Correction: Deoxycholic acid supplementation impairs glucose homeostasis in mice.

[This corrects the article DOI: 10.1371/journal.pone.0200908.].

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.

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.

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.

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.

RIPK3-mediated inflammation is a conserved ß cell response to ER stress.

Islet inflammation is an important etiopathology of type 2 diabetes; however, the underlying mechanisms are not well defined. Using complementary experimental models, we discovered RIPK3-dependent IL1B induction in ß cells as an instigator of islet inflammation. In cultured ß cells, ER stress activated RIPK3, leading to NF-kB-mediated proinflammatory gene expression. In a zebrafish muscle insulin resistance model, overnutrition caused islet inflammation, ß cell dysfunction, and loss in an ER stress-, ripk3-, and il1b-dependent manner. In mouse islets, high-fat diet triggered the IL1B expression in ß cells before macrophage recruitment in vivo, and RIPK3 inhibition suppressed palmitate-induced ß cell dysfunction and Il1b expression in vitro. Furthermore, in human islets grafted in hyperglycemic mice, a marked increase in ER stress, RIPK3, and NF-kB activation in ß cells were accompanied with murine macrophage infiltration. Thus, RIPK3-mediated induction of proinflammatory mediators is a conserved, previously unrecognized ß cell response to metabolic stress and a mediator of the ensuing islet inflammation.

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.

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.

TGR5 Protects Against Colitis in Mice, but Vertical Sleeve Gastrectomy Increases Colitis Severity.

BACKGROUND AND AIMS: Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is the most effective long-term treatment for obesity. However, there are conflicting reports on the effect of bariatric surgery on inflammatory bowel disease (IBD). Bariatric surgery increases bile acid concentrations, which can decrease inflammation by signaling through the bile acid receptor, TGR5. TGR5 signaling protects against chemically induced colitis in mice. VSG increases circulating bile acid concentrations to increase TGR5 signaling, which contributes to improved metabolic regulation after VSG. Therefore, we investigated the effect of VSG on chemically induced colitis development and the role of TGR5 in this context. METHODS: VSG or sham surgery was performed in high fat diet-fed male Tgr5+/+ and Tgr5-/- littermates. Sham-operated mice were food restricted to match their body weight to VSG-operated mice. Colitis was induced with 2.5% dextran sodium sulfate (DSS) in water post-operatively. Body weight, energy intake, fecal scoring, colon histopathology, colonic markers of inflammation, goblet cell counts, and colonic microRNA-21 levels were assessed. RESULTS: VSG decreased body weight independently of genotype. Consistent with previous work, genetic ablation of TGR5 increased the severity of DSS-induced colitis. Notably, despite the effect of VSG to decrease body weight and increase TGR5 signaling, VSG increased the severity of DSS-induced colitis. VSG-induced increases in colitis were associated with increased colonic expression of TNF-α, IL-6, MCP-1, and microRNA-21. CONCLUSIONS: While our data demonstrate that TGR5 protects against colitis, they also demonstrate that VSG potentiates chemically induced colitis in mice. These data suggest that individuals undergoing VSG may be at increased risk for developing colitis; however, further study is needed.

Requirement for sphingosine kinase 1 in mediating phase 1 of the hypotensive response to anandamide in the anaesthetised mouse.

In the isolated rat carotid artery, the endocannabinoid anandamide induces endothelium-dependent relaxation via activation of the enzyme sphingosine kinase (SK). This generates sphingosine-1-phosphate (S1P) which can be released from the cell and activates S1P receptors on the endothelium. In anaesthetised mice, anandamide has a well-characterised triphasic effect on blood pressure but the contribution of SK and S1P receptors in mediating changes in blood pressure has never been studied. Therefore, we assessed this in the current study. The peak hypotensive response to 1 and 10 mg/kg anandamide was measured in control C57BL/6 mice and in mice pretreated with selective inhibitors of SK1 (BML-258, also known as SK1-I) or SK2 ((R)-FTY720 methylether (ROMe), a dual SK1/2 inhibitor (SKi) or an S1P1 receptor antagonist (W146). Vasodilator responses to S1P were also studied in isolated mouse aortic rings. The hypotensive response to anandamide was significantly attenuated by BML-258 but not by ROMe. Antagonising S1P1 receptors with W146 completely blocked the fall in systolic but not diastolic blood pressure in response to anandamide. S1P induced vasodilation in denuded aortic rings was blocked by W146 but caused no vasodilation in endothelium-intact rings. This study provides evidence that the SK1/S1P regulatory-axis is necessary for the rapid hypotension induced by anandamide. Generation of S1P in response to anandamide likely activates S1P1 to reduce total peripheral resistance and lower mean arterial pressure. These findings have important implications in our understanding of the hypotensive and cardiovascular actions of cannabinoids.

Rethinking Bile Acid Metabolism and Signaling for Type 2 Diabetes Treatment.

PURPOSE OF REVIEW: Herein, we review the role of FXR and TGR5 in the regulation of hepatic bile acid metabolism, with a focus on how our understanding of bile acid metabolic regulation by these receptors has evolved in recent years and how this improved understanding may facilitate targeting bile acids for type 2 diabetes treatment. RECENT FINDINGS: Bile acid profile is a key regulator of metabolic homeostasis. Inhibition of expression of the enzyme that is required for cholic acid synthesis and thus determines bile acid profile, Cyp8b1, may be an effective target for type 2 diabetes treatment. FXR and, more recently, TGR5 have been shown to regulate bile acid metabolism and Cyp8b1 expression and, therefore, may provide a mechanism with which to target bile acid profile for type 2 diabetes treatment. Inhibition of Cyp8b1 expression is a promising therapeutic modality for type 2 diabetes; however, further work is needed to fully understand the pathways regulating Cyp8b1 expression.

Deoxycholic acid supplementation impairs glucose homeostasis in mice.

Bile acids are critical contributors to the regulation of whole body glucose homeostasis; however, the mechanisms remain incompletely defined. While the hydrophilic bile acid subtype, ursodeoxycholic acid, has been shown to attenuate hepatic endoplasmic reticulum (ER) stress and thereby improve glucose regulation in mice, the effect of hydrophobic bile acid subtypes on ER stress and glucose regulation in vivo is unknown. Therefore, we investigated the effect of the hydrophobic bile acid subtype, deoxycholic acid (DCA), on ER stress and glucose regulation. Eight week old C57BL/6J mice were fed a high fat diet supplemented with or without DCA. Glucose regulation was assessed by oral glucose tolerance and insulin tolerance testing. In addition, circulating bile acid profile and hepatic insulin and ER stress signaling were measured. DCA supplementation did not alter body weight or food intake, but did impair glucose regulation. Consistent with the impairment in glucose regulation, DCA increased the hydrophobicity of the circulating bile acid profile, decreased hepatic insulin signaling and increased hepatic ER stress signaling. Together, these data suggest that dietary supplementation of DCA impairs whole body glucose regulation by disrupting hepatic ER homeostasis in mice.

ß Cell GLP-1R Signaling Alters α Cell Proglucagon Processing after Vertical Sleeve Gastrectomy in Mice.

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), causes high rates of type 2 diabetes remission and remarkable increases in postprandial glucagon-like peptide-1 (GLP-1) secretion. GLP-1 plays a critical role in islet function by potentiating glucose-stimulated insulin secretion; however, the mechanisms remain incompletely defined. Therefore, we applied a murine VSG model to an inducible ß cell-specific GLP-1 receptor (GLP-1R) knockout mouse model to investigate the role of the ß cell GLP-1R in islet function. Our data show that loss of ß cell GLP-1R signaling decreases α cell GLP-1 expression after VSG. Furthermore, we find a ß cell GLP-1R-dependent increase in α cell expression of the prohormone convertase required for the production of GLP-1 after VSG. Together, the findings herein reveal two concepts. First, our data support a paracrine role for α cell-derived GLP-1 in the metabolic benefits observed after VSG. Second, we have identified a role for the ß cell GLP-1R as a regulator of α cell proglucagon processing.

The Role of O-GlcNAcylation in Perivascular Adipose Tissue Dysfunction of Offspring of High-Fat Diet-Fed Rats.

Perivascular adipose tissue (PVAT), which reduces vascular contractility, is dysfunctional in the male offspring of rats fed a high-fat diet (HFD), partially due to a reduced NO bioavailability. O-GlcNAcylation of eNOS decreases its activity, thus we investigated the role of O-GlcNAcylation in the prenatal programming of PVAT dysfunction. Female Sprague-Dawley rats were fed either a control (10% fat) or an obesogenic HFD (45% fat) diet for 12 weeks prior to mating, and throughout pregnancy and lactation. Offspring were weaned onto the control diet and were killed at 12 and 24 weeks of age. Mesenteric arteries from the 12-week-old offspring of HFD dams (HFDO) contracted less to U46619; these effects were mimicked by glucosamine in control arteries. PVAT from 12- and 24-week-old controls, but not from HFDO, exerted an anticontractile effect. Glucosamine attenuated the anticontractile effect of PVAT in the vessels from controls but not from HFDO. AMP-activated protein kinase (AMPK) activation (with A769662) partially restored an anticontractile effect in glucosamine-treated controls and HFDO PVAT. Glucosamine decreased AMPK activity and expression in HFDO PVAT, although phosphorylated eNOS expression was only reduced in that from males. The loss of anticontractile effect of HFDO PVAT is likely to result from increased O-GlcNAcylation, which decreased AMPK activity and, in males, decreased NO bioavailability.