Functional Analysis of Novel Candidate Regulators of Insulin Secretion in the MIN6 Mouse Pancreatic β Cell Line

PLoS One. 2016 Mar 17;11(3):e0151927. doi: 10.1371/journal.pone.0151927. eCollection 2016.

Abstract

Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic β cells is important for understanding and treating diabetes. The pancreatic β cell line, MIN6, retains GSIS but gradually loses it in long-term culture. The MIN6 subclone, MIN6c4, exhibits well-regulated GSIS even after prolonged culture. We previously used DNA microarray analysis to compare gene expression in the parental MIN6 cells and MIN6c4 cells and identified several differentially regulated genes that may be involved in maintaining GSIS. Here we investigated the potential roles of six of these genes in GSIS: Tmem59l (Transmembrane protein 59 like), Scgn (Secretagogin), Gucy2c (Guanylate cyclase 2c), Slc29a4 (Solute carrier family 29, member 4), Cdhr1 (Cadherin-related family member 1), and Celsr2 (Cadherin EGF LAG seven-pass G-type receptor 2). These genes were knocked down in MIN6c4 cells using lentivirus vectors expressing gene-specific short hairpin RNAs (shRNAs), and the effects of the knockdown on insulin expression and secretion were analyzed. Suppression of Tmem59l, Scgn, and Gucy2c expression resulted in significantly decreased glucose- and/or KCl-stimulated insulin secretion from MIN6c4 cells, while the suppression of Slc29a4 expression resulted in increased insulin secretion. Tmem59l overexpression rescued the phenotype of the Tmem59l knockdown MIN6c4 cells, and immunostaining analysis indicated that the TMEM59L protein colocalized with insulin and GM130, a Golgi complex marker, in MIN6 cells. Collectively, our findings suggested that the proteins encoded by Tmem59l, Scgn, Gucy2c, and Slc29a4 play important roles in regulating GSIS. Detailed studies of these proteins and their functions are expected to provide new insights into the molecular mechanisms involved in insulin secretion.

MeSH terms

  • Animals
  • Blotting, Western
  • Cadherins / physiology
  • Cell Line
  • Female
  • Fluorescent Antibody Technique
  • Gene Knockdown Techniques
  • Genes, Regulator / physiology
  • Glucose / physiology
  • Insulin / metabolism*
  • Insulin / physiology
  • Insulin Secretion
  • Insulin-Secreting Cells / metabolism*
  • Insulin-Secreting Cells / physiology
  • Membrane Proteins / physiology
  • Membrane Transport Proteins / physiology
  • Mice
  • Mice, Inbred C57BL
  • Receptors, Enterotoxin
  • Receptors, Guanylate Cyclase-Coupled / physiology
  • Receptors, Peptide / physiology
  • Reverse Transcriptase Polymerase Chain Reaction
  • Secretagogins / physiology

Substances

  • Cadherins
  • Celsr2 protein, mouse
  • Insulin
  • Membrane Proteins
  • Membrane Transport Proteins
  • Receptors, Peptide
  • SCGN protein, mouse
  • Secretagogins
  • Tmem59l protein, mouse
  • equilibrative nucleoside transporter-4, mouse
  • Gucy2c protein, mouse
  • Receptors, Enterotoxin
  • Receptors, Guanylate Cyclase-Coupled
  • Glucose

Grant support

The authors have no support or funding to report.