RESUMEN
Elevated levels of fasting insulin release and insufficient glucose-stimulated insulin secretion (GSIS) are hallmarks of diabetes. Studies have established cross-talk between integrin signaling and insulin activity, but more details of how integrin-dependent signaling impacts the pathophysiology of diabetes are needed. Here, we dissected integrin-dependent signaling pathways involved in the regulation of insulin secretion in ß-cells and studied their link to the still debated autocrine regulation of insulin secretion by insulin/insulin-like growth factor (IGF) 2-AKT signaling. We observed for the first time a cooperation between different AKT isoforms and focal adhesion kinase (FAK)-dependent adhesion signaling, which either controlled GSIS or prevented insulin secretion under fasting conditions. Indeed, ß-cells form integrin-containing adhesions, which provide anchorage to the pancreatic extracellular matrix and are the origin of intracellular signaling via FAK and paxillin. Under low-glucose conditions, ß-cells adopt a starved adhesion phenotype consisting of actin stress fibers and large peripheral focal adhesion. In contrast, glucose stimulation induces cell spreading, actin remodeling, and point-like adhesions that contain phospho-FAK and phosphopaxillin, located in small protrusions. Rat primary ß-cells and mouse insulinomas showed an adhesion remodeling during GSIS resulting from autocrine insulin/IGF2 and AKT1 signaling. However, under starving conditions, the maintenance of stress fibers and the large adhesion phenotype required autocrine IGF2-IGF1 receptor signaling mediated by AKT2 and elevated FAK-kinase activity and ROCK-RhoA levels but low levels of paxillin phosphorylation. This starved adhesion phenotype prevented excessive insulin granule release to maintain low insulin secretion during fasting. Thus, deregulation of the IGF2 and adhesion-mediated signaling may explain dysfunctions observed in diabetes.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/metabolismo , Integrinas/metabolismo , Transducción de Señal , Actinas/metabolismo , Animales , Comunicación Autocrina , Adhesión Celular/efectos de los fármacos , Quinasa 1 de Adhesión Focal/metabolismo , Glucosa/farmacología , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Tirfostinos/farmacología , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
KEY POINTS: Fractalkine receptor antagonist inhibited neutrophil recruitment to masseter muscles and exacerbated fatigability during masticatory activity. Fractalkine-mediated neutrophil recruitment is required for both upregulation of myokines (CXCL1, interleukin-6) and enhanced GLUT4 translocation in response to masticatory activity. Fractalkine and intercellular adhesion molecule-1 expression in endothelial cells increased in response to masticatory activity. In vitro experiments demonstrated that contracting myotubes lack the ability to upregulate fractalkine but revealed that endothelial fractalkine upregulation is induced using a conditioned medium of contracting myotubes. ABSTRACT: Physical exercise stimulates neutrophil recruitment within working skeletal muscle, although its underlying mechanisms remain ill-defined. By employing a masticatory behaviour (gnawing) model, we demonstrate the importance of intramuscular paracrine and autocrine systems that are triggered by muscle contractile activity and reliant upon fractalkine/CX3CL1-mediated signals. These signals were revealed to be required for achieving proper GLUT4 translocation and glucose uptake to meet the glucose demands for fatigue alleviation. Specifically, fractalkine expression and neutrophil recruitment both increased in the masseter muscle tissues upon masticatory activity. Importantly, a fractalkine antagonist inhibited neutrophil accumulation and exacerbated fatigability during masticatory activity. We found that fractalkine-dependent neutrophil recruitment is required for both upregulation of myokines (i.e. CXCL1 and interleukin-6) and enhanced GLUT4 translocation in response to gnawing activity. Immunofluorescence analysis of masseter muscles demonstrated that fractalkine and intercellular adhesion molecule-1 expression are both upregulated in endothelial cells but not in myofibres. The in vitro exercise model further revealed that contractile activity failed to stimulate fractalkine upregulation in myotubes, implying that fractalkine is not a myokine (myofibre-derived factor). Nevertheless, endothelial fractalkine expression was markedly stimulated by a conditioned medium from the contracting myotubes. Moreover, intercellular adhesion molecule-1, a key adhesion molecule for neutrophils, was upregulated in endothelial cells by fractalkine. Taken together, our findings strongly suggest that endothelial fractalkine serves as a key factor for organizing a physiologically beneficial intramuscular microenvironment by recruiting neutrophils in response to relatively mild exercise (i.e. masticatory muscle activity).
