Identify of blood glucose metabolism regulation pathways and related proteins in the db/db mouse model through iTRAQ quantitative mass spectrometry.

Abstract

Background: Diabetes cardiomyopathy (DCM) has become the main cause of death of diabetes patients due to heart failure. As the initial unclear symptoms and complex underlying pathological mechanisms, it presents significant challenges for early diagnosis. It is essential to explore valuable biomarkers to enhance our understanding involved in DCM.

Methods: Twelve-week-old db/db model mice (diabetes group) and normal mice (control group) were maintained in a specific pathogen-free (SPF) environment. Body weight, blood glucose, and insulin levels were measured regularly. At 26 weeks, cardiac tissue was collected to assess oxidative stress, inflammatory factors, and fibrosis markers, followed by histopathological examination. Meanwhile, iTRAQ-based quantitative mass spectrometry was employed to identify differentially expressed proteins (DEPs) in cardiac tissue. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) methods were utilized to analyze their biological functions and signaling pathways. Furthermore, specific proteins and their associated signaling pathways related to blood glucose regulation were validated in cardiac tissue and myocardial cells using western blot and immunofluorescence analysis.

Results: The mice in the control group were active and exhibited healthy growth, whereas those in the diabetes group showed increased food and water intake. Furthermore, there were significant elevations in blood glucose concentration, insulin levels, and body weight in the diabetes group. Histopathological examinations revealed that the myocardium in the diabetes group was markedly hypertrophic due to persistent hyperglycemia, accompanied by muscle fiber disarray, nuclear damage, and a significant increase in the expression of oxidative stress and inflammatory factors. Mass spectrometry analysis identified a total of 107 DEPs, comprising 83 up-regulated and 24 down-regulated proteins. Notably, the most significant difference was observed in the regulation of the glycogen metabolism enzyme (PYGM). GO and KEGG analyses indicated that the DEPs were primarily involved in glycogen metabolism, catalysis, and oxidative stress, with signaling pathways related to fatty acid metabolism, the PPAR pathway, insulin resistance, and the tricarboxylic acid cycle. Subsequent immunofluorescence and western blot analysis confirmed that hyperglycemia inhibits the PI3K/AKT signaling pathway and upregulates the expression of PYGM. Conversely, the knockout of PYGM significantly enhanced the activity of the PI3K/AKT signaling pathway.

Conclusion: The research suggests that the inhibition of PYGM enhances the activity of the PI3K/AKT signaling pathway in patients with DCM, and then help to reduce blood glucose levels. This molecular mechanism exerts a protective effect on DCM. These findings highlight the potential of targeting PYGM as a novel biomarker for the early diagnosis, which may serve as an effective therapeutic strategy.