1,8-Cineole Ameliorates Steatosis of Pten Liver Specific KO Mice via Akt Inactivation

Hepatocyte-specific Phosphatase and tensin homolog (Pten)-knockout (KO) mice exhibit hepatic lesions analogous to non-alcoholic steatohepatitis (NASH). 1,8-cineole is a monoterpene oxide and it has several biological effects including hepatoprotective effects. In this study we revealed that 1,8-cineole ameliorates NASH of Pten KO mice. Pten KO mice were assigned to a control group without any medication or to a 1,8-cineole group injected with 50 mg/kg i.p. twice per week for eight weeks. At eight weeks, livers from each group were processed to measure triglyceride (TG) content, gene expression analysis, western blot analysis, and histological examination including Oil red O staining. 1,8-cineole ameliorated hepatic steatosis in Pten KO mice, revealed by TG content and Oil red O staining. Moreover, 1,8-cineole downregulated collagen 1a1 expression and improved liver fibrosis. Thus, 1,8-cineole has potential as a candidate to treat NASH by inactivating the Akt/PI3-kinase pathway.


1,8-Cineole Ameliorates NASH in Pten KO Mice
Eight-week continuous intraperitoneal injection of 1,8-cineole ameliorated lipid accumulation of livers compared to the control group. The body weight, liver weight, liver/body weight %, and serum parameters are listed on Table 1. No significant weight loss was observed between the two groups. Liver weight was significantly lower in the 1,8-cineole group. Serum cholesterol of the 1,8-cineole group was significantly decreased compared to the control group. Serum insulin was significantly increased in the 1,8-cineole group. Figure 1a shows gross appearance of liver, HE staining, Oil red O staining, and Sirius red staining. In the 1,8-cineole group, lipid droplet and fibrotic area were decreased compared to the control group. Figure 1b shows the Oil red O positive area and that of the 1,8-cineole group was significantly lower than the control group. Figure 1c shows triglyceride content and Figure 1d shows cholesterol content of the liver. Both triglyceride and cholesterol contents were significantly decreased in the 1,8-cineole group compared to the control group.    (T-Akt), phospho mTOR (P-mTOR), total mTOR (mTOR), Phospho-protein phosphatase type 2A (P-PP2A), total PP2A (PP2A), phospho-insulin receptor (P-insulin receptor), total insulin receptor (insulin receptor) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the 1,8-cineole group, FASN, P-Akt, P-mTOR, and P-PP2A were decreased. P-insulin receptor was increased in the 1,8-cineole group. Densitometry of FASN/GAPDH (b), phospho/total Akt (c), phospho/total mTOR (d), phospho/total PP2A (e), and phospho/total insulin receptor (f) in the two groups (n = 3). In the 1,8-cineole group, Akt and PP2A were significantly dephosphorylated, and the insulin receptor was significantly activated. * p < 0.05 vs. control.

