RESUMO
AIMS/HYPOTHESIS: Genome-wide studies have uncovered multiple independent signals at the RREB1 locus associated with altered type 2 diabetes risk and related glycaemic traits. However, little is known about the function of the zinc finger transcription factor Ras-responsive element binding protein 1 (RREB1) in glucose homeostasis or how changes in its expression and/or function influence diabetes risk. METHODS: A zebrafish model lacking rreb1a and rreb1b was used to study the effect of RREB1 loss in vivo. Using transcriptomic and cellular phenotyping of a human beta cell model (EndoC-ßH1) and human induced pluripotent stem cell (hiPSC)-derived beta-like cells, we investigated how loss of RREB1 expression and activity affects pancreatic endocrine cell development and function. Ex vivo measurements of human islet function were performed in donor islets from carriers of RREB1 type 2 diabetes risk alleles. RESULTS: CRISPR/Cas9-mediated loss of rreb1a and rreb1b function in zebrafish supports an in vivo role for the transcription factor in beta cell mass, beta cell insulin expression and glucose levels. Loss of RREB1 also reduced insulin gene expression and cellular insulin content in EndoC-ßH1 cells and impaired insulin secretion under prolonged stimulation. Transcriptomic analysis of RREB1 knockdown and knockout EndoC-ßH1 cells supports RREB1 as a novel regulator of genes involved in insulin secretion. In vitro differentiation of RREB1KO/KO hiPSCs revealed dysregulation of pro-endocrine cell genes, including RFX family members, suggesting that RREB1 also regulates genes involved in endocrine cell development. Human donor islets from carriers of type 2 diabetes risk alleles in RREB1 have altered glucose-stimulated insulin secretion ex vivo, consistent with a role for RREB1 in regulating islet cell function. CONCLUSIONS/INTERPRETATION: Together, our results indicate that RREB1 regulates beta cell function by transcriptionally regulating the expression of genes involved in beta cell development and function.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animais , Humanos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Glucose/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra/genéticaRESUMO
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/metabolismoRESUMO
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 , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Fígado/metabolismo , Camundongos , Proinsulina/sangue , Proinsulina/metabolismo , Locos de Características Quantitativas , TranscriptomaRESUMO
The intersection of genome-wide association analyses with physiological and functional data indicates that variants regulating islet gene transcription influence type 2 diabetes (T2D) predisposition and glucose homeostasis. However, the specific genes through which these regulatory variants act remain poorly characterized. We generated expression quantitative trait locus (eQTL) data in 118 human islet samples using RNA-sequencing and high-density genotyping. We identified fourteen loci at which cis-exon-eQTL signals overlapped active islet chromatin signatures and were coincident with established T2D and/or glycemic trait associations. At some, these data provide an experimental link between GWAS signals and biological candidates, such as DGKB and ADCY5. At others, the cis-signals implicate genes with no prior connection to islet biology, including WARS and ZMIZ1. At the ZMIZ1 locus, we show that perturbation of ZMIZ1 expression in human islets and beta-cells influences exocytosis and insulin secretion, highlighting a novel role for ZMIZ1 in the maintenance of glucose homeostasis. Together, these findings provide a significant advance in the mechanistic insights of T2D and glycemic trait association loci.