Asunto(s)
Células Endoteliales/citología , Transportador de Glucosa de Tipo 4/metabolismo , Músculo Esquelético/fisiología , Neutrófilos/citología , Condicionamiento Físico Animal , Animales , Células Cultivadas , Ratones , Contracción Muscular , Fibras Musculares Esqueléticas/fisiologíaRESUMEN
Ischaemia impairs organ quality during preservation in a time-dependent manner, due to a lack of oxygen supply. Its impact on pancreas and islet transplantation outcome has been demonstrated by a correlation between cold ischaemia time and poor islet isolation efficiency. Our goal in the present study was to improve pancreas and islet quality using a novel natural oxygen carrier (M101, 2 g/L), which has been proven safe and efficient in other clinical applications, including kidney transplantation, and for several pre-clinical transplantation models. When M101 was added to the preservation solution of rat pancreas during ischaemia, a decrease in oxidative stress (ROS), necrosis (HMGB1), and cellular stress pathway (p38 MAPK)activity was observed. Freshly isolated islets had improved function when M101 was injected in the pancreas. Additionally, human pancreases exposed to M101 for 3 hours had an increase in complex 1 mitochondrial activity, as well as activation of AKT activity, a cell survival marker. Insulin secretion was also up-regulated for isolated islets. In summary, these results demonstrate a positive effect of the oxygen carrier M101 on rat and human pancreas during preservation, with an overall improvement in post-isolation islet quality.
Asunto(s)
Isquemia Fría , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Preservación de Órganos/métodos , Estrés Oxidativo/efectos de los fármacos , Páncreas , Animales , Supervivencia Celular/efectos de los fármacos , Complejo I de Transporte de Electrón/metabolismo , Proteína HMGB1/metabolismo , Humanos , Secreción de Insulina/efectos de los fármacos , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Masculino , Necrosis/prevención & control , Soluciones Preservantes de Órganos , Oxígeno/metabolismo , Páncreas/citología , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND & AIMS: Glucose-dependent insulinotropic peptide (GIP) induces production of interleukin 6 (IL6) by adipocytes. IL6 increases production of glucagon-like peptide (GLP)-1 by L cells and α cells, leading to secretion of insulin from ß cells. We investigated whether GIP regulates GLP1 and glycemia via IL6. METHODS: We obtained samples of human pancreatic islets and isolated islets from mice; human α cells and ß cells were sorted by flow cytometry and incubated with GIP. Islets were analyzed by quantitative polymerase chain reaction and immunohistochemistry. BKS.Cg-Dock7m+/+ Leprdb/J db/db mice (diabetic mice) and db/+ mice, as well as C57BL/6J IL6-knockout mice (IL6-KO) and C57BL/6J mice with the full-length Il6 gene (controls), were fed a chow or a high-fat diet; some mice were given injections of recombinant GIP, IL6, GLP, a neutralizing antibody against IL6 (anti-IL6), lipopolysaccharide, and/or IL1B. Mice were given a glucose challenge and blood samples were collected and analyzed. RESULTS: Incubation of mouse and human pancreatic α cells with GIP induced their production of IL6, leading to production of GLP1 and insulin secretion from pancreatic islets. This did not occur in islets from IL6-KO mice or in islets incubated with anti-IL6. Incubation of islets with IL1B resulted in IL6 production but directly reduced GLP1 production. Incubation of mouse islets with the sodium glucose transporter 2 inhibitor dapagliflozin induced production of GLP1 and IL6. Injection of control mice with GIP increased plasma levels of GLP1, insulin, and glucose tolerance; these effects were amplified in mice given lipopolysaccharide but reduced in IL6-KO mice or in mice given anti-IL6. Islets from diabetic mice had increased levels of IL1B and IL6, compared with db/+ mice, but injection of GIP did not lead to production of GLP1 or reduce glycemia. CONCLUSIONS: In studies of pancreatic islets from human beings and mice, we found that GIP induces production of IL6 by α cells, leading to islet production of GLP1 and insulin. This process is regulated by inflammation, via IL1B, and by sodium glucose transporter 2. In diabetic mice, increased islet levels of IL6 and IL1B might increase or reduce the production of GLP1 and affect glycemia.
