Mechanism of AMPK-mediated apoptosis of rat gastric smooth muscle cells under high glucose condition.

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)-ß3, CaMKII, CaMKIV and eukaryotic translation initiation factor 4E binding protein1 (4E-BP1) in rat gastric smooth muscle cells.

Correction to: Effects of AMPK on Apoptosis and Energy Metabolism of Gastric Smooth Muscle Cells in Rats with Diabetic Gastroparesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effects of AMPK on Apoptosis and Energy Metabolism of Gastric Smooth Muscle Cells in Rats with Diabetic Gastroparesis.

This study aimed to investigate the effect of AMPK on apoptosis and energy metabolism of gastric smooth muscle cells in diabetic rats and to explore the role of AMPK in the pathogenesis of diabetic gastroparesis (DGP). After establishment of a diabetic rat model, rats were divided into normal control (NC), 4-week (DM4W), 6-week (DM6W), and 8-week (DM8W) diabetic model groups. The gastric residual pigment ratio, intestinal transit rate, and intestinal propulsion rate in each group were detected to confirm the successful establishment of the DGP model. The spontaneous contraction in isolated gastric smooth muscle strips of the NC and DM8W groups was experimentally observed. The expression of phospho-AMPK, AMPK, phospho-LKB1, LKB1, phospho-TAK1, TAK1, and CaMMKß in rat gastric smooth muscle tissues was detected by western blot analysis; ADP, AMP, ATP contents, and the energy charge were detected using Elisa; and apoptosis of gastric smooth muscle cells was detected by flow cytometry. The rat gastric smooth muscle cells were cultured in vitro, and treated with an AMPK inhibitor and an agonist. At 24 and 48 h, the effects of AMPK on apoptosis and energy metabolism of gastric smooth muscle cells were observed. Reduced spontaneous contractions, AMPK activation, cell apoptosis, and energy metabolism disorders were observed in gastric smooth muscle tissues of a diabetic rat, and AMPK activation was associated with an increased ratio of ADP/ATP, AMP/ATP, LKB1 activity, and CaMMKß expression. From in vitro cell culture experiments, we found that AMPK activation of high-glucose conditions promoted cell apoptosis. Inhibition of AMPK had no obvious effect on apoptosis at the early stage with high glucose, but the inhibitory effect was significant at the late stage with high glucose. AMPK can regulate both mitochondrial metabolism and glycolysis pathways under high-glucose conditions. During the early stage with high glucose, AMPK was the main promotion factor of the mitochondrial metabolism pathway, but did not increase the ATP production, AMPK also promoted the glycolysis pathway. During the late stage with high glucose, AMPK was a major inhibitor of the mitochondrial pathway, and still played a role in promoting the glycolytic pathway, which acted as the main regulator. Apoptosis and energy metabolism disorders were present in gastric smooth muscle cells during the occurrence of DGP. Under high-glucose condition, AMPK was activated, which can promote apoptosis, change the energetic metabolism pathway of cells, inhibit mitochondrial energy metabolism, and promote glycolysis.

Effects of insulin-like growth factor-1 on endoplasmic reticulum stress and autophagy in rat gastric smooth muscle cells cultured at different glucose concentrations in vitro.

