From Insulin Measurement to Partial Exocytosis Model: Advances in Single Pancreatic Beta Cell Amperometry over Four Decades

Abstract

Single cell Amperometry (SCA) is a powerful, sensitive, high temporal resolution electrochemical technique used to quantify secreted molecular messengers from individual cells and vesicles. This technique has been extensively applied to study the process of exocytosis, and it has also been applied, albeit less frequently, to investigate insulin exocytosis from single pancreatic beta cells. Insufficient insulin release can lead to diabetes, a chronic lifestyle disorder that affects millions of people worldwide. This review aims to summarize and highlight electrochemical measurements of insulin via monitoring its secretion from beta cells by SCA with micro- and nanoelectrodes since the 1990s and to explain how and why serotonin is used as a proxy for monitoring insulin during exocytosis from single beta cells. Finally, we describe how the combination of SCA measurements with the intracellular vesicle impact electrochemical cytometry (IVIEC) technique has led to important findings regarding fractional release types in beta cells. These findings, reported recently, have opened a new window in the study of pore formation, exocytosis from single vesicles, and the mechanisms of insulin secretion. This sensitive cellular electroanalysis approach should help in the development of novel therapeutic strategies targeting diabetes in the future.

Figure 1

(a) TEM image of a single pancreatic beta cell with many vesicles (granules). (b) Schematic of a secretory vesicle showing the structure and components. (Reprinted from ref () copyright 2010 Elsevier).

Figure 2

Graphical illustration of single-cell amperometry (SCA) measurements of exocytosis using a disc carbon fiber electrode (DCFE, left) positioned on the cell surface, and vesicle content analysis via IVIEC (Intracellular vesicle impact electrochemical cytometry) using a nanotip carbon fiber electrode (NCFE) placed within the cytoplasm of a single beta cell. (Reprinted from ref () copyright 2021 Wiley-VCH).

Figure 3

(A) Amperometric spike recordings from a single cell after glucose stimulation, representing the first report of insulin exocytosis measurement using a micro carbon electrode modified with a Ru-O/CN-Ru film. (Reprinted from ref () American Chemical Society copyright 1993). (B) Detection of insulin secretion from single beta cells of different species using a modified carbon fiber electrode with a Ru-O/CN-Ru film: (B.1) Canine beta cells stimulated with 200 μM tolbutamide. (B.2) Porcine beta cells stimulated with 200 μM tolbutamide. (B.3) Mouse beta cells stimulated with 30 mM K⁺. (B.4) INS-1 beta cells stimulated with 30 mM K⁺. (Reprinted from ref () American Chemical Society copyright 1999). (C) Bielectrode amperometric analysis and their recordings obtained from a single cell. The relative positions of the two electrodes (Numbered 1 and 2) are indicated by the drawings below the recordings. Note that major current spike activity was observed only when the electrodes were positioned in the middle of the cell and toward the lower left quadrant. (Reprinted from ref (),\ Springer copyright 2000).

Figure 4

(A) Amperometric recording displaying the oxidation of preloaded 5-HT from vesicles of mouse primary beta cells using the intracellular vesicle impact electrochemical cytometry (IVIEC) technique (inset: amplified amperometric current spike indicated by a red star in the trace). (B) Diagrammatic representation showing the experimental configuration for the carbon nanotip electrode positioned inside a single cell for IVIEC measurements. (C) Diagrammatic representation of the carbon disk-shaped electrode on the surface of a single cell for SCA measurements. (D) Representative amperometric trace from SCA due to the oxidation of secreted 5-HT from a single primary mouse beta cell (inset: amplified amperometric current spike marked with a red star in the trace). (E) Histograms presenting the frequency of quantified secreted 5-HT from vesicles during exocytosis by SCA (Blue, Number of cells: 26) and the frequency of quantified stored 5-HT within vesicles by IVIEC techniques (Red, Number of cells: 51). (F) Plot showing the mean and median number of molecules detected by SCA and IVIEC (Inset: calculated Fraction of Release (FR) of 5-HT in the tested single mouse beta cell). (Reprinted from ref () copyright 2021 Wiley-VCH).