Arginase Inhibition Ameliorates Hepatic Metabolic Abnormalities in Obese Mice

Objectives We examined whether arginase inhibition influences hepatic metabolic pathways and whole body adiposity in diet-induced obesity. Methods and Results After obesity induction by a high fat diet (HFD), mice were fed either the HFD or the HFD with an arginase inhibitor, Nω-hydroxy-nor-L-arginine (nor-NOHA). Nor-NOHA significantly prevented HFD-induced increases in body, liver, and visceral fat tissue weight, and ameliorated abnormal lipid profiles. Furthermore, nor-NOHA treatment reduced lipid accumulation in oleic acid-induced hepatic steatosis in vitro. Arginase inhibition increased hepatic nitric oxide (NO) in HFD-fed mice and HepG2 cells, and reversed the elevated mRNA expression of hepatic genes in lipid metabolism. Expression of phosphorylated 5′ AMPK-activated protein kinase α was increased by arginase inhibition in the mouse livers and HepG2 cells. Conclusions Arginase inhibition ameliorated obesity-induced hepatic lipid abnormalities and whole body adiposity, possibly as a result of increased hepatic NO production and subsequent activation of metabolic pathways involved in hepatic triglyceride metabolism and mitochondrial function.


Introduction
Arginase metabolizes L-arginine to urea and L-ornithine, and has been implicated in the regulation of nitric oxide (NO) synthesis. Arginase potentially inhibits the production of NO by competition with NO synthase (NOS) for the substrate L-arginine [1]. Several lines of evidence have consistently demonstrated that increased arginase activity is associated with endothelial dysfunction in various experimental models of hypertension, atherosclerosis, vascular disease, and aging [2][3][4], and these associations are mainly derived from impaired production of NO. Recently, upregulation of arginase and/or increased arginase activity have also been described in metabolic disorders such as type 2 diabetes mellitus (T2DM) in animals [5,6] , and humans [7]. More recently, it was reported that arginase blockade improved endothelial dysfunction in coronary artery disease (CAD) patients with T2DM [8], suggesting arginase as a promising therapeutic target for DMinduced vascular treatment. These studies also reported that the effects were enhanced by increased bioavailability of NO through shunting L-arginine from the arginase pathway to the endothelial NOS pathway [8].
Obesity is a serious health problem that continuously increases the morbidity and mortality of a variety of acute and chronic diseases [9,10]. Several studies have reported that L-arginine supplementation prevents obesity and obesity-related metabolic complications [11,12]. Previously, we observed significant upregulations of arginase I in the peripheral blood mononuclear cells (PBMCs) of overweight/obese individuals [13]. In line with this, increased physiological levels of NO could ameliorate all of the adverse features of metabolic complications in obese animals [14]. The apparent metabolic interrelationship between arginine and NO availability raises the question as to whether arginase blockade and the subsequent increase in NO availability might improve obesity and related metabolic conditions.
In the present study, we hypothesized that chronic inhibition of arginase prevents diet-induced obesity. Since previous studies convincingly demonstrated that obesity induces metabolic dysregulation in the liver, such as abnormalities of glucose and lipid homeostasis, which ultimately produces hepatic steatosis [15], we evaluated the effects of the arginase inhibitor N v -hydroxy-nor-Larginine (nor-NOHA) on hepatic metabolic pathways in obese mice induced by a high fat diet (HFD). This approach ultimately allowed us to test whether arginase inhibition demonstrates an anti-obesity effect. This study provides novel information regarding how arginase inhibition may exert protective properties against hepatic lipid abnormalities induced by obesity and addresses arginase inhibition as a potential therapeutic agent for obesity and related metabolic complications.
This animal study was approved by the Institutional Animal Care and Use Committee as governed by the National Institute of Health's ''Guide for the Care and Use of Laboratory Animals and by the Committee on Animal Experimentation and Ethics of Korea University'' (KUIACUC-2013-48). Four-week-old male C57BL/6 mice were purchased from DBL (Chungbuk, Korea) and used for the experiment. After a one-week adaptation period, the animals were randomly assigned into control (ND, n = 10), HFD (40% fat for total energy, n = 10), and HFD treated with arginase inhibitor nor-NOHA (HFD with nor-NOHA, n = 10) groups for 12 weeks. Diet composition for each group was presented in Table S1. For obesity induction, all mice in the HFD and HFD with nor-NOHA groups were fed the same HFDs for 7 weeks. These mice were then placed on either only HFD sham gavaged or HFD orally gavaged with 40 mg kg 21 nor-NOHA (Bachem; Bubendorf, Switzerland) dissolved in 0.9% NaCl solution for 5 weeks. Daily food intake and weekly body weight gain were routinely recorded throughout the experimental period.

