The role of AMPKα2 in the HFD-induced nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic steatosis, inflammation and liver fibrosis and has become one of the leading causes of hepatocellular carcinoma and liver failure. However, the underlying molecular mechanism of hepatic steatosis and the progression to nonalcoholic steatohepatitis (NASH) are not fully understood. Herein, we discovered that AMPKα2 catalytic subunit showed reduced expression in the liver following high fat diet (HFD) feeding to mice. Importantly, knockout of AMPKα2 in mice aggravated NAFLD, hepatic steatosis, inflammation and fibrosis. On the other hand, hepatocyte-targeted overexpression of AMPKα2 prevented or reversed NAFLD indications. In vivo mechanistic studies revealed that increased phosphorylation of IKKα/β and NF-κB in HFD-fed AMPKα2^−/− mice compared to WT mice, and treatment of these mouse cohorts with an inhibitor of NF-κB signaling for 4 weeks, effectively attenuated the progression of steatohepatitis and metabolic disorder features. In summary, AMPKα2 provides a protective role in the process of hepatic steatosis to NASH progression through suppression of liver NF-κB signaling.     

Hypoxia inducible factor-1 promotes liver fibrosis in nonalcoholic fatty liver disease by activating PTEN/p65 signaling pathway

Obesity is a major contributor to the development of steatohepatitis and fibrosis from nonalcoholic fatty liver disease (NAFLD). Hypoxia aggravates progression of NAFLD. In mice on high-fat diet (HFD), hepatic steatosis leads to liver tissue hypoxia, evidenced by accumulation of hypoxia inducible factor-1-alpha (HIF-1α), which is a central regulator of the global response to hypoxia. Hepatocyte cell signaling is an important factor in hepatic fibrogenesis. We here hypothesize that HIF-1α knockout in hepatocyte may protect against liver fibrosis. We first found that HFD led to 80% more hepatic collagen deposition than Hif1a^−/−hep mice, which was confirmed by a-SMA staining of liver tissue. Body weight and liver weight were similar between groups. We then found the increasing HIF1a expression and decreasing PTEN expression in the mice on HFD and in PA-treated HepG2 cells. Finally, we found that HIF1 mediated PTEN/nfkb-p65 pathway plays an important role in the development of NAFLD to liver fibrosis. Collectively, these results identify a novel HIF1a/PTEN/NF-κ Bp65 signaling pathway in NAFLD, which could be targeted for the therapy.     

Therapeutic Role of Ursolic Acid on Ameliorating Hepatic Steatosis and Improving Metabolic Disorders in High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease Rats

Background

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver diseases around the world, and is closely associated with obesity, diabetes, and insulin resistance. Ursolic acid (UA), an ubiquitous triterpenoid with multifold biological roles, is distributed in various plants. This study was conducted to investigate the therapeutic effect and potential mechanisms of UA against hepatic steatosis in a high-fat diet (HFD)-induced obese non-alcoholic fatty liver disease (NAFLD) rat model.

Methodology/Principal Findings

Obese NAFLD model was established in Sprague-Dawley rats by 8-week HFD feeding. Therapeutic role of UA was evaluated using 0.125%, 0.25%, 0.5% UA-supplemented diet for another 6 weeks. The results from both morphologic and histological detections indicated that UA significantly reversed HFD-induced hepatic steatosis and liver injury. Besides, hepatic peroxisome proliferator-activated receptor (PPAR)-α was markedly up-regulated at both mRNA and protein levels by UA. Knocking down PPAR-α significantly inhibited the anti-steatosis role of UA in vitro. HFD-induced adverse changes in the key genes, which participated in hepatic lipid metabolism, were also alleviated by UA treatment. Furthermore, UA significantly ameliorated HFD-induced metabolic disorders, including insulin resistance, inflammation and oxidative stress.

Conclusions/Significance

These results demonstrated that UA effectively ameliorated HFD-induced hepatic steatosis through a PPAR-α involved pathway, via improving key enzymes in the controlling of lipids metabolism. The metabolic disorders were accordingly improved with the decrease of hepatic steatosis. Thereby, UA could be a promising candidate for the treatment of NAFLD.     

