Genetic and Biochemical basis of Advanced Glycation End Products induced Micro- and Macrovascular Complications in Type 2 Diabetes Mellitus

Abstract

Type 2 Diabetes Mellitus (T2DM) is commonly defined as a complex metabolic disorder marked by hyperglycemia, insulin insufficiency and inflammation. Hyperglycemia complemented by oxidative stress leads to dicarbonyl stress which causes cellular dysfunction and formation of Advanced Glycation End Products (AGEs) resulting in a myriad of vascular complications in T2DM. Methylglyoxal is a potent, highly reactive precursor of all AGEs which is detoxified by Glyoxalase-1. The study focused on Glyoxalase-1 (Glo-1), a critical component of the Glyoxalase system, which serves as the body's primary defense against dicarbonyl stress. Reduced expression or activity levels of Glo-1 have been associated with various diseases, including Type 2 Diabetes Mellitus (T2DM) and its vascular complications. To identify the most damaging missense or nonsynonymous single nucleotide polymorphisms (nsSNPs) in the Glo-1 gene, a computational approach was employed. Several bioinformatic methods were utilized to pinpoint missense SNPs that have the potential to compromise the structural and functional integrity of Glo-1 while being evolutionarily conserved. One specific missense SNP (rs1038747749), resulting in the substitution of Arginine with Glutamine at position 38, was identified as particularly significant. This mutation occurs at key sites within the Glo 1 protein, including the active site, glutathione binding site, and the dimeric interface. Structurally, the mutation replaces a small, neutrally charged Glutamine with a positively charged Arginine. Before conducting molecular dynamics simulations, comparative modeling of both the wild-type and mutant (R38Q) Glo-1 proteins was performed. The results of this modeling indicated that rs1038747749 has a detrimental effect on the stability, rigidity, compactness, hydrogen bonds, and interactions of the Glo-1 protein. This negative impact on various structural and functional parameters was observed during the computational analysis. This study also investigated the interaction of Glo-1 with markers of hyperglycemia and oxidative stress under three states/modalities of dicarbonyl stress: Glo-1 downregulation, Genetic and Biochemical basis of AGEs induced Vascular Complications in Type 2 Diabetes Mellitus xvii Glo-1 expression and exogenous MGO accumulation. The three models of dicarbonyl stress were achieved via siRNA mediated downregulation of Glo-1 under normo- and hyperglycemic conditions in HMEC-1 cell line; Glo-1 expressing transgenic rats and MGO treated mice respectively. In our investigation of siRNA mediated Glo-1 downregulation in HMEC-1 cell line, we observed an upregulation of VCAM expression (p-value < 0.001), and a similar observation was also made in transgenic rats expressing Glo-1 where expression of VCAM was also upregulated (p-value 0.0125). Additionally, we observed a substantial rise in TXNIP expression (p-value 0.008) concomitant to Glo-1 expression under diabetic conditions. These results provide credence to the hypothesis that VCAM is a key marker for diabetic vascular complications and also highlights that TXNIP and Glo 1 are working in synergy perpetuating dicarbonyl stress underlying chronic hyperglycemia which eventually lead to vascular complications in Type 2 Diabetes Mellitus.