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Pancreatic islet transplantation has therapeutic potential in type 1 diabetes and is also an established therapy in chronic pancreatitis. However, the long-term transplant outcomes are modest. Identifying indicators of graft function will aid the preservation of transplanted islets and glycemic control. We analyzed beta cell prohormone peptide levels in a retrospective cohort of total pancreatectomy autologous islet transplant patients (n = 28). Proinsulin-to-C-peptide (PI/C) and proIAPP-to-total IAPP (proIAPP/IAPP) ratios measured at 3 months post-transplant were significantly higher in patients who remained insulin dependent at 1 year follow-up. In an immuno-deficient mouse model of human islet transplantation, recipient mice that later became hyperglycemic displayed significantly higher PI/C ratios than mice that remained normoglycemic. Histological analysis of islet grafts showed reduced proportional insulin- and proinsulin-positive area, but elevated glucagon-positive area in grafts that experienced greater secretory demand. Increased prohormone convertase 1/3 was detected in glucagon-positive cells, and glucagon-like peptide 1 (GLP-1) area was elevated in grafts from mice that displayed hyperglycemia or elevated plasma PI/C ratios, demonstrating intra-islet incretin production in metabolically challenged human islet grafts. These data indicate that in failing grafts, alpha cell prohormone processing is likely altered, and incomplete beta cell prohormone processing may be an early indicator of insulin dependency.
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Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Animales , Péptido C , Glucagón , Humanos , Insulina , Ratones , Proinsulina , Estudios RetrospectivosRESUMEN
Amyloid formation contributes to the development of progressive metabolic and neurodegenerative diseases, while also serving functional roles in host defense. Emerging evidence suggests that as amyloidogenic peptides populate distinct aggregation states, they interact with different combinations of pattern recognition receptors (PRRs) to direct the phenotype and function of tissue-resident and infiltrating innate immune cells. We review recent evidence of innate immunomodulation by distinct forms of amyloidogenic peptides produced by mammals (humans, non-human primates), bacteria, and fungi, as well as the corresponding cell-surface and intracellular PRRs in these interactions, in human and mouse models. Our emerging understanding of peptide aggregate-innate immune cell interactions, and the factors regulating the balance between amyloid function and pathogenicity, might aid the development of anti-amyloid and immunomodulating therapies.
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Proteínas Amiloidogénicas/metabolismo , Inmunidad Innata , Inmunomodulación , Péptidos/metabolismo , Amiloide/inmunología , Amiloide/metabolismo , Proteínas Amiloidogénicas/inmunología , Amiloidosis/etiología , Amiloidosis/metabolismo , Amiloidosis/patología , Animales , Biomarcadores , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Monocitos/inmunología , Monocitos/metabolismo , Péptidos/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de SeñalRESUMEN
Type 1 diabetes results from defects in immune self-tolerance that lead to inflammatory infiltrate in pancreatic islets, beta cell dysfunction and T cell-mediated killing of beta cells. Although therapies that broadly inhibit immunity show promise to mitigate autoinflammatory damage caused by effector T cells, these are unlikely to permanently reset tolerance or promote regeneration of the already diminished pool of beta cells. An emerging concept is that certain populations of immune cells may have the capacity to both promote tolerance and support the restoration of beta cells by supporting proliferation, differentiation and/or regeneration. Here we will highlight three immune cell types-macrophages, regulatory T cells and innate lymphoid cells-for which there is evidence of dual roles of immune regulation and tissue regeneration. We explore how findings in this area from other fields might be extrapolated to type 1 diabetes and highlight recent discoveries in the context of type 1 diabetes. We also discuss technological advances that are supporting this area of research and contextualise new therapeutic avenues to consider for type 1 diabetes.
