Skin cells are vulnerable to oxidative stress-induced senescence, which may lead to abnormal aging or aging-related disorders. Therefore, strategies that can ameliorate oxidative stress-induced senescence are expected to protect skin from damage, holding the promise of treating skin diseases in the clinic. This study aims to investigate whether caffeine, a well-known purine alkaloid, is able to prevent skin from oxidative stress-induced senescence, and to explore the underlying molecular mechanisms.Methods: A free radical inducer 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) was used to induce oxidative stress and cellular senescence in both transformed skin cells and in normal human epidermal keratinocytes (NHEKs). Ultraviolet (UV) irradiation was established as the in vivo oxidative stress model in mouse skin tissues. Cellular senescence was determined by SA β-galactosidase staining, immunofluorescence and western blotting. Activation of autophagy was confirmed by western blotting, immunofluorescence, and transmission electron microscopy. Reactive oxygen species (ROS) detection by commercial kits, gene knockdown by RNA interference (RNAi) and receptor activation/inactivation by agonist/antagonist treatment were applied in mechanistic experiments.Results: We report that AAPH induced senescence in both transformed skin cells and in NHEKs. Similarly, UV irradiation induced senescence in mouse skin tissues. Remarkably, low dose of caffeine (<10 μM) suppressed cellular senescence and skin damage induced by AAPH or UV. Mechanistically, caffeine facilitated the elimination of ROS by activating autophagy. Using a combination of RNAi and chemical treatment, we demonstrate that caffeine activates autophagy through a series of sequential events, starting from the inhibition of its primary cellular target adenosine A2a receptor (A2AR) to an increase in the protein level of Sirtuin 3 (SIRT3) and to the activation of 5' adenosine monophosphate-activated protein kinase (AMPK). Oral administration of caffeine increased the protein level of SIRT3, induced autophagy, and reduced senescence and tissue damage in UV-irradiated mouse skin. On the other hand, co-administration with autophagy inhibitors attenuated the protective effect of caffeine on UV-induced skin damage in mice.Conclusion: The results reveal that caffeine protects skin from oxidative stress-induced senescence through activating the A2AR/SIRT3/AMPK-mediated autophagy. Our study not only demonstrated the beneficial effect of caffeine using both in vitro and in vivo models, but also systematically investigated the underlying molecular mechanisms. These discoveries implicate the potential of caffeine in the protection of skin disease.

Praziquantel (PZQ), prescribed as a racemic mixture, is the most readily available drug to treat schistosomiasis. In the present study, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based metabolomics was employed to decipher the metabolic pathways and enantioselective metabolic differences of PZQ. Many phase I and four new phase II metabolites were found in urine and feces samples of mice 24h after dosing indicating that the major metabolic reaction encompassed oxidation, dehydrogenation, and glucuronidation. Differences in the formation of all these metabolites were observed between (R)-PZQ and (S)-PZQ. In an in vitro phase I incubation system, the major involvement of CYP3A, CYP2C9, and CYP2C19 in the metabolism of PZQ, and CYP3A, CYP2C9, and CYP2C19 exhibited different catalytic activity towards the PZQ enantiomers. Apparent Km and Vmax differences were observed in the catalytic formation of three mono-oxidized metabolites by CYP2C9 and CYP3A4 further supporting the metabolic differences for PZQ enantiomers. Molecular docking showed that chirality resulted in differences in location and conformation, which likely accounts for the metabolic differences. In conclusion, in silico, in vitro, and in vivo methods revealed the enantioselective metabolic profile of praziquantel.

Methionine- and choline-deficient diet (MCD) is a model for nonalcoholic steatohepatitis (NASH) in rodents. However, the mechanism of NASH development by dietary methionine/choline deficiency remains undetermined. To elucidate the early metabolic changes associated with MCD-NASH, serum metabolomic analysis was performed using mice treated with MCD and control diet for three days and one week, revealing significant increases in oleic and linoleic acids after MCD treatment. These increases were correlated with reduced body weight and white adipose tissue (WAT) mass, increased phosphorylation of hormone-sensitive lipase, and up-regulation of genes encoding carboxylesterase 3 and β2-adrenergic receptor in WAT, indicating accelerated lipolysis in adipocytes. The changes in serum fatty acids and WAT by MCD treatment were reversed by methionine supplementation, and similar alterations were detected in mice fed a methionine-deficient diet (MD), thus demonstrating that dietary methionine deficiency enhances lipolysis in WAT. MD treatment decreased glucose and increased fibroblast growth factor 21 in serum, thus exhibiting a similar metabolic phenotype as the fasting response. Comparison between MCD and choline-deficient diet (CD) treatments suggested that the addition of MD-induced metabolic alterations, such as WAT lipolysis, to CD-induced hepatic steatosis promotes liver injury. Collectively, these results demonstrate an important role for dietary methionine deficiency and WAT lipolysis in the development of MCD-NASH.

