Lipopolysaccharides mediate autophagy of mouse insulinoma βtc6 cells in high glucose state

doi:10.12307/2022.609

Abstract

Abstract: BACKGROUND: Our previous studies have confirmed that diabetes mellitus promotes the occurrence and development of periodontitis. However, it is unclear whether periodontitis can promote the development of diabetes.
OBJECTIVE: To explore the molecular mechanism by which periodontitis promotes the development of diabetes mellitus.
METHODS: Mouse βtc6 insulinoma cells cultured in vitro were divided into control group, glucose group, lipopolysaccharide group, and glucose+lipopolysaccharide group. The cells in the latter three groups were treated with different concentrations of glucose (0, 25, 50, 100 mmol/L) and lipopolysaccharide (0, 10, 20, 40 mg/L) alone or in combination for 12 hours. Insulin secretion was measured in each group. In addition, 3-methyladenine (an autophagy inhibitor, 5 μmol/L) and rapamycin (an autophagy activator, 10 mmol/L) were used to intervene with the phosphatidylinositol 3-kinase/rotein kinase B/the mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. The cells were then further divided into rapamycin+lipopolysaccharide group, rapamycin+glucose group, rapamycin+glucose+lipopolysaccharide group, 3-methyladenine+lipopolysaccharide group, 3-methyladenine+glucose group, and 3-methyladenine+glucose+lipopolysaccharide group. The cell counting kit-8 method was used to detect the proliferation of βtc6 cells. Fluorescent probes were used to detect the amount of reactive oxygen species in βtc6 cells. Production of autophagosomes in βtc6 cells was observed by transmission electron microscopy. Western blot method was used to detect the expression of autophagy proteins and PI3K/AKT/mTOR signaling pathway-related proteins.
RESULTS AND CONCLUSION: The insulin secretion was out of control as the glucose concentration exceeded 50 mmol/L (P > 0.05), whereas the insulin secretion was inhibited as the lipopolysaccharide concentration exceeded 20 mg/L (P < 0.05). After addition of 3-methyladenine, the expression levels of Becline1, p-PI3K, p-AKT/AKT, p-AKT/AKT, p-AKT/AKT, and p-PI3K were significantly decreased in the 3-methyladenine+glucose group, 3-methyladenine+lipopolysaccharide group, and 3-methyladenine+lipopolysaccharide+glucose group compared with the control group. While the expression of p62 protein showed the opposite trend (P < 0.05). After addition of rapamycin, the expression of Becline1, p-PI3K, and p-mTOR/mTOR was significantly increased in comparison with the control group (P < 0.05). These findings indicate that lipopolysaccharides can induce excessive autophagy of pancreatic β cells and downregulate insulin secretion. This mechanism may be related to the silencing of PI3K/AKT/mTOR signaling pathway.

Key words: periodontitis, diabetes mellitus, lipopolysaccharide, autophagy, 3-methyladenine, PI3K/AKT/mTOR signaling pathway

CLC Number:

Cai Zhiguo, Du Shasha, Yang Kun, Zhao Na, Liu Qi. Lipopolysaccharides mediate autophagy of mouse insulinoma βtc6 cells in high glucose state[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(20): 3127-3132.

Figures/Tables 7

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