The Influence Of Insulin Signaling On The Adipogenic Cells Dedifferentiation With Different Genotype Mice

Recent study had demonstrated that visceral or subcutaneous white mature fat cells can naturally generate to the occurrence of dedifferentiation in vitro culture conditions without exogenous insulin. However, the addition of insulin can maintain its original phenotype and inhibit the dedifferentiation. Therefore, I hypothesized that the insulin signaling was related to mature fat cells dedifferentiation. Nevertheless, the specific mechanism of adipocyte dedifferentiation affected by insulin signaling needed further exploring.By analyzing the influence of insulin signaling on the adipogenic cells dedifferentiation in obese and non obese mice, we investigated the mechanism of visceral white fat cell dedifferentiation of mice in different physiological states, in the morphology and molecular leve.C57BL/6J WT, TNF-?-/-, Leprdb/dband DT male mice were chosen as experimental materias.The first two kinds of mice are non obese, while the later two kinds of mice are genetically obese.Firstly, the adipose-derived stem cells were induced to differentiate into mature fat cells by classical ‘cocktail-method'. Then we used different methods to make mature fat cells dedifferentiate and set up three groups of experiments. The simple control group was initially developed by removing insulin after 10 days of fat induction with cells cultured in complete growth medium containing 10% fetal bovine serum(containing unknown amounts of insulin).The intervention group was firstly formed by removing the insulin and adding the insulin signaling pathway inhibitor(OSI-906, linstinib) with cells cultured in complete growth medium containing 10% fetal bovine serum. The DMSO control group was developed by removing the insulin and adding the OSI-906 solvent(DMSO) with cells cultured in complete growth medium containing 10% fetal bovine serum. After the start of the three groups of cells to dedifferentiate,in the corresponding culture conditions, we continue to cultivate 8 daysMoreover, we were in order to detect whether the dedifferentiated fat cells(DFATCs)reobtained stem cell characteristics, the dedifferentiated fat cells were induced into adipogenic cells and osteoblast cell.Finally, by handing out the data of morphology, gene and protein expression, we investigated the effects of insulin signaling on adipogenic cells dedifferentiation of mice with different genotypes. The results were as follows:1. We isolated and purified the WT ADSCs to identify the key stages in the process of adipogenic cells dedifferentiation. The five stages(D0, D10, DD3, DD8 and RD10) wereidentified by the morphological changes of the cells.2. We chose different concentrations(0, 0.1, 0.3, 1 ?M) of OSI-906 to intervene ADSCs adipogenic differentiation of WT ADSCs. Data of morphology and protein expression showed that the minimal OSI-906 concentration completely inhibites the insulin signaling pathway in the condition of the presence of in insulin and serum. The results showed that the concentration was 0.3 ?M.3. Morphological detection results were listed as below:(1) ADSCs were induced into mature fat cells after 10 days.(2) As time went on, mature fat cells gradually began to dedifferentiate and would complete the dedifferentiation after 8 days.(3) The rate of dedifferentiation: the intervention group>the control group, the simple control group?the DMSO control group.(4) The DFAT cells of three groups were induced into mature fat cells after 10 days, and the results of three groups had no difference, significantly.(5) The morphological detection results of WT, TNF-?-/-, Leprdb/dband DT ADSCs were basically consistent.4. The expression of CD105 were up-regulated. The DFATCs of WT mice were induced into osteoblast cell in day 21. The result of alizarin red staining was positive, which indicated DFATCs could reacquire stem cell characteristics.5. In the dedifferentiation process of adipogenic cells, the mRNA level of insulin signaling pathway relative factor(AKT, GLUT-4) did not change. The expression of adipogenesis relative factor(C/EBP?, PPAR?, adiponectin and SREBP-1) were down-regulated. The expression of lipolysis key factor(ATGL) were up-regulated early and down-regulated later. No notable difference was found between simple control group and DMSO control group. In contrast to control group, OSI-906 intervention group could increase the mRNA expression of ATGL, and inhibite the m RNA expression of C/EBP?, PPAR?, adiponectin and SREBP-1.In WT, TNF-?-/-, Leprdb/db and DT mice, the changes in expression of the gene transcription level were generally consistent. The main difference was that the relative expression of the detected molecule(the same period and the same group) was somewhat different.6. In the dedifferentiation process of adipogenic cells, the activation level of p-AKT(Thr308) decreased significantly. The protein expression of GLUT-4 increased early and decreased later on. The protein level of SREBP-1 and adiponectin kept decreasing to the detection limit in the early stage of dedifferentiation(DD3). No significant difference was found between simple control group and DMSO control group. In contrast to control group, OSI-906 intervention group down-regulate the activation level of p-AKT(Thr308).In WT, TNF-?-/-, Leprdb/db and DT mice, the changes in the expression of the gene translation level were generally consistent. The main difference was that the relative expression of thedetected molecule(the same period and the same group) was somewhat different.In conclusion, the lack of insulin signaling could influence adipogenic cells dedifferentiation of obese(Leprdb/db and DT) and non obese(WT and TNF-?-/-) mice. The lack of insulin signaling could inhibite the expression of SREBP-1, PPAR? and C/EBP?, which promoted the expression of lipolysis key molecules and fat stem cell markers. AS a result, it promoted the dedifferentiation of adipogenic cells. The effect of insulin signaling on the mice adipogenic cells dedifferentiation was independent of the mice genotype and obesity.