TRPM7 is a crucial regulator of pancreatic endocrine development and high-fat-diet-induced ß-cell proliferation.

The melastatin subfamily of the transient receptor potential channels (TRPM) are regulators of pancreatic ß-cell function. TRPM7 is the most abundant islet TRPM channel; however, the role of TRPM7 in ß-cell function has not been determined. Here, we used various spatiotemporal transgenic mouse models to investigate how TRPM7 knockout influences pancreatic endocrine development, proliferation and function. Ablation of TRPM7 within pancreatic progenitors reduced pancreatic size, and α-cell and ß-cell mass. This resulted in modestly impaired glucose tolerance. However, TRPM7 ablation following endocrine specification or in adult mice did not impact endocrine expansion or glucose tolerance. As TRPM7 regulates cell proliferation, we assessed how TRPM7 influences ß-cell hyperplasia under insulin-resistant conditions. ß-Cell proliferation induced by high-fat diet was significantly decreased in TRPM7-deficient ß-cells. The endocrine roles of TRPM7 may be influenced by cation flux through the channel, and indeed we found that TRPM7 ablation altered ß-cell Mg2+ and reduced the magnitude of elevation in ß-cell Mg2+ during proliferation. Together, these findings revealed that TRPM7 controls pancreatic development and ß-cell proliferation, which is likely due to regulation of Mg2+ homeostasis.

Liraglutide increases islet Ca2+ oscillation frequency and insulin secretion by activating hyperpolarization-activated cyclic nucleotide-gated channels.

AIM: To determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN) channels impact glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) modulation of islet Ca2+ handling and insulin secretion. METHODS: The impact of liraglutide (GLP-1 analogue) on islet Ca2+ handling, HCN currents and insulin secretion was monitored with fluorescence microscopy, electrophysiology and enzyme immunoassays, respectively. Furthermore, liraglutide-mediated ß-to-δ-cell cross-communication was assessed following selective ablation of either mouse islet δ or ß cells. RESULTS: Liraglutide increased ß-cell Ca2+ oscillation frequency in mouse and human islets under stimulatory glucose conditions. This was dependent in part on liraglutide activation of HCN channels, which also enhanced insulin secretion. Similarly, liraglutide activation of HCN channels also increased ß-cell Ca2+ oscillation frequency in islets from rodents exposed to a diabetogenic diet. Interestingly, liraglutide accelerated Ca2+ oscillations in a majority of islet δ cells, which showed synchronized Ca2+ oscillations equivalent to ß cells; therefore, we assessed if either cell type was driving this liraglutide-mediated islet Ca2+ response. Although δ-cell loss did not impact liraglutide-mediated increase in ß-cell Ca2+ oscillation frequency, ß-cell ablation attenuated liraglutide-facilitated acceleration of δ-cell Ca2+ oscillations. CONCLUSION: The data presented here show that liraglutide-induced stimulation of islet HCN channels augments Ca2+ oscillation frequency. As insulin secretion oscillates with ß-cell Ca2+ , these findings have important implications for pulsatile insulin secretion that is probably enhanced by liraglutide activation of HCN channels and therapeutics that target GLP-1Rs for treating diabetes. Furthermore, these studies suggest that liraglutide as well as GLP-1-based therapies enhance δ-cell Ca2+ oscillation frequency and somatostatin secretion kinetics in a ß-cell-dependent manner.

Tetraspanin-7 regulation of L-type voltage-dependent calcium channels controls pancreatic ß-cell insulin secretion.

