Preliminary Study On The Effect Of PQQ And Its Modification On Insulin Signaling Pathway In C2C12 Cells

Insulin resistance refers to the emergence of insulin resistance,although the body's blood insulin concentration remains unchanged or increased,the body's sensitivity to insulin is significantly reduced.Insulin resistance can lead to various diseases such as type 2 diabetes,cardiovascular diseases.Metformin has been the drug of choice for the treatment of type 2 diabetes.Its mechanism of action is mainly related to activating AMPK,inhibiting the hepatocyte's glucagon signal to inhibiting the liver output,but metformin can cause lactic acidosis and stomach.Intestinal reaction.The side effects of drugs are inevitable,and researchers are always looking for more efficient drugs.Peripheral insulin can cross the blood-brain barrier through the specific transport system into the central nervous system.If the use of glucose in the central nervous system of the insulin receptor is hindered,it will disturb the transmission of signals in the central nervous system and lead to abnormal cognitive function,eventually leading to the development of Alzheimer's disease.Therefore,some experts also claim that Alzheimer's disease is called type 3 diabetes.Pyrroloquinoline quinone(PQQ)is an effective growth factor in bacteria and higher organisms.Preclinical studies have shown that in the absence of PQQ,animals exhibit developmental delay,decreased immunity,impaired fertility,and the most important is the reduction in the number of mitochondria in the tissue,while adding PQQ in the diet can reverse this phenomenon to restore system function,increasing the number of mitochondria and energy efficiency.Mitochondrial dysfunction has a close relationship with the occurrence of type 2 diabetes,but the mechanism is still not clear.It has been reported that PQQ can influence the insulin signaling pathway by inhibiting the activity of protein tyrosine phosphatase B1,and that PQQ can also affect glucose tolerance in rats.AMPK(AMP-dependent protein kinase)is a key molecule in the regulation of bio-energy metabolism and is the core of the study of diabetes and other metabolic-related diseases.Genetic and pharmacological studies have shown that AMPK is essential for the body to maintain glucose balance,and that activation of AMPK by drug molecules can ameliorate the metabolic imbalance caused by type 2 diabetes.Therefore,we speculate that PQQ may affect insulin signaling through activation of AMPK and increase insulin sensitivity.We have modified PQQ to generate a series of derivatives and screened them for the effect of PQQ on diabetes is not obvious.Finding which can significantly activate the remodeling of the AMPK protein and study its role in the insulin signaling pathway.We studied the PQQ remodelling at the cellular level to investigate the role of PQQ remodeling in insulin sensitivity and selected C2C12 and N2a-tau cells as model cells for study.Experiment content and results:1.Determine the optimal concentration and optimal time of PQQ in H292 cells and C2C12 cells,which is mainly determined by Western Blot detection of P-AMPK protein expression,and finally determine the optimal time was 8h and the optimal concentration was 5?M of PQQ in H292 cells.In C2C12 cells,the optimal time for PQQ was 4h,and the optimal concentration was 50?M.2.Selecting effective drugs from the deconstructor and repeating experiments in C2Cl2 cells to detect the protein expression of P-AMPK and P-S6 under different concentrations of drugs.The results showed that the effect of 143 was the most obvious.Therefore,143 was selected as a novel compound for research.3.The effect of 143 on the activity of C2C12 cells was detected by MTS.The results showed that at low concentrations,cell growth was promoted,and as the concentration increased,cell growth began to be inhibited,and using Origin 8 software to analyze the median effective concentration of it was 10.42 ?M.4.In C2C12 cells treated with different concentrations of 143,the P-Akt activity was detected.In the condition of serum starvation and no serum starvation,after adding different concentrations of insulin for stimulation,it was found that 143 can increase the expression of P-Akt and activate the pathway of insulin signaling.5.The above experiment shows that 143 can activate AMPK to inhibit p70S6K and then activate Akt,but it is unclear whether P-AMPK activation is a direct or indirect effect,we use AICAR and Compound.C conducted further research and found that 143 activation of P-AMPK was not a direct effect in C2C12 cells.6.Insulin can not only maintain the balance of blood sugar in the periphery,but also regulate the blood sugar in the central nervous system.Therefore,we examined the effect of 143 on protein expression in the insulin signaling pathway in N2a cells.As a result,it was found that in N2a cells 143 can activate P-AMPK and P-S6,but there is no activation of P-Akt which is inconsistent with the results in C2C12 cells.Conclusion:We initially confirmed that 143 can effectively increase insulin sensitivity,probably by activating P-AMPK and inhibiting p70S6K,then negatively feeding PI3K-Akt signaling pathway.It has a certain potential in the treatment of glucose metabolism,which provides new drugs for the treatment and prevention of type 2 diabetes.