Sample collection (blood and tissues) and measurement of weight
After 12 weeks, the final body weights were measured and the mice were sacrificed after a 12 h fast. Following this, they were anesthetized with 40 mg kg 21 zoletilie (Virbac; Carros, France) mixed with 10 mg kg 21 rompun (Bayerkorea; Seoul, Korea); blood samples were collected from the abdominal inferior vena cava, then transferred to vacutainer tubes with EDTA. The centrifuged plasma was aliquoted and stored in a freezer at 280uC until analysis. Liver and visceral adipose tissues (i.e., epididymal, perirenal, retroperitoneal, and mesenteric fat) were extracted, washed with 1X phosphate-buffered saline (PBS), weighed (in g), and then rapidly frozen with liquid nitrogen or put into 10% formaldehyde solution and stored in the freezer at 280uC.
Analysis of lipid, phosphorylated 59 adenosine monophosphate-activated protein kinase (p-AMPK)a and histology Plasma concentrations of total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-c) were measured enzymatically using commercial kits (Asan Pharmaceutical; Seoul, Korea). Hepatic lipids were extracted using the method developed by Folch [16]. The concentrations of TC and TG in the hepatic-lipid extracts were measured using the same enzymatic test kits used for the plasma analysis. p-AMPKa (Thr172) from liver tissue lysates was measured using a commercial kit (Abcam; MA, USA). Tissue samples of the epididymal fat pads and liver were fixed with a 10% formaldehyde solution and embedded in paraffin. Five-millimeter sections were cut, stained with hematoxylin-eosin, viewed with an optical microscope (Nikon; Tokyo, Japan), and photographed at a final magnification of 4006.
RNA extraction from animal liver and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) To analyze the mRNA expression of genes in liver tissue, total RNA was extracted from 50 mg of liver with the QIAzolLysis reagent of the RNeasy Lipid Tissue Mini Kit (Qiagen; USA) according to the manufacturer's protocol. cDNA was synthesized from 1 mg of RNA using oligo-dT and Superscript II reverse transcriptase (Invitrogen; USA). One microgram of cDNA was subjected to quantitative real-time PCR amplification using the SYBR Green PCR Kit (Qiagen). The sequences of the designed primers are shown in Table 1. PCR conditions were: 15 min at 95uC, followed by 40 cycles at 94uC for 30 s, 58uC for 20 s, and 7uC for 30 s (Step One Plus; Applied Biosystems; Foster City, CA, USA). GAPDH was used as the control in the comparative cycle threshold (Ct) method.

Cell culture and oleic acid-induced steatosis
The human hepatoma HepG2 cells were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA) and cultured in high-glucose Dulbecco's modified Eagle medium (DMEM; Gibco; Eggenstein, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS) containing 100,000 Units/L penicillamine and 100 mg/L streptomycin (Gibco). The cells were maintained at 37uC in a humidified atmosphere of 95% air and 5% CO 2 . After 24 h, the cells were serum-starved overnight and treated with varying concentrations of oleic acid (OLA; Sigma-Aldrich; St. Louis, MO, USA) for 24 h. Control cells were treated with OLA-free medium containing ethanol.

Cell viability assay
The HepG2 cells (1610 5 cells) were serum-starved overnight and treated with varying concentrations (0-2.0 mM) of OLA (Sigma-Aldrich) for 24 h. Control cells were treated with OLAfree medium containing ethanol. Cell viability was measured by adding 1 mg/mL of 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to each well and incubating at 37uC for 1 h. After incubation, absorbance was measured at a wavelength of 570 nm with a NanoQuant microplate reader (Tecan Trading AG; Switzerland). This assay was repeated three times.

