Obesity-induced Non-alcoholic Fatty Liver Disease Research Articles

The antianginal ranolazine fails to improve glycaemia in obese liver-specific pyruvate dehydrogenase deficient male mice.

Recent studies have demonstrated that stimulating pyruvate dehydrogenase (PDH, gene Pdha1), the rate-limiting enzyme of glucose oxidation, can reverse obesity-induced non-alcoholic fatty liver disease (NAFLD), which can be achieved via treatment with the antianginal ranolazine. Accordingly, our aim was to determine whether ranolazine's ability to mitigate obesity-induced NAFLD and hyperglycaemia requires increases in hepatic PDH activity. We generated liver-specific PDH-deficient (Pdha1Liver-/- ) mice, which were provided a high-fat diet for 12 weeks to induce obesity. Pdha1Liver-/- mice and their albumin-Cre (AlbCre ) littermates were randomized to treatment with either vehicle control or ranolazine (50 mg/kg) once daily via oral gavage during the final 5weeks, following which we assessed glucose and pyruvate tolerance. Pdha1Liver-/- mice exhibited no overt phenotypic differences (e.g. adiposity, glucose tolerance) when compared to their AlbCre littermates. Of interest, ranolazine treatment improved glucose tolerance and mildly reduced hepatic triacylglycerol content in obese AlbCre mice but not in obese Pdha1Liver-/- mice. The latter was independent of changes in hepatic mRNA expression of genes involved in regulating lipogenesis. Liver-specific PDH deficiency is insufficient to promote an NAFLD phenotype. Nonetheless, hepatic PDH activity partially contributes to how the antianginal ranolazine improves glucose tolerance and alleviates hepatic steatosis in obesity.

Hepatocyte Kctd17 Inhibition Ameliorates Glucose Intolerance and Hepatic Steatosis Caused by Obesity-induced Chrebp Stabilization

Background & AimsObesity predisposes to type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD), but underlying mechanisms are incompletely understood. Potassium channel tetramerization domain-containing protein 17 (Kctd17) levels are increased in livers from obese mice and humans. In this study, we investigated the mechanism of increased Kctd17 and whether it is causal to obesity-induced metabolic complications. MethodsWe transduced Rosa26-LSL-Cas9 knockin mice with AAV8-TBG-Cre (Control), AAV8-U6-Kctd17 sgRNA-TBG-Cre (L-Kctd17), AAV8-U6-Oga sgRNA-TBG-Cre (L-Oga), or AAV8-U6-Kctd17/Oga sgRNA-TBG-Cre (DKO). We fed mice a high-fat diet (HFD) and assessed for hepatic glucose and lipid homeostasis. We generated Kctd17, O-GlcNAcase (Oga), or Kctd17/Oga-knockout hepatoma cells by CRISPR-Cas9, and Kctd17-directed antisense oligonucleotide to test therapeutic potential in vivo. We analyzed transcriptomic data from patients with NAFLD. ResultsHepatocyte Kctd17 expression was increased in HFD-fed mice due to increased Srebp1c activity. HFD-fed L-Kctd17 or Kctd17 antisense oligonucleotide–treated mice show improved glucose tolerance and hepatic steatosis, whereas forced Kctd17 expression caused glucose intolerance and hepatic steatosis even in lean mice. Kctd17 induced Oga degradation, resulting in increasing carbohydrate response element-binding protein (Chrebp) protein, so concomitant Oga knockout negated metabolic benefits of hepatocyte Kctd17 deletion. In patients with NAFLD, KCTD17 messenger RNA was positively correlated with expression of Chrebp target and other lipogenic genes. ConclusionsSrebp1c-induced hepatocyte Kctd17 expression in obesity disrupted glucose and lipid metabolism by stabilizing Chrebp, and may represent a novel therapeutic target for obesity-induced T2D and NAFLD.

An improved method for isolation and flow cytometric characterization of intrahepatic leukocytes from fatty and fibrotic liver tissues.

