Abstract
The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labeling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.
Highlights
Type 2 diabetes is characterized by insufficient functional β-cell m ass[1]
Human β cells respond to glucose[16,17,18], fatty acids12, serpinB17, dual-specificity tyrosine-regulated kinase 1A (DYRK1A) inhibitors including h armine[19,20,21], and heparin-binding epidermal growth factor-like growth factor (HB-EGF)[22], proliferation rates are very low compared to rodents and published data on the efficacy of human β-cell mitogens are often highly v ariable[23]
In this study we evaluated the capacity of harmine, glucose and HB-EGF to stimulate β-cell proliferation in isolated human islets using a multi-faceted approach involving intact and dispersed islet cultures, flow cytometry and immunohisto/cytochemistry and several proliferation and cell type-specific markers
Summary
Type 2 diabetes is characterized by insufficient functional β-cell m ass[1]. In non-diabetic obese individuals and during pregnancy, accretion of insulin secretory capacity per β cell combined with β-cell mass expansion maintains glucose homeostasis by balancing levels of circulating insulin and insulin sensitivity. Human β cells respond to glucose[16,17,18], fatty acids12, serpinB17, dual-specificity tyrosine-regulated kinase 1A (DYRK1A) inhibitors including h armine[19,20,21], and heparin-binding epidermal growth factor-like growth factor (HB-EGF)[22], proliferation rates are very low compared to rodents and published data on the efficacy of human β-cell mitogens are often highly v ariable[23]. In this study we assessed the mitogenic properties of harmine, glucose and HB-EGF in both intact and dispersed human islets comparing immunochemistry to flow cytometry to differentiate proliferating β and non-β cell types, including glucagon+ and cytokeratin-19 (CK19)+ cells
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