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1.
Nat Genet ; 51(11): 1596-1606, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31676859

RESUMO

A rare loss-of-function allele p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8), which is enriched in Western Finland, protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, particularly when compared with individuals matched for the genotype of a common T2D-risk allele in SLC30A8, p.Arg325. In genome-edited human induced pluripotent stem cell (iPSC)-derived ß-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human ß cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function similar to isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aimed at maintaining insulin secretion capacity in T2D.

2.
Nat Immunol ; 20(3): 350-361, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30718914

RESUMO

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.


Assuntos
Agamaglobulinemia/imunologia , Linfócitos B/imunologia , Proteínas de Transporte de Cátions/imunologia , Zinco/imunologia , Agamaglobulinemia/genética , Agamaglobulinemia/metabolismo , Animais , Linfócitos B/metabolismo , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Pré-Escolar , Citosol/imunologia , Citosol/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Linhagem , Zinco/metabolismo
3.
Nat Rev Endocrinol ; 14(12): 694-704, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30310153

RESUMO

Findings from the past 10 years have placed the glucagon-secreting pancreatic α-cell centre stage in the development of diabetes mellitus, a disease affecting almost one in every ten adults worldwide. Glucagon secretion is reduced in patients with type 1 diabetes mellitus, increasing the risk of insulin-induced hypoglycaemia, but is enhanced in type 2 diabetes mellitus, exacerbating the effects of diminished insulin release and action on blood levels of glucose. A better understanding of the mechanisms underlying these changes is therefore an important goal. RNA sequencing reveals that, despite their opposing roles in the control of blood levels of glucose, α-cells and ß-cells have remarkably similar patterns of gene expression. This similarity might explain the fairly facile interconversion between these cells and the ability of the α-cell compartment to serve as a source of new ß-cells in models of extreme ß-cell loss that mimic type 1 diabetes mellitus. Emerging data suggest that GABA might facilitate this interconversion, whereas the amino acid glutamine serves as a liver-derived factor to promote α-cell replication and maintenance of α-cell mass. Here, we survey these developments and their therapeutic implications for patients with diabetes mellitus.

4.
Proc Natl Acad Sci U S A ; 115(32): E7642-E7649, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30038024

RESUMO

SLC30A8 encodes a zinc transporter that is primarily expressed in the pancreatic islets of Langerhans. In ß-cells it transports zinc into insulin-containing secretory granules. Loss-of-function (LOF) mutations in SLC30A8 protect against type 2 diabetes in humans. In this study, we generated a knockin mouse model carrying one of the most common human LOF mutations for SLC30A8, R138X. The R138X mice had normal body weight, glucose tolerance, and pancreatic ß-cell mass. Interestingly, in hyperglycemic conditions induced by the insulin receptor antagonist S961, the R138X mice showed a 50% increase in insulin secretion. This effect was not associated with enhanced ß-cell proliferation or mass. Our data suggest that the SLC30A8 R138X LOF mutation may exert beneficial effects on glucose metabolism by increasing the capacity of ß-cells to secrete insulin under hyperglycemic conditions.


Assuntos
Diabetes Mellitus Tipo 2/genética , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Transportador 8 de Zinco/genética , Alelos , Animais , Glicemia , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Humanos , Hiperglicemia/sangue , Hiperglicemia/induzido quimicamente , Hiperglicemia/metabolismo , Mutação com Perda de Função , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/farmacologia , Receptor de Insulina/antagonistas & inibidores , Receptor de Insulina/metabolismo , Transportador 8 de Zinco/metabolismo
5.
J Clin Invest ; 128(8): 3369-3381, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29864031

RESUMO

Neuronatin (Nnat) is an imprinted gene implicated in human obesity and widely expressed in neuroendocrine and metabolic tissues in a hormone- and nutrient-sensitive manner. However, its molecular and cellular functions and precise role in organismal physiology remain only partly defined. Here we demonstrate that mice lacking Nnat globally or specifically in ß cells display impaired glucose-stimulated insulin secretion leading to defective glucose handling under conditions of nutrient excess. In contrast, we report no evidence for any feeding or body weight phenotypes in global Nnat-null mice. At the molecular level neuronatin augments insulin signal peptide cleavage by binding to the signal peptidase complex and facilitates translocation of the nascent preprohormone. Loss of neuronatin expression in ß cells therefore reduces insulin content and blunts glucose-stimulated insulin secretion. Nnat expression, in turn, is glucose-regulated. This mechanism therefore represents a novel site of nutrient-sensitive control of ß cell function and whole-animal glucose homeostasis. These data also suggest a potential wider role for Nnat in the regulation of metabolism through the modulation of peptide processing events.