Assuntos
Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Insulina/genética , Fatores de Transcrição/genética , Diabetes Mellitus Tipo 2/patologia , Éxons , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Locos de Características Quantitativas/genética , Transdução de Sinais , Fatores de Transcrição/biossínteseRESUMO
We previously cataloged putative autocrine/paracrine signaling loops in pancreatic islets, including factors best known for their roles in axon guidance. Emerging evidence points to nonneuronal roles for these factors, including the Slit-Roundabout receptor (Robo) family, in cell growth, migration, and survival. We found SLIT1 and SLIT3 in both beta cells and alpha cells, whereas SLIT2 was predominantly expressed in beta cells. ROBO1 and ROBO2 receptors were detected in beta and alpha cells. Remarkably, even modest knockdown of Slit production resulted in significant beta-cell death, demonstrating a critical autocrine/paracrine survival role for this pathway. Indeed, recombinant SLIT1, SLIT2, and SLIT3 decreased serum deprivation, cytokine, and thapsigargin-induced cell death under hyperglycemic conditions. SLIT treatment also induced a gradual release of endoplasmic reticulum luminal Ca(2+), suggesting a unique molecular mechanism capable of protecting beta cells from endoplasmic reticulum stress-induced apoptosis. SLIT treatment was also associated with rapid actin remodeling. SLITs potentiated glucose-stimulated insulin secretion and increased the frequency of glucose-induced Ca(2+) oscillations. These observations point to unexpected roles for local Slit secretion in the survival and function of pancreatic beta cells. Because diabetes results from a deficiency in functional beta-cell mass, these studies may contribute to therapeutic approaches for improving beta-cell survival and function.
Assuntos
Sobrevivência Celular/fisiologia , Diabetes Mellitus/fisiopatologia , Glicoproteínas/metabolismo , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais/fisiologia , Actinas/metabolismo , Análise de Variância , Animais , Benzimidazóis , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Immunoblotting , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas RoundaboutRESUMO
AIMS/HYPOTHESIS: There are potential advantages to the low-temperature (-196 °C) banking of isolated islets, including the maintenance of viable islets for future research. We therefore assessed the in vitro and in vivo function of islets cryopreserved for nearly 20 years. METHODS: Human islets were cryopreserved from 1991 to 2001 and thawed between 2012 and 2014. These were characterised by immunostaining, patch-clamp electrophysiology, insulin secretion, transcriptome analysis and transplantation into a streptozotocin (STZ)-induced mouse model of diabetes. RESULTS: The cryopreservation time was 17.6 ± 0.4 years (n = 43). The thawed islets stained positive with dithizone, contained insulin-positive and glucagon-positive cells, and displayed levels of apoptosis and transcriptome profiles similar to those of freshly isolated islets, although their insulin content was lower. The cryopreserved beta cells possessed ion channels and exocytotic responses identical to those of freshly isolated beta cells. Cells from a subset of five donors demonstrated similar perifusion insulin secretion profiles pre- and post-cryopreservation. The transplantation of cryopreserved islets into the diabetic mice improved their glucose tolerance but did not completely normalise their blood glucose levels. Circulating human insulin and insulin-positive grafts were detectable at 10 weeks post-transplantation. CONCLUSIONS/INTERPRETATION: We have demonstrated the potential for long-term banking of human islets for research, which could enable the use of tissue from a large number of donors with future technologies to gain new insight into diabetes.
Assuntos
Criopreservação , Ilhotas Pancreáticas/fisiologia , Bancos de Tecidos , Adulto , Animais , Diabetes Mellitus Experimental/terapia , Exocitose/fisiologia , Feminino , Proteínas de Homeodomínio/genética , Humanos , Insulina/sangue , Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Canais Iônicos/metabolismo , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Transcriptoma/genéticaRESUMO
PI3Kγ, a G-protein-coupled type 1B phosphoinositol 3-kinase, exhibits a basal glucose-independent activity in ß-cells and can be activated by the glucose-dependent insulinotropic polypeptide (GIP). We therefore investigated the role of the PI3Kγ catalytic subunit (p110γ) in insulin secretion and ß-cell exocytosis stimulated by GIP. We inhibited p110γ with AS604850 (1 µmol/liter) or knocked it down using an shRNA adenovirus or siRNA duplex in mouse and human islets and ß-cells. Inhibition of PI3Kγ blunted the exocytotic and insulinotropic response to GIP receptor activation, whereas responses to the glucagon-like peptide-1 or the glucagon-like peptide-1 receptor agonist exendin-4 were unchanged. Downstream, we find that GIP, much like glucose stimulation, activates the small GTPase protein Rac1 to induce actin remodeling. Inhibition of PI3Kγ blocked these effects of GIP. Although exendin-4 could also stimulate actin remodeling, this was not prevented by p110γ inhibition. Finally, forced actin depolymerization with latrunculin B restored the exocytotic and secretory responses to GIP during PI3Kγ inhibition, demonstrating that the loss of GIP-induced actin depolymerization was indeed limiting insulin exocytosis.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucose/metabolismo , Insulina/metabolismo , Actinas/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Dioxóis/química , Eletrofisiologia , Exocitose , Humanos , Concentração Inibidora 50 , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neuropeptídeos/metabolismo , Estrutura Terciária de Proteína , Tiazolidinedionas/química , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in ß-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that ß-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 ß-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (KATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP(+) ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-ß-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.
