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Potential conflict of interest: Nothing to report.
Obesity is associated with chronic inflammation and contributes to the development of insulin resistance and nonalcoholic fatty liver disease. The suppressor of cytokine signaling-3 (SOCS3) protein is increased in inflammation and is thought to contribute to the pathogenesis of insulin resistance by inhibiting insulin and leptin signaling. Therefore, we studied the metabolic effects of liver-specific SOCS3 deletion in vivo. We fed wild-type (WT) and liver-specific SOCS3 knockout (SOCS3 LKO) mice either a control diet or a high-fat diet (HFD) for 6 weeks and examined their metabolic phenotype. We isolated hepatocytes from WT and SOCS3 LKO mice and examined the effects of tumor necrosis factor α and insulin on Akt phosphorylation and fatty acid metabolism and lipogenic gene expression. Hepatocytes from control-fed SOCS3 LKO mice were protected from developing tumor necrosis factor α–induced insulin resistance but also had increased lipogenesis and expression of sterol response element–binding protein-1c target genes. Lean SOCS3 LKO mice fed a control diet had enhanced hepatic insulin sensitivity; however, when fed an HFD, SOCS3 LKO mice had increased liver fat, inflammation, and whole-body insulin resistance. SOCS3 LKO mice fed an HFD also had elevated hypothalamic SOCS3 and fatty acid synthase expression and developed greater obesity due to increased food intake and reduced energy expenditure. Conclusion: Deletion of SOCS3 in the liver increases liver insulin sensitivity in mice fed a control diet but paradoxically promotes lipogenesis, leading to the development of nonalcoholic fatty liver disease, inflammation, and obesity. (HEPATOLOGY 2010.)
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Obesity is associated with type 2 diabetes and the metabolic syndrome and is a major cause of morbidity and mortality. The metabolic syndrome is characterized by nonalcoholic fatty liver disease (NAFLD), dyslipidemia, and insulin resistance and it is currently believed that chronic low-grade inflammation in obesity may be an important contributing factor by impairing insulin sensitivity.1 The molecular events mediating obesity, insulin resistance, and NAFLD are currently incompletely understood.
Suppressor of cytokine signaling (SOCS) proteins (SOCS1 through SOCS7 and cytokine-inducible SH-2–containing protein) are induced by proinflammatory cytokines and regulate cytokine signaling through the Janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway.2 SOCS3 expression in obesity may be increased due to elevated inflammatory cytokines such as interleukin-6 (IL-6)3 and tumor necrosis factor alpha (TNF-α).4 Several members of the SOCS family, including SOCS1,5-8 SOCS3,3, 4, 8-11 SOCS6,5, 12 and SOCS7,13 have been implicated in insulin resistance. SOCS3 inhibits insulin signaling in several ways. It binds to the insulin receptor3, 9 and prevents its association with insulin receptor substrate-1 (IRS1) and IRS2.3, 10 In addition, by binding to IRS1 and IRS2 via the SOCS box, SOCS3 may also target IRS proteins for proteasomal degradation.8 SOCS3 is also important in the development of obesity-related leptin resistance in the hypothalamus14, 15 and skeletal muscle.16
In the liver, short-term overexpression of SOCS3 in vivo worsened insulin resistance,10 whereas suppression of SOCS3 using small interfering RNA ameliorated insulin resistance in obese, diabetic db/db mice.10 Torisu et al.17 demonstrated that mice with liver-specific deletion of SOCS3 (SOCS3 LKO) have improved liver insulin sensitivity but surprisingly also develop obesity and systemic insulin resistance. In the current study, we have shown that SOCS3 LKO mice have increased liver lipogenesis, which exacerbates the development of obesity-related fatty liver, inflammation, and insulin resistance. These factors contribute to the development of obesity, which is due to reduced energy expenditure and increased food intake. Taken together, these findings reveal a novel role for liver SOCS3 as an important negative regulator of not only insulin sensitivity but also lipogenesis and energy balance, highlighting the intricate cross-talk between the liver and whole-body energy metabolism.
