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

AIM: To determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN) channels impact glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) modulation of islet Ca(2+) handling and insulin secretion. METHODS: The impact of liraglutide (GLP-1 analogue) on islet Ca(2+) handling, HCN currents and insulin secretion was monitored with fluorescence microscopy, electrophysiology and enzyme immunoassays, respectively. Furthermore, liraglutide-mediated β-to-δ-cell cross-communication was assessed following selective ablation of either mouse islet δ or β cells. RESULTS: Liraglutide increased β-cell Ca(2+) 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 β-cell Ca(2+) oscillation frequency in islets from rodents exposed to a diabetogenic diet. Interestingly, liraglutide accelerated Ca(2+) oscillations in a majority of islet δ cells, which showed synchronized Ca(2+) oscillations equivalent to β cells; therefore, we assessed if either cell type was driving this liraglutide-mediated islet Ca(2+) response. Although δ-cell loss did not impact liraglutide-mediated increase in β-cell Ca(2+) oscillation frequency, β-cell ablation attenuated liraglutide-facilitated acceleration of δ-cell Ca(2+) oscillations. CONCLUSION: The data presented here show that liraglutide-induced stimulation of islet HCN channels augments Ca(2+) oscillation frequency. As insulin secretion oscillates with β-cell Ca(2+), 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 Ca(2+) oscillation frequency and somatostatin secretion kinetics in a β-cell-dependent manner.

Maturity-onset diabetes of the young (MODY) is a heterogeneous group of monogenic disorders of impaired pancreatic β cell function. The mechanisms underlying MODY include β cell KATP channel dysfunction (e.g., KCNJ11 [MODY13] or ABCC8 [MODY12] mutations); however, no other β 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 β 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.

Perturbations in Ca2+ homeostasis contribute to β-cell failure in diabetes, limiting insulin secretion and exacerbating hyperglycemia. TALK-1 is the most islet-restricted and most abundantly expressed K+ channel of the β-cell. ER TALK-1 channels provide K+ countercurrent for [Ca2+]ER release, thereby tuning [Ca2+]ER storage and release. During β-cell Ca2+ influx, TALK-1-enhanced [Ca2+]ER release leads to activation of the hyperpolarizing Ca2+ activated K+ channels, thus increasing the silent phase duration between Ca2+ oscillations. A gain-of-function mutation in TALK-1(L114P) causes MODY, by disrupting cytoplasmic and ER Ca2+ handling. However, the ability to probe ER TALK-1 channels as a therapeutic target to normalize β-cell Ca2+ handling and reduce ER stress in diabetes has not been determined. Here, using a high throughput fluorescence-based thallium (TI+) flux assay (z’ =0.64) in a TALK-1 overexpression cell line, we identified 80 TALK-1 selective inhibitors from a small molecule library of 50,000 compounds. 4 potent inhibitors that also inhibited the ER membrane TALK-1 channels were identified by monitoring cyclopiazonic acid-induced [Ca2+]ER release (e.g. K2P16-i7(IC50=5.3μM); 63.36% [Ca2+]ER recovery, p-value=0.02). We also tested these inhibitors for their efficacy in controlling mouse islet [Ca2+]ER storage as well as cytosolic Ca2+ oscillations. TALK-1 inhibitors significantly increased islet [Ca2+]ER storage and Ca2+ oscillation frequency (e.g.K2P16-i7; [Ca2+]ER AUC=60.9a.u., peaks/min=0.45 vs. DMSO; [Ca2+]ER AUC=49.7a.u., peaks/min= 0.17, p-value Disclosure A. Y. Nakhe: None. P. Dadi: None. D. Westover: None. E. Days: None. J. Denton: None. D. Jacobson: None.

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 β- 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., Graphical abstract Image 1, Highlights • Lactate reduces islet α-cell Ca2+ entry under low glucose conditions. • Lactate does not alter β- or δ-cell Ca2+ handling under low glucose conditions. • Lactate enters islet α-cells through monocarboxylate transporters. • Lactate hyperpolarizes islet α-cell membrane potential by activating KATP channels. • Lactate reduces mouse and human islet glucagon 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 β-cell TSPAN-7 regulates Ca2+ handling and hormone secretion. We find that TSPAN-7 reduces β-cell glucose-stimulated Ca2+ entry, slows Ca2+ oscillation frequency and decreases glucose-stimulated insulin secretion. TSPAN-7 controls β-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 β-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 β- 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 β-cells. Additionally, mouse β-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 β-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 β-cells and from a heterologous expression system. Finally, TSPAN-7 KD in human β-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 β-cell L-type CaV channels is a key determinant of β-cell glucose-stimulated Ca2+ entry and thus the set-point of GSIS.

The melastatin subfamily of the transient receptor potential channels (TRPM) are regulators of pancreatic β-cell function. TRPM7 is the most abundant islet TRPM channel; however, the role of TRPM7 in β-cell function has not been determined. Here, we utilized 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, as well as α-cell and β-cell mass. This resulted in impaired glucose tolerance due to decreased serum insulin levels. However, ablation of TRPM7 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 β-cell hyperplasia under insulin resistant conditions. β-cell proliferation induced by high-fat diet was significantly decreased in TRPM7 deficient β-cells. The endocrine roles of TRPM7 may be influenced by cation flux through the channel, and indeed we find that TRPM7 ablation alters β-cell intracellular Mg2+. Together, these findings reveal that TRPM7 controls pancreatic progenitor expansion and β-cell proliferation, which is likely due to regulation of Mg2+homeostasis.SummaryThis manuscript identifies a critical developmental role for TRPM7 channels in pancreatic progenitor cells. The manuscript also determines that TRPM7 plays a key role in β-cell proliferation under insulin-resistant conditions.