Assuntos
Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo , Acrilatos/farmacologia , Difosfato de Adenosina/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Glucose/genética , Humanos , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Masculino , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transportadores de Ácidos Monocarboxílicos , NADP/genética , NADP/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Post-translational modification by the small ubiquitin-like modifier-1 (SUMO1) limits insulin secretion from ß-cells by inhibiting insulin exocytosis and glucagon-like peptide-1 (GLP-1) receptor signalling. The secretion of glucagon from α-cells is regulated in a manner opposite to that of insulin; it is inhibited by elevated glucose and GLP-1, and increased by adrenergic signalling. We therefore sought to determine whether SUMO1 modulates mouse and human α-cell function. Action potentials (APs), ion channel function and exocytosis in single α-cells from mice and humans, identified by glucagon immunostaining, and glucagon secretion from intact islets were measured. The effects of SUMO1 on α-cell function and the respective inhibitory and stimulatory effects of exendin 4 and adrenaline were examined. Upregulation of SUMO1 increased α-cell AP duration, frequency and amplitude, in part as a result of increased Ca(2+) channel activity that led to elevated exocytosis. The ability of SUMO1 to enhance α-cell exocytosis was cAMP-dependent and resulted from an increased L-type Ca(2+) current and a shift away from exocytosis dependent on non-L-type channels, an effect that was mimicked by knockdown of the deSUMOylating enzyme sentrin/SUMO-specific protease-1 (SENP1). Finally, although SUMO1 prevented GLP-1 receptor-mediated inhibition of α-cell Na(+) channels and single-cell exocytosis, it failed to prevent the exendin 4-mediated inhibition of glucagon secretion. Consistent with its cAMP dependence, however, SUMO1 enhanced α-cell exocytosis and glucagon secretion stimulated by adrenaline. Thus, by contrast with its inhibitory role in ß-cell exocytosis, SUMO1 is a positive regulator of α-cell exocytosis and glucagon secretion under conditions of elevated cAMP.
Assuntos
AMP Cíclico/metabolismo , Exocitose , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Proteína SUMO-1/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Cisteína Endopeptidases , Endopeptidases/genética , Endopeptidases/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Células Secretoras de Glucagon/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Glucagon/agonistas , Receptores de Glucagon/antagonistas & inibidores , Proteína SUMO-1/genética , Sódio/metabolismoRESUMO
Posttranslational modification by the small ubiquitin-like modifier (SUMO) peptides, known as SUMOylation, is reversed by the sentrin/SUMO-specific proteases (SENPs). While increased SUMOylation reduces ß-cell exocytosis, insulin secretion, and responsiveness to GLP-1, the impact of SUMOylation on islet cell survival is unknown. Mouse islets, INS-1 832/13 cells, or human islets were transduced with adenoviruses to increase either SENP1 or SUMO1 or were transfected with siRNA duplexes to knockdown SENP1. We examined insulin secretion, intracellular Ca²âº responses, induction of endoplasmic reticulum stress markers and inducible nitric oxide synthase (iNOS) expression, and apoptosis by TUNEL and caspase 3 cleavage. Surprisingly, upregulation of SENP1 reduces insulin secretion and impairs intracellular Ca²âº handling. This secretory dysfunction is due to SENP1-induced cell death. Indeed, the detrimental effect of SENP1 on secretory function is diminished when two mediators of ß-cell death, iNOS and NF-κB, are pharmacologically inhibited. Conversely, enhanced SUMOylation protects against IL-1ß-induced cell death. This is associated with reduced iNOS expression, cleavage of caspase 3, and nuclear translocation of NF-κB. Taken together, these findings identify SUMO1 as a novel antiapoptotic protein in islets and demonstrate that reduced viability accounts for impaired islet function following SENP1 up-regulation.