Downregulation of microRNA-451 in non-alcoholic steatohepatitis inhibits fatty acid-induced proinflammatory cytokine production through the AMPK/AKT pathway

Mechanisms associated with the progression of non-alcoholic fatty liver disease (NAFLD) remain unclear. We attempted to identify the pattern of altered gene expression at different time points in a high fat diet (HFD)-induced NAFLD mouse model. The early up-regulated genes are mainly involved in the innate immune responses, while the late up-regulated genes represent the inflammation processes. Although recent studies have shown that microRNAs play important roles in hepatic metabolic functions, the pivotal role of microRNAs in the progression of NAFLD is not fully understood. We investigated the functions of miR-451, which was identified as a target gene in the inflammatory process in NAFLD. miR-451 expression was significantly decreased in the palmitate (PA)-exposed HepG2 cells and in liver tissues of HFD-induced non-alcoholic steatohepatitis (NASH) mice. Its decreased expressions were also observed in liver specimens of NASH patients. In vitro analysis of the effect of miR-451 on proinflammatory cytokine provided evidence for negative regulation of PA-induced interleukin (IL)-8 and tumor necrosis factor-alpha (TNF-α) production. Furthermore, miR-451 over-expression inhibited translocation of the PA-induced NF-κB p65 subunit into the nucleus. Our result showed that Cab39 is a direct target of miRNA-451 in steatotic cells. Further study showed that AMPK activated through Cab39 inhibits NF-κB transactivation induced in steatotic HepG2 cells. miR-451 over-expression in steatotic cells significantly suppressed PA-induced inflammatory cytokine. These results provide new insights into the negative regulation of miR-451 in fatty acid-induced inflammation via the AMPK/AKT pathway and demonstrate potential therapeutic applications for miR-451 in preventing the progression from simple steatosis to severely advanced liver disease.     

Grape pomace reduces the severity of non-alcoholic hepatic steatosis and the development of steatohepatitis by improving insulin sensitivity and reducing ectopic fat deposition in mice

While non-alcoholic fatty liver disease (NAFLD) represents the common cause of chronic liver disease, specific therapies are currently unavailable. The wine industry produces millions of tons of residue (pomace), which contains high levels of bioactive phytochemicals. The aim of this study was to clarify the potential benefits of grape pomace for the treatment of NAFLD at different levels of severity, and to clarify the mechanism of action. C57Bl/6 mice were given high fat diet (HFD) or western diet (WD) as models of obesity and hepatic steatosis or steatohepatitis, respectively, with or without pomace supplementation (50–250 mg/day). Pomace inhibited food intake, and reduced serum leptin and body weight gain. Ectopic fat deposition was reduced, while white adipose tissue mass was preserved. In addition, pomace improved glucose tolerance and insulin sensitivity, prevented the development of adipose tissue inflammation, and reduced hepatic steatosis. Higher expression of genes involved in fatty acids transport and oxidation was observed in adipose tissue, while lipogenic genes were attenuated in the liver of pomace-treated mice. In WD-fed mice, pomace reduced the severity of hepatic steatosis and inflammation and improved blood lipid profile, but was ineffective in reversing hepatic damage of advanced NASH. In conclusion, pomace improved insulin sensitivity and reduced ectopic fat deposition, leading to a healthier metabolic profile. Pomace may hold the potential as a supplement with beneficial health outcomes for the prevention and treatment of hepatic steatosis and other obesity-related pathologies.     

E3 ubiquitin ligase Grail promotes hepatic steatosis through Sirt1 inhibition

In obese adults, nonalcoholic fatty liver disease (NAFLD) is accompanied by multiple metabolic dysfunctions. Although upregulated hepatic fatty acid synthesis has been identified as a crucial mediator of NAFLD development, the underlying mechanisms are yet to be elucidated. In this study, we reported upregulated expression of gene related to anergy in lymphocytes (GRAIL) in the livers of humans and mice with hepatic steatosis. Grail ablation markedly alleviated the high-fat diet-induced hepatic fat accumulation and expression of genes related to the lipid metabolism, in vitro and in vivo. Conversely, overexpression of GRAIL exacerbated lipid accumulation and enhanced the expression of lipid metabolic genes in mice and liver cells. Our results demonstrated that Grail regulated the lipid accumulation in hepatic steatosis via interaction with sirtuin 1. Thus, Grail poses as a significant molecular regulator in the development of NAFLD.Subject terms: Cell signalling, Metabolic disorders     

Effects of Sodium Butyrate and Its Synthetic Amide Derivative on Liver Inflammation and Glucose Tolerance in an Animal Model of Steatosis Induced by High Fat Diet

Background & Aims

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease. Insulin resistance (IR) appears to be critical in its pathogenesis. We evaluated the effects of sodium butyrate (butyrate) and its synthetic derivative N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA) in a rat model of insulin resistance and steatosis induced by high-fat diet (HFD).

