B3galt5 functions as a PXR target gene and regulates obesity and insulin resistance by maintaining intestinal integrity.

Pregnane X receptor (PXR) has been reported to regulate glycolipid metabolism. The dysfunction of intestinal barrier contributes to metabolic disorders. However, the role of intestinal PXR in metabolic diseases remains largely unknown. Here, we show that activation of PXR by tributyl citrate (TBC), an intestinal-selective PXR agonist, improves high fat diet (HFD)-induced obesity. The metabolic benefit of intestinal PXR activation is associated with upregulation of ß-1,3 galactosyltransferase 5 (B3galt5). Our results reveal that B3galt5 mainly expresses in the intestine and is a direct PXR transcriptional target. B3galt5 knockout exacerbates HFD-induced obesity, insulin resistance and inflammation. Mechanistically, B3galt5 is essential to maintain the integrity of intestinal mucus barrier. B3galt5 ablation impairs the O-glycosylation of mucin2, destabilizes the mucus layer, and increases intestinal permeability. Furthermore, B3galt5 deficiency abolishes the beneficial effect of intestinal PXR activation on metabolic disorders. Our results suggest the intestinal-selective PXR activation regulates B3galt5 expression and maintains metabolic homeostasis, making it a potential therapeutic strategy in obesity.

Reactive Oxygen Species-Responsive Delivery of a Notch Inhibitor to Alleviate Nonalcoholic Steatohepatitis by Inhibiting Hepatic de Novo Lipogenesis and Inflammation.

With the increased prevalence of nonalcoholic steatohepatitis (NASH) in the world, effective pharmacotherapy in clinical practice is still lacking. Previous studies have shown that dibenzazepine (DBZ), a Notch inhibitor, could alleviate NASH development in a mouse model. However, low bioavailability, poor water solubility, and extrahepatic side effects restrict its clinical application. To overcome these barriers, we developed a reactive oxygen species (ROS)-sensitive nanoparticle based on the conjugation of bilirubin to poly(ethylene glycol) (PEG) chains, taking into account the overaccumulation of hepatic ROS in the pathologic state of nonalcoholic steatohepatitis (NASH). The PEGylated bilirubin can self-assemble into nanoparticles in an aqueous solution and encapsulate insoluble DBZ into its hydrophobic cavity. DBZ nanoparticles (DBZ Nps) had good stability, rapidly released DBZ in response to H2O2, and effectively scavenged intracellular ROS of hepatocytes. After systemic administration, DBZ Nps could accumulate in the liver of the NASH mice, extend persistence in circulation, and improve the bioavailability of DBZ. Furthermore, DBZ Nps significantly improved glucose intolerance, relieved hepatic lipid accumulation and inflammation, and ameliorated NASH-induced liver fibrosis. Additionally, DBZ Nps had no significant extrahepatic side effects. Taken together, our results highlight the potential of the ROS-sensitive DBZ nanoparticle as a promising therapeutic strategy for NASH.

Glutaredoxin 1 regulates cholesterol metabolism and gallstone formation by influencing protein S-glutathionylation.

OBJECTIVE: Cholesterol gallstone disease (CGD) is closely related to cholesterol metabolic disorder. Glutaredoxin-1 (Glrx1) and Glrx1-related protein S-glutathionylation are increasingly being observed to drive various physiological and pathological processes, especially in metabolic diseases such as diabetes, obesity and fatty liver. However, Glrx1 has been minimally explored in cholesterol metabolism and gallstone disease. METHODS: We first investigated whether Glrx1 plays a role in gallstone formation in lithogenic diet-fed mice using immunoblotting and quantitative real-time PCR. Then a whole-body Glrx1-deficient (Glrx1-/-) mice and hepatic-specific Glrx1-overexpressing (AAV8-TBG-Glrx1) mice were generated, in which we analyzed the effects of Glrx1 on lipid metabolism upon LGD feeding. Quantitative proteomic analysis and immunoprecipitation (IP) of glutathionylated proteins were performed. RESULTS: We found that protein S-glutathionylation was markedly decreased and the deglutathionylating enzyme Glrx1 was greatly increased in the liver of lithogenic diet-fed mice. Glrx1-/- mice were protected from gallstone disease induced by a lithogenic diet because their biliary cholesterol and cholesterol saturation index (CSI) were reduced. Conversely, AAV8-TBG-Glrx1 mice showed greater gallstone progression with increased cholesterol secretion and CSI. Further studies showed that Glrx1-overexpressing greatly altered bile acid levels and/or composition to increase intestinal cholesterol absorption by upregulating Cyp8b1. In addition, liquid chromatography-mass spectrometry and IP analysis revealed that Glrx1 also affected the function of asialoglycoprotein receptor 1 (ASGR1) by mediating its deglutathionylation, thereby altering the expression of LXRα and controlling cholesterol secretion. CONCLUSION: Our findings present novel roles of Glrx1 and Glrx1-regulated protein S-glutathionylation in gallstone formation through the targeting of cholesterol metabolism. Our data advises Glrx1 significantly increased gallstone formation by simultaneously increase bile-acid-dependent cholesterol absorption and ASGR1- LXRα-dependent cholesterol efflux. Our work suggests the potential effects of inhibiting Glrx1 activity to treat cholelithiasis.

