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1.
Diabetes ; 73(8): 1255-1265, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38985991

RESUMO

Inducible pluripotent stem cell-derived human ß-like cells (BLCs) hold promise for both therapy and disease modeling, but their generation remains challenging and their functional analyses beyond transcriptomic and morphological assessments remain limited. Here, we validate an approach using multicellular and single-cell electrophysiological tools to evaluate function of BLCs from pioneer protocols that can be easily adapted to more differentiated BLCs. The multi-electrode arrays (MEAs) measuring the extracellular electrical activity revealed that BLCs, like primary ß-cells, are electrically coupled and produce slow potential (SP) signals that are closely linked to insulin secretion. We also used high-resolution single-cell patch clamp measurements to capture the exocytotic properties, and characterize voltage-gated sodium and calcium currents, and found that they were comparable with those in primary ß- and EndoC-ßH1 cells. The KATP channel conductance is greater than in human primary ß-cells, which may account for the limited glucose responsiveness observed with MEA. We used MEAs to study the impact of the type 2 diabetes-protective SLC30A8 allele (p.Lys34Serfs50*) and found that BLCs with this allele have stronger electrical coupling activity. Our data suggest that BLCs can be used to evaluate the functional impact of genetic variants on ß-cell function and coupling.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células Secretoras de Insulina , Transportador 8 de Zinco , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Transportador 8 de Zinco/genética , Transportador 8 de Zinco/metabolismo , Diferenciação Celular , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Técnicas de Patch-Clamp , Fenômenos Eletrofisiológicos
2.
bioRxiv ; 2023 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-37905040

RESUMO

iPSC-derived human ß-like cells (BLC) hold promise for both therapy and disease modelling, but their generation remains challenging and their functional analyses beyond transcriptomic and morphological assessments remain limited. Here, we validate an approach using multicellular and single cell electrophysiological tools to evaluate BLCs functions. The Multi-Electrode Arrays (MEAs) measuring the extracellular electrical activity revealed that BLCs are electrically coupled, produce slow potential (SP) signals like primary ß-cells that are closely linked to insulin secretion. We also used high-resolution single-cell patch-clamp measurements to capture the exocytotic properties, and characterize voltage-gated sodium and calcium currents. These were comparable to those in primary ß and EndoC-ßH1 cells. The KATP channel conductance is greater than in human primary ß cells which may account for the limited glucose responsiveness observed with MEA. We used MEAs to study the impact of the type 2 diabetes protective SLC30A8 allele (p.Lys34Serfs*50) and found that BLCs with this allele have stronger electrical coupling. Our data suggest that with an adapted approach BLCs from pioneer protocol can be used to evaluate the functional impact of genetic variants on ß-cell function and coupling.

3.
EMBO Mol Med ; 15(9): e16858, 2023 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-37490001

RESUMO

Hyperreactive platelets are commonly observed in diabetic patients indicating a potential link between glucose homeostasis and platelet reactivity. This raises the possibility that platelets may play a role in the regulation of metabolism. Pancreatic ß cells are the central regulators of systemic glucose homeostasis. Here, we show that factor(s) derived from ß cells stimulate platelet activity and platelets selectively localize to the vascular endothelium of pancreatic islets. Both depletion of platelets and ablation of major platelet adhesion or activation pathways consistently resulted in impaired glucose tolerance and decreased circulating insulin levels. Furthermore, we found platelet-derived lipid classes to promote insulin secretion and identified 20-Hydroxyeicosatetraenoic acid (20-HETE) as the main factor promoting ß cells function. Finally, we demonstrate that the levels of platelet-derived 20-HETE decline with age and that this parallels with reduced impact of platelets on ß cell function. Our findings identify an unexpected function of platelets in the regulation of insulin secretion and glucose metabolism, which promotes metabolic fitness in young individuals.