Genetic and acquired abnormalities contribute to pancreatic β-cell failure in diabetes. Transcription factors Hnf4α (MODY1) and FoxO1 are respective examples of these two components, and are known to act through β-cell-specific enhancers. However, their relationship is unclear. Here we show by genome-wide interrogation of chromatin modifications that FoxO1 ablation in mature β-cells leads to increased selection of FoxO1 enhancers by Hnf4α. To model the functional significance we generated single and compound knockouts of FoxO1 and Hnf4α in β-cells. Single knockout of either gene impaired insulin secretion in mechanistically distinct fashions. Surprisingly, the defective β-cell secretory function of either single mutant in hyperglycemic clamps and isolated islets treated with various secretagogues, was completely reversed in double mutants. Gene expression analyses revealed the reversal of β-cell dysfunction with an antagonistic network regulating glycolysis, including β-cell “disallowed” genes; and that a synergistic network regulating protocadherins emerged as likely mediators of the functional restoration of insulin secretion. The findings provide evidence of antagonistic epistasis as a model of gene/environment interactions in the pathogenesis of β-cell dysfunction.

Tetraspanin7-mediated inhibition of β-cell voltage-dependent Ca2+ channels regulates glucose-stimulated insulin secretion. Glucose-stimulated insulin secretion (GSIS) is regulated by Ca2+ influx into pancreatic β-cells through voltage-dependent Ca2+ channels (VDCCs). As some tetraspanin (TSPAN) transmembrane proteins control Ca2+ handling they may also modulate GSIS. TSPAN7 is the most abundant TSPAN in mouse and human islets. TSPAN7 autoantibodies are also found in type 1 diabetic serum, suggesting a link between TSPAN7 and diabetes. The aim herein was to determine if TSPAN7 controls β-cell Ca2+ handling and insulin secretion. Immunofluorescence imaging of mouse and human pancreatic slices indicated that TSPAN7 is highly expressed in β- and α-cells. shRNA-mediated TSPAN7 knockdown (KD) in mouse and human β-cells augmented glucose-stimulated Ca2+ influx (by 32.3±9.5% and 33.8±15.1% respectively) as well as KCl-stimulated Ca2+ influx (45 mM; 34.5±9.6%, human). Mouse β-cell Ca2+ oscillation frequency was also accelerated by TSPAN7 KD (by 29.4±10.1%). As TSPANs interact with and regulate VDCCs, we examined the effect of TSPAN7 on VDCC currents. TSPAN7 KD increased peak L-type VDCC currents in mouse and human β-cells (by 4.1±1.3 pA/pF and 2.0±0.9 pA/pF respectively). Heterologous expression of TSPAN7 with L-type VDCCs in HEK293 cells also decreased VDCC currents (CaV1.2: 4.3±1.8 pA/pF; CaV1.3: 17.5±5.4 pA/pF) and reduced KCl-stimulated (50 mM) Ca2+ influx (CaV1.2: 51.0±7.1%; CaV1.3: 28.4±7.5%). Furthermore, TSPAN7 coimmunoprecipitated with CaV1.2 and CaV1.3 from primary human β-cells and when heterologously expressed in HEK293 cells. Finally, TSPAN7 KD in human β-cells increased basal (5.6 mM glucose; 8.3±5.5 ng*hr-1) and stimulated (45 mM KCl+14 mM glucose; 20.4±17.1 ng*hr-1) insulin secretion. These findings reveal that TSPAN7 modulation of β-cell L-type VDCCs limits glucose-stimulated Ca2+ influx and insulin secretion. Disclosure M. Dickerson: None. P. Dadi: None. R.B. Butterworth: None. D. Jacobson: None. Funding American Diabetes Association (1-17-IBS-024 to D.J.); National Institutes of Health (T32DK101003, DK097392, DK115620); Vanderbilt University Medical Center Diabetes Research and Training Center (P60DK20593)

Insulin levels are inadequate to maintain euglycemia in patients with type-2 diabetes (T2D), which is due in part to perturbations in pancreatic ꞵ-cell Ca2+ homeostasis. TALK-1 K+ channels are key regulators of ꞵ-cell electrical excitability, Ca2+ handling and glucose-stimulated insulin secretion (GSIS). KCNK16, which encodes TALK-1 is the most abundant ꞵ-cell K+ channel transcript and also the most islet-restricted ion channel transcript. Furthermore, a nonsynonymous coding sequence polymorphism in KCNK16 causes a predisposition for developing T2D. However, the ability to utilize TALK-1 as a therapeutic target to normalize ꞵ-cell Ca2+ homeostasis in T2D has not been determined. Here, we optimized a high throughput fluorescence-based thallium (TI+) flux assay (z’ =0.64) to screen for TALK-1 inhibitors, which resulted in identification of 286 TALK-1 inhibitors with potent drug-like properties (from a 50,000 compound screen). Of these hits, 78 exhibit TALK-1 selectivity and do not influence the activity of other K+ channels including TALK-2, TREK-2, TASK-1, TASK-3, KATP or hERG channels. We then performed dose response curve analyses and identified 9 potent inhibitors with IC50s < 2.5μM that inhibit TALK-1 activity (>65%). To test the efficacy of the inhibitors for their ability to augment islet function, we assessed their impact on mouse islet Ca2+ handling. TALK-1 inhibitors significantly increased islet Ca2+ oscillation frequency (e.g., VU0233408, 2.47 peaks/min vs. DMSO, 1.82 peaks/min, p-value= 0.011), similar to what was previously observed in TALK-1 KO islets. Moreover, the inhibitors did not increase glucose-stimulated Ca2+ oscillation frequency in TALK-1 KO islets. Thus, pharmacological inhibition of TALK-1 is predicted to increase GSIS. In conclusion, we have identified the first potent and selective TALK-1 inhibitors that can be utilized to assess the role of TALK-1 during human ꞵ-cell GSIS under physiological and diabetic conditions. Disclosure A.Y. Nakhe: None. P. Dadi: None. D. Westover: None. E. Days: None. J. Denton: None. D. Jacobson: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK115620VDC); Vanderbilt University Diabetes and Research Training Center (P60DK20593)