The animal ethics committee of St. Vincent's Health approved all procedures. The generation of SOCS3 LKO mice have been described previously (mice were a gift from Prof. Warren Alexander, Walter and Eliza Hall Institute of Medical Research, Australia).18 Male littermates were randomly placed on a chow diet (8% kcal/fat) or a high-fat diet (HFD, 45% kcal/fat, Specialty Feeds, Australia) from 6 weeks of age for 6 weeks. Mice were injected intraperitoneally with recombinant IL-6 (1 μg/kg body weight; a gift from Dr. Richard Simpson, Ludwig Institute, Australia) or saline, and tissues were collected 2 hours later. Hepatocytes were prepared by the collagenase perfusion method19 from 10-week-old chow-fed wild-type (WT) and SOCS3 LKO mice and incubated the following day with either vehicle or TNFα (10 ng/mL; R&D Systems, Minneapolis, MN) for 2 hours before the addition of vehicle or insulin (1 nM) for 4 hours (messenger RNA [mRNA] analysis) or 2 minutes (Akt phosphorylation). Lipogenesis was assessed by injecting mice with [3H]H2O (0.5 Ci/kg) for 1 hour or by incubating hepatocytes with serum-free Medium 199–containing [1-14C]acetate (0.5 μCi/mL) (Amersham Biosciences, UK) and 0.5 mM unlabeled sodium acetate in the presence or absence of insulin.19, 20 Hypothalamic sections were dissected as described.16 For insulin signaling, 0.5 U/kg body weight of insulin or saline was injected into the inferior vena cava of overnight fasted mice. Tissues were harvested 10 minutes later for analysis. Intraperitoneal glucose tolerance tests were conducted, following a 6-hour fast, with 1.0 g/kg body weight of D-glucose in saline and blood glucose monitored by tail tip bleeding.16 Euglycemic-hyperinsulinemic clamps were performed in conscious mice.21, 22 Voluntary physical activity, resting energy expenditure, and substrate oxidation rates were measured by indirect calorimetry.23
Gene expression analysis was completed using quantitative real-time polymerase chain reaction (RT-qPCR; Rotorgene 3000; Corbett Research, Australia) using Assay-on-Demand gene expression kits (Applied Biosystems).16 Lipid and protein analyses were completed as previously described.16, 20 Insulin and plasma adiponectin were measured by ELISA and adipokines (leptin, TNFα, resistin, tPAI-1) measured by BioPlex assay (Linco Research, Inc.).16, 20 NEFA (Wako Pure Chemicals, Osaka, Japan), serum triglycerides and free glycerol (Sigma) were measured as per manufacturer's recommendations.16, 20 Liver microarray analysis was completed using publicly available expression data for SOCS3 LKO and control livers obtained from the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/), series GSE369.18 The Limma package24 of the R Project for Statistical Computing (www.r-project. org) was employed to perform quantile normalization of probe intensity data, and to identify significantly differentially expressed genes.
Data are presented as mean ± standard error of the mean (SEM). Data were analyzed using a two-way analysis of variance with a Bonferroni post hoc test (GraphPad PRISM, version 4.0). P < 0.05 was considered as significant.
Increased Obesity in HFD-Fed SOCS3 LKO Mice.
We validated that deletion of the SOCS3 gene was limited to the liver in mice acutely injected with interleukin-6 (Fig. 1A and Supporting Information Fig. 1). As expected, the HFD increased liver SOCS3 expression in littermate control floxed WT mice but not in SOCS3 LKO mice (Fig. 1A). SOCS1 and SOCS3 are highly homologous, but we found no difference in HFD induction of SOCS1 between genotypes (data not shown). On a control chow diet, SOCS3 LKO mice weighed the same as WT littermates. However, when fed an HFD (from 6 weeks of age onward), they gained more weight (Fig. 1B). Epididymal fat pad weights were significantly larger in absolute terms (Fig. 1C) and as percentage of total body mass (data not shown) in SOCS3 LKO mice fed an HFD indicating that the increased weight gain in SOCS3 LKO mice was due to increased adiposity.
To assess the mechanisms contributing to increased weight gain in SOCS3 LKO mice we measured food intake and energy expenditure. On an HFD, SOCS3 LKO mice consumed significantly more food per day (Fig. 1D), even when corrected for body mass (data not shown), and expended less energy (Fig. 1E). Glucose oxidation rate was reduced in chow-fed and tended to be reduced in HFD-fed SOCS3 LKO mice (data not shown). There was no difference in the rates of fat oxidation over a 24-hour light/dark cycle on either diet (data not shown). Taken together, these data suggest that increased adiposity in SOCS3 LKO mice on an HFD was due to reduced daily energy expenditure and increased caloric intake.