Assuntos
Células Secretoras de Insulina , Humanos , Secreção de Insulina , Insulina/metabolismo , Plaquetas , Glucose/metabolismo
4.
medRxiv ; 2023 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-37090505

RESUMO

Patients with type 2 diabetes vary in their response to currently available therapeutic agents (including GLP-1 receptor agonists) leading to suboptimal glycemic control and increased risk of complications. We show that human carriers of hypomorphic T2D-risk alleles in the gene encoding peptidyl-glycine alpha-amidating monooxygenase (PAM), as well as Pam-knockout mice, display increased resistance to GLP-1 in vivo. Pam inactivation in mice leads to reduced gastric GLP-1R expression and faster gastric emptying: this persists during GLP-1R agonist treatment and is rescued when GLP-1R activity is antagonized, indicating resistance to GLP-1's gastric slowing properties. Meta-analysis of human data from studies examining GLP-1R agonist response (including RCTs) reveals a relative loss of 44% and 20% of glucose lowering (measured by glycated hemoglobin) in individuals with hypomorphic PAM alleles p.S539W and p.D536G treated with GLP-1R agonist. Genetic variation in PAM has effects on incretin signaling that alters response to medication used commonly for treatment of T2D.

5.
Diabet Med ; 39(12): e14984, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36264270

RESUMO

BACKGROUND: Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate ß-cell L-type Ca2+ channel activity. However, the role of Tspan7 in pancreatic ß-cell function is not yet fully understood. METHODS: Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. ß-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using mRNA-sequencing and quantitative PCR. RESULTS: Tspan7 is expressed in insulin-containing granules of pancreatic ß-cells and glucagon-producing α-cells. Tspan7 knockout mice (Tspan7y/- mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan7y/- islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan7y/- ß-cells, but ß-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+ . TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM K+ . Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent ß-cells, exocytotic Ca2+ sensitivity was reduced in a human ß-cell line (EndoC-ßH1) following Tspan7 KD. CONCLUSION: Tspan7 is involved in the regulation of Ca2+ -dependent exocytosis in ß-cells. Its function is more significant in human ß-cells than their rodent counterparts.


Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Humanos , Camundongos , Exocitose/fisiologia , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Tetraspaninas/genética , Tetraspaninas/metabolismo
7.
Cell Rep ; 34(5): 108703, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33535042

RESUMO

Using chromatin conformation capture, we show that an enhancer cluster in the STARD10 type 2 diabetes (T2D) locus forms a defined 3-dimensional (3D) chromatin domain. A 4.1-kb region within this locus, carrying 5 T2D-associated variants, physically interacts with CTCF-binding regions and with an enhancer possessing strong transcriptional activity. Analysis of human islet 3D chromatin interaction maps identifies the FCHSD2 gene as an additional target of the enhancer cluster. CRISPR-Cas9-mediated deletion of the variant region, or of the associated enhancer, from human pancreas-derived EndoC-ßH1 cells impairs glucose-stimulated insulin secretion. Expression of both STARD10 and FCHSD2 is reduced in cells harboring CRISPR deletions, and lower expression of STARD10 and FCHSD2 is associated, the latter nominally, with the possession of risk variant alleles in human islets. Finally, CRISPR-Cas9-mediated loss of STARD10 or FCHSD2, but not ARAP1, impairs regulated insulin secretion. Thus, multiple genes at the STARD10 locus influence ß cell function.


Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Humanos
9.
Nat Commun ; 11(1): 467, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980626

RESUMO

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived ß-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.


Assuntos
Corantes Fluorescentes , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Linhagem Celular , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Receptor do Peptídeo Semelhante ao Glucagon 1/deficiência , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Células HEK293 , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Transdução de Sinais , Distribuição Tecidual
10.
Nat Genet ; 51(11): 1596-1606, 2019 11.
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.


Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/prevenção & controle , Glucose/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Transportador 8 de Zinco/metabolismo , Adolescente , Adulto , Idoso , Diabetes Mellitus Tipo 2/patologia , Feminino , Genótipo , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Ilhotas Pancreáticas/patologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Transportador 8 de Zinco/genética
11.
Nat Commun ; 10(1): 139, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30635569

RESUMO

Hypoglycaemia (low plasma glucose) is a serious and potentially fatal complication of insulin-treated diabetes. In healthy individuals, hypoglycaemia triggers glucagon secretion, which restores normal plasma glucose levels by stimulation of hepatic glucose production. This counterregulatory mechanism is impaired in diabetes. Here we show in mice that therapeutic concentrations of insulin inhibit glucagon secretion by an indirect (paracrine) mechanism mediated by stimulation of intra-islet somatostatin release. Insulin's capacity to inhibit glucagon secretion is lost following genetic ablation of insulin receptors in the somatostatin-secreting δ-cells, when insulin-induced somatostatin secretion is suppressed by dapagliflozin (an inhibitor of sodium-glucose co-tranporter-2; SGLT2) or when the action of secreted somatostatin is prevented by somatostatin receptor (SSTR) antagonists. Administration of these compounds in vivo antagonises insulin's hypoglycaemic effect. We extend these data to isolated human islets. We propose that SSTR or SGLT2 antagonists should be considered as adjuncts to insulin in diabetes therapy.


Assuntos
Diabetes Mellitus/patologia , Glucagon/metabolismo , Hipoglicemia/patologia , Insulina/metabolismo , Transportador 2 de Glucose-Sódio/metabolismo , Somatostatina/metabolismo , Animais , Compostos Benzidrílicos/farmacologia , Glicemia/análise , Diabetes Mellitus/tratamento farmacológico , Feminino , Células Secretoras de Glucagon/efeitos dos fármacos , Glucosídeos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Insulina/genética , Receptores de Somatostatina/antagonistas & inibidores , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia
12.
Wellcome Open Res ; 4: 150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31976379

RESUMO

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-ßH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes ( INS, IDE, PAM) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1-KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1-KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8-KO cells demonstrated no reduction in K ATP channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-ßH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.

13.
Biochim Biophys Acta Biomembr ; 1861(3): 670-676, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30579961

RESUMO

Neurotransmitter and hormone exocytosis depends on SNARE protein transmembrane domains and membrane lipids but their interplay is poorly understood. We investigated the interaction of the structure of VAMP2, a vesicular transmembrane SNARE protein, and membrane lipid composition by infrared spectroscopy using either the wild-type transmembrane domain (TMD), VAMP2TM22, or a peptide mutated at the central residues G100/C103 (VAMP2TM22VV) previously identified by us as being critical for exocytosis. Our data show that the structure of VAMP2TM22, in terms of α-helices and ß-sheets is strongly influenced by peptide/lipid ratios, by lipid species including cholesterol and by membrane surface charges. Differences observed in acyl chain alignments further underscore the role of the two central small amino acid residues G100/C103 within the transmembrane domain during lipid rearrangements in membrane fusion.


Assuntos
Lipídeos de Membrana/fisiologia , Proteína 2 Associada à Membrana da Vesícula/química , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Membrana Celular/metabolismo , Exocitose/fisiologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fusão de Membrana/fisiologia , Lipídeos de Membrana/farmacologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/genética , Estrutura Terciária de Proteína , Proteínas SNARE/química , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética
14.
Sci Rep ; 8(1): 16994, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451893

RESUMO

Limited access to human islets has prompted the development of human beta cell models. The human beta cell lines EndoC-ßH1 and EndoC-ßH2 are increasingly used by the research community. However, little is known of their electrophysiological and secretory properties. Here, we monitored parameters that constitute the glucose-triggering pathway of insulin release. Both cell lines respond to glucose (6 and 20 mM) with 2- to 3-fold stimulation of insulin secretion which correlated with an elevation of [Ca2+]i, membrane depolarisation and increased action potential firing. Similar to human primary beta cells, KATP channel activity is low at 1 mM glucose and is further reduced upon increasing glucose concentration; an effect that was mimicked by the KATP channel blocker tolbutamide. The upstroke of the action potentials reflects the activation of Ca2+ channels with some small contribution of TTX-sensitive Na+ channels. The repolarisation involves activation of voltage-gated Kv2.2 channels and large-conductance Ca2+-activated K+ channels. Exocytosis presented a similar kinetics to human primary beta cells. The ultrastructure of these cells shows insulin vesicles composed of an electron-dense core surrounded by a thin clear halo. We conclude that the EndoC-ßH1 and -ßH2 cells share many features of primary human ß-cells and thus represent a useful experimental model.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Exocitose , Glucose/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Células Cultivadas , Fenômenos Eletrofisiológicos , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Edulcorantes/farmacologia
15.
Nat Genet ; 50(8): 1122-1131, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30054598