BackgroundMaturity-onset diabetes of the young (MODY) is a heterogeneous group of monogenic disorders of impaired glucose-stimulated insulin secretion (GSIS). Mechanisms include β-cell KATPchannel dysfunction (e.g.,KCNJ11(MODY13) orABCC8(MODY12) mutations); however, no other β-cell channelopathies have been identified in MODY.MethodsA four-generation family with autosomal dominant non-obese, non-ketotic antibody-negative diabetes, without mutations in known MODY genes, underwent exome sequencing. Whole-cell and single-channel K+currents, Ca2+handling, and GSIS were determined in cells expressing either mutated or wild-type (WT) protein.ResultsWe identified a novel non-synonymous genetic mutation inKCNK16(NM_001135105: c.341T>C, p.Leu114Pro) segregating with MODY.KCNK16is the most abundant and β-cell-restricted K+channel transcript and encodes the two-pore-domain K+channel TALK-1. Whole-cell K+currents in transfected HEK293 cells demonstrated drastic (312-fold increase) gain-of-function with TALK-1 Leu144Pro vs. WT, due to greater single channel activity. Glucose-stimulated cytosolic Ca2+influx was inhibited in mouse islets expressing TALK-1 Leu114Pro (area under the curve [AUC] at 20mM glucose: Leu114Pro 60.1 vs. WT 89.1;P=0.030) and less endoplasmic reticulum calcium storage (cyclopiazonic acid-induced release AUC: Leu114Pro 17.5 vs. WT 46.8;P=0.008). TALK-1 Leu114Pro significantly blunted GSIS compared to TALK-1 WT in both mouse (52% decrease,P=0.039) and human (38% decrease,P=0.019) islets.ConclusionsOur data identify a novel MODY-associated gene,KCNK16; with a gain-of-function mutation limiting Ca2+influx and GSIS. A gain-of-function common polymorphism inKCNK16is associated with type 2 diabetes (T2DM); thus, our findings have therapeutic implications not only forKCNK16-associated MODY but also for T2DM.

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 β-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., Highlights • The two-pore domain K+ channel TALK-1 is expressed in islet and gastric δ-cells. • TALK-1 channels set δ-cell ER Ca2+ stores, regulating CICR and somatostatin release. • TALK-1 control of δ-cell function tunes α-cell Ca2+ handling and glucagon secretion. • Glucose enhances δ-cell Ca2+ signals and coordinates δ-cell function with β-cells.

We used mice lacking Abcc8 , a key component of the β-cell K ATP -channel, to analyze the effects of a sustained elevation in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) on β-cell identity and gene expression. Lineage tracing analysis revealed the conversion of β-cells lacking Abcc8 into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified Abcc8 −/− β-cells confirmed an increase in Ppy gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca 2+ signaling, the maintenance of β-cell identity, and cell adhesion. The expression of S100a6 and S100a4 , two highly upregulated genes, is closely correlated with membrane depolarization, suggesting their use as markers for an increase in [Ca 2+ ] i . Moreover, a bioinformatics analysis predicts that many of the dysregulated genes are regulated by common transcription factors, one of which, Ascl1 , was confirmed to be directly controlled by Ca 2+ influx in β-cells. Interestingly, among the upregulated genes is Aldh1a3 , a putative marker of β-cell dedifferentiation, and other genes associated with β-cell failure. Taken together, our results suggest that chronically elevated β-cell [Ca 2+ ] i in Abcc8 −/− islets contributes to the alteration of β-cell identity, islet cell numbers and morphology, and gene expression by disrupting a network of Ca 2+ -regulated genes.

Glucose-stimulated insulin secretion from pancreatic β-cells is controlled by ATP-regulated potassium (K(ATP)) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) subunits. The K(ATP) channel-opener diazoxide is FDA-approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular K(ATP) 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 K(ATP) channels. VU0071063 induces hyperpolarization of β-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.

During Glucose-Stimulated Insulin Secretion (GSIS), β-cells exhibit oscillations in cytosolic Ca2+ ([Ca2+]c) and endoplasmic reticulum (ER) Ca2+ ([Ca2+]ER), which are regulated by the two-pore domain K+ channel, TALK-1. TALK-1 is the most abundant β-cell K+ channel and it controls Ca2+ homeostasis by modulating K+ permeability at both the plasma and ER membrane. However, how TALK-1 modulates ER stress and β-cell failure during the pathogenesis of diabetes has not been determined. To understand how TALK-1 controls β-cell function under diabetic conditions, we assessed how a mutation in TALK-1 (ND) which causes neonatal diabetes, impacts TALK-1 channel activity and [Ca2+]ER handling. While plasmallemal TALK-1 activity was inhibited with the ND mutation, [Ca2+]ER storage was significantly reduced by the ND mutation compared to control TALK-1. This suggests that TALK-1 ND enhances TALK-1 activity on the ER membrane leading to reduced [Ca2+]ER storage. Reducing [Ca2+]ER enhances ER stress, which would be expected to cause β-cell dysfunction. Indeed, TALK-1 ND significantly reduces β-cell insulin secretion compared to control. To further investigate the role of TALK-1 we developed a β-cell-specific TALK-1 KO mouse model (β-TALK1-KO). On a high fat diet, β-TALK1-KO mice show improved glucose tolerance compared to controls. Mitochondrial calcium ([Ca2+]mito) was also examined because it is modulated by [Ca2+]ER; we found that β-TALK1-KO mice have increased [Ca2+]mito. As reactive oxygen species (ROS) production is a byproduct of mitochondrial respiration, we investigated if TALK-1 affects ROS production. TALK1 KO islets were found to have reduced ROS production compared to wild type islets. Together, these data indicate that increased TALK-1 channel activity reduces [Ca2+]ER, [Ca2+]mito, and increases ROS; suggesting that TALK-1 plays an important role in modulating the β-cell responses to stress associated with diabetic conditions. Disclosure S.M. Graff: None. P. Dadi: None. C.E. Ibsen: None. M. Dickerson: None. K.L. Jordan: None. D. Jacobson: None.

Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes. Exome sequencing of a non-obese four-generation family with autosomal dominant non-ketotic antibody-negative diabetes, who were negative for mutations in known MODY genes, identified a novel coding variant in KCNK16 (NM_001135105: c.341T>C, p.Leu114Pro) affecting a highly conserved base and predicted to be damaging. KCNK16 encodes TALK-1, a pancreatic β-cell potassium channel which modulates β-cell excitability, controlling Ca2+ influx and insulin secretion. Activation of TALK-1 reduces insulin secretion. A coding gain-of-function variant in KCNK16 is associated with type 2 diabetes in multi-ethnic genome-wide association studies. Notably, TALK-1 is not sensitive to sulfonylureas. HEK-293 cells were transfected with either wild-type or Leu114Pro TALK-1 constructs using a CMV promoter and including P2A mCherry to enable confirmation of successful transfection. Whole cell cross-membrane voltage clamping, with voltage ramped from -120mV to 60mV, demonstrated a drastic increase in current with mutant TALK-1 compared to wild-type, consistent with marked gain-of-function and likely to reduce insulin secretion. Our data suggest that KCNK16 may be a novel MODY gene. If confirmed, this has therapeutic implications not only for these individuals but potentially for novel management of type 2 diabetes. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Pancreatic α-cells exhibit oscillations in cytosolic Ca2+(Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+coscillations have not been elucidated. As β-cell Ca2+coscillations are regulated in part by Ca2+-activated K+(Kslow) currents, this work investigated the role of Kslowin α-cell Ca2+handling and GCG secretion. α-Cells displayed Kslowcurrents 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 Kslowwas 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 Kslowwith 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+cwas 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+cfollowing prolonged Kslowinhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslowactivation provides a hyperpolarizing influence on α-cell Vmthat sustains Ca2+entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+cis elevated during secretagogue stimulation, Kslowactivation helps to preserve GCG secretion.

In the developing pancreas, transient Neurog3-expressing progenitors give rise to four major islet-cell types, α, β, δ, and γ; when and how the Neurog3(+) cells choose cell-fate is unknown. Using single-cell RNAseq, trajectory analysis, and combinatorial lineage tracing, we showed here that the Neurog3(+) cells co-expressing Myt1 (i.e., Myt1(+)Neurog3(+)) were biased towards β-cell fate; while those not simultaneously expressing Myt1 (Myt1(-)Neurog3(+)) favored α-fate. Myt1 manipulation only marginally affected α- vs. β-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 over-expression or Arx enhancer hyper-methylation favored β-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.

Calcium influx into pancreatic α-cells through voltage-dependent calcium channels (VDCCs) is required for glucagon (GCG) secretion and the mechanisms that mediate this process become defective during the pathogenesis of diabetes. Potassium channels are key regulators of membrane potential (Vm), and thus, modulate VDCC activity. However, the role of calcium-activated potassium (KCa) channels in α-cell calcium handling and GCG secretion have not been determined. The aim of this study was to investigate the importance of KCa channels to α-cell electrical excitability, calcium handling, and GCG secretion. α-cells display a KCa current, which is partially inhibited by blocking small conductance KCa (SK) or big conductance KCa (BK) channels. Inhibition of P/Q-type VDCCs, sarco/endoplasmic reticulum (ER) calcium-ATPases (SERCAs), and KATP channels also partially inhibit this current. It is likely that calcium influx through VDCCs triggers calcium-induced calcium release (CICR) from ER calcium stores activating α-cell KCa channels. The inhibition of SK channels transiently increases calcium influx into α-cells at 1 mM glucose; however, over time intracellular calcium decreases. This may be due to voltage-dependent inactivation of α-cell VDCCs. Interestingly, SK channel inhibition reduces GCG secretion at 1 mM glucose while SST secretion is unaffected. Thus, it is likely that this is an intrinsic α-cell effect. These findings demonstrate that α-cells possess a KCa current and that its activation is important for α-cell electrical activity, Ca2+ handling, and GCG secretion. Disclosure M. Dickerson: None. M.K. Altman: None. P. Dadi: None. N.C. Vierra: None. D. Jacobson: None.

Cytokines present during low-grade inflammation contribute to β-cell dysfunction and diabetes. Cytokine signaling disrupts β-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 β-cell Ca2+ handling remain unknown. Here we investigated the role of K+ currents in cytokine-mediated β-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 β-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 β-cells by facilitating increased Ca2+ content to maintain GSIS.

Summary Islet β cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that β cells of newborns secrete more insulin than adults in response to similar intracellular Ca 2+ concentrations, suggesting differences in the Ca 2+ sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca 2+ binding paralog of the β cell Ca 2+ sensor Syt7, increased by ∼8-fold during β 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-βH1 cells, a human β cell line. These findings reveal the role that altered Ca 2+ sensing plays in regulating β cell maturation.

