Metabolic Effects of TNF(alpha), HIV Protease Inhibitor, and TZD Treatment on Primary Adipocytes

摘要

Type II diabetes (T2D) has been characterized as epidemic disease. While T2D is not life-threatening, it affects many major organs, therefore, leading to many serious complications such as CVD, neuropathy, nephropathy, and eye damage etc. Further investigations on disease pathology and treatment effects are needed in preventing T2D complications. Adipocyte dysfunction is one of the hallmarks of T2D and a high risk factor for CVD, however, much of our knowledge of adipocyte metabolism is derived from rodent cell models and through individual endpoint assays (e.g. glucose uptake), and may not translate to comprehensive human biology. Metabolomics characterization of perturbation effects of human adipocytes would provide rich metabolic pathway information for studying diabetes and facilitating drug discovery and therapeutics. Differentiated adipocyte cells, derived from adult mesenchymal stem cells, were cultured in adipogenic maintenance medium containing 10 mM glucose, 120 pM insulin, and then followed by 4 or 7 days of treatments. TNF(alpha), thiazolidinedione (TZD), and HIV protease inhibitor were used to stimulate and inhibit lipolysis, and destroy mitochondria function respectively. Three additional treatments with metformin, rapamycin, and rapamycin + TNF(alpha), were conducted to assess and classify overall treatment effects. Metabolites from collected cells were extracted with 80percent methanol and dried under N_(2). The dry metabolite extract was reconstituted with recon-solution containing internal standards and analyzed by a 10-min LC/MS/MS based method, which simultaneously measure more than 200 metabolites representing multiple metabolic pathways. Principal component analysis on metabolomics data from adipocytes shows separation in metabolic consequence resulted from TZD, TNF(alpha) and HIV protease inhibitor treatment. Interestingly, the difference resulted from different treatment compound is found to be smaller, compared to the difference resulting from treatment times or different donor. Hierarchical cluster analysis shows metformin and rapamycin result in metabolic phenotypes closer to TZD as expected. Metabolic phenotyping and clustering analysis also reveals TNF(alpha) has larger metabolic impact than rapamycin when combined in treatment. Metabolomics analysis generated sets of metabolites which changed upon TNF(alpha), TZD, and HIV protease inhibitor treatments. More metabolite levels increased after TNF(alpha) treatment, more metabolite decreased after TZD treatment, and fewer metabolites changed under HIV protease inhibitor treatment. Comparison of altered metabolites under TNF(alpha) and TZD treatment shows the majority of the altered metabolites are different, with a small set of metabolites in common across the two treatments. The list of altered metabolites will be presented. Further data analysis at the pathway level shows TNF(alpha) and TZD have an opposite impact on: TCA, OxPhos, Nicotinate/Nicotinamide Metabolism, Pyruvate Metabolism, Pentose/Glucuronate interconvention, Ala and Asp metabolism, and glycophospholipid metabolism. Neither treatment affected nucleotide metabolism & glycolysis. In addition, only TZD treatment results in changes of pentose phosphate pathway, and only TNF(alpha) treatment leads to observed effects on urea cycle, glutathione metabolism. No clear metabolic pathway alteration was observed after HIV protease inhibitor treatment under current experiment condition, longer treatment time may be needed.