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Diabetes Mellitus Tipo 1/inmunología , Islotes Pancreáticos/inmunología , Linfocitos/inmunología , Macrófagos/inmunología , Linfocitos T Reguladores/inmunología , Salud , Humanos , Inmunidad InnataRESUMEN
BACKGROUND: The resident immune population of pancreatic islets has roles in islet development, beta cell physiology, and the pathology of diabetes. These roles have largely been attributed to islet macrophages, comprising 90% of islet immune cells (in the absence of islet autoimmunity), and, in the case of type 1 diabetes, to infiltrating autoreactive T cells. In adipose, tissue-resident and recruited T and B cells have been implicated in the development of insulin resistance during diet-induced obesity and ageing, but whether this is paralleled in the pancreatic islets is not known. Here, we investigated the non-macrophage component of resident islet immune cells in islets isolated from C57BL/6 J male mice during ageing (3 to 24 months of age) and following similar weight gain achieved by 12 weeks of 60% high fat diet. Immune cells were also examined by flow cytometry in cadaveric non-diabetic human islets. RESULTS: Immune cells comprised 2.7 ± 1.3% of total islet cells in non-diabetic mouse islets, and 2.3 ± 1.7% of total islet cells in non-diabetic human islets. In 3-month old mice on standard diet, B and T cells each comprised approximately 2-4% of the total islet immune cell compartment, and approximately 0.1% of total islet cells. A similar amount of T cells were present in non-diabetic human islets. The majority of islet T cells expressed the αß T cell receptor, and were comprised of CD8-positive, CD4-positive, and regulatory T cells, with a minor population of γδ T cells. Interestingly, the number of islet T cells increased linearly (R2 = 0.9902) with age from 0.10 ± 0.05% (3 months) to 0.38 ± 0.11% (24 months) of islet cells. This increase was uncoupled from body weight, and was not phenocopied by a degree similar weight gain induced by high fat diet in mice. CONCLUSIONS: This study reveals that T cells are a part of the normal islet immune population in mouse and human islets, and accumulate in islets during ageing in a body weight-independent manner. Though comprising only a small subset of the immune cells within islets, islet T cells may play a role in the physiology of islet ageing.
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Unfortunately, the human islet checklist was omitted from the electronic supplementary material (ESM) linked to this paper.
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AIMS/HYPOTHESIS: Peptide hormones are first synthesised as larger, inactive precursors that are converted to their active forms by endopeptidase cleavage and post-translational modifications, such as amidation. Recent, large-scale genome-wide studies have suggested that two coding variants of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), are associated with impaired insulin secretion and increased type 2 diabetes risk. We aimed to elucidate the role of PAM in modulating beta cell peptide amidation, beta cell function and the development of diabetes. METHODS: PAM transcript and protein levels were analysed in mouse islets following induction of endoplasmic reticulum (ER) or cytokine stress, and PAM expression patterns were examined in human islets. To study whether haploinsufficiency of PAM accelerates the development of diabetes, Pam+/- and Pam+/+ mice were fed a low-fat diet (LFD) or high-fat diet (HFD) and glucose homeostasis was assessed. Since aggregates of the PAM substrate human islet amyloid polypeptide (hIAPP) lead to islet inflammation and beta cell failure, we also investigated whether PAM haploinsufficiency accelerated hIAPP-induced diabetes and islet amyloid formation in Pam+/- and Pam+/+ mice with beta cell expression of hIAPP. RESULTS: Immunostaining revealed high expression of PAM in alpha, beta and delta cells in human pancreatic islets. Pam mRNA and PAM protein expression were reduced in mouse islets following administration of an HFD, and in isolated islets following induction of ER stress with thapsigargin, or cytokine stress with IL-1ß, IFN-γ and TFN-α. Despite Pam+/- only having 50% PAM expression and enzyme activity as compared with Pam+/+ mice, glucose tolerance and body mass composition were comparable in the two models. After 24 weeks of HFD, both Pam+/- and Pam+/+ mice had insulin resistance and impaired glucose tolerance, but no differences in glucose tolerance, insulin sensitivity or plasma insulin levels were observed in PAM haploinsufficient mice. Islet amyloid formation and beta cell function were also similar in Pam+/- and Pam+/+ mice with beta cell expression of hIAPP. CONCLUSIONS/INTERPRETATION: Haploinsufficiency of PAM in mice does not accelerate the development of diet-induced obesity or hIAPP transgene-induced diabetes.
