RESUMO
The gain-of-function mutation in the TALK-1 K+ channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of ß-cell electrical activity and glucose-stimulated insulin secretion. The KCNK16 gene encoding TALK-1 is the most abundant and ß-cell-restricted K+ channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the CD-1 (ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell ß-cell K+ currents resulting in blunted glucose-stimulated Ca2+ entry and loss of glucose-induced Ca2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impairs glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet insulin secretion during development. These data suggest that TALK-1 is an islet-restricted target for the treatment for diabetes.
Assuntos
Diabetes Mellitus Tipo 2 , Glucagon , Glucose , Secreção de Insulina , Camundongos Endogâmicos C57BL , Animais , Masculino , Camundongos , Animais Recém-Nascidos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Glucagon/metabolismo , Glucose/metabolismo , Homeostase , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Secreção de Insulina/genética , Ilhotas Pancreáticas/metabolismo , Mutação , Canais de Potássio/metabolismo , Canais de Potássio/genéticaRESUMO
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.
Assuntos
Diabetes Mellitus , Células Secretoras de Insulina , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Diabetes Mellitus/metabolismo , Dieta , Retículo Endoplasmático/metabolismo , Glucose/metabolismo , Homeostase , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Camundongos Knockout , Mitocôndrias/metabolismoRESUMO
The gain-of-function mutation in the TALK-1 K + channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of ß-cell electrical activity and glucose-stimulated insulin secretion (GSIS). The KCNK16 gene encoding TALK-1, is the most abundant and ß-cell-restricted K + channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the mixed C57BL/6J:CD-1(ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell ß-cell K + currents resulting in blunted glucose-stimulated Ca 2+ entry and loss of glucose-induced Ca 2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impaired glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet hormone secretion during development. These data strongly suggest that TALK-1 is an islet-restricted target for the treatment of diabetes.
RESUMO
A variant originated from Oldenlandia affinis asparaginyl ligase, OaAEP1-C247A, has emerged as an ideal tool for protein labeling. However, its preparation was laborious and time-consuming. It is recombinantly produced as a zymogen, requiring acid activation and four chromatographic steps; despite these extensive steps, the catalytically active enzyme exhibited only moderate purity. Here, we report a novel preparation protocol, in which the cap and catalytically active core domains are produced as separate entities. The active enzyme can be obtained in two chromatographic steps, immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC), with no acid activation required, thereby shortening the purification procedure from at least 2 days to less than 6 h. In addition to the original C247A mutation which enhanced reaction with various amino nucleophiles, an extra D29E mutation was introduced to prevent self-cleavage, which led to noticeable improvements in homogeneity and activity of the enzyme. Indeed, the resulting "split AEP" (i.e., core domain of OaAEP1-D29E/C247A) exhibited improved catalytic efficiency constant (kcat/KM) that was found to be â¼3-fold higher than that of the original acid-activated counterpart (OaAEP1-C247A). Furthermore, we described a protein labeling protocol that couples the enzymatic reaction with an irreversible chemical transformation, thereby enabling high conversion of labeled protein with a lowered amount of reagent. Precisely, an alternative Asn-Cys-Leu (NCL) recognition sequence was used for substrate recognition. As the byproduct contains an N-terminal cysteine, it can be transformed into an inert 1,2 aminothiol motif by reacting with formylphenyl boronic acid (FPBA). Finally, the opportunities and challenges associated with the use of asparaginyl ligase are discussed.
Assuntos
Cisteína Endopeptidases , Proteínas , Catálise , LigasesRESUMO
Preceramic polymers (PCPs) are a group of specialty macromolecules that serve as precursors for generating inorganics, including ceramic carbides, nitrides, and borides. PCPs represent interesting synthetic challenges for chemists due to the elements incorporated into their structure. This group of polymers is also of interest to engineers as PCPs enable the processing of polymer-derived ceramic products including high-performance ceramic fibers and composites. These finished ceramic materials are of growing significance for applications that experience extreme operating environments (e.g., aerospace propulsion and high-speed atmospheric flight). This Review provides an overview of advances in the synthesis and postpolymerization modification of macromolecules forming nonoxide ceramics. These PCPs include polycarbosilanes, polysilanes, polysilazanes, and precursors for ultrahigh-temperature ceramics. Following our review of PCP synthetic chemistry, we provide examples of the application and processing of these polymers, including their use in fiber spinning, composite fabrication, and additive manufacturing. The principal objective of this Review is to provide a resource that bridges the disciplines of synthetic chemistry and ceramic engineering while providing both insights and inspiration for future collaborative work that will ultimately drive the PCP field forward.
