Angiotensinogen in hepatocytes contributes to Western diet-induced liver steatosis
2019; Elsevier BV; Volume: 60; Issue: 12 Linguagem: Inglês
10.1194/jlr.m093252
ISSN1539-7262
AutoresXinran Tao, Jiabing Rong, Hong Lü, Alan Daugherty, Peng Shi, Changle Ke, Zhaocai Zhang, Yinchuan Xu, Jianan Wang,
Tópico(s)Lipid metabolism and biosynthesis
ResumoNonalcoholic fatty liver disease (NAFLD) is considered as a liver manifestation of metabolic disorders. Previous studies indicate that the renin-angiotensin system (RAS) plays a complex role in NAFLD. As the only precursor of the RAS, decreased angiotensinogen (AGT) profoundly impacts RAS bioactivity. Here, we investigated the role of hepatocyte-derived AGT in liver steatosis. AGT floxed mice (hepAGT+/+) and hepatocyte-specific AGT-deficient mice (hepAGT−/−) were fed a Western diet and a normal laboratory diet for 12 weeks, respectively. Compared with hepAGT+/+ mice, Western diet-fed hepAGT−/− mice gained less body weight with improved insulin sensitivity. The attenuated severity of liver steatosis in hepAGT−/− mice was evidenced by histologic changes and reduced intrahepatic triglycerides. The abundance of SREBP1 and its downstream molecules, acetyl-CoA carboxylase and FASN, was suppressed in hepAGT−/− mice. Furthermore, serum derived from hepAGT+/+ mice stimulated hepatocyte SREBP1 expression, which could be diminished by protein kinase B (Akt)/mammalian target of rapamycin (mTOR) inhibition in vitro. Administration of losartan did not affect diet-induced body weight gain, liver steatosis severity, and hepatic p-Akt, p-mTOR, and SREBP1 protein abundance in hepAGT+/+ mice. These data suggest that attenuation of Western diet-induced liver steatosis in hepAGT−/− mice is associated with the alternation of the Akt/mTOR/SREBP-1c pathway. Nonalcoholic fatty liver disease (NAFLD) is considered as a liver manifestation of metabolic disorders. Previous studies indicate that the renin-angiotensin system (RAS) plays a complex role in NAFLD. As the only precursor of the RAS, decreased angiotensinogen (AGT) profoundly impacts RAS bioactivity. Here, we investigated the role of hepatocyte-derived AGT in liver steatosis. AGT floxed mice (hepAGT+/+) and hepatocyte-specific AGT-deficient mice (hepAGT−/−) were fed a Western diet and a normal laboratory diet for 12 weeks, respectively. Compared with hepAGT+/+ mice, Western diet-fed hepAGT−/− mice gained less body weight with improved insulin sensitivity. The attenuated severity of liver steatosis in hepAGT−/− mice was evidenced by histologic changes and reduced intrahepatic triglycerides. The abundance of SREBP1 and its downstream molecules, acetyl-CoA carboxylase and FASN, was suppressed in hepAGT−/− mice. Furthermore, serum derived from hepAGT+/+ mice stimulated hepatocyte SREBP1 expression, which could be diminished by protein kinase B (Akt)/mammalian target of rapamycin (mTOR) inhibition in vitro. Administration of losartan did not affect diet-induced body weight gain, liver steatosis severity, and hepatic p-Akt, p-mTOR, and SREBP1 protein abundance in hepAGT+/+ mice. These data suggest that attenuation of Western diet-induced liver steatosis in hepAGT−/− mice is associated with the alternation of the Akt/mTOR/SREBP-1c pathway. Nonalcoholic fatty liver disease (NAFLD) is defined as excessive liver lipid accumulation (triglyceride content more than 5% of liver weight) excluding other competing causes of hepatic steatosis (1.Chalasani N. Younossi Z. Lavine J.E. Charlton M. Cusi K. Rinella M. Harrison S.A. Brunt E.M. Sanyal A.J. The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.Hepatology. 2018; 67: 328-357Crossref PubMed Scopus (3275) Google Scholar). With a prevalence among adults reaching about 25%, NAFLD is now the most common liver disease worldwide (2.Younossi Z.M. Koenig A.B. Abdelatif D. Fazel Y. Henry L. Wymer M. Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes.Hepatology. 2016; 64: 73-84Crossref PubMed Scopus (5294) Google Scholar). Highly accompanied by obesity and dyslipidemia, NAFLD is recognized as a typical liver manifestation of metabolic syndrome, which may arise from insulin resistance (3.Asrih M. Jornayvaz F.R. Metabolic syndrome and nonalcoholic fatty liver disease: is insulin resistance the link?.Mol. Cell. Endocrinol. 2015; 418: 55-65Crossref PubMed Scopus (218) Google Scholar). Additionally, NAFLD is also considered as a major contributor to cardiovascular disease and obesity-related comorbidities (4.Tessari P. Coracina A. Cosma A. Tiengo A. Hepatic lipid metabolism and non-alcoholic fatty liver disease.Nutr. Metab. Cardiovasc. Dis. 2009; 19: 291-302Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 5.Loria P. Lonardo A. Bellentani S. Day C.P. Marchesini G. Carulli N. Non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease: an open question.Nutr. Metab. Cardiovasc. Dis. 2007; 17: 684-698Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Previous studies have shed a light on the effect of the renin-angiotensin system (RAS) on NAFLD. It has been suggested that inhibition or deletion of critical components of the RAS, including renin, angiotensin converting enzyme, or angiotensin type 1 receptor (AT1R), may protect rodents from diet-induced liver steatosis (6.Nabeshima Y. Tazuma S. Kanno K. Hyogo H. Chayama K. Deletion of angiotensin II type I receptor reduces hepatic steatosis.J. Hepatol. 2009; 50: 1226-1235Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 7Hussain S.A. Utba R.M. Assumaidaee A.M. Effects of azilsartan, aliskiren or their combination on high fat diet-induced non-alcoholic liver disease model in rats.Med. Arch. 2017; 71: 251-255Crossref PubMed Scopus (5) Google Scholar, 8Takahashi N. Li F. Hua K. Deng J. Wang C.H. Bowers R.R. Bartness T.J. Kim H.S. Harp J.B. Increased energy expenditure, dietary fat wasting, and resistance to diet-induced obesity in mice lacking renin.Cell Metab. 2007; 6: 506-512Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 9.Frantz E.D. Penna-de-Carvalho A. Batista Tde M. Aguila M.B. Mandarim-de-Lacerda C.A. Comparative effects of the renin-angiotensin system blockers on nonalcoholic fatty liver disease and insulin resistance in C57BL/6 mice.Metab. Syndr. Relat. Disord. 2014; 12: 191-201Crossref PubMed Scopus (16) Google Scholar). However, results from previous studies have been equivocal. For instance, Verbeek et al. (10.Verbeek J. Spincemaille P. Vanhorebeek I. Van den Berghe G. Vander Elst I. Windmolders P. van Pelt J. van der Merwe S. Bedossa P. Nevens F. et al.Dietary intervention, but not losartan, completely reverses non-alcoholic steatohepatitis in obese and insulin resistant mice.Lipids Health Dis. 2017; 16: 46Crossref PubMed Scopus (18) Google Scholar) reported that losartan, an angiotensin receptor blocker, failed to improve NAFLD in obese mice. It was also suggested that telmisartan, another angiotensin receptor blocker, significantly ameliorated liver triglyceride accumulation via partial PPARγ agonism, rather than RAS inhibition (11.Sugimoto K. Qi N.R. Kazdova L. Pravenec M. Ogihara T. Kurtz T.W. Telmisartan but not valsartan increases caloric expenditure and protects against weight gain and hepatic steatosis.Hypertension. 