Study of protein biomarkers, metabolites and metals in type 2 diabetes in humans and cell culture system

Diabetes mellitus is a complex metabolic disorder, characterized by hyperglycemia, resulting due to the defects in insulin secretion or action or both. Diabetes has different types owing to the underlying sub-clinical and molecular etiology; and type 2 diabetes (T2D) is the predominant form of diabetes (~90% of all diabetics). T2D generally develops at the age of ?40 years and is strongly linked to obesity due to unhealthy food intake and/or physical inactivity. Prior to the appearance of clinical symptoms, T2D starts to develop through an asymptomatic phase of 7-10 years as prediabetes, which remains undiagnosed clinically. At clinical stage, it is diagnosed by blood glucose, which is a late marker, and substantial damage has already been caused to different tissues/organs at the molecular levels. In the uncontrolled diabetes, patients develop severe complications (e.g. blindness, stroke, heart attack, kidney failure, lower limb amputations etc). Hence, there is a need to discover/identify and validate new biomarkers for T2D or its main precursor obesity, before the development of clinically irreversible symptoms. These biomarkers could be used either as early indicators to monitor the progress of pathological process or as novel drug targets to arrest the disease at early time points during sub-clinical stages. Therefore, in this study, we aimed to identify and/or validate protein and analyte (metabolite/metal) based biomarkers for T2D or its major precursor obesity. In this regard, we collected blood and urine samples from non-diabetic (ND) controls and T2D patients. In the protein based biomarker study, alpha-1 antitrypsin (A1AT) which is an acute phase protein (secreted by the liver during inflammation) was validated in human serum samples. A1AT was selected because it was the strongly linked biomarker from a proteomic study in a mouse model (ob/ob mouse model, leptin deficient) of obesity and T2D. Hence, levels of A1AT were measured in obese diabetics and controls by ELISA and quantitative Western Blot (qWB) analysis, and it was found to decrease linearly with obesity in controls but not in diabetics. Moreover, A1AT relationship with leptin was investigated in human serum samples and in the liver (HepG2) cell lines in a functional genomics approach by gene expression (qPCR and qWB) analysis of A1AT. It was found, that A1AT is regulated by leptin stimulation, and thus leptin plays a role in the JAK-STAT signaling pathway, which is activated to control inflammation, and in obesity there is also a low-grade inflammation. Urine samples were analyzed using a metabolomics approach by proton NMR spectrometry and it was found that there were significant differences in some of the metabolite resonances, which differentiated diabetics from the controls.Similarly, these samples were also analyzed to see the changes in metallevels in diabetes. In this study, several metals (e.g. Zn, Mn, Mg, Fe, Cu, Cr, Ni, Pb) were analyzed using atomic absorption spectrophotometry. Significant differences were found for some of the metals associated with diabetes.\ud In conclusion, this study resulted in some new information about the biology of A1AT in obesity and its regulation by leptin, as well as differences of some metals and metabolites in diabetes. This knowledge would help in future to early diagnose, manage or treat obesity or type 2 diabetes more effectively, thus reducing the associated morbidity/mortality