Type 2 diabetes incidence increases with age, while β-cell replication declines. The transcription factor FoxM1 is required for β-cell replication in various situations, and its expression declines with age. We hypothesized that increased FoxM1 activity in aged β-cells would rejuvenate proliferation. Induction of an activated form of FoxM1 was sufficient to increase β-cell mass and proliferation in 12-month-old male mice after just 2 weeks. Unexpectedly, at 2 months of age, induction of activated FoxM1 in male mice improved glucose homeostasis with unchanged β-cell mass. Cells expressing activated FoxM1 demonstrated enhanced glucose-stimulated Ca2+ influx, which resulted in improved glucose tolerance through enhanced β-cell function. Conversely, our laboratory has previously demonstrated that mice lacking FoxM1 in the pancreas display glucose intolerance or diabetes with only a 60% reduction in β-cell mass, suggesting that the loss of FoxM1 is detrimental to β-cell function. Ex vivo insulin secretion was therefore examined in size-matched islets from young mice lacking FoxM1 in β-cells. Foxm1-deficient islets indeed displayed reduced insulin secretion. Our studies reveal that activated FoxM1 increases β-cell replication while simultaneously enhancing insulin secretion and improving glucose homeostasis, making FoxM1 an attractive therapeutic target for diabetes.

Two-pore domain K+ (K2P) channels play an important role in tuning β-cell glucose-stimulated insulin secretion (GSIS). The K2P channel TWIK-related alkaline pH-activated K2P (TALK)-1 is linked to type 2 diabetes risk through a coding sequence polymorphism (rs1535500); however, its physiological function has remained elusive. Here, we show that TALK-1 channels are expressed in mouse and human β-cells, where they serve as key regulators of electrical excitability and GSIS. We find that the rs1535500 polymorphism, which results in an alanine-to-glutamate substitution in the C-terminus of human TALK-1, increases channel activity. Genetic ablation of TALK-1 results in β-cell membrane potential depolarization, increased islet Ca2+ influx, and enhanced second-phase GSIS. Moreover, mice lacking TALK-1 channels are resistant to high-fat diet–induced elevations in fasting glycemia. These findings reveal TALK-1 channels as important modulators of second-phase insulin secretion and suggest a clinically relevant mechanism for rs1535500, which may increase type 2 diabetes risk by limiting GSIS.

Glucose regulation of pancreatic α-cell Ca(2+) entry through voltage-dependent Ca(2+) channels is essential for normal glucagon secretion and becomes defective during the pathogenesis of diabetes mellitus. The 2-pore domain K(+) channel, TWIK-related acid-sensitive K(+) channel 1 (TASK-1), is an important modulator of membrane voltage and Ca(2+) entry. However, its role in α-cells has not been determined. Therefore, we addressed how TASK-1 channels regulate α-cell electrical activity, Ca(2+) entry, and glucagon secretion. We find that TASK-1 channels expressed in human and rodent α-cells are blocked by the TASK-1 channel inhibitor A1899. Alpha-cell 2-pore domain K(+) currents were also significantly reduced after ablation of mouse α-cell TASK-1 channels. Inhibition of TASK-1 channels with A1899 caused plasma membrane potential depolarization in both human and mouse α-cells, which resulted in increased electrical excitability. Moreover, ablation of α-cell TASK-1 channels increased α-cell electrical excitability under elevated glucose (11 mM) conditions compared with control α-cells. This resulted in significantly elevated α-cell Ca(2+) influx when TASK-1 channels were inhibited in the presence of high glucose (14 mM). However, there was an insignificant change in α-cell Ca(2+) influx after TASK-1 inhibition in low glucose (1 mM). Glucagon secretion from mouse and human islets was also elevated specifically in high (11 mM) glucose after acute TASK-1 inhibition. Interestingly, mice deficient for α-cell TASK-1 showed improvements in both glucose inhibition of glucagon secretion and glucose tolerance, which resulted from the chronic loss of α-cell TASK-1 currents. Therefore, these data suggest an important role for TASK-1 channels in limiting α-cell excitability and glucagon secretion during glucose stimulation.

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 β cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine β cells did not affect β 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 β 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 β cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation.

Cytokines present during low-grade inflammation contribute to β-cell dysfunction and diabetes. Cytokine signaling disrupts β-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 β-cell Ca2+ handling remain unknown. Here we investigated the role of K+ currents in cytokine-mediated β-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 β-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 β-cells by facilitating increased Ca2+ content to maintain GSIS.

Ca2+ handling by the endoplasmic reticulum (ER) serves critical roles controlling pancreatic β-cell function and becomes perturbed during the pathogenesis of diabetes. ER Ca2+ homeostasis is determined by ion movements across the ER membrane, including K+ flux through K+ channels. Here, we demonstrated that K+ flux through ER-localized TALK-1 channels facilitated Ca2+ release from the ER in mouse and human β-cells. We found that β-cells from mice lacking TALK-1 exhibited reduced basal cytosolic Ca2+ and increased ER Ca2+ concentrations, suggesting reduced ER Ca2+ leak. These changes in Ca2+ homeostasis were presumably due to TALK-1-mediated ER K+ flux, because we recorded K+ currents mediated by functional TALK-1 channels on the nuclear membrane, which is continuous with the ER. Moreover, overexpression of K+-impermeable TALK-1 channels in HEK293 cells did not reduce ER Ca2+ stores. Reduced ER Ca2+ content in β-cells is associated with ER stress and islet dysfunction in diabetes, and islets from TALK-1-deficient mice fed a high-fat diet showed reduced signs of ER stress, suggesting that TALK-1 activity exacerbated ER stress. Our data establish TALK-1 channels as key regulators of β-cell ER Ca2+, and suggest that TALK-1 may be a therapeutic target to reduce ER Ca2+ handling defects in β-cells during the pathogenesis of diabetes.