BACKGROUND AND PURPOSENoscapine is a promising anti-tumour agent. The purpose of the present study was to describe the metabolic map and investigate the bioactivation of noscapine. EXPERIMENTAL APPROACHUltra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry-based metabolomics was used to analyse the in vitro incubation mixtures, urine and faeces samples from mice treated with noscapine. Recombinant drug-metabolizing enzymes were employed to identify those involved in noscapine metabolism. Hepatic GSH levels and serum biochemistry were also carried out to determine reactive metabolites of noscapine. KEY RESULTSSeveral novel phase I metabolites of noscapine were detected after oral gavage of mice, including an N-demethylated metabolite, two hydroxylated metabolites, one metabolite undergoing both demethylation and cleavage of the methylenedioxy group and a bis-demethylated metabolite. Additionally, several novel glucuronides were detected, and their structures were elucidated through MS/MS fragmentology. Recombinant enzymes screening showed the involvement of several cytochromes P450, flavin-containing mono-oxygenase 1 and the UDP-glucuronosyltransferases UGT1A1, UGT1A3, UGT1A9 and UGT2B7, in noscapine metabolism. In vitro glutathione trapping revealed the existence of an ortho-quinone reactive intermediate formed through further oxidation of a catechol metabolite. However, this bioactivation process of noscapine does not occur in vivo. Similar to this result, altered glutathione levels in liver and serum biochemistry revealed no evidence of hepatic damage, thus indicating that, at least in mice, noscapine does not induce hepatotoxicity through bioactivation. CONCLUSIONS AND IMPLICATIONSA comprehensive metabolic map and bioactivation evaluation provides important information for the development of noscapine as an anti-tumour drug.

ABSTRACT:Magnolol is a food additive that is often found in mints and gums. Human exposure to this compound can reach a high dose; thus, characterization of magnolol disposition in humans is very important. Previous studies indicated that magnolol can undergo extensive glucuronidation in humans in vivo. In this study, in vitro assays were used to characterize the glucuronidation pathway in human liver and intestine. Assays with recombinant human UDP-glucuronosyltransferase enzymes (UGTs) revealed that multiple UGT isoforms were involved in magnolol glucuronidation, including UGT1A1, -1A3, -1A7, -1A8, -1A9, -1A10, and -2B7. Magnolol glucuronidation by human liver microsomes (HLM), human intestine microsomes (HIM), and most recombinant UGTs exhibited strong substrate inhibition kinetics. The degree of substrate inhibition was relatively low in the case of UGT1A10, whereas the reaction catalyzed by UGT1A9 followed biphasic kinetics. Chemical inhibition studies and the relative activity factor (RAF) approach were used to identify the individual UGTs that played important roles in magnolol glucuronidation in HLM and HIM. The results indicate that UGT2B7 is mainly responsible for the reaction in HLM, whereas UGT2B7 and UGT1A10 are significant contributors in HIM. In summary, the current study clarifies the glucuronidation pathway of magnolol and demonstrates that the RAF approach can be used as an efficient method for deciphering the roles of individual UGTs in a given glucuronidation pathway in the native tissue that is catalyzed by multiple isoforms with variable and atypical kinetics.