RESUMO

The molecular mechanisms underpinning susceptibility loci for type 2 diabetes (T2D) remain poorly understood. Coding variants in peptidylglycine α-amidating monooxygenase (PAM) are associated with both T2D risk and insulinogenic index. Here, we demonstrate that the T2D risk alleles impact negatively on overall PAM activity via defects in expression and catalytic function. PAM deficiency results in reduced insulin content and altered dynamics of insulin secretion in a human ß-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in ß-cell function, and establish molecular mechanisms for T2D risk alleles at this locus.


Assuntos
Amidina-Liases/genética , Diabetes Mellitus Tipo 2/genética , Secreção de Insulina/genética , Células Secretoras de Insulina/patologia , Oxigenases de Função Mista/genética , Alelos , Animais , Linhagem Celular , Predisposição Genética para Doença , Células HEK293 , Humanos , Insulina/genética , Camundongos , Polimorfismo de Nucleotídeo Único
16.
Stem Cell Res ; 29: 220-231, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29734117

RESUMO

Recent studies have reported significant advances in the differentiation of human pluripotent stem cells to clinically relevant cell types such as the insulin producing beta-like cells and motor neurons. However, many of the current differentiation protocols lead to heterogeneous cell cultures containing cell types other than the targeted cell fate. Genetically modified human pluripotent stem cells reporting the expression of specific genes are of great value for differentiation protocol optimization and for the purification of relevant cell populations from heterogeneous cell cultures. Here we present the generation of human induced pluripotent stem cell (iPSC) lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterization of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. Cell sorting and gene expression profiling by RNA sequencing revealed that NKX6.1-positive cells from pancreatic differentiations closely resemble human beta cells. Furthermore, functional characterization of the isolated cells demonstrated that glucose-stimulated insulin secretion is mainly confined to the NKX6.1-positive cells. We expect that the NKX6.1-GFP iPSC lines and the results presented here will contribute to the further refinement of differentiation protocols and characterization of hPSC-derived beta cells and motor neurons for disease modelling and cell replacement therapies.


Assuntos
Diferenciação Celular , Genes Reporter , Loci Gênicos , Proteínas de Fluorescência Verde , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina/metabolismo , Neurônios Motores/metabolismo , Linhagem Celular , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Neurônios Motores/citologia
17.
Cell Rep ; 22(4): 1054-1066, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29420171

RESUMO

Spatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2σ subunit (AP2σ) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2σ mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2σ mutations reduced CaSR signaling via Gαq/11 and Gαi/o pathways. The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by Gαq/11. Thus, compartmental bias for CaSR-mediated Gαq/11 endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.


Assuntos
Complexo 2 de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Sinalização do Cálcio/genética , Endossomos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores de Detecção de Cálcio/genética , Humanos , Mutação
18.
Cell Calcium ; 68: 45-61, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29129207

RESUMO

Secretory vesicle exocytosis is a fundamental biological event and the process by which hormones (like insulin) are released into the blood. Considerable progress has been made in understanding this precisely orchestrated sequence of events from secretory vesicle docked at the cell membrane, hemifusion, to the opening of a membrane fusion pore. The exact biophysical and physiological regulation of these events implies a close interaction between membrane proteins and lipids in a confined space and constrained geometry to ensure appropriate delivery of cargo. We consider some of the still open questions such as the nature of the initiation of the fusion pore, the structure and the role of the Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (SNARE) transmembrane domains and their influence on the dynamics and regulation of exocytosis. We discuss how the membrane composition and protein-lipid interactions influence the likelihood of the nascent fusion pore forming. We relate these factors to the hypothesis that fusion pore expansion could be affected in type-2 diabetes via changes in disease-related gene transcription and alterations in the circulating lipid profile. Detailed characterisation of the dynamics of the fusion pore in vitro will contribute to understanding the larger issue of insulin secretory defects in diabetes.