Methods

After weaning, young male Sprague-Dawley rats were divided into 4 groups receiving different diets for 6 weeks: 1. control group (standard diet); 2. HFD; 3. HFD plus butyrate (20 mg/kg/die) and 4. HFD plus FBA (42.5 mg/Kg/die, the equimolecular dose of butyrate). Liver tissues of the rats were analyzed by Western blot and real-time PCR. Insulin resistance, liver inflammation and Toll-like pattern modifications were determined.

Results

Evaluation of these two preparations of butyrate showed a reduction of liver steatosis and inflammation in HFD fed animals. The compounds showed a similar potency in the normalisation of several variables, such as transaminases, homeostasis model assessment for insulin resistance index, and glucose tolerance. Both treatments significantly reduced hepatic TNF-α expression and restored GLUTs and PPARs, either in liver or adipose tissue. Finally, FBA showed a higher potency in reducing pro-inflammatory parameters in the liver, via suppression of Toll-like receptors and NF-κB activation.

Conclusions

Our results demonstrated a protective effect of butyrate in limiting molecular events underlying the onset of IR and NAFLD, suggesting a potential clinical relevance for this substance. In particular, its derivative, FBA, could represent an alternative therapeutic option to sodium butyrate, sharing a comparable efficacy, but a better palatability and compliance.     

Rapamycin-mediated CD36 translational suppression contributes to alleviation of hepatic steatosis

Rapamycin, a mammalian target of rapamycin (mTOR)-specific inhibitor, has the effect of anti-lipid deposition on non-alcoholic fatty liver disease (NAFLD), but the mechanisms with which rapamycin alleviates hepatic steatosis are not fully disclosed. CD36 is known to facilitate long-chain fatty acid uptake and contribute to NAFLD progression. Hepatic CD36 expression is closely associated with hepatic steatosis, while mTOR pathway is involved in CD36 translational control. This study was undertaken to investigate whether rapamycin alleviates hepatic steatosis via the inhibition of mTOR pathway-dependent CD36 translation. Human hepatoblastoma HepG2 cells were treated with palmitate and C57BL/6J mice were fed with high fat diet (HFD) to induce hepatic steatosis. Hepatic CD36 protein expression was significantly increased with lipid accumulation in palmitate-treated HepG2 cells or HFD-fed C57BL/6J mice. Rapamycin reduced hepatic steatosis and CD36 protein expression, but it had no influence on CD36 mRNA expression. Rapamycin had no effect on CD36 protein stability, but it significantly decreased CD36 translational efficiency. We further confirmed that rapamycin inhibited the phosphorylation of mTOR and its downstream translational regulators including p70 ribosomal protein S6 kinase (p70S6K), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor 4E (eIF4E). This study demonstrates that rapamycin inhibits hepatic CD36 translational efficiency through the mTOR pathway, resulting in reduction of CD36 protein expression and alleviation of hepatic steatosis.     

Attenuation by a Vigna nakashimae extract of nonalcoholic fatty liver disease in high-fat diet-fed mice

A Vigna nakashimae (VN) extract has been shown to have antidiabetic and anti-obesity effects. However, the mechanism underlying the effect of a VN extract on hepatic inflammation and endoplasmic reticulum (ER) stress remains unclear. In the present study, we investigated how a VN extract protects against the development of non-alcoholic fatty liver disease (NAFLD). A VN extract for 12 weeks reduced the body weight, serum metabolic parameters, cytokines, and hepatic steatosis in high-fat diet (HFD)-fed mice. A VN extract decreased HFD-induced hepatic acetyl CoA carboxylase and glucose transporter 4 expressions. In addition to the levels of high-mobility group box 1 and receptor for advanced glycation, the hepatic expression of ATF4 and caspase-3 was also reduced by a VN extract. Thus, these data indicate that a chronic VN extract prevented NAFLD through multiple mechanisms, including inflammation, ER stress, and apoptosis in the liver.     

