Angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis

The classical axis of renin-angiotensin system (RAS), angiotensin (Ang)-converting enzyme (ACE)/Ang II/AT1, contributes to the development of non-alcoholic fatty liver disease (NAFLD). However, the role of bypass axis of RAS (Angiotensin-converting enzyme 2 (ACE2)/Ang-(1–7)/Mas) in hepatic steatosis is still unclear. Here we showed that deletion of ACE2 aggravates liver steatosis, which is correlated with the increased expression of hepatic lipogenic genes and the decreased expression of fatty acid oxidation-related genes in the liver of ACE2 knockout (ACE2−/y) mice. Meanwhile, oxidative stress and inflammation were also aggravated in ACE2−/y mice. On the contrary, overexpression of ACE2 improved fatty liver in db/db mice, and the mRNA levels of fatty acid oxidation-related genes were up-regulated. In vitro, Ang-(1–7)/ACE2 ameliorated hepatic steatosis, oxidative stress and inflammation in free fatty acid (FFA)-induced HepG2 cells, and what’s more, Akt inhibitors reduced ACE2-mediated lipid metabolism. Furthermore, ACE2-mediated Akt activation could be attenuated by blockade of ATP/P2 receptor/Calmodulin (CaM) pathway. These results indicated that Ang-(1–7)/ACE2/Mas axis may reduce liver lipid accumulation partly by regulating lipid-metabolizing genes through ATP/P2 receptor/CaM signaling pathway. Our findings support the potential role of ACE2/Ang-(1–7)/Mas axis in prevention and treatment of hepatic lipid metabolism.


Results
Deletion of ACE2 Aggravates the Development of Hepatic Steatosis in ACE2 −/y Mice. ACE2 −/y mice developed a prominent fatty liver phenotype featuring a pale liver appearance caused by extensive fat accumulation. Results from the histological examination and Oil-Red-O staining of hepatic sections showed an extensive hepatic lipid deposition in the livers of ACE2 −/y mice and a reduced accumulation in wild-type (WT) mice livers (Fig. 1A). Consistently, liver weights and liver TG contents were significantly higher in ACE2 −/y mice than in WT mice (Fig. 1B,C), but the cholesterol contents did not change significantly (Fig. 1D).

Overexpression of ACE2 Ameliorates Hepatic Steatosis in db/db Mice.
To further investigate the effect of ACE2 in vivo, ACE2 was overexpressed in the liver of db/db mice to evaluate its role in lipid metabolism. Compared with Ad-GFP-treated mice, Ad-ACE2-treated mice exhibited a significant reduction in fat accumulation in the liver (Fig. 3A). Moreover, serum Ang II, TG and Alanine aminotransferase (ALT) levels were decreased by hepatic ACE2 overexpression (Fig. 3B-D), with little change in serum cholesterol and Aspartate aminotransferase (AST) levels (Fig. 3C,D). The amelioration of plasma lipid profiles after ACE2 overexpression suggested that ACE2 might affect whole-body insulin sensitivity. To test whether ACE2 improved glucose metabolism, a glucose tolerance tests (GTT) was performed. Compared with Ad-GFP-treated mice, insulin sensitivity was significantly improved in Ad-ACE2-treated mice (Supporting Fig. S1A, S1B).
Next, we investigate the expression levels of lipid metabolism-related genes. The mRNA levels of fatty acid oxidation-related genes, CPT-1α, PPARα and PPARγ were up-regulated, while little change was observed in PGC-1α and MCAD mRNA levels in the liver of Ad-ACE2-treated mice (Fig. 3E). We also wondered whether ACE2 alters the expression of lipogenesis genes. However, the expression of lipogenesis genes did not exhibit significant difference between Ad-GFP-treated and Ad-ACE2-treated groups (Supporting Fig. S1C). All together, these results indicated that ACE2 could significantly improve hepatic steatosis in db/db mice.   Quantitative assay of (B) AngII, (C) triglyceride and cholesterol, (D) ALT and AST levels in ACE2overexpressing db/db mice. *P < 0.05versus db/db + GFP mice (n = 6). (E) Relative gene expression levels of fatty acid oxidation-related genes (PGC-1α, CPT-1α, PPARα, PPARγ and MCAD). *P < 0.05 versus db/ db + GFP by Student's t test.
To further explore these findings, we then investigated the effect of ACE2 in FFA-induced HepG2 cells. The expression of ACE2 in ACE2-overexpressing HepG2 cells was detected (Supporting Fig. S2). Similarly, lipid accumulation was significantly suppressed in control or FFA-induced ACE2-overexpressing HepG2 cells (Fig. 4C).
To detect whether the inhibitory effects of ACE2 on lipid accumulation are regulated by Akt, ACE2-overexpressing HepG2 cells were treated with Akt inhibitors (Triciribine (API-2) and MK-2206). Interestingly, the expression of ACCα , SREBP-1c, LXRα and FAS were increased in API-2-treated and MK-2206-treated groups (Fig. 4E). These results indicated that treatment of ACE2-overexpressing HepG2 cells with Akt inhibitors may reduce ACE2-mediated lipid metabolism in vitro.
The western blot results further documented that ACE2 regulated the expressions of genes related to oxidative stress signaling and inflammation. The expressions of marker genes for oxidative stress signaling (Gpx1, catalase and SOD2) were increased, while proinflammatory cytokines (TNF-α , MCP-1 and IL-8) were reduced (Fig. 5B). These results suggested that ACE2 could protect against oxidative stress and inflammation in HepG2 cells.

