Negative feedback synchronizes islets of Langerhans.

Insulin is released from the pancreas in pulses with a period of ~ 5 min. These oscillatory insulin levels are essential for proper liver utilization and perturbed pulsatility is observed in type 2 diabetes. What coordinates the many islets of Langerhans throughout the pancreas to produce unified oscillations of insulin secretion? One hypothesis is that coordination is achieved through an insulin-dependent negative feedback action of the liver onto the glucose level. This hypothesis was tested in an in vitro setting using a microfluidic system where the population response from a group of islets was input to a model of hepatic glucose uptake, which provided a negative feedback to the glucose level. This modified glucose level was then delivered back to the islet chamber where the population response was again monitored and used to update the glucose concentration delivered to the islets. We found that, with appropriate parameters for the model, oscillations in islet activity were synchronized. This approach demonstrates that rhythmic activity of a population of physically uncoupled islets can be coordinated by a downstream system that senses islet activity and supplies negative feedback. In the intact animal, the liver can play this role of the coordinator of islet activity.

Synchronization of mouse islets of Langerhans by glucose waveforms.

Pancreatic islets secrete insulin in a pulsatile manner, and the individual islets are synchronized, producing in vivo oscillations. In this report, the ability of imposed glucose waveforms to synchronize a population of islets was investigated. A microfluidic system was used to deliver glucose waveforms to ∼20 islets while fura 2 fluorescence was imaged. All islets were entrained to a sinusoidal waveform of glucose (11 mM median, 1 mM amplitude, and a 5-min period), producing synchronized oscillations of fura 2 fluorescence. During perfusion with constant 11 mM glucose, oscillations of fura 2 fluorescence were observed in individual islets, but the average signal was nonoscillatory. Spectral analysis and a synchronization index (λ) were used to measure the period of fura 2 fluorescence oscillations and evaluate synchronization of islets, respectively. During perfusion with glucose waveforms, spectral analysis revealed a dominant frequency at 5 min, and λ, which can range from 0 (unsynchronized) to 1 (perfect synchronization), was 0.78 ± 0.15. In contrast, during perfusion with constant 11 mM glucose, the main peak in the spectral analysis corresponded to a period of 5 min but was substantially smaller than during perfusion with oscillatory glucose, and the average λ was 0.52 ± 0.09, significantly lower than during perfusion with sinusoidal glucose. These results indicated that an oscillatory glucose level synchronized the activity of a heterogeneous islet population, serving as preliminary evidence that islets could be synchronized in vivo through oscillatory glucose levels produced by a liver-pancreas feedback loop.

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

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.

Simultaneous capillary electrophoresis competitive immunoassay for insulin, glucagon, and islet amyloid polypeptide secretion from mouse islets of Langerhans.

A capillary electrophoresis competitive immunoassay was developed for the simultaneous quantitation of insulin, glucagon, and islet amyloid polypeptide (IAPP) secretion from islets of Langerhans. Separation buffers and conditions were optimized for the resolution of fluorescein isothiocyanate (FITC)-labeled glucagon and IAPP immunoassay reagents, which were excited with the 488 nm line of an Ar(+) laser and detected at 520 nm with a photomultiplier tube (PMT). Cy5-labeled insulin immunoassay reagents were excited by a 635 nm laser diode module and detected at 700 nm with a separate PMT. Optimum resolution was achieved with a 20mM carbonate separation buffer at pH 9.0 using a 20 cm effective separation length with an electric field of 500 V/cm. Limits of detection for insulin, glucagon, and IAPP were 2, 3, and 3 nM, respectively. This method was used to monitor the simultaneous secretion of these peptides from as few as 14 islets after incubation in 4, 11, and 20 mM glucose for 6h. For insulin and IAPP, a statistically significant increase in secretion levels was observed, while glucagon levels were significantly reduced in the 4 and 11 mM glucose conditions. To further demonstrate the utility of the assay, the Ca(2+)-dependent secretion of these peptides was demonstrated which agreed with published reports. The ability to examine the secretion of multiple peptides may allow for the determination of regulation of secretory processes within islets of Langerhans.