AMPK Pathway-Mediated Autophagy Regulates Osteoclast Formation And Function In High Glucose Conditions

Objective:The richness of material life and the extension of human life expectancy make the threat of chronic diseases to health occupy a more prominent position.Among them,diabetes mellitus(DM)is a typical representative disease.Diabetic Osteoporosis(DO)is one of the most important complications of diabetes.Osteoporosis(OP)is a disease characterized by increased bone resorption and reduced bone formation.The decreasing bone mass and the change of micro-structure in bone tissue result in increased bone fragility and increased fracture risk.Hyperglycaemia is a common feature of type 1 and type 2 diabetes and may be a factor that increases the risk of fracture.It is generally accepted that glucose promotes osteoclast formation and enhances its function,but studies by some scholars have shown that high glucose inhibits osteoclasts.Therefore,there is still controversy over the effect of high glucose on osteoclasts.Autophagy,a process that is primarily responsible for the elimination of damaged proteins and organelles,is now widely recognized as an anti-aging program.Additionally,autophagy is also considered a kind of stress response mechanism to maintain homeostasis.Autophagy also plays an important role in osteoclasts.Beclin-1,an autophagy protein,acts on RANKL-related osteoclast formation by inducing the expression of ROS and NFATc1 genes,and knockout the osteoclasts of Atg5,Atg7 and LC3 decreased the cell-specific protein CTSK secretion and specific folds structure.AMPK pathway is a major metabolic regulatory pathway,its activation or not in response to the amount of glucose related.At the same time,this pathway can regulate autophagy by regulating the downstream m TORC1 and ULK1 elements.Therefore,the mainly aims of the study is to investigate the effects of high glucose on osteoclast formation and function;the role of autophagy on the formation and function of osteoclasts in high glucose environment and observe the changes of related signal pathways;and to explore the molecular mechanisms of the high glucose environmental effects on the formation and function of osteoclasts.Methods: CCK8 assays detected the effects of different concentrations of glucose(5.5,10.5,15.5,20.5,25.5,and 30.5 m M/L)at various time points(days 1 to 6)on theviability of RAW264.7 cells;Western Blotting method was used to detect the expression of CTSK in osteoclasts which induced by different concentrations of glucose at the fourth day;the antitartaric acid phosphatase staining kit was used to detect the effect of different concentrations of glucose on the mumbers of osteoclasts induced at the fourth day;the effects of different concentrations of glucose on osteoclast absorption at the fourth day were examined using hydroxyapatite matrix bone resorption plate.Western blotting was detected the expression of p-AMPK,AMPK,p-m TOR,m TOR,p-ULK1,ULK1,LC3I/II and P62 which are AMPK pathway related proteins and autophagy-related proteins of osteoclasts in low glucose and high glucose concentrations in absence or presence of AMPK pathway inhibitor Compound C;western blotting was used to detect autophagy-related protein Beclin-1,LC3I/II and P62 of osteoclasts induced by low glucose(5.5m M/L)and high glucose(30.5m M/L);the number of osteoclasts autophagosomes induced at the fourth day in low and high glucose concentrations was observed by a transmission electron microscope;the number of LC3 puncta in osteoclasts at the fourth day in low and high glucose concentrations was observed by immunofluorescence staining;western blotting was detected the effects of autophagic inhibitors 3-methyladenine(3-MA)and bafilomycin A1(baf A1)and autophagic activation rapamycin(rapa)on the expression of LC3I/II,P62 and CTSK on the fourth day,and antitartaric acid phosphatase staining kit was used to detected the number of osteoclasts induced at the fourth day.Results: There were no significant effects on the viability of RAW264.7 cells from six different concentrations of glucose medium from 5.5 m M/L to 30.5 m M/L,but it was found that as the concentration of glucose increased,the expression of osteoclast function protein CTSK,the number of osteoclasts and bone absorption areas gradually decreased.After low-glucose condition(5.5m M/L)and high-glucose condition(30.5m M/L)induction medium induced osteoclast formation for 4 days,the expression level of AMPK/m TOR/ULK1 signaling pathway in osteoclasts under high glucose condition was lower than that in the low glucose condition.AMPK pathway inhibitor Compound C can inhibit the expression of autophagy protein LC3 II and increase the expression of P62 in the low-or high-glucose condition.After 4 days of osteoclast formation induced by low glucose and high glucose condition,the expression of autophagy-related proteinsBeclin-1 and LC3 II were increased and the expression of P62 was decreased under low glucose condition compared with the high glucose ccondition.Transmission electron microscopy and immunofluorescence staining revealed that the number of autophagosomes in osteoclasts was lower in low-glucose condition than in high-glucose condition.Using autophagy inhibitors 3-MA and baf A1 and the autophagy agonist Rapa on the osteoclastogenesis process,it was found that 3-MA and baf A1 can inhibit the expression of CTSK and osteoclast formation,while Rapa can promote the expression of CTSK and osteoclast formation compared with the control group.Conclusion: 1.Changes in glucose concentration did not affect RAW264.7 cell viability,but high glucose inhibited the formation and function of osteoclasts.2.AMPK/m TOR/ULK1 signaling pathway was inhibited in a high glucose condition and it was involved in the regulation of autophagy in osteoclasts.3.High glucose condition inhibited autophagy during the formation of osteoclasts,thereby weakening the formation and function of osteoclasts.