6.
Nat Commun ; 9(1): 1602, 2018 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-29686402

RESUMO

Glucagon-like peptide-1 receptor (GLP-1R) activation promotes insulin secretion from pancreatic beta cells, causes weight loss, and is an important pharmacological target in type 2 diabetes (T2D). Like other G protein-coupled receptors, the GLP-1R undergoes agonist-mediated endocytosis, but the functional and therapeutic consequences of modulating GLP-1R endocytic trafficking have not been clearly defined. Here, we investigate a series of biased GLP-1R agonists with variable propensities for GLP-1R internalization and recycling. Compared to a panel of FDA-approved GLP-1 mimetics, compounds that retain GLP-1R at the plasma membrane produce greater long-term insulin release, which is dependent on a reduction in ß-arrestin recruitment and faster agonist dissociation rates. Such molecules elicit glycemic benefits in mice without concomitant increases in signs of nausea, a common side effect of GLP-1 therapies. Our study identifies a set of agents with specific GLP-1R trafficking profiles and the potential for greater efficacy and tolerability as T2D treatments.

7.
Metallomics ; 10(2): 229-239, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29431830

RESUMO

Zinc homeostasis is essential for normal cellular function, and defects in this process are associated with a number of diseases including type 2 diabetes (T2D), neurological disorders and cardiovascular disease. Thus, variants in the SLC30A8 gene, encoding the vesicular/granular zinc transporter ZnT8, are associated with altered insulin release and increased T2D risk while the zinc importer ZIP12 is implicated in pulmonary hypertension. In light of these, and findings in other diseases, recent efforts have focused on the development of refined sensors for intracellular free zinc ions that can be targeted to subcellular regions including the cytosol, endoplasmic reticulum (ER), secretory granules, Golgi apparatus, nucleus and the mitochondria. Here, we discuss recent advances in Zn2+ probe engineering and their applications to the measurement of labile subcellular zinc pools in different cell types.

8.
Diabetes ; 67(3): 385-399, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29284659

RESUMO

The glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) is a key target for type 2 diabetes (T2D) treatment. Because endocytic trafficking of agonist-bound receptors is one of the most important routes for regulation of receptor signaling, a better understanding of this process may facilitate the development of new T2D therapeutic strategies. Here, we screened 29 proteins with known functions in G protein-coupled receptor trafficking for their role in GLP-1R potentiation of insulin secretion in pancreatic ß-cells. We identify five (clathrin, dynamin1, AP2, sorting nexins [SNX] SNX27, and SNX1) that increase and four (huntingtin-interacting protein 1 [HIP1], HIP14, GASP-1, and Nedd4) that decrease insulin secretion from murine insulinoma MIN6B1 cells in response to the GLP-1 analog exendin-4. The roles of HIP1 and the endosomal SNX1 and SNX27 were further characterized in mouse and human ß-cell lines and human islets. While HIP1 was required for the coupling of cell surface GLP-1R activation with clathrin-dependent endocytosis, the SNXs were found to control the balance between GLP-1R plasma membrane recycling and lysosomal degradation and, in doing so, determine the overall ß-cell incretin responses. We thus identify key modulators of GLP-1R trafficking and signaling that might provide novel targets to enhance insulin secretion in T2D.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endocitose , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Células Secretoras de Insulina/metabolismo , Sistemas do Segundo Mensageiro , Nexinas de Classificação/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Endocitose/efeitos dos fármacos , Exenatida , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Incretinas/farmacologia , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/ultraestrutura , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Peptídeos/farmacologia , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Nexinas de Classificação/antagonistas & inibidores , Nexinas de Classificação/genética , Técnicas de Cultura de Tecidos , Peçonhas/farmacologia
9.
Diabetes Obes Metab ; 19 Suppl 1: 30-41, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28466490