Lipotoxicity and steatohepatitis in an overfed mouse model for non-alcoholic fatty liver disease

The major risk factors for non-alcoholic fatty liver disease (NAFLD) are obesity, insulin resistance and dyslipidemia. The cause for progression from the steatosis stage to the inflammatory condition (non-alcoholic steatohepatitis (NASH)) remains elusive at present. Aim of this study was to test whether the different stages of NAFLD as well as the associated metabolic abnormalities can be recreated in time in an overfed mouse model and study the mechanisms underlying the transition from steatosis to NASH.Male C57Bl/6J mice were subjected to continuous intragastric overfeeding with a high-fat liquid diet (HFLD) for different time periods. Mice fed a solid high-fat diet (HFD) ad libitum served as controls. Liver histology and metabolic characteristics of liver, white adipose tisue (WAT) and plasma were studied.Both HFD-fed and HFLD-overfed mice initially developed liver steatosis, but only the latter progressed in time to NASH. NASH coincided with obesity, hyperinsulinemia, loss of liver glycogen and hepatic endoplasmatic reticulum stress. Peroxisome proliferator-activated receptor γ (Pparγ), fibroblast growth factor 21 (Fgf21), fatty acid binding protein (Fabp) and fatty acid translocase (CD36) were induced exclusively in the livers of the HFLD-overfed mice. Inflammation, reduced adiponectin expression and altered expression of genes that influence adipogenic capacity were only observed in WAT of HFLD-overfed mice.In conclusion: this dietary mouse model displays the different stages and the metabolic settings often found in human NAFLD. Lipotoxicity due to compromised adipose tissue function is likely associated with the progression to NASH, but whether this is cause or consequence remains to be established.     

Liraglutide modulates gut microbiota and reduces NAFLD in obese mice

Metabolic disorders such as insulin resistance and diabetes are associated with obesity and nonalcoholic fatty liver disease (NAFLD). The aggressive form of a fatty liver disease may progress to cirrhosis and hepatocellular carcinoma. Furthermore, recent studies demonstrated that there is a dysbiosis in the gut microbiota associated with early stages of metabolic disease. Therefore, the identification and repurposing of drugs already used to treat insulin resistance may be an excellent option for other disorders. We evaluated the effect of liraglutide on obesity, NAFLD and gut microbiota modulation in two different animal models of obesity: the ob/ob mice and the high-fat diet (HFD)-fed mice. Liraglutide treatment induced significant weight loss in both obesity models, showed improvements in glycemic parameters and reduced inflammatory cell infiltration in the cecum and the liver. In ob/ob mice, the liraglutide treatment was able to reduce the accumulation of liver fat by 78% and reversed steatosis in the HFD mice. The gut microbiota analysis showed that liraglutide changed the overall composition as well as the relative abundance of weight-relevant phylotypes such as a reduction of Proteobacteria and an increase of Akkermansia muciniphila in the treated HFD group. We show that liraglutide can lead to weight loss and gut microbiota modulations, and is associated with an improvement of NAFLD. Furthermore, by generating a profile of the intestinal microbiota, we compiled a list of potential bacterial targets that may modulate metabolism and induce a metabolic profile that is considered normal or clinically controlled.     

Targeting the gut microbiota with resveratrol: a demonstration of novel evidence for the management of hepatic steatosis

Resveratrol is a natural polyphenol that has been reported to reduce the risk of obesity and nonalcoholic fatty liver disease (NAFLD). Recent evidence has demonstrated that the gut microbiota plays an important role in the protection against NAFLD and other metabolic diseases. The present study aimed to investigate the relationship between the gut microbiota and the beneficial effects of resveratrol on the amelioration of NAFLD in mice. We observed marked decreases in body weight and liver steatosis and improved insulin resistance in high-fat diet (HFD)-fed mice treated with resveratrol. Furthermore, we found that resveratrol treatment alleviated NAFLD in HFD-fed mice by improving the intestinal microenvironment, including gut barrier function and gut microbiota composition. On the one hand, resveratrol improved gut intestinal barrier integrity through the repair of intestinal mucosal morphology and increased the expression of physical barrier- and physiochemical barrier-related factors in HFD-fed mice. On the other hand, in HFD-fed mice, resveratrol supplementation modulated the gut bacterial composition. The resveratrol-induced gut microbiota was characterized by a decreased abundance of harmful bacteria, including Desulfovibrio, Lachnospiraceae_NK4A316_group and Alistipes, as well as an increased abundance of short-chain fatty acid (SCFA)-producing bacteria, such as Allobaculum, Bacteroides and Blautia. Moreover, transplantation of the HFDR-microbiota into HFD-fed mice sufficiently decreased body weight, liver steatosis and low-grade inflammation and improved hepatic lipid metabolism. Collectively, resveratrol would provide a potentially dietary intervention strategy against NAFLD through modulating the intestinal microenvironment.     