Oil Red O staining and quantification of TG content
The HepG2 cells (2610 5 cells) were seeded on each well of a 48well culture plate. After 24 h, the cells were serum-starved overnight. The next day, they were treated with varying amounts of OLA (0, 0.5, 1, and 1.5 mM) or 1.5 mM OLA with nor-NOHA (0, 10, 50 mM) for 24 h. The treated cells were washed with PBS and fixed with 10% formalin for 1 h at room temperature. Subsequently, the cells were washed with 60% isopropanol, stained with Oil Red O for 10 min at room temperature, and washed 4 times with distilled water. Images for each dish were captured using a microscope (Olympus Corporation; Tokyo, Japan). Isopropanol (100%) was added to the cells, and after 10 min, absorbance was measured at 500 nm with a spectrophotometer (PerkinElmer; Waltham, MA, USA). AdipoRed assay reagent (Lonza; Walkersville, MD, USA) was used for the quantification of TG content according to the manufacturer's protocol.

Analysis of nitric oxide and western blot
NO from the liver tissue and supernatant of the treated cells was measured using a Nitrate/Nitrite Colorimetric Assay kit (Cayman Chemical; Ann Arbor, MI, USA). Total protein was isolated from liver tissues by homogenization in cold radioimmunoprecipitation assay (RIPA) lysis buffer (Amresco; Solon, OH, USA) containing protease inhibitors (Roche Diagnostics; Mannheim, Germany) and phosphatase inhibitors (Sigma-Aldrich). Total protein was extracted from 1 6 10 6 HepG2 cells using a cold lysis buffer (40 mM HEPES, pH 7.5, 120 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing a protease inhibitor cocktail (Roche Diagnostics). Western blot analysis was performed using specific antibodies for AMPKa, phospho-AMPKa (Thr172) (Cell Signaling Technology; Danvers, MA, USA) and arginase 1, eNOS, and b-actin (Santacruz Biotechnology, CA, USA).

Arginase activity assay
Arginase activity was measured using cell lysates and homogenates prepared from liver. The cells and tissues were lysed in cold buffer (50 mM Tris-HCl, PH 7.5, 0.1 mM EDTA, and protease inhibitors) at 4uC and then centrifuged for 20 min at 14,000 g. The supernatants were used using the Quantichrome arginase assay kit (BioAssay Systems, Hayward, CA).

Statistical analysis
Statistical analysis was performed using SPSS 21.0 (Statistical Package for the Social Science, SPSS Inc.; Chicago, IL, USA). The results are presented as mean 6 S.E. and the differences among the experimental groups were analyzed using one-way analysis of variance (ANOVA) with Duncan's multiple range test and p,0.05 as the criterion of significance.

Arginase inhibition prevents diet-induced obesity and reduces liver weight
As expected, the body weight of HFD-fed mice was significantly increased compared with that of ND-fed mice at the end of the 7week obesity induction period. There were significant differences in body weight gains between HFD-fed mice and HFD with nor-NOHA-fed mice over the additional 5 weeks (Fig. 1A). HFD with nor-NOHA-fed mice showed a remarkably lower body weight gain compared to mice fed HFD alone (Fig. 1B), although the amount of total food intake was significantly higher in HFD with nor-NOHA-fed mice than in HFD-fed mice. At termination of the 12-week period, the HFD-fed mice showed significantly increased weights in all adipose tissues compared with ND-fed mice (Fig. 1C). HFD with nor-NOHA-fed mice showed significantly reduced adipose tissue weights compared with the HFD-fed mice, and, furthermore, had significantly reduced sizes of epididymal fat adipocytes (Fig. 1E). As for the liver, HFD with nor-NOHA-fed mice showed significantly lower liver weights and marked decreases in lipid accumulation compared to HFD-fed mice (Fig. 1F).

