High-content screening identifies a role for Na ⁺ channels in insulin production

Abstract

Insulin production is the central feature of functionally mature and differentiated pancreatic β-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote β-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 β-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of β-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect β-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and β-cell differentiation status. In particular, our unbiased screen identified a novel role for a β-cell sodium channel gene in insulin production.

Oestrogen receptor β mediates the actions of bisphenol-A on ion channel expression in mouse pancreatic beta cells

“…While no alterations in GSIS are observed in Scn9a −/− mice, these mice present higher insulin content relative to wild-type counterparts [12]. Furthermore, Na v 1.7 channel inhibition increases both Ins1 and Ins2 mRNA expression [12]. These data suggest that Na v 1.7 channels may be associated with insulin biosynthesis.…”

“…Alternatively, the Na v 1.7 channel, encoded by Scn9a, corresponds to 85% of the Na + current [30], although its role is yet to be unveiled as the Na v 1.7 channel is inactivated at physiological membrane potentials. While no alterations in GSIS are observed in Scn9a −/− mice, these mice present higher insulin content relative to wild-type counterparts [12]. Furthermore, Na v 1.7 channel inhibition increases both Ins1 and Ins2 mRNA expression [12].…”

“…Mouse beta cells express the voltageactivated Na + channels (Na v channels) Na v 1.3 and Na v 1.7. Na v 1.3 participates in the depolarisation of the action potential and affects insulin secretion [11]; Na v 1.7 does not participate in insulin release, yet it might play a role in insulin production [12] and beta cell survival [13]. The repolarisation phase between bursts of action potential is due to small-conductance Ca 2+ -activated K + channels (K Ca 2.3 channels) [6,14,15].…”

Modulation of Ionic Channels and Insulin Secretion by Drugs and Hormones in Pancreatic Beta Cells

“…Changes in the activity of Nav1.7 channels may be linked to beta-cell dysfunction and the susceptibility of developing painful neuropathy, because this channel is also expressed in dorsal root ganglion neurons (Hoeijmakers et al, 2014). Recently, it was shown that Nav1.7 channels are linked to insulin production, because pancreatic islets from Scn9a2/2 mice exhibit increased insulin content compared with their wild-type counterparts (Szabat et al, 2015). The latter also offers new perspectives for drugs like carbamazepine, which inhibits the channel and dose dependently increases the mRNA expression of both insulin genes (Ins1 and Ins 2), as well as Pdx1 (a marker of pancreatic beta cells) in isolated islets from mice (Szabat et al, 2015).…”

The quest to make fully functional human pancreatic beta cells from embryonic stem cells: climbing a mountain in the clouds

“…Both of the 2014 landmark papers made use of highthroughput screening, and although this is a powerful technique, some might wonder whether it has taken us off the path of mimicking pancreas development. Despite this concern, high-throughput efforts to identify factors that increase insulin production and promote beta cell differentiation continue in our group and elsewhere [45].…”