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).

Keywords: beta cell function; concentration-response; glucose-stimulated insulin secretion; islet assessment; perifusion; stimulation index; type 1 diabetes.

Figure 1

Dynamic GSIS in isolated human and murine islets. Average of all experimental data collected for free murine and human islets perifused using a low (3 mM; G3, 8 min) → high (11 mM; G11, 20 min) → low (3 mM; G3, 15 min) incoming glucose stimulation (plus 10 min KCl followed by G3) as shown. Automated PERI4-02 multichannel perifusion apparatus used (samples collected every minute; 0.1 mL/min flow rate, ~100 IEQ per channel). Data are average ± SEM from multiple isolations (n = 25 and 34 total samples for murine and human, respectively).

Figure 2

Variability of dynamic GSIS in isolated human and mouse islets. All experimental data for human (A) and mouse (B) islets perifused as before (G3 → G11 → G3 → KCl, see Figure 1) shown together with their overall average (thick black line; n = 34 and 25, respectively).

Figure 3

Concentration dependence of insulin secretion in murine and human islets. Summary of experimental data for free human (A) and murine islets (B) perifused using standard equipment and parallel stepwise incoming glucose stimulations (3 mM → 5/7/9/11/16.7/30 mM → 3 mM as indicated; plus 10 min KCl and 10 min low; corrected to ~100 IEQ per chamber; n = 3–12 per group). Averages of all data obtained in our labs for the G3 → G11 protocol are included as dashed gray lines for reference.

Figure 4

Scatter plot of insulin secretion in response to different glucose concentrations. Data points represent the average insulin secreted by each individual human (A) and mouse (B) islet sample (pg/min/IEQ; first- and second phase combined). Lines indicate group average and SD.

Figure 5

Concentration-response of insulin secretion. (A) Average insulin secretion rate (first- and second phase) at different glucose steps expressed as percent of maximum value (at G30) and fitted with a sigmoidal dose response function (Hill function, Equation 1). Compared to human islets, mouse islets have a similar Hill slope (n = 3.4 ± 0.4 vs. 3.2 ± 0.4), but a right-shifted response (half-maximal concentration C₅₀ = 13.7 ± 0.6 vs. 7.9 ± 0.4 mM). (B) Best-fit sigmoid function describing percent insulin secretion rate in function of the high glucose challenge for human islets (Equation 1; n = 3.2, C₅₀ = 7.9 mM) overlapped with its first and second derivative (right axis) and some commonly used stimulation indices (SIs). See text for details.