A review of the role of ethanol-induced adipose tissue dysfunction in alcohol-associated liver disease.

Alcohol-associated liver disease (AALD) encompasses a spectrum of liver diseases that includes simple steatosis, steatohepatitis, fibrosis, and cirrhosis. The adverse effects of alcohol in liver and the mechanisms by which ethanol (EtOH) promotes liver injury are well studied. Although liver is known to be the primary organ affected by EtOH exposure, alcohol's effects on other organs are also known to contribute significantly to the development of liver injury. It is becoming increasingly evident that adipose tissue (AT) is an important site of EtOH action. Both AT storage and secretory functions are altered by EtOH. For example, AT lipolysis, stimulated by EtOH, contributes to chronic alcohol-induced hepatic steatosis. Adipocytes secrete a wide variety of biologically active molecules known as adipokines. EtOH alters the secretion of these adipokines from AT, which include cytokines and chemokines that exert paracrine effects in liver. In addition, the level of EtOH-metabolizing enzymes, in particular, CYP2E1, rises in the AT of EtOH-fed mice, which promotes oxidative stress and/or inflammation in AT. Thus, AT dysfunction characterized by increased AT lipolysis and free fatty acid mobilization and altered secretion of adipokines can contribute to the severity of AALD. Of note, moderate EtOH exposure results in AT browning and activation of brown adipose tissue which, in turn, can promote thermogenesis. In this review article, we discuss the direct effects of EtOH consumption in AT and the mechanisms by which EtOH impacts the functions of AT, which, in turn, increases the severity of AALD in animal models and humans.

Blockade of thromboxane A2 signaling attenuates ethanol-induced myocardial inflammatory response in mice.

BACKGROUND: Alcohol-associated cardiomyopathy (ACM) is a cardiac muscle disease characterized by inflammation and oxidative stress. Thromboxane-prostanoid receptor (TP-R) plays an important role in the pathogenesis of cardiovascular disease. Herein, we hypothesize that TP-R mediates alcohol-induced early cardiac injury. METHODS: Eight-week-old male C57BL/6 wild-type mice were fed a chronic ethanol (ET) or control diet (CON) for 10 days followed by a single binge of ethanol or maltose-dextrin through oral gavage. A cohort of ethanol-fed mice received SQ 29,548 (SQ), a TP-R antagonist. RNA sequencing, real-time PCR, and western blot analysis were performed on left ventricle to investigate alterations in genes and/or proteins mediating oxidative stress, inflammation, and cardiac remodeling. Sirius Red staining was performed to measure myocardial fibrosis. RESULTS: RNA-sequencing analysis of myocardium from CON and ET groups identified 142 genes that were significantly altered between the two groups. In particular, the gene expression of thioredoxin-interacting protein (TXNIP), a component of NLR family pyrin domain containing 3 (NLRP3) signaling, which mediates oxidative stress and inflammatory response, was upregulated in response to ethanol exposure. The myocardial protein levels of TP-R and thromboxane A2 synthase were increased upon alcohol exposure. Ethanol increased the levels of 4-hydroxynonenal, a marker of oxidative stress, with a concomitant increase in the protein levels of TXNIP and NLRP3, and administration of SQ attenuated these effects. Additionally, ethanol increased the protein levels of pro-inflammatory mediators, including tumor necrosis factor alpha and the NLRP3 downstream product, secretory interleukin 1 beta, and SQ blunted these effects. Finally, the Sirius red staining of the myocardium revealed an increase in collagen deposition in ethanol-fed mice which was attenuated by TP-R antagonism. CONCLUSION: This study demonstrates that ethanol promotes the NLRP3 signaling pathway within the myocardium, leading to a pro-inflammatory milieu that potentially initiates early myocardial remodeling, and TP-R antagonism attenuates this effect.

A Pilot Study on the Proteomics Profile of Serum Exosome-Enriched Extracellular Vesicles from Normal versus Individuals with Obesity-Related Insulin Resistance.