SOCS3 LKO Mice Have Enhanced Hepatic Insulin Sensitivity on a Chow Diet but Greater Hepatic Insulin Resistance when Fed an HFD.
We measured serum glucose and insulin concentrations as well as glucose tolerance and found that on a chow diet SOCS3 LKO mice were comparable to WT littermates (Fig. 2A-C). In contrast, HFD-fed SOCS3 LKO mice developed hyperglycemia (Fig. 2A) and a greater degree of hyperinsulinemia (Fig. 2B) and glucose intolerance (Fig. 2D) than WT littermates. These data suggest that deletion of liver SOCS3 accelerates the onset of HFD-induced insulin resistance.
To assess the specific contribution of hepatic versus peripheral insulin resistance to glucose homeostasis in SOCS3 LKO mice we conducted euglycemic-hyperinsulinemic clamps. Basal glucose turnover was similar between the two groups of mice on both diets (Supporting Table 1). In chow fed mice the glucose infusion rate (GIR), and the glucose disposal rate (GDR) were not different between groups (Fig. 3A,B). As anticipated, the HFD significantly reduced both GIR and GDR relative to chow fed mice, but this reduction was significantly greater in SOCS3 LKO mice (Fig. 3A,B), indicating increased peripheral insulin resistance relative to HFD fed WT littermates, a finding consistent with the increased weight gain and body mass. Consistent with the findings in the clamp study, Akt phosphorylation in muscle following a bolus of insulin was reduced (data not shown) and skeletal muscle triglyceride and diglyceride content were increased (data not shown) in SOCS3 LKO mice.
Hepatic glucose production (HGP) during the clamp was lower in chow-fed SOCS3 LKO mice (Fig. 3C,D), indicating enhanced hepatic insulin sensitivity. The HFD increased HGP during the clamp in WT mice (Fig. 3C) but surprisingly, SOCS3 LKO mice had a higher HGP and reduced suppression (Fig. 3C,D), than WT littermates. These data indicate that the deletion of liver SOCS3 exacerbates HFD-induced hepatic insulin resistance.
To further study these effects, we examined insulin signaling in the liver following a bolus injection of insulin. IRS1 phosphorylation, IRS1-associated PI-3 kinase and Akt phosphorylation were all higher in chow-fed SOCS3 LKO mice (Fig. 4A-C), consistent with the enhanced HGP suppression seen during the clamp. In contrast to control-fed mice, but consistent with the clamp data, insulin signaling was significantly attenuated in HFD-fed SOCS3 LKO mice (Fig. 4A-C); indicating greater hepatic insulin resistance had developed despite the absence of SOCS3. Consistent with changes in insulin signaling and HGP, the expression of the major gluconeogenic enzymes Pck1 (phosphoenolpyruvate carboxykinase 1) and G6pc (glucose 6 phosphatase) were reduced by insulin in chow-fed SOCS3-deficient livers (Fig. 4D). Similar findings were also observed in isolated hepatocytes (Supporting Fig. 2A,B). In contrast, Pck1 and G6pc expression were significantly higher in both WT and SOCS3 LKO mice fed an HFD (Fig. 4D). These data indicate that on a chow diet, deletion of SOCS3 enhances insulin sensitivity by increasing IRS1 phosphorylation, but when mice are challenged with an HFD, factors independent of SOCS3 lead to hepatic insulin resistance.
Studies in adipocytes have demonstrated that TNFα induces insulin resistance by increasing SOCS3 expression4, 10, 11; therefore, we studied the role of TNFα in hepatocytes from WT and SOCS3 LKO mice. As anticipated, SOCS3 expression was increased in WT but not SOCS3 LKO hepatocytes in response to insulin (∼70%) and TNFα (∼100%) (data not shown). TNFα blunted the ability of insulin to increase Akt phosphorylation in WT but not SOCS3 LKO hepatocytes (Fig. 4E). These data when combined with previous reports10, 17 show that SOCS3 is a negative regulator of liver insulin signaling and suggest that insulin resistance in HFD-fed SOCS3LKO mice is independent of TNFα.