Flow cytometry is an imperative tool to characterize alterations in a wide range of immune cell populations during inflammatory conditions and disease states that affect the liver such as the obesity-induced non-alcoholic fatty liver disease and liver fibrosis. Identification and quantification of immune cell subsets from the liver is critically dependent on efficient isolation of intrahepatic leukocytes. The isolation of leukocytes from fatty and fibrotic livers and processing the cells for flow cytometry can be challenging with respect to cell yields, purity and most importantly, the level of autofluorescence resulting from fat deposition. Here, we describe an efficient method for isolating intrahepatic leukocytes from mice fed with high fat diet and propose a strategy to alleviate autofluorescence during phenotyping by multicolor flowcytometry. We also describe a gating strategy for robust identification of granulocytes, pro-inflammatory, anti-inflammatory and transitional state monocyte subsets, dendritic cells, B cell, T lymphocyte subpopulations and NK cell subsets. Overall, the procedures described here will allow simultaneous processing of several samples while ensuring reproducible cell isolation and efficient noise reduction required for reliable characterization of intrahepatic leukocytes from the fatty liver tissues.

Loss of TRIM67 Attenuates the Progress of Obesity-Induced Non-Alcoholic Fatty Liver Disease.

Obesity is considered as a major cause for the development and progress of non-alcoholic fatty liver disease (NAFLD), which is one of the most prevalent chronic liver diseases worldwide. However, molecular mechanisms that implicate in obesity-driven pathophysiology of NAFLD are not well defined. Here, we report a tripartite motif (TRIM) protein family member—TRIM67—that is hardly expressed in liver but is inducible on obese conditions. Enhanced expression of TRIM67 activates hepatic inflammation to disturb lipid metabolic homeostasis and promote the progress of NAFLD induced by obesity, while the deficiency in TRIM67 is protective against these pathophysiological processes. Finally, we show that the important transcription coactivator PGC-1α implicates in the response of hepatic TRIM67 to obesity.

233-LB: Ranolazine Fails to Improve Glycemia in Obese Liver-Specific Pyruvate Dehydrogenase–Deficient Mice

Background: Nonalcoholic fatty liver disease (NAFLD) increases risk for obesity-related insulin resistance and type 2 diabetes. Recent studies have demonstrated that stimulating pyruvate dehydrogenase (PDH) , the rate-limiting enzyme in glucose oxidation, can reverse obesity-induced NAFLD. Moreover, our lab has observed that treatment with the antianginal drug, ranolazine, augments hepatic PDH activity, leading to a reversal of obesity-induced glucose intolerance and NAFLD in mice. Our aim herein was to determine whether ranolazine's ability to mitigate obesity-induced NAFLD and hyperglycemia requires increases in hepatic PDH activity. Methods: We generated liver-specific PDH deficient (PDHLiver-/-) mice by crossing floxed Pdha1 mice with albumin-Cre (Alb-Cre) mice. PDHLiver-/- and Alb-Cre mice were placed on a high-fat diet for 12-weeks to induce obesity, whereas their lean controls were fed a low-fat diet. Mice were randomized to treatment with either vehicle control or ranolazine (50 mg/kg) once daily via oral gavage for 5-weeks, following which we assessed glucose homeostasis and hepatic steatosis. Results: Lean PDHLiver-/- mice exhibited no overt phenotypic differences (e.g. adiposity, glucose tolerance) from their Alb-Cre littermates, though a mild but nonsignificant worsening of pyruvate tolerance was observed. Similar findings were observed in obese PDHLiver-/- mice compared to their Alb-Cre littermates. Of interest, ranolazine treatment produced mild improvements in glucose tolerance in obese Alb-Cre mice, but not in obese PDHLiver-/- mice. However, this improvement in ranolazine treated Alb-Cre mice appears to be independent of decreases in hepatic triacylglycerol content. Conclusions: Although hepatic PDH activity appears to contribute to the glucose-lowering actions of ranolazine, this appears to be dissociated from its actions on hepatic steatosis, and impaired hepatic PDH activity per se is not sufficient to produce an NAFLD phenotype. Disclosure C. T. Saed: None. S. Tabatabaei dakhili: None. A. A. Greenwell: None. J. S. F. Chan: None. K. Yang: None. K. Gopal: None. F. Eaton: None. J. R. Ussher: None.