Effects of arginase inhibition on lipids and NO in plasma and the liver
As shown in Table 2, arginase inhibition by HFD with nor-NOHA significantly decreased the hepatic TG content, whereas it did not affect the hepatic TC content. HFD with nor-NOHA-fed mice displayed significantly lower levels of circulating TG levels compared to those of the control group. With respect to hepatic NO (nitrate+nitrite) concentrations, elevated levels of hepatic NO in HFD-fed mice were even more significantly increased by nor-NOHA ( Table 2).

Effects of arginase inhibition on hepatic lipid metabolism in HFD-fed mice
In order to elucidate the underlying mechanism of metabolic effects of arginase inhibition by nor-NOHA, we first analyzed expression levels of key genes known to control hepatic TG metabolism including de novo lipogenesis and fatty acid oxidation. The results showed that the HFD significantly increased mRNA abundance of peroxisome proliferator-activated receptor (PPAR)-c2 ( Fig. 2A), adipose differentiation-related protein (ADRP) (Fig. 2C), and stearoyl-CoA desaturase-1 (SCD-1) (Fig. 2D), and these changes in transcript levels caused by the HFD were reversed by nor-NOHA to levels observed in the ND-fed mice (Fig. 2). By contrast, the HFD did not affect mRNA abundance of sterol regulatory element-binding protein (SREBP) 1 C (Fig. 2B), fatty acid synthase (FAS) (Fig. 2F), acetyl-CoA carboxylase (ACC) (Fig. 2E) and carnitine palmitoyltransferase (CPT)-1a (Fig. 2H). In addition, the HFD with nor-NOHA significantly increased hepatic mRNA abundance of PPAR-a compared to the HFD alone (Fig. 2G).

Effects of arginase inhibition on hepatic mitochondrial function in HFD-fed mice
Based on the systemic effects of arginase inhibition on body weight and fat mass, we analyzed the expression of genes involved in hepatic mitochondrial function. The HFD remarkably reduced mRNA abundance of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1a (Fig. 3A) and PGC-1b (Fig. 3B) compared to mice in the ND group, and these changes were reversed by arginase inhibition from nor-NOHA.   Effects of arginase inhibition on the activation of hepatic AMPKa in HFD-fed mice To determine whether the activated AMPKa pathway is required for hepatic mitochondrial function and lipid metabolism improved by arginase inhibition, we investigated that expression of phosphorylated AMPKa at Thr172 in the livers of ND-and HFDfed mice. Phosphorylation of AMPKa in HFD-fed mice was decreased. However, arginase inhibition dramatically increased phosphorylated AMPKa in HFD with nor-NOHA-fed mice compared to HFD-fed mice (Fig. 4).

Inhibitory effect of nor-NOHA on lipid accumulation in OLA-induced hepatic steatosis in vitro
In order to demonstrate that the changes in hepatic phenotypes are not due to secondary weight beneficial effects but are the direct effect of nor-NOHA, we attempted to investigate the direct effects of nor-NOHA in HepG2 cells with OLA-induced hepatic steatosis in vitro. To examine the effect of OLA and nor-NOHA on viability in HepG2 cells, first, cells were treated with varying concentrations of OLA and nor-NOHA for 24 h, and then assayed using the MTT assay. The MTT results indicated that treatment of OLA at a dose ranging from 0.2 mM to 2 mM and nor-NOHA at a dose ranging from 1 mM to 100 mM did not affect cell viability (Fig. 5A for OLA, data not shown for nor-NOHA). When the HepG2 cells were cultured at various concentrations of OLA (ethanol, 0.5, 1, 1.5 mM) for 24 h to develop an in vitro model of hepatic steatosis, intracellular lipid was accumulated in a dose-dependent manner based on TG content and Oil Red O staining (Fig. 5B). Using a dose of 1.5 mM of OLA, we investigated the effects of nor-NOHA on lipid accumulation, NO release, and AMPKa activation. We found that nor-NOHA treatment markedly diminished the lipid accumulation in HepG2 cells. In addition, nor-NOHA dramatically reduced the intracellular TG content (%) at all doses ranging from 10 mM to 50 mM (Fig. 5C). When the NO level from the supernatant of the treated cells was measured, decreased levels of NO in OLA-induced steatosis HepG2 cells was significantly increased by nor-NOHA at both 10 mM and 50 mM (Fig. 5D). Furthermore, nor-NOHA significantly enhanced phosphorylated AMPKa at a dose of 10 mM (Fig. 5F). Arginase inhibition by nor-NOHA for 30 min reduced arginase activity in OLA-treated HepG2 cells in a dose dependent manner (Fig. 5E), whereas nor-NOHA did not affect the protein expressions of arginase 1 and eNOS in OLA-treated HepG2 cells (Fig. 5G).

