Aims: Glucokinase (GCK) acts as the glucose sensor in maintaining glucose homeostasis. The inactivating mutation of the GCK gene leads to glucokinase-maturity onset diabetes of the young (GCK-MODY). This study aims to gain further insights into the molecular alterations triggered by GCK partial inactivation in hepatocytes, potentially underlying the favorable prognosis of GCK-MODY.
Main methods: A GCK knockdown HepG2 cell model was established, and the integration of proteomics and metabolomics was used to gain a comprehensive understanding of the molecular pathway changes caused by GCK inactivation in the liver.
Key findings: Proteomic analysis identified 257 differential proteins. KEGG pathway enrichment analysis showed that protein expression changes in the GCK knockdown group were significantly enriched in central carbon metabolism, the TCA cycle, amino acid metabolism and the oxidative phosphorylation pathway. Among them, enzymes in the TCA cycle (PC, IDH2, SDH) were significantly downregulated in GCK-knockdown group. Targeted metabolomics revealed that in the GCK knockdown hepatocytes, TCA cycle intermediates were significantly decreased, including pyruvate, oxaloacetate, citrate and succinic acid, and three metabolites increased including glycine, betaine and homocysteine. These metabolic alterations in turn reduced the accumulation of reactive oxygen species in GCK knockdown hepatocytes. Correlation analysis indicated that TCA cycle metabolites were positively correlated with proteins involved in the TCA cycle, carbon metabolism, glycolysis, Ras signaling, fibrosis and inflammation.
Significance: In conclusion, GCK knockdown reduced TCA cycle flux and oxidative stress in hepatocytes by influencing the levels of key transcription factors and enzymes, providing a comprehensive understanding of the effects of GCK partial inactivation on liver metabolism and molecular mechanisms.
Keywords: GCK-MODY; Glucokinase; Liver; Metabolomics; Proteomics; TCA cycle.
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