Glutaredoxin-1 alleviates acetaminophen-induced liver injury by decreasing its toxic metabolites.

Excessive N-acetyl-p-benzoquinone imine (NAPQI) formation is a starting event that triggers oxidative stress and subsequent hepatocyte necrosis in acetaminophen (APAP) overdose caused acute liver failure (ALF). S-glutathionylation is a reversible redox post-translational modification and a prospective mechanism of APAP hepatotoxicity. Glutaredoxin-1 (Glrx1), a glutathione-specific thioltransferase, is a primary enzyme to catalyze deglutathionylation. The objective of this study was to explored whether and how Glrx1 is associated with the development of ALF induced by APAP. The Glrx1 knockout mice (Glrx1-/-) and liver-specific overexpression of Glrx1 (AAV8-Glrx1) mice were produced and underwent APAP-induced ALF. Pirfenidone (PFD), a potential inducer of Glrx1, was administrated preceding APAP to assess its protective effects. Our results revealed that the hepatic total protein S-glutathionylation (PSSG) increased and the Glrx1 level reduced in mice after APAP toxicity. Glrx1-/- mice were more sensitive to APAP overdose, with higher oxidative stress and more toxic metabolites of APAP. This was attributed to Glrx1 deficiency increasing the total hepatic PSSG and the S-glutathionylation of cytochrome p450 3a11 (Cyp3a11), which likely increased the activity of Cyp3a11. Conversely, AAV8-Glrx1 mice were defended against liver damage caused by APAP overdose by inhibiting the S-glutathionylation and activity of Cyp3a11, which reduced the toxic metabolites of APAP and oxidative stress. PFD precede administration upregulated Glrx1 expression and alleviated APAP-induced ALF by decreasing oxidative stress. We have identified the function of Glrx1 mediated PSSG in liver injury caused by APAP overdose. Increasing Glrx1 expression may be investigated for the medical treatment of APAP-caused hepatic injury.

MANF in POMC Neurons Promotes Brown Adipose Tissue Thermogenesis and Protects Against Diet-Induced Obesity.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an emerging regulator in metabolic control. Hypothalamic proopiomelanocortin (POMC) neurons play critical roles in maintaining whole-body energy homeostasis. Whether MANF in POMC neurons is required for the proper regulation of energy balance remains unknown. Here, we showed that mice lacking MANF in POMC neurons were more prone to develop diet-induced obesity. In addition, the ablation of MANF induced endoplasmic reticulum (ER) stress and leptin resistance in the hypothalamus, reduced POMC expression and posttranslational processing, and ultimately decreased sympathetic nerve activity and thermogenesis in brown adipose tissue (BAT). Conversely, MANF overexpression in hypothalamic POMC neurons attenuated ER stress, increased POMC expression and processing, and then stimulated sympathetic innervation and activity in BAT, resulting in increased BAT thermogenesis, thus protecting mice against dietary obesity. Overall, our findings provide evidence that MANF is required for POMC neurons to combat obesity.

Mesencephalic astrocyte-derived neurotrophic factor alleviates non-alcoholic steatohepatitis induced by Western diet in mice.