The effects of brewers’ spent grain on high-fat diet-induced fatty liver

Obesity drives nonalcoholic fatty liver disease (NAFLD). This study investigated the effects of dietary brewers’ spent grain (BSG) supplementation on obesity-induced NAFLD. Mice fed a high-fat diet supplemented with 30% BSG (HFD30) had reduced body weight and decreased plasma total cholesterol (TC) concentrations compared with HFD-fed mice. Retroperitoneal white adipose tissue (RWAT) and liver weights were reduced. Consistent with reduced hepatic triacylglycerol, TC, and non-esterified fatty acid concentrations, HFD30-fed mice showed reduced hepatic steatosis. 3-hydroxy-3-methylglutaryl-CoA reductase and low-density lipoprotein receptor genes were increased, whereas carnitine palmitoyltransferase 1 alpha, ATP-binding cassette subfamily A member 1 (Abca1), and cholesterol 7 alpha-hydroxylase genes were upregulated in the liver of HFD30-fed mice. Abca1 gene expression was also increased in epididymal WAT and RWAT of HFD30-fed mice. BSG supplementation increased and decreased fecal fat and bile acid concentrations, respectively. Taken together, BSG supplementation reduced HFD-induced hepatic lipid accumulation by increasing fatty acid oxidation and bile acid synthesis in the liver as well as decreasing lipid absorption in the intestine.

Serum amyloid A1 exacerbates hepatic steatosis via TLR4-mediated NF-κB signaling pathway

ObjectiveChronic inflammatory response plays a prominent role in obesity-related nonalcoholic fatty liver disease (NAFLD). However, the intrahepatic triggering mechanism of inflammation remains obscure. This study aimed to elucidate the role of serum amyloid A1 (SAA1), an acute-phase response protein, in the obesity-induced hepatic inflammation and NAFLD. MethodsMale mice were fed a high fat diet (HFD) for 16 weeks, and insulin resistance, hepatic steatosis, and inflammation in mice were monitored. Murine SAA1/2 was genetically manipulated to investigate the role of SAA1 in NAFLD. ResultsWe found that SAA1 was increased in the NAFLD liver in both humans and mice. Knockout of SAA1/2 or knockdown of hepatic SAA1/2 promoted energy expenditure and alleviated HFD-induced metabolic disorder, hepatic steatosis, and inflammation. Endogenous overexpression of SAA1 in hepatocytes by adeno-associated virus 8 (AAV8) transfection aggravated overnutrition-associated gain of body weight, insulin resistance, hepatic lipid accumulation, and liver injury, which were markedly alleviated by knockout of murine toll-like receptor 4 (TLR4). Mechanistically, SAA1 directly bound with TLR4/myeloid differentiation 2 (MD2) to induce TLR4 internalization, leading to the activation of nuclear factor (NF)-κB signaling and production of both SAA1 and other inflammatory cytokines, including interleukin (IL)-6 and C–C chemokine ligand (CCL2) in hepatocytes. Administration of HFD mice with an AAV8-shRNA-SAA1/2 showed a therapeutic effect on hepatic inflammation and NAFLD progression. ConclusionsThese results demonstrate that SAA1 triggers hepatic steatosis and intrahepatic inflammatory response by forming a SAA1/TLR4/NF-κB/SAA1 feedforward regulatory circuit, which, in turn, leads to NAFLD progression. SAA1 may act as a potential target for the disease intervention.

Peony seed oil protects against obesity-induced non-alcoholic fatty liver disease via modulation of endoplasmic reticulum stress


 
 
 
 Purpose: To investigate the effect of Peony seed oil (PO) on non-alcoholic fatty liver disease (NAFLD), and the underlying mechanism of action.
 Methods: A mouse model of NAFLD was established using high-fat diet (HFD) for 12 weeks. Serum levels of triacylglycerol (TG), total cholesterol (TC), glucose and free fatty acids (FFAs) were determined. Activities of aspartate aminotransferase (AST) and alanine amino transferase (ALT) were assayed in liver homogenates, while mRNA and protein expressions of GRP78, x-box binding protein 1 (XBP1) and phosphorylated inositol-requiring enzyme 1 (p-IRE1α) were assayed in liver tissue using real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were also determined by qRT-PCR.
 Results: HFD diet led to significant increases in body weight as well as serum TG, TC, glucose and FFA levels. However, treatment with PO significantly reversed the effect of HFD on these parameters (p < 0.05). The mRNA expression levels of TNF- α, IL-6 and IL-1β were significantly higher in HFD control group than in normal control group, but were significantly reduced after PO treatment (p < 0.05). The protein expression levels of GRP78, XBP-1 and p-IRE1α significantly increased in the liver of NAFLD group, when compared with normal control rats, but were significantly downregulated by treatment with PO (p < 0.05).
 Conclusion: PO attenuates HFD-induced NAFLD via inactivation of endoplasmic reticulum stress (ERS) pathway. Thus, PO has potentials for the treatment of NAFLD but further studies are required to ascertain this.
 