Assuntos
Exocitose , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Fusão de Membrana , Animais , Diabetes Mellitus/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura
19.
J Mol Endocrinol ; 59(3): R121-R140, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28811318

RESUMO

Islet amyloid polypeptide (IAPP) forms cytotoxic oligomers and amyloid fibrils in islets in type 2 diabetes (T2DM). The causal factors for amyloid formation are largely unknown. Mechanisms of molecular folding and assembly of human IAPP (hIAPP) into ß-sheets, oligomers and fibrils have been assessed by detailed biophysical studies of hIAPP and non-fibrillogenic, rodent IAPP (rIAPP); cytotoxicity is associated with the early phases (oligomers/multimers) of fibrillogenesis. Interaction with synthetic membranes promotes ß-sheet assembly possibly via a transient α-helical molecular conformation. Cellular hIAPP cytotoxicity can be activated from intracellular or extracellular sites. In transgenic rodents overexpressing hIAPP, intracellular pro-apoptotic signals can be generated at different points in ß-cell protein synthesis. Increased cellular trafficking of proIAPP, failure of the unfolded protein response (UPR) or excess trafficking of misfolded peptide via the degradation pathways can induce apoptosis; these data indicate that defects in intracellular handling of hIAPP can induce cytotoxicity. However, there is no evidence for IAPP overexpression in T2DM. Extracellular amyloidosis is directly related to the degree of ß-cell apoptosis in islets in T2DM. IAPP fragments, fibrils and multimers interact with membranes causing disruption in vivo and in vitro These findings support a role for extracellular IAPP in ß-sheet conformation in cytotoxicity. Inhibitors of fibrillogenesis are useful tools to determine the aberrant mechanisms that result in hIAPP molecular refolding and islet amyloidosis. However, currently, their role as therapeutic agents remains uncertain.


Assuntos
Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Animais , Apoptose , Membrana Celular/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Espaço Extracelular/metabolismo , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Espaço Intracelular/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Terapia de Alvo Molecular , Agregados Proteicos , Agregação Patológica de Proteínas , Ligação Proteica , Multimerização Proteica , Transdução de Sinais , Relação Estrutura-Atividade
20.
Sci Rep ; 7(1): 2835, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28588281

RESUMO

Exocytosis depends on cytosolic domains of SNARE proteins but the function of the transmembrane domains (TMDs) in membrane fusion remains controversial. The TMD of the SNARE protein synaptobrevin2/VAMP2 contains two highly conserved small amino acids, G100 and C103, in its central portion. Substituting G100 and/or C103 with the ß-branched amino acid valine impairs the structural flexibility of the TMD in terms of α-helix/ß-sheet transitions in model membranes (measured by infrared reflection-absorption or evanescent wave spectroscopy) during increase in protein/lipid ratios, a parameter expected to be altered by recruitment of SNAREs at fusion sites. This structural change is accompanied by reduced membrane fluidity (measured by infrared ellipsometry). The G100V/C103V mutation nearly abolishes depolarization-evoked exocytosis (measured by membrane capacitance) and hormone secretion (measured biochemically). Single-vesicle optical (by TIRF microscopy) and biophysical measurements of ATP release indicate that G100V/C103V retards initial fusion-pore opening, hinders its expansion and leads to premature closure in most instances. We conclude that the TMD of VAMP2 plays a critical role in membrane fusion and that the structural mobility provided by the central small amino acids is crucial for exocytosis by influencing the molecular re-arrangements of the lipid membrane that are necessary for fusion pore opening and expansion.


Assuntos
Sequência Conservada , Exocitose , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Proteína 2 Associada à Membrana da Vesícula/química , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Sequência de Aminoácidos , Animais , Técnicas de Silenciamento de Genes , Hormônios/metabolismo , Modelos Biológicos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Ratos , Proteína 2 Associada à Membrana da Vesícula/genética
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