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Amidina-Liasas/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Oxigenasas de Función Mixta/genética , Amidina-Liasas/fisiología , Animales , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Progresión de la Enfermedad , Epistasis Genética/fisiología , Femenino , Predisposición Genética a la Enfermedad , Haploinsuficiencia , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Polipéptido Amiloide de los Islotes Pancreáticos/fisiología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxigenasas de Función Mixta/fisiología , Ratas , Ratas Endogámicas Lew , Factores de RiesgoRESUMEN
Epidemiologic studies have reported relationships between maternal high folate and/or low B12 status during pregnancy and greater adiposity and insulin resistance in children. The goal of this study was to determine the effects of maternal folic acid supplementation (10 mg/kg diet), with (50 µg/kg diet) and without B12, on adult female offspring adiposity and glucose homeostasis. Female C57BL/6J mice were fed 1 of 3 diets from weaning and throughout breeding, pregnancy, and lactation: control (2 mg/kg diet folic acid, 50 µg/kg diet B12), supplemental folic acid with no B12 (SFA-B12), or supplemental folic acid with adequate B12 (SFA+B12). Female offspring were weaned onto the control diet or a Western diet (45% energy fat, 2 mg/kg diet folic acid, 50 µg/kg diet B12) for 35 wk. After weaning, control diet-fed offspring with SFA-B12 dams had fasting hyperglycemia, glucose intolerance, lower ß cell mass, and greater islet hepatocyte nuclear factor 1 homeobox α and nuclear receptor subfamily 1 group H member 3 mRNA than did offspring from control dams. In Western diet-fed offspring, those with SFA-B12 dams had lower fasting blood glucose and plasma insulin concentrations, and were smaller than control offspring. Our findings suggest that maternal folic acid supplementation with B12 deficiency during pregnancy/lactation programs the metabolic health of adult female offspring but is dependent on offspring diet.-Henderson, A. M., Tai, D. C., Aleliunas, R. E., Aljaadi, A. M., Glier, M. B., Xu, E. E., Miller, J. W., Verchere, C. B., Green, T. J., Devlin, A. M. Maternal folic acid supplementation with vitamin B12 deficiency during pregnancy and lactation affects the metabolic health of adult female offspring but is dependent on offspring diet.
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Dieta , Ácido Fólico/metabolismo , Lactancia/fisiología , Fenómenos Fisiologicos Nutricionales Maternos/fisiología , Deficiencia de Vitamina B 12/metabolismo , Animales , Femenino , Resistencia a la Insulina , Ratones Endogámicos C57BL , Obesidad/metabolismo , Embarazo , DesteteRESUMEN
Biosynthesis of peptide hormones by pancreatic islet endocrine cells is a tightly orchestrated process that is critical for metabolic homeostasis. Like neuroendocrine peptides, insulin and other islet hormones are first synthesized as larger precursor molecules that are processed to their mature secreted products through a series of proteolytic cleavages, mediated by the prohormone convertases Pc1/3 and Pc2, and carboxypeptidase E. Additional posttranslational modifications including C-terminal amidation of the ß-cell peptide islet amyloid polypeptide (IAPP) by peptidyl-glycine α-amidating monooxygenase (Pam) may also occur. Genome-wide association studies (GWAS) have showed genetic linkage of these processing enzymes to obesity, ß-cell dysfunction, and type 2 diabetes (T2D), pointing to their important roles in metabolism and blood glucose regulation. In both type 1 diabetes (T1D) and T2D, and in the face of metabolic or inflammatory stresses, islet prohormone processing may become impaired; indeed elevated proinsulin:insulin (PI:I) ratios are a hallmark of the ß-cell dysfunction in T2D. Recent studies suggest that genetic or acquired defects in proIAPP processing may lead to the production and secretion of incompletely processed forms of proIAPP that could contribute to T2D pathogenesis, and additionally that impaired processing of both PI and proIAPP may be characteristic of ß-cell dysfunction in T1D. In islet α-cells, the prohormone proglucagon is normally processed to bioactive glucagon by Pc2 but may express Pc1/3 under certain conditions leading to production of GLP-1(7-36NH2 ). A better understanding of how ß-cell processing of PI and proIAPP, as well as α-cell processing of proglucagon, are impacted by genetic susceptibility and in the face of diabetogenic stresses, may lead to new therapeutic approaches for improving islet function in diabetes.