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-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 β-cells is poorly understood, especially in vivo. Here, we report that mice with conditional inhibition of CaMKII in β-cells show significantly impaired glucose tolerance due to decreased GSIS. Moreover, β-cell CaMKII inhibition dramatically exacerbates glucose intolerance following exposure to a high fat diet. The impairment of islet GSIS by β-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 β-cells with CaMKII inhibition, as well as in primary wild-type β-cells treated with a peptide inhibitor of CaMKII. The levels of basal β-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 β-cell Ca(2+) signals that enhance GSIS under physiological and pathological conditions.

Elevated fasting blood glucose (FBG) is associated with increased risk for the development of type 2 diabetes and cardiovascular-associated mortality. Genome-wide association studies (GWAS) have linked polymorphisms in G6PC2 with variations in FBG and body fat, although not insulin sensitivity or glucose tolerance. G6PC2 encodes an islet-specific, endoplasmic reticulum–resident glucose-6-phosphatase catalytic subunit. A combination of in situ perfused pancreas, in vitro isolated islet, and in vivo analyses were used to explore the function of G6pc2 in mice. G6pc2 deletion had little effect on insulin sensitivity and glucose tolerance, whereas body fat was reduced in female G6pc2 knockout (KO) mice on both a chow and high-fat diet, observations that are all consistent with human GWAS data. G6pc2 deletion resulted in a leftward shift in the dose-response curve for glucose-stimulated insulin secretion (GSIS). As a consequence, under fasting conditions in which plasma insulin levels were identical, blood glucose levels were reduced in G6pc2 KO mice, again consistent with human GWAS data. Glucose-6-phosphatase activity was reduced, whereas basal cytoplasmic calcium levels were elevated in islets isolated from G6pc2 KO mice. These data suggest that G6pc2 represents a novel, negative regulator of basal GSIS that acts by hydrolyzing glucose-6-phosphate, thereby reducing glycolytic flux.

We previously identified the G6PC2 locus as a strong determinant of fasting plasma glucose (FPG) and showed that a common G6PC2 intronic single nucleotide polymorphism (SNP) (rs560887) and two common G6PC2 promoter SNPs (rs573225 and rs13431652) are highly associated with FPG. However, these promoter SNPs have complex effects on G6PC2 fusion gene expression, and our data suggested that only rs13431652 is a potentially causative SNP. Here we examine the effect of rs560887 on G6PC2 pre-mRNA splicing and the contribution of an additional common G6PC2 promoter SNP, rs2232316, to the association signal.Minigene analyses were used to characterise the effect of rs560887 on G6PC2 pre-mRNA splicing. Fusion gene and gel retardation analyses characterised the effect of rs2232316 on G6PC2 promoter activity and transcription factor binding. The genetic association of rs2232316 with FPG variation was assessed using regression adjusted for age, sex and BMI in 4,220 Europeans with normal FPG.The rs560887-G allele was shown to enhance G6PC2 pre-mRNA splicing, whereas the rs2232316-A allele enhanced G6PC2 transcription by promoting Foxa2 binding. Genetic analyses provide evidence for association of the rs2232316-A allele with increased FPG (β = 0.04 mmol/l; p = 4.3 × 10(-3)) as part of the same signal as rs560887, rs573225 and rs13431652.As with rs13431652, the in situ functional data with rs560887 and rs2232316 are in accord with the putative function of G6PC2 in pancreatic islets, and suggest that all three are potentially causative SNPs that contribute to the association between G6PC2 and FPG.

We used mice lacking Abcc8, a key component of the β-cell KATP-channel, to analyze the effects of a sustained elevation in the intracellular Ca2+ concentration ([Ca2+]i) on β-cell identity and gene expression. Lineage tracing analysis revealed the conversion of β-cells lacking Abcc8 into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified Abcc8−/− β-cells confirmed an increase in Ppy gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca2+ signaling, the maintenance of β-cell identity, and cell adhesion. The expression of S100a6 and S100a4, two highly upregulated genes, is closely correlated with membrane depolarization, suggesting their use as markers for an increase in [Ca2+]i. Moreover, a bioinformatics analysis predicts that many of the dysregulated genes are regulated by common transcription factors, one of which, Ascl1, was confirmed to be directly controlled by Ca2+ influx in β-cells. Interestingly, among the upregulated genes is Aldh1a3, a putative marker of β-cell dedifferentiation, and other genes associated with β-cell failure. Taken together, our results suggest that chronically elevated β-cell [Ca2+]i in Abcc8−/− islets contributes to the alteration of β-cell identity, islet cell numbers and morphology, and gene expression by disrupting a network of Ca2+-regulated genes.

A series of structurally related diphenol aldimines (DPAs) were synthesized. These aldimines involve different substitution patterns of their phenolic groups, for the purpose of optimizing their ability to inhibit ATP synthase. The inhibitory effects of these DPA compounds were evaluated using purified F1 and membrane-bound F1F0 E. coli ATP synthase. Structure-activity relationship studies of these di-phenol compounds showed that maximum inhibition was achieved when both phenolic groups are either in the meta-positions (DPA-7, IC50 = 2.0 μM), or in the ortho-positions (DPA-9, IC50 = 5.0 μM). The lowest ATP synthase inhibition was found to be when the phenolic groups are both in the para-positions (DPA-2, IC50 = 100.0 μM). Results also show that the inhibitory effects of these compounds on ATPase are completely reversible. Identical inhibition patterns of both the purified F1 and the membrane bound F1F0 enzyme were observed. Study of E. coli cell growth showed that these diphenol aldimines effectively inhibit both ATP synthesis and cell growth.