Discussion
Accumulating evidence has demonstrated the role of NO in the regulation of the inter-organ metabolism of energy substrates and the pathological outcomes in metabolic abnormalities [14]. NO, which is synthesized from L-arginine by NOS, participates in multiple cell signaling pathways as a signaling molecule, and therefore regulates the metabolism of energy substrates in a celland tissue-dependent manner [1]. Since NOS competes with arginase for L-arginine in producing NO, increased arginase activity and/or arginase-mediated L-arginine depletion provides a mechanism for the metabolic consequences caused by a lack of NO bioavailability. Based on the reciprocal interactions between arginase and eNOS in endothelial dysfunction, increased activity and expression of arginase have been demonstrated in several pathological cardiovascular conditions in T2DM, including hypertension, atherosclerosis, myocardial ischemia, and vascular dysfunction [17]. Although the importance of arginase in endothelial dysfunction and atherosclerosis has been demonstrated, the role of arginase in obesity and obesity-related complications has never been evaluated.
In the present study, we found that nor-NOHA ameliorated hepatic metabolic abnormalities in diet-induced obese mice, and we further demonstrated its systemic effects on body weight and amount of adipose tissue. Specifically, nor-NOHA significantly reduced HFD-induced elevations in hepatic TG content, which was also observed in OLA hepatic steatosis in vitro. Moreover, nor-NOHA further increased hepatic NO concentration in HFDinduced obesity, which was also demonstrated in HepG2 cells. In order to elucidate the underlying mechanism for these results, the expression of several genes to control hepatic TG metabolism, including de novo lipogenesis and fatty acid oxidation, was investigated.
SREBP-1c is the master gene known to be responsible for lipid accumulation in the liver induced by an HFD [18], thus exacerbating hepatic steatosis. On the other hand, several studies have demonstrated that activation of PPAR-c2 in hepatocytes is sufficient to initiate hepatic steatosis through mechanisms involving activation of lipogenic genes, de novo lipogenesis, and Table 2. Effect of nor-NOHA on body weight, food intake, lipid profile and NO in plasma and the liver among the experimental groups.
Groups ND (n = 10) HFD (n = 10) HFD With nor-NOHA (n = 10) increased hepatic TG concentrations [19], even independently of SREBP-1c activation [20]. Although under normal circumstances PPAR-c2 is only minimally expressed in hepatocytes, it is nonetheless involved in the development of hepatic steatosis by modulating fatty acid transport and the TG incorporation pathway [20]. Enhanced lipogenesis in HFD-fed mice, as shown in the present study, seems to be mainly derived from upregulation of PPAR-c2, independent of SREBP-1c. This is further supported by the results showing that the mRNAs of target genes for PPAR-c2, ADRP, and SCD-1 were increased in the livers of HFD-fed mice, whereas SREBP-1c and the mRNAs of its target genes, FAS and ACC, were not altered. The increased TG accumulation in the livers of HFD-fed mice was likely due to an increase in the expression of ADRP and SCD-1. Interestingly, nor-NOHA resulted in significant reductions in mRNA levels of PPAR-c2, ADRP, and SCD-1 in HFD-fed mice without alterations in mRNA levels of SREBP-1c, FAS, and ACC-1, Consistent with previous speculation [21,22], mRNA abundance of ACC and FAS was not affected in either the HFD-or HFD with nor-NOHA-fed mice, which might be due to a regulatory feedback mechanism in response to HFD feeding. ADRP is the droplet protein where lipids are sequestered in hepatocytes, and its expression is increased in cells expressing PPAR-c2 [23]. SCD-1 encodes for the rate-limiting enzyme in monounsaturated fatty acid (MUFA) synthesis, which is a key component in the formation of TG, cholesterol esters, and phospholipids [24]. Our results showing downregulations of ADRP on lipid droplets and SCD-1 by nor-NOHA in the liver indicate that arginase inhibition may exert its inhibitory effects on TG formation and development of fatty liver by modulating ADRP and SCD-1 through regulation of PPAR-c2. It has