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Carboxipeptidasa H/fisiología , Islotes Pancreáticos/metabolismo , Proproteína Convertasa 1/fisiología , Proproteína Convertasa 2/fisiología , Amidina-Liasas/metabolismo , Células Secretoras de Glucagón/metabolismo , Humanos , Insulina/biosíntesis , Células Secretoras de Insulina/metabolismo , Oxigenasas de Función Mixta/metabolismo , Proinsulina/metabolismoRESUMEN
AIMS/HYPOTHESIS: A contributor to beta cell failure in type 2 diabetes and islet transplants is amyloid formation by aggregation of the beta cell peptide, islet amyloid polypeptide (IAPP). Similar to the proinsulin processing pathway that generates insulin, IAPP is derived from a prohormone precursor, proIAPP, which requires cleavage by prohormone convertase (PC) 1/3 and PC2 in rodent pancreatic beta cells. We hypothesised that loss of PC2 would promote beta cell death and dysfunction in a rodent model of human beta cell proIAPP overexpression. METHODS: We generated an islet transplant model wherein immune-deficient mouse models of diabetes received islets expressing amyloidogenic human proIAPP and lacking PC2, leading to restoration of normoglycaemia accompanied by increased secretion of human proIAPP. Blood glucose levels were analysed for up to 16 weeks in transplant recipients and grafts were assessed for islet amyloid and beta cell number and death. RESULTS: Hyperglycaemia (blood glucose >16.9 mmol/l) returned in 94% of recipients of islets expressing human proIAPP and lacking PC2, whereas recipients of islets that express human proIAPP and normal PC2 levels remained normoglycaemic for at least 16 weeks. Islet graft failure was accompanied by a â¼20% reduction in insulin-positive cells, yet the degree of amyloid deposition and beta cell apoptosis was similar to those of controls expressing human proIAPP with functional PC2 levels. CONCLUSIONS/INTERPRETATION: PC2 deficiency in transplanted mouse islets expressing human proIAPP promotes beta cell loss and graft failure. Our data suggest that impaired NH2-terminal processing and increased secretion of human proIAPP promote beta cell failure.