 
 

Corn peptides improved obesity-induced non-alcoholic fatty liver disease through relieving lipid metabolism, insulin resistance and oxidative stress.

Non-alcoholic fatty liver disease (NAFLD) is increasingly threatening human health. The remarkable effects of corn peptides (CPs) as bioactive peptides on liver protection have attracted much attention. Nevertheless, the specific effect of CPs on NAFLD remains unclear. The present study was designed to investigate the efficacy of CPs in the prevention and auxiliary treatment of high-fat diet (HFD)-induced NAFLD in SD rats, and puerarin was used as the positive control. SD rats were fed a high-fat diet to establish the NAFLD rat model, and LO2 cells were treated with a high concentration of fructose to simulate the NAFLD cell model. NAFLD was comprehensively examined in terms of body weight, liver function markers, serum biochemistry and liver histology. Protein expression was determined using western blot analysis. The results of animal experiments showed that CPs could effectively inhibit the rate of weight gain, reduce the blood lipid level and liver index, and enhance glucose tolerance. The results of cell experiments showed that CPs could effectively reduce the accumulation of lipids in LO2 cells and inhibit the accumulation of reactive oxygen species (ROS). In addition, CPs could markedly reduce liver lipid accumulation in the liver cell and liver tissue, as further evidenced by the reduced expression of SREBP-1c in human non-tumour hepatic (LO2) cells. Meanwhile, the increased expression of SIRT1/PPAR-α and Nrf2/HO-1 pathways under the pretreatment of CPs in LO2 cells indicated that CPs could markedly relieve high fat-induced fatty liver injury, regulate insulin sensitivity, and reduce production of ROS. The results of in vivo and in vitro experiments demonstrated that CPs provided potential prevention and auxiliary treatment for NAFLD through reducing lipid accumulation, alleviating insulin resistance, and inhibiting oxidative stress. This study investigated the biological activity of CPs and laid the theoretical basis for the development of CP-based functional foods and dietary supplements.

Hepatic MIR20B promotes nonalcoholic fatty liver disease by suppressing PPARA.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation and imbalances in lipid metabolism in the liver. Although nuclear receptors (NRs) play a crucial role in hepatic lipid metabolism, the underlying mechanisms of NR regulation in NAFLD remain largely unclear. Using network analysis and RNA-seq to determine the correlation between NRs and microRNA in human NAFLD patients, we revealed that MIR20B specifically targets PPARA. MIR20B mimic and anti-MIR20B were administered to human HepG2 and Huh-7 cells and mouse primary hepatocytes as well as high-fat diet (HFD)- or methionine-deficient diet (MCD)-fed mice to verify the specific function of MIR20B in NAFLD. We tested the inhibition of the therapeutic effect of a PPARα agonist, fenofibrate, by Mir20b and the synergic effect of combination of fenofibrate with anti-Mir20b in NAFLD mouse model. We revealed that MIR20B specifically targets PPARA through miRNA regulatory network analysis of nuclear receptor genes in NAFLD. The expression of MIR20B was upregulated in free fatty acid (FA)-treated hepatocytes and the livers of both obesity-induced mice and NAFLD patients. Overexpression of MIR20B significantly increased hepatic lipid accumulation and triglyceride levels. Furthermore, MIR20B significantly reduced FA oxidation and mitochondrial biogenesis by targeting PPARA. In Mir20b-introduced mice, the effect of fenofibrate to ameliorate hepatic steatosis was significantly suppressed. Finally, inhibition of Mir20b significantly increased FA oxidation and uptake, resulting in improved insulin sensitivity and a decrease in NAFLD progression. Moreover, combination of fenofibrate and anti-Mir20b exhibited the synergic effect on improvement of NAFLD in MCD-fed mice. Taken together, our results demonstrate that the novel MIR20B targets PPARA, plays a significant role in hepatic lipid metabolism, and present an opportunity for the development of novel therapeutics for NAFLD. This research was funded by Korea Mouse Phenotyping Project (2016M3A9D5A01952411), the National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1F1A1061267, 2018R1A5A1024340, NRF-2021R1I1A2041463, 2020R1I1A1A01074940, 2016M3C9A394589324), and the Future-leading Project Research Fund (1.210034.01) of UNIST.