been reported that activation of PPAR-a displays antiobesity effects and beneficial effects on the management of hepatic steatosis [25,26]. It is generally accepted that these effects can be attributed to PPAR-a-induced transcriptional activation of the many genes involved in b-oxidation of fatty acids, which are predominantly found in the liver [27]. Although there was a marked upregulation of PPAR-a in HFD with nor-NOHA-fed mice, mRNA levels of CPT-1a, the oxidative enzyme for fatty acid b-oxidation [28], were not affected. This suggests that reduced hepatic TG accumulation in HFD with nor-NOHA-fed mice probably did not occur through activation of fatty acid boxidation. Defects in mitochondrial energy metabolism have been suggested to cause hepatic steatosis, insulin resistance, and Figure 3. Effects of the nor-NOHA, on the mRNA levels of several genes in the liver. Total RNA was extracted from each liver and subjected to real-time PCR analyses using primers specific for PGC-1a (A), and PGC-1b (B). The results are expressed as mean 6 SE of mice, and differences between groups were tested by analysis of variance (ANOVA) with Duncan's multiple range test. The same letter indicates no significant difference between two groups (p,0.05). doi:10.1371/journal.pone.0103048.g003 Figure 4. Effect of the nor-NOHA on levels of phosphorylated AMPKa at Thr172 (p-AMPKa) in the liver. Densitometric analysis of p-AMPKa protein levels, using an enzyme-linked immunosorbent assay (ELISA) kit (upper) and protein levels of p-AMPKa and AMPK levels, using a representative western blot image (lower). The results are expressed as mean 6 SE of mice, and differences between groups were tested by analysis of variance (ANOVA) with Duncan's multiple range test. The same letter indicates no significant difference between two groups (p,0.05). doi:10.1371/journal.pone.0103048.g004 T2DM [29][30][31]. Based on this information, effects of arginase inhibition by nor-NOHA on key genes involved in hepatic mitochondrial bioenergetics were also tested. In the present study, nor-NOHA significantly reversed the reduced PGC-1b expression in HFD-fed mice, whereas the effect on PGC-1a was not significant. PGC-1 is involved in mitochondrial biogenesis, adaptive thermogenesis, fatty acid b-oxidation, and hepatic gluconeogenesis [31], and is a key transcriptional cofactor regulating expression of PPAR-a [29], which in turn stimulates the expression of NRF-1 [31]. In addition, several previous studies have proposed that PGC-1b has a stronger ability to regulate mitochondrial function than PGC-1a [32]. The present study demonstrates that arginase inhibition by nor-NOHA improved mitochondrial function through the upregulation of mainly PGC-1b. It may be that enhanced NO bioavailability resulting from arginase inhibition activates expression of PGC-1 [14], thereby enhancing mitochondrial biogenesis and oxidative phosphorylation. Collectively, it can be speculated that arginase inhibition attenuates HFD-induced hepatic steatosis through modulation in the hepatic expression of genes involved in lipogenesis and mitochondrial function.
Previous studies indicate that AMPKa, which is a member of the metabolic sensor protein kinase family, is involved in regulating vascular function by increasing the activation of eNOS [33] and is downregulated in abnormal metabolic complications such as T2DM and obesity [34]. Indeed, it was reported that vascular eNOS and AMPKa levels are reduced in HFD-fed rats [35]. In addition, AMPKa stimulates expression of PGC-1a [36] and mitochondrial biogenesis [37] as well as fatty acid oxidation [38]. Therefore, it is likely that AMPKa possibly in cooperation with NO [36] plays a key role in regulating lipid metabolism through controlling fatty acid oxidation and activating mitochondrial function. In the present study, the phosphorylation level of AMPKa at Thr172 was significantly up-regulated following arginase inhibition in the livers of HFD-fed mice, suggesting that the NO-AMPKa pathway may be involved in the beneficial effects of arginase inhibition on obesity-induced hepatic abnormalities. This is further supported by our in vitro findings clearly showing The results are expressed as mean 6 SE of at least three