Concentration-Dependency and Time Profile of Insulin Secretion: Dynamic Perifusion Studies With Human and Murine Islets

Abstract

The detailed characterization and quantification of the kinetics of glucose-stimulated insulin secretion (GSIS) by normal pancreatic islets is of considerable interest for characterizing β-cell dysfunction, assessing the quality of isolated islets, and improving the design of artificial pancreas devices. Here, we performed dynamic evaluation of GSIS by human and mouse islets at high temporal resolution (every minute) in response to different glucose steps using an automated multichannel perifusion instrument. In both species, insulin responses were biphasic (a transient first-phase peak followed by a sustained second-phase), and the amount of insulin released showed a sigmoid-type dependence on glucose concentration. However, compared to murine islets, human islets have (1) a less pronounced first-phase response, (2) a flat secretion rate during second-phase response, (3) a left-shifted concentration response (reaching half-maximal response at 7.9 ± 0.4 vs. 13.7 ± 0.6 mM), and (4) an ~3-fold lower maximal secretion rate (8.3 ± 2.3 vs. 23.9 ± 5.1 pg/min/islet at 30 mM glucose). These results can be used to establish a more informative protocol for the calculation of the stimulation index, which is widely used for islet assessment in both research and clinical applications, but without an accepted standard or clear evidence as to what low- to high-glucose steps can provide better characterization of islet function. Data obtained here suggest that human islet functionality might be best characterized with a dynamic stimulation index obtained with a glucose step from a low of 4–5 to a high of 14–17 mM (e.g., G4 → G16).

The Effect of Recovery Warm-up Time Following Cold Storage on the Dynamic Glucose-stimulated Insulin Secretion of Isolated Human Islets

“…Insulin concentrations were determined using commercially available human insulin ELISA kits (Mercodia Inc., Winston Salem, NC, USA) and converted to μg/l using the formula provided by the manufacturer (1 µg/l = 23 mU/l). Because accurate assessment of islet mass (IEQ) is challenging, to account for possible differences among parallel channels, values were adjusted by up to 30% based on the response to KCl as described before using the area under the curve (AUC) in each column for normalization 36 , 37 , 48 , 49 , 56 , 57 . All responses are scaled to 100 IEQ.…”

“…Additional assays that evaluate β-cell fractional viability and content as well as function are available. They can offer meaningful information predictive of in vivo function 36 – 38 and are essential to ensure progress in our understanding of islet function and pathogenesis of T1D 39 , 40 . However, due to the lack of standardized protocols, the need for highly specialized equipment, and the time required to obtain results, they are not currently utilized as lot release criteria.…”

“…Although not part of currently accepted final islet product release criteria, such studies represent the most complex in vitro assay to assess the quality and function of isolated pancreatic islets and provide considerably more physiological data than those obtainable from static GSIS and corresponding stimulation indices (SIs). Improved equipment and analytical techniques now allow the quantitative assessment of insulin release kinetics with customizable temporal resolution and under fully controllable incoming concentrations of glucose and/or other secretagogues of interest 36 , 37 , 44 – 49 . Dynamic perifusion studies are now routinely used to assess the quality and function of islets 46 , 50 , 51 , and microfluidic chip technologies make possible even the quantitative monitoring of single islet insulin secretion with high time resolution 52 , 53 .…”

“…However, various nonstandardized systems and protocols are being used including variations in (i) low- (basal) and high- (stimulating) glucose concentrations (e.g., 3 mM→11 mM, 5.6 mM→16.7 mM, 2.8 mM→28 mM, and others), (ii) exposure times to stimulating glucose (typically, 10 to 30 min), (iii) flow rates (e.g., 30 to 1,000 μl/min), (iv) oxygen concentrations (from 21% atmospheric up to 95%), (v) quantity of islets per channel, (vi) analytical methods used to quantify insulin concentrations (enzyme-linked immunosorbent assay [ELISA], radioimmunoassay, immunochemiluminometric assay, and others), (vii) measurement units used to express results (pg/IEQ/min, ng/100islets/min, mU/l, μU/ml/ngDNA, μU/ml/ngDNA, relative values compared to baseline, percent insulin content, and others), and (viii) perifusion systems utilized. As a first step toward more informative and standardized perifusion assays, we have previously investigated the suitability of various glucose steps utilized to assess islets 37 .…”

Glucose transporters in pancreatic islets

“…However, it must be noted that these experiments were performed with dissociated human islets that contained approximately 52% β-cells; hence, the author's conclusion on glucose transport in β-cells needs to be considered cautiously [152]. Perifusion experiments revealed a left-shifted GSIS profile of human islets compared to mouse islets [1,114]. In comparison to their murine counterparts, human islets secreted more insulin at low glucose concentrations (5.4 mmol/l) but showed a decreased stimulation index at high glucose levels (16.7 mmol/l) [22,114] (Fig.…”