Asunto(s)
Polipéptido Inhibidor Gástrico/metabolismo , Péptido 1 Similar al Glucagón/biosíntesis , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Interleucina-6/metabolismo , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Ratones , Ratones Endogámicos C57BLRESUMEN
OBJECTIVE: The contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-LeuTAA) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-LeuTAA, on mitochondrial metabolism and pancreatic islet functions. METHODS: We used antisense oligonucleotides to reduce mt-tRF-LeuTAA levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-LeuTAA inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-LeuTAA silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays. RESULTS: Our study unveils a modulation of mt-tRF-LeuTAA levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-LeuTAA interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-LeuTAA in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-LeuTAA impairs insulin secretion of rat and human pancreatic ß-cells. CONCLUSIONS: Our findings indicate that mt-tRF-LeuTAA interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion.
Asunto(s)
Secreción de Insulina , Células Secretoras de Insulina , Mitocondrias , Animales , Ratas , Humanos , Mitocondrias/metabolismo , Células Secretoras de Insulina/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Masculino , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética , Ratones , Ratas Wistar , Transporte de ElectrónRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease mainly characterized by the hepatic accumulation of lipid inducing a deregulation of ß-oxidation. Its advanced form is non-alcoholic steatohepatitis (NASH), which, in addition to lipid accumulation, induces hepatocellular damage, oxidative stress and fibrosis that can progress to cirrhosis and to its final stage: hepatocellular carcinoma (HCC). To date, no specific therapeutic treatment exists. The implications of organ crosstalk have been highlighted in many metabolic disorders, such as diabetes, metabolic-associated liver diseases and obesity. Skeletal muscle, in addition to its role as a reservoir and consumer of energy and carbohydrate metabolism, is involved in this inter-organs' communication through different secreted products: myokines, exosomes and enzymes, for example. Interestingly, resistance exercise has been shown to have a beneficial impact on different metabolic pathways, such as lipid oxidation in different organs through their secreted products. In this review, we will mainly focus on myokines and their effects on non-alcoholic fatty liver disease, and their complication: non-alcoholic steatohepatitis and HCC.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Hígado/metabolismo , Fibrosis , LípidosRESUMEN
The regulation of insulin secretion is under control of a complex inter-organ/cells crosstalk involving various metabolites and/or physical connections. In this review, we try to illustrate with current knowledge how ß-cells communicate with other cell types and organs in physiological and pathological contexts. Moreover, this review will provide a better understanding of the microenvironment and of the context in which ß-cells exist and how this can influence their survival and function. Recent studies showed that ß-cell insulin secretion is regulated also by a direct and indirect inter-organ/inter-cellular communication involving various factors, illustrating the idea of "the hidden face of the iceberg". Moreover, any disruption on the physiological communication between ß-cells and other cells or organs can participate on diabetes onset. Therefore, for new anti-diabetic treatments' development, it is necessary to consider the entire network of cells and organs involved in the regulation of ß-cellular function and no longer just ß-cell or pancreatic islet alone. In this context, we discuss here the intra-islet communication, the ß-cell/skeletal muscle, ß-cell/adipose tissue and ß-cell/liver cross talk.