The anti-anginal ranolazine does not confer beneficial actions against hepatic steatosis in male mice subjected to high-fat diet and streptozotocin-induced type 2 diabetes.

Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of excess fat in the liver in the absence of alcohol and increases one's risk for both diabetes and cardiovascular disease (e.g., angina). We have shown that the second-line anti-anginal therapy, ranolazine, mitigates obesity-induced NAFLD, and our aim was to determine whether these actions of ranolazine also extend to NAFLD associated with type 2 diabetes (T2D). Eight-week-old male C57BL/6J mice were fed either a low-fat diet or a high-fat diet for 15 weeks, with a single dose of streptozotocin (STZ; 75 mg/kg) administered in the high-fat diet-fed mice at 4 weeks to induce experimental T2D. Mice were treated with either vehicle control or ranolazine during the final 7 weeks (50 mg/kg once daily). We assessed glycemia via monitoring glucose tolerance, insulin tolerance, and pyruvate tolerance, whereas hepatic steatosis was assessed via quantifying triacylglycerol content. We observed that ranolazine did not improve glycemia in mice with experimental T2D, while also having no impact on hepatic triacylglycerol content. Therefore, the salutary actions of ranolazine against NAFLD may be limited to obese individuals but not those who are obese with T2D.

Functional multispectral optoacoustic tomography imaging of hepatic steatosis development in mice

The increasing worldwide prevalence of obesity, fatty liver diseases and the emerging understanding of the important roles lipids play in various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. We investigated the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non‐invasively visualize lipids in laboratory mice around the 930nm spectral range. Using an obesity‐induced non‐alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label‐free mode. Moreover, we demonstrate the efficacy of using the real‐time clearance kinetics of indocyanine green (ICG) in the liver, monitored by MSOT, as a biomarker to evaluate the organ’s function and assess the severity of NAFLD. This study establishes MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD.

Ziziphus spina-christi (L.) fortified with Camellia sinensis mediates apoptosis, Notch-1 signaling, and mitigates obesity-induced non-alcoholic fatty liver.

The habit of drinking tea is highly prevalent and could be utilized to introduce more health benefits through fortification with medicinal plants. The purpose of this analysis was to assess the nutritional quality and health benefits of fortified Ziziphus tea (ZT) with green tea (GT) against obesity and non-alcoholic fatty liver disease (NAFLD). Proximate analysis and sensory evaluation were carried out on the fortified tea. In the in vivo study, 15 SD rats were used for each group. Flow cytometry was utilized for caspase 3 analysis. ELISA was used for the detection of tumor necrosis factor-alpha and adiponectin levels. Real-time PCR was used to detect Notch-1 and Hes-1 gene expression. The composition of fortified (GT+ZT) showed a significant improvement in the nutritional value represented by the increase in overall protein, crude fat, crude fiber, ash, carbohydrate, mineral contents, and antioxidant capacity. Treatment with GT+ZT restored the disturbance in body weight, lipid profile, liver function, glucose, insulin sensitivity index, and oxidative status. It reversed the changes in TNF-α and adiponectin levels. Their protective effects against NAFLD were indicated by the inhibition of hepatic caspase-3 activity, suppression of Notch-1, and Hes-1 gene expression and amelioration of high-fat diet (HFD)-induced histological alterations. Collectively, our findings, elucidate the precise mechanism of fortified ZT+GT for the attenuation of obesity-induced metabolic disorders and NAFLD via regulating lipolysis, TNF-α, adiponectin, apoptosis, and Notch-1 signaling pathways, and provide a foundation for an easily implemented healthy habit of drinking. PRACTICAL APPLICATIONS: The incorporation of bioactive compounds into functional foods is a growing market. Consumer attention in well-being has increased rapidly toward a fortified diet that provides additional health effects. The fortified (GT+ZT) tea may potentially serve as an easily implemented healthy drinking habit to prevent and manage obesity and NAFLD and reduce the risk of other diseases. Fortification with ZT improved the health-promoting functionality of GT through the enhancement of total protein, carbohydrates, antioxidant, and mineral contents. This was reflected by their synergetic therapeutic activity in ameliorating the disturbance in obesity-related disorders and NAFLD via regulating lipolysis, inflammation, oxidative stress, apoptosis, and Notch-1 signaling pathways. Therefore, (GT+ZT) could be considered functional foods which attribute to functional improvement and reduction in disease risk.