RESUMO

Ca2+ is the key intracellular regulator of insulin secretion, acting in the ß-cell as the ultimate trigger for exocytosis. In response to high glucose, ATP-sensitive K+ channel closure and plasma membrane depolarization engage a sophisticated machinery to drive pulsatile cytosolic Ca2+ changes. Voltage-gated Ca2+ channels, Ca2+ -activated K+ channels and Na+ /Ca2+ exchange all play important roles. The use of targeted Ca2+ probes has revealed that during each cytosolic Ca2+ pulse, uptake of Ca2+ by mitochondria, endoplasmic reticulum (ER), secretory granules and lysosomes fine-tune cytosolic Ca2+ dynamics and control organellar function. For example, changes in the expression of the Ca2+ -binding protein Sorcin appear to provide a link between ER Ca2+ levels and ER stress, affecting ß-cell function and survival. Across the islet, intercellular communication between highly interconnected "hubs," which act as pacemaker ß-cells, and subservient "followers," ensures efficient insulin secretion. Loss of connectivity is seen after the deletion of genes associated with type 2 diabetes (T2D) and follows metabolic and inflammatory insults that characterize this disease. Hubs, which typically comprise ~1%-10% of total ß-cells, are repurposed for their specialized role by expression of high glucokinase (Gck) but lower Pdx1 and Nkx6.1 levels. Single cell-omics are poised to provide a deeper understanding of the nature of these cells and of the networks through which they communicate. New insights into the control of both the intra- and intercellular Ca2+ dynamics may thus shed light on T2D pathology and provide novel opportunities for therapy.


Assuntos
Sinalização do Cálcio , Comunicação Celular , Ilhotas Pancreáticas/metabolismo , Modelos Biológicos , Animais , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Exocitose , Junções Comunicantes/enzimologia , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/enzimologia , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Via Secretória
10.
J Biol Chem ; 292(21): 8892-8906, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28377501

RESUMO

Heterozygous mutations in the human paired box gene PAX6 lead to impaired glucose tolerance. Although embryonic deletion of the Pax6 gene in mice leads to loss of most pancreatic islet cell types, the functional consequences of Pax6 loss in adults are poorly defined. Here we developed a mouse line in which Pax6 was selectively inactivated in ß cells by crossing animals with floxed Pax6 alleles to mice expressing the inducible Pdx1CreERT transgene. Pax6 deficiency, achieved by tamoxifen injection, caused progressive hyperglycemia. Although ß cell mass was preserved 8 days post-injection, total insulin content and insulin:chromogranin A immunoreactivity were reduced by ∼60%, and glucose-stimulated insulin secretion was eliminated. RNA sequencing and quantitative real-time PCR analyses revealed that, although the expression of key ß cell genes, including Ins2, Slc30a8, MafA, Slc2a2, G6pc2, and Glp1r, was reduced after Pax6 deletion, that of several genes that are usually selectively repressed ("disallowed") in ß cells, including Slc16a1, was increased. Assessed in intact islets, glucose-induced ATP:ADP increases were significantly reduced (p < 0.05) in ßPax6KO versus control ß cells, and the former displayed attenuated increases in cytosolic Ca2+ Unexpectedly, glucose-induced increases in intercellular connectivity were enhanced after Pax6 deletion, consistent with increases in the expression of the glucose sensor glucokinase, but decreases in that of two transcription factors usually expressed in fully differentiated ß-cells, Pdx1 and Nkx6.1, were observed in islet "hub" cells. These results indicate that Pax6 is required for the functional identity of adult ß cells. Furthermore, deficiencies in ß cell glucose sensing are likely to contribute to defective insulin secretion in human carriers of PAX6 mutations.


Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Fator de Transcrição PAX6/biossíntese , Trifosfato de Adenosina/genética , Animais , Humanos , Camundongos , Camundongos Knockout , Fator de Transcrição PAX6/genética
11.
Brain ; 140(4): 928-939, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28334855

RESUMO

A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an ∼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4.4 at D4S2971; θ = 0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus, segregating as expected within the kindred and not found in a homozygous state in 300 Bedouin controls. We showed that SLC30A9 (solute carrier family 30 member 9; also known as ZnT-9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH-SY5Y cells overexpressing SLC30A9 fused to enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum, not co-localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signalling. However, using dual-luciferase reporter assay in SH-SY5Y cells we showed that Wnt signalling was not affected by the mutation. Based on protein modelling, the identified mutation is expected to affect SLC30A9's highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn2+ measurements in HEK293 cells overexpressing wild-type and mutant SLC30A9 showed lower zinc concentration within mutant rather than wild-type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is through defective function of this novel activity of SLC30A9 rather than by a defect in its previously described role in transcriptional activation of Wnt signalling.


Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Ciclo Celular/genética , Neuropatia Hereditária Motora e Sensorial/genética , Homeostase/genética , Deficiência Intelectual/genética , Nefropatias/genética , Proteínas Nucleares/genética , Zinco/metabolismo , Idade de Início , Árabes , Mapeamento Cromossômico , Consanguinidade , Citosol/metabolismo , Citosol/ultraestrutura , Feminino , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Lactente , Masculino , Mutação , Linhagem , Síndrome , Via de Sinalização Wnt/genética
12.
Am J Hum Genet ; 100(2): 238-256, 2017 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-28132686

RESUMO

Genetic variants near ARAP1 (CENTD2) and STARD10 influence type 2 diabetes (T2D) risk. The risk alleles impair glucose-induced insulin secretion and, paradoxically but characteristically, are associated with decreased proinsulin:insulin ratios, indicating improved proinsulin conversion. Neither the identity of the causal variants nor the gene(s) through which risk is conferred have been firmly established. Whereas ARAP1 encodes a GTPase activating protein, STARD10 is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer protein family. By integrating genetic fine-mapping and epigenomic annotation data and performing promoter-reporter and chromatin conformational capture (3C) studies in ß cell lines, we localize the causal variant(s) at this locus to a 5 kb region that overlaps a stretch-enhancer active in islets. This region contains several highly correlated T2D-risk variants, including the rs140130268 indel. Expression QTL analysis of islet transcriptomes from three independent subject groups demonstrated that T2D-risk allele carriers displayed reduced levels of STARD10 mRNA, with no concomitant change in ARAP1 mRNA levels. Correspondingly, ß-cell-selective deletion of StarD10 in mice led to impaired glucose-stimulated Ca2+ dynamics and insulin secretion and recapitulated the pattern of improved proinsulin processing observed at the human GWAS signal. Conversely, overexpression of StarD10 in the adult ß cell improved glucose tolerance in high fat-fed animals. In contrast, manipulation of Arap1 in ß cells had no impact on insulin secretion or proinsulin conversion in mice. This convergence of human and murine data provides compelling evidence that the T2D risk associated with variation at this locus is mediated through reduction in STARD10 expression in the ß cell.


Assuntos
Diabetes Mellitus Tipo 2/genética , Insulina/metabolismo , Fosfoproteínas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Alelos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Diabetes Mellitus Tipo 2/sangue , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica , Variação Genética , Homeostase , Humanos , Insulina/sangue , Células Secretoras de Insulina/metabolismo , Fígado/metabolismo , Camundongos , Proinsulina/sangue , Proinsulina/metabolismo , Locos de Características Quantitativas , Transcriptoma
13.
Nutr Metab (Lond) ; 13: 46, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27390586

RESUMO

BACKGROUND: The human SLC30A8 gene encodes the secretory granule-localised zinc transporter ZnT8 whose expression is chiefly restricted to the endocrine pancreas. Single nucleotide polymorphisms (SNPs) in the human SLC30A8 gene have been associated, through genome-wide studies, with altered type 2 diabetes risk. In addition to a role in the control of insulin release, recent studies involving targeted gene ablation from the pancreatic α cell (Solomou et al., J Biol Chem 290(35):21432-42) have also implicated ZnT8 in the control of glucagon release. Up to now, however, the possibility that increased levels of the transporter in these cells may impact glucagon secretion has not been explored. METHODS: Here, we use a recently-developed reverse tetracyline transactivator promoter-regulated ZnT8 transgene to drive the over-expression of human ZnT8 selectively in the α cell in adult mice. Glucose homeostasis and glucagon secretion were subsequently assessed both in vivo during hypoglycemic clamps and from isolated islets in vitro. RESULTS: Doxyclin-dependent human ZnT8 mRNA expression was apparent in both isolated islets and in fluorescence-activated cell sorting- (FACS) purified α cells. Examined at 12 weeks of age, intraperitoneal glucose (1 g/kg) tolerance was unchanged in transgenic mice versus wild-type littermates (n = 8-10 mice/genotype, p > 0.05) and sensitivity to intraperitoneal insulin (0.75U/kg) was similarly unaltered in transgenic animals. In contrast, under hyperinsulinemic-hypoglycemic clamp, a ~45 % (p < 0.001) reduction in glucose infusion rate was apparent, and glucagon release was significantly (~40 %, p < 0.01) impaired, in transgenic mice. Correspondingly, examined in vitro, glucagon secretion was significantly reduced (~30 %, p < 0.05) from transgenic versus control islets at low, stimulatory glucose concentrations (1 mM, p < 0.05) but not at high glucose (17 mM) glucose (p > 0.05). Over-expression of ZnT8 in glucagonoma-derived αTC1-9 cells increased granule free Zn(2+) concentrations consistent with a role for Zn(2+) in this compartment in the action of ZnT8 on glucagon secretion. CONCLUSIONS: Increased ZnT8 expression, and a likely increase in intragranular free Zn(2+) concentration, is deleterious in pancreatic α cells for stimulated glucagon release. These data provide further evidence that type 2 diabetes-associated polymorphisms in the SLC30A8/ZnT8 gene may act in part via alterations in glucagon release and suggest that ZnT8 activation may restrict glucagon release in some settings.

