO was first tested in primary mouse hepatocytes as described for other ASOs.20Yu X.X. Murray S.F. Pandey S.K. Booten S.L. Bao D. Song X.Z. Kelly S. Chen S. McKay R. Monia B.P. Bhanot S. Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.Hepatology. 2005; 42: 362-371Google Scholar, 23Crooke R.M. Graham M.J. Lemonidis K.M. Whipple C.P. Koo S. Perera R.J. An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis.J Lipid Res. 2005; 46: 872-884Google Scholar Experiments were performed in accordance with the Institutional Animal Care and Use Committee guidelines and approvals. Six-week-old female Lepob/ob(B6.V-Lepob/J) and wild-type C57BL/6J mice were purchased from the Jackson Laboratories (Bar Harbor, ME). The mice were housed (n = 5 per cage) under 12-hour light–dark cycle (light on at 6 AM) and ambient temperature 22°C, and allowed free access to water and food. Lepob/ob mice were fed regular rodent chow (Lab Diet 5001, Richmond, IN). C57Bl/6J mice were placed on high-fat diet (45 kcal% fat; Research Diet, New Brunswick, NJ).18Takahashi N. Patel H.R. Qi Y. Dushay J. Ahima R.S. Divergent effects of leptin in mice susceptible or resistant to obesity.Horm Metab Res. 2002; 34: 691-697Google Scholar ASO treatment was initiated after a week of acclimation. ADRP and control ASOs were injected with sterile-filtered saline (50 mg/kg intraperitoneal [IP] twice weekly) for 4 weeks. Our preliminary studies demonstrated that this dose was optimum in decreasing lipids without inducing toxicity. Body weight and food intake were measured twice weekly. Tail blood glucose was measured weekly (One Touch Ultra glucometer; Johnson & Johnson, Arlington, TX).18Takahashi N. Patel H.R. Qi Y. Dushay J. Ahima R.S. Divergent effects of leptin in mice susceptible or resistant to obesity.Horm Metab Res. 2002; 34: 691-697Google Scholar, 24Qi Y. Takahashi N. Hileman S.M. Patel H.R. Berg A.H. Pajvani U.B. Scherer P.E. Ahima R.S. Adiponectin acts in the brain to decrease body weight.Nat Med. 2004; 10: 524-529Google Scholar Glucose tolerance test was done in DIO mice 3 weeks after ADRP ASO vs. control ASO treatment. The mice were fasted overnight and given 1.5 g/kg 20% glucose IP. Glucose was measured in tail blood at time 0 (before IP glucose), 15, 30, 60, 90, and 120 minutes. For the insulin tolerance test, DIO and Lepob/ob mice were fasted for 4 hours. Humulin 0.75 U/kg (Eli Lilly, Indianapolis, IN) was injected IP, and blood glucose was measured at times of 0, 15, 30, 60, and 90 minutes. To assess very low density lipoprotein secretion, the mice were fasted for 4 hours, and received 1 g/kg Poloxamer 407 IP.25Millar J.S. Cromley D.A. McCoy M.G. Rader D.J. Billheimer J.T. Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339.J Lipid Res. 2005; 46: 2023-2028Google Scholar Tail blood was drawn at time 0, and 1, 2, and 3 hours later, and serum was prepared for triglyceride measurement.19Takahashi N. Qi Y. Patel H.R. Ahima R.S. A novel aminosterol reverses diabetes and fatty liver disease in obese mice.J Hepatol. 2004; 41: 391-398Abstract Full Text Full Text PDF Scopus (34) Google Scholar Energy expenditure and fuel oxidation were assessed using indirect calorimetry (Oxymax; Columbus Instruments, Columbus, OH).18Takahashi N. Patel H.R. Qi Y. Dushay J. Ahima R.S. Divergent effects of leptin in mice susceptible or resistant to obesity.Horm Metab Res. 2002; 34: 691-697Google Scholar, 19Takahashi N. Qi Y. Patel H.R. Ahima R.S. A novel aminosterol reverses diabetes and fatty liver disease in obese mice.J Hepatol. 2004; 41: 391-398Abstract Full Text Full Text PDF Scopus (34) Google Scholar After the last ADRP- or control ASO injection, Lepob/ob and DIO mice were habituated in single cages for 3 days, and the oxygen consumption and carbon dioxide production were measured for 4 hours using an air flow of 500 mL/min, sample flow 400 mL/min, and ambient temperature 22°C. The respiratory quotient (respiratory quotient = carbon dioxide production/oxygen consumption) ranges from 0.7 for fat oxidation, to 1.0 for carbohydrate oxidation. Locomotor behavior was assessed simultaneously during calorimetry using photobeams.19Takahashi N. Qi Y. Patel H.R. Ahima R.S. A novel aminosterol reverses diabetes and fatty liver disease in obese mice.J Hepatol. 