Asunto(s)
Diabetes Mellitus , Células Secretoras de Insulina , Islotes Pancreáticos , Diabetes Mellitus/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismoRESUMEN
Circular RNAs (circRNAs) are class of non-coding RNA, which are characterized by a covalently closed loop structure. Functionally they can act on cellular physiology, notably by sponging microRNAs (miR), regulating gene expression or interacting with binding protein. To date, circRNAs might represent an interesting, underexploited avenue for new target discovery for therapeutic applications, especially in the liver. The first characteristic of non-alcoholic fatty liver disease (NAFLD) is hepatic cholesterol accumulation, followed by its advanced form of the affection, nonalcoholic steatohepatitis (NASH), due to the occurrence of lobular inflammation, irreversible fibrosis, and in some cases hepatocellular carcinoma (HCC). Therefore, studies have investigated the importance of the dysregulation of circRNAs in the onset of metabolic disorders. In this review, we summarize the potential role of circRNAs in the development of metabolic diseases associated with the liver such as NAFLD or NASH, and their potential to become therapeutic strategies for these pathologies.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/epidemiología , ARN Circular/genética , Neoplasias Hepáticas/etiologíaRESUMEN
The need to consume adequate dietary protein to preserve physical function during ageing is well recognized. However, the effect of protein intakes on glucose metabolism is still intensively debated. During age-related estrogen withdrawal at the time of the menopause, it is known that glucose homeostasis may be impaired but the influence of dietary protein levels in this context is unknown. The aim of the present study is to elucidate the individual and interactive effects of estrogen deficiency and suboptimal protein intake on glucose homeostasis in a preclinical model involving ovariectomy (OVX) and a 13 week period of a moderately reduced protein intake in 7 month-old ageing rats. To investigate mechanisms of action acting via the pancreas-liver-muscle axis, fasting circulating levels of insulin, glucagon, IGF-1, FGF21 and glycemia were measured. The hepatic lipid infiltration and the protein expression of GLUT4 in the gastrocnemius were analyzed. The gene expression of some hepatokines, myokines and lipid storage/oxidation related transcription factors were quantified in the liver and the gastrocnemius. We show that, regardless of the estrogen status, moderate dietary protein restriction increases fasting glycemia without modifying insulinemia, body weight gain and composition. This fasting hyperglycemia is associated with estrogen status-specific metabolic alterations in the muscle and liver. In estrogen-replete (SHAM) rats, GLUT4 was down-regulated in skeletal muscle while in estrogen-deficient (OVX) rats, hepatic stress-associated hyperglucagonemia and high serum FGF21 were observed. These findings highlight the importance of meeting dietary protein needs to avoid disturbances in glucose homeostasis in ageing female rats with or without estrogen withdrawal.
Asunto(s)
Dieta con Restricción de Proteínas , Estrógenos , Animales , Glucemia/metabolismo , Proteínas en la Dieta , Femenino , Homeostasis , Lípidos , RatasRESUMEN
Impaired pancreatic ß-cell function and insulin secretion are hallmarks of type 2 diabetes. miRNAs are short, noncoding RNAs that silence gene expression vital for the development and function of ß cells. We have previously shown that ß cell-specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels. Nevertheless, the function of this miRNA in ß cells is unclear. We hypothesized that miR-125b-5p expression is regulated by glucose and that this miRNA mediates some of the deleterious effects of hyperglycemia in ß cells. Here, we show that islet miR-125b-5p expression is upregulated by glucose in an AMPK-dependent manner and that short-term miR-125b-5p overexpression impairs glucose-stimulated insulin secretion (GSIS) in the mouse insulinoma MIN6 cells and in human islets. An unbiased, high-throughput screen in MIN6 cells identified multiple miR-125b-5p targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Inactivation of miR-125b-5p in the human ß-cell line EndoCß-H1 shortened mitochondria and enhanced GSIS, whereas mice overexpressing miR-125b-5p selectively in ß cells (MIR125B-Tg) were hyperglycemic and glucose intolerant. MIR125B-Tg ß cells contained enlarged lysosomal structures and had reduced insulin content and secretion. Collectively, we identify miR-125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion.
Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , MicroARNs , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Células Secretoras de Insulina/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Type 2 diabetes is associated with defects in insulin signaling and the resulting abnormal glucose and lipid metabolism. The complexity of insulin signaling cascades is highlighted by the existence of multiple isoforms of target proteins implicated in metabolic and gene-regulatory events. We utilized siRNA to decipher the specific role of predominant insulin receptor substrates and Akt isoforms expressed in human skeletal muscle. Gene silencing revealed specialized roles of insulin signaling cascades to metabolic endpoints. IRS-1 and Akt2 were required for myoblast differentiation and glucose metabolism, whereas IRS-2 and Akt1 were dispensable. A key role of IRS-2 and Akt1 in lipid metabolism was revealed, highlighting reciprocal relationships between metabolic pathways. Unraveling the isoform-specific regulation of glucose and lipid metabolism by key elements along insulin signaling cascades through siRNA-mediated gene silencing in human tissues will facilitate the discovery of novel targets for the treatment of diabetes and related metabolic disorders.