Assuntos
Apoptose , Regulação para Baixo , Endopeptidases/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Sumoilação , Regulação para Cima , Animais , Sinalização do Cálcio , Linhagem Celular , Células Cultivadas , Cisteína Endopeptidases , Endopeptidases/química , Endopeptidases/genética , Humanos , Secreção de Insulina , Células Secretoras de Insulina/citologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Interferência de RNA , Ratos , Proteínas Recombinantes/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Técnicas de Cultura de TecidosRESUMO
Physiological variability in pancreatic cell type gene regulation and the impact on diabetes risk is poorly understood. In this study we mapped gene regulation in pancreatic cell types using single cell multiomic (joint RNA-seq and ATAC-seq) profiling in 28 non-diabetic donors in combination with single cell data from 35 non-diabetic donors in the Human Pancreas Analysis Program. We identified widespread associations with age, sex, BMI, and HbA1c, where gene regulatory responses were highly cell type- and phenotype-specific. In beta cells, donor age associated with hypoxia, apoptosis, unfolded protein response, and external signal-dependent transcriptional regulators, while HbA1c associated with inflammatory responses and gender with chromatin organization. We identified 10.8K loci where genetic variants were QTLs for cis regulatory element (cRE) accessibility, including 20% with lineage- or cell type-specific effects which disrupted distinct transcription factor motifs. Type 2 diabetes and glycemic trait associated variants were enriched in both phenotype- and QTL-associated beta cell cREs, whereas type 1 diabetes showed limited enrichment. Variants at 226 diabetes and glycemic trait loci were QTLs in beta and other cell types, including 40 that were statistically colocalized, and annotating target genes of colocalized QTLs revealed genes with putatively novel roles in disease. Our findings reveal diverse responses of pancreatic cell types to phenotype and genotype in physiology, and identify pathways, networks, and genes through which physiology impacts diabetes risk.
RESUMO
Population level variation and molecular mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized despite ramifications for personalized nutrition. Here, we define prototypical insulin secretion dynamics in response to the three macronutrients in islets from 140 cadaveric donors, including those diagnosed with type 2 diabetes. While islets from the majority of donors exhibited the expected relative response magnitudes, with glucose being highest, amino acid moderate, and fatty acid small, 9% of islets stimulated with amino acid and 8% of islets stimulated with fatty acids had larger responses compared with high glucose. We leveraged this insulin response heterogeneity and used transcriptomics and proteomics to identify molecular correlates of specific nutrient responsiveness, as well as those proteins and mRNAs altered in type 2 diabetes. We also examine nutrient-responsiveness in stem cell-derived islet clusters and observe that they have dysregulated fuel sensitivity, which is a hallmark of functionally immature cells. Our study now represents the first comparison of dynamic responses to nutrients and multi-omics analysis in human insulin secreting cells. Responses of different people's islets to carbohydrate, protein, and fat lay the groundwork for personalized nutrition. ONE-SENTENCE SUMMARY: Deep phenotyping and multi-omics reveal individualized nutrient-specific insulin secretion propensity.
RESUMO
Population-level variation and mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized. We defined prototypical insulin secretion responses to three macronutrients in islets from 140 cadaveric donors, including those with type 2 diabetes. The majority of donors' islets exhibited the highest insulin response to glucose, moderate response to amino acid, and minimal response to fatty acid. However, 9% of donors' islets had amino acid responses, and 8% had fatty acid responses that were larger than their glucose-stimulated insulin responses. We leveraged this heterogeneity and used multi-omics to identify molecular correlates of nutrient responsiveness, as well as proteins and mRNAs altered in type 2 diabetes. We also examined nutrient-stimulated insulin release from stem cell-derived islets and observed responsiveness to fat but not carbohydrate or protein-potentially a hallmark of immaturity. Understanding the diversity of insulin responses to carbohydrate, protein, and fat lays the groundwork for personalized nutrition.