SOCS3 LKO Mice Have Increased Lipogenesis and Develop NAFLD When Fed an HFD.
Because the excess accumulation of lipids can impair hepatic insulin sensitivity (for review, see Savage et al.25) we hypothesized that this may have contributed to the reduced liver insulin sensitivity of HFD-fed SOCS3 LKO mice. We found that HFD-fed SOCS3 LKO mice had increased liver weights (data not shown) and hematoxylin and eosin (H&E) staining of liver sections revealed greater deposition of lipid droplets and portal inflammation compared to WT controls (Fig. 5A). Other features of NAFLD such as fibrosis, Mallory's hyaline, or hepatocellular ballooning were not present. Biochemical analysis demonstrated increased levels of liver TG (Fig. 5B) and DG (Fig. 5C, P = 0.06) in HFD-fed SOCS3 LKO mice. There was no difference in the amount of glycogen or ceramide (data not shown). In order to assess whether the steatosis seen in the absence of hepatic SOCS3 was a consequence of increased de novo lipogenesis or a suppression of oxidative genes, we conducted gene microarrays of chow-fed SOCS3 LKO mice liver and found that stearoyl-coenzyme A (CoA) decarboxylase-1 (SCD-1) was up-regulated (Supporting Table 2) a finding confirmed via RT-qPCR (Fig. 5D). SCD-1 is a downstream target of sterol response element–binding protein-1c (SREBP-1c), a transcription factor that also regulates the expression of GPAT-1 and FASn. Consistent with increases in liver lipogenic gene expression chow-fed SOCS3 LKO mice also had increased lipogenesis in vivo (Supporting Information Fig. 3). In agreement with previous findings, HFD increased hepatic lipogenic gene expression and this effect was much more dramatic in SOCS3 LKO mice (Fig. 5D). There was no difference in the expression of oxidative enzymes in the microarray (Supporting Information Table 2) or in subsequent RT-qPCR analysis of Ppara (peroxisome proliferator-activated receptor alpha), Pparg (peroxisome proliferator-activated receptor gamma), Cpt1 (carnitine palmitoyl transferase-1), Pgc1a (PPARγ transcriptional coactivator 1α), Mcad (medium chain acyl-CoA dehydrogenase), Cytochrome C (Cytc), Cs (citrate synthase) Ucp2 (uncoupling protein 2), Acox1 (acyl-CoA oxidase), Cyp4a10 (cytochrome P450 4a10), or Cyp4a14 (cytochrome P450 4a14) (Supporting Information Table 3).
STAT3 may be a negative regulator of lipogenesis, potentially by inhibiting SREBP1c expression.26 Therefore, we examined STAT3 phosphorylation in livers of chow and HFD fed mice, under the same conditions in which lipogenic gene expression was determined. We found that there was no effect of genotype or diet on STAT3 phosphorylation despite hypersensitivity of SOCS3 LKO mice to IL-6 stimulation (Supporting Information Fig. 4A). Despite differences in STAT3 phosphorylation following IL-6 stimulation lipogenic gene expression between WT and SOCS3 LKO mice was similar (Supporting Information Fig. 4B). These findings in conjunction with findings that STAT3 LKO mice do not develop steatosis despite obesity and hyperinsulinemia27 suggest that STAT3 is not a critical factor driving lipogenic gene expression.
In addition to the suppression of hepatic gluconeogenesis, insulin also promotes lipogenesis.28 To examine whether the increased deposition of lipid in SOCS3 LKO mice was due to changes in liver metabolism independently of the hormonal milieu or neuronal inputs, we measured rates of fatty acid synthesis and oxidation in hepatocytes from WT and SOCS3 LKO mice with and without insulin. Hepatocytes from SOCS3 LKO mice had dramatically increased rates of fatty acid synthesis into DG (Fig. 6A) and TG (Fig. 6B) basally, a difference which was maintained in response to insulin. Rates of fatty acid oxidation were not different between genotypes (Fig. 6C). Consistent with increased rates of fatty acid synthesis and gene expression analysis in vivo the expression of Srebp-1c, GPAT-1, FASn, and SCD-1 were all up-regulated in hepatocytes from SOCS3 LKO mice, an effect which was independent of insulin (Fig. 6D).
Inflammation and Increased Hypothalamic SOCS3 and FASn in SOCS3 LKO Mice Fed an HFD.