KEY POINTS: Tetraspanin (TSPAN) proteins regulate many biological processes, including intracellular calcium (Ca2+ ) handling. TSPAN-7 is enriched in pancreatic islet cells; however, the function of islet TSPAN-7 has not been identified. Here, we characterize how ß-cell TSPAN-7 regulates Ca2+ handling and hormone secretion. We find that TSPAN-7 reduces ß-cell glucose-stimulated Ca2+ entry, slows Ca2+ oscillation frequency and decreases glucose-stimulated insulin secretion. TSPAN-7 controls ß-cell function through a direct interaction with L-type voltage-dependent Ca2+ channels (CaV 1.2 and CaV 1.3), which reduces channel Ca2+ conductance. TSPAN-7 slows activation of CaV 1.2 and accelerates recovery from voltage-dependent inactivation; TSPAN-7 also slows CaV 1.3 inactivation kinetics. These findings strongly implicate TSPAN-7 as a key regulator in determining the set-point of glucose-stimulated Ca2+ influx and insulin secretion. ABSTRACT: Glucose-stimulated insulin secretion (GSIS) is regulated by calcium (Ca2+ ) entry into pancreatic ß-cells through voltage-dependent Ca2+ (CaV ) channels. Tetraspanin (TSPAN) transmembrane proteins control Ca2+ handling, and thus they may also modulate GSIS. TSPAN-7 is the most abundant islet TSPAN and immunostaining of mouse and human pancreatic slices shows that TSPAN-7 is highly expressed in ß- and α-cells; however, the function of islet TSPAN-7 has not been determined. Here, we show that TSPAN-7 knockdown (KD) increases glucose-stimulated Ca2+ influx into mouse and human ß-cells. Additionally, mouse ß-cell Ca2+ oscillation frequency was accelerated by TSPAN-7 KD. Because TSPAN-7 KD also enhanced Ca2+ entry when membrane potential was clamped with depolarization, the effect of TSPAN-7 on CaV channel activity was examined. TSPAN-7 KD enhanced L-type CaV currents in mouse and human ß-cells. Conversely, heterologous expression of TSPAN-7 with CaV 1.2 and CaV 1.3 L-type CaV channels decreased CaV currents and reduced Ca2+ influx through both channels. This was presumably the result of a direct interaction of TSPAN-7 and L-type CaV channels because TSPAN-7 coimmunoprecipitated with both CaV 1.2 and CaV 1.3 from primary human ß-cells and from a heterologous expression system. Finally, TSPAN-7 KD in human ß-cells increased basal (5.6 mM glucose) and stimulated (45 mM KCl + 14 mM glucose) insulin secretion. These findings strongly suggest that TSPAN-7 modulation of ß-cell L-type CaV channels is a key determinant of ß-cell glucose-stimulated Ca2+ entry and thus the set-point of GSIS.

Glucose-mediated inhibition of calcium-activated potassium channels limits α-cell calcium influx and glucagon secretion.

Pancreatic α-cells exhibit oscillations in cytosolic Ca2+ (Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+c oscillations have not been elucidated. As ß-cell Ca2+c oscillations are regulated in part by Ca2+-activated K+ (Kslow) currents, this work investigated the role of Kslow in α-cell Ca2+ handling and GCG secretion. α-Cells displayed Kslow currents that were dependent on Ca2+ influx through L- and P/Q-type voltage-dependent Ca2+ channels (VDCCs) as well as Ca2+ released from endoplasmic reticulum stores. α-Cell Kslow was decreased by small-conductance Ca2+-activated K+ (SK) channel inhibitors apamin and UCL 1684, large-conductance Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+ (IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell Kslow with apamin depolarized membrane potential ( Vm) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca2+ influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca2+ influx. Total α-cell Ca2+c was similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca2+c following prolonged Kslow inhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslow activation provides a hyperpolarizing influence on α-cell Vm that sustains Ca2+ entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+c is elevated during secretagogue stimulation, Kslow activation helps to preserve GCG secretion.

Structure-Activity Relationships, Pharmacokinetics, and Pharmacodynamics of the Kir6.2/SUR1-Specific Channel Opener VU0071063.