Assuntos
Diabetes Mellitus Tipo 2 , Secreção de Insulina , Insulina , Ilhotas Pancreáticas , Proteômica , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Masculino , Feminino , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pessoa de Meia-Idade , Nutrientes/metabolismo , Adulto , Glucose/metabolismo , Idoso , Ácidos Graxos/metabolismoRESUMO
Comprehensive molecular and cellular phenotyping of human islets can enable deep mechanistic insights for diabetes research. We established the Human Islet Data Analysis and Sharing (HI-DAS) consortium to advance goals in accessibility, usability, and integration of data from human islets isolated from donors with and without diabetes at the Alberta Diabetes Institute (ADI) IsletCore. Here we introduce HumanIslets.com, an open resource for the research community. This platform, which presently includes data on 547 human islet donors, allows users to access linked datasets describing molecular profiles, islet function and donor phenotypes, and to perform various statistical and functional analyses at the donor, islet and single-cell levels. As an example of the analytic capacity of this resource we show a dissociation between cell culture effects on transcript and protein expression, and an approach to correct for exocrine contamination found in hand-picked islets. Finally, we provide an example workflow and visualization that highlights links between type 2 diabetes status, SERCA3b Ca2+-ATPase levels at the transcript and protein level, insulin secretion and islet cell phenotypes. HumanIslets.com provides a growing and adaptable set of resources and tools to support the metabolism and diabetes research community.
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Triton X-100 (TX-100) is a nonionic detergent frequently used at millimolar concentrations to disrupt cell membranes and solubilize proteins. At low micromolar concentrations, TX-100 has been reported to inhibit the function of potassium channels. Here, we have used electrophysiological and functional techniques to examine the effects of TX-100 on another class of ion channels, L-type voltage-operated calcium channels (VOCCs). TX-100 (30 nmol·L(-1) to 3 µmol·L(-1)) caused reversible concentration-dependent inhibition of recombinant L-type VOCC (CaV 1.2) currents and of native L-type VOCC currents recorded from rat vascular smooth muscle cells and cardiac myocytes, and murine and human pancreatic ß-cells. In functional studies, TX-100 (165 nmol·L(-1) to 3.4 µmol·L(-1)) caused concentration-dependent relaxation of rat isolated mesenteric resistance arteries prestimulated with phenylephrine or KCl. This effect was independent of the endothelium. TX-100 (1.6 µmol·L(-1)) inhibited depolarization-induced exocytosis in both murine and human isolated pancreatic ß-cells. These data indicate that at concentrations within the nanomolar to low micromolar range, TX-100 significantly inhibits L-type VOCC activity in a number of cell types, an effect paralleled by inhibition of cell functions dependent upon activation of these channels. This inhibition occurs at concentrations below those used to solubilize proteins and may compromise the use of solutions containing TX-100 in bioassays.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Endotélio Vascular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Octoxinol/farmacologia , Animais , Linhagem Celular , Endotélio Vascular/metabolismo , Exocitose/efeitos dos fármacos , Células HEK293 , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenilefrina/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Vasodilatação/efeitos dos fármacosRESUMO
The endocrine pancreas is critically important in the regulation of energy metabolism, with defective insulin secretion from pancreatic islet ß-cells a major contributing factor to the development of type 2 diabetes. Small ubiquitin-like modifier (SUMO) proteins have been demonstrated to covalently modify a wide range of target proteins, mediating a broad range of cellular processes. While the effects of SUMOylation on ß-cell gene transcription have been previously reviewed, recent reports indicate roles for SUMO outside of the nucleus. In this review we shall focus on the reported non-nuclear roles of SUMOylation in the regulation of ß-cells, including SUMOylation as a novel signaling pathway in the acute regulation of insulin secretion.
