A novel optical tracer for VMAT2 applied to live cell measurements of vesicle maturation in cultured human β-cells

Sci Rep. 2019 Apr 1;9(1):5403. doi: 10.1038/s41598-019-41891-x.

Abstract

The islet β-cells integrate external signals to modulate insulin secretion to better regulate blood glucose levels during periods of changing metabolic demand. The vesicular monoamine transporter type 2 (VMAT2), an important regulator of CNS neurotransmission, has an analogous role in the endocrine pancreas as a key control point of insulin secretion, with additional roles in regulating β-cell differentiation and proliferation. Here we report on the synthesis and biological characterisation of a fluorescent ligand for VMAT2 suitable for live cell imaging. Staining for VMAT2 and dopamine in live β-cell cultures show colocalisation in specific vesicles and reveal a heterogeneous population with respect to cell size, shape, vesicle number, size, and contents. Staining for VMAT2 and zinc ion, as a surrogate for insulin, reveals a wide range of vesicle sizes. Immunohistochemistry shows larger β-cell vesicles enriched for proinsulin, whereas smaller vesicles predominantly contain the processed mature insulin. In β-cell cultures obtained from nondiabetic donors, incubation at non-stimulatory glucose concentrations promotes a shift in vesicle diameter towards the more mature insulin vesicles at the expense of the larger immature insulin secretory vesicle population. We anticipate that this probe will be a useful reagent to identify living β-cells within complex mixtures for further manipulation and characterisation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cells, Cultured
  • Dopamine / chemistry
  • Dopamine / metabolism
  • Glucose / pharmacology
  • HEK293 Cells
  • Humans
  • Indicators and Reagents / chemistry
  • Insulin Secretion / drug effects
  • Insulin-Secreting Cells / metabolism*
  • Islets of Langerhans / cytology
  • Islets of Langerhans / metabolism
  • Microscopy, Fluorescence
  • Optical Imaging / methods*
  • Secretory Vesicles / metabolism*
  • Vesicular Monoamine Transport Proteins / chemistry
  • Vesicular Monoamine Transport Proteins / metabolism*
  • Zinc / chemistry
  • Zinc / metabolism

Substances

  • Indicators and Reagents
  • SLC18A2 protein, human
  • Vesicular Monoamine Transport Proteins
  • Glucose
  • Zinc
  • Dopamine