14.
Arch Biochem Biophys ; 611: 79-85, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27262257

RESUMO

Zn2+ ions are essential for the normal processing and storage of insulin and altered pancreatic insulin content is associated with all forms of diabetes mellitus. Work of the past decade has identified variants in the human SLC30A8 gene, encoding the zinc transporter ZnT8 which is expressed highly selectively on the secretory granule of pancreatic islet ß and α cells, as affecting the risk of Type 2 Diabetes. Here, we review the regulation and roles of Zn2+ ions in islet cells, the mechanisms through which SLC30A8 variants might affect glucose homeostasis and diabetes risk, and the novel technologies including recombinant targeted zinc probes and knockout mice which have been developed to explore these questions.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Zinco/fisiologia , Animais , Citoplasma/metabolismo , Diabetes Mellitus Tipo 2/genética , Estudo de Associação Genômica Ampla , Homeostase , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Metalotioneína/metabolismo , Camundongos , Transportador 8 de Zinco
15.
Am J Physiol Endocrinol Metab ; 311(2): E488-507, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27329800

RESUMO

Single nucleotide polymorphisms (SNPs) close to the VPS13C, C2CD4A and C2CD4B genes on chromosome 15q are associated with impaired fasting glucose and increased risk of type 2 diabetes. eQTL analysis revealed an association between possession of risk (C) alleles at a previously implicated causal SNP, rs7163757, and lowered VPS13C and C2CD4A levels in islets from female (n = 40, P < 0.041) but not from male subjects. Explored using promoter-reporter assays in ß-cells and other cell lines, the risk variant at rs7163757 lowered enhancer activity. Mice deleted for Vps13c selectively in the ß-cell were generated by crossing animals bearing a floxed allele at exon 1 to mice expressing Cre recombinase under Ins1 promoter control (Ins1Cre). Whereas Vps13c(fl/fl):Ins1Cre (ßVps13cKO) mice displayed normal weight gain compared with control littermates, deletion of Vps13c had little effect on glucose tolerance. Pancreatic histology revealed no significant change in ß-cell mass in KO mice vs. controls, and glucose-stimulated insulin secretion from isolated islets was not altered in vitro between control and ßVps13cKO mice. However, a tendency was observed in female null mice for lower insulin levels and ß-cell function (HOMA-B) in vivo. Furthermore, glucose-stimulated increases in intracellular free Ca(2+) were significantly increased in islets from female KO mice, suggesting impaired Ca(2+) sensitivity of the secretory machinery. The present data thus provide evidence for a limited role for changes in VPS13C expression in conferring altered disease risk at this locus, particularly in females, and suggest that C2CD4A may also be involved.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Intolerância à Glucose/genética , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas/genética , Animais , Western Blotting , Cálcio/metabolismo , Diabetes Mellitus Tipo 2/genética , Feminino , Células Secretoras de Glucagon/patologia , Insulina/metabolismo , Resistência à Insulina , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Knockout , Pâncreas/patologia , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Fatores Sexuais
16.
Diabetes ; 65(5): 1268-82, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26861785