Discussion
In this study, we demonstrated that 1,8-cineole administration improved steatosis and fibrosis seen in Pten KO mice. We confirmed the decreased hepatic TG and cholesterol accumulation in these mice by lipid analysis. 1,8-cineole treatment is involved in many pathways and genes related to synthesis of lipid and hepatic fibrosis confirmed by western blot and RT-PCR.
Hyperactivation of Akt is the main reason of steatosis, fibrosis, and carcinogenesis of Pten KO mice. By activating PP2A, LXR alpha and downstream Abca 1, 1,8-cineole inactivated Akt and finally reduced steatosis and fibrosis of the Pten KO mice. Pten is a tumor suppressor gene which inactivates Akt. Hyperactivation of Akt causes steatosis, fibrosis and carcinogenesis [4]. Induced activation of Akt also causes steatosis in the murine liver [14]. In clinical study, mutations of Pten cause insulin hypersensitivity and obesity and the insulin hypersensitivity could be explained by the presence of enhanced insulin signaling through the PI3K-Akt pathway, as evidenced by increased Akt phosphorylation [15]. In our study, insulin concentration in the control group was lower than that in the 1,8-cineole group. Serum glucose levels were almost equal between the two groups. That means 1,8-cineole reduced the insulin hypersensitivity of Pten KO mice. As a result, G6P expression and phospho insulin receptor were activated in the 1,8-cineole group. In a previous study, we found that 1,8-cineole inactivated Akt in the human colorectal cancer cell line RKO [12]. Akt activation is tightly controlled by counteraction of PTEN, PP2A, and pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) [16]. It has long been known that PP2A negatively regulates Akt activity in various systems [16]. In this study, dephosphorylation of PP2A, which means activation of PP2A, causes inactivation of Akt and downstream mTOR, as observed in the 1,8-cineole group. LXR alpha activation upregulates Abca1 and Abcg1. These molecules inactivate Akt in the lipid raft of the cellular membrane [17]. T0901317 is an agonist of LXR alpha. By inactivating Akt in the prostate cancer cell line, T0901317 is expected to be a chemotherapeutic agent [17]. Activation of LXR results in increased HDL levels and net whole body cholesterol loss [18]. There is a report that LXR activation by T0901317 exacerbated steatosis in high fat diet treated mice [19]. 1,8-cineole is reported to activate LXR alpha, Abca1, and Abcg1 in the murine macrophage cell line RAW 246.7 [20]. In our study, expression of LXR alpha and downstream Abca1 was elevated in 1,8-cineole mouse liver. One of the reasons for Akt inactivation by 1,8-cineole would be due to LXR alpha activation, but the relationship between LXR alpha and steatosis is still controversial.
FGF21 induces hepatic fatty acid oxidation by transcriptional regulation of key enzymes of fatty acid oxidation [21]. Dasarathy et al. revealed that plasma FGF21 concentration was higher in NASH than in controls [22]. FGF21 is also activated by hepatocarcinogenesis and hepatic stress [23]. FGF21 is an Akt dependent myokine [24], and LXR alpha activation is reported to reduce FGF 21 [25]. In our study, FGF 21 expression was decreased in the 1,8-cineole group, a result of Akt inactivation and LXR alpha activation.
In the NASH liver, fibrosis progresses and liver cirrhosis occurs [2]. There is a clinical evidence that hepatic Akt expression correlates with advanced fibrosis in patients with chronic hepatitis C infection [26]. Higher expression of Akt exacerbates liver fibrosis. 1,8-cineole treatment decreased fibrotic area stained by Sirius red in the liver, and COL1A1 expression was decreased.
1,8-cineole was reported to have various pharmacological effects, such as smooth muscle relaxant, anti-inflammation, antioxidant and hypotension [6][7][8][9][10][11]. Hepatoprotection was suggested to be associated with a reduction in TNF-α serum concentration [27]. Lima et al. reported that 1,8-cineole ameliorates cerulein-induced acute pancreatitis in mice by oral administration of 1,8-cineole from 100 to 400 mg/kg [28]. The effects of acute and subacute toxicity of 1,8-cineole in Kunming mice were studied. After acute oral administration, the LD50 value (95% Cl) was 3849 mg/kg. In the subacute toxicity study, there were no significant differences in body weight and relative organ weight between the control group and the 1,8-cineole treatment groups for 30 days with up to 192.45 mg/kg oral administration [29]. Although there are few reports on i.p. administration, 50 mg/kg of 1,8-cineole is assumed to be safe. Essential oil of Rosemary, which contains about 40% 1,8-cineole, was reported to have a hepatoprotective effect against CCl4 [30]. In our study, 1,8-cineole not only ameliorates steatosis, but also reduced fibrosis in Pten KO mice.