2006; 47: 1003-1009Crossref PubMed Scopus (144) Google Scholar). This controversial evidence has implicated the complexity of the RAS as well as the interaction among its components; thus, manipulating one certain component of the RAS may trigger diverse changes. Moreover, unexpected side effects of RAS intervention would interfere with the outcomes. Angiotensinogen (AGT) is a polypeptide consisting of 485 amino acid residues (12.Lu H. Cassis L.A. Kooi C.W. Daugherty A. Structure and functions of angiotensinogen.Hypertens. Res. 2016; 39: 492-500Crossref PubMed Scopus (98) Google Scholar). It can be cleaved to generate angiotensin I, angiotensin II (Ang II), and other angiotensin peptides of the RAS (13.Jeunemaitre X. Soubrier F. Kotelevtsev Y.V. Lifton R.P. Williams C.S. Charru A. Hunt S.C. Hopkins P.N. Williams R.R. Lalouel J.M. et al.Molecular basis of human hypertension: role of angiotensinogen.Cell. 1992; 71: 169-180Abstract Full Text PDF PubMed Scopus (1713) Google Scholar). AGT is expressed in multiple organs including brain, kidney, and adipose tissues; however, AGT derived from hepatocytes alone contributes up to 90% of circulating AGT (14.Lu H. Wu C. Howatt D.A. Balakrishnan A. Moorleghen J.J. Chen X. Zhao M. Graham M.J. Mullick A.E. Crooke R.M. et al.Angiotensinogen exerts effects independent of angiotensin II.Arterioscler. Thromb. Vasc. Biol. 2016; 36: 256-265Crossref PubMed Scopus (49) Google Scholar). As a unique substrate of the RAS, reduction of AGT profoundly impacts RAS activation. Therefore, it is reasonable to evaluate the overall impact of AGT deletion on the influence of the RAS on NAFLD. A previous study has revealed that high-fat diet-fed global AGT KO mice showed reduced body weight and less adipose tissue (15.Massiera F. Seydoux J. Geloen A. Quignard-Boulange A. Turban S. Saint-Marc P. Fukamizu A. Negrel R. Ailhaud G. Teboul M. Angiotensinogen-deficient mice exhibit impairment of diet-induced weight gain with alteration in adipose tissue development and increased locomotor activity.Endocrinology. 2001; 142: 5220-5225Crossref PubMed Scopus (135) Google Scholar). However, whether AGT deficiency affects diet-induced liver steatosis has not been reported. Recently, Lu et al. (14.Lu H. Wu C. Howatt D.A. Balakrishnan A. Moorleghen J.J. Chen X. Zhao M. Graham M.J. Mullick A.E. Crooke R.M. et al.Angiotensinogen exerts effects independent of angiotensin II.Arterioscler. Thromb. Vasc. Biol. 2016; 36: 256-265Crossref PubMed Scopus (49) Google Scholar) have reported that both pharmaceutical and genetic repression of hepatocyte-specific AGT protected mice from Western diet-induced liver triglyceride deposition. Yet, the underlying mechanism remains unclear. Hence, the current study was designed to figure out the role of the RAS in development of NAFLD using a hepatocyte-specific AGT-deficient (hepAGT−/−) mouse model. By comparing hepatic changes between hepAGT+/+ and hepAGT−/− mice, we aimed to elucidate the impact of hepatocyte-derived AGT on NAFLD and its potential mechanisms. AGT floxed mice with and without transgenic Alb-Cre of C57BL/6 background were kindly provided by Dr. Alan Daugherty's laboratory in Saha Cardiovascular Research Center, University of Kentucky. The hepAGT−/− mice were generated by crossing Alb-Cre+/− AGT floxed mice (male) to AGT floxed mice (female) as described previously (14.Lu H. Wu C. Howatt D.A. Balakrishnan A. Moorleghen J.J. Chen X. Zhao M. Graham M.J. Mullick A.E. Crooke R.M. et al.Angiotensinogen exerts effects independent of angiotensin II.Arterioscler. Thromb. Vasc. Biol. 2016; 36: 256-265Crossref PubMed Scopus (49) Google Scholar). As expected, the mRNA and protein abundance of AGT were significantly decreased in hepAGT−/− mice compared with hepAGT+/+ littermates (supplemental Fig. S1). In the current study, all mice were maintained in a barrier facility on a light:dark cycle of 12:12 h (ambient temperature of 23°C) and fed a normal mouse laboratory diet. At the age of 8–10 weeks, male hepAGT−/− and hepAGT+/+ mice were divided into