Structural and Regulatory Analysis of Angptl8 Using Computational Approaches and the Formal Verification of its Biological Regulatory Network in Insulin Resistance

In this thesis, the regulation of ANGPTL8 has been deciphered at different regulatory levels using computational systems-level approaches in order to gain insights into its physiological and pathological mechanism of action. ANGPTL8 is a recently identified novel hormone and an atypical member of Angiopoietin like (ANGPTL) protein family. Dual nature of ANGPTL8 due to its ability to regulate both glucose and lipid metabolic pathways led to its emergence as a novel molecular player for the possible treatment of Diabetes Mellitus and other related metabolic disorders. Initial research efforts investigating its physiological and pathological roles partly elucidated its functional role in the regulation of metabolic machinery and insulin resistance. Thus, this thesis was designed to provide a clear understanding of the regulation of ANGPTL8 during homeostasis and compensatory insulin resistance. For this purpose, an initial check was performed to verify the association of ANGPTL8 in different murine models of compensatory insulin resistance exhibiting normoglycemia using transcriptomics data analysis. Furthermore, a systematic methodology was employed to construct a regulatory pathway of ANGPTL8 utilizing its reported interaction data. Since, none of the major pathways databases held any interaction data regarding ANGPTL8, this step was a pre-requisite for aiding future genomics studies for which pathways analysis is an integral part of the workflow. This pathway was made available to the research community through WikiPathways (an open source pathways database) and it allowed the visualization of ANGPTL8’s regulation with respect to other genes and proteins in different pathways. This pathway also aided in understanding the complex interplay of novel hormones, genes and proteins in metabolic disorders with regards to ANGPTL8. Moreover, an integrated pathways based analysis was performed on a hepatic transcriptomics data set to identify the co-expressed genes with ANGPTL8, investigate their occurrence in the known pathways, find overrepresented gene ontology classes in these genes and identify the disease linked SNPs in them. The results aided in identification of genes at different levels of regulation regarding ANGPTL8 and have implications for follow up experimental studies. This could aid in identifying additional protein partners and mutual role of these genes in leading to pathogenesis of Diabetes Mellitus and associated metabolic disorders. The signaling dynamics involved in the homeostasis and insulin resistance by ANGPTL8 were further analyzed with the formal approaches of modeling of biological regulatory networks. For this purpose, a discrete mode of ANGPTL8 associated BRN based on kinetic logic of René Thomas’ was constructed and its kinetic parameters were estimated using a model checking approach. This model was converted into a stochastic Petri net in order to simulate its time dependent dynamics. The results of these models provided insights on the role of ANGPTL8 in maintaining normoglycemia through the AKT→ FOXO, AKT→ GSK3-β and AKT→ ChREBP signaling. These results supported the hypothesis on the role of ANGPTL8 in supplementing the insulin signaling pathway during insulin resistance and aggravating the pathogenesis upon its loss. Thus, the results of this study supports its role as a glucose lowering agent and have therapeutic implications for the treatment of Diabetes Mellitus and related disorders. Furthermore, the structural characterization of ANGPTL8 was performed using several computational approaches which was verified after thorough protein-protein interaction analysis based on the available experimental knowledge and the new inferences. The finally selected structure of ANGPTL8 is based on the structure of human p85β-iSH2 domain (PDBentry:3mtt). The human p85β-iSH2 domain is one of the subunits of the regulatory domain (p85) of Phosphatidylinositol3-kinase (PI3K) protein. The protein-protein interaction analysis of ANGPTL8 with its binding partner Lipoprotein lipase revealed a model in which ANGPTL8 inhibits it by steric block of catalytic site and thus interfering with its function. This result is comparable with a similar family member ANGPTL4. In conclusion, this thesis enhanced our current understanding about the functional and structural characterization of ANGPTL8 and will further contribute to its assessment as a drug candidate.