Pyruvate Dehydrogenase as a Therapeutic Target for Nonalcoholic Fatty Liver Disease.

Excess caloric intake combined with a sedentary lifestyle in the general population has greatly increased the prevalence of obesity and nonalcoholic fatty liver disease (NAFLD), which is defined as the accumulation of excess fat in the liver in the absence of alcohol abuse or other attributable causes such as infection with hepatitis C. Furthermore, NAFLD increases the risk for insulin resistance, type 2 diabetes (T2D), and cardiovascular disease, while currently having no approved therapy to counteract its pathology. Thus, increasing efforts to understand the mechanisms responsible for NAFLD have been pursued in preclinical studies, in the hopes of developing novel therapies that can prevent the progression of insulin resistance and/or T2D. The pathology of NAFLD is multifactorial, with proposed mechanisms including inflammation, oxidative stress, and mitochondrial dysfunction to name a few. The latter remains a subject of ongoing debate, but may be attributed to impaired hepatic fatty acid oxidation, thereby increasing the accumulation of triacylglycerol within hepatocytes. More recent studies have also demonstrated that the mitochondrial dysfunction in NAFLD may also encompass impairments in glucose oxidation, despite oxidative energy production having minimal contribution to overall glucose/pyruvate metabolism in the liver. Accordingly, strategies to reverse this defect in glucose oxidation can ameliorate hepatic steatosis and improve glucose homeostasis. We will review herein the evidence supporting impaired hepatic glucose oxidation as a mechanism of NAFLD, while discussing the validity of pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation, as a potential target for NAFLD. In addition, we will discuss potential mechanisms of action by which increased hepatic PDH activity and subsequent glucose oxidation can reverse the pathology of obesity-induced NAFLD, as well as opportunities to target this pathway with clinical agents.

Tomatidine ameliorates obesity-induced nonalcoholic fatty liver disease in mice

Tomatidine is isolated from the leaves and green fruits of some plants in the Solanaceae family, and has been reported to have anti-inflammatory and antitumor effects. Previous studies have found that tomatidine decreases hepatic lipid accumulation via regulation of vitamin D receptor and activation of AMP-activated protein kinase (AMPK) phosphorylation. However, whether tomatidine reduces weight gain and improves nonalcoholic fatty liver disease (NAFLD) remains unclear. In this study, we investigated how tomatidine ameliorates NAFLD in obese mice and evaluated the regulatory mechanism of lipogenesis in hepatocytes. Male C57BL/6 mice were fed a high-fat diet (HFD) to induce obesity and NAFLD, and treated with tomatidine via intraperitoneal injection. In vitro, FL83B hepatocytes were incubated with oleic acid and treated with tomatidine to evaluate lipid metabolism. Our results demonstrate that tomatidine significantly decreases body weight and fat weight compared to HFD-fed mice. In addition, tomatidine decreased hepatic lipid accumulation and improved hepatocyte steatosis in HFD-induced obese mice. We also found that tomatidine significantly regulated serum total cholesterol, fasting blood glucose, low-density lipoprotein, and triglyceride levels, but the serum high-density lipoprotein and adiponectin concentrations were higher than in the HFD-fed obese mice. In vivo and in vitro, tomatidine significantly suppressed the expression of fatty acid synthase and transcription factors involved in lipogenesis, and increased the expression of adipose triglyceride lipase. Tomatidine promoted the sirtuin 1 (sirt1)/AMPK signaling pathway to increase lipolysis and β-oxidation in fatty liver cells. These findings suggest that tomatidine potentially ameliorates obesity and acts against hepatic steatosis by regulating lipogenesis and the sirt1/AMPK pathway.