either the normal laboratory diet group or the Western diet group. Western diet is defined as a diet enriched with saturated fat (42% calories from fat, Diet #TD.88137; Harlan Teklad). Body weight and food consumption were checked weekly. All animal studies were approved by the Animal Care and Use Committee of Zhejiang University in accordance with the Chinese guidelines for the care and use of laboratory animals. An intraperitoneal glucose tolerance test (IPGTT) and an insulin tolerance test (ITT) were performed as described (16.Nagy C. Einwallner E. Study of in vivo glucose metabolism in high-fat diet-fed mice using oral glucose tolerance test (OGTT) and insulin tolerance test (ITT).J. Vis. Exp. 2018; 131doi:10.3791/56672.Google Scholar). Briefly, for the IPGTT, mice were fasted overnight (16 h) and then fasting blood glucose concentration measurement was performed. Next, normal saline containing 20% (w/v) glucose (2 g glucose per kilogram body weight) was intraperitoneally injected into each mouse, and blood samples were collected at 15, 30, 60, and 120 min after injection for further blood glucose measurement. For the ITT, after a 5 h fast, the fasting blood glucose concentration of each individual mouse was measured. Insulin (Novolin R; Novo Nordisk, Bagsvaerd, Denmark) was then injected intraperitoneally (0.75 unit of insulin per kilogram body weight). The concentration of blood glucose was measured at 15, 30, 60, and 120 min after insulin injection. The IPGTT and ITT results were analyzed as the incremental area under the blood glucose curve (for IPGTT) and area under the curve of percent change from baseline glucose (for ITT) for interpretation of insulin sensitivity, respectively. Mouse blood samples were collected via right ventricle with EDTA (1.8 mg/ml) after 5 h fasting and then centrifuged at 400 g at 4°C for 20 min to separate plasma. Plasma insulin concentration was measured by insulin ELISA kit (10-1247-01; Mercodia, Uppsala, Sweden) following the manufacturer's instructions. Plasma triglyceride concentration was measured using a triglyceride measurement kit (E1003; Applygen Technologies Inc., Beijing, China). Plasma alanine transaminase (ALT) and aspartate transaminase (AST) activity was detected by ALT assay kit (C009-2; Njjcbio, Jiangsu, China) and AST assay kit (C010-2; Njjcbio), respectively. Liver tissues were snap-frozen in liquid nitrogen at euthanization, placed in a −80°C refrigerator, and analysis was performed within a month. The liver tissue samples were homogenized by tissue lysis buffer using tissue triglyceride or cholesterol assay kits (E1013 for tissue triglyceride assay, E1015 for tissue free cholesterol assay, and E1016 for tissue total cholesterol assay; Applygen Technologies Inc.), and the measurement of liver tissue triglyceride, free cholesterol, and cholesteryl ester concentrations was further carried out following the manufacturer's manuals. The concentration of tissue cholesteryl ester was calculated as follows: Concentration (cholesteryl ester) = Concentration (total cholesterol) – Concentration (free cholesterol). Total mRNA was extracted from liver tissue by TRIzol (TRIzol reagent; Invitrogen) and reverse-transcribed to cDNA using PrimeScript™ RT Master Mix (Perfect Real Time) (RR036A; Takara, Beijing, China). The sequences of primers utilized in the current study are listed in Table 1. Quantitative real-time PCR (qPCR) assays were performed using One Step TB Green™ PrimeScript™ RT-PCR kit (Perfect Real Time) (RR066A; Takara). The mRNA abundance of target genes was normalized to β-actin and analyzed using the ΔΔCt method for quantification.TABLE 1.Primer sequences for qPCRGeneForward Primer (5′→3′)Reverse Primer (5′→3′)mAgtGTACAGACAGCACCCTACTTCACGTCACGGAGAAGTTGTTβ-actinAGATCAAGATCATTGCTCCTCCTACGCAGCTCAGTAACAGTCCmSrebf1GGGCAAGTACACAGGAGGACAGATCTCTGCCAGTGTTGCCmFasnAAGCAGGCACACACAATGGAAGTGTTCGTTCCTCGGAGTGmAccGCGATACACTCTGGTGCTCACCCAGGGAAACCAGGATATTmScd1CATGCGATCTATCCGTCGGTCCTCCAGGCACTGGAACATAGmDgat2AGTGGCAATGCTATCATCATCGTAAGGAATAAGTGGGAACCAGAmLxrGCTGCTTCGTGACCCACTATCTGTCTCCCCATCTCACCCAmCd36GCCAAGCTATTGCGACATGATCAGATCCGAACACAGCGTAGAmUcp2TCTGCACTCCTGTGTTCTCCTTAGAAAATGGCTGGGAGACGAmSlc27a2ATCGTGGTTGGGGCTACTTTAGTTTGGTTTCTGCGGTGTGTTGmSlc27a5GAGGGCAATGTGGGCTTAATGAGGCTCTGCTGTCTCTATGTCmCide-aTGACATTCATGGGATTGCAGACGGCCAGTTGTGATGACTAAGACmCide-bGCTGCTACGTGGAGTGCTAAACAACATCCCACTCTTGGGGmCide-cATGGACTACGCCATGAAGTCTCGGTGCTAACACGACAGGGmPparγTCCTCATCTCAGAGGGCCAAATGTCCTCGATGGGCTTCACmLipcCATTTTCCTGGTGTTCTGCATCTTAGCAAGCCATCCACCGACmMgllTGGAAAAGTGGCGACATGAGTCTTTAGGCCCTGTTTCCATTmPparαCACGATGCTGTCCTCCTTGATGCCAGGCCGATCTCCAmFgf21AGCATACCCCATCCCTGACTAGGAGACTTTCTGGACTGCGmAcox1ACCTTCCCTTTCTTGCTTTGCGCTTTCCTGTGATTTC.TGGTGTmCpt1aATGGACCCCACAACAACGGTCATCAGCAACCGGCCCAAAmAcadlCCGATTGCCAGCTAATGCCTTGCTTCACGTAGTTCCTGGTmPPARgc1aCGCCGTGTGATTTACGTTGGGCTGTCTCCATCATCCCGC Open table in a new tab Total protein from hepatocytes and liver were extracted using RIPA lysis buffer (P0013B; Beyotime, Shanghai, China). The proteins were separated by SDS-PAGE gel, then transferred to PVDF membranes (Millipore, Darmstadt, Germany), and immunoblotted by primary antibodies according to the manufacturers' manuals. The primary antibodies were detected by a HRP-conjugated secondary antibody from the appropriate species and reacted with Immobilon Western Chemiluminescent HRP Substrate (Millipore). Primary antibodies against the following proteins were used: AGT (IBL-America #JP28101, RRID: AB_2341481), SREBP1 (Abcam #ab28481, RRID: AB_778069), acetyl-CoA carboxylase (ACC) (Cell Signaling Technology #3676, RRID: AB_2219397), FASN (Abcam #ab128856, RRID: AB_11143234), phospho-protein kinase B (Akt) (Ser473) (Cell Signaling Technology #4060, RRID: AB_2315049), Akt (pan) (Cell Signaling Technology #4691, RRID: AB_915783), β-actin (KC-5A08, Aksomics Inc., Shanghai, China), cluster of differentiation 36 (CD36) (Abcam #ab133625, RRID: AB_2716564), LC3A/B (Cell Signaling Technology #4108, RRID: AB_2137703), P62 (Cell Signaling Technology #5114, RRID: AB_10624872), ATG7 (Cell Signaling Technology #8558, RRID: AB_10831194), and ATG12 (Cell Signaling Technology #4180, RRID: AB_1903898). Livers were dissociated and then embedded in proper embedding medium. Paraffin-embedded sections at 3–4 μm thickness were stained with H&E. In addition, liver tissue crystal sections embedded with Optimal Cutting Temperature Compound (4583; Sakura) were stained with Oil Red O. The images were visualized by phase-contrast light microscopy. Mice were fasted for 5 h and then treated with bafilomycin A1 (S1413; Selleck Chemicals; 2.5 mg/kg body weight) or vehicle (DMSO, D2650; Sigma, Saint Louis, MO) via intraperitoneal injection. Three and one-half hours after injection, the mice were euthanized and liver microtubule-associated protein 1 light chain 3 (LC3)-II protein abundance was quantified using Western blotting for assessment of hepatic autophagic flux as previously described (17.Xiao C. Wang K. Xu Y. Hu H. Zhang N. Wang Y. Zhong Z. Zhao J. Li Q. Zhu D. et al.Transplanted mesenchymal stem cells reduce autophagic flux in infarcted hearts via the exosomal transfer of miR-125b.Circ. Res. 2018; 123: 564-578Crossref PubMed Scopus (146) Google Scholar). Mitochondria in liver were isolated using a tissue mitochondria isolation kit (C3606; Beyotime). Briefly, 100 mg of fresh liver tissue were collected, washed, and minced in ice-cold PBS. Then the tissue pieces were transferred to a precooled glass homogenizer with 1 ml of mitochondria