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1.
Proc Natl Acad Sci U S A ; 117(16): 9022-9031, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32284404

RESUMO

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13 Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic ß-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in ß-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13 translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in ß-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic ß-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting.


Assuntos
Diabetes Mellitus Tipo 1/genética , Células Secretoras de Insulina/imunologia , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT1/genética , Regiões 3' não Traduzidas/genética , Sobrevivência Celular/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/virologia , Predisposição Genética para Doença , Células HEK293 , Humanos , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/virologia , Células Jurkat , Poli I-C/imunologia , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , RNA Viral/imunologia , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima/imunologia
2.
Artigo em Inglês | MEDLINE | ID: mdl-32210914

RESUMO

Background and Aims: The synthetic atypical cannabinoid Abn-CBD, a cannabidiol (CBD) derivative, has been recently shown to modulate the immune system in different organs, but its impact in obesity-related meta-inflammation remains unstudied. We investigated the effects of Abn-CBD on metabolic and inflammatory parameters utilizing a diet-induced obese (DIO) mouse model of prediabetes and non-alcoholic fatty liver disease (NAFLD). Materials and Methods: Ten-week-old C57Bl/6J mice were fed a high-fat diet for 15 weeks, following a 2-week treatment of daily intraperitoneal injections with Abn-CBD or vehicle. At week 15 mice were obese, prediabetic and developed NAFLD. Body weight and glucose homeostasis were monitored. Mice were euthanized and blood, liver, adipose tissue and pancreas were collected and processed for metabolic and inflammatory analysis. Results: Body weight and triglycerides profiles in blood and liver were comparable between vehicle- and Abn-CBD-treated DIO mice. However, treatment with Abn-CBD reduced hyperinsulinemia and markers of systemic low-grade inflammation in plasma and fat, also promoting white adipose tissue browning. Pancreatic islets from Abn-CBD-treated mice showed lower apoptosis, inflammation and oxidative stress than vehicle-treated DIO mice, and beta cell proliferation was induced. Furthermore, Abn-CBD lowered hepatic fibrosis, inflammation and macrophage infiltration in the liver when compared to vehicle-treated DIO mice. Importantly, the balance between hepatocyte proliferation and apoptosis was improved in Abn-CBD-treated compared to vehicle-treated DIO mice. Conclusions: These results suggest that Abn-CBD exerts beneficial immunomodulatory actions in the liver, pancreas and adipose tissue of DIO prediabetic mice with NAFLD, thus protecting tissues. Therefore, Abn-CBD and related compounds could represent novel pharmacological strategies for managing obesity-related metabolic disorders.

3.
Metabolism ; 104: 154137, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31904355

RESUMO

Type 1 diabetes mellitus (T1DM) is an autoimmune disease that targets the destruction of islet beta-cells resulting in insulin deficiency, hyperglycemia and death if untreated. Despite advances in medical devices and longer-acting insulin, there is still no robust therapy to substitute and protect beta-cells that are lost in T1DM. Attempts to refrain from the autoimmune attack have failed to achieve glycemic control in patients highlighting the necessity for a paradigm shift in T1DM treatment. Paradoxically, beta-cells are present in T1DM patients indicating a disturbed equilibrium between the immune attack and beta-cell regeneration reminiscent of unresolved wound healing that under normal circumstances progression towards an anti-inflammatory milieu promotes regeneration. Thus, the ultimate T1DM therapy should concomitantly restore immune self-tolerance and replenish the beta-cell mass similar to wound healing. Recently the agonistic activation of the nuclear receptor LRH-1/NR5A2 was shown to induce immune self-tolerance, increase beta-cell survival and promote regeneration through a mechanism of alpha-to-beta cell phenotypic switch. This trans-regeneration process appears to be facilitated by a pancreatic anti-inflammatory environment induced by LRH-1/NR5A2 activation. Herein, we review the literature on the role of LRH1/NR5A2 in immunity and islet physiology and propose that a cross-talk between these cellular compartments is mandatory to achieve therapeutic benefits.