2004; 41: 391-398Abstract Full Text Full Text PDF Scopus (34) Google Scholar Body composition was measured using dual emission X-ray absorptiometry (PIXImus DEXA; General Electric, Madison, WI).18Takahashi N. Patel H.R. Qi Y. Dushay J. Ahima R.S. Divergent effects of leptin in mice susceptible or resistant to obesity.Horm Metab Res. 2002; 34: 691-697Google Scholar, 19Takahashi N. Qi Y. Patel H.R. Ahima R.S. A novel aminosterol reverses diabetes and fatty liver disease in obese mice.J Hepatol. 2004; 41: 391-398Abstract Full Text Full Text PDF Scopus (34) Google Scholar Three days after the last ADRP ASO or control treatment, tail blood glucose was measured, after which the mice were euthanized at 1200–1300 hours and blood was obtained via cardiac puncture. Samples of liver, perigonadal fat, and hindquarter muscle were rapidly dissected, frozen in liquid nitrogen, and stored at −80°C. Serum triglyceride, nonesterified fatty acids, cholesterol, and β-hydroxybutyric acid were measured using colorimetric assays.19Takahashi N. Qi Y. Patel H.R. Ahima R.S. A novel aminosterol reverses diabetes and fatty liver disease in obese mice.J Hepatol. 2004; 41: 391-398Abstract Full Text Full Text PDF Scopus (34) Google Scholar, 24Qi Y. Takahashi N. Hileman S.M. Patel H.R. Berg A.H. Pajvani U.B. Scherer P.E. Ahima R.S. Adiponectin acts in the brain to decrease body weight.Nat Med. 2004; 10: 524-529Google Scholar Glucose, insulin, leptin, and adiponectin were measured using enzyme immunoassays as previously described.19Takahashi N. Qi Y. Patel H.R. Ahima R.S. A novel aminosterol reverses diabetes and fatty liver disease in obese mice.J Hepatol. 2004; 41: 391-398Abstract Full Text Full Text PDF Scopus (34) Google Scholar, 24Qi Y. Takahashi N. Hileman S.M. Patel H.R. Berg A.H. Pajvani U.B. Scherer P.E. Ahima R.S. Adiponectin acts in the brain to decrease body weight.Nat Med. 2004; 10: 524-529Google Scholar The HOMA index, a measure of insulin resistance, was calculated based on the glucose and insulin concentrations.26Cai D. Yuan M. Frantz D.F. Melendez P.A. Hansen L. Lee J. Shoelson S.E. Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB.Nat Med. 2005; 11: 183-190Google Scholar, 27Matthews D.R. Hosker J.P. Rudenski A.S. Naylor B.A. Treacher D.F. Turner R.C. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man.Diabetologia. 1985; 28: 412-419Google Scholar Lipids were extracted from livers for triglyceride and cholesterol measurements.19Takahashi N. Qi Y. Patel H.R. Ahima R.S. A novel aminosterol reverses diabetes and fatty liver disease in obese mice.J Hepatol. 2004; 41: 391-398Abstract Full Text Full Text PDF Scopus (34) Google Scholar RNA was extracted from liver, fat, and muscle using Trizol reagent (Invitrogen, Carlsbad, CA), and expression of lipogenic and lipolytic genes and S3-12 and tumor necrosis factor-alpha (TNF-α) was analyzed using real-time polymerase chain reaction (ABI Prism; Applied Biosystems, Foster City, CA).19Takahashi N. Qi Y. Patel H.R. Ahima R.S. A novel aminosterol reverses diabetes and fatty liver disease in obese mice.J Hepatol. 2004; 41: 391-398Abstract Full Text Full Text PDF Scopus (34) Google Scholar The level of mRNA expression was normalized to 36B4.19Takahashi N. Qi Y. Patel H.R. Ahima R.S. A novel aminosterol reverses diabetes and fatty liver disease in obese mice.J Hepatol. 