isolation buffer and homogenized with 10 strokes at medium speed, and then centrifuged at 600 g at 4°C for 5 min. The supernatant was then transferred to a new tube and centrifuged at 3,500 g at 4°C for 10 min. Then the supernatant was carefully transferred to a new tube for cytoplasmic protein quantitation. The remaining mitochondrial pellets were washed with 1 ml of isolation buffer and resuspended in 100 μl of mitochondrial preserving solution on ice for further analysis. The fatty acid oxidation capacity of isolated liver mitochondria was measured using an Oxygraph-2k machine (O2k; OROBOROS Instruments, Innsbruck, Austria). Briefly, the isolated liver mitochondria in 100 μl of preserving solution were added to the chamber with 2 ml of MiR05 [respiration media containing 0.5 mM EGTA, 3 mM MgCl2·6H2O, 60 mM potassium lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, and 1 g/l fatty acid-free BSA (pH 7.1)]. Malate (0.25 mM), palmitoylcarnitine (2.5 μM) (P4509; Sigma), octanoylcarnitine (0.2 mM) (0605; TOCRIS Bioscience), and ADP+Mg2+ (1.25 mM) were used as substrates for measuring β-oxidation. The capacity of mitochondrial fatty acid oxidation was interpreted by the maximal oxygen consumption rate, which was calculated as the difference of oxygen flux between pre- and post-treatment with 0.5 μM titration of rotenone (45656; Sigma). Losartan was continuously administered to mice for 12 weeks. Briefly, Model 2006 Alzet mini-osmotic pumps (Durect Corporation) were implanted subcutaneously into male hepAGT+/+ mice aged 8–10 weeks to deliver either 0.9% saline (vehicle) or losartan (61188; Sigma; 10 mg/kg/day). The osmotic pumps were equipped following the manufacturer's instructions. Pumps were replaced every 6 weeks post first implantation. The isolation and culture of primary hepatocytes were performed as described (18.Klaunig J.E. Goldblatt P.J. Hinton D.E. Lipsky M.M. Chacko J. Trump B.F. Mouse liver cell culture. I. Hepatocyte isolation.In Vitro. 1981; 17: 913-925Crossref PubMed Scopus (344) Google Scholar, 19.Klaunig J.E. Goldblatt P.J. Hinton D.E. Lipsky M.M. Trump B.F. Mouse liver cell culture. II. Primary culture.In Vitro. 1981; 17: 926-934Crossref PubMed Scopus (88) Google Scholar). Briefly, under anesthesia, the hepatic portal vein and inferior vena cava of each mouse were fully exposed. The liver was sufficiently perfused with D-Hank's balanced salt solution via the hepatic portal vein. The liver was next perfused with 50 ml of prewarmed digestive solution containing 0.85 mg/ml type II collagenase (17101015; Gibco). Then, the whole liver was taken down and rinsed in DMEM containing 10% FBS in a sterile dish. Hepatocytes were liberated by tearing the liver capsule. The suspension was filtered by sterile 100 μm strainer and centrifuged at 50 g at 4°C for 2 min. The supernatant was carefully discarded and the cell pellets were resuspended with DMEM containing 10% FBS and centrifuged at 50 g at 4°C for 2 min. After repeating this step one more time, hepatocyte pellets were then resuspended with DMEM containing 10% FBS and plated to culture plates precoated with rat tail collagen. The plating medium was replaced with serum-free DMEM 5 h after plating, and hepatocytes were cultured overnight before subsequent experiments. All experiments were finished within 48 h after plating. Primary hepatocytes were incubated with DMEM containing 0.5 mM palmitic acid and 10 nM insulin (Novolin R; Novo Nordisk) to induce steatosis. In the serum stimulation experiments, the above medium was added with 10% serum from hepAGT+/+ and hepAGT−/− mice, respectively. Serum used in this experiment was obtained from hepAGT+/+ and hepAGT−/− mice fed a normal laboratory diet after 5 h of fasting. For Akt and mammalian