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Amiloide/metabolismo , Células Secretoras de Insulina/metabolismo , Proproteína Convertasa 2/metabolismo , Amiloide/genética , Animales , Glucemia/metabolismo , Western Blotting , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Trasplante de Islotes Pancreáticos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Proinsulina/metabolismo , Proproteína Convertasa 1/genética , Proproteína Convertasa 1/metabolismo , Proproteína Convertasa 2/genéticaRESUMEN
Aggregation of islet amyloid polypeptide (IAPP) contributes to beta cell dysfunction in type 2 diabetes and islet transplantation. Like other amyloidogenic peptides, human IAPP induces macrophage IL-1ß secretion by stimulating both the synthesis and processing of proIL-1ß, a pro-inflammatory cytokine that (when chronically elevated) impairs beta cell insulin secretion. We sought to determine the specific mechanism of IAPP-induced proIL-1ß synthesis. Soluble IAPP species produced early during IAPP aggregation provided a Toll-like-receptor-2- (TLR2-) dependent stimulus for NF-κB activation in HEK 293 cells and bone marrow-derived macrophages (BMDMs). Non-amyloidogenic rodent IAPP and thioflavin-T-positive fibrillar amyloid produced by human IAPP aggregation failed to activate TLR2. Blockade of TLR6 but not TLR1 prevented hIAPP-induced TLR2 activation, consistent with stimulation of a TLR2/6 heterodimer. TLR2 and its downstream adaptor protein MyD88 were required for IAPP-induced cytokine production by BMDMs, a process that is partially dependent on autoinduction by IL-1. BMDMs treated with soluble but not fibrillar IAPP provided a TLR2-dependent priming stimulus for ATP-induced IL-1ß secretion, whereas late IAPP aggregates induced NLRP3-dependent IL-1ß secretion by LPS-primed macrophages. Moreover, inhibition of TLR2 and depletion of islet macrophages prevented up-regulation of Il1b and Tnf expression in human IAPP-expressing transgenic mouse islets. These data suggest participation by both soluble and fibrillar aggregates in IAPP-induced islet inflammation. IAPP-induced activation of TLR2 and secretion of IL-1 may be important therapeutic targets to prevent amyloid-associated beta cell dysfunction.
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Inmunidad Innata , Células Secretoras de Insulina/inmunología , Polipéptido Amiloide de los Islotes Pancreáticos/inmunología , Agregación Patológica de Proteínas/inmunología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Células HEK293 , Humanos , Células Secretoras de Insulina/patología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Type 2 diabetes (T2D) is characterized by a gradual decline in pancreatic beta cell function that determines the progressive course of the disease. While beta-cell failure is an important contributor to hyperglycaemia, chronic hyperglycaemia itself is also detrimental for beta-cell function, probably by inducing prolonged secretory stress on the beta cell as well as through direct glucotoxic mechanisms that have not been fully defined. For years, research has been carried out in search of therapies targeting hyperglycaemia that preserve long-term beta-cell function in T2D, a quest that is still ongoing. Current strategies aim to improve glycaemic control, either by promoting endogenous insulin secretion, such as sulfonylureas, or by mechanisms that may impact the beta cell indirectly, for example, providing beta-cell rest through insulin treatment. Although overall long-term success is limited with currently available interventions, in this review we argue that strategies that induce beta-cell rest have considerable potential to preserve long-term beta-cell function. This is based on laboratory-based studies involving human islets as well as clinical studies employing intensive insulin therapy, thiazolidinediones, bariatric surgery, short-acting glucagon-like peptide (GLP)-1 receptor agonists and a promising new class of diabetes drugs, sodium-glucose-linked transporter (SGLT)-2 inhibitors. Nevertheless, a lack of long-term clinical studies that focus on beta-cell function for the newer glucose-lowering agents, as well as commonly used combination therapies, preclude a straightforward conclusion; this gap in our knowledge should be a focus of future studies.
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Complicaciones de la Diabetes/prevención & control , Diabetes Mellitus Tipo 2/terapia , Medicina Basada en la Evidencia , Hiperglucemia/prevención & control , Hipoglucemia/prevención & control , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Terapia Combinada/efectos adversos , Terapia Combinada/tendencias , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Dieta para Diabéticos , Quimioterapia Combinada/efectos adversos , Ejercicio Físico , Estilo de Vida Saludable , Humanos , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/uso terapéutico , Insulina/efectos adversos , Insulina/uso terapéutico , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacosRESUMEN
Obesity is associated with insulin resistance and inflammation thought to be caused by a visceral adipose tissue (VAT)-localized reduction in immunoregulatory cells and increase in proinflammatory immune cells. We previously found that VAT regulatory T cells (Tregs) normally express high levels of IL-10 and that expression of this cytokine in VAT Tregs is specifically reduced in mice fed a high-fat diet. In this study, we further investigated the phenotype of VAT Tregs and found that the majority of IL-10-expressing Tregs in the VAT of lean mice also expressed the ST2 chain of the IL-33R. In addition to high expression of IL-10, ST2(+) Tregs in lean VAT expressed higher proportions of Th2-associated proteins, including GATA3 and CCR4, and Neuropillin-1 compared with ST2(-) Tregs. The proportion of ST2(+) Tregs in VAT was severely diminished in obese mice that had been fed a high-fat/sucrose diet, and this effect could be completely reversed by treatment with IL-33. IL-33 treatment also reversed VAT inflammation in obese mice and resulted in a reduction of hyperinsulinemia and insulin resistance. These data suggest that IL-33 contributes to the maintenance of the normal pool of ST2(+) Tregs in the VAT, and that therapeutic administration of IL-33 results in multiple anti-obesity effects, including the reversal of VAT inflammation and alleviation of insulin resistance.
