Measurement of the entrainment window of islets of Langerhans by microfluidic delivery of a chirped glucose waveform

Abstract

Within single islets of Langerhans, the endocrine portion of the pancreas, intracellular metabolites, as well as insulin secretion, oscillate with a period of ∼5 min. In vivo, pulsatile insulin oscillations are also observed with periods ranging from 5-15 minutes. In order for oscillations of insulin to be observed in vivo, the majority of islets in the pancreas must synchronize their output. It is known that populations of islets can be synchronized via entrainment of the individual islets to low amplitude glucose oscillations that have periods close to islets' natural period. However, the range of glucose periods and amplitudes that can entrain islets has not been rigorously examined. To find the range of glucose periods that can entrain islets, a microfluidic system was utilized to produce and deliver a chirped glucose waveform to populations of islets while their individual intracellular [Ca(2+)] ([Ca(2+)]i) oscillations were imaged. Waveforms with amplitudes of 0.5, 1, and 1.5 mM above a median value of 11 mM were applied while the period was swept from 20-2 min. Oscillations of [Ca(2+)]i resonated the strongest when the period of the glucose wave was within 2 min of the natural period of the islets, typically close to 5 min. Some examples of 1 : 2 and 2 : 1 entrainment were observed during exposure to long and short glucose periods, respectively. These results shed light on the dynamic nature of islet behavior and may help to understand dynamics observed in vivo.

Fig. 1. Microfluidic device design

The design of the microfluidic chip is shown on the left side of the figure. The points labelled “A” and “B” were used as inputs for BSS containing 3 and 13 mM glucose, respectively. The flow rates from these two inputs were set by the heights of their respective solutions above the microfluidic system. At the flow-splitting region, outlined by the dashed box, the input solutions were split to three outlets; the two outlets on the side were sent to a “Waste” reservoir while the centre outlet was sent to the “Islet chamber”. The ratio of the two solutions entering the middle outlet was dictated by the flow rate ratio of the two input solutions. Because the two input solutions were connected via a pulley and fixed length of cord, the total flow rate entering the centre channel remained constant. On the right side of the figure are photographs taken of the flow-splitting region when fluorescein and buffer were being input to the system. The top image shows when the buffer had a higher flow rate than fluorescein; the middle image shows when the two solutions had equal flow rates; the bottom image shows when fluorescein had a higher flow rate than the buffer solution. The two solutions entering the centre outlet mixed to homogeneity prior to delivery to the islet chamber.

Fig. 2. Entrainment of islets using a 20 to 2 min chirped glucose waveform at 1 mM amplitude

The top shows the average [Ca²⁺]_i response (black line) from 7 islets with error bars corresponding to ± 1 SD. The applied glucose concentration (red line) is shown on the right y-axis. The spectrogram of the average [Ca²⁺]_i trace is shown on the bottom with the experimental time scale on the bottom x-axis, the period of the glucose wave on the top x-axis, and the measured [Ca²⁺]_i oscillation period on the left y-axis. The scale bar for the spectrogram is shown on the right of the figure with red being the largest magnitude and blue being the smallest. The solid white line corresponds to how the period of the applied glucose wave was swept in time. The dashed white lines are used to indicate the region of entrainment from 5.4 to 3.4 min.

Fig. 3. Response to constant glucose level

The top shows the average (black line) and ± 1 SD of the [Ca²⁺]_i for 7 islets exposed to a constant glucose level (red) throughout the experiment. The spectrogram of the average [Ca²⁺]_i is shown below and does not show any major frequency component. The scale of the spectrogram is the same as that in Fig. 2.

Fig. 4. A chirped glucose wave from 2 to 20 min with 1 mM amplitude

The experiment shown is the application of a chirped waveform with the period of the glucose increasing from 2 to 20 min. The average ± 1 SD [Ca²⁺]_i from 7 islets and its corresponding spectrogram are shown on the top and bottom, respectively. The lines on the spectrogram are similar to those described for Fig. 2.

Fig. 5. Entrainment of islets using a chirped glucose waveform at 1.5 mM amplitude

The average ± 1 SD [Ca²⁺]_i from 6 islets is shown at the top of the figure. The data is shown starting with a glucose concentration of 11 mM for 6 min followed by a 60 min chirped wave. Similar to Fig. 2, the period of the waveform was swept from 20 - 2 min, but a 1.5 mM amplitude was used. The islets were entrained 1:1 between 7.2 and 2.9 min (dashed white lines).

Fig. 6. A chirped glucose wave at 0.5 mM amplitude

The average ± 1 SD [Ca²⁺]_i from 5 islets is shown in the upper trace. A chirped glucose wave was applied that swept the periods from 20 - 2 min with 0.5 mM amplitude. The entrained region was observed between 4.2 – 2.6 min.

Fig. 7. Single islet entrainment data

Two [Ca²⁺]_i traces and their corresponding spectrograms from single islets are shown where the glucose period was swept from 20 - 2 min at a 1 mM amplitude. In (A) the islet had a T_n ~4 min and was entrained 1:1 from 27 - 45 min as seen by the [Ca²⁺]_i oscillation period following the upper white line. The lower white line corresponds to 2:1 entrainment and the islet follows this line from 55 – 62 min. The spectrogram in (B) shows that the islet had a T_n ~3 min and upon application of the chirped wave, followed the 1:1 line from 13 – 65 min, corresponding to glucose periods of 9.8 - 2.0 min.