Glucose Metabolism, Islet Architecture, and Genetic Homogeneity in Imprinting of [Ca2+]i and Insulin Rhythms in Mouse Islets

We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca2+]i) that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed “islet imprinting.” We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J) as well as outbred mouse strains (Swiss-Webster; CD1). Second, imprinting was observed in NAD(P)H oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca2+]i oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a KATP-channel opener that blocks [Ca2+]i influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca2+]i oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca2+]i oscillations. Lastly, to test whether the imprinted [Ca2+]i patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca2+]i oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon

A Practical Guide to Rodent Islet Isolation and Assessment

Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes

[Ca²⁺]_i flux and insulin release patterns show mouse-to-mouse imprinting.

<p>(A) [Ca²⁺]_i flux and insulin release traces from islets taken from three different mice (labeled accordingly). Displayed oscillation frequency averages are 9 min (Mouse 1), 4.5 min (Mouse 2), and 15 s (Mouse 6). Periods were calculated using local minimum values. Insulin oscillations from mouse 6 were faster than the measured temporal resolution (22 s) of the chip, causing under sampling of secretion dynamics. (B) Comparison of average [Ca²⁺]_i and insulin oscillation periods from each animal. Data sets are n≥6 islets and error bars are ±1 standard deviation. (C) Plot of average [Ca²⁺]_i versus insulin for each mouse. The linear relationship of data points suggests good agreement of oscillation frequencies (R² = 0.98; p<0.0001).</p

Effects of weight gain with age on imprinting.

<p>(A–B) Representative examples of islet [Ca²⁺]_i patterns in 11 mM glucose among lean mice weighing <30 g (A) and aged/large mice weighing >40 g (B). (C) Mean period of oscillations among 10 lean and 3 aged/large Swiss-Webster mice. Mean weight and mean period of islet [Ca²⁺]_i oscillations differed between groups (p<0.001).</p

Both inbred and outbred mice display islet imprinting.

<p>(A–B) Two representative C57BL/6J mice out of a group of 9 displayed very different [Ca²⁺]_i oscillation patterns. Three representative islets from Mouse 6 display slow oscillations (A, period: 3.9±0.2 minutes, n = 12 islets total) and three representative islets from Mouse 5 display fast oscillations (B, period: 0.9±0.6 minutes, n = 9 islets total). One trace shown in B (bottom) shows a clear ‘slow component’ that was representative of n = 4 islets from Mouse 5 (period: 5.4±0.1 minutes). (C–D) The variation in the period of [Ca²⁺]_i oscillations indicates distinct differences from mouse to mouse for the inbred B6 strain (C) and the outbred CD-1 strain (D), as shown by one-way ANOVA (p<1.0e-24). Boxes drawn around Mouse 6 and Mouse 5 in (C) are described above.</p

Dispersed beta cells do not display frank imprinting.

<p>(A) Representative examples of oscillatory patterns from 3 individual beta cells (A) and 3 islets (B) taken from the same mouse (Mouse 8 as indicated by the box in C). (C–D) Mean period ± SEM from 12 sets of beta cells (C) and corresponding islets (D) from the same mouse. Beta cells displayed longer period and also a greater degree of variability in their periods as noted by the large standard deviations they exhibited compared to islets. A total of 137 beta cells and 109 islets were recorded among 12 mice. One-way ANOVA indicates differences among beta-cell periods (P<0.01) and substantial differences among islet periods (p<1.0e-25) from mouse to mouse. (E) Scatter plot showing the relationship between oscillatory periods of beta cells and islets among the 12 mice studied (R² = 0.39, p = 0.22).</p