Glucose-stimulated insulin secretion from pancreatic ß-cells is controlled by ATP-regulated potassium (KATP) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) subunits. The KATP channel-opener diazoxide is FDA-approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular KATP channels and other ion channels. The development of more specific openers would provide critically needed tool compounds for probing the therapeutic potential of Kir6.2/SUR1 activation. Here, we characterize a novel scaffold activator of Kir6.2/SUR1 that our group recently discovered in a high-throughput screen. Optimization efforts with medicinal chemistry identified key structural elements that are essential for VU0071063-dependent opening of Kir6.2/SUR1. VU0071063 has no effects on heterologously expressed Kir6.1/SUR2B channels or ductus arteriole tone, indicating it does not open vascular KATP channels. VU0071063 induces hyperpolarization of ß-cell membrane potential and inhibits insulin secretion more potently than diazoxide. VU0071063 exhibits metabolic and pharmacokinetic properties that are favorable for an in vivo probe and is brain penetrant. Administration of VU0071063 inhibits glucose-stimulated insulin secretion and glucose-lowering in mice. Taken together, these studies indicate that VU0071063 is a more potent and specific opener of Kir6.2/SUR1 than diazoxide and should be useful as an in vitro and in vivo tool compound for investigating the therapeutic potential of Kir6.2/SUR1 expressed in the pancreas and brain.

Inhibition of pancreatic ß-cell Ca2+/calmodulin-dependent protein kinase II reduces glucose-stimulated calcium influx and insulin secretion, impairing glucose tolerance.

Glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells is caused by Ca(2+) entry via voltage-dependent Ca(2+) channels. CaMKII is a key mediator and feedback regulator of Ca(2+) signaling in many tissues, but its role in ß-cells is poorly understood, especially in vivo. Here, we report that mice with conditional inhibition of CaMKII in ß-cells show significantly impaired glucose tolerance due to decreased GSIS. Moreover, ß-cell CaMKII inhibition dramatically exacerbates glucose intolerance following exposure to a high fat diet. The impairment of islet GSIS by ß-cell CaMKII inhibition is not accompanied by changes in either glucose metabolism or the activities of KATP and voltage-gated potassium channels. However, glucose-stimulated Ca(2+) entry via voltage-dependent Ca(2+) channels is reduced in islet ß-cells with CaMKII inhibition, as well as in primary wild-type ß-cells treated with a peptide inhibitor of CaMKII. The levels of basal ß-cell cytoplasmic Ca(2+) and of endoplasmic reticulum Ca(2+) stores are also decreased by CaMKII inhibition. In addition, CaMKII inhibition suppresses glucose-stimulated action potential firing frequency. These results reveal that CaMKII is a Ca(2+) sensor with a key role as a feed-forward stimulator of ß-cell Ca(2+) signals that enhance GSIS under physiological and pathological conditions.

Direct activation of ß-cell KATP channels with a novel xanthine derivative.

ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet ß-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The KATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved Kir6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing KATP channels. Here we report the serendipitous discovery in a high-throughput screen of a novel activator of Kir6.2/SUR1: VU0071063 [7-(4-(tert-butyl)benzyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione]. The xanthine derivative rapidly and dose-dependently activates Kir6.2/SUR1 with a half-effective concentration (EC50) of approximately 7 µM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1- over SUR2A-containing Kir6.1 or Kir6.2 channels, as well as Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native Kir6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic ß cells. VU0071063 represents a novel tool/compound for investigating ß-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry.

The MODY-associated KCNK16 L114P mutation increases islet glucagon secretion and limits insulin secretion resulting in transient neonatal diabetes and glucose dyshomeostasis in adults.

The gain-of-function mutation in the TALK-1 K + channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of ß-cell electrical activity and glucose-stimulated insulin secretion (GSIS). The KCNK16 gene encoding TALK-1, is the most abundant and ß-cell-restricted K + channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the mixed C57BL/6J:CD-1(ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell ß-cell K + currents resulting in blunted glucose-stimulated Ca 2+ entry and loss of glucose-induced Ca 2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impaired glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet hormone secretion during development. These data strongly suggest that TALK-1 is an islet-restricted target for the treatment of diabetes.

The MODY-associated KCNK16 L114P mutation increases islet glucagon secretion and limits insulin secretion resulting in transient neonatal diabetes and glucose dyshomeostasis in adults.