Assuntos
Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/terapia , Inflamação/patologia , Ilhotas Pancreáticas/fisiologia , Animais , Humanos , Ilhotas Pancreáticas/patologia , Regeneração
4.
Int J Mol Sci ; 20(24)2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817798

RESUMO

Gestational diabetes mellitus (GDM), a metabolic disease that develops with the increase in insulin resistance during late pregnancy, is currently one of the most common complications affecting pregnancy. The polygenic nature of GDM, together with the interplay between different genetic variants with nutritional and environmental factors has hindered the full understanding of the etiology of this disease. However, an important genetic overlap has been found with type 2 diabetes mellitus (T2DM) and, as in the case of T2DM, most of the identified loci are associated with ß-cell function. Early detection of GDM and adequate interventions to control the maternal glycemia are necessary to avoid the adverse outcomes for both the mother and the offspring. The in utero exposure to the diabetic milieu predispose these children for future diseases, among them T2DM, originating a vicious circle implicated in the increased prevalence of both GDM and T2DM. The involvement of inflammatory processes in the development of GDM highlights the importance of pancreatic ß-cell factors able to favor the adaptation processes required during gestation, concomitantly with the protection of the islets from an inflammatory milieu. In this regard, two members of the Pax family of transcription factors, PAX4 and PAX8, together with the chromatin remodeler factor HMG20A, have gained great relevance due to their involvement in ß-cell mass adaptation together with their anti-inflammatory properties. Mutations in these factors have been associated with GDM, highlighting these as novel candidates for genetic screening analysis in the identification of women at risk of developing GDM.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Diabetes Gestacional/metabolismo , Diabetes Gestacional/fisiopatologia , Ilhotas Pancreáticas/fisiologia , Glicemia/metabolismo , Feminino , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Fator de Transcrição PAX8/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Gravidez
5.
Aging (Albany NY) ; 11(18): 7746-7779, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31518338

RESUMO

An inverse correlation between thyroid hormone levels and longevity has been reported in several species and reduced thyroid hormone levels have been proposed as a biomarker for healthy aging and metabolic fitness. However, hypothyroidism is a medical condition associated with compromised health and reduced life expectancy. Herein, we show, using wild-type and the Pax8 ablated model of hypothyroidism in mice, that hyperthyroidism and severe hypothyroidism are associated with an overall unhealthy status and shorter lifespan. Mild hypothyroid Pax8 +/- mice were heavier and displayed insulin resistance, hepatic steatosis and increased prevalence of liver cancer yet had normal lifespan. These pathophysiological conditions were precipitated by hepatic mitochondrial dysfunction and oxidative damage accumulation. These findings indicate that individuals carrying mutations on PAX8 may be susceptible to develop liver cancer and/or diabetes and raise concerns regarding the development of interventions aiming to modulate thyroid hormones to promote healthy aging or lifespan in mammals.

7.
Sci Rep ; 9(1): 9515, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266981

RESUMO

Most studies in type 1 diabetes (T1D) have focused on the loss of the pancreatic beta-cell population. However, despite the involvement of the alpha-cell in the aetiology and complications of T1D, little is known about the regulation of the pancreatic alpha-cell mass in this disease. The need for a better understanding of this process is further emphasized by recent findings suggesting that alpha-cells may constitute a potential reservoir for beta-cell regeneration. In this study, we characterized the pancreatic alpha-cell mass and its regulatory processes in the transgenic RIP-B7.1 mice model of experimental autoimmune diabetes (EAD). Diabetic mice presented insulitis, hyperglycaemia, hypoinsulinemia and hyperglucagonemia along with lower pancreatic insulin content. While alpha-cell mass and pancreatic glucagon content were preserved at the early-onset of EAD, both parameters were reduced in the advanced phase. At both stages, alpha-cell size, proliferation and ductal neogenesis were up-regulated, whereas apoptosis was almost negligible. Interestingly, we found an increase in the proportion of glucagon-containing cells positive for insulin or the beta-cell transcription factor PDX1. Our findings suggest that pancreatic alpha-cell renewal mechanisms are boosted during the natural course of EAD, possibly as an attempt to maintain the alpha-cell population and/or to increase beta-cell regeneration via alpha-cell transdifferentiation.

8.
Genes (Basel) ; 10(5)2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31072002

RESUMO

The high prevalence of type 2 diabetes mellitus (T2DM), together with the fact that current treatments are only palliative and do not avoid major secondary complications, reveals the need for novel approaches to treat the cause of this disease. Efforts are currently underway to identify therapeutic targets implicated in either the regeneration or re-differentiation of a functional pancreatic islet ß-cell mass to restore insulin levels and normoglycemia. However, T2DM is not only caused by failures in ß-cells but also by dysfunctions in the central nervous system (CNS), especially in the hypothalamus and brainstem. Herein, we review the physiological contribution of hypothalamic neuronal and glial populations, particularly astrocytes, in the control of the systemic response that regulates blood glucose levels. The glucosensing capacity of hypothalamic astrocytes, together with their regulation by metabolic hormones, highlights the relevance of these cells in the control of glucose homeostasis. Moreover, the critical role of astrocytes in the response to inflammation, a process associated with obesity and T2DM, further emphasizes the importance of these cells as novel targets to stimulate the CNS in response to metabesity (over-nutrition-derived metabolic dysfunctions). We suggest that novel T2DM therapies should aim at stimulating the CNS astrocytic response, as well as recovering the functional pancreatic ß-cell mass. Whether or not a common factor expressed in both cell types can be feasibly targeted is also discussed.