The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of 17 bioflavonoid compounds on purified F1 or membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were morin (IC50 approximately 0.07 mM)>silymarin (IC50 approximately 0.11 mM)>baicalein (IC50 approximately 0.29 mM)>silibinin (IC50 approximately 0.34 mM)>rimantadin (IC50 approximately 2.0 mM)>amantidin (IC50 approximately 2.5 mM)>epicatechin (IC50 approximately 4.0 mM). Inhibition by hesperidin, chrysin, kaempferol, diosmin, apigenin, genistein, or rutin was partial in the range of 40-60% and inhibition by galangin, daidzein, or luteolin was insignificant. The main skeleton, size, shape, geometry, and position of functional groups on inhibitors played important role in the effective inhibition of ATP synthase. In all cases inhibition was found fully reversible and identical in both F1Fo membrane preparations and isolated purified F1. ATPase and growth assays suggested that the bioflavonoid compounds used in this study inhibited F1-ATPase as well as ATP synthesis nearly equally, which signifies a link between the beneficial effects of dietary bioflavonoids and their inhibitory action on ATP synthase.

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-beta-D glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the gamma-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the gamma-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-beta-d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely ( approximately 0 residual activity), inhibition by other compounds was partial with approximately 20% residual activity by quercetin, approximately 50% residual activity by quercetin-3-beta-D glucoside, and approximately 60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC(50) approximately 14 microM) followed by quercetin (IC(50) approximately 33 microM), quercetin-3-beta-D glucoside (IC(50) approximately 71 microM), resveratrol (IC(50) approximately 94 microM), quercitrin (IC(50) approximately 120 microM). Inhibition was identical in both F(1)F(o) membrane preparations as well as in isolated purified F(1). In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-beta-d glucoside inhibited only ATPase activity and not ATP synthesis.

Glucose-stimulated insulin secretion (GSIS) relies on β-cell Ca2+ influx, which is modulated by the two-pore-domain K+ (K2P) channel, TALK-1. A gain-of-function polymorphism in KCNK16, the gene encoding TALK-1, increases risk for developing type-2 diabetes. While TALK-1 serves an important role in modulating GSIS, the regulatory mechanism(s) that control β-cell TALK-1 channels are unknown. Therefore, we employed a membrane-specific yeast two-hybrid (MYTH) assay to identify TALK-1-interacting proteins in human islets, which will assist in determining signaling modalities that modulate TALK-1 function. Twenty-one proteins from a human islet cDNA library interacted with TALK-1. Some of these interactions increased TALK-1 activity, including intracellular osteopontin (iOPN). Intracellular OPN is highly expressed in β-cells and is upregulated under pre-diabetic conditions to help maintain normal β-cell function; however, the functional role of iOPN in β-cells is poorly understood. We found that iOPN colocalized with TALK-1 in pancreatic sections and coimmunoprecipitated with human islet TALK-1 channels. As human β-cells express two K+ channel-forming variants of TALK-1, regulation of these TALK-1 variants by iOPN was assessed. At physiological voltages iOPN activated TALK-1 transcript variant 3 channels but not TALK-1 transcript variant 2 channels. Activation of TALK-1 channels by iOPN also hyperpolarized resting membrane potential (Vm) in HEK293 cells and in primary mouse β-cells. Intracellular OPN was also knocked down in β-cells to test its effect on β-cell TALK-1 channel activity. Reducing β-cell iOPN significantly decreased TALK-1 K+ currents and increased glucose-stimulated Ca2+ influx. Importantly, iOPN did not affect the function of other K2P channels or alter Ca2+ influx into TALK-1 deficient β-cells. These results reveal the first protein interactions with the TALK-1 channel and found that an interaction with iOPN increased β-cell TALK-1 K+ currents. The TALK-1/iOPN complex caused Vm hyperpolarization and reduced β-cell glucose-stimulated Ca2+ influx, which is predicted to inhibit GSIS.

ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet β-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 β cells. VU0071063 represents a novel tool/compound for investigating β-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry.

Human beta-cell insulin secretion is dependent on Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs). K+ channels modulate the beta-cell membrane potential, which influences VDCC activity. The most abundant K+ channel of the human islet is the two-pore domain K+ (K2P) channel TALK-1; however, its function is unknown. We characterized TALK-1 currents in primary mouse beta-cells and find that K2P currents are significantly reduced after genetic ablation of TALK-1. To identify mechanisms that regulate TALK-1 channel activity, we used a Tl+-sensitive fluorescent screen to assess if TALK-1 is regulated by kinases. Cells expressing TALK-1 channels were monitored for changes in Tl+ flux in response to co-expression with a constitutively active kinase (examining a total of 192 kinases), revealing 9 kinases that modify Tl+ flux through TALK-1. Interestingly, three phosphatidylinositol phosphate kinases (PIKs) including PIK3CG, PI4K2B, and PIKFYVE, increased TALK-1 channel activity. To determine if activation of TALK-1 channels by PIKs is due to build-up of PIPs, we depleted membrane PIP2 with a rapamycin-recruitable phosphatase and found that TALK-1 channel activity was reduced. As charged residues of other K+ channels mediate PIP2 sensitivity, we assessed if PIP2 influences TALK-1 channel activity through a charged residue enriched domain in the C-terminus. We found that neutralization of these charged residues significantly reduced PIP2 activation of TALK-1 channels. Importantly, PIP2 introduced via patch pipette into primary mouse beta-cells also significantly enhanced TALK-1 currents. As glucose induces Ca2+-dependent oscillations in beta-cell PIP2, we then assessed how TALK-1 regulates islet Ca2+ dynamics. We find that Ca2+ oscillation frequency is significantly increased in islets lacking TALK-1, which results in enhanced glucose-stimulated insulin secretion (GSIS). These data provide compelling evidence that TALK-1 influences GSIS by limiting excitability, and suggests a novel mechanism for phospholipid-dependent regulation of beta-cell Ca2+ entry.