Calcium supplementation relieves high-fat diet-induced liver steatosis by reducing energy metabolism and promoting lipolysis

Nonalcoholic fatty liver disease (NAFLD) is a chronic disease affecting the health of many people worldwide. Previous studies have shown that dietary calcium supplementation may alleviate NAFLD, but the underlying mechanism is not clear. In this study investigating the effect of calcium on hepatic lipid metabolism, 8-week-old male C57BL/6J mice were divided into four groups (n = 6): (1) mice given a normal chow containing 0.5% calcium (CN0.5), (2) mice given a normal chow containing 1.2% calcium (CN1.2), (3) mice given a high-fat diet (HFD) containing 0.5% calcium (HFD0.5), and (4) mice fed a HFD containing 1.2% calcium (HFD1.2). To understand the underlying mechanism, cells were treated with oleic acid and palmitic acid to mimic the HFD conditions in vitro. The results showed that calcium alleviated the increase in triglyceride accumulation induced by oleic acid and/or palmitic acid in HepG2, AML12, and primary hepatocyte cells. Our data demonstrated that calcium supplementation alleviated HFD-induced hepatic steatosis through increased liver lipase activity, proving calcium is involved in the regulation of hepatic lipid metabolism. Moreover, calcium also increased the level of glycogen in the liver, and at the same time had the effect of reducing glycolysis and promoting glucose absorption. Calcium addition increased calcium levels in the mitochondria and cytoplasm. Taken together, we concluded that calcium supplementation could relieve HFD-induced hepatic steatosis by changing energy metabolism and lipase activity.     

Hepatic deficiency of selenoprotein S exacerbates hepatic steatosis and insulin resistance

Nonalcoholic fatty liver disease (NAFLD) is closely associated with insulin resistance (IR) and type 2 diabetes mellitus (T2DM), which are all complex metabolic disorders. Selenoprotein S (SelS) is an endoplasmic reticulum (ER) resident selenoprotein involved in regulating ER stress and has been found to participate in the occurrence and development of IR and T2DM. However, the potential role and mechanism of SelS in NAFLD remains unclear. Here, we analyzed SelS expression in the liver of high-fat diet (HFD)-fed mice and obese T2DM model (db/db) mice and generated hepatocyte-specific SelS knockout (SelS^H-KO) mice using the Cre-loxP system. We showed that hepatic SelS expression levels were significantly downregulated in HFD-fed mice and db/db mice. Hepatic SelS deficiency markedly increased ER stress markers in the liver and caused hepatic steatosis via increased fatty acid uptake and reduced fatty acid oxidation. Impaired insulin signaling was detected in the liver of SelS^H-KO mice with decreased phosphorylation levels of insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/Akt), which ultimately led to disturbed glucose homeostasis. Meanwhile, our results showed hepatic protein kinase Cɛ (PKCɛ) activation participated in the negative regulation of insulin signaling in SelS^H-KO mice. Moreover, the inhibitory effect of SelS on hepatic steatosis and IR was confirmed by SelS overexpression in primary hepatocytes in vitro. Thus, we conclude that hepatic SelS plays a key role in regulating hepatic lipid accumulation and insulin action, suggesting that SelS may be a potential intervention target for the prevention and treatment of NAFLD and T2DM.Subject terms: Metabolic syndrome, Obesity     

