Glycogen metabolic dysfunction in T2DM with MASLD: linking α-hydroxybutyrate to GYS2 downregulation

This study aimed to investigate the molecular characteristics of T2DM with MASLD in a C57 mouse model, using multi-omics techniques to identify key nutritional metabolites that drive metabolic dysfunction. The goal was to reveal how metabolic disturbances are linked to transcriptional reprogramming, providing a new theoretical basis for nutritional biomarker discovery and targeted metabolic interventions.

In this study, a mouse model of T2DM with MASLD was established using a high-fat diet (HFD) combined with intraperitoneal injection of low-dose streptozotocin (STZ). The mice were randomly allocated into three groups: a healthy control group, a MASLD-only group (HFD), and a T2DM with MASLD group (HFD + 40 mg/kg STZ, ip). Serum untargeted metabolomic analysis and hepatic transcriptomic sequencing were performed to identify significantly differential metabolites and transcripts between groups, specifically focusing on α-hydroxybutyrate (α-HB) and glycogen synthase 2 (GYS2). Multi-omics data integration was conducted via Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Pearson correlation analysis. For the validation experiment, both normal mice and T2DM with MASLD mice were further divided into two subgroups, with one subgroup from each cohort receiving α-HB intervention to investigate its effects on GYS2 expression and the progression of T2DM with MASLD.

Metabolomic and transcriptomic analyses revealed that α-HB levels were significantly elevated in T2DM with MASLD mice, and this elevation showed a significant correlation with the downregulation of GYS2. Validation experiments demonstrated that, compared with normal mice, the T2DM with MASLD mice induced by a high-fat diet combined with STZ injection exhibited significantly increased fasting blood glucose (13.14 ± 0.44 vs. 5.08 ± 0.30 mmol/L, p < 0.0001), insulin levels (47.41 ± 1.38 vs. 12.38 ± 1.29 μU/mL, p < 0.0001), serum triglycerides (4.75 ± 0.13 vs. 1.27 ± 0.13 mmol/L, p < 0.0001), and hepatic triglycerides (61.97 ± 3.53 vs. 17.13 ± 1.01 mg/g, p < 0.0001), along with elevated inflammatory factors. GYS2 was significantly downregulated in T2DM with MASLD mice. Compared with saline-treated T2DM with MASLD mice, α-HB-treated T2DM with MASLD mice showed a further downregulation of hepatic GYS2 expression (p = 0.0011). Following α-HB intervention, T2DM with MASLD mice showed a further downregulation of hepatic GYS2 expression compared to saline-treated T2DM with MASLD mice (p = 0.0011). This was accompanied by increased fasting blood glucose (14.70 ± 0.37 vs. 13.14 ± 0.44 mmol/L, p = 0.0230) and insulin levels (53.16 ± 0.90 vs. 47.41 ± 1.38 μU/mL, p = 0.0086), as well as reduced hepatic glycogen synthesis (21.60 ± 0.97 vs. 35.34 ± 1.40 mg/mg, p < 0.0001). Concurrently, serum triglycerides (5.63 ± 0.50 vs. 4.75 ± 0.13 mmol/L, p = 0.0160), hepatic triglycerides (70.53 ± 0.69 vs. 61.97 ± 3.53 mg/g, p < 0.0398), and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) (9.69 ± 0.62 vs. 45.70 ± 2.50 ng/L, p = 0.0013) and interleukin-6 (IL-6) (55.87 ± 5.39 vs. 37.68 ± 0.78 ng/L, p = 0.0029) were all significantly elevated.

This study reveals that α-HB, a metabolite elevated under conditions of nutrient overload and insulin resistance, plays an important role in the progression of T2DM with MASLD. It may contribute to a vicious cycle by impairing hepatic energy storage and flux, likely through affecting the activity and expression of GYS2. This finding provides a nutritional metabolism perspective for understanding the T2DM with MASLD comorbidity and suggests α-HB as a potential target for nutritional strategies or precision nutrition approaches.