SLC25A13 (citrin or aspartate-glutamate carrier 2) is located in the mitochondrial membrane in the liver and its genetic deficiency causes adult-onset type II citrullinemia (CTLN2). CTLN2 is one of the urea cycle disorders characterized by sudden-onset hyperammonemia due to reduced argininosuccinate synthase activity. This disorder is frequently accompanied with hepatosteatosis in the absence of obesity and ethanol consumption. However, the precise mechanism of steatogenesis remains unclear. The expression of genes associated with fatty acid (FA) and triglyceride (TG) metabolism was examined using liver samples obtained from 16 CTLN2 patients and compared with 7 healthy individuals. Although expression of hepatic genes associated with lipogenesis and TG hydrolysis was not changed, the mRNAs encoding enzymes/proteins involved in FA oxidation (carnitine palmitoyl-CoA transferase 1α, medium- and very-long-chain acyl-CoA dehydrogenases, and acyl-CoA oxidase 1), very-low-density lipoprotein secretion (microsomal TG transfer protein), and FA transport (CD36 and FA-binding protein 1), were markedly suppressed in CTLN2 patients. Serum concentrations of ketone bodies were also decreased in these patients, suggesting reduced mitochondrial β-oxidation activity. Consistent with these findings, the expression of peroxisome proliferator-activated receptor α (PPARα), a master regulator of hepatic lipid metabolism, was significantly down-regulated. Hepatic PPARα expression was inversely correlated with severity of steatosis and circulating ammonia and citrulline levels. Additionally, phosphorylation of c-Jun-N-terminal kinase was enhanced in CTLN2 livers, which was likely associated with lower hepatic PPARα. Collectively, down-regulation of PPARα is associated with steatogenesis in CTLN2 patients. These findings provide a novel link between urea cycle disorder, lipid metabolism, and PPARα.

Objectives This study aimed to investigate the associations between trimethylamine N-oxide (TMAO) and related metabolites in early pregnancy and the risk of gestational diabetes mellitus (GDM). Design A prospective cohort of 22,302 pregnant women from 2010 to 2012 in Tianjin, China, was used to perform a nested case-control study. A total of 243 women with GDM and 243 women without GDM matched by maternal age (±1 year) were used as cases and controls, respectively. Conditional logistic regression and restricted cubic spline were used to examine the full-range risk associations between individual TMAOs metabolites at the first antenatal care visit with GDM. Trimethylamine conversion ratio (TMAR) was defined as trimethylamine (TMA)/its precursors, and trimethylamine N-oxide conversion ratio (TMAOR) was defined as TMAO/TMA. An additive interaction between high TMAR and low TMAOR indicates a state of TMA accumulation, and a mathematical interaction between high TMAR and high TMAOR indicates accumulation of TMAO. Results TMA was linearly associated with GDM, whereas TMA precursors and TMAO were inversely associated with GDM with clear threshold effects, i.e., 16 nmol/mL for TMAO, 200 nmol/mL for betaine, 112 nmol/mL for l-carnitine, and 110 and 270 nmol/mL for cholinechloride (a U-shaped relationship). Copresence of TMAR >0.35 and TMAOR ≤0.15 was associated with a markedly higher OR (11.16; 95% CI, 5.45 to 22.8), compared with TMAR >0.35 only (OR = 1.71; 95% CI, 0.42 to 6.95) or TMAOR ≤0.15 only (OR = 2.06; 95% CI, 1.09 to 3.90), with a significant additive interaction. However, the mathematical interaction was nonsignificant. Conclusions TMAO metabolites in the early pregnancy were associated with the risk of GDM, whereas TMA was more likely to play a causal role in GDM.

Background Bile acid metabolism plays an important role in metabolism but it is uncertain whether bile acid metabolites in early pregnancy are associated with risk of gestational diabetes mellitus (GDM). Methods We organized a 1:1 case-control study nested in a prospective cohort of 22,302 pregnant women recruited from 2010 to 2012 in China: 243 women with GDM were matched with 243 non-GDM controls on age (±1 year). Conditional logistic regression and restricted cubic spline were used to examine full-range associations of bile acid metabolites with GDM. Findings All the 9 detectable bile acids were inversely associated with the risk of GDM, among them, 8 in nonlinear and one in largely linear manners in multivariable analysis. Glycoursodeoxycholic acid (GUDCA) at ≤0.07 nmol/mL and deoxycholic acid (DCA) at ≤0.28 nmol/mL had threshold effects and their decreasing levels below the cutoff points were associated with rapid rises in the risk of GDM. In traditional risk factor model, the stepwise procedure identified that GUDCA ≤ 0.07 nmol/mL and DCA ≤ 0.280 nmol/mL were still significant (OR: 6.84, 95%CI: 1.10–42.48 & 2.06, 1.26–3.37), while other bile acids were not. Inclusion of the two bile acids in the model increased the area under operating characteristic's curve from 0.69 to 0.76 (95% CI: 0.71–0.80) ( P < .05). Interpretation Serum GUDCA ≤ 0.07 nmol/mL and DCA ≤ 0.28 nmol/mL in early pregnancy were independently associated with increased risk of GDM in Chinese pregnant women. Funding Talent Recruitment Scheme grant of Tianjin Medical University and National Key Research and Development Program, etc.