Excessive lipid accumulation, inflammation, and fibrosis in the liver are the major characteristics of non-alcoholic steatohepatitis (NASH). Mesencephalic astrocyte-derived neurotrophic factor (MANF) plays an important role in metabolic homeostasis, raising the possibility that it is involved in NASH. Here, we reduced and increased MANF levels in mice in order to explore its influence on hepatic triglyceride homeostasis, inflammation, and fibrosis during NASH progression. The MANF expression was decreased in Western diet-induced NASH mice. In vivo, liver-specific MANF knockout exacerbated hepatic lipid accumulation, inflammation, and fibrosis of mice induced by Western diet, while liver-specific MANF overexpression mitigated these NASH pathogenic features. In vitro, knocking down MANF in primary hepatocyte cultures aggravated hepatic steatosis and inflammation, which MANF overexpression markedly attenuated. Studies in vitro and in vivo suggested that MANF regulated hepatic lipid synthesis by modulating SREBP1 expression. Inhibiting SREBP1 in primary hepatocytes blocked lipid accumulation after MANF knockdown. MANF overexpression reversed LXRs agonist GW3965 induced SREBP1 and LIPIN1 expression. MANF decreased the expression of pro-inflammatory cytokines by inhibiting NF-κB phosphorylation. These results suggest that MANF can protect against NASH by regulating SREBP1 expression and NF-κB signaling.

Mesencephalic astrocyte-derived neurotrophic factor protects against paracetamol -induced liver injury by inhibiting PERK-ATF4-CHOP signaling pathway.

Paracetamol (APAP), an over-the-counter drug, is normally safe within the therapeutic dose range but can cause irreversible liver damage after an overdose. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress protein and plays a crucial role in metabolic disease. However, the role of MANF in APAP-induced acute hepatotoxicity is still unknown. We used hepatocyte-specific MANF-knockout mice and hepatocyte-specific MANF transgenic mice to investigate the role of hepatocyte-derived MANF in APAP-induced acute liver injury. MANF deficiency was associated with a decreased expression of detoxification enzymes, aggravated glutathione depletion and apoptosis in hepatocytes. Mechanistically, MANF knockout significantly increased PERK-eIF2α-ATF4-CHOP signaling pathway. Blockade of PERK abolished MANF deficiency-over-induced hepatotoxicity after APAP administration. Conversely, hepatocyte-specific MANF overexpression attenuated APAP-induced hepatotoxicity by downregulating the PERK-eIF2α-ATF4-CHOP signaling pathway. Thus, hepatocyte-derived MANF may play a protective role in APAP-induced hepatotoxicity.

[The role of SIRT6 in nonalcoholic steatohepatitis].

SIRT6, a member of the silencing information regulatory protein family, is a nicotinamide adenine dinucleotide-dependent histone deacetylase and an ADP-ribose transferase enzyme. It plays an important role in fundamental physiological and pathological processes, including lipid metabolism, inflammation, oxidative stress and fibrosis, and is considered as a potential therapeutic target for metabolic syndrome. SIRT6 knockout mice displayed severe fatty liver, and the expression of SIRT6 in the liver of nonalcoholic steatohepatitis (NASH) mice was significantly lower than that of normal mice. Overexpression of SIRT6 significantly ameliorated NASH-induced liver damage. It is suggested that SIRT6 may play a key role in protecting against NASH. In this paper, we review the important regulatory functions of SIRT6 in the occurrence and development of NASH.

Farnesylthiosalicylic Acid-Loaded Albumin Nanoparticle Alleviates Renal Fibrosis by Inhibiting Ras/Raf1/p38 Signaling Pathway.

BACKGROUND: Renal fibrosis is the common pathway in chronic kidney diseases progression to end-stage renal disease, but to date, no clinical drug for its treatment is approved. It has been demonstrated that the inhibitor of proto-oncogene Ras, farnesylthiosalicylic acid (FTS), shows therapeutic potential for renal fibrosis, but its application was hindered by the water-insolubility and low bioavailability. Hence, in this study, we improved these properties of FTS by encapsulating it into bovine serum albumin nanoparticles (AN-FTS) and tested its therapeutic effect in renal fibrosis. METHODS: AN-FTS was developed using a classic emulsification-solvent ultrasonication. The pharmacokinetics of DiD-loaded albumin nanoparticle were investigated in SD rats. The biodistribution and therapeutic efficacy of AN-FTS was assessed in a mouse model of renal fibrosis induced by unilateral ureteral obstruction (UUO). RESULTS: AN-FTS showed a uniform spherical shape with the size of 100.6 ± 1.12 nm and PDI < 0.25. In vitro, AN-FTS displayed stronger inhibitory effects on the activation of renal fibroblasts cells NRK-49F than free FTS. In vivo, AN-FTS showed significantly higher peak concentration and area under the concentration-time curve. After intravenous administration to UUO-induced renal fibrosis mice, AN-FTS accumulated preferentially in the fibrotic kidney, and alleviated renal fibrosis and inflammation significantly more than the free drug. Mechanistically, the improved anti-fibrosis effect of AN-FTS was associated with greater inhibition in renal epithelial-to-mesenchymal transformation process via Ras/Raf1/p38 signaling pathway. CONCLUSION: The study reveals that AN-FTS is capable of delivering FTS to fibrotic kidney and showed superior therapeutic efficacy for renal fibrosis.

