Abstract
To observe changes in AMP-activated protein kinase (AMPK) activity and phosphorylation changes in AMPK signaling pathway in gastric smooth muscle cells of rats with diabetic gastroparesis (DGP), investigate the effect of AMPK on apoptosis and explore the underlying mechanism. After establishing rat model of DGP, rats were divided into normal control (NC) and DGP groups. The phosphorylation changes in AMPK pathway were detected by AMPK Signaling Phospho-Antibody Array, and the apoptosis-related proteins were determined. Rat gastric smooth muscle cells were cultured in vitro under different glucose conditions, and divided into normal and high glucose groups. The AMPK activity and intracellular Ca2+ changes in cells were observed. After AMPK silencing, cells were divided into high glucose-24h, high glucose-48h and high glucose-48h+siRNA groups. Changes in expression of apoptosis-related proteins were observed. AMPK activity and apoptosis rates were both increased in gastric smooth muscle tissues in DGP rats (P<0.05, P<0.001, respectively). A total of 14 apoptosis-related differentially phosphorylated proteins were identified. Under high-glucose condition, AMPK activity and intracellular Ca2+ concentrations in rat gastric smooth muscle cells were increased (both P<0.05). After AMPK silencing, p53 expression was decreased, Akt and p70 S6 ribosomal protein kinase (p70S6K) activities were were increased, Bcl-2 expression was increased, CaMKII activity was decreased in the high glucose-48h group. Under high-glucose condition, activated AMPK can directly or indirectly promote cells apoptosis by regulating the expression and activity of p53, Akt, p70S6K, Protein kinase A (PKA), Phospholipidol C (PLC)-β3, CaMKII, CaMKIV and eukaryotic translation initiation factor 4E binding protein1 (4E-BP1) in rat gastric smooth muscle cells.
Highlights
Diabetic gastroparesis (DGP) is one of the common complications of diabetes, which is characterized by decreased gastric motility-induced delayed gastric emptying
In order to explore the effect of AMP-activated protein kinase (AMPK) on apoptosis of gastric smooth muscle cells in DGP rats and the underlying mechanism, we first prepared a rat model of DGP diabetes, and observed the apoptosis of gastric smooth muscle cells in DGP rats, the results showed that the apoptosis rate of gastric smooth muscle cells in DGP rats was significantly higher than that in the normal control group, which suggested that apoptosis is involved in the development of DGP
The results showed that AMPK activity was higher in normal glucose-48h group than that in normal glucose-24h group, which was higher in high glucose-48h group than that in high glucose-24h group, but AMPK activity was lower in high glucose-24h group than that in normal glucose-24h group, as well as in high glucose-48h group than that in normal glucose-48h group, the results indicated that AMPK activity was gradually increased with time prolonged under either normal nor high glucose conditions, the AMPK activity was increased in a time-dependent manner
Summary
Diabetic gastroparesis (DGP) is one of the common complications of diabetes, which is characterized by decreased gastric motility-induced delayed gastric emptying. Gastric smooth muscle motility is regulated by nerve and body fluids, the contribution of the functional status of gastric smooth muscle cells in regulating smooth muscle motility cannot be neglected. The contractile activity of gastric smooth muscle is the power source of gastric emptying, and the effective gastric smooth muscle cell numbers and sufficient energy supply are critical for gastric smooth muscle contraction. Finding certain factors affecting gastric smooth muscle cell apoptosis and determining the relevant mechanisms have a positive role in the treatment of DGP and even diabetes. AMP-activated protein kinase (AMPK) is expressed in various tissues and organs and can be activated by multiple stimuli. AMPK plays an important role in regulation of many physiological and pathological
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