Overexpression of ACE2 Activates Akt through the ATP/P2 receptor/CaM Pathway. Considering
Ang II can induce production of mitochondrial reactive oxygen species and ACE2 −/y mice displayed elevated levels of oxidative stress 12,17 , the impact of ACE2 on mitochondria function was examined by detecting cellular ATP content. Expectedly, ACE2 overexpression elevated intracellular and extracellular ATP levels in HepG2 cells (Fig. 6A).

Discussion
The classical pathway of RAS, ACE/AngII/AT1, modulates and contributes to the development of NAFLD 4,5,8 . On the other hand, Ang-(1-7) exerts an important role of anti-obesity by Mas receptor 11,[18][19][20] . The present study is the first to clarify the possible mechanisms of ACE2/Ang-(1-7)/Mas regulate hepatic steatosis. We show that deletion of ACE2 aggravated hepatic steatosis, oxidative stress and inflammation in ACE2 −/y mice. On the contrary, overexpression of ACE2 improved hyperglycemia and fatty liver in db/db mice. In vitro study, Ang-(1-7)/ACE2 ameliorated hepatic steatosis, oxidative stress and inflammation in FFA-induced HepG2 cells. Notably, Akt inhibitors reduced ACE2-mediated lipid metabolism, and what's more, ACE2-mediated Akt activation can be attenuated by blockade of ATP/P2 receptor/CaM pathway. Taken together, Ang-(1-7)/ACE2/Mas axis may reduce liver lipid accumulation partly by regulating lipid-metabolizing genes through ATP/P2 receptor/CaM signaling pathway. The reduction of oxidative stress and inflammation may also be involved in the amelioration of hepatic steatosis.
Multiple metabolic pathways lead to the development of hepatic steatosis, including increased lipogenesis and lipolysis, and decreased fatty acid oxidation 21 . Key transcriptional regulators such as LXRα and SREBP-1c coordinately control lipogenesis 22 . LXRα and SREBP-1c increase the expression of key lipogenic genes, including those for FAS, SCD1 and ACC 23 . ACC1 converts acetyl-CoA to malonyl-CoA, and inhibits fatty acid entry into the mitochondria reducing β -oxidation. FAS utilizes both acetyl-CoA and malonyl-CoA to form palmitic acid (C16:0). Besides, adiponectin appears to have a pivotal role in improving fatty acid oxidation and decreasing fatty acid synthesis 24 . The liver has adiponectin receptors, and their stimulations lead to increased fatty acid β -oxidation and thereby decreased hepatic TG content. Aberrant induction of these factors may contribute to hepatic steatosis. In the present study, the changes of these genes in ACE2 −/y mice and ACE2-overexpressing HepG2 cells support the idea that ACE2 ameliorates hepatic steatosis.
Several studies demonstrated that the excessive production of reactive oxygen species and altered redox balance promoted hepatic lipid accumulation 5,25,26 . Our previous study documented that the ROS levels were increased in the liver of ACE2 −/y mice, whereas in HepG2 cells, Ang-(1-7) could protect against oxidative stress by inhibiting NADPH oxidase expression 12 . Acting to protect against oxidative stress is a complex system of enzymatic antioxidants (SOD, GPX, glutathione reductase, catalase) and non-enzymatic antioxidants (glutathione (GSH), vitamins C and D) 27 . In the present study, the increased lipid accumulation in the liver may be involved in the decrease in the expressions of enzymatic antioxidants genes and the resulting increased ROS production.   The expression of oxidative stress signaling in ACE2 −/y mice and FFA-induced ACE2-overexpressing HepG2 cells were in agreement with previous data.
Numerous studies have reported an association between NAFLD and systemic inflammatory stress 28,29 . Steatosis is a necessary step in the pathogenesis of NAFLD, but inflammation is required to bring about steatohepatitis, which leads to chronic damage to hepatocytes and progressive fibrosis. It has been demonstrated that elevated proinflammatory cytokines expression, as TNF-α , promoted hepatic lipid accumulation 30 . Several studies showed that many RAS components are expressed in the liver meddling metabolic and inflammatory processes 5,31-33 . The increased expression of Ang II induces NAFLD and modulates inflammatory cell recruitment into the liver during liver injury 4,5 . In addition, recent study has shown RAS as a potent mediator in the activation of inflammatory mechanisms involved in obesity 18,31 . Our current findings are consistent with these data and document for the first time that the inflammatory levels increased in the liver of ACE2 −/y mice, whereas in HepG2 cells, ACE2 could protect against inflammatory stress by inhibiting proinflammatory cytokines expression.
