[LC3-mediated autophagy promotes the occurrence of diabetic gastroparesis by targeting SIRT1].

OBJECTIVES: Diabetic gastroparesis (DGP) is a common complication of diabetes mellitus, and its pathogenesis is complex and has not yet been fully elucidated. This study aims to investigate whether microtubule-associated protein 1 light chain 3 (LC3)-mediated autophagy promotes the progression of DGP by targeting sirtuin 1 (SIRT1).

METHODS: Rat gastric smooth muscle cells (GSMCs) were treated with 35 mmol/L glucose to establish an in vitro model of DGP. Autophagy was inhibited using 3-methyladenine (3-MA). The apoptosis rate, trypan blue-positive cell rate, and cell viability were detected. The expression levels of SIRT1, autophagy-related markers (LC3 II, LC3 I, Beclin-1, and p62), and apoptosis-related proteins [B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and cleaved/non-cleaved caspase-3] were analyzed. The interaction between LC3 II and SIRT1 was validated by co-immunoprecipitation. Six- to 8-week-old SD rats were randomly divided into 4 groups: A normal group, a negative control group, a model group, and a model + 3-MA group (n=6 per group). Gastric pigment retention rate and intestinal propulsion rate were measured, and the levels of SIRT1 and apoptosis-related proteins were detected. Terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was performed to assess the level of apoptosis in gastric tissues.

RESULTS: High glucose treatment increased the apoptosis rate and trypan blue-positive cell rate in GSMCs while reducing cell viability. Meanwhile, high glucose also downregulated the expression levels of SIRT1, Bcl-2, and p62, and upregulated the LC3 II/I ratio, Beclin-1, Bax, and cleaved caspase-3 (all P<0.05). Treatment with 3-MA reversed the effects of high glucose on GSMCs. In GSMCs treated with high glucose and 3-MA, SIRT1 silencing increased the apoptosis rate and trypan blue-positive cell rate while reducing cell viability. The LC3 II/I ratio, Beclin-1, Bax, and cleaved caspase-3 were significantly increased (all P<0.05), whereas the expression levels of SIRT1, Bcl-2, and p62 were significantly decreased (P<0.05). In addition, high glucose treatment decreased nuclear SIRT1 levels (P<0.05) while increasing cytoplasmic SIRT1 levels. Treatment with 3-MA reversed the effects of high glucose on nuclear and cytoplasmic SIRT1 expression. Co-immunoprecipitation results showed that SIRT1 interacted with LC3. Disruption of the LC3-SIRT1 interaction increased nuclear SIRT1 levels while decreasing cytoplasmic SIRT1 levels. Compared with the negative control group, rats in the model group showed a higher gastric pigment retention rate and a lower intestinal propulsion rate. The number of TUNEL-positive cells in gastric tissue was significantly higher in the model group than in the negative control group. In addition, the expression levels of Bax and cleaved caspase-3 were higher in the model group (P<0.05), whereas the expression levels of SIRT1 and Bcl-2 were lower (all P<0.05). Treatment with 3-MA reversed these changes.

CONCLUSIONS: High glucose can downregulate SIRT1 expression in an LC3 II-dependent manner, thereby inducing apoptosis of GSMCs and promoting the progression of DGP.