RESUMO
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.
Assuntos
Células Secretoras de Insulina , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Sódio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Somatostatina/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
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.
Assuntos
Ilhotas Pancreáticas , Liraglutida , Animais , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Liraglutida/farmacologia , CamundongosRESUMO
Impaired pancreatic ß-cell function and insulin secretion are hallmarks of type 2 diabetes. miRNAs are short, noncoding RNAs that silence gene expression vital for the development and function of ß cells. We have previously shown that ß cell-specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels. Nevertheless, the function of this miRNA in ß cells is unclear. We hypothesized that miR-125b-5p expression is regulated by glucose and that this miRNA mediates some of the deleterious effects of hyperglycemia in ß cells. Here, we show that islet miR-125b-5p expression is upregulated by glucose in an AMPK-dependent manner and that short-term miR-125b-5p overexpression impairs glucose-stimulated insulin secretion (GSIS) in the mouse insulinoma MIN6 cells and in human islets. An unbiased, high-throughput screen in MIN6 cells identified multiple miR-125b-5p targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Inactivation of miR-125b-5p in the human ß-cell line EndoCß-H1 shortened mitochondria and enhanced GSIS, whereas mice overexpressing miR-125b-5p selectively in ß cells (MIR125B-Tg) were hyperglycemic and glucose intolerant. MIR125B-Tg ß cells contained enlarged lysosomal structures and had reduced insulin content and secretion. Collectively, we identify miR-125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , MicroRNAs , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Células Secretoras de Insulina/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
The extent of grey matter demyelination and neurodegeneration in the progressive multiple sclerosis (PMS) brains at post-mortem associates with more severe disease. Regional tissue atrophy, especially affecting the cortical and deep grey matter, including the thalamus, is prognostic for poor outcomes. Microglial and complement activation are important in the pathogenesis and contribute to damaging processes that underlie tissue atrophy in PMS. We investigated the extent of pathology and innate immune activation in the thalamus in comparison to cortical grey and white matter in blocks from 21 cases of PMS and 10 matched controls. Using a digital pathology workflow, we show that the thalamus is invariably affected by demyelination and had a far higher proportion of active inflammatory lesions than forebrain cortical tissue blocks from the same cases. Lesions were larger and more frequent in the medial nuclei near the ventricular margin, whilst neuronal loss was greatest in the lateral thalamic nuclei. The extent of thalamic neuron loss was not associated with thalamic demyelination but correlated with the burden of white matter pathology in other forebrain areas (Spearman r = 0.79, p < 0.0001). Only thalamic neuronal loss, and not that seen in other forebrain cortical areas, correlated with disease duration (Spearman r = -0.58, p = 0.009) and age of death (Spearman r = -0.47, p = 0.045). Immunoreactivity for the complement pattern recognition molecule C1q, and products of complement activation (C4d, Bb and C3b) were elevated in thalamic lesions with an active inflammatory pathology. Complement regulatory protein, C1 inhibitor, was unchanged in expression. We conclude that active inflammatory demyelination, neuronal loss and local complement synthesis and activation in the thalamus, are important to the pathological and clinical disease outcomes of PMS.
Assuntos
Esclerose Múltipla Crônica Progressiva , Esclerose Múltipla , Atrofia/patologia , Ativação do Complemento , Substância Cinzenta/patologia , Humanos , Esclerose Múltipla/patologia , Esclerose Múltipla Crônica Progressiva/patologia , Tálamo/patologiaRESUMO
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.