RESUMO

Encoding acyl-CoA thioesterase-7 (Acot7) is one of ∼60 genes expressed ubiquitously across tissues but relatively silenced, or disallowed, in pancreatic ß-cells. The capacity of ACOT7 to hydrolyze long-chain acyl-CoA esters suggests potential roles in ß-oxidation, lipid biosynthesis, signal transduction, or insulin exocytosis. We explored the physiological relevance of ß-cell-specific Acot7 silencing by re-expressing ACOT7 in these cells. ACOT7 overexpression in clonal MIN6 and INS1(832/13) ß-cells impaired insulin secretion in response to glucose plus fatty acids. Furthermore, in a panel of transgenic mouse lines, we demonstrate that overexpression of mitochondrial ACOT7 selectively in the adult ß-cell reduces glucose tolerance dose dependently and impairs glucose-stimulated insulin secretion. By contrast, depolarization-induced secretion was unaffected, arguing against a direct action on the exocytotic machinery. Acyl-CoA levels, ATP/ADP increases, membrane depolarization, and Ca(2+) fluxes were all markedly reduced in transgenic mouse islets, whereas glucose-induced oxygen consumption was unchanged. Although glucose-induced increases in ATP/ADP ratio were similarly lowered after ACOT7 overexpression in INS1(832/13) cells, changes in mitochondrial membrane potential were unaffected, consistent with an action of Acot7 to increase cellular ATP consumption. Because Acot7 mRNA levels are increased in human islets in type 2 diabetes, inhibition of the enzyme might provide a novel therapeutic strategy.


Assuntos
Regulação para Baixo , Ácidos Graxos não Esterificados/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Palmitoil-CoA Hidrolase/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular Tumoral , Células Clonais , Feminino , Intolerância à Glucose/enzimologia , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos , Palmitoil-CoA Hidrolase/genética , Ratos , Proteínas Recombinantes/metabolismo , Caracteres Sexuais , Técnicas de Cultura de Tecidos , Regulação para Cima
17.
Mol Endocrinol ; 30(1): 77-91, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26584158

RESUMO

Zinc transporter 8 (ZnT8), encoded by SLC30A8, is chiefly expressed within pancreatic islet cells, where it mediates zinc (Zn(2+)) uptake into secretory granules. Although a common nonsynonymous polymorphism (R325W), which lowers activity, is associated with increased type 2 diabetes (T2D) risk, rare inactivating mutations in SLC30A8 have been reported to protect against T2D. Here, we generate and characterize new mouse models to explore the impact on glucose homeostasis of graded changes in ZnT8 activity in the ß-cell. Firstly, Slc30a8 was deleted highly selectively in these cells using the novel deleter strain, Ins1Cre. The resultant Ins1CreZnT8KO mice displayed significant (P < .05) impairments in glucose tolerance at 10 weeks of age vs littermate controls, and glucose-induced increases in circulating insulin were inhibited in vivo. Although insulin release from Ins1CreZnT8KO islets was normal, Zn(2+) release was severely impaired. Conversely, transgenic ZnT8Tg mice, overexpressing the transporter inducibly in the adult ß-cell using an insulin promoter-dependent Tet-On system, showed significant (P < .01) improvements in glucose tolerance compared with control animals. Glucose-induced insulin secretion from ZnT8Tg islets was severely impaired, whereas Zn(2+) release was significantly enhanced. Our findings demonstrate that glucose homeostasis in the mouse improves as ß-cell ZnT8 activity increases, and remarkably, these changes track Zn(2+) rather than insulin release in vitro. Activation of ZnT8 in ß-cells might therefore provide the basis of a novel approach to treating T2D.


Assuntos
Proteínas de Transporte de Cátions/genética , Intolerância à Glucose/genética , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Proteínas de Transporte de Cátions/metabolismo , Intolerância à Glucose/metabolismo , Homeostase , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Transgênicos , Vesículas Secretórias/metabolismo , Transportador 8 de Zinco
18.
Proc Nutr Soc ; 75(1): 61-72, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26365743