target of rapamycin (mTOR) signaling inhibition, primary hepatocytes were pretreated with 10 μM of MK2206 dihydrochloride (MK2206) (HY-10358; MCE) for 2 h and 20 nM of rapamycin (S1039; Selleck Chemicals) for 30 min to inhibit Akt and mTOR activity, respectively. All quantitative normal data were presented as mean ± SD. Comparison between groups was performed via Student's t-test (comparison between two groups), one-way ANOVA or two-way ANOVA (comparison for more than two groups) for data that passed the normality test. Data that did not pass the normality test were expressed as median (minimum, maximum) and analyzed by ANOVA on ranks. Statistical analyses were completed using Sigma Plot 12.0 software. P-values less than 0.05 were considered statistically significant. Eight- to ten-week-old male hepAGT−/− and hepAGT+/+ mice were fed a normal laboratory diet or Western diet, respectively, for 12 weeks. Body weight and food intake were measured weekly. No difference in body weight was observed among all groups at baseline when fed a normal laboratory diet. However, after 3 weeks of Western diet feeding, hepAGT−/− mice started to exhibit significantly less body weight gain than hepAGT+/+ mice (25.1 ± 1.3 g for hepAGT−/− vs. 28.8 ± 2.3 g for hepAGT+/+, P < 0.01). At the end of the 12 week Western diet feeding, the body weight gain of hepAGT−/− mice was significantly lower than that of hepAGT+/+ counterparts (27.9 ± 0.2 g for hepAGT−/− vs. 38.2 ± 0.7 g for hepAGT+/+, P < 0.001) (Fig. 1A, B). In addition, hepAGT−/− mice also displayed reduction in Western diet-induced adipose tissue increase (supplemental Fig. S2G–M). However, total food consumption was not affected by deficiency of hepatocyte-specific AGT either in normal diet or Western diet cohorts (supplemental Fig. S2A, B). These data support that loss of hepatocyte-derived AGT prevents Western diet-induced obesity. Obesity is usually associated with insulin resistance, which serves as a significant characteristic of metabolic disorders. Therefore, the plasma insulin concentration of hepAGT+/+ and hepAGT−/− mice was measured. Western diet induced an elevation of plasma insulin in hepAGT+/+ mice, which could be attenuated by deletion of hepatic AGT (Fig. 1C). We then performed IPGTT and ITT to assess systemic glucose tolerance and insulin sensitivity, respectively. When fed a normal laboratory diet, the blood glucose level was not different between hepAGT−/− and hepAGT+/+ mice in both IPGTT and ITT tests (Fig. 1D, G; supplemental Fig. S2C–F). In cohorts of mice fed a Western diet, the incremental area under the curve of the blood glucose curve in the IPGTT was reduced, and the inverse area under the curve of the blood glucose curve in the ITT was increased in the hepAGT−/− group compared with the hepAGT+/+ group (Fig. 1E, F, H, I; supplemental Fig. S2C–F), which implicates that glucose tolerance and insulin sensitivity were improved in hepAGT−/− mice. Collectively, all these data reveal that hepatocyte-derived AGT deficiency prevents Western diet-induced body weight gain with improved insulin sensitivity. Liver steatosis is a typical hepatic manifestation of metabolic disorders; therefore, we next evaluated the effect of hepatocyte-specific AGT deficiency on Western diet-induced liver steatosis. Twelve weeks of Western diet feeding induced a remarkable liver enlargement in hepAGT+/+ mice relative to hepAGT−/− mice (Fig. 2A). Accordingly, the liver weight as well as liver-body weight ratio were significantly lower in hepAGT−/− mice than in hepAGT+/+ mice (Fig. 2B, C; supplemental Fig. S3A). The prominent feature of liver steatosis is excessive lipid accumulation in liver tissue. H&E and Oil Red O staining displayed obvious reductions of hepatocyte