Multi-omics profiling of collagen-induced arthritis mouse model reveals early metabolic dysregulation via SIRT1 axis.

Rheumatoid arthritis (RA) is characterized by joint infiltration of immune cells and synovial inflammation which leads to progressive disability. Current treatments improve the disease outcome, but the unmet medical need is still high. New discoveries over the last decade have revealed the major impact of cellular metabolism on immune cell functions. So far, a comprehensive understanding of metabolic changes during disease development, especially in the diseased microenvironment, is still limited. Therefore, we studied the longitudinal metabolic changes during the development of murine arthritis by integrating metabolomics and transcriptomics data. We identified an early change in macrophage pathways which was accompanied by oxidative stress, a drop in NAD+ level and induction of glucose transporters. We discovered inhibition of SIRT1, a NAD-dependent histone deacetylase and confirmed its dysregulation in human macrophages and synovial tissues of RA patients. Mining this database should enable the discovery of novel metabolic targets and therapy opportunities in RA.

ADP-dependent glucokinase regulates energy metabolism via ER-localized glucose sensing.

Modulation of energy metabolism to a highly glycolytic phenotype, i.e. Warburg effect, is a common phenotype of cancer and activated immune cells allowing increased biomass-production for proliferation and cell division. Endoplasmic reticulum (ER)-localized ADP-dependent glucokinase (ADPGK) has been shown to play a critical role in T cell receptor activation-induced remodeling of energy metabolism, however the underlying mechanisms remain unclear. Therefore, we established and characterized in vitro and in vivo models for ADPGK-deficiency using Jurkat T cells and zebrafish. Upon activation, ADPGK knockout Jurkat T cells displayed increased cell death and ER stress. The increase in cell death resulted from a metabolic catastrophe and knockout cells displayed severely disturbed energy metabolism hindering induction of Warburg phenotype. ADPGK knockdown in zebrafish embryos led to short, dorsalized body axis induced by elevated apoptosis. ADPGK hypomorphic zebrafish further displayed dysfunctional glucose metabolism. In both model systems loss of ADPGK function led to defective N- and O-glycosylation. Overall, our data illustrate that ADPGK is part of a glucose sensing system in the ER modulating metabolism via regulation of N- and O-glycosylation.

Maleic Acid--but Not Structurally Related Methylmalonic Acid--Interrupts Energy Metabolism by Impaired Calcium Homeostasis.

Maleic acid (MA) has been shown to induce Fanconi syndrome via disturbance of renal energy homeostasis, though the underlying pathomechanism is still under debate. Our study aimed to examine the pathomechanism underlying maleic acid-induced nephrotoxicity. Methylmalonic acid (MMA) is structurally similar to MA and accumulates in patients affected with methymalonic aciduria, a defect in the degradation of branched-chain amino acids, odd-chain fatty acids and cholesterol, which is associated with the development of tubulointerstitial nephritis resulting in chronic renal failure. We therefore used MMA application as a control experiment in our study and stressed hPTECs with MA and MMA to further validate the specificity of our findings. MMA did not show any toxic effects on proximal tubule cells, whereas maleic acid induced concentration-dependent and time-dependent cell death shown by increased lactate dehydrogenase release as well as ethidium homodimer and calcein acetoxymethyl ester staining. The toxic effect of MA was blocked by administration of single amino acids, in particular L-alanine and L-glutamate. MA application further resulted in severe impairment of cellular energy homeostasis on the level of glycolysis, respiratory chain, and citric acid cycle resulting in ATP depletion. As underlying mechanism we could identify disturbance of calcium homeostasis. MA toxicity was critically dependent on calcium levels in culture medium and blocked by the extra- and intracellular calcium chelators EGTA and BAPTA-AM respectively. Moreover, MA-induced cell death was associated with activation of calcium-dependent calpain proteases. In summary, our study shows a comprehensive pathomechanistic concept for MA-induced dysfunction and damage of human proximal tubule cells.

Cross-reactivity of acetylfentanyl and risperidone with a fentanyl immunoassay.

Fentanyl and its analogs, such as acetylfentanyl, have become a concern for potential abuse. Fentanyl compliance monitoring and urine drug testing are becoming increasingly necessary; however, a limited number of fentanyl immunoassays have been validated for clinical use. The purpose of this study was to validate the use of the DRI® fentanyl immunoassay, determine the potential cross-reactivity of acetylfentanyl and other pharmaceuticals, and investigate acetylfentanyl use in San Francisco. All urine toxicology samples from patients presenting to the emergency department were analyzed using the fentanyl immunoassay for 4 months. Positive samples were analyzed qualitatively using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for fentanyl, fentanyl metabolites, fentanyl analogs and greater than 200 common drugs and metabolites. Subsequently, quantitative analysis was performed using LC-tandem mass spectrometry (LC-MS-MS). Acetylfentanyl, risperidone and 9-hydroxyrisperidone were found to cross-react with the fentanyl immunoassay. No acetylfentanyl was detected in our emergency department patient population. The fentanyl immunoassay demonstrated 100% diagnostic sensitivity in a subset of urines tested; however, the specificity was only 86% due to seven false-positive samples observed. Five of the seven samples were positive for risperidone and 9-hydroxyrisperidone. The DRI® fentanyl immunoassay can be used to screen for fentanyl or acetylfentanyl; however, confirmatory testing should be performed for all samples that screen positive.

Glycine ameliorates liver injury and vitamin D deficiency induced by bile duct ligation.

BACKGROUND: Patients with chronic liver disease had lower serum concentrations 25-hydroxyvitamin D (25OHD). Glycine, a nonessential amino acid, exerts anti-inflammatory, cytoprotective, and immunomodulatory properties. This study aimed to establish a tandem mass spectrometry assay to measure 25OHD in guinea pigs serum and to investigate the effects of glycine against the liver damage induced by bile duct ligation (BDL). METHODS: BDL was performed on male guinea pigs. Glycine, alanine, serine or tyrosine was given by intraperitoneal injection. The animals were sacrificed and examined at 7 and 14 days after BDL. Serum concentrations of total bilirubin and aminotransferase were measured. Serum concentrations of 25OHD2 and 25OHD3 were measured by API 5000 mass spectrometer. In addition, oxidative stress was assessed by serum ischemia-modified albumin (IMA) and hepatic malondialdehyde (MDA), and apoptosis by hepatic caspase 3 activities. RESULTS: Serum 25OHD concentrations were decreased around 50% in the BDL group at days 7 and 14 post ligation, compared to sham (mean 65.3 ng/ml, p<0.005). Glycine but not other amino acid treatment blunted the reduced serum 25OHD (52.6 ng/ml, p<0.05) resulting from BDL. The concentrations of 25OHD were negatively associated with concentrations of IMA (r=-0.305, p<0.05) and caspase 3 (r=-0.562, p<0.0001). At day-14 post ligation, glycine treatment also ameliorated liver damage indicated by serum AST (p<0.005), ALT (p<0.05) and hepatic caspase 3 activities (p<0.05) and oxidative stress. CONCLUSION: Our results indicate that glycine may protect against BDL-induced liver injury through attenuation of oxidative stress, apoptosis and the vitamin D deficiency.