Assuntos
Proliferação de Células/genética , Dieta Hiperlipídica , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Pâncreas/crescimento & desenvolvimento , Pâncreas/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Intolerância à Glucose/genética , Homeostase/genética , Magnésio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Cátion TRPM/genéticaRESUMO
Recent efforts have demonstrated that the morphology of ceramics can be manipulated to control both their deformation mechanism and mechanical performance. However, precise control of the ceramic nanostructure is still difficult to achieve. Biotemplating, leading to biomorphic materials, provides a facile route to manipulate the nanostructure of the resulting materials, and the use of melanin as a coating provides a new route to biotemplated materials. Melanin is underutilized for structural materials partly due to the cost of procuring it from natural sources and the inability to control the shape and sizes of melanin particles. Taking a combined synthetic biology and chemical synthesis approach, we report the melanization of Escherichia coli and its subsequent silanization and functionalization with preceramic polymers to make novel biomorphic silicon-based ceramic materials. Graft-to and graft-from reactions were used to append preceramic polymers to the melanin, followed by pyrolysis under argon. Samples were analyzed by FTIR, XRD, XPS, and TEM and found to retain the shape and size of the original cells with high fidelity. The homogeneity of coverage and yield of the resulting ceramic materials depended on the type of grafting reaction. This work provides a promising proof-of-concept that bacterial-templated ceramics can be readily made and opens a host of possibilities for further studies and applications.
Assuntos
Melaninas , Polímeros , Cerâmica , SilícioRESUMO
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.
Assuntos
Sinalização do Cálcio , Diabetes Mellitus Tipo 2 , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Glicemia/metabolismo , Canalopatias/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Mutação com Ganho de Função , Humanos , Potenciais da Membrana/fisiologia , CamundongosRESUMO
AIMS/HYPOTHESIS: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. METHODS: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. RESULTS: Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. CONCLUSIONS/INTERPRETATION: Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans. DATA AVAILABILITY: The datasets generated and/or analysed during the current study are available in the Biorxiv repository ( www.biorxiv.org/content/10.1101/2020.05.18.099200v1 ). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576 ) and ProteomeXchange ( www.ebi.ac.uk/pride/archive/projects/PXD021597 ) repositories, respectively.
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Glicemia/metabolismo , Diabetes Mellitus Tipo 2/genética , Homeostase/genética , Células Secretoras de Insulina/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Biomarcadores/sangue , Glicemia/genética , Feminino , Hormônio Foliculoestimulante/sangue , Genótipo , Humanos , Insulina/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Hipófise/metabolismo , Caracteres Sexuais , Aumento de Peso , Peixe-Zebra/sangue , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/sangue , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Hemorrhagic shock (HS) and trauma induce endothelial barrier compromise, inflammation, and aberrant clotting. We have shown that fresh human platelets (Plts) and Plt extracellular vesicles mitigate vascular leak in murine models of injury. Here, we investigate the potential of freeze-dried platelets (FDPlts) to attenuate pulmonary vascular permeability, decrease inflammation, and promote clotting in a murine model of HS. METHODS: Human FDPlts were characterized using in vitro assays of Plt marker expression, aggregation, coagulation, and endothelial cell permeability. An intravital model of vascular injury in the mouse cremaster muscle was used to assess the ability of FDPlts to incorporate into clots. Mouse groups subjected to controlled hemorrhage for 90 minutes were (1) lactated Ringer solution (LR), (2) FDPlts, (3) fresh human Plts, (4) murine whole blood (WB), and (5) shams (only instrumented). Hemorrhagic shock mouse endpoints included coagulation, pulmonary vascular permeability, and lung injury. RESULTS: Freeze-dried Plts expressed Plt-specific markers and retained functionality similar to fresh Plts. In in vitro assays of Plt aggregation, differences were noted. In vivo, FDPlts and Plts were found to incorporate into clots in postcapillary venules in the mouse cremaster muscle. Hemorrhagic shock mice resuscitated with LR displayed increased pulmonary vascular permeability compared with sham (sham, 686.6 ± 359.7; shock-LR, 2,637 ± 954.7; p = 0.001), and treatment with FDPlts or WB attenuated permeability compared with shock: shock-FDPlts, 1,328 ± 462.6 (p = 0.05), and shock-WB, 1,024 ± 370.5 (p = 0.0108). However, human Plts (Days 1-3) did not attenuate vascular leak in HS mice compared with shock-LR (shock-Plts, 3,601 ± 1,581; p = 0.33). CONCLUSION: FDPlts contribute to clot formation similar to fresh human Plts. FDPlts also attenuated vascular permeability in vitro and in vivo. Mouse WB resuscitation but not fresh human Plts attenuated vascular permeability after HS. These data suggest that the effect of FDPlts may be a suitable alternative to fresh Plts in modulating hemostasis and the endotheliopathy associated with injury.