The gain-of-function mutation in the TALK-1 K+ channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of ß-cell electrical activity and glucose-stimulated insulin secretion. The KCNK16 gene encoding TALK-1 is the most abundant and ß-cell-restricted K+ channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the CD-1 (ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell ß-cell K+ currents resulting in blunted glucose-stimulated Ca2+ entry and loss of glucose-induced Ca2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impairs glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet insulin secretion during development. These data suggest that TALK-1 is an islet-restricted target for the treatment for diabetes.

TALK-1-mediated alterations of ß-cell mitochondrial function and insulin secretion impair glucose homeostasis on a diabetogenic diet.

Mitochondrial Ca2+ ([Ca2+]m) homeostasis is critical for ß-cell function and becomes disrupted during the pathogenesis of diabetes. [Ca2+]m uptake is dependent on elevations in cytoplasmic Ca2+ ([Ca2+]c) and endoplasmic reticulum Ca2+ ([Ca2+]ER) release, both of which are regulated by the two-pore domain K+ channel TALK-1. Here, utilizing a novel ß-cell TALK-1-knockout (ß-TALK-1-KO) mouse model, we found that TALK-1 limited ß-cell [Ca2+]m accumulation and ATP production. However, following exposure to a high-fat diet (HFD), ATP-linked respiration, glucose-stimulated oxygen consumption rate, and glucose-stimulated insulin secretion (GSIS) were increased in control but not TALK1-KO mice. Although ß-TALK-1-KO animals showed similar GSIS before and after HFD treatment, these mice were protected from HFD-induced glucose intolerance. Collectively, these data identify that TALK-1 channel control of ß-cell function reduces [Ca2+]m and suggest that metabolic remodeling in diabetes drives dysglycemia.

Gi/o protein-coupled receptor inhibition of beta-cell electrical excitability and insulin secretion depends on Na+/K+ ATPase activation.

Gi/o-coupled somatostatin or α2-adrenergic receptor activation stimulated ß-cell NKA activity, resulting in islet Ca2+ fluctuations. Furthermore, intra-islet paracrine activation of ß-cell Gi/o-GPCRs and NKAs by δ-cell somatostatin secretion slowed Ca2+ oscillations, which decreased insulin secretion. ß-cell membrane potential hyperpolarization resulting from Gi/o-GPCR activation was dependent on NKA phosphorylation by Src tyrosine kinases. Whereas, ß-cell NKA function was inhibited by cAMP-dependent PKA activity. These data reveal that NKA-mediated ß-cell membrane potential hyperpolarization is the primary and conserved mechanism for Gi/o-GPCR control of electrical excitability, Ca2+ handling, and insulin secretion.

Antagonistic epistasis of Hnf4α and FoxO1 metabolic networks through enhancer interactions in ß-cell function.

OBJECTIVE: Genetic and acquired abnormalities contribute to pancreatic ß-cell failure in diabetes. Transcription factors Hnf4α (MODY1) and FoxO1 are respective examples of these two components and act through ß-cell-specific enhancers. However, their relationship is unclear. METHODS: In this report, we show by genome-wide interrogation of chromatin modifications that ablation of FoxO1 in mature ß-cells enriches active Hnf4α enhancers according to a HOMER analysis. RESULTS: To model the functional significance of this predicted unusual enhancer utilization, we generated single and compound knockouts of FoxO1 and Hnf4α in ß-cells. Single knockout of either gene impaired insulin secretion in mechanistically distinct fashions as indicated by their responses to sulfonylurea and calcium fluxes. Surprisingly, the defective ß-cell secretory function of either single mutant in hyperglycemic clamps and isolated islets treated with various secretagogues was completely reversed in double mutants lacking FoxO1 and Hnf4α. Gene expression analyses revealed distinct epistatic modalities by which the two transcription factors regulate networks associated with reversal of ß-cell dysfunction. An antagonistic network regulating glycolysis, including ß-cell "disallowed" genes, and a synergistic network regulating protocadherins emerged as likely mediators of the functional restoration of insulin secretion. CONCLUSIONS: The findings provide evidence of antagonistic epistasis as a model of gene/environment interactions in the pathogenesis of ß-cell dysfunction.