The purpose of the study was to observe changes in endoplasmic reticulum stress (ERS)- and autophagy-related proteins in gastric smooth muscle tissues of diabetic rats with gastroparesis, investigate the effect of insulin-like growth factor 1 (IGF-1) on ERS and autophagy in rat gastric smooth muscle cells cultured under different glucose concentrations, and explore the influence of IGF-1 on development of diabetic gastroparesis (DGP). After establishing a rat model of DGP, rats were divided into normal control (NC) and 6-week diabetic model (DM6W) groups. Expression of ERS-related and autophagy-related proteins was detected by western blot analysis and immunofluorescence assay in rat gastric smooth muscle tissue and in vitro-cultured rat gastric smooth muscle cells exposed to different glucose concentrations and treatment with IGF-1 for 24 or 48 h. Changes in glucose-regulated-protein-78 (GRP78), growth arrest and DNA damage-inducible gene 153 (CHOP), and microtubule-associated protein 1A/1B light chain 3B (LC3) expression levels were detected by western blot analysis, and GRP78 and LC3 expression were examined by confocal laser-scanning microscopy. In vivo expression levels of GRP78, CHOP, and LC3 were significantly higher in the DM6W group compared with the NC group (p < 0.001). Twenty-four hours after cells were cultured at different glucose concentrations in vitro, expression of GRP78, CHOP, and LC3II/I was significantly higher in the high glucose-treated group compared with the normal glucose group (p < 0.05). After IGF-1 intervention, CHOP and GRP78 expression were significantly higher in the normal glucose + IGF-1 group compared with the normal glucose group (p < 0.01), while no significant difference was found between high glucose and high glucose + IGF-1 groups. LC3II/I expression was significantly lower in the normal glucose + IGF-1 group compared with the normal glucose group, and was significantly lower in the high glucose and high glucose + IGF-1 groups (p < 0.05). After 48 h of culture, CHOP expression was significantly higher and LC3II/I expression was significantly lower in the high glucose group compared with the normal glucose group (p < 0.05), but no significant change in GRP78 expression was observed between these two groups. After IGF-1 intervention, there was no difference in CHOP or GRP78 expression between normal glucose + IGF-1 and normal glucose groups. However, CHOP and GRP78 expression were significantly lower in the high glucose + IGF-1 group compared with the high glucose group (p < 0.05). There was no significant difference in LC3II/I expression between normal glucose + IGF-1 and normal glucose groups, or high glucose + IGF-1 and high glucose groups. Results of confocal laser-scanning microscopy showed significantly lower expression of LC3II/I in the high glucose + IGF-1 group compared with the high glucose group (p < 0.05). ERS and autophagy were involved in the occurrence of DGP. IGF-1 exerted an inhibitory effect on ERS in rat gastric smooth muscle cells cultured under high glucose conditions, and this inhibitory effect increased with time. IGF-1 inhibited the level of autophagy in rat gastric smooth muscle cells cultured under high glucose conditions at early stages, which may be achieved through inhibition of ERS.

The role of CNP-mediated PKG/PKA-PLCß pathway in diabetes-induced gastric motility disorder.

Our previous work demonstrated that the C-type natriuretic peptide (CNP)/cyclic guanosine monophosphate (cGMP)/cyclic adenosine monophosphate (cAMP) pathway in gastric antrum smooth muscle of rats with diabetes was upregulated and played an important role in the development of diabetic gastric dysmotility. Our goal for this study was to explore the downstream signaling pathways of CNP. We found that the expressions of protein kinase G (PKG) and protein kinase A (PKA) in gastric smooth muscle tissue of rats with diabetes were significantly upregulated. The expressions of ß-type phospholipase C 3(PLCß3) and ß-type phospholipase C 1(PLCß1) protein were reduced, whereas Phosphor-PLCß3Ser1105 (P-PLCß3Ser1105) was increased. The inhibitory effect of CNP on gastric antral smooth muscle in diabetic rats was significantly greater than in the normal group. The content of trisphosphate inositol (IP3) in the gastric antral smooth muscle of rats with diabetes was significantly lower than that of the normal group. After blocking PKA with N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89, a blockage PKA), the effect of CNP on the production of IP3 was decreased, while blocking PKG with KT5823 (a blockage PKG) simultaneously, and CNP can no longer reduce the IP3 production. CNP promoted the phosphorylation of PLCß3Ser1105, thereby inhibiting the activity of PLCß3 in gastric smooth muscle tissue of rats with diabetes; this effect can be abolished by blocking PKA and PKG. These results suggested that CNP can decrease IP3 level in gastric smooth muscle cells and thus inhibit gastric smooth muscle contraction through PKG/PKA-PLCß pathway, which may play an important role in the development of diabetic gastroparesis.