vacuolation and lipid-loading in liver tissues of hepAGT−/− mice as compared with those of hepAGT+/+ mice when fed a Western diet (Fig. 2G–J). Next, the contents of lipid deposits in hepatic tissues were quantified. Consistent with the histological changes, the contents of the intrahepatic lipid profile including triglycerides, free cholesterol, and cholesteryl ester were remarkably lower in Western diet-fed hepAGT−/− mice versus the hepAGT+/+ counterparts (Fig. 2D–F; supplemental Fig. S3B). Liver steatosis can induce hepatic injury. Thus, we further measured the activity of plasma ALT and AST. Western diet increased the plasma ALT concentrations in hepAGT+/+ mice but not in hepAGT−/− mice (supplemental Fig. S3C). However, plasma AST concentrations were not different between hepAGT−/− and hepAGT+/+ mice (supplemental Fig. S3D). Collectively, our results indicate that hepatocyte-specific AGT deficiency attenuates Western diet-induced liver steatosis. Hepatic triglyceride accumulation usually results from the imbalance between lipid import and consumption. A decrease in lipid export, fatty acid oxidation, lipolysis, and/or even autophagy can result in impaired lipid expenditure. To better understand the mechanisms by which hepatic AGT deletion influenced hepatic steatosis, liver mRNA expression of critical genes involved in the above-mentioned bio-processes was quantified by qPCR. No statistical differences in the expression of specific genes related to thermogenesis (Ucp2), lipolysis (Lipc), and fatty acid oxidation (Mgll, Pparα, Acox1, Cpt1a, Acadl, and Pparg1cα) were identified between hepAGT+/+ and hepAGT−/− mice irrespective of diet (Fig. 3A,supplemental Fig. S4A). Mitochondria are the pivotal organelles for energy metabolism in hepatocytes; therefore, we compared respiratory function of hepatic mitochondria from both hepAGT+/+ mice and hepAGT−/− mice. Neither hepatocyte mitochondrial fatty acid oxidation capacity (Fig. 3B, C) nor total respiratory function (supplemental Fig. S4B, C) was affected by hepatic AGT deletion, suggesting that hepatocyte-derived AGT may not contribute to mitochondrial lipid utilization. Lipids are mainly exported from liver via lipoprotein secretion, which contributes to plasma triglycerides. However, the levels of plasma triglycerides in all groups were not different regardless of genotype or diet treatment, indicating that hepatocyte-derived AGT might not affect lipoprotein secretion. (Fig. 3D). Autophagy is a conserved quality-control process that participates in lipid droplet degradation, whose dysfunction contributes to the pathophysiology of NAFLD. Therefore, to verify whether liver autophagy is affected by hepatocyte-specific AGT deletion, the abundance of autophagy-related proteins was detected. However, a difference in protein abundance of hepatic P62, LC3-II, ATG7, and ATG12 was not identified between hepAGT+/+ mice and hepAGT−/− mice (Fig. 3E). In addition, to further investigate the dynamics of hepatic autophagy, hepAGT+/+ mice and hepAGT−/− mice were treated with bafilomycin A1, an autophagy-blocking agent. The results support that hepatic AGT deficiency does not affect hepatic autophagic flux (Fig. 3F, G). In summary, these findings indicate that hepatic AGT exerts minor effects on liver fatty acid utilization. Given that the above findings suggested that the absence of hepatocyte-derived AGT exerted minimal effects on liver fatty acid utilization, we further investigated to determine whether hepatic AGT deficiency affected pathways involved in lipid importation, including fatty acid uptake, lipid biosynthesis, and lipid storage. CD36 and fatty acid transport proteins (FATPs) are major contributor
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