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Resistencia a la Insulina , Interleucinas/inmunología , Grasa Intraabdominal/inmunología , Obesidad/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Dieta Alta en Grasa/efectos adversos , Citometría de Flujo , Inflamación/inmunología , Resistencia a la Insulina/inmunología , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Autoimmune destruction of insulin-producing ß cells in type 1 diabetes and islet transplantation involves a variety of immune pathways but is primarily mediated by self-reactive T cells. Chemokines can modulate local immune responses in inflammation and tumors by recruiting immune cells. We have reported that expression of the chemokine CCL22 in pancreatic ß cells in the NOD mouse prevents autoimmune attack by recruiting T regulatory cells (Tregs), protecting mice from diabetes. In this study we show that invariant NKT cells are also recruited to CCL22-expressing islet transplants and are required for CCL22-mediated protection from autoimmunity. Moreover, CCL22 induces an influx of plasmacytoid dendritic cells, which correlates with higher levels of IDO in CCL22-expressing islet grafts. In addition to its chemotactic properties, we found that CCL22 activates Tregs and promotes their ability to induce expression of IDO by dendritic cells. Islet CCL22 expression thus produces a tolerogenic milieu through the interplay of Tregs, invariant NKT cells, and plasmacytoid dendritic cells, which results in suppression of effector T cell responses and protection of ß cells. The immunomodulatory properties of CCL22 could be harnessed for prevention of graft rejection and type 1 diabetes as well as other autoimmune disorders.
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Quimiocina CCL22/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Animales , Quimiocina CCL22/genética , Quimiotaxis/genética , Quimiotaxis/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Inmunomodulación/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Islotes Pancreáticos/metabolismo , Trasplante de Islotes Pancreáticos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Transducción Genética , Trasplantes/inmunología , Trasplantes/metabolismoRESUMEN
AIMS/HYPOTHESIS: Islet amyloid, a pathological feature of type 2 diabetes, forms from the aggregation of islet amyloid polypeptide (IAPP), a beta cell peptide that is produced and co-secreted with insulin. Cholesterol regulates amyloid-ß processing, deposition and clearance, promoting amyloidogenesis in the brain. ATP-binding cassette transporter 1 (ABCA1) is a cholesterol efflux transporter that when absent increases and when overexpressed reduces brain amyloid-ß deposition in mouse models of Alzheimer's disease. We examined whether alterations in ABCA1 expression and islet cholesterol content could also modulate islet amyloidogenesis. METHODS: Thioflavin S staining for amyloid was performed in islets isolated from mice with beta cell expression of human IAPP (hIAPP (Tg/o)) and cultured for 8 days following cholesterol loading, microRNA-33 overexpression (to reduce ABCA1 expression) or palmitate treatment in the presence or absence of ABCA1 overexpression or mevastatin treatment (to reduce cholesterol synthesis). hIAPP (Tg/o) mice were crossed with beta cell-specific Abca1-knockout mice (hIAPP (Tg/o) Abca1 (ßKO)) and glucose tolerance and amyloid formation were assessed. RESULTS: Cholesterol loading and microRNA-33-induced reduction in islet ABCA1 expression increased Thioflavin S-positive amyloid in hIAPP (Tg/o) islets. Palmitate treatment also increased amyloid formation and this was reduced by both ABCA1 overexpression and mevastatin treatment. hIAPP (Tg/o) Abca1 (ßKO) mice had increased islet cholesterol, accompanied by fasting hyperglycaemia, glucose intolerance, impaired in vivo insulin secretion and an increased islet proinsulin:insulin ratio. Amyloid area was increased in cultured hIAPP (Tg/o) Abca1 (ßKO) islets compared with hIAPP (Tg/o) controls. CONCLUSIONS/INTERPRETATION: These data suggest that elevations in islet cholesterol may lead to increases in IAPP aggregation and islet amyloid formation, further worsening beta cell function and glucose homeostasis.