OBJECTIVE: Circulating exosome-enriched extracellular vesicles (EVs) have drawn considerable importance in obesity-related insulin-resistance (IR). We sought to compare the proteomics profile of serum exosomes from normal individuals and those with obesity and IR. METHODS: We isolated serum exosomes from male subjects with obesity and insulin resistance (Ob-IR, HOMA-IR > 2.0) and lean/overweight insulin-sensitive (Normal (N), HOMA-IR < 2.0) individuals. The differential protein expression between the two groups was detected by a label-free quantitative mass spectrometry analysis followed by GO annotation and ingenuity pathway analysis (IPA). RESULTS: We identified 23 upregulated and 46 downregulated proteins between Ob-IR and N groups. Some of these proteins are involved in altering insulin signaling (VPS13C, TBC1D32, TTR, and ADIPOQ), inflammation (NFκB and CRP), and B-cell proliferation/activation (IGLV4-69, IGKV1D-13, and IGHV4-28). GO analysis revealed that the differentially expressed proteins (DEPs) are mainly involved in regulating immune cell activation and are located in extracellular space. IPA analysis showed that top molecules mediating IR, inflammation and B-cell activation were upregulated in Ob-IR subjects compared to N subjects. CONCLUSIONS: Serum exosomal proteins can be used as biomarkers to identify the future risk of diabetes and a therapeutic target to prevent or slow down the progression of diabetes in high-risk individuals.

Myristic Acid Supplementation Aggravates High Fat Diet-Induced Adipose Inflammation and Systemic Insulin Resistance in Mice.

Saturated fatty acids (SFAs) are considered to be detrimental to human health. One of the SFAs, myristic acid (MA), is known to exert a hypercholesterolemic effect in mice as well as humans. However, its effects on altering adipose tissue (AT) inflammation and systemic insulin resistance (IR) in obesity are still unclear. Here, we sought to determine the effects of a high fat (HF) diet supplemented with MA on obesity-associated metabolic disorders in mice. Wild-type C57BL/6 mice were fed a HF diet in the presence or absence of 3% MA for 12 weeks. Plasma lipids, plasma adipokines, AT inflammation, systemic IR, glucose homeostasis, and hepatic steatosis were assessed. The body weight and visceral adipose tissue (VAT) mass were significantly higher in mice receiving the HF+MA diet compared to HF diet-fed controls. Plasma total cholesterol levels were marginally increased in HF+MA-fed mice compared to controls. Fasting blood glucose was comparable between HF and HF+MA-fed mice. Interestingly, the plasma insulin and HOMA-IR index, a measure of insulin resistance, were significantly higher in HF+MA-fed mice compared to HF controls. Macrophage and inflammatory markers were significantly elevated in the AT and AT-derived stromal vascular cells upon MA feeding. Moreover, the level of circulating resistin, an adipokine promoting insulin resistance, was significantly higher in HF+MA-fed mice compared with HF controls. The insulin tolerance test revealed that the IR was higher in mice receiving the MA supplementation compared to HF controls. Moreover, the glucose tolerance test showed impairment in systemic glucose homeostasis in MA-fed mice. Analyses of liver samples showed a trend towards an increase in liver TG upon MA feeding. However, markers of oxidative stress and inflammation were reduced in the liver of mice fed an MA diet compared to controls. Taken together, our data suggest that chronic administration of MA in diet exacerbates obesity-associated insulin resistance and this effect is mediated in part, via increased AT inflammation and increased secretion of resistin.

Lauric Acid versus Palmitic Acid: Effects on Adipose Tissue Inflammation, Insulin Resistance, and Non-Alcoholic Fatty Liver Disease in Obesity.