NAFLD is known to drive hepatic inflammation; therefore, we assessed the expression of proinflammatory cytokines in the liver and serum. Consistent with changes in liver fat, HFD but not chow-fed SOCS3 LKO mice had increases in liver inflammation (TNFα, IL-6, and the macrophage marker F4/80) and circulating levels of IL-6 and tPAI-1 (Fig. 7A and Table 1). Serum free-fatty acids (NEFA), triglycerides (TG), and adiponectin levels were not different between genotypes (Table 1).
Table 1. Metabolic Parameters and Adipocytokine Levels in SOCS3 LKO Mice
Several recent studies have demonstrated that systemic inflammation can lead to leptin resistance.14, 29-31 In addition, hyperinsulinemia has been shown to increase SOCS39 and FASn expression,32 which in turn increases appetite and reduces energy expenditure.33, 34 Therefore, to examine the mechanisms contributing to the increased food intake and reduced energy expenditure in HFD-fed SOCS3 LKO mice, we measured hypothalamic expression of FASn, SOCS3, the orexigenic neuropeptides neuropeptide Y (NPY) and Agouti-related protein (AgRP), and the anorexigenic neuropeptide pro-opiomelanocortin (POMC). Consistent with normal food intake and energy expenditure in chow-fed mice, there was no difference in gene expression between groups (Fig. 7B). However, in SOCS3 LKO mice fed an HFD we found increased hypothalamic FASn and SOCS3 expression and a trend toward increased NPY (P = 0.08) and AgRP (P = 0.10) expression (Fig. 7B). These findings suggest that increased liver steatosis and subsequent inflammation and hyperinsulinemia may lead to increased hypothalamic SOCS3 and FASn expression which could contribute to the hyperphagia, reduced energy expenditure, and subsequent weight gain observed in HFD-fed SOCS3 LKO mice (Fig. 7C).
Previous studies have reported improved insulin sensitivity with SOCS3 deletion.10, 11, 17, 26 In agreement with these, we found that deletion of SOCS3 in the liver of chow-fed animals improved insulin signaling, resulting in enhanced suppression of hepatic glucose production. Consistent with this we found that deletion of SOCS3 protected against acute TNFα-induced insulin resistance. Given these findings, and those of other previous reports,17, 26 we anticipated that SOCS3 LKO mice would be protected against HFD-induced hepatic steatosis and insulin resistance. Instead, we found that SOCS3 deficiency promoted liver lipid accumulation, inflammation, and the development of more severe skeletal muscle and hepatic insulin resistance. This heightened inflammation and hyperinsulinemia was associated with increased hypothalamic expression of SOCS3 and FASn, which may have increased appetite and decreased energy expenditure, further exacerbating the obesity and systemic insulin resistance in HFD-fed SOCS3 LKO mice.
Our findings confirm those of a previous study17 but our additional findings lead us to quite different conclusions. Similar to Torisu et al.,17 we found greater insulin sensitivity in young mice lacking hepatic SOCS3. However, Torisu et al. did not find hepatic insulin resistance, steatosis, or increased hepatic lipogenesis in HFD-fed mice. Through clamp studies of hepatic glucose production in chow-fed and HFD-fed SOCS3 LKO mice, we found that SOCS3 LKO mice developed greater hepatic insulin resistance when challenged with an HFD. To clarify the mechanisms contributing to the perturbations in whole-body glucose homeostasis and energy partitioning, we performed food intake studies and calorimetry and found that SOCS3 LKO mice consumed more food and also expended less energy. Furthermore, we found biochemical evidence for hypothalamic changes (increased SOCS3 and FASn) consistent with the increased food consumption and reduced energy expenditure. These extrahepatic changes are particularly interesting because they are distant from the genetic alteration in the mice that is confined to hepatic SOCS3 deletion. No evidence of SOCS3 deletion outside the liver was found; in fact, hypothalamic SOCS3 was increased. We hypothesize that the metabolic deterioration and development of NAFLD seen with the HFD is connected to the increased lipogenic capacity of the liver from SOCS3 LKO mice, which leads to steatosis, inflammation, and in turn causes the perturbations to appetite and energy expenditure (Fig. 7C).
SOCS3 LKO mice were prone to NAFLD when fed an HFD due to increased lipogenesis. This finding was supported by studies in isolated hepatocytes that persisted even in the absence of insulin and other circulating hormones. Therefore, in vivo in mice fed an HFD the combined effects of the absence of liver SOCS3 driving the expression of SCD-1 and GPAT-1 and a system primed with substrate (elevated fatty acids and hyperglycemia) would favor conditions that would be expected to promote the development of NAFLD. This increase in lipids, especially reactive lipids such as DG,35 would in turn trigger activation of serine/threonine kinases and inflammation capable of impairing insulin signalling independently of SOCS3 (for review, see Erion and Shulman36). These findings are supported by other mouse studies demonstrating that GPAT-1 overexpression leads to hepatic steatosis and insulin resistance37 whereas the deletion of GPAT-138 or SCD-139 reverses the effects of obesity on these parameters. Importantly this same relationship between elevated SCD1 activity and liver DG is also observed in humans with NAFLD.40
Our findings are very different to those of Ueki et al.26 who showed that transient knockdown of SOCS3 using small interfering RNA in livers of obese db/db mice reduced steatosis and improved hepatic insulin sensitivity. Ueki et al.26 suggested that the reduced steatosis in db/db mice was attributed to enhanced STAT3 phosphorylation and reduction in SREBP1c expression. Instead, we found that liver STAT3 phosphorylation was not different between chow and HFD-fed WT and SOCS LKO mice. These data when combined with findings showing that STAT3 LKO mice do not develop hepatic steatosis when challenged with an HFD27 suggest that STAT3 may not be a critical regulator of lipogenesis under conditions of diet-induced obesity.
The development of greater obesity in HFD-fed SOCS3 LKO mice was unexpected. To elucidate the mechanisms contributing to the increased adiposity we performed food intake studies and calorimetry and found that SOCS3 LKO mice fed an HFD exhibited increased food consumption and reduced caloric expenditure compared with controls. Previous studies have shown that oligonucleotide inhibition of SCD-1 in the liver of obese animals not only rescues hepatic steatosis but also reduces food intake, increases energy expenditure and improves hepatic insulin sensitivity.41, 42 One mechanism by which this may occur is by reducing inflammation,43 which as recent studies have shown,14, 29-31 plays an important role in the development of hypothalamic leptin resistance and obesity. Therefore, our findings of increased inflammation and elevated hypothalamic expression of SOCS3 and orexigenic neuropeptides, NPY and AgRP, in HFD-fed but not chow-fed SOCS3 LKO mice is consistent with the role of liver inflammation regulating appetite and energy expenditure. Another possibility for the increased weight gain in HFD SOCS LKO mice may be related to hyperinsulinemia which increases the expression of FASn, an important regulator of appetite and energy expenditure.34 Lastly, it remains possible that energy expenditure and appetite could be altered by liver-specific effects on the vagus nerve44, 45 or by altering the expression of a circulating factor such as the soluble leptin receptor,46 however we believe these possibilities are relatively unlikely because we only observed differences when SOCS3 LKO mice were fed an HFD.
In conclusion, we have shown that hepatic SOCS3 is a physiological regulator of insulin signaling in vivo and that it is involved in the maintenance of hepatic insulin sensitivity even in the absence of overt inflammation as found in lean chow-fed mice and unstimulated hepatocytes. Although the deletion of liver SOCS3 enhances hepatic insulin sensitivity, in the presence of an obesogenic milieu of hyperglycemia and elevated fatty acids it promotes the development of NAFLD and inflammation, factors which may contribute to the development of obesity and systemic insulin resistance. Because liver-specific deletion of SOCS3 proved to be detrimental when animals are made obese through feeding an HFD, it is conceivable that targeting SOCS3 both centrally and in multiple peripheral tissues (liver, muscle, and fat) simultaneously may prove a more effective strategy. These studies highlight the complexity of targeting insulin sensitivity to tackle the primary obstacles encountered in treating type 2 diabetes and obesity, namely insulin and leptin resistance. Further studies clarifying the cross-talk between liver metabolism and central regulation of appetite and energy expenditure are clearly warranted.
The authors thank Dr. P. Bhathal, (an experienced liver histopathologist, Melbourne Pathology, Melbourne, Australia) for reviewing the H&E-stained sections of the liver. We also thank Dr. Anne Johnston for editing of the manuscript.