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Transportador 1 de Casete de Unión a ATP/deficiencia , Amiloidosis/metabolismo , Amiloidosis/patología , Colesterol/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Amiloide/metabolismo , Animales , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Lovastatina/análogos & derivados , Lovastatina/farmacología , Masculino , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Palmitatos/farmacología , RatasRESUMEN
AIMS/HYPOTHESIS: Aggregation of islet amyloid polypeptide (IAPP) to form amyloid contributes to beta cell dysfunction in type 2 diabetes. Human but not non-amyloidogenic rodent IAPP induces islet macrophage proIL-1ß synthesis. We evaluated the effect of IL-1 receptor antagonist (IL-1Ra) on islet inflammation and dysfunction in a mouse model of type 2 diabetes with amyloid formation. METHODS: Lean and obese male mice (A/a or A(vy)/A at the agouti locus, respectively) with or without beta cell human IAPP expression (hIAPP(Tg/0)) were treated with PBS or IL-1Ra (50 mg kg(-1) day(-1)) from 16 weeks of age. Intraperitoneal glucose and insulin tolerance tests were performed after 8 weeks. Pancreases were harvested for histology and gene expression analysis. RESULTS: Aggregation of human IAPP was associated with marked upregulation of proinflammatory gene expression in islets of obese hIAPP(Tg/0) mice, together with amyloid deposition and fasting hyperglycaemia. IL-1Ra improved glucose tolerance and reduced plasma proinsulin:insulin in both lean and obese hIAPP(Tg/0) mice with no effect on insulin sensitivity. The severity and prevalence of islet amyloid was reduced by IL-1Ra in lean hIAPP (Tg/0) mice, suggesting a feed-forward mechanism by which islet inflammation promotes islet amyloid at the early stages of disease. IL-1Ra limited Il1a, Il1b, Tnf and Ccl2 expression in islets from obese hIAPP(Tg/0) mice, suggesting an altered islet inflammatory milieu. CONCLUSIONS/INTERPRETATION: These data provide the first in vivo evidenceusing a transgenic mouse model with amyloid deposits resembling those found in human isletsthat IAPP-induced beta cell dysfunction in type 2 diabetes may be mediated by IL-1. Anti-IL-1 therapies may limit islet inflammation and dysfunction associated with amyloid formation.