Animals
Pten flox/flox mice (129Ola_C57BL6/J F2), generated as previously described [31], were mated to AlbCre transgenic mice (C57BL6/J background; The Jackson Laboratory, Bar Harbor, ME, USA) [32] in which expression of Cre is controlled by the promoter of the hepatocyte-specific gene Albumin. Offspring carrying AlbCre and two copies of the floxed Pten allele (AlbCrePtenflox/flox) were used in this study as homozygous mutant (Pten KO) mice [33]. Animal experiments were carried out in a humane manner after receiving approval from the Institutional Animal Experiment Committee of the University of Tsukuba (identification code 14-377, 2 September 2014) and in accordance with the Regulation for Animal Experiments at our university and the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Treatment
Twelve-week-old male Pten KO mice were randomly divided into two groups: control group, mice without any treatment; and the 1,8-cineole group, mice with 50 mg/kg of 1,8-cineole administered twice a week i.p. for eight weeks. In each group, six to eight mice were used. At 20 weeks of age, the mice were sacrificed and specimens collected.

Histology and Oil Red O Staining
Formalin-fixed tissues were embedded in paraffin using standard procedures. Sections (4 μm thick) were cut and stained with either hematoxylin-eosin (HE) for standard microscopy or Sirius red stain to show fibrosis. To visualize lipids, frozen sections (5 μm thick) were stained with Oil Red O (Nakarai Tesque Inc., Kyoto, Japan) and counterstained with hematoxylin.

Biochemical Analyses of Liver Extracts and Serum
Levels of triglyceride (TG) (Abnova, Taipei, Taiwan) and cholesterol (Wako Pure Chemical Industries, Osaka, Japan) in total lipids extract of the liver and alanine aminotransferase (ALT), TG, cholesterol, glucose (Fuji Film, Tokyo, Japan), and insulin (Mercodia, Uppsala, Sweden) were determined by colorimetric, or enzymatic assays.

Western Blot Analysis
For western blot analysis, total protein extracts of control and 1,8-cineole group mice livers were obtained, then separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA). The following antibodies were used as primary antibodies: total Akt, phosphoserine 473 Akt, phospho and total mammalian target of rapamycin (mTOR) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, MA, USA). Purified mouse anti-FASN antibody was purchased from BD Biosciences (San Jose, CA, USA). Phospho (Tyr 972) and total insulin receptor were purchased from Merck Millipore (Darmstadt, Germany). Phospho and total PP2A were purchased from Abcam (Cambridge, MA, USA). Secondary goat anti rabbit and goat anti mouse antibodies conjugated with horseradish peroxidase were purchased from Cell Signaling Technology. Immunoblots were analyzed by enhanced chemiluminescence. Densitometry of phospho/total Akt was calculated by Image J [34].

Total RNA Extraction
For reverse transcription-polymerase chain reaction (RT-PCR), total RNA was extracted from 50 mg liver samples in in vitro and in vivo experiments. Total RNA was isolated from whole cells using a NucleoSpin ® RNA II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. RNA concentrations were determined by measuring the absorbance at 260/280 nm with a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The synthesis of complementary DNA was performed using AMV reverse transcriptase (Promega, Madison, WI, USA) and random primers (Takara Bio Inc., Shiga, Japan). Briefly, a mixture of 1 mM dNTPs (Fermentas Life Sciences, Burlingame, ON, Canada), 0.025 μg/mL random primers, 0.25 U/mL reverse transcriptase, and 500 ng of total RNA was incubated at 30 °C for 10 min, 37 °C for 60 min, 95 °C for 5 min and at 4 °C before storage at −80 °C.

Statistical Analysis
All data are expressed as the mean ± SD of samples. Comparisons between the two groups were made using Mann-Whitney's U test. In all cases, a p-value <0.05 was considered significant.

Conclusions
In conclusion, these findings demonstrate that 1,8-cineole might exert its hepatoprotective activity by reducing steatosis and fibrosis in Pten KO mice in vitro and in vivo. 1,8-cineole shows promise as a strong and safe therapeutic agent for NASH.