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.

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.

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.

Akt signals upstream of L-type calcium channels to optimize insulin secretion.

OBJECTIVES: Both the serine/threonine protein kinase Akt and the voltage-gated L-type calcium channel act as important players in glucose-stimulated insulin secretion. Akt recruits the L-type calcium channel to and maintains them in the plasma membrane. This study aimed to characterize the role of L-type calcium channels in mediation of Akt signaling in glucose-stimulated insulin secretion. METHODS: Insulin secretion was evaluated in rat pancreatic islets and INS-1 pancreatic ß cells by a standard insulin radioimmunoassay. RESULTS: Akt inhibition effectively abrogates not only glucose-stimulated but also potassium depolarization-stimulated insulin secretion from rat islets, the latter critically relying on the voltage-gated calcium channel-mediated Ca(2+) influx without involvement of glucose metabolism. Likewise, Akt inhibition also reduces both glucose-stimulated and potassium depolarization-stimulated insulin secretion from INS-1 cells. Importantly, pharmacological ablation of L-type calcium channels partially blocks Akt inhibition-induced reduction in glucose-stimulated insulin secretion but completely prevents that in potassium depolarization-evoked insulin release from INS-1 cells. Furthermore, Akt inhibition does not influence calcium ionophore A23187-induced insulin secretion from INS-1 cells, which occurred without involvement of L-type calcium channels. CONCLUSIONS: Akt signals upstream of L-type calcium channels to optimize glucose-stimulated insulin secretion.

Subthreshold α2-adrenergic activation counteracts glucagon-like peptide-1 potentiation of glucose-stimulated insulin secretion.

The pancreatic ß cell harbors α2-adrenergic and glucagon-like peptide-1 (GLP-1) receptors on its plasma membrane to sense the corresponding ligands adrenaline/noradrenaline and GLP-1 to govern glucose-stimulated insulin secretion. However, it is not known whether these two signaling systems interact to gain the adequate and timely control of insulin release in response to glucose. The present work shows that the α2-adrenergic agonist clonidine concentration-dependently depresses glucose-stimulated insulin secretion from INS-1 cells. On the contrary, GLP-1 concentration-dependently potentiates insulin secretory response to glucose. Importantly, the present work reveals that subthreshold α2-adrenergic activation with clonidine counteracts GLP-1 potentiation of glucose-induced insulin secretion. This counteractory process relies on pertussis toxin- (PTX-) sensitive Gi proteins since it no longer occurs following PTX-mediated inactivation of Gi proteins. The counteraction of GLP-1 potentiation of glucose-stimulated insulin secretion by subthreshold α2-adrenergic activation is likely to serve as a molecular mechanism for the delicate regulation of insulin release.

Glutamate is a positive autocrine signal for glucagon release.

An important feature of glucose homeostasis is the effective release of glucagon from the pancreatic alpha cell. The molecular mechanisms regulating glucagon secretion are still poorly understood. We now demonstrate that human alpha cells express ionotropic glutamate receptors (iGluRs) that are essential for glucagon release. A lowering in glucose concentration results in the release of glutamate from the alpha cell. Glutamate then acts on iGluRs of the AMPA/kainate type, resulting in membrane depolarization, opening of voltage-gated Ca(2+) channels, increase in cytoplasmic free Ca(2+) concentration, and enhanced glucagon release. In vivo blockade of iGluRs reduces glucagon secretion and exacerbates insulin-induced hypoglycemia in mice. Hence, the glutamate autocrine feedback loop endows the alpha cell with the ability to effectively potentiate its own secretory activity. This is a prerequisite to guarantee adequate glucagon release despite relatively modest changes in blood glucose concentration under physiological conditions.