2004; 41: 391-398Abstract Full Text Full Text PDF Scopus (34) Google Scholar For immunoblotting, liver, muscle, and epidydimal fat samples were homogenized in lysis buffer containing 50 mmol/L Tris-HCl (pH 7.4), 250 mmol/L mannitol, 0.5% (w/v) Triton X-100, 50 mmol/L NaF, 1 mmol/L sodium pyrophosphate, 1 mmol/L benzamidine, and 1 mmol/L phenylmethanesulfonyl fluoride supplemented with complete protein inhibition cocktail tablet from Roche (Penzberg, Germany). Protein extracts were separated by 4%–12% NuPAGE Bis-Tris gel (Invitrogen) and transferred to nitrocellulose membranes using semidry transfer cells (Bio-Rad Laboratories, Hercules, CA). After 1 hour of blocking with Tris-buffered saline with 0.1% (vol/vol) Tween 20 containing 3% (wt/vol) nonfat dried milk, membranes were incubated with guinea pig polyclonal antibody against ADRP (Fitzgerald, Concord, MA) or TIP47 (1:1000 dilution) overnight at 4°C, washed 3 times with Tris-buffered saline with 0.1% (vol/vol) Tween 20, incubated with horseradish peroxidase-conjugated goat anti guinea pig IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 1 hour at room temperature, and visualized with enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK). The levels of ADRP were normalized to β-actin or GAPDH (1:1000) (Cell Signaling, Beverly, MA) detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology Inc.). Film autoradiograms were analyzed using laser densitometry and Image J (National Institutes of Health, Bethesda, MD). Liver samples from mice treated with saline vehicle, control ASO, or ADRP ASO were fixed in 10% buffered formalin overnight, embedded in paraffin, sectioned, and processed for hematoxylin-eosin staining or immunohistochemistry. ADRP immunostaining was detected using guinea pig polyclonal anti-ADRP 1:2000 (Fitzgerald Industries International Inc.) and Cy2-conjugated donkey anti-guinea pig antibody 1:800 (Jackson Immuno-Research, West Grove, PA). Nuclear staining was detected using 4′,6-diamidino-2-phenylindole dihydrochloride 1:10,000. The slides were examined under brightfield and fluorescence microscopy (Nikon E600); images were captured using a Cool Snap CF digital camera (BD Biosciences Bioimaging, Rockville, MD). Lepob/ob mice treated with ADRP or control ASO were studied by hyperinsulinemic-euglycemic clamp as previously described.28Qi Y. Nie Z. Lee Y.S. Singhal N.S. Scherer P.E. Lazar M.A. Ahima R.S. Loss of resistin improves glucose homeostasis in leptin deficiency.Diabetes. 2006; 55: 3083-3090Google Scholar An indwelling catheter was inserted in the right internal jugular vein and extended to the right atrium. Four days after recovery, the mice were fasted for 4 hours, placed in restrainers, and administered a bolus injection of 5 μCi of [3-3H]-glucose followed by continuous intravenous (IV) infusion at 0.05 μCi/min. After baseline glucose measurements, a priming dose of regular insulin 40 mU/kg (Humulin; Eli Lilly, Indianapolis, IN) was given IV followed by continuous infusion at 20 mU/kg/min. This insulin dose increases plasma insulin by 10-fold. Blood glucose was maintained at 120–140 mg/dL using a variable infusion of 30% glucose. At the end of the 120-minute clamp, 10 μCi 2-deoxy-D-[1-14C]glucose was injected to estimate glucose uptake. The mice were euthanized, and liver, perigonadal fat, brown adipose tissue, and soleus muscle were excised, frozen immediately in liquid nitrogen, and stored at –80°C for subsequent analysis of glucose uptake.28Qi Y. Nie Z. Lee Y.S. Singhal N.S. Scherer P.E. Lazar M.A. Ahima R.S. Loss of resistin improves glucose homeostasis in leptin deficiency.Diabetes. 2006; 55: 3083-3090Google Scholar, 29Rajala M.W. Qi Y. Patel H.R. Takahashi N. Banerjee R. Pajvani U.B. Sinha M.K. Gingerich R.L. Scherer P.E. Ahima R.S. Regulation of resistin expression and circulating levels in obesity, diabetes, and fasting.Diabetes. 2004; 53: 1671-1679Google Scholar, 30Banerjee R.R. Rangwala S.M. Shapiro J.S. Rich A.S. Rhoades B. Qi Y. Wang J. Rajala M.W. Pocai A. Scherer P.E. Steppan C.M. Ahima R.S. Obici S. Rossetti L. Lazar M.A. Regulation of fasted blood glucose by resistin.Science. 