Chronic activation of PPARα with fenofibrate reduces autophagic proteins in the liver of mice independent of FGF21.

Autophagy is a catabolic mechanism to degrade cellular components to maintain cellular energy levels during starvation, a condition where PPARα may be activated. Here we report a reduced autophagic capacity in the liver following chronic activation of PPARα with fenofibrate (FB) in mice. Chronic administration of the PPARα agonist FB substantially reduced the levels of multiple autophagy proteins in the liver (Atg3, Agt4B, Atg5, Atg7 and beclin 1) which were associated with a decrease in the light chain LC3II/LC3I ratio and the accumulation of p62. This was concomitant with an increase in the expression of lipogenic proteins mSREBP1c, ACC, FAS and SCD1. These effects of FB were completely abolished in PPARα-/- mice but remained intact in mice with global deletion of FGF21, a key downstream mediator for PPARα-induced effects. Further studies showed that decreased the content of autophagy proteins by FB was associated with a significant reduction in the level of FoxO1, a transcriptional regulator of autophagic proteins, which occurred independently of both mTOR and Akt. These findings suggest that chronic stimulation of PPARα may suppress the autophagy capacity in the liver as a result of reduced content of a number of autophagy-associated proteins independent of FGF21.

Fibroblast growth factor 21 improves hepatic insulin sensitivity by inhibiting mammalian target of rapamycin complex 1 in mice.

UNLABELLED: Among the 22 fibroblast growth factors (FGFs), FGF21 has now emerged as a key metabolic regulator. However, the mechanism whereby FGF21 mediates its metabolic actions per se remains largely unknown. Here, we show that FGF21 represses mammalian target of rapamycin complex 1 (mTORC1) and improves insulin sensitivity and glycogen storage in a hepatocyte-autonomous manner. Administration of FGF21 in mice inhibits mTORC1 in the liver, whereas FGF21-deficient mice display pronounced insulin-stimulated mTORC1 activation and exacerbated hepatic insulin resistance (IR). FGF21 inhibits insulin- or nutrient-stimulated activation of mTORC1 to enhance phosphorylation of Akt in HepG2 cells at both normal and IR condition. TSC1 deficiency abrogates FGF21-mediated inhibition of mTORC1 and augmentation of insulin signaling and glycogen synthesis. Strikingly, hepatic ßKlotho knockdown or hepatic hyperactivation of mTORC1/ribosomal protein S6 kinase 1 abrogates hepatic insulin-sensitizing and glycemic-control effects of FGF21 in diet-induced insulin-resistant mice. Moreover, FGF21 improves methionine- and choline-deficient diet-induced steatohepatitis. CONCLUSIONS: FGF21 acts as an inhibitor of mTORC1 to control hepatic insulin action and maintain glucose homeostasis, and mTORC1 inhibition by FGF21 has the therapeutic potential for treating IR and type 2 diabetes. (Hepatology 2016;64:425-438).

FGF21 maintains glucose homeostasis by mediating the cross talk between liver and brain during prolonged fasting.

Hepatic gluconeogenesis is a main source of blood glucose during prolonged fasting and is orchestrated by endocrine and neural pathways. Here we show that the hepatocyte-secreted hormone fibroblast growth factor 21 (FGF21) induces fasting gluconeogenesis via the brain-liver axis. Prolonged fasting induces activation of the transcription factor peroxisome proliferator-activated receptor α (PPARα) in the liver and subsequent hepatic production of FGF21, which enters into the brain to activate the hypothalamic-pituitary-adrenal (HPA) axis for release of corticosterone, thereby stimulating hepatic gluconeogenesis. Fasted FGF21 knockout (KO) mice exhibit severe hypoglycemia and defective hepatic gluconeogenesis due to impaired activation of the HPA axis and blunted release of corticosterone, a phenotype similar to that observed in PPARα KO mice. By contrast, intracerebroventricular injection of FGF21 reverses fasting hypoglycemia and impairment in hepatic gluconeogenesis by restoring corticosterone production in both FGF21 KO and PPARα KO mice, whereas all these central effects of FGF21 were abrogated by blockage of hypothalamic FGF receptor-1. FGF21 acts directly on the hypothalamic neurons to activate the mitogen-activated protein kinase extracellular signal-related kinase 1/2 (ERK1/2), thereby stimulating the expression of corticotropin-releasing hormone by activation of the transcription factor cAMP response element binding protein. Therefore, FGF21 maintains glucose homeostasis during prolonged fasting by fine tuning the interorgan cross talk between liver and brain.

Acetaminophen induced gender-dependent liver injury and the involvement of GCL and GPx.

Acetaminophen (AP) is widely used as the antipyretic and analgesic drug in clinic, and it can induce serious liver injury in the case of excessive abuse. The present study showed that AP (400 mg/kg) induced obvious liver injury, while in male mice the hepatotoxicity induced by AP was much more serious than in female mice as indicated by the results of alanine aminotransferase (ALT) activity and reduced glutathione (GSH) amount. Further, the enzymatic activity and protein expression of glutamate-cysteine ligase (GCL) and glutathione peroxidase (GPx) were all higher in female mice liver than in male after the administration of AP (200 mg/kg). Meanwhile, AP (10 mM) decreased GCL and GPx activity in isolated mouse hepatocytes in the time-dependent manner, while the inhibitors of GCL and GPx can augment AP induced-cytotoxicity. Taken together, our results demonstrate the gender-related liver injury induced by AP and the important role of GCL and GPx in regulating such hepatotoxicity.

The difference of glutathione antioxidant system in newly weaned and young mice liver and its involvement in isoline-induced hepatotoxicity.

Cellular glutathione antioxidant system plays important roles in counteracting hepatotoxins-induced oxidative stress injury. The present study was designed to observe the differences of this system in newly weaned and young mice liver and its involvement in the susceptibility to isoline-induced liver injury. Our results showed that liver reduced glutathione (GSH) amounts were higher in newly weaned mice than young mice. Glutamate-cysteine ligase (GCL) activity was higher in newly weaned mice due to the higher expression of catalytic subunit of GCL (GCLC) protein and mRNA. However, the activities of glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) were higher in young mice liver, which might be due to the higher expression of GR, GPx-1, and GST-Pi proteins. Next, the results of AST analysis and histopathological evaluation showed that newly weaned mice demonstrated more severe liver injury induced by isoline. Furthermore, liver GSH amounts and the activities of GR, GPx, and GST were all lower in newly weaned mice than young mice after treated with isoline. Depletion of cellular GSH by D,L -buthionine-(S, R)-sulfoximine (BSO) aggravated isoline-induced cytotoxicity, while N-acetyl-l cysteine (NAC) ameliorated such cytotoxicity. Furthermore, the inhibitors of GR, GPx, and GST all aggravated isoline-induced cytotoxicity. In conclusion, our results demonstrated the differences of glutathione antioxidant system between newly weaned and young mice liver. Meanwhile, our results also revealed age-dependent liver injury induced by isoline for the first time, which might be due to the different responses of glutathione antioxidant system to isoline between newly weaned and young mice.

Gender-related difference in liver injury induced by Dioscorea bulbifera L. rhizome in mice.

The present study was undertaken to investigate the gender-related liver injury induced by Dioscorea bulbifera L. (DB), a traditional medicinal plant, in mice, and further explored its hepatotoxic chemical compound. Serum and liver tissue samples were collected at 0, 4, 8, 12 h, after mice were administrated orally with 640 mg/kg ethyl acetate extracts (EF) isolated from DB. After treatments, serum alanine transaminase (ALT) and aspartate transaminase (AST) activities were both significantly elevated. Liver lipid peroxidation (LPO) level increased, while glutathione amounts, glutathione-S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) activities all decreased in the time-dependent manner. Further analysis demonstrated that ALT and AST activities in female mice were significantly lower than those in male. Meanwhile, liver glutathione amounts and CAT activity in female mice after giving EF for 12 h were both higher than those in male. Further, comparing the liver injury induced by Diosbulbin B isolated from DB with that induced by EF on the basis of chemical analysis for the amounts of Diosbulbin B in EF of DB, we found that Diosbulbin B could be the main hepatotoxic chemical compound in DB. Taken together, our results show that DB can induce gender-related liver oxidative stress injury in mice, and its main hepatotoxic chemical compound is Diosbulbin B, for the first time.

The gender-dependent difference of liver GSH antioxidant system in mice and its influence on isoline-induced liver injury.

Intracellular reduced glutathione (GSH) antioxidant system is crucial for counteracting oxidative stress-induced liver injury. The present study was designed to observe the gender-dependent difference of GSH antioxidant system and its influence on hepatotoxic pyrrolizidine alkaloid (HPA) isoline-induced liver injury. Lower activities and protein expressions of glutamate-cysteine ligase (GCL) and glutathione peroxidase (GPx) were found in male mice livers than in female. Isoline is a natural HPA, our further results showed that male mice demonstrated more higher serum ALT/AST levels, less GSH amounts, lower GCL and GPx activities and proteins induced by isoline as compared to female. N-acetyl-l-cysteine (NAC), which is the precursor of cellular GSH biosynthesis, ameliorated liver injury induced by isoline. l-Buthionine-(S, R)-sulfoximine (BSO) and mercaptosuccinic acid (MA), inhibitors of GCL and GPx, both augmented isoline-induced cytotoxicity in cultured mice hepatocytes. BSO and MA also increased other natural HPAs clivorine and senecionine-induced cytotoxicity. Taken together, our results demonstrated the higher GCL and GPx activities in female mice, which indicated their crucial roles in regulating the resistance of liver injury induced by hepatotoxins in female. Meanwhile, our results also revealed the female-resistant liver injury induced by HPAs for the first time.

Acetaminophen-induced cytotoxicity on human normal liver L-02 cells and the protection of antioxidants.

In vitro cell models, which can partially mimic in vivo responses, offer potentially sensitive tools for toxicological assessment. The objective of this study was to explore the possible mechanisms of acetaminophen (AP)-induced toxicity in human normal liver L-02 cells. The expression of the CYP2E1 enzyme, which is reported to transform AP to its toxic metabolites, was higher in L-02 than in Hep3B cells. Further cell viability and reduced glutathione (GSH) depletion after AP treatment were examined. After exposure to AP for 24 h, cell viability decreased in a concentration-dependent manner. Concentration-dependent GSH depletion was also observed after AP treatment for 48 h, indicating oxidative stress had occurred in L-02 cells. The effects of D, L-buthionine-(S, R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, and N-acetylcysteine (NAC), a precursor of GSH synthesis, on the cytotoxicity induced by AP were also investigated. BSO aggravated the cytotoxicity induced by AP while NAC ameliorated such cell death. Further results showed that 10 mM AP caused cell apoptosis after 48 h treatment based on the DNA fragmentation assay and western blot of caspase-3 activation, respectively. In addition, the protective effects of various well-known antioxidants against AP-induced hepatotoxicity were observed. Taken together, these results indicate that oxidative stress and cellular apoptosis are involved in AP-induced toxicity in human normal liver L-02 cells, and this cell line is a suitable in vitro cell model for AP hepatotoxicity study.