Assuntos
Núcleo Celular/fisiologia , Células Secretoras de Insulina/fisiologia , Proteína SUMO-1/fisiologia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Transdução de SinaisRESUMO
Designated a pandemic in March 2020, the spread of severe acute respiratory syndrome virus 2 (SARS-CoV2), the virus responsible for coronavirus disease 2019 (COVID-19), led to new guidelines and restrictions being implemented for individuals, businesses, and societies in efforts to limit the impacts of COVID-19 on personal health and healthcare systems. Here we report the impacts of the COVID-19 pandemic on pancreas processing and islet isolation/distribution outcomes at the Alberta Diabetes Institute IsletCore, a facility specializing in the processing and distribution of human pancreatic islets for research. While the number of organs processed was significantly reduced, organ quality and the function of cellular outputs were minimally impacted during the pandemic when compared to an equivalent period immediately prior. Despite the maintained quality of isolated islets, feedback from recipient groups was more negative. Our findings suggest this is likely due to disrupted distribution which led to increased transit times to recipient labs, particularly those overseas. Thus, to improve overall outcomes in a climate of limited research islet supply, prioritization of tissue recipients based on likely tissue transit times may be needed.
Assuntos
COVID-19 , Ilhotas Pancreáticas , COVID-19/epidemiologia , Humanos , Pandemias , RNA Viral , SARS-CoV-2RESUMO
Pancreatic islet beta cells are essential for maintaining glucose homeostasis. To understand the impact of aging on beta cells, we performed meta-analysis of single-cell RNA sequencing datasets, transcription factor (TF) regulon analysis, high-resolution confocal microscopy, and measured insulin secretion from nondiabetic donors spanning most of the human life span. This revealed the range of molecular and functional changes that occur during beta cell aging, including the transcriptional deregulation that associates with cellular immaturity and reorganization of beta cell TF networks, increased gene transcription rates, and reduced glucose-stimulated insulin release. These alterations associate with activation of endoplasmic reticulum (ER) stress and autophagy pathways. We propose that a chronic state of ER stress undermines old beta cell structure function to increase the risk of beta cell failure and type 2 diabetes onset as humans age.
RESUMO
OBJECTIVE: Identifying the transcripts which mediate genetic association signals for type 2 diabetes (T2D) is critical to understand disease mechanisms. Studies in pancreatic islets support the transcription factor ZMIZ1 as a transcript underlying a T2D GWAS signal, but how it influences T2D risk is unknown. METHODS: ß-Cell-specific Zmiz1 knockout (Zmiz1ßKO) mice were generated and phenotypically characterised. Glucose homeostasis was assessed in Zmiz1ßKO mice and their control littermates on chow diet (CD) and high fat diet (HFD). Islet morphology and function were examined by immunohistochemistry and in vitro islet function was assessed by dynamic insulin secretion assay. Transcript and protein expression were assessed by RNA sequencing and Western blotting. In islets isolated from genotyped human donors, we assessed glucose-dependent insulin secretion and islet insulin content by static incubation assay. RESULTS: Male and female Zmiz1ßKO mice were glucose intolerant with impaired insulin secretion, compared with control littermates. Transcriptomic profiling of Zmiz1ßKO islets identified over 500 differentially expressed genes including those involved in ß-cell function and maturity, which we confirmed at the protein level. Upon HFD, Zmiz1ßKO mice fail to expand ß-cell mass and become severely diabetic. Human islets from carriers of the ZMIZ1-linked T2D-risk alleles have reduced islet insulin content and glucose-stimulated insulin secretion. CONCLUSIONS: ß-Cell Zmiz1 is required for normal glucose homeostasis. Genetic variation at the ZMIZ1 locus may influence T2D-risk by reducing islet mass expansion upon metabolic stress and the ability to maintain a mature ß-cell state.