It has been previously reported that reduced hepatic ATP levels are associated with the development of NAFLD in rats 34 . ATP has been demonstrated to be a signaling molecule to activate the PI3K/Akt signaling pathway [35][36][37] . The ATP receptors (P2X and P2Y receptors) mediate such action. P2X receptors are permeable to calcium, which are ligand-gated ion channels 37 . P2Y receptors are G-protein-coupled receptors which stimulate PLC to increase IP3 production, resulting in calcium release from internal stores 37 . Increased cytosolic free calcium activates CaM, leading to the activation of the PI3K/Akt pathway 38,39 . In the present study, we found that the P2 receptor antagonists (PPADS) completely abolished ACE2 induced Akt activation, which reveals a critical role of ATP receptors in this process. Furthermore, Akt activation was also significantly blocked by IP3R antagonist (2-APB), CaM antagonist (CPZ) and depletion of extracellular calcium in HepG2 cells. These data reveal the possible roles of ATP/P2 receptor/CaM signal pathway in ACE2-mediated hepatic insulin resistance and liver lipid accumulation in HepG2 cells. What is more, these findings offer another plausible explanation for the molecular basis of the relationship between the ACE2/Ang-(1-7)/Mas axis and liver glucose/lipid metabolism.
Insulin resistance and steatosis is closely associated in the development of NAFLD 40 . Elevated hepatic FFAs worsen insulin resistance. The accompanying increase in oxidative stress and proinflammatory cytokine production following liver lipid accumulation causes a further increase in insulin resistance, thereby establishing a vicious cycle 3 . Fatty acid synthesis cross-talks with insulin signaling, and Akt plays a pivotal role in glucose and lipid metabolism signaling pathway 41 . Akt has been found to not only regulate gluconeogenesis and glycogenolysis, but also play a critical role in the regulation of liver lipid metabolism. Activation of Akt represses lipogenesis and prevents excessive lipid deposition in the liver [42][43][44] . In addition, the increased phosphorylation level of Akt in the liver is associated with the amelioration of steatosis in diabetic mice 45 . In our more recently research on the effect of ACE2/Ang-(1-7)/Mas axis on hepatic insulin resistance, the phosphorylation levels of Akt increased markedly in ACE2-overexpressing cells, while significantly inhibited in the liver of ACE2 −/y mice. As expected, Akt inhibitors can reduce ACE2-mediated lipid metabolism. Furthermore, hepatic overexpression of ACE2 improved hyperglycemia and fatty liver in db/db Mice. Together with these two studies, the mechanism underlying glucose and lipid metabolism could involve the ability of ACE2/Ang-(1-7) to activate Akt.
One of the limitations of the current study is that we did not get positive results from high fat-induced ACE2 −/y mice (data not shown), these may due to the new finding function of ACE2 as a key regulator of dietary amino acid homeostasis, innate immunity, gut microbial ecology, and transmissible susceptibility to colitis 46 , which may affect the metabolism of high fat diet. These need to be confirmed in further systematic experiment; maybe hepatic specific knockout ACE2 mice can solve this issue.
In summary, as described in Fig. 7, our study demonstrates that ACE2/Ang-(1-7)/Mas axis activation plays an important role in hepatic lipid metabolism through a mechanism that could involve regulation of lipid-metabolizing genes through ATP/P2 receptor/CaM signaling pathway, and reduction of oxidative stress and inflammation. Together with our more recently research on ACE2/Ang-(1-7)/Mas axis can inhibit hepatic insulin resistance, our current findings suggest that ACE2/Ang-(1-7)/Mas axis is a potential target for drug therapy for NAFLD.