Assuntos
Encéfalo/metabolismo , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Doenças Metabólicas/metabolismo , Animais , Astrócitos/metabolismo , Metabolismo Energético , Homeostase , Humanos
9.
Diabetes ; 68(1): 109-118, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30352879

RESUMO

Transient Pax8 expression was reported in mouse islets during gestation, whereas a genome-wide linkage and admixture mapping study highlighted PAX8 as a candidate gene for diabetes mellitus (DM). We sought the significance of PAX8 expression in mouse and human islet biology. PAX8 was induced in gestating mouse islets and in human islets treated with recombinant prolactin. Global gene expression profiling of human and mouse islets overexpressing the corresponding species-specific PAX8 revealed the modulation of distinct genetic pathways that converge on cell survival. Accordingly, apoptosis was reduced in PAX8-overexpressing islets. These findings support that PAX8 could be a candidate gene for the study of gestational DM (GDM). PAX8 was genotyped in patients with GDM and gestational thyroid dysfunction (GTD), a pathology commonly found in patients with mutations on PAX8 A novel missense PAX8 mutation (p.T356M, c.1067C>T) was identified in a female diagnosed with GDM and GTD as well as in her father with type 2 DM but was absent in control patients. The p.T356M variant did not alter protein stability or cellular localization, whereas its transactivation activity was hindered. In parallel, a retrospective clinical analysis uncovered that a pregnant female harboring a second PAX8 mutation (p.P25R, c.74C>G) previously reported to cause congenital hypothyroidism also developed GDM. These data indicate that increased expression of PAX8 affects islet viability and that PAX8 could be considered as a candidate gene for the study of GDM.


Assuntos
Diabetes Gestacional/metabolismo , Fator de Transcrição PAX8/metabolismo , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Diabetes Gestacional/genética , Feminino , Genótipo , Teste de Tolerância a Glucose , Humanos , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Mutação/genética , Mutação de Sentido Incorreto/genética , Fator de Transcrição PAX8/genética , Linhagem , Gravidez , Estudos Retrospectivos
10.
Curr Opin Pharmacol ; 43: 1-10, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30048825

RESUMO

The high prevalence of diabetes mellitus (DM) in our society, together with the fact that current treatments are only palliative and do not prevent the development of life threatening side effects, highlights the urgent need for novel therapies targeting the root cause of the disease. Independent of the etiology of DM, the definitive therapeutic approach will imply the restitution of an adequate functional ß-cell mass capable of compensating for the insulin demand of the organism. The recent demonstration of heterogeneity within the islets as well as their innate plasticity has encouraged the development of studies aiming at potentiation of the regenerative capacity of islets. In this regard, factors implicated in pancreas ontogeny as well as in the adaptation processes that take place in the islets under situations of increased insulin demand have gained much interest. One of these factors is the transcription factor PAX4, required for ß-cell formation during embryonic development and implicated in adult ß-cell adaptation under stress situations. Here we review the therapeutic potential of PAX4 as well as its downstream targets for the development of novel treatments for DM.


Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus/tratamento farmacológico , Desenho de Fármacos , Proteínas de Homeodomínio/metabolismo , Hipoglicemiantes/uso terapêutico , Células Secretoras de Insulina/efeitos dos fármacos , Fatores de Transcrição Box Pareados/metabolismo , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Plasticidade Celular , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Difusão de Inovações , Humanos , Hipoglicemiantes/efeitos adversos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Terapia de Alvo Molecular , Fenótipo , Transdução de Sinais/efeitos dos fármacos
11.
Nat Commun ; 9(1): 1488, 2018 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-29662071

RESUMO

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.


Assuntos
Comunicação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/terapia , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Fenalenos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Insulina/metabolismo , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/imunologia , Estreptozocina , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Transplante Heterólogo
12.
Cell Death Dis ; 9(3): 279, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29449530

RESUMO

HMG20A (also known as iBRAF) is a chromatin factor involved in neuronal differentiation and maturation. Recently small nucleotide polymorphisms (SNPs) in the HMG20A gene have been linked to type 2 diabetes mellitus (T2DM) yet neither expression nor function of this T2DM candidate gene in islets is known. Herein we demonstrate that HMG20