RESUMO

Zinc is an important micronutrient, essential in the diet to avoid a variety of conditions associated with malnutrition such as diarrhoea and alopecia. Lowered circulating levels of zinc are also found in diabetes mellitus, a condition which affects one in twelve of the adult population and whose treatments consume approximately 10 % of healthcare budgets. Zn2+ ions are essential for a huge range of cellular functions and, in the specialised pancreatic ß-cell, for the storage of insulin within the secretory granule. Correspondingly, genetic variants in the SLC30A8 gene, which encodes the diabetes-associated granule-resident Zn2+ transporter ZnT8, are associated with an altered risk of type 2 diabetes. Here, we focus on (i) recent advances in measuring free zinc concentrations dynamically in subcellular compartments, and (ii) studies dissecting the role of intracellular zinc in the control of glucose homeostasis in vitro and in vivo. We discuss the effects on insulin secretion and action of deleting or over-expressing Slc30a8 highly selectively in the pancreatic ß-cell, and the role of zinc in insulin signalling. While modulated by genetic variability, healthy levels of dietary zinc, and hence normal cellular zinc homeostasis, are likely to play an important role in the proper release and action of insulin to maintain glucose homeostasis and lower diabetes risk.

19.
Nature ; 524(7565): 356-60, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26258299

RESUMO

The typical response of the adult mammalian pulmonary circulation to a low oxygen environment is vasoconstriction and structural remodelling of pulmonary arterioles, leading to chronic elevation of pulmonary artery pressure (pulmonary hypertension) and right ventricular hypertrophy. Some mammals, however, exhibit genetic resistance to hypoxia-induced pulmonary hypertension. We used a congenic breeding program and comparative genomics to exploit this variation in the rat and identified the gene Slc39a12 as a major regulator of hypoxia-induced pulmonary vascular remodelling. Slc39a12 encodes the zinc transporter ZIP12. Here we report that ZIP12 expression is increased in many cell types, including endothelial, smooth muscle and interstitial cells, in the remodelled pulmonary arterioles of rats, cows and humans susceptible to hypoxia-induced pulmonary hypertension. We show that ZIP12 expression in pulmonary vascular smooth muscle cells is hypoxia dependent and that targeted inhibition of ZIP12 inhibits the rise in intracellular labile zinc in hypoxia-exposed pulmonary vascular smooth muscle cells and their proliferation in culture. We demonstrate that genetic disruption of ZIP12 expression attenuates the development of pulmonary hypertension in rats housed in a hypoxic atmosphere. This new and unexpected insight into the fundamental role of a zinc transporter in mammalian pulmonary vascular homeostasis suggests a new drug target for the pharmacological management of pulmonary hypertension.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Animais Congênicos , Arteríolas/metabolismo , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Bovinos , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Cromossomos de Mamíferos/genética , Doença Crônica , Feminino , Técnicas de Silenciamento de Genes , Homeostase , Humanos , Hipertensão Pulmonar/genética , Hipóxia/genética , Espaço Intracelular/metabolismo , Masculino , Músculo Liso Vascular/citologia , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos WKY , Zinco/metabolismo
20.
ACS Chem Biol ; 10(9): 2126-34, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26151333

RESUMO

Zn(2+) plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn(2+) homeostasis and the putative signaling role of Zn(2+), various fluorescent sensors have been developed that allow monitoring of Zn(2+) concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn(2+) sensors have been most widely applied, the eCALWY sensors developed by our group and the ZapCY sensors developed by Palmer and co-workers. Both have been successfully used to measure cytosolic free Zn(2+), but distinctly different concentrations have been reported when using these sensors to measure Zn(2+) concentrations in the ER and mitochondria. Here, we report the development of a versatile alternative FRET sensor containing a de novo Cys2His2 binding pocket that was created on the surface of the donor and acceptor fluorescent domains. This eZinCh-2 sensor binds Zn(2+) with a high affinity that is similar to that of eCALWY-4 (Kd = 1 nM at pH 7.1), while displaying a substantially larger change in emission ratio. eZinCh-2 not only provides an attractive alternative for measuring Zn(2+) in the cytosol but was also successfully used for measuring Zn(2+) in the ER, mitochondria, and secretory vesicles. Moreover, organelle-targeted eZinCh-2 can also be used in combination with the previously reported redCALWY sensors to allow multicolor imaging of intracellular Zn(2+) simultaneously in the cytosol and the ER or mitochondria.


Assuntos
Citosol/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Zinco/análise , Sítios de Ligação , Cátions Bivalentes/análise , Cátions Bivalentes/metabolismo , Citosol/química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , Modelos Moleculares , Imagem Óptica/métodos , Zinco/metabolismo
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