Competing Interests: MKS and DRP work for Lexicon Pharmaceuticals and generated the mice described in this study. There are no pending patents, products in development or marketed products associated with these mice or this study. This relationship between Lexicon and Vanderbilt does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. Funding: The Vanderbilt Hormone Assay & Analytical Services Core and the Vanderbilt Islet Procurement and Analysis Core are both supported by National Institutes of Health (NIH) grants DK20593, to the Vanderbilt Diabetes Research Training Center (VDRTC), and DK59637, to the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC). The authors also thank personnel in the MMPC for advice on performing glucose tolerance tests and Mary Dawes for performing electron microscopy. The electron microscopy core is part of the VUMC Cell Imaging Shared Resource, which is supported by NIH grants CA68485, DK20593, DK58404, HD15052, DK59637 and EY08126. Research in the laboratory of RO was supported by NIH grant DK92589. Research in the laboratory of JCH was supported by the American Diabetes Association (9901-116), NIH grant DK052068, Juvenile Diabetes Research Association grant 4-2007-1056 and the Barbara Davis Center Diabetes and Endocrinology Research Center (P30 DK57516). Research in the laboratory of DWP was supported by NIH grant DK085064. Research in the laboratory of DAJ was supported by K01 DK081666. Research in the laboratory of OPM was supported by NIH grants DK043748 and DK078188. SAS was supported by NIH grant DK080193 and JDRF grant 1-2008-1021. The Vanderbilt Hormone Assay & Analytical Services Core, the Vanderbilt Islet Procurement and Analysis Core and the Vanderbilt Cell Imaging Shared Resource are all supported by NIH grant P60 DK20593, to the Vanderbilt Diabetes Research Training Center (VDRTC). LDP was supported by the Vanderbilt Molecular Endocrinology Training Program grant 5T32 DK07563. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Glucose stimulation of the pancreatic β-cell depolarizes the membrane potential to allow activation of voltage dependent calcium channels resulting in action potential (AP) firing, calcium influx, and insulin secretion. Two-pore-domain potassium (K2P) channels regulate the membrane potential from where AP firing occurs in many neurons. However, a role of K2P channels in regulating the pancreatic β-cell membrane potential is unknown and thus we addressed expression and function of the TASK K2P channels in the mouse β-cell. We find that TASK-1 and TASK-3 channels are expressed in mouse β-cells. Furthermore, β-cell TASK-like currents are sensitive to external pH, showing inhibition with acidic pH and stimulation with alkaline pH. Interestingly, glucose regulates β-cell intracellular pH causing acidic conditions in low glucose and alkaline conditions in high glucose, which may serve to regulate β-cell TASK channel activity. Increasing glucose from 2 to 14 mM caused polarization of the β-cell membrane potential whereas reducing glucose from 14 to 2 mM caused membrane depolarization by 6.1 +/- 2.2 mV when KATP was inhibited with tolbutamide. Similarly extracellular alkalinization (pH 8) resulted in polarization of the β-cell membrane potential by 9.19 +/-2.0 mV, whereas extracellular acidification (pH 6) caused membrane depolarization by 9.84 +/-2.9 mV. Anesthetic compounds that activate (halothane) or inhibit (lidocaine) TASK channels were evaluated for their ability to regulate β-cell membrane potential. Treatment of islets with lidocaine depolarized the β-cell membrane potential by 6.2 +/- 1.5 mV and halothane treatment resulted in β-cell membrane polarization. Both lidocaine and halothane have been shown to cause perturbations in human glucose homeostasis. Thus, this data implicate important roles for TASK channels in regulating the β-cell membrane potential, which may influence anesthetic and pH/glucose induced modulation of insulin secretion.

Glucose regulation of pancreatic alpha-cell Ca2+ entry through voltage-dependent Ca2+ channels is essential for normal glucagon secretion and becomes defective during the pathogenesis of diabetes mellitus. The two-pore-domain potassium (K2P) channel, TASK-1, is an important modulator of membrane voltage and Ca2+ entry, however, its role in alpha-cells has not been determined. Therefore, we addressed how TASK-1 channels modulate alpha-cell electrical activity, calcium entry and glucagon secretion. We find that TASK-1 channels are expressed in human and rodent alpha-cells. Furthermore, alpha-cell K2P currents are blocked by the specific and potent TASK-1 channel inhibitor A1899. Alpha-cell K2P currents are also significantly reduced following ablation of mouse alpha-cell TASK-1 channels. Inhibition of TASK-1 channels with A1899 causes membrane potential depolarization in both the human and mouse alpha-cells, which results in increased Ca2+ influx. While A1899 augments alpha-cell Ca2+ influx under low (1 mM) and high (14 mM) glucose conditions, treatment of mouse and human islets with A1899 only increases glucagon secretion under elevated (14 mM) glucose conditions. Therefore, these data suggest an important role for TASK-1 channels in limiting alpha-cell excitability and glucagon secretion during glucose stimulation. Interestingly, we also find that TASK-1 expression is reduced during the progression of type-2 diabetes mellitus in human islets. Thus, decreased TASK-1 channel activity may exacerbate the defective glucose inhibition of glucagon secretion observed in type-2 diabetes mellitus.