Temporal Transcriptome Analysis Reveals Dynamic Gene Expression Patterns Driving ß-Cell Maturation.

Newly differentiated pancreatic ß cells lack proper insulin secretion profiles of mature functional ß cells. The global gene expression differences between paired immature and mature ß cells have been studied, but the dynamics of transcriptional events, correlating with temporal development of glucose-stimulated insulin secretion (GSIS), remain to be fully defined. This aspect is important to identify which genes and pathways are necessary for ß-cell development or for maturation, as defective insulin secretion is linked with diseases such as diabetes. In this study, we assayed through RNA sequencing the global gene expression across six ß-cell developmental stages in mice, spanning from ß-cell progenitor to mature ß cells. A computational pipeline then selected genes differentially expressed with respect to progenitors and clustered them into groups with distinct temporal patterns associated with biological functions and pathways. These patterns were finally correlated with experimental GSIS, calcium influx, and insulin granule formation data. Gene expression temporal profiling revealed the timing of important biological processes across ß-cell maturation, such as the deregulation of ß-cell developmental pathways and the activation of molecular machineries for vesicle biosynthesis and transport, signal transduction of transmembrane receptors, and glucose-induced Ca2+ influx, which were established over a week before ß-cell maturation completes. In particular, ß cells developed robust insulin secretion at high glucose several days after birth, coincident with the establishment of glucose-induced calcium influx. Yet the neonatal ß cells displayed high basal insulin secretion, which decreased to the low levels found in mature ß cells only a week later. Different genes associated with calcium-mediated processes, whose alterations are linked with insulin resistance and deregulation of glucose homeostasis, showed increased expression across ß-cell stages, in accordance with the temporal acquisition of proper GSIS. Our temporal gene expression pattern analysis provided a comprehensive database of the underlying molecular components and biological mechanisms driving ß-cell maturation at different temporal stages, which are fundamental for better control of the in vitro production of functional ß cells from human embryonic stem/induced pluripotent cell for transplantation-based type 1 diabetes therapy.

A KCNK16 mutation causing TALK-1 gain of function is associated with maturity-onset diabetes of the young.

Maturity-onset diabetes of the young (MODY) is a heterogeneous group of monogenic disorders of impaired pancreatic ß cell function. The mechanisms underlying MODY include ß cell KATP channel dysfunction (e.g., KCNJ11 [MODY13] or ABCC8 [MODY12] mutations); however, no other ß cell channelopathies have been associated with MODY to date. Here, we have identified a nonsynonymous coding variant in KCNK16 (NM_001135105: c.341T>C, p.Leu114Pro) segregating with MODY. KCNK16 is the most abundant and ß cell-restricted K+ channel transcript, encoding the two-pore-domain K+ channel TALK-1. Whole-cell K+ currents demonstrated a large gain of function with TALK-1 Leu114Pro compared with TALK-1 WT, due to greater single-channel activity. Glucose-stimulated membrane potential depolarization and Ca2+ influx were inhibited in mouse islets expressing TALK-1 Leu114Pro with less endoplasmic reticulum Ca2+ storage. TALK-1 Leu114Pro significantly blunted glucose-stimulated insulin secretion compared with TALK-1 WT in mouse and human islets. These data suggest that KCNK16 is a previously unreported gene for MODY.

Lactate activation of α-cell KATP channels inhibits glucagon secretion by hyperpolarizing the membrane potential and reducing Ca2+ entry.

