Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Apr 20;24(4):525-531.e4.
doi: 10.1016/j.chembiol.2017.03.001. Epub 2017 Mar 30.

A Ratiometric Sensor for Imaging Insulin Secretion in Single β Cells

Martina Schifferer  1 , Dmytro A Yushchenko  2 , Frank Stein  1 , Andrey Bolbat  1 , Carsten Schultz  3
Affiliations

A Ratiometric Sensor for Imaging Insulin Secretion in Single β Cells

Martina Schifferer et al. Cell Chem Biol. .

Abstract

Despite the urgent need for assays to visualize insulin secretion there is to date no reliable method available for measuring insulin release from single cells. To address this need, we developed a genetically encoded reporter termed RINS1 based on proinsulin superfolder GFP (sfGFP) and mCherry fusions for monitoring insulin secretion. RINS1 expression in MIN6 β cells resulted in proper processing yielding single-labeled insulin species. Unexpectedly, glucose or drug stimulation of insulin secretion in β cells led to the preferential release of the insulin-sfGFP construct, while the mCherry-fused C-peptide remained trapped in exocytic granules. This physical separation was used to monitor glucose-stimulated insulin secretion ratiometrically by total internal reflection fluorescence microscopy in single MIN6 and primary mouse β cells. Further, RINS1 enabled parallel monitoring of pulsatile insulin release in tolbutamide-treated β cells, demonstrating the potential of RINS1 for investigations of antidiabetic drug candidates at the single-cell level.

Keywords: TIRF; biosensor; calcium; fluorescence; glucose; granule; insulin; mCherry; oscillation; potassium channel; superfolder GFP; tolbutamide.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1
Figure 1
Proinsulin-Based RINS1 Sensor Design and Expression in β Cells (A) Cartoon of RINS1 and of its non-cleavable mutant RINS1mut. (B) Confocal images of MIN6 β cells expressing RINS1. Enlargement of the selected square (top right). Merged images (top), single channels (bottom), mCherry (magenta), sfGFP (green). Scale bar, 5 μm (top left), 1 μm (top right). See also Figure S1. (C) Quantification (box and whisker plot) of the colocalization of RINS1 mCherry-only with granule marker phogrin (blue, n = 168) and sfGFP in RINS1 (red, n = 164) by Pearson coefficient. (D) Western analysis of MIN6 lysates using an anti-insulin antibody. Cells were transfected by RINS1 or mut, non-transfected cells served as controls. Anti-insulin western blotting showed bands for proinsulin RINS1 (66 kDa), sfGFP-insulin (36 kDa).
Figure 2
Figure 2
Monitoring Stimulated Insulin Secretion in Single β Cells (A) Cartoon demonstrating glucose-stimulated secretion in β cells expressing RINS1. SG, secretory granule; GLUT2, glucose transporter 2; KATP, ATP-sensitive potassium-channel; VDC, voltage-dependent channel. (B–E) MIN6 cell-expressing RINS1. (B) Confocal microscopy of MIN6 before (top) and after (bottom) addition of 20 mM glucose when sfGFP-tagged insulin (green) released from the cell while C-peptide-mCherry (magenta) stayed in the granules. Scale bar, 5 μm. (C) Whole-field of view quantification of RINS1 sfGFP, mCherry, and sfGFP/mCherry ratio channels observed over time by confocal microscopy. sfGFP (green), mCherry (magenta), and sfGFP/mCherry (blue). n = 19. (D) TIRF imaging of RINS1-expressing MIN6 cells before (top) and after (bottom) addition of 20 mM glucose. See also Figure S2. (E) Quantification of TIRF imaging data of MIN6 cells expressing RINS1 (red, n = 20) and RINS1mut (blue, n = 16) stimulated after 60 s with 20 mM glucose (black line). Traces for sfGFP, mCherry, and sfGFP/mCherry ratio channels are shown. (C, E) Error bars represent standard error of the mean.
Figure 3
Figure 3
Drug-Mediated Modulation of Insulin Secretion Visualized by RINS1 in MIN6 Cells Imaged by TIRFM (A and B) The effect of tolbutamide incubation (100 μM, 30 min) on insulin secretion at the single-cell level. (A) Example TIRF images of RINS1-bearing granules in a single β cell at defined time points. For a full movie, see the Supplemental Information. Scale bar, 2 μm. (B) sfGFP (green), mCherry (magenta) intensity, and ratio sfGFP/mCherry (blue) changes over time. Arrow heads indicate time points of images in (A). (C) Schematic drawing of sulfonylurea drugs affecting insulin secretion. Tolbutamide (red) inhibits KATP channels, leading to depolarization and calcium influx even in the absence of glucose. (D) MIN6 cell-expressing RINS1 and B-GECO treated with tolbutamide (100 μM, 30 min) and stimulated by glucose after 60 s. B-GECO (blue) indicating calcium levels and RINS1 sfGFP/mCherry ratio (black) of for a single cell is plotted.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ammala C., Larsson O., Berggren P.O., Bokvist K., Juntti-Berggren L., Kindmark H., Rorsman P. Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells. Nature. 1991;353:849–852. - PubMed
    1. Ammala C., Ashcroft F.M., Rorsman P. Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells. Nature. 1993;363:356–358. - PubMed
    1. Aronson D.E., Costantini L.M., Snapp E.L. Superfolder GFP is fluorescent in oxidizing environments when targeted via the sec translocon. Traffic. 2011;12:543–548. - PMC - PubMed
    1. Burns S.M., Vetere A., Walpita D., Dancik V., Khodier C., Perez J., Clemons P.A., Wagner B.K., Altshuler D. High-throughput luminescent reporter of insulin secretion for discovering regulators of pancreatic Beta-cell function. Cell Metab. 2015;21:126–137. - PubMed
    1. Cheng K., Delghingaro-Augusto V., Nolan C.J., Turner N., Hallahan N., Andrikopoulos S., Gunton J.E. High passage MIN6 cells have impaired insulin secretion with impaired glucose and lipid oxidation. PLoS One. 2012;7:e40868. - PMC - PubMed

MeSH terms