Asunto(s)
Silenciador del Gen/fisiología , Fosfoproteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , ARN Interferente Pequeño/genética , Células Cultivadas , Femenino , Glucosa/metabolismo , Humanos , Insulina/fisiología , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Lípidos/fisiología , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Oxidación-Reducción , Palmitatos/metabolismo , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/fisiologíaRESUMEN
The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased ß and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for α cell development. We conclude that Pax6 is critical for α cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and α cell differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica/fisiología , Células Secretoras de Glucagón/metabolismo , Proteínas de Homeodominio/metabolismo , Modelos Biológicos , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Elementos de Respuesta/fisiología , Animales , Línea Celular , Proteínas del Ojo/genética , Células Secretoras de Glucagón/citología , Proteínas de Homeodominio/genética , Humanos , Ratones , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proglucagón/biosíntesis , Proglucagón/genética , Ratas , Proteínas Represoras/genéticaRESUMEN
We have determined the lipid, protein and miRNA composition of skeletal muscle (SkM)-released extracellular vesicles (ELVs) from Ob/ob (OB) vs wild-type (WT) mice. The results showed that atrophic insulin-resistant OB-SkM released less ELVs than WT-SkM, highlighted by a RAB35 decrease and an increase in intramuscular cholesterol content. Proteomic analyses of OB-ELVs revealed a group of 37 proteins functionally connected, involved in lipid oxidation and with catalytic activities. OB-ELVs had modified contents for phosphatidylcholine (PC 34-4, PC 40-3 and PC 34-0), sphingomyelin (Sm d18:1/18:1) and ceramides (Cer d18:1/18:0) and were enriched in cholesterol, likely to alleviated intracellular accumulation. Surprisingly many ELV miRNAs had a nuclear addressing sequence, and targeted genes encoding proteins with nuclear activities. Interestingly, SkM-ELV miRNA did not target mitochondria. The most significant function targeted by the 7 miRNAs altered in OB-ELVs was lipid metabolism. In agreement, OB-ELVs induced lipid storage in recipient adipocytes and increased lipid up-take and fatty acid oxidation in recipient muscle cells. In addition, OB-ELVs altered insulin-sensitivity and induced atrophy in muscle cells, reproducing the phenotype of the releasing OB muscles. These data suggest for the first time, a cross-talk between muscle cells and adipocytes, through the SkM-ELV route, in favor of adipose tissue expansion.
Asunto(s)
Homeostasis , Resistencia a la Insulina , Lípidos/análisis , MicroARNs/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/patología , Tejido Adiposo , Animales , Exosomas/genética , Exosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Proteínas Musculares/genética , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Proteoma/análisis , Proteoma/metabolismoRESUMEN
Obesity and type 2 diabetes present partially overlapping phenotypes with systemic inflammation as a common feature, raising the hypothesis that elevated cytokine levels may contribute to peripheral insulin resistance as well as the decreased beta cell functional mass observed in type 2 diabetes. In healthy humans, TNF-alpha infusion induces skeletal muscle insulin resistance. We now explore the impact of TNF-alpha on primary beta cell function and the underlying signaling pathways. Human and rat primary beta cells were sorted by FACS and cultured for 24 h +/- 20 ng/ml TNF-alpha to explore the impact on apoptosis, proliferation, and short-term insulin secretion (1 h, 2.8 mm glucose followed by 1 h, 16.7 mm glucose at the end of the 24-h culture period) as well as key signaling protein phosphorylation and expression. Prior exposure to TNF-alpha for 24 h inhibits glucose-stimulated insulin secretion from primary beta cells. This is associated with a decrease in glucose-stimulated phosphorylation of key proteins in the insulin signaling pathway including Akt, AS160, and other Akt substrates, ERK as well as the insulin receptor. Strikingly, TNF-alpha treatment decreased IRS-2 protein level by 46 +/- 7% versus control, although mRNA expression was unchanged. While TNF-alpha treatment increased MAP4K4 mRNA expression by 33 +/- 5%, knockdown of MAP4K4 by siRNA-protected beta cells against the detrimental effects of TNF-alpha on both insulin secretion and signaling. We thus identify MAP4K4 as a key upstream mediator of TNF-alpha action on the beta cell, making it a potential therapeutic target for preservation of beta cell function in type 2 diabetes.