OBJECTIVE: Elevations in pancreatic α-cell intracellular Ca2+ ([Ca2+]i) lead to glucagon (GCG) secretion. Although glucose inhibits GCG secretion, how lactate and pyruvate control α-cell Ca2+ handling is unknown. Lactate enters cells through monocarboxylate transporters (MCTs) and is also produced during glycolysis by lactate dehydrogenase A (LDHA), an enzyme expressed in α-cells. As lactate activates ATP-sensitive K+ (KATP) channels in cardiomyocytes, lactate may also modulate α-cell KATP. Therefore, this study investigated how lactate signaling controls α-cell Ca2+ handling and GCG secretion. METHODS: Mouse and human islets were used in combination with confocal microscopy, electrophysiology, GCG immunoassays, and fluorescent thallium flux assays to assess α-cell Ca2+ handling, Vm, KATP currents, and GCG secretion. RESULTS: Lactate-inhibited mouse (75 ± 25%) and human (47 ± 9%) α-cell [Ca2+]i fluctuations only under low-glucose conditions (1 mM) but had no effect on ß- or δ-cells [Ca2+]i. Glyburide inhibition of KATP channels restored α-cell [Ca2+]i fluctuations in the presence of lactate. Lactate transport into α-cells via MCTs hyperpolarized mouse (14 ± 1 mV) and human (12 ± 1 mV) α-cell Vm and activated KATP channels. Interestingly, pyruvate showed a similar KATP activation profile and α-cell [Ca2+]i inhibition as lactate. Lactate-induced inhibition of α-cell [Ca2+]i influx resulted in reduced GCG secretion in mouse (62 ± 6%) and human (43 ± 13%) islets. CONCLUSIONS: These data demonstrate for the first time that lactate entry into α-cells through MCTs results in KATP activation, Vm hyperpolarization, reduced [Ca2+]i, and inhibition of GCG secretion. Thus, taken together, these data indicate that lactate either within α-cells and/or elevated in serum could serve as important modulators of α-cell function.

Neurog3-Independent Methylation Is the Earliest Detectable Mark Distinguishing Pancreatic Progenitor Identity.

In the developing pancreas, transient Neurog3-expressing progenitors give rise to four major islet cell types: α, ß, δ, and γ; when and how the Neurog3+ cells choose cell fate is unknown. Using single-cell RNA-seq, trajectory analysis, and combinatorial lineage tracing, we showed here that the Neurog3+ cells co-expressing Myt1 (i.e., Myt1+Neurog3+) were biased toward ß cell fate, while those not simultaneously expressing Myt1 (Myt1-Neurog3+) favored α fate. Myt1 manipulation only marginally affected α versus ß cell specification, suggesting Myt1 as a marker but not determinant for islet-cell-type specification. The Myt1+Neurog3+ cells displayed higher Dnmt1 expression and enhancer methylation at Arx, an α-fate-promoting gene. Inhibiting Dnmts in pancreatic progenitors promoted α cell specification, while Dnmt1 overexpression or Arx enhancer hypermethylation favored ß cell production. Moreover, the pancreatic progenitors contained distinct Arx enhancer methylation states without transcriptionally definable sub-populations, a phenotype independent of Neurog3 activity. These data suggest that Neurog3-independent methylation on fate-determining gene enhancers specifies distinct endocrine-cell programs.

TALK-1 reduces delta-cell endoplasmic reticulum and cytoplasmic calcium levels limiting somatostatin secretion.