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Amiloide/metabolismo , Interleucina-1/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Animales , Humanos , Inmunohistoquímica , Inflamación/metabolismo , Interleucina-1/genética , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones TransgénicosRESUMEN
Thioredoxin-interacting protein (TXNIP) is up-regulated by glucose and diabetes and plays a critical role in glucotoxicity, inflammation, and beta-cell apoptosis, whereas we have found that TXNIP deficiency protects against diabetes. Interestingly, human islet amyloid polypeptide (IAPP) is also induced by glucose, aggregates into insoluble amyloid fibrils found in islets of most individuals with type 2 diabetes and promotes inflammation and beta-cell cytotoxicity. However, so far no connection between TXNIP and IAPP signaling had been reported. Using TXNIP gain and loss of function experiments, INS-1 beta-cells and beta-cell-specific Txnip knock-out mice, we now found that TXNIP regulates IAPP expression. Promoter analyses and chromatin-immunoprecipitation assays further demonstrated that TXNIP increases IAPP expression at the transcriptional level, and we discovered that TXNIP-induced FoxA2 (forkhead box A2) transcription factor expression was conferring this effect by promoting FoxA2 enrichment at the proximal FoxA2 site in the IAPP promoter. Moreover, we found that TXNIP down-regulates miR-124a expression, a microRNA known to directly target FoxA2. Indeed, miR-124a overexpression led to decreased FoxA2 expression and IAPP promoter occupancy and to a significant reduction in IAPP mRNA and protein expression and also effectively inhibited TXNIP-induced IAPP expression. Thus, our studies have identified a novel TXNIP/miR-124a/FoxA2/IAPP signaling cascade linking the critical beta-cell signaling pathways of TXNIP and IAPP and thereby provide new mechanistic insight into an important aspect of transcriptional regulation and beta-cell biology.
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Proteínas Portadoras/fisiología , Factor Nuclear 3-beta del Hepatocito/fisiología , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , MicroARNs/fisiología , Tiorredoxinas/fisiología , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Línea Celular , Regulación hacia Abajo , Humanos , Ratones , Ratones Noqueados , Ratas , Tiorredoxinas/genética , Transcripción Genética/fisiologíaRESUMEN
Type 2 diabetes is characterized by both peripheral insulin resistance and reduced insulin secretion by beta-cells. The reasons for beta-cell dysfunction in this disease are incompletely understood but may include the accumulation of toxic lipids within this cell type. We examined the role of Abca1, a cellular cholesterol transporter, in cholesterol homeostasis and insulin secretion in beta-cells. Mice with specific inactivation of Abca1 in beta-cells had markedly impaired glucose tolerance and defective insulin secretion but normal insulin sensitivity. Islets isolated from these mice showed altered cholesterol homeostasis and impaired insulin secretion in vitro. We found that rosiglitazone, an activator of the peroxisome proliferator-activated receptor-gamma, which upregulates Abca1 in beta-cells, requires beta-cell Abca1 for its beneficial effects on glucose tolerance. These experiments establish a new role for Abca1 in beta-cell cholesterol homeostasis and insulin secretion, and suggest that cholesterol accumulation may contribute to beta-cell dysfunction in type 2 diabetes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Glucosa/metabolismo , Homeostasis/fisiología , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Tiazolidinedionas/farmacología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Animales , Colesterol/metabolismo , Homeostasis/efectos de los fármacos , Secreción de Insulina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
OBJECTIVES: Human embryonic stem cell (hESC; SC)-derived pancreatic ß cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCß cells with varying degrees of maturation, these cells still differ from deceased donor human ß cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCß cell functional maturation. METHODS: To monitor SCß cell maturation in vitro, we created an IAPP-2A-mScar and INSULIN-2A-EGFP dual fluorescent reporter (INS2A-EGFP/+;IAPP2A-mScarlet/+) hESC line using CRISPR/Cas9. Pluripotent SC were then differentiated using a 7-stage protocol to islet-like cells. Immunohistochemistry, flow cytometry, qPCR, GSIS and electrophysiology were used to characterise resulting cell populations. RESULTS: We observed robust expression of EGFP and mScarlet fluorescent proteins in insulin- and IAPP-expressing cells without any compromise to their differentiation. We show that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period, and that a subset of insulin-expressing cells remain IAPP-free. Compared to this IAPP-free population, we show these insulin- and IAPP-expressing cells are less polyhormonal, more glucose-sensitive, and exhibit decreased action potential firing in low (2.8 mM) glucose. CONCLUSIONS: The INS2A-EGFP/+;IAPP2A-mScarlet/+ hESC line provides a useful tool for tracking populations of maturing hESC-derived ß cells in vitro. This tool has already been shared with 3 groups and is freely available to all.
RESUMEN
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.