Phosphorylation of eIF2\u03b1 signaling pathway attenuates obesity-induced non-alcoholic fatty liver disease in an ER stress and autophagy-dependent manner

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder and frequently exacerbates in postmenopausal women. In NAFLD, the endoplasmic reticulum (ER) plays an important role in lipid metabolism, in which salubrinal is a selective inhibitor of eIF2α de-phosphorylation in response to ER stress. To determine the potential mechanism of obesity-induced NAFLD, we employed salubrinal and evaluated the effect of ER stress and autophagy on lipid metabolism. Ninety-five female C57BL/6 mice were randomly divided into five groups: standard chow diet, high-fat (HF) diet, HF with salubrinal, HF with ovariectomy, and HF with ovariectomy and salubrinal. All mice except for SC were given HF diet. After the 8-week obesity induction, salubrinal was subcutaneously injected for the next 8 weeks. The expression of ER stress and autophagy markers was evaluated in vivo and in vitro. Compared to the normal mice, the serum lipid level and adipose tissue were increased in obese mice, while salubrinal attenuated obesity by blocking lipid disorder. Also, the histological severity of hepatic steatosis and fibrosis in the liver and lipidosis was suppressed in response to salubrinal. Furthermore, salubrinal inhibited ER stress by increasing the expression of p-eIF2α and ATF4 with a decrease in the level of CHOP. It promoted autophagy by increasing LC3II/I and inhibiting p62. Correlation analysis indicated that lipogenesis in the development of NAFLD was associated with ER stress. Collectively, we demonstrated that eIF2α played a key role in obesity-induced NAFLD, and salubrinal alleviated hepatic steatosis and lipid metabolism by altering ER stress and autophagy through eIF2α signaling.

Saturated Fatty Acids Promote Hepatocytic Senecence through Regulation of miR-34a/Cyclin-Dependent Kinase 6.

Obesity increases intracellular lipid accumulation in hepatocytes, which can induce non-alcoholic fatty liver disease (NAFLD). With progression of NAFLD, a sizable fraction of patients develop non-alcoholic steatohepatitis (NASH), eventually leading to cirrhosis and hepatocellular carcinoma (HCC). The mechanism involved in obesity-induced NAFLD remains unclear. Free fatty acids and high-fat diets, which induce hepatocyte senescence, are major risk factors for NAFLD. Therefore in this study, the mechanism of lipotoxicity-induced hepatocyte senescence is investigated. The mice are fed a high-fat diet (HFD) and BNL CL.2 cells are treated with palmitate acid (PA) to establish in vivo and in vitro models of lipotoxicity, respectively. SA-β-gal staining is used to analyze the positively stained senescent hepatocytes. The results show that both PA and HFD induce cellular senescence. Real-time-PCR quantitative analysis reveals that miR-34a is significantly upregulated in the liver tissues of the HFD mice and in the PA-treated BNL CL.2 cells. Western blotting analysis shows that cyclin-dependent kinase inhibitor 1 (CDKN1, also known as p21) is upregulated, while cyclin-dependent kinase 6 (CDK6) is downregulated. Further investigation of the mechanism reveals that CDK6 is a target of miR-34a, which binds to the 3' UTR of CDK6 and inhibits its expression. The findings reveal that miR-34a is upregulated in a high-fat environment in the liver, and induces hepatocyte senescence by targeting CDK6. The miR-34a-CDK6 signaling axis may promote NAFLD development in a high-fat environment and therefore represents a potential target for NAFLD therapy.

Western Diet Causes Obesity-Induced Nonalcoholic Fatty Liver Disease Development by Differentially Compromising the Autophagic Response