Coconut oil, rich in medium-chain saturated fatty acids (MCSFA), in particular, lauric acid (LA), is known to exert beneficial metabolic effects. Although LA is the most abundant saturated fatty acid in coconut oil, the specific role of LA in altering obesity-related metabolic disorders remains unknown. Here, we examined the effects of supplementing a high fat (HF) diet with purified LA on obesity-associated metabolic derangements in comparison with palmitic acid (PA), a long-chain saturated fatty acid. Male C57BL/6 mice were fed a control chow diet (CD) or an HF diet supplemented with 3% LA (HF + LA) or PA (HF + PA) for 12 wk. Markers of adipose tissue (AT) inflammation, systemic insulin resistance (IR), and hepatic steatosis, were assessed. The body weight and total fat mass were significantly higher in both HF + LA and HF + PA diet-fed groups compared to CD controls. However, the visceral adipose tissue (VAT) mass was significantly higher (p < 0.001) in HF + LA-fed mice compared to both CD as well as HF + PA-fed mice. Interestingly, markers of AT inflammation were promoted to a lesser extent in HF + LA-fed mice compared to HF + PA-fed mice. Thus, immunohistochemical analysis of VAT showed an increase in MCP-1 and IL-6 staining in HF + PA-fed mice but not in HF + LA-fed mice compared to CD controls. Further, the mRNA levels of macrophage and inflammatory markers were significantly higher in HF + PA-fed mice (p < 0.001) whereas these markers were increased to a lesser extent in HF + LA-fed group. Of note, the insulin tolerance test revealed that IR was significantly increased only in HF + PA-fed mice but not in HF + LA-fed group compared to CD controls. While liver triglycerides were increased significantly in both HF + PA and HF + LA-fed mice, liver weight and plasma markers of liver injury such as alanine aminotransferase and aspartate aminotransferase were increased significantly only in HF + PA-fed mice but not in HF + LA-fed mice. Taken together, our data suggest that although both LA and PA increased AT inflammation, systemic IR, and liver injury, the extent of metabolic derangements caused by LA was less compared to PA in the setting of high fat feeding.

Lack of Augmenter of Liver Regeneration Disrupts Cholesterol Homeostasis of Liver in Mice by Inhibiting the AMPK Pathway.

It is well known that excessive cholesterol accumulation within hepatocytes deteriorates nonalcoholic fatty liver disease (NAFLD). Augmenter of liver regeneration (ALR) has been reported to alleviate NAFLD through anti-apoptosis; however, whether ALR could protect liver from cholesterol-induced NAFLD remains unclear. Mice with heterozygous deletion of Gfer (the gene for ALR, Gfer +/-) were generated, and liver steatosis was induced by either choline-deficient ethionine-supplemented, methionine choline-deficient diet for 4 weeks, or high-fat diet for 16 weeks. The results showed that Gfer +/- mice developed a more severe fatty liver phenotype than Gfer +/+ mice. The livers of Gfer +/- mice exhibited a higher concentration of cholesterol and low-density lipoprotein compared with the normal mice. Transcriptome-based analysis predicts low-density lipoprotein receptor (LDLR) primarily involved in the metabolic pathway. The experiments further indicate that cholesterol accumulation within hepatocytes is closely associated with enhancing the expression of LDLR and activation of sterol regulatory element binding protein 2 (SREBP2). Because adenosine monophosphate-activated protein kinase (AMPK) is a critical regulator of SREBP2 activation, we measured whether the activity of AMPK was regulated by ALR. We found that knockdown of ALR expression inhibited the phosphorylation of LKB1, an upstream activator of AMPK, followed by AMPK inactivation and SREBP2 maturation/nuclear translocation, leading to extensive cholesterol accumulation. Meanwhile, cellular oxidative stress increased as a result of ALR knockdown, indicating that ALR might also have a role in suppressing reactive oxygen species production. Conclusion: Our results confirm that ALR regulates cholesterol metabolism and alleviates hepatic steatosis probably through the LKB1-AMPK-SREBP2-LDLR pathway in vivo and in vitro, providing a putative mechanism for combating fatty liver disease.

Deficiency in augmenter of liver regeneration accelerates liver fibrosis by promoting migration of hepatic stellate cell.

BACKGROUND: Augmenter of liver regeneration (ALR) protects liver from various injuries, however, the association of ALR with liver fibrosis, particularly its effect on hepatic stellate cells (HSC), remains unclear. In this study, we investigated the impact of ALR on the activation of HSC, a pivotal event in occurrence of liver fibrosis. METHODS: Liver fibrosis was induced in vivo in mice with heterozygous ALR knockdown (ALR-KD) by administration of CCl4 or bile duct ligation. The effect of ALR-KD and ALR-overexpression on liver fibrosis was studied in mice and in HSC cells as well. RESULTS: Hepatic collagen deposition and expression of α-smooth muscle actin (α-SMA) were significantly increased in the ALR-KD mice compared to wild-type mice. In vitro, ALR-shRNA resulted in the activation of HSC cell line (LX-2). Furthermore, ALR-shRNA promoted LX-2 cell migration, accompanied by increased filamentous actin (F-actin) assembly. The ALR-KD-mediated increase in HSC migration was associated with mitochondrial fusion, resulting in mitochondria elongation and enhancing ATP production. In contrast, ALR transfection (ALR-Tx) decelerated HSC migration and inhibited F-actin assembly, concomitantly enhancing mitochondrial fission and reducing ATP synthesis. Mechanically, stimulation of HSC migration by ALR-shRNA was attributed to the increased mitochondrial Ca2+ influx in HSCs. Treatment of ALR-shRNA-cells with Ruthenium Red (RuR), a specific inhibitor of mitochondrial calcium uniporter (MCU), significantly suppressed mitochondrial Ca2+ influx, HSC migration, mitochondrial fusion and ATP production. ALR-KD-induced HSC migration was verified in vitro in primary mouse HSCs. CONCLUSION: Inhibition of ALR expression aggravates liver fibrosis, probably via promoting HSC migration and mitochondrial fusion.