Methods
Animal. ACE2 knockout (ACE2 KO, ACE2 −/y ) mice were a gift from Prof. Dr. Josef Penninger from Institute for Molecular Biotechnology GmbH. In every experiment, ACE2 KO mice were identified by PCR on genomic DNA extracted from tail biopsies. Male db/db mice at the age of 6 weeks were purchased from Nanjing biological medicine research institute affiliated to Nanjing medical university. db/db mice were maintained on the BKS background. ACE2 KO mice were identified by PCR using genomic DNA extracted from tail biopsies. All of the mice were maintained on a 12 h light/dark cycle. All of the experiments in this study were performed using male ACE2 −/y mice and their age-and sex-matched wild-type (WT) littermates, and were fed with standard chow. All

Overexpression of ACE2 in HepG2 cells and Mouse Liver.
To overexpress ACE2 in the liver, the adenovirus coding for rat ACE2 (rACE2) upstream of an enhanced green fluorescent protein (eGFP) reporter gene (Ad-rACE2-eGFP) or with the eGFP virus alone (Ad-eGFP) were purchased from SinoGenoMax (Beijing, China), and were injected into db/db mice (male, 5 to 7-week-old) by way of the tail vein (5 × 10 8 particle forming units (pfu) in a total volume of 100 μ L of 0.9% wt/vol saline). At the 6th day post-virus injection, glucose tolerance tests (GTT) were performed. On the 7th day, the fed animals were sacrificed for experimental analysis. To over express ACE2 in HepG2 cells, HepG2 cells were infected with 1.0 × 10 4 pfu Ad-ACE2 or Ad-GFP for 48 hours.
Biochemical Assays. Serum triglyceride, cholesterol, aspartate aminotransferase and alanine aminotransferase levels were measured using enzymatic kits (Biosino, China) by automatic biochemical analyzer (Hitachi 7160, Japanese). Ang II in serum was measured by radioimmunoassay (BEIJING SINO-UK INSTITUTE OF BIOLOGICAL TECHNOLOGY, china).

RNA Extraction and Quantitative
Real-time RT-PCR. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. A total of 500ng of RNA was used as the template for the first-strand cDNA synthesis using ReverTraAceqPCR RT Kit (TOYOBO, Osaka, Japan) in accordance with the manufacturer's protocol. The transcripts were quantified using Light Cycler 480Real-Time PCR system (Roche, Basel, Switzerland). Primers were designed using Primer Quest (Integrated DNA Technologies, Inc).
Histological Analysis. The tissue samples were fixed in 10% formaldehyde for 1 hour at 4 °C, and the fixed specimens were then dehydrated and embedded in paraffin according to standard procedure. The specimens were cut into 5μ m thick, and stained with Oil-Red-O for lipid accumulation. The sections were then observed with a microscope (200 × ) and analyzed on four random fields/slides. Determination of Triphosphate (ATP) Content. Cultured cells were lysed in a lysis buffer provided by ATP-Lite Assay Kit (Vigorous Biotechnology Beijing). The medium of cultured cells was also collected for ATP determination. The ATP content was measured (nmol) and normalized by protein concentration (nmol/mg) in the same sample and presented as a percentage of the control.
Statistical Analysis. All of the data are presented as the mean ± SD. The data were analyzed by Student's t test or one-way ANOVA (with Bonferroni post-hoc tests to compare replicate means) when appropriate. Statistical comparisons were performed using Prism5 (GraphPad Software, San Diego, CA). P values less than 0.05 were considered to be statistically significant. Representative results from at least three independent experiments are shown unless otherwise stated.