Nonalcoholic fatty liver disease (NAFLD) is characterized by the development of steatosis, which can ultimately compromise liver function. Mitochondria are key players in obesity-induced metabolic disorders; however, the distinct role of hypercaloric diet constituents in hepatic cellular oxidative stress and metabolism is unknown. Male mice were fed either a high-fat (HF) diet, a high-sucrose (HS) diet or a combined HF plus HS (HFHS) diet for 16 weeks. This study shows that hypercaloric diets caused steatosis; however, the HFHS diet induced severe fibrotic phenotype. At the mitochondrial level, lipidomic analysis showed an increased cardiolipin content for all tested diets. Despite this, no alterations were found in the coupling efficiency of oxidative phosphorylation and neither in mitochondrial fatty acid oxidation (FAO). Consistent with unchanged mitochondrial function, no alterations in mitochondrial-induced reactive oxygen species (ROS) and antioxidant capacity were found. In contrast, the HF and HS diets caused lipid peroxidation and provoked altered antioxidant enzyme levels/activities in liver tissue. Our work provides evidence that hepatic oxidative damage may be caused by augmented levels of peroxisomes and consequently higher peroxisomal FAO-induced ROS in the early NAFLD stage. Hepatic damage is also associated with autophagic flux impairment, which was demonstrated to be diet-type dependent. The HS diet induced a reduction in autophagosomal formation, while the HF diet reduced levels of cathepsins. The accumulation of damaged organelles could instigate hepatocyte injuries and NAFLD progression.

High-altitude chronic hypoxia ameliorates obesity-induced non-alcoholic fatty liver disease in mice by regulating mitochondrial and AMPK signaling

AimsHigh-fat intake induces obesity and non-alcoholic fatty liver disease (NAFLD). However, high-altitude chronic hypoxia might alleviate NAFLD progression through improved mitochondrial function and AMP-activated protein kinase (AMPK) signaling. We hypothesized that high-altitude chronic hypoxia would have protective effects against NAFLD development. Main methodsC57BL/6J mice were randomly divided into control (normal diet and altitude 50 m), CHH (normal diet and altitude 4300 m), HFD (high-fat diet and altitude 50 m), and HFD-CHH (high-fat diet and altitude 4300 m) groups. After being maintained for 8 weeks under the appropriate conditions, mice were evaluated. Key findingsThe degree of liver lipid accumulation and expression of the lipid synthesis-related genes acetyl-CoA carboxylase1 (ACC1), fatty acid synthesis (FAS), and sterol regulatory element binding protein-1c (SREBP-1c) were reduced in the HFD-CHH group; however, expression of the lipolysis-related gene carnitine palmitoyl transferase 1 (CPT1) was increased. Furthermore, in addition to increased expression of mitochondrial biogenesis-related genes, mitochondrial respiratory function and mitochondrial DNA content were elevated in the HFD-CHH group compared to those in the HFD group. The HFD-CHH group also exhibited significantly increased antioxidation activity and decreased reactive oxygen species production (P < 0.05). Finally, AMPK signaling in the liver was activated and the expression of phosphorylated-AMPK (P-AMPK) was significantly increased in the HFD-CHH group. SignificanceCollectively, our findings suggest that high altitude-induced hypoxia might improve impaired mitochondrial function and activate AMPK signaling in obesity-induced NAFLD. High-altitude chronic hypoxia could be a new treatment strategy for obesity-induced NAFLD.

Mice Deficient in the IL-1\u03b2 Activation Genes Prtn3, Elane, and Casp1 Are Protected Against the Development of Obesity-Induced NAFLD

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. Inflammatory pathways contribute to disease pathogenesis; however, regulation of the underlying mechanism is not completely understood. IL-1β, a pro-inflammatory cytokine, participates in the development and progression of NAFLD. To become bioactive, IL-1β requires enzymatic processing. Mechanisms that activate IL-1β include the classical NLRP3 inflammasome-caspase-1 and the neutrophil serine proteases, neutrophil elastase, and proteinase-3. Several studies have shown that both caspase-1 and the neutrophil serine proteases are important for NAFLD development. However, it is unknown whether these pathways interact and if they have a synergistic effect in promoting NAFLD. In the present study, we developed a novel and unique mouse model by intercrossing caspase-1/11 knockout mice with neutrophil elastase/proteinase-3 double knockout mice. Subsequently, these mice were examined regarding the development of high-fat diet–induced NAFLD. Our results show that mice deficient in caspase-1, neutrophil elastase, and proteinase-3 were protected from developing diet-induced weigh gain, liver steatosis, and adipose tissue inflammation when compared with controls. We conclude that pathways that process pro-IL-1β to bioactive IL-1β play an important role in promoting the development of NAFLD and obesity-induced inflammation. Targeting these pathways could have a therapeutic potential in patients with NAFLD.