Preclinical Evaluation of an Epidermal Growth Factor Receptor-Targeted Doxorubicin-Peptide Conjugate: Toxicity, Biodistribution, and Efficacy in Mice.

Doxorubicin (DOX) is known to induce apoptosis and necrosis in healthy tissue resulting in unwanted toxicities. To improve the ability of DOX to more specifically target tumors and minimize undesirable side effects, conjugation of DOX with epidermal growth factor receptor (EGFR)--binding peptide (DOX-EBP) has been developed to deliver DOX to EGFR-overexpressing neoplastic cells. Here, we investigated whether DOX-EBP was able to reduce toxicity and enhance anticancer efficacy in vivo through receptor-mediated targeted delivery system. Nude mice were treated with DOX or DOX-EBP to estimate general toxicity, normal tissue damage, biodistribution, and antitumor efficacy. In addition, the expression levels of EGFR in tumor tissues and normal organs were investigated by Western blotting, and their mRNA expression was analyzed by reverse transcription PCR. This study demonstrated that DOX-EBP was able to effectively decrease the distribution of DOX in normal tissues without EGFR overexpressing and reduce DOX-induced toxicity. On the other hand, the research also confirmed that DOX-EBP was able to preferentially accumulate DOX in EGFR-overexpressing tumor tissues and showed the enhanced anticancer efficacy over free DOX. DOX-EBP could be used for receptor-targeted chemotherapy with less toxicity and greater efficacy of tumor cells overexpressing EGFR. DOX-EBP conjugate is a good therapeutic agent for cancer treatment.

Targeted delivery of doxorubicin through conjugation with EGF receptor-binding peptide overcomes drug resistance in human colon cancer cells.

BACKGROUND AND PURPOSE: Induction of multidrug resistance by doxorubicin (DOX), together with non-specific toxicities, has restricted DOX-based chemotherapy. Recently, we demonstrated that DOX conjugated with an EGF receptor-binding peptide (DOX-EBP) had enhanced anticancer efficacy and reduced systemic toxicity when targeting EGF receptor-overexpressing tumours. Here we investigated whether DOX-EBP is able to overcome drug resistance and the underlying molecular mechanisms. EXPERIMENTAL APPROACH: DOX-resistant SW480/DOX cells were derived from non-resistant SW480 cells by stepwise exposure to increasing concentrations of DOX, and P-glycoprotein overexpression induced by DOX was confirmed by Western blotting. Cytotoxicity and intracellular distribution of drugs were evaluated by MTT assay and fluorescence microscopy respectively. EGF receptor-mediated endocytosis was determined in EGF receptor and endocytosis inhibition assays. Drug accumulation in tumour cells and murine xenografts was determined by HPLC. KEY RESULTS: The cytotoxicity and accumulation of DOX-EBP in SW480/DOX cells were almost the same as in SW480 cells, but those of free DOX were reduced. DOX-EBP accumulation was prevented by inhibitors of both EGF receptors and endocytosis, suggesting EGF receptors mediate endocytotic uptake. Tumour accumulation of DOX-EBP was significantly higher than free DOX in mice, and the levels of DOX-EBP were similar in DOX-resistant and non-resistant tumour tissues. Importantly, DOX-EBP, but not free DOX, was effective at inhibiting solid tumour growth and increased survival rate in both sensitive and resistant models. CONCLUSION AND IMPLICATIONS: DOX-EBP can overcome DOX resistance of tumour cells and increase in vivo antitumour efficacy. Therefore, it has the potential to be a potent therapeutic agent for treating drug-resistant cancers.