Date Published: February 6, 2019
Publisher: Public Library of Science
Author(s): Joel E. Adablah, Ryan Vinson, Michael G. Roper, Richard Bertram, Wataru Nishimura.
Pulsatile insulin secretion into the portal vein from the many pancreatic islets of Langerhans is critical for efficient glucose homeostasis. The islets are themselves endogenous oscillators, but since they are not physically coupled it is not obvious how their oscillations are synchronized across the pancreas. It has been proposed that synchronization of islets is achieved through periodic activity of intrapancreatic ganglia, and indeed there are data supporting this proposal. Postganglionic nerves are cholinergic, and their product, acetylcholine, can influence islet β-cells through actions on M3 muscarinic receptors which are coupled to Gq type G-proteins. In addition, the neurons secrete several peptide hormones that act on β-cell receptors. The data supporting synchronization via intrapancreatic ganglia are, however, limited. In particular, it has not been shown that trains of muscarinic pulses are effective at synchronizing islets in vitro. Also, if as has been suggested, there is a ganglionic pacemaker driving islets to a preferred frequency, no neural circuitry for this pacemaker has been identified. In this study, both points are addressed using a microfluidic system that allows for the pulsed application of the muscarinic agonist carbachol. We find that murine islets are entrained and synchronized over a wide range of frequencies when the carbachol pulsing is periodic, adding support to the hypothesis that ganglia can synchronize islets in vivo. We also find that islet synchronization is very effective even if the carbachol pulses are applied at random times. This suggests that a neural pacemaker is not needed; all that is required is that islets receive occasional coordinated input from postganglionic neurons. The endogenous rhythmic activity of the islets then sets the frequency of the islet population rhythm, while the input from ganglia acts only to keep the islet oscillators in phase.
The rodent endocrine pancreas contains thousands to tens of thousands of islets of Langerhans , while a human may have over 3 million . Islets contain 500–2000 cells, with each cell type releasing particular hormones in a glucose-dependent manner . The majority of these cells are insulin-secreting β-cells that, like other cells, metabolize glucose leading to an increase in the ratio of ATP/ADP. Unlike most other cells, β-cells express KATP ion channels that are inactivated by the increased ATP/ADP ratio, and the resulting decrease in hyperpolarizing K+ current depolarizes the cell membrane . This depolarization opens voltage-gated Ca2+ channels allowing for the influx of Ca2+ that triggers insulin secretion into the blood [5,6]. Insulin promotes the absorption of glucose into fat, skeletal muscle, and liver cells .
The microfluidic platform employed for this study facilitated precise and automated delivery of glucose and the various CCh profiles to groups of 3–4 islets. Multiple experiments were performed to investigate the effects of different CCh profiles. Although the layout of the microfluidic channels has been described previously, the use of the piezoelectric transducer in conjunction with the flow rate sensors was not. We found that this active flow rate control was much better at rapidly generating pulses and maintaining stable flow rates than passive methods. The reagents took 48 s (SD 1) to travel the 75 mm long channel from the inputs to the islet chamber. This dispersive flow resulted in attenuation of the 10 μM CCh pulses by 43.5% (SD 0.4). Thus, the concentration felt by the islets was ~5.7 μM. This value is lower than the those used in other reports [25,26] although, as shown below, it was still large enough to induce responses in all islets tested.
We have demonstrated that periodic pulses of CCh are capable of entraining pancreatic islet Ca2+ oscillations with stimulus periods ranging from 2 to 10 min. There was a clear preference for a ~5 min response period, which is the natural period of most individual islet oscillators. Further, all islets subjected to the CCh pulses with a 5 min rest time responded with Ca2+ oscillations of 5 min periodicity. One third of the islets stimulated at a higher frequency (2 min rest time) responded by oscillating with a period of 4 min. Fourteen of the 20 islets stimulated at a low frequency (10 min stimulus period) responded with oscillations of 5 min period, due to 1:2 entrainment. It is evident, then, that a cholinergic agonist like CCh, or the related ACh, could be an effective islet synchronizer if applied periodically.
We have demonstrated that oscillations in islet activity, as measured through the intracellular Ca2+ concentration, can be entrained by the periodic application of the cholinergic agonist CCh. This is true for a range of stimulus periods from 2 to 10 min. In the case of the 2-min stimulus period there is often 2:1 entrainment, with one Ca2+ pulse for every other CCh stimulus. In the case of the 10-min stimulus period there is often 1:2 entrainment with two Ca2+ pulses per stimulus.