independent experimental results, and differences between groups were tested by analysis of variance (ANOVA) with Duncan's multiple range test. The same letter indicates no significant difference between two groups (p,0.05). doi:10.1371/journal.pone.0103048.g005 that arginase inhibition was able to increase NO release and the expression of phosphorylated AMPKa in OLA-induced hepatic steatosis. Also, arginase inhibition by nor-NOHA reduced arginase activity in OLA-treated HepG2 cells in a dose dependent manner, whereas nor-NOHA did not affect the protein expressions of arginase 1 and eNOS. These indicate that the effect of nor-NOHA on NO release was possibly derived from inhibition of arginase activity, not from changes in protein expressions. Of particular interest is the finding that nor-NOHA elicited systemic effects by marked reductions in body weight and fat mass in diet-induced obesity. The mechanism by which arginase inhibition prevents diet-induced obesity was unclear; however, the arginine-NO pathway might be involved in the regulation of whole-body metabolism. Whether the systemic effects shown in the present study were due to arginase inhibition working primarily through enhanced NO production or through an increased bioavailability of arginine remains to be determined, although co-treatment of NOS inhibitor with nor-NOHA has been shown to significantly reverse reduced lipid accumulation by arginase inhibition in OLAinduced hepatic steatosis ( Figure S1). Furthermore, since we evaluated the effects of arginase inhibition in established obese animals, we cannot exclude the possibility that longer, chronic arginase inhibition than that tested here could elicit much greater systemic effects.
In conclusion, we found that arginase inhibition showed general improvement in hepatic lipid metabolism and was sufficient to reduce whole body adiposity. The plausible mechanism may be that arginase inhibition increased hepatic NO production, which in turn activated the metabolic pathways involved in hepatic TG metabolism and mitochondrial function. However, caution is needed to interpret data for involvement of NO since we did not test the combined effects of arginase inhibition and NOS inhibition on weight loss. Also, the data regarding the effects of nor-NOHA in a normal diet are required for a clear interpretation of the results. Considering that even a modest weight loss in obese subjects is associated with a significant reduction of cardiovascular risks and diabetes [39], arginase inhibition may be a novel means to reduce body fat, and therefore shows therapeutic potential for human health application. Figure S1 Effect of cotreatment of nor-NOHA and L-NAME on lipid accumulation in OLA-induced hepatic steatosis in HepG2 cells. The HepG2 cells (2610 5 cells) were seeded on each well of a 48-well culture plate. After 24 h, the cells were serum-starved overnight. The next day, they were treated with 1.5 mM OLA cotreated with 5 mM nor-NOHA using 25 mM L-NAME (N Gnitro-L-arginine methyl ester) for 24 h. The co-treated cells were washed with PBS and fixed with 10% formalin for 1 h at room temperature. Subsequently, the cells were washed with 60% isopropanol, stained with (B) Oil Red O for 10 min at room temperature, and washed 4 times with distilled water. Images for each dish were captured using a microscope (4006 magnification, Olympus Corporation; Tokyo, Japan). Isopropanol (100%) was added to the cells, and after 10 min, absorbance was measured at 500 nm with a spectrophotometer (PerkinElmer; Waltham, MA, USA). (A) AdipoRed assay reagent (Lonza; Walkersville, MD, USA) was used for the quantification of TG content according to the manufacturer's protocol. The data represent the mean percentage levels compared with ethanol-treated cells. The results are expressed as mean 6 SE of at least three independent experimental results, and differences between groups were tested by analysis of variance (ANOVA) with Duncan's multiple range test. The same letter indicates no significant difference between two groups (p,0.05). The result showed that co-treatment of 25 mM L-NAME with nor-NOHA significantly reversed reduced lipid accumulation in OLA-induced hepatic steatosis by arginase inhibition. (DOCX)