Assuntos
Plaquetas/fisiologia , Permeabilidade Capilar/fisiologia , Modelos Animais de Doenças , Células Endoteliais/fisiologia , Liofilização , Hemostasia/fisiologia , Pulmão/irrigação sanguínea , Transfusão de Plaquetas , Choque Hemorrágico/terapia , Trombose/sangue , Animais , Humanos , Camundongos , Choque Hemorrágico/sangueRESUMO
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.
Assuntos
Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/fisiologia , Glucagon/fisiologia , Células Secretoras de Glucagon/fisiologia , Glucose/farmacologia , Humanos , Ilhotas Pancreáticas/metabolismo , Canais KATP/metabolismo , Ácido Láctico/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pâncreas/metabolismo , Cultura Primária de Células , Ácido Pirúvico/farmacologiaRESUMO
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.
Assuntos
Células Secretoras de Glucagon , Células Secretoras de Insulina , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , CamundongosRESUMO
AIMS/HYPOTHESIS: Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. METHODS: Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. RESULTS: Glucose-stimulated mitochondrial Ca2+ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (ßMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in ßMcu-KO animals. ßMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young ßMcu-KO (<12 weeks), but not in older animals vs WT mice. CONCLUSIONS/INTERPRETATION: MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in ßMcu-KO mice remain to be established.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Glucose/metabolismo , Secreção de Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The activation of neuronal plasma membrane Ca2+ channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca2+ channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts. Activated purified CaMKIIα also directly binds Shank3 between residues 829 and 1130. Mutation of Shank3 residues 949Arg-Arg-Lys951 to three alanines disrupts CaMKII binding in vitro and CaMKII association with Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and LTCCs is important for increased phosphorylation of the nuclear CREB transcription factor and expression of c-Fos induced by depolarization of cultured hippocampal neurons. Thus, this novel CaMKII-Shank3 interaction is essential for the initiation of a specific long-range signal from LTCCs in the plasma membrane to the nucleus that is required for activity-dependent changes in neuronal gene expression during learning and memory.SIGNIFICANCE STATEMENT Precise neuronal expression of genes is essential for normal brain function. Proteins involved in signaling pathways that underlie activity-dependent gene expression, such as CaMKII, Shank3, and L-type calcium channels, are often mutated in multiple neuropsychiatric disorders. Shank3 and CaMKII were previously shown to bind L-type calcium channels, and we show here that Shank3 also binds to CaMKII. Our data show that each of these interactions is required for depolarization-induced phosphorylation of the CREB nuclear transcription factor, which stimulates the expression of c-Fos, a neuronal immediate early gene with key roles in synaptic plasticity, brain development, and behavior.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Núcleo Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Transdução de Sinais/fisiologiaRESUMO
Pancreatic α-cells exhibit oscillations in cytosolic Ca2+ (Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+c oscillations have not been elucidated. As ß-cell Ca2+c oscillations are regulated in part by Ca2+-activated K+ (Kslow) currents, this work investigated the role of Kslow in α-cell Ca2+ handling and GCG secretion. α-Cells displayed Kslow currents that were dependent on Ca2+ influx through L- and P/Q-type voltage-dependent Ca2+ channels (VDCCs) as well as Ca2+ released from endoplasmic reticulum stores. α-Cell Kslow was decreased by small-conductance Ca2+-activated K+ (SK) channel inhibitors apamin and UCL 1684, large-conductance Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+ (IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell Kslow with apamin depolarized membrane potential ( Vm) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca2+ influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca2+ influx. Total α-cell Ca2+c was similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca2+c following prolonged Kslow inhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslow activation provides a hyperpolarizing influence on α-cell Vm that sustains Ca2+ entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+c is elevated during secretagogue stimulation, Kslow activation helps to preserve GCG secretion.