Asunto(s)
Glucosa/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Secreción de Insulina , Células Secretoras de Insulina/citología , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismoRESUMEN
Islet transplantation is one of the most efficient cell therapies used in clinics and could treat a large proportion of patients with diabetes. However, it is limited by the high requirement of pancreas necessary to provide the sufficient surviving islet mass in the hepatic tissue and restore normoglycaemia. Reduction in organ procurement requirements could be achieved by extrahepatic transplantation using a biomaterial that enhances islet survival and function. We report a plasma-supplemented hydroxypropyl methylcellulose (HPMC) hydrogel, engineered specifically using a newly developed technique for intra-omental islet infusion, known as hOMING (h-Omental Matrix Islet filliNG). The HPMC hydrogel delivered islets with better performance than that of the classical intrahepatic infusion. After the validation of the HPMC suitability for islets in vivo and in vitro, plasma supplementation modified the rheological properties of HPMC without affecting its applicability with hOMING. The biomaterial association was proven to be more efficient both in vitro and in vivo, with better islet viability and function than that of the current clinical intrahepatic delivery technique. Indeed, when the islet mass was decreased by 25% or 35%, glycaemia control was observed in the group of plasma-supplemented hydrogels, whereas no regulation was observed in the hepatic group. Plasma gelation, observed immediately post infusion, decreased anoïkis and promoted vascularisation. To conclude, the threshold mass for islet transplantation could be decreased using HPMC-Plasma combined with the hOMING technique. The simplicity of the hOMING technique and the already validated use of its components could facilitate its transfer to clinics. STATEMENT OF SIGNIFICANCE: One of the major limitations for the broad deployment of current cell therapy for brittle type 1 diabetes is the islets' destruction during the transplantation process. Retrieved from their natural environment, the islets are grafted into a foreign tissue, which triggers massive cell loss. It is mandatory to provide the islets with an 3D environment specifically designed for promoting isletimplantation to improve cell therapy outcomes. For this aim, we combined HPMC and plasma. HPMC provides suitable rheological properties to the plasma to be injectable and be maintained in the omentum. Afterwards, the plasma polymerises around the graft in vivo, thereby allowing their optimal integration into their transplantation site. As a result, the islet mass required to obtain glycaemic control was reduced by 35%.
Asunto(s)
Diabetes Mellitus Experimental/cirugía , Excipientes/farmacología , Control Glucémico/métodos , Hidrogeles/farmacología , Derivados de la Hipromelosa/farmacología , Trasplante de Islotes Pancreáticos , Animales , Difusión , Excipientes/química , Hidrogeles/química , Derivados de la Hipromelosa/química , Islotes Pancreáticos/citología , Masculino , Epiplón/cirugía , Oxígeno/química , Oxígeno/metabolismo , Ratas Endogámicas Lew , Ratas Wistar , ViscosidadRESUMEN
Aims: Nicotinamide adenine dinucleotide phosphate oxidases (NOX-es) produce reactive oxygen species and modulate ß-cell insulin secretion. Islets of type 2 diabetic subjects present elevated expression of NOX5. Here, we sought to characterize regulation of NOX5 expression in human islets in vitro and to uncover the relevance of NOX5 in islet function in vivo using a novel mouse model expressing NOX5 in doxycycline-inducible, ß-cell-specific manner (RIP/rtTA/NOX5 mice). Results:In situ hybridization and immunohistochemistry employed on pancreatic sections demonstrated NOX5 messenger ribonucleic acid (mRNA) and protein expressions in human islets. In cultures of dispersed islets, NOX5 protein was observed in somatostatin-positive (δ) cells in basal (2.8 mM glucose) conditions. Small interfering ribonucleic acid (siRNA)-mediated knockdown of NOX5 in human islets cultured in basal glucose concentrations resulted in diminished glucose-induced insulin secretion (GIIS) in vitro. However, when islets were preincubated in high (16.7 mM) glucose media for 12 h, NOX5 appeared also in insulin-positive (ß) cells. In vivo, mice with ß-cell NOX5 expression developed aggravated impairment of GIIS compared with control mice when challenged with 14 weeks of high-fat diet. Similarly, in vitro palmitate preincubation resulted in more severe reduction of insulin release in islets of RIP/rtTA/NOX5 mice compared with their control littermates. Decreased insulin secretion was most distinct in response to theophylline stimulation, suggesting impaired cyclic adenosine monophosphate (cAMP)-mediated signaling due to increased phosphodiesterase activation. Innovation and Conclusions: Our data provide the first insight into the complex regulation and function of NOX5 in islets implying an important role for NOX5 in δ-cell-mediated intraislet crosstalk in physiological circumstances but also identifying it as an aggravating factor in ß-cell failure in diabetic conditions.