OBJECTIVE: Single-cell RNA sequencing studies have revealed that the type-2 diabetes associated two-pore domain K+ (K2P) channel TALK-1 is abundantly expressed in somatostatin-secreting δ-cells. However, a physiological role for TALK-1 in δ-cells remains unknown. We previously determined that in ß-cells, K+ flux through endoplasmic reticulum (ER)-localized TALK-1 channels enhances ER Ca2+ leak, modulating Ca2+ handling and insulin secretion. As glucose amplification of islet somatostatin release relies on Ca2+-induced Ca2+ release (CICR) from the δ-cell ER, we investigated whether TALK-1 modulates δ-cell Ca2+ handling and somatostatin secretion. METHODS: To define the functions of islet δ-cell TALK-1 channels, we generated control and TALK-1 channel-deficient (TALK-1 KO) mice expressing fluorescent reporters specifically in δ- and α-cells to facilitate cell type identification. Using immunofluorescence, patch clamp electrophysiology, Ca2+ imaging, and hormone secretion assays, we assessed how TALK-1 channel activity impacts δ- and α-cell function. RESULTS: TALK-1 channels are expressed in both mouse and human δ-cells, where they modulate glucose-stimulated changes in cytosolic Ca2+ and somatostatin secretion. Measurement of cytosolic Ca2+ levels in response to membrane potential depolarization revealed enhanced CICR in TALK-1 KO δ-cells that could be abolished by depleting ER Ca2+ with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors. Consistent with elevated somatostatin inhibitory tone, we observed significantly reduced glucagon secretion and α-cell Ca2+ oscillations in TALK-1 KO islets, and found that blockade of α-cell somatostatin signaling with a somatostatin receptor 2 (SSTR2) antagonist restored glucagon secretion in TALK-1 KO islets. CONCLUSIONS: These data indicate that TALK-1 reduces δ-cell cytosolic Ca2+ elevations and somatostatin release by limiting δ-cell CICR, modulating the intraislet paracrine signaling mechanisms that control glucagon secretion.

Cytokine-mediated changes in K+ channel activity promotes an adaptive Ca2+ response that sustains ß-cell insulin secretion during inflammation.

Cytokines present during low-grade inflammation contribute to ß-cell dysfunction and diabetes. Cytokine signaling disrupts ß-cell glucose-stimulated Ca2+ influx (GSCI) and endoplasmic reticulum (ER) Ca2+ ([Ca2+]ER) handling, leading to diminished glucose-stimulated insulin secretion (GSIS). However, cytokine-mediated changes in ion channel activity that alter ß-cell Ca2+ handling remain unknown. Here we investigated the role of K+ currents in cytokine-mediated ß-cell dysfunction. Kslow currents, which control the termination of intracellular Ca2+ ([Ca2+]i) oscillations, were reduced following cytokine exposure. As a consequence, [Ca2+]i and electrical oscillations were accelerated. Cytokine exposure also increased basal islet [Ca2+]i and decreased GSCI. The effect of cytokines on TALK-1 K+ currents were also examined as TALK-1 mediates Kslow by facilitating [Ca2+]ER release. Cytokine exposure decreased KCNK16 transcript abundance and associated TALK-1 protein expression, increasing [Ca2+]ER storage while maintaining 2nd phase GSCI and GSIS. This adaptive Ca2+ response was absent in TALK-1 KO islets, which exhibited decreased 2nd phase GSCI and diminished GSIS. These findings suggest that Kslow and TALK-1 currents play important roles in altered ß-cell Ca2+ handling and electrical activity during low-grade inflammation. These results also reveal that a cytokine-mediated reduction in TALK-1 serves an acute protective role in ß-cells by facilitating increased Ca2+ content to maintain GSIS.

Synaptotagmin 4 Regulates Pancreatic ß Cell Maturation by Modulating the Ca2+ Sensitivity of Insulin Secretion Vesicles.

Islet ß cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that ß cells of newborns secrete more insulin than adults in response to similar intracellular Ca2+ concentrations, suggesting differences in the Ca2+ sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca2+ binding paralog of the ß cell Ca2+ sensor Syt7, increased by ∼8-fold during ß cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-ßH1 cells, a human ß cell line. These findings reveal the role that altered Ca2+ sensing plays in regulating ß cell maturation.

Cystic fibrosis-related diabetes is caused by islet loss and inflammation.

Cystic fibrosis-related (CF-related) diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting approximately 35% of adults with CF. However, the underlying causes of CFRD are unclear. Here, we examined cystic fibrosis transmembrane conductance regulator (CFTR) islet expression and whether the CFTR participates in islet endocrine cell function using murine models of ß cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine ß cells did not affect ß cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein and electrical activity were not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion, with few changes in key islet-regulatory transcripts. Furthermore, approximately 65% of ß cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is caused by ß cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation.