The anterior chamber of the eye technology and its anatomical, optical, and immunological bases.

The anterior chamber of the eye (ACE) is distinct in its anatomy, optics, and immunology. This guarantees that the eye perceives visual information in the context of physiology even when encountering adverse incidents like inflammation. In addition, this endows the ACE with the special nursery bed iris enriched in vasculatures and nerves. The ACE constitutes a confined space enclosing an oxygen/nutrient-rich, immune-privileged, and less stressful milieu as well as an optically transparent medium. Therefore, aside from visual perception, the ACE unexpectedly serves as an excellent transplantation site for different body parts and a unique platform for noninvasive, longitudinal, and intravital microimaging of different grafts. On the basis of these merits, the ACE technology has evolved from the prototypical through the conventional to the advanced version. Studies using this technology as a versatile biomedical research platform have led to a diverse range of basic knowledge and in-depth understanding of a variety of cells, tissues, and organs as well as artificial biomaterials, pharmaceuticals, and abiotic substances. Remarkably, the technology turns in vivo dynamic imaging of the morphological characteristics, organotypic features, developmental fates, and specific functions of intracameral grafts into reality under physiological and pathological conditions. Here we review the anatomical, optical, and immunological bases as well as technical details of the ACE technology. Moreover, we discuss major achievements obtained and potential prospective avenues for this technology.

Multiple Inositol Polyphosphate Phosphatase Compartmentalization Separates Inositol Phosphate Metabolism from Inositol Lipid Signaling.

Multiple inositol polyphosphate phosphatase (MINPP1) is an enigmatic enzyme that is responsible for the metabolism of inositol hexakisphosphate (InsP6) and inositol 1,3,4,5,6 pentakisphosphate (Ins(1,3,4,5,6)P5 in mammalian cells, despite being restricted to the confines of the ER. The reason for this compartmentalization is unclear. In our previous studies in the insulin-secreting HIT cell line, we expressed MINPP1 in the cytosol to artificially reduce the concentration of these higher inositol phosphates. Undocumented at the time, we noted cytosolic MINPP1 expression reduced cell growth. We were struck by the similarities in substrate preference between a number of different enzymes that are able to metabolize both inositol phosphates and lipids, notably IPMK and PTEN. MINPP1 was first characterized as a phosphatase that could remove the 3-phosphate from inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). This molecule shares strong structural homology with the major product of the growth-promoting Phosphatidyl 3-kinase (PI3K), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and PTEN can degrade both this lipid and Ins(1,3,4,5)P4. Because of this similar substrate preference, we postulated that the cytosolic version of MINPP1 (cyt-MINPP1) may not only attack inositol polyphosphates but also PtdIns(3,4,5)P3, a key signal in mitogenesis. Our experiments show that expression of cyt-MINPP1 in HIT cells lowers the concentration of PtdIns(3,4,5)P3. We conclude this reflects a direct effect of MINPP1 upon the lipid because cyt-MINPP1 actively dephosphorylates synthetic, di(C4:0)PtdIns(3,4,5)P3 in vitro. These data illustrate the importance of MINPP1's confinement to the ER whereby important aspects of inositol phosphate metabolism and inositol lipid signaling can be separately regulated and give one important clarification for MINPP1's ER seclusion.

Tectorigenin targets PKACα to promote GLUT4 expression in skeletal muscle and improve insulin resistance in vitro and in vivo.

The decreased expression and dysfunction of glucose transporter 4 (GLUT4), the insulin-responsive glucose transporter, are closely related to the occurrence of insulin resistance (IR). To improve the expression of GLUT4 may represent a promising strategy to prevent and treat IR and type 2 diabetes (T2DM). Here, we demonstrate that the natural compound tectorigenin (TG) enhances GLUT4 expression, glucose uptake and insulin responsiveness via activating AMP-activated protein kinase (AMPK)/myocyte enhancer factor 2 (MEF2) signaling in both normal and IR skeletal muscle cells and tissues. Accordingly, prophylactic and therapeutic uses of TG can significantly ameliorate IR and hyperglycemia in T2DM mice. Mechanistically, we identify protein kinase A catalytic subunit α (PKACα) as the target of TG to increase GLUT4 expression and TG-PKACα binding promotes the dissociation of PKACα from the regulatory subunits, leading to the activation of PKA/AMPK signaling. PKACα knockdown in local quadriceps muscles almost completely abolished the therapeutic effects of TG on IR and T2DM, as well as the enhancement on AMPK signaling and GLUT4 expression in skeletal muscle. This study supports TG as a new drug candidate to treat IR and its related diseases, but also enriches our knowledge of PKA signaling in glucose metabolism in skeletal muscle.

In Vivo CaV3 Channel Inhibition Promotes Maturation of Glucose-Dependent Ca2+ Signaling in Human iPSC-Islets.

CaV3 channels are ontogenetically downregulated with the maturation of certain electrically excitable cells, including pancreatic ß cells. Abnormally exaggerated CaV3 channels drive the dedifferentiation of mature ß cells. This led us to question whether excessive CaV3 channels, retained mistakenly in engineered human-induced pluripotent stem cell-derived islet (hiPSC-islet) cells, act as an obstacle to hiPSC-islet maturation. We addressed this question by using the anterior chamber of the eye (ACE) of immunodeficient mice as a site for recapitulation of in vivo hiPSC-islet maturation in combination with intravitreal drug infusion, intravital microimaging, measurements of cytoplasmic-free Ca2+ concentration ([Ca2+]i) and patch clamp analysis. We observed that the ACE is well suited for recapitulation, observation and intervention of hiPSC-islet maturation. Intriguingly, intraocular hiPSC-islet grafts, retrieved intact following intravitreal infusion of the CaV3 channel blocker NNC55-0396, exhibited decreased basal [Ca2+]i levels and increased glucose-stimulated [Ca2+]i responses. Insulin-expressing cells of these islet grafts indeed expressed the NNC55-0396 target CaV3 channels. Intraocular hiPSC-islets underwent satisfactory engraftment, vascularization and light scattering without being influenced by the intravitreally infused NNC55-0396. These data demonstrate that inhibiting CaV3 channels facilitates the maturation of glucose-activated Ca2+ signaling in hiPSC-islets, supporting the notion that excessive CaV3 channels as a developmental error impede the maturation of engineered hiPSC-islet insulin-expressing cells.

Inositol hexakisphosphate primes syndapin I/PACSIN 1 activation in endocytosis.

Endocytosis is controlled by a well-orchestrated molecular machinery, where the individual players as well as their precise interactions are not fully understood. We now show that syndapin I/PACSIN 1 is expressed in pancreatic ß cells and that its knockdown abrogates ß cell endocytosis leading to disturbed plasma membrane protein homeostasis, as exemplified by an elevated density of L-type Ca2+ channels. Intriguingly, inositol hexakisphosphate (InsP6) activates casein kinase 2 (CK2) that phosphorylates syndapin I/PACSIN 1, thereby promoting interactions between syndapin I/PACSIN 1 and neural Wiskott-Aldrich syndrome protein (N-WASP) and driving ß cell endocytosis. Dominant-negative interference with endogenous syndapin I/PACSIN 1 protein complexes, by overexpression of the syndapin I/PACSIN 1 SH3 domain, decreases InsP6-stimulated endocytosis. InsP6 thus promotes syndapin I/PACSIN 1 priming by CK2-dependent phosphorylation, which endows the syndapin I/PACSIN 1 SH3 domain with the capability to interact with the endocytic machinery and thereby initiate endocytosis, as exemplified in ß cells.

Intracameral Microimaging of Maturation of Human iPSC Derivatives into Islet Endocrine Cells.

We exploited the anterior chamber of the eye (ACE) of immunodeficient mice as an ectopic site for both transplantation and microimaging of engineered surrogate islets from human induced pluripotent stem cells (hiPSC-SIs). These islets contained a majority of insulin-expressing cells, positive or negative for PDX1 and NKX6.1, and a minority of glucagon- or somatostatin-positive cells. Single, non-aggregated hiPSC-SIs were satisfactorily engrafted onto the iris. They underwent gradual vascularization and progressively increased their light scattering signals, reflecting the abundance of zinc-insulin crystal packaged inside mature insulin secretory granules. Intracameral hiPSC-SIs retrieved from recipients showed enhanced insulin immunofluorescence in correlation with the parallel increase in overall vascularization and light backscattering during the post-transplantation period. This approach enables longitudinal, nondestructive and intravital microimaging of cell fates, engraftment, vascularization and mature insulin secretory granules of single hiPSC-SI grafts, and may offer a feasible and reliable means to screen compounds for promoting in vivo hiPSC-SI maturation.

Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels.

ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

Enhanced expression of ß cell CaV3.1 channels impairs insulin release and glucose homeostasis.

Voltage-gated calcium 3.1 (CaV3.1) channels are absent in healthy mouse ß cells and mediate minor T-type Ca2+ currents in healthy rat and human ß cells but become evident under diabetic conditions. Whether more active CaV3.1 channels affect insulin secretion and glucose homeostasis remains enigmatic. We addressed this question by enhancing de novo expression of ß cell CaV3.1 channels and exploring the consequent impacts on dynamic insulin secretion and glucose homeostasis as well as underlying molecular mechanisms with a series of in vitro and in vivo approaches. We now demonstrate that a recombinant adenovirus encoding enhanced green fluorescent protein-CaV3.1 subunit (Ad-EGFP-CaV3.1) efficiently transduced rat and human islets as well as dispersed islet cells. The resulting CaV3.1 channels conducted typical T-type Ca2+ currents, leading to an enhanced basal cytosolic-free Ca2+ concentration ([Ca2+]i). Ad-EGFP-CaV3.1-transduced islets released significantly less insulin under both the basal and first phases following glucose stimulation and could no longer normalize hyperglycemia in recipient rats rendered diabetic by streptozotocin treatment. Furthermore, Ad-EGFP-CaV3.1 transduction reduced phosphorylated FoxO1 in the cytoplasm of INS-1E cells, elevated FoxO1 nuclear retention, and decreased syntaxin 1A, SNAP-25, and synaptotagmin III. These effects were prevented by inhibiting CaV3.1 channels or the Ca2+-dependent phosphatase calcineurin. Enhanced expression of ß cell CaV3.1 channels therefore impairs insulin release and glucose homeostasis by means of initial excessive Ca2+ influx, subsequent activation of calcineurin, consequent dephosphorylation and nuclear retention of FoxO1, and eventual FoxO1-mediated down-regulation of ß cell exocytotic proteins. The present work thus suggests an elevated expression of CaV3.1 channels plays a significant role in diabetes pathogenesis.

IgGs from patients with amyotrophic lateral sclerosis and diabetes target CaVα2δ1 subunits impairing islet cell function and survival.

Patients with amyotrophic lateral sclerosis (ALS) often show hallmarks of type 2 diabetes mellitus (T2DM). However, the causal link between ALS and T2DM has remained a mystery. We now demonstrate that 60% of ALS patients with T2DM (ALS-T2DM) have sera that exaggerated K+-induced increases in cytosolic free Ca2+ concentration ([Ca2+]i) in mouse islet cells. The effect was attributed to the presence of pathogenic immunoglobulin Gs (IgGs) in ALS-T2DM sera. The pathogenic IgGs immunocaptured the voltage-dependent Ca2+ (CaV) channel subunit CaVα2δ1 in the plasma membrane enhancing CaV1 channel-mediated Ca2+ influx and [Ca2+]i, resulting in impaired mitochondrial function. Consequently, impairments in [Ca2+]i dynamics, insulin secretion, and cell viability occurred. These data reveal that patients with ALS-T2DM carry cytotoxic ALS-T2DM-IgG autoantibodies that serve as a causal link between ALS and T2DM by immunoattacking CaVα2δ1 subunits. Our findings may lay the foundation for a pharmacological treatment strategy for patients suffering from a combination of these diseases.

Hypericin maintians PDX1 expression via the Erk pathway and protects islet ß-cells against glucotoxicity and lipotoxicity.

A decrease in islet ß-cell mass is closely associated with the development and progression of diabetes. Therefore, protection against ß-cell loss is an essential measure to prevent and treat diabetes. In this study, we investigated the protective effects of non-photoactivated hypericin, a natural compound, on ß-cells both in vitro and in vivo. In vitro, hypericin greatly improved INS-1 cell viability under high-glucose and high-fatty-acid conditions by inhibiting glucotoxicity- and lipotoxicity-induced apoptosis and nitric oxide (NO) production. Then, we further demonstrated that hypericin elicited its protective effects against glucotoxicity and lipotoxicity in INS-1 cells by attenuating the reduction in pancreatic duodenal homeobox-1 (PDX1) expression and Erk activity. In vivo, prophylactic or therapeutic use of hypericin inhibited islet ß-cell apoptosis and enhanced the anti-oxidative ability of pancreatic tissue in high-fat/high-sucrose (HFHS)-fed mice, thus alleviating ß-cell loss and maintaining or improving ß-cell mass and islet size. More importantly, hypericin treatment decreased fasting blood glucose, improved glucose intolerance and insulin intolerance, and alleviated hyperinsulinaemia in HFHS-fed mice. Therefore, hypericin showed preventive and therapeutic effects against HFHS-induced onset of type II diabetes in mice. Hypericin possesses great potential for development as an anti-diabetes drug in the future.

Dracorhodin perchlorate protects pancreatic ß-cells against glucotoxicity- or lipotoxicity-induced dysfunction and apoptosis in vitro and in vivo.

Glucotoxicity or lipotoxicity leads to hyperglycemia and insulin secretion deficiency, which are important causes for the onset of type 2 diabetes mellitus (T2DM). Thus, the restoration of ß-cell function is a long-sought goal in diabetes research. Previous studies have implicated pancreatic and duodenal homeobox 1 gene (Pdx1) in ß-cell function and insulin secretion. In this study, we established a Pdx1 promoter-dependent luciferase system and identified the natural compound dracorhodin perchlorate (DP) as an effective promotor of Pdx1 expression. We further demonstrated that DP could significantly inhibit ß-cell apoptosis induced by 33 mm glucose or 200 µm palmitate by interfering with endoplasmic reticulum stress and mitochondrial pathways and enhance insulin secretion as well. These effects were associated with enhanced activities of Erk1/2, which in turn promoted Pdx1 expression and increased the ratio of Bcl2/Bax, since inhibition of the Erk1/2 pathway abolished the DP-induced expression of Pdx1 and suppression of apoptosis. In addition, our in vivo results in diabetic mice indicated that DP treatment lowered blood glucose, raised insulin levels, enhanced Pdx1 expression and increased islet size and number in the pancreas of diabetic mice. Our findings suggest that Pdx1 is a potential target molecule of DP in the treatment of T2DM via the inhibition of glucotoxicity- or lipotoxicity- induced ß-cell apoptosis and the attenuation of insulin secretion dysfunction.

The eye as a novel imaging site in diabetes research.

Diabetes develops due to deficient functional ß cell mass, insulin resistance, or both. Yet, various challenges in understanding the mechanisms underlying diabetes development in vivo remain to be overcome owing to the lack of appropriate intravital imaging technologies. To meet these challenges, we have exploited the anterior chamber of the eye (ACE) as a novel imaging site to understand diabetes basics and clinics in vivo. We have developed a technology platform transplanting pancreatic islets into the ACE where they later on can be imaged non-invasively for long time. It turns out that the ACE serves as an optimal imaging site and provides implanted islets with an oxygen-rich milieu and an immune-privileged niche where they undergo optimal engraftment, rich vascularization and dense innervation, preserve organotypic features and live with satisfactory viability and functionality. The ACE technology has led to a series of significant observations. It enables in vivo microscopy of islet cytoarchitecture, function and viability in the physiological context and intravital imaging of a variety of pathological events such as autoimmune insulitis, defects in ß cell function and mass and insulin resistance during diabetes development in a real-time manner. Furthermore, application of the ACE technology in humanized mice and non-human primates verifies translational and clinical values of the technology. In this article, we describe the ACE technology in detail, review accumulated knowledge gained by means of the ACE technology and delineate prospective avenues for the ACE technology.

Blocking Ca2+ Channel ß3 Subunit Reverses Diabetes.

Voltage-gated Ca2+ channels (Cav) are essential for pancreatic beta cell function as they mediate Ca2+ influx, which leads to insulin exocytosis. The ß3 subunit of Cav (Cavß3) has been suggested to regulate cytosolic Ca2+ ([Ca2+]i) oscillation frequency and insulin secretion under physiological conditions, but its role in diabetes is unclear. Here, we report that islets from diabetic mice show Cavß3 overexpression, altered [Ca2+]i dynamics, and impaired insulin secretion upon glucose stimulation. Consequently, in high-fat diet (HFD)-induced diabetes, Cavß3-deficient (Cavß3-/-) mice showed improved islet function and enhanced glucose tolerance. Normalization of Cavß3 expression in ob/ob islets by an antisense oligonucleotide rescued the altered [Ca2+]i dynamics and impaired insulin secretion. Importantly, transplantation of Cavß3-/- islets into the anterior chamber of the eye improved glucose tolerance in HFD-fed mice. Cavß3 overexpression in human islets also impaired insulin secretion. We thus suggest that Cavß3 may serve as a druggable target for diabetes treatment.

TLR3-/4-Priming Differentially Promotes Ca(2+) Signaling and Cytokine Expression and Ca(2+)-Dependently Augments Cytokine Release in hMSCs.

In human mesenchymal stem cells (hMSCs), toll-like receptor 3 (TLR3) and TLR4 act as key players in the tissue repair process by recognizing their ligands and stimulating downstream processes including cytokine release. The mechanisms of TLR3- and TLR4-mediated cytokine releases from hMSCs remain uncertain. Here, we show that exposure to the TLR3 agonist polyinosinic-polycytidylic acid (poly(I:C)) or incubation with the TLR4 agonist lipopolysaccharide (LPS) increased the mRNA expression levels of TLR3, TLR4 and cytokines in hMSCs. Poly(I:C) exposure rather than LPS incubation not only elevated inositol 1,4,5-triphosphate receptor (IP3R) expression and IP3R-mediated Ca(2+) release, but also promoted Orai and STIM expression as well as store-operated Ca(2+) entry into hMSCs. In addition, we also observed that 21 Ca(2+) signaling genes were significantly up-regulated in response to TLR3 priming of hMSCs by RNA sequencing analysis. Both poly(I:C) and LPS exposure enhanced cytokine release from hMSCs. The enhanced cytokine release vanished upon siRNA knockdown and chelation of intracellular Ca(2+). These data demonstrate that TLR3- and TLR4-priming differentially enhance Ca(2+) signaling and cytokine expression, and Ca(2+) -dependently potentiates cytokine release in hMSCs.

Photoactivation of hypericin decreases the viability of RINm5F insulinoma cells through reduction in JNK/ERK phosphorylation and elevation of caspase-9/caspase-3 cleavage and Bax-to-Bcl-2 ratio.

Insulinomas cause neuroglycopenic symptoms, permanent neurological damage and even death. Current available therapies cannot satisfactorily treat malignant insulinomas and some benign insulinomas. The promising phototherapeutic results and harmless side effects of hypericin in some cancer treatments prompted us to explore possible anti-growth activity of photoactivated hypericin against RINm5F insulinoma cells and underlying mechanisms. We now show that detectable and maximal internalization of hypericin in RINm5F insulinoma cells occurred in 20 and 60 min respectively. Hypericin was considerably associated with the plasma membrane, appreciably localized in the sub-plasma membrane region and substantially accumulated in the cytoplasm. Photoactivated hypericin decreased the viability of RINm5F insulinoma cells due to its anti-proliferative and apoptotic actions. Photoactivation of hypericin inhibited cell proliferation reflected by decreased expression of the proliferation marker Ki-67 and cell-cycle arrest in the G0/G1-phase. The anti-proliferative effect resulted from down-regulation of phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK). Photoactivated hypericin triggered apoptosis through activation of caspase-3 and caspase-9 and elevation of the Bax-to B-cell lymphoma 2 (Bcl-2) ratio. The findings lay a solid foundation for implementation of hypericin-mediated photodynamic therapy in treatment of insulinomas.

CaV1.2 and CaV1.3 channel hyperactivation in mouse islet ß cells exposed to type 1 diabetic serum.

The voltage-gated Ca(2+) (CaV) channel acts as a key player in ß cell physiology and pathophysiology. ß cell CaV channels undergo hyperactivation subsequent to exposure to type 1 diabetic (T1D) serum resulting in increased cytosolic free Ca(2+) concentration and thereby Ca(2+)-triggered ß cell apoptosis. The present study was aimed at revealing the subtypes of CaV1 channels hyperactivated by T1D serum as well as the biophysical mechanisms responsible for T1D serum-induced hyperactivation of ß cell CaV1 channels. Patch-clamp recordings and single-cell RT-PCR analysis were performed in pancreatic ß cells from CaV1 channel knockout and corresponding control mice. We now show that functional CaV1.3 channels are expressed in a subgroup of islet ß cells from CaV1.2 knockout mice (CaV1.2(-/-)). T1D serum enhanced whole-cell CaV currents in islet ß cells from CaV1.3 knockout mice (CaV1.3(-/-)). T1D serum increased the open probability and number of functional unitary CaV1 channels in CaV1.2(-/-) and CaV1.3(-/-) ß cells. These data demonstrate that T1D serum hyperactivates both CaV1.2 and CaV1.3 channels by increasing their conductivity and number. These findings suggest CaV1.2 and CaV1.3 channels as potential targets for anti-diabetes therapy.

Ionic mechanisms in pancreatic ß cell signaling.

The function and survival of pancreatic ß cells critically rely on complex electrical signaling systems composed of a series of ionic events, namely fluxes of K(+), Na(+), Ca(2+) and Cl(-) across the ß cell membranes. These electrical signaling systems not only sense events occurring in the extracellular space and intracellular milieu of pancreatic islet cells, but also control different ß cell activities, most notably glucose-stimulated insulin secretion. Three major ion fluxes including K(+) efflux through ATP-sensitive K(+) (KATP) channels, the voltage-gated Ca(2+) (CaV) channel-mediated Ca(2+) influx and K(+) efflux through voltage-gated K(+) (KV) channels operate in the ß cell. These ion fluxes set the resting membrane potential and the shape, rate and pattern of firing of action potentials under different metabolic conditions. The KATP channel-mediated K(+) efflux determines the resting membrane potential and keeps the excitability of the ß cell at low levels. Ca(2+) influx through CaV1 channels, a major type of ß cell CaV channels, causes the upstroke or depolarization phase of the action potential and regulates a wide range of ß cell functions including the most elementary ß cell function, insulin secretion. K(+) efflux mediated by KV2.1 delayed rectifier K(+) channels, a predominant form of ß cell KV channels, brings about the downstroke or repolarization phase of the action potential, which acts as a brake for insulin secretion owing to shutting down the CaV channel-mediated Ca(2+) entry. These three ion channel-mediated ion fluxes are the most important ionic events in ß cell signaling. This review concisely discusses various ionic mechanisms in ß cell signaling and highlights KATP channel-, CaV1 channel- and KV2.1 channel-mediated ion fluxes.

Differential regulation of mouse pancreatic islet insulin secretion and Smad proteins by activin ligands.

AIMS/HYPOTHESIS: Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells is regulated by paracrine factors, the identity and mechanisms of action of which are incompletely understood. Activins are expressed in pancreatic islets and have been implicated in the regulation of GSIS. Activins A and B signal through a common set of intracellular components, but it is unclear whether they display similar or distinct functions in glucose homeostasis. METHODS: We examined glucose homeostatic responses in mice lacking activin B and in pancreatic islets derived from these mutants. We compared the ability of activins A and B to regulate downstream signalling, ATP production and GSIS in islets and beta cells. RESULTS: Mice lacking activin B displayed elevated serum insulin levels and GSIS. Injection of a soluble activin B antagonist phenocopied these changes in wild-type mice. Isolated pancreatic islets from mutant mice showed enhanced GSIS, which could be rescued by exogenous activin B. Activin B negatively regulated GSIS and ATP production in wild-type islets, while activin A displayed the opposite effects. The downstream mediator Smad3 responded preferentially to activin B in pancreatic islets and beta cells, while Smad2 showed a preference for activin A, indicating distinct signalling effects of the two activins. In line with this, overexpression of Smad3, but not Smad2, decreased GSIS in pancreatic islets. CONCLUSIONS/INTERPRETATION: These results reveal a tug-of-war between activin ligands in the regulation of insulin secretion by beta cells, and suggest that manipulation of activin signalling could be a useful strategy for the control of glucose homeostasis in diabetes and metabolic disease.

Apolipoprotein CIII hyperactivates ß cell CaV1 channels through SR-BI/ß1 integrin-dependent coactivation of PKA and Src.

Apolipoprotein CIII (ApoCIII) not only serves as an inhibitor of triglyceride hydrolysis but also participates in diabetes-related pathological events such as hyperactivation of voltage-gated Ca(2+) (CaV) channels in the pancreatic ß cell. However, nothing is known about the molecular mechanisms whereby ApoCIII hyperactivates ß cell CaV channels. We now demonstrate that ApoCIII increased CaV1 channel open probability and density. ApoCIII enhanced whole-cell Ca(2+) currents and the CaV1 channel blocker nimodipine completely abrogated this enhancement. The effect of ApoCIII was not influenced by individual inhibition of PKA, PKC, or Src. However, combined inhibition of PKA, PKC, and Src counteracted the effect of ApoCIII, similar results obtained by coinhibition of PKA and Src. Moreover, knockdown of ß1 integrin or scavenger receptor class B type I (SR-BI) prevented ApoCIII from hyperactivating ß cell CaV channels. These data reveal that ApoCIII hyperactivates ß cell CaV1 channels through SR-BI/ß1 integrin-dependent coactivation of PKA and Src.

Inositol hexakisphosphate suppresses excitatory neurotransmission via synaptotagmin-1 C2B domain in the hippocampal neuron.

Inositol hexakisphosphate (InsP(6)) levels rise and fall with neuronal excitation and silence, respectively, in the hippocampus, suggesting potential signaling functions of this inositol polyphosphate in hippocampal neurons. We now demonstrate that intracellular application of InsP(6) caused a concentration-dependent inhibition of autaptic excitatory postsynaptic currents (EPSCs) in cultured hippocampal neurons. The treatment did not alter the size and replenishment rate of the readily releasable pool in autaptic neurons. Intracellular exposure to InsP(6) did not affect spontaneous EPSCs or excitatory amino acid-activated currents in neurons lacking autapses. The InsP(6)-induced inhibition of autaptic EPSCs was effectively abolished by coapplication of an antibody to synaptotagmin-1 C2B domain. Importantly, preabsorption of the antibody with a GST-WT synaptotagmin-1 C2B domain fragment but not with a GST-mutant synaptotagmin-1 C2B domain fragment that poorly reacted with the antibody impaired the activity of the antibody on the InsP(6)-induced inhibition of autaptic EPSCs. Furthermore, K(+) depolarization significantly elevated endogenous levels of InsP(6) and occluded the inhibition of autaptic EPSCs by exogenous InsP(6). These data reveal that InsP(6) suppresses excitatory neurotransmission via inhibition of the presynaptic synaptotagmin-1 C2B domain-mediated fusion via an interaction with the synaptotagmin Ca(2+)-binding sites rather than via interference with presynaptic Ca(2+) levels, synaptic vesicle trafficking, or inactivation of postsynaptic ionotropic glutamate receptors. Therefore, elevated InsP(6) in activated neurons serves as a unique negative feedback signal to control hippocampal excitatory neurotransmission.