We successfully extracted hADSCs from human adipose tissue according to the method reported in the literature [14, 15] and characterized the phenotypes of hADSCs through flow cytometry. After that, we used a simple chemical method not involving insulin to differentiate hADSCs into IPCs in vitro. In order to assess the function of IPCs, we tested the glucose-induced insulin secretion of IPCs and beta cells in vitro. Our data show that regardless of whether they were stimulated for 30 min or 1 h, the beta cells could release a certain amount of insulin after stimulation with high or low glucose concentrations. However, only when stimulated for 1 h in low glucose concentrations did IPCs secrete a little bit of insulin. The results indicate that IPCs can secrete insulin in response to glucose stimulation, similar to, but not as well as beta cells. Even though we only compared beta cells and one kind of IPC which was derived from one source using one differentiation method, our results made evident the difference in physiological function between these IPCs and beta cells. This evidence led to the question: ‘What were the reasons for the difference between IPCs and beta cells?’ We conjectured that these differences were due to the differences in cellular structure. To confirm our hypothesis, we first used AFM to detect cell surface ultrastructure of beta cells and IPCs.
AFM images indicated the changes in morphological properties of IPCs and beta cells stimulated by glucose. The morphologies of IPCs and beta cells were similar to each other, as observed via AFM. They all were polygonal and contained visibly porous features in the cytoplasm. AFM is a common method used to observe cell morphology. However, few studies have reported that these porous structures existed naturally on the cell surface [16–20]. Pores on the cell surface generally appeared after treatment with some drugs [21, 22]. Nevertheless, the pores observed after drug treatment were not the same as the porous structures we detected. The porous structures in the IPCs and beta cells were organized and well distributed around the nuclei. The pores that appear after drug treatment are dispersed and isolated. Kim et al. deemed that these isolated holes on the cell surface after drug treatment might be one form of cell apoptosis . Additionally, we speculated that these uniform holes arranged in the cytoplasmic membrane might be dependent onto the type of cells. Beta cells are endocrine cells, while IPCs are artificially synthesized copies of endocrine cells. The pores in the cytoplasmic membrane might be indicative of the exocytosis process through which the hormone is released into the extracellular space.
Simultaneously, we used AFM to compare the cell membrane particle size and Ra of the membrane surface before or after glucose stimulation of IPCs and beta cells. Our results revealed that both membrane particle size and Ra of beta cells were larger than those of IPCs. When both two groups of endocrine cells were stimulated by glucose, the membrane particle size and Ra were higher than those not stimulated, except for IPCs that were stimulated for 30 min with low glucose concentration. The magnitude of cellular Ra, as well as the types, structure, and quantity of membrane protein molecules, directly influenced the inclines and declines of the membrane surface . We speculated that the reason for the lower membrane particle size and Ra in IPCs might be due to their lower membrane protein content. The cell membrane accomplishes its biological function through membrane liquidity, and exocytosis is one of the functions that depend on membrane liquidity [24, 25]. IPCs and beta cells secreted insulin through exocytosis. In the meantime, their plasma membranes were replenished via membrane liquidity. We inferred that the change in membrane liquidity might cause the increase in cell membrane particle size and Ra after glucose stimulation.
Beta cells secrete insulin through exocytosis. In beta cells, actin filaments form a dense network under plasma membrane. This actin network acts as a barricade, preventing passive diffusion of insulin follicles to the plasma membrane. Thus, the actin network ultimately lessens insulin secretion via reduction of exocytosis . On the contrary, F-actin depolymerization can increase exocytosis, which increases insulin secretion. We proposed that the pores we observed that were located in the cytoplasmic membrane were one of the characteristics of insulin exocytosis, and increased evidence of porous structures may be related to the enhancement of insulin exocytosis.
To prove that exocytosis had been enhanced after glucose stimulation of IPCs and beta cells, we demonstrated that without glucose stimulation, the actin network underneath the plasma membrane was continuous and dense. After glucose stimulation, the actin network depolymerized and became discontinuous. After F-actin depolymerization, inhibition of exocytosis was relieved and insulin secretion increased. Interestingly, in the IPCs group, the cortical actin network did not depolymerize in low glucose concentrations after 30 min of stimulation. The actin network became discontinuous and depolymerized only after low-glucose stimulation for 1 h.