Insulin resistance (Insulin Resistance, IR) refers to the decreased sensitivity of insulin target tissue (adipose tissue, skeletal muscle, liver) to insulin. In the early stage, islet
The insulin signal pathway is closely related to the occurrence of insulin resistance. Changes in the activity of key signal molecules in the insulin signal pathway will affect the transmission of insulin signal, and then affect the biological activity of insulin, thus affecting the occurrence of insulin resistance and T2DM. A large number of studies have confirmed that there are two main pathways of insulin postreceptor signal transduction: one is the IRS-1-PI3-K-Akt pathway; the other is RAS mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) pathway. MAPK pathway is mainly related to gene transcriptional regulation, and insulin mainly mediates its metabolic regulation through the PI3-K pathway. As the main signal pathway of insulin, IRS-1-PI3-K-Akt pathway is involved in the physiological mechanism of glucose metabolism in vivo [
At present, more than 50 kinds of lysosomal membrane proteins have been found [
3T3-L1 adipocytes were purchased from the Cell Resource Center of Shanghai Academy of Life Sciences, Chinese Academy of Sciences, while C2-C12 myoblasts and HEPA1-6 hepatoma cells were purchased from ATCC cell bank. 3T3-L1 adipocytes, C2-C12 myoblasts, and HEPA1-6 hepatoma cells were cultured in a DMEM medium containing 10%FBS in an incubator at 37°C and 5% CO2. When the cells grew to the logarithmic phase and the density was 80%, they could be used in the experiment. The above three kinds of cells were transferred to a six-well plate and cultured to about 60% of the cell density, and the lentivirus prepared in advance for knocking out sidt2 and having puro resistance was added to the culture medium. In order to make the lentivirus better-transfected cells, the cells were not treated within 48 hours, and the culture medium could be replaced after 48 hours. 72 hours after the virus was added, the new medium was replaced, and an appropriate amount of puro was added to it to screen cells (the amount of puros varied according to different cell lines). At the same time, it was necessary to add polybrene, to increase the transfection efficiency of the virus. The cells were screened by adding puro for three consecutive days, and the screened cells were cultured normally.
The normal cultured cells were subcultured in a six-well plate, and the cells were in good condition with a density of about 60%. The cells were incubated with 10-3 mmol/l insulin for 15 minutes, and then replaced with a proper amount of culture solution and preconfigured glucose solution, so that each well cell in the six-well plate was placed in a medium with different glucose concentrations (HEPA1-6 cells, 3T3-L1 cells: 5.5, 36.1, 50, 70, 85, 125 mmol/l, C2-C12 cells: 5.5, 11.5, 25, 45, 60, 100 mmol/l). After being incubated in the incubator of 37°C and 5%CO2 for 24 hours, the liquid transfer gun absorbed the culture solution from each hole and put it into the 1.5 ml EP tube. According to the instructions of the glucose detection kit (Nanjing Jiancheng Institute of Biological Engineering), the concentration of glucose in different holes was detected and analyzed.
The total protein was extracted with high-efficiency RIPA cell lysate (Solarbio, China), and the concentration of total protein was determined by Nanodrop2000 ultramicrospectrophotometer. Total cell lysates were separated on 10% SDS-PAGE and blotted with the following primary antibodies: rabbit anti-
Data were presented as the
The puromycin-resistant cell lines infected by the virus were selected according to the above methods for culture, and the cell proteins were extracted and verified by Western blotting and analyzed (as shown in Figure
WB results of three cell lines, the leftmost three holes are the control group, and the right three holes are the sidt2 elimination group (
We use the above cell culture methods to culture cells and induce them to differentiate and mature. The glucose solution was added to the cell culture medium to change the glucose concentration of the living environment. 24 hours later, the glucose detection kit was used to detect the remaining glucose concentration in the culture solution, so as to calculate and evaluate the glucose uptake of the cells. As shown in Figure
Glucose uptake of three cell lines (
The phosphorylation of insulin receptor substrate-1 serine 307 (insulin receptor substrate-1 Ser307, IRS-1 Ser307) plays an important negative role in insulin signal transduction. After IRS-1Ser307 phosphorylation, the interaction between IRS-1 and insulin receptor (insulin receptor, InsR) is weakened, and the level of tyrosine phosphorylation of IRS-1 under insulin stimulation is decreased, which further affects the downstream transduction of insulin signal. In the three cell lines, the expression of PIRS-1 (ser307) phosphorylated protein in the sidt2 knockout group was significantly higher than that in the control group, as shown in Figure
WB results of three cell lines (
The P85 regulatory subunit of phosphatidylinositol-3 kinase (phosphatidylinositol3-kinase, PI3K) plays an important role in activating the activity of PI3K. As shown in Figure
WB results of three cell lines (
AKT is the downstream gene of the IRS-1 signal pathway and the target gene of PI3-K. It has two phosphorylation sites, ser473 and thr308. Phosphorylation can promote the transfer of GLUT-4 from the capsule to the cytoplasm and promote glucose uptake and utilization. Activation of AKT phosphorylation sites can prevent or reduce insulin resistance in cells. As shown in Figure
WB results of three cell lines (
The occurrence of T2DM is a multicause, multistep complex process, and its pathogenesis is also affected by many factors. One of the most important pathophysiological changes is the insulin target tissue (mainly liver, muscle) producing insulin resistance or accompanied by insufficient insulin secretion. Therefore, maintaining the normal function of islet
The main physiological function of insulin is to regulate the uptake and utilization of glucose by the target tissue. Measuring the glucose uptake rate of adipocytes, myoblasts, and hepatocellular carcinoma cells stimulated by insulin in vitro is an effective method to evaluate insulin sensitivity. In this study, we found that 3T3-L1 adipocytes, C2-C12 myoblasts, and HEPA1-6 hepatoma cells all showed decreased glucose uptake stimulated by insulin after deletion of sidt2 gene, suggesting that sidt2 gene is involved in the occurrence of insulin resistance. What is the specific mechanism? From the point of view of the insulin signal transduction pathway, the obstacle of the IRS signal pathway is an important pathway of insulin resistance. Serine phosphorylation of IRS-1 is considered to be a negative regulator of the IRS-1 signaling pathway. Phosphorylation of serine 307 of IRS-1 inhibits tyrosine phosphorylation of IRS-1 and leads to insulin resistance. In this study, the sidt2 gene promotes the expression of PIRS-1 (ser307), which is consistent with the decrease of insulin-stimulated glucose uptake caused by the deletion of the sidt2 gene [
In conclusion, we found that sidt2 gene knockout can cause insulin resistance in peripheral tissue, which may be achieved by affecting the expression of key proteins in the insulin signal transduction pathway. In future experimental studies, we can overexpress the sidt2 gene, and then observe whether insulin resistance is corrected and how the expression of key proteins in the insulin signal transduction pathway will change. In addition, whether the sidt2 gene is involved in other mechanisms of insulin resistance needs to be further studied.
Data is available.
The authors declare that they have no conflicts of interest.
This work was supported by the National Natural Science Foundation of China (grant No.81200632); The Innovation training program for college students in Anhui Province, China (S201910368035); Scientific research project of middle and young people in Wannan Medical College (WK201906).