Asunto(s)
Islotes Pancreáticos/metabolismo , NADPH Oxidasa 5/genética , Animales , Células Cultivadas , Dieta Alta en Grasa/efectos adversos , Femenino , Humanos , Secreción de Insulina/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , NADPH Oxidasa 5/metabolismoRESUMEN
Fine-tuning of insulin release from pancreatic ß-cells is essential to maintain blood glucose homeostasis. Here, we report that insulin secretion is regulated by a circular RNA containing the lariat sequence of the second intron of the insulin gene. Silencing of this intronic circular RNA in pancreatic islets leads to a decrease in the expression of key components of the secretory machinery of ß-cells, resulting in impaired glucose- or KCl-induced insulin release and calcium signaling. The effect of the circular RNA is exerted at the transcriptional level and involves an interaction with the RNA-binding protein TAR DNA-binding protein 43 kDa (TDP-43). The level of this circularized intron is reduced in the islets of rodent diabetes models and of type 2 diabetic patients, possibly explaining their impaired secretory capacity. The study of this and other circular RNAs helps understanding ß-cell dysfunction under diabetes conditions, and the etiology of this common metabolic disorder.
Asunto(s)
Secreción de Insulina/genética , Insulina/genética , Intrones , ARN Circular/metabolismo , Animales , Señalización del Calcio , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ratones , ARN Circular/genética , RatasRESUMEN
All cells export part of their intracellular content into the extracellular space through the release of various types of extracellular vesicles (EVs). They are synthetized either from the budding of the plasma membrane [i.e., microparticles (MPs, 150-300 nm size)] or from the late endosomes in which intraluminal vesicles progressively (ILVs) accumulate during their maturation into multivesicular bodies (MVBs). ILVs are then released into the extracellular space through MVB fusion with the plasma membrane [i.e., exosomes (50-100 nm size)]. In the context of metabolic diseases, recent data have highlighted the role of EVs in inflammation associated with pancreas dysfunction, adipose tissue homeostasis, liver steatosis, inflammation, and skeletal muscle (SkM) insulin resistance (IR). Among these insulin-sensitive tissues, SkM is the largest organ in human and is responsible for whole-body glucose disposal and locomotion. Therefore, understanding the contribution of SkM-EVs in the development of diabetes/obesity/dystrophy/,-related diseases is a hot topic. In this review, we have summarized the role of SkM-EVs in muscle physiology and in the development of metabolic diseases and identify important gaps that have to be filled in order to have more precise information on SkM-EVs biological actions and to understand the functions of the different subpopulations of SkM-EVs on the whole-body homeostasis.
RESUMEN
Regenerative medicine based on cell therapy represents a new hope for curing disease. Current obstacles include proper in vivo validation of the efficiency of the therapy. For transfer to the recipient body, cells often need to be combined with biomaterials, especially hydrogels. However, validation of the efficacy of such a graft requires the right environment, the right hydrogel, and the right recipient site. The omentum might be such a site. Based on the example of islet transplantation, we developed the hOMING (h-Omental Matrix Islet filliNG) technique, which consists of the injection of the graft inside the tissue, in between the omental layers, to improve islet implantation and survival. To achieve this, islets have to be embedded in a hydrogel with a viscosity that enables its injection using an atraumatic needle. Syringes are loaded with a combination of hydrogel and islets. Several injections are performed inside the omental tissue at different entry points, and the deposition of the islet/hydrogel mixture is made along a line. We tested the feasibility of this innovative approach using dextran beads. The beads were well spread throughout the omental tissue, in close proximity to blood vessels. To test the efficacy of the graft, we transplanted islets into diabetic rats and perform a metabolic follow-up over two months. The transplanted islets exhibited a high rate of re-vascularization around and inside islets, and reversed diabetes. The hOMING technique could be applicable for other types of hydrogel or cell therapy, for cells with high metabolic activity.