Niacin increases diet-induced hepatic steatosis in B6129 mice

Nonalcoholic fatty liver disease (NAFLD) is a very common disorder affecting between 20 and 30% of adults in the United States. However, there is no effective pharmacotherapy for treating NAFLD. Niacin, a water-soluble vitamin (B3), at pharmacological doses, decreases hepatic triglyceride (TG) content in NAFLD through inhibition of diacylglycerol acyltransferase 2, a key enzyme that catalyzes the final step in TG synthesis. Alternatively, some studies indicate that niacin induces fatty liver in high-fat diet (HFD)-fed rats. Therefore, in this study we investigated whether niacin is beneficial in treating NAFLD in two strains of mice, C57BL/6J (B6) and B6129SF2/J (B6129) mice, with 20 weeks of HFD feeding. Niacin treatment was started from week 5 until the end of the study. Niacin treatment increased normalized liver weight, hepatic TG content and NAFLD score in HFD-fed B6129 mice but had no impact on B6 mice. Metabolomics analysis revealed that in B6129 mice, 4-hydroxyphenylpyruvic acid (4-HPP), which is associated with fatty acid oxidation, did not change with HFD feeding but significantly decreased with niacin treatment. Lipidomics analysis discovered that the abundance of phosphocholine (PC), which is critical for very low-density lipoprotein (VLDL)–TG production and secretion, was decreased in HFD-fed B6129 with niacin treatment. In conclusion, niacin had no impact on diet-induced NAFLD development in B6 mice but potentiated hepatic steatosis in HFD-fed B6129 mice due to impaired fatty acid oxidation and decreased VLDL-TG production and secretion.     

Disruption of tumor necrosis factor alpha receptor 1 signaling accelerates NAFLD progression in mice upon a high-fat diet

Obesity is accompanied by a low-grade inflammation state, characterized by increased proinflammatory cytokines levels such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β). In this regard, there exists a lack of studies in hepatic tissue about the role of TNFα receptor 1 (TNFR1) in the context of obesity and insulin resistance during the progression of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to evaluate the effects of high-caloric feeding (HFD) (40% fat, for 16 weeks) on liver inflammation-induced apoptosis, insulin resistance, hepatic lipid accumulation and its progression toward nonalcoholic steatohepatitis (NASH) in TNFR1 knock-out and wild-type mice. Mechanisms involved in HFD-derived IL-1β release and impairment of insulin signaling are still unknown, so we determined whether IL-1β affects liver insulin sensitivity and apoptosis through TNFα receptor 1 (TNFR1)-dependent pathways. We showed that knocking out TNFR1 induces an enhanced IL-1β plasmatic release upon HFD feed. This was correlated with higher hepatic and epididymal white adipose tissue mRNA levels. In vivo and in vitro assays confirmed an impairment in hepatic insulin signaling, in part due to IL-1β-induced decrease of AKT activation and diminution of IRS1 levels, followed by an increase in inflammation, macrophage (resident and recruited) accumulation, hepatocyte apoptotic process and finally hepatic damage. In addition, TNFR1 KO mice displayed higher levels of pro-fibrogenic markers. TNFR1 signaling disruption upon an HFD leads to an accelerated progression from simple steatosis to a more severe phenotype with many NASH features, pointing out a key role of TNFR1 in NAFLD progression.     

Intermittent hypoxia upregulates hepatic heme oxygenase-1 and ferritin-1, thereby limiting hepatic pathogenesis in rats fed a high-fat diet

Non-alcoholic fatty liver disease (NAFLD) is prevalent in patients with sleep apnea syndrome (SAS). Intermittent hypoxia (IH) and a high-fat diet (HFD) reproduce SAS and NAFLD, respectively, in rodents. In this study, rats were fed either an HFD or a standard diet (SD) for 2 weeks, and breathed either IH air or normoxic air for 4 days (early phase) or 6 weeks (late phase), with the same diets maintained during the exposure. HFD increased hepatic lipid accumulation, as detected by oil-red staining and triglyceride content. However, IH exposure reversed the hepatic steatosis at the late phase in these HFD-rats. IH exposure also increased hepatic expression of HO-1 and iron-binding protein ferritin-1 at the late phase, in association with increase in serum iron, bilirubin, and hepatic levels of lipid peroxides, such as 4-hydroxy-2-nonenal (HNE). IH exposure increased serum levels of hemoglobin (Hb) at the early phase and immunofluorescence of Hb and HO-1 in CD68-positive Kupffer cells (KCs) at the late phase. These findings support that IH induces erythrocytosis, erythro-phagocytosis, and generation of Hb in the KCs. The Hb promotes HO-1 expression in KCs, thereby produces iron, bilirubin, and carbon monoxide (CO). The iron would be either sequestrated by ferritin-1, transferred to the bone marrow for erythropoiesis, or would produce hydroxyradicals and HNE in the liver of rats fed an HFD. HNE might also contribute to the upregulation of HO-1, transferrin-1, and IκB, thereby limiting hepatic steatosis and inflammation via inhibition of nuclear factor κB (NFκB) activation.