2004; 303: 1195-1198Google Scholar The effects of ADRP ASO on various parameters were analyzed using Student's t test; P < .05 was considered significant. Administration of the ADRP ASO via twice weekly IP injections for 4 weeks decreased ADRP mRNA levels in Lepob/ob mouse liver by 55% (Figure 1A), and ADRP protein by 75% (Figure 1B and C). The control ASO did not alter ADRP expression, similar to saline injection (Figure 1B–C). Immunostaining of liver sections revealed a drastic reduction in ADRP content after administration of ADRP ASO (Figure 1D–F). Likewise, ADRP ASO treatment reduced ADRP mRNA and protein levels in DIO mice (Figure 1G–L). The ADRP ASO did not affect ADRP expression in adipose tissue or muscle in Lepob/ob mice (Figure 2A and B) and DIO mice (data not shown), confirming previous reports showing that peripheral ASO administration acts primarily in the liver.20Yu X.X. Murray S.F. Pandey S.K. Booten S.L. Bao D. Song X.Z. Kelly S. Chen S. McKay R. Monia B.P. Bhanot S. Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.Hepatology. 2005; 42: 362-371Google Scholar, 22Savage D.B. Choi C.S. Samuel V.T. Liu Z.X. Zhang D. Wang A. Zhang X.M. Cline G.W. Yu X.X. Geisler J.G. Bhanot S. Monia B.P. Shulman G.I. Reversal of diet-induced hepatic steatosis and hepatic insulin resistance by antisense oligonucleotide inhibitors of acetyl-CoA carboxylases 1 and 2.J Clin Invest. 2006; 116: 817-824Google Scholar, 23Crooke R.M. Graham M.J. Lemonidis K.M. Whipple C.P. Koo S. Perera R.J. An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis.J Lipid Res. 2005; 46: 872-884Google Scholar Perilipin was not detectable in liver. ADRP ASO treatment did not alter expression of TIP47 (Figure 2C) and S3-12 (Figure 2D) in the liver, or TNF-α in liver and adipose tissue compared to controls (Figure 2E and F). Histologic examination did not reveal inflammatory infiltrate on liver sections from ADRP ASO and control mice.Figure 2Lack of effect of ADRP ASO on (A and B) ADRP protein in white adipose tissue (WAT) and skeletal muscle, and (C) TIP47 protein in liver detected by Western blot, and mRNA expression of (D) S3-12 and (E and F) TNF-α mRNA expression in liver and WAT.View Large Image Figure ViewerDownload Hi-res image Download (PPT) After 4 weeks of treatment, ADRP ASO decreased hepatic triglyceride content in Lepob/ob mice by 50%, but cholesterol did not change substantially (Figure 3A and B). Serum ALT levels fell by 90% in Lepob/ob mice (Figure 3C), arguing against hepatic toxicity. Histologic analysis confirmed a drastic reduction in hepatic steatosis in Lepob/ob mice mice after ADRP ASO treatment compared to saline vehicle and control ASO (Figure 3D–F). Similarly, ADRP ASO decreased hepatic triglyceride content, but not cholesterol, in DIO mice (Figure 3G and H). Serum ALT levels were reduced by ADRP ASO treatment compared with vehicle and control ASO (Figure 3I). We examined the effects of ADRP reduction on energy balance, body composition, and serum chemistry (Table 1). The administration of the ADRP ASO did not affect food intake or energy expenditure in either Lepob/ob or DIO mice. The respiratory quotient did not decrease after ADRP ASO treatment (Table 1). ADRP ASO did not affect body weight in Lepob/ob and DIO mice; however, body fat measured by DEXA decreased (Table 1). In DIO mice, the reduction in body fat was associated with a decrease in leptin (Table 1). Serum triglyceride concentration fell in response to ADRP ASO in Lepob/ob and DIO mice, but cholesterol, nonesterified fatty acids and β-hydroxybutyric acid were not affected (Table 1). ADRP ASO did not change the adiponectin level in Lepob/ob mice, although there was an increase in adiponectin in DIO mice, but this was not significant (Table 1). In contrast to the liver, ADRP ASO did not affect the expression of lipogenic and lipolytic genes in adipose tissue (data not shown).Table 1Effects of ADRP ASO Treatment in Lepob/ob and Diet-Induced Obese (DIO) MiceLepob/obDIOCo
Referência(s)