Pancreatic Stellate Cells Aggravates The Detrimental Effects Of High Glucose On Beta-Cell

Part 1:Pancreatic stellate cells aggravates the detrimental effects of high glucose on beta-cellBackground and aims:The mechanisms of β-cell failure in type 2 diabetes mellitus (T2DM) have not been fully explained. Several recent studies observed pancreatic stellate cells (PSCs) were present in the diabetic islets, but their potential role in β-cell failure was unknown. We here assess the effects of PSCs on β-cell function and glucose homeostasis in vivo and in vitro.Materials and methods:PSCs were transplanted into the pancreas of normoglycemic Wistar and hyperglycemic Goto-Kakizaki (GK) rats. Sixteen weeks post-transplantation, β-cell function was assessed by glucose tolerance tests and measurements of HbA1c. Immunofluorescent staining for insulin, TUNEL and Masson’s trichromestain were performed to assess the β-cell mass, apoptosis and fibrosis, respectively. The in vitro effects of PSCs conditioned medium (PSCs-CM) and/or high concentration of glucose on INS-1 cell function was assessed by measuring insulin secretion, INS-1 cell survival, apoptosis and endoplasmic reticulum stress (ER stress) associated CHOP expression.Results:PSCs transplantation exacerbated the impaired β-cell function in GK rats, leading to increased blood glucose and HbA1c levels, decreased insulin secretion, reduced β-cell mass and increased islet apoptosis and fibrosis. In contrast, PSCs transplantation had no significant effects in Wistar rats. In vitro, PSCs-CM caused impaired INS-1 cell viability, and increased apoptosis, which were more pronounced in the presence of high glucose. The deleterious effects of PSCs on β-cells were associated with increased CHOP expression.Conclusion:Our study demonstrates that PSCs induce β-cell failure in vitro and in vivo, suggesting that they may play a potential role in the progression of T2DM.Part 2:The effect of rat islet stellate cells on beta-cell functionBackground and aims:The central role of PSCs in pancreatic fibrogenesis is well established. However, the mechanism responsible for the islet fibrosis presenting in the late stage of T2DM has not been fully elucidated. This study was designed to determine whether the endocrine pancreatic islets contain cells resembling PSCs.Materials and methods:Rat ISC was isolated from freshly isolated rat islets, while rat PSC used as a positive control was isolated from pancreatic tissue. Immunofluorescence visualization of markers for PSC (α-smooth muscle actin (α-SMA), vimentin, glial fibrillary acidic protein (GFAP)) was used to characterize the rat ISC. Cell counting kit-8 (CCK-8) was used to assess the proliferation of ISC. The wound-healing assay and the transwell migration were used to assess the migration capacity of ISC. Immunofluorescence and western blot against collagen types Ⅰ (col-Ⅰ), collagen types Ⅱ (col-Ⅲ) and fibronectin (FN) were performed to identify extracellular matrix (ECM) component synthesized by ISC. The in vitro effects of ISC on INS-1 cell was assessed by measuring INS-1 cell survival. Furthermore, ISC-ECM was isolated to assessed its effects on islet viability.Results:PSCs were isolated from pancreas using standard explants techniques. A similar method was used to acquire ISCs. Adherent ISCs with a stellate, angular morphology migrated from the edge of cultured islets within 48 h of primary culture. ISCs contained fewer lipid droplets than equivalent PSCs, and their rapid disappearance accompanied by the increased expression of α-SMA suggested that ISCs were more rapidly activated than PSCs in vitro. They expressed α-SMA, vimentin, GFAP and were positive for ECM components col-Ⅰ, col-Ⅲ and FN, all of which are characteristics of classical PSCs. However, ISCs differed from PSCs by having reduced rates of proliferation and migration in vitro. ISC impaired INS-1 cell viability, which were more pronounced in the presence of high glucose. The deleterious effects of ISC on β-cells were associated with ISC-ECM, which induced obvious destruction on islet.Conclusion:Our in vitro study shows that isolated islets contain a population of stellate cells which are phenotypically similar but not identical to PSCs. In view of the established role of PSCs in pancreatic fibrosis, we suggest that these may contribute to islet fibrosis in T2DM.Part 3:The effect of human islet stellate cells on isletBackground and aims:Our previous research reported islet stellate cell (ISC) exhibiting similar appearance to classical pancreatic stellate cell (PSC) was isolated from freshly isolated rat islets, which may contribute to the islet fibrosis in type 2 diabetes mellitus (T2DM). This study was designed to determine whether human islets also contain ISC, and if so, whether human ISC is different from human PSC.Materials and methods:Using standard explants techniques, human ISC was isolated from freshly isolated human islets, while human PSC used as a positive control was isolated from pancreatic tissue. Immunofluorescence visualization of markers for PSC (α-smooth muscle actin (a-SMA), desmin, vimentin, glial fibrillary acidic protein (GFAP)) was used to characterize the human ISC. Cell counting kit-8 (CCK-8) was used to assess the proliferation of ISC. The wound-healing assay and the transwell migration were used to assess the migration capacity of ISC. Immunofluorescence against collagen types Ⅰ (col-Ⅰ), collagen types Ⅲ (col-Ⅲ) and fibronectin (FN) was performed to identify extracellular matrix (ECM) component synthesized by ISC. The in vitro effects of ISC on human islet function was assessed by measuring insulin secretion, islet survival. Furthermore, ISC-ECM was isolated to assessed its effects on islet viability.Results:After about 24 h in culture, an increasing number of ISC with triangular shape began to grow out from human islets. The passaged ISC expressed α-SMA, desmin, vimentin, GFAP and was positive for col-Ⅰ, col-Ⅲ and FN. Compared with PSC, the proliferation and migration ability of ISC was significantly slower than those of PSC. ISC increased islet viability and insulin secretion, but the ISC-ECM still exerted deleterious effects on islet.Conclusion:Similar to our previous rat experiment, the current study shows that human islets also contain ISC which is phenotypically similar but not identical to human PSC. Activated human ISC is a high active ECM-producing cell type, indicating human ISC may contribute to islet fibrosis in patients with T2DM.