Hepatocyte-specific deletion of Nlrp6 in mice exacerbates the development of non-alcoholic steatohepatitis.

OBJECTIVE: Previous studies have established that deficiency in Nucleotide-binding and oligomerization domain (NOD)-like receptor family pyrin domain containing 6 (Nlrp6) changes the configuration of the gut microbiota, which leads to hepatic steatosis. Here, we aimed to determine the hepatic function of Nlrp6 in lipid metabolism and inflammation and its role in the development of non-alcoholic steatohepatitis (NASH). METHODS: Nlrp6Loxp/Loxp and hepatocyte-specific Nlrp6-knockout mice were fed a high-fat diet (HFD) or methionine-choline deficient (MCD) diet to induce fatty liver or steatohepatitis, respectively. Primary hepatocytes were isolated to further explore the underlying mechanisms in vitro. In addition, we used adenovirus to overexpress Nlrp6 in ob/ob mice to demonstrate its role in NASH. RESULTS: Hepatic Nlrp6 expression was downregulated in NASH patients and in obese mice. Hepatocyte-specific Nlrp6 deficiency promoted HFD- or MCD diet-induced lipid accumulation and inflammation, whereas Nlrp6 overexpression in ob/ob mice had beneficial effects. In vitro studies demonstrated that knockdown of Nlrp6 aggravated hepatic steatosis and inflammation in hepatocytes, but its overexpression markedly attenuated these abnormalities. Moreover, both in vitro and in vivo study demonstrated that Nlrp6 inhibited Cd36-mediated lipid uptake. Nlrp6 deficiency-enhanced fatty acid uptake was blocked by a Cd36 inhibitor in hepatocytes. Nlrp6 ablation increased the expression of proinflammatory cytokines, likely as a result of increased NF-κB phosphorylation and activation. Mechanistically, Nlrp6 promoted the degradation of transforming growth factor-ß-activated kinase 1 (TAK1)-binding protein 2/3 (TAB2/3) via a lysosomal-dependent pathway, which suppressed NF-κB activation. CONCLUSIONS: Nlrp6 may play a key role in the pathological process of NASH by inhibiting Cd36 and NF-κB pathways. It may be a potential therapeutic target for NASH.

Mesencephalic astrocyte-derived neurotrophic factor alleviates alcohol induced hepatic steatosis via activating Stat3-mediated autophagy.

Alcoholic fatty liver disease (AFLD) is induced by alcohol consumption and may progress to more severe liver diseases such as alcoholic steatohepatitis, fibrosis and cirrhosis, and even hepatocellular carcinoma. Mesencephalic astrocyte-derived neurotrophic factor (MANF) participates in maintaining lipid homeostasis. However, the role of MANF in the pathogenesis of AFLD remains unclear. We established an AFLD mouse model following the US National Institute on Alcohol Abuse and Alcoholism procedure. Both mRNA and protein levels of MANF were significantly increased in the chronic binge alcohol feeding model. Liver-specific knockout of MANF aggravated hepatic lipid accumulation. Similarly, liver-specific overexpression of MANF alleviated AFLD in mouse livers. MANF affected hepatic lipid metabolism by modulating autophagy. The levels of LC3-II and Atg5-Atg12 were decreased in mouse livers with MANF liver-specific knockout and increased with MANF liver-specific overexpression. Furthermore, MANF changed the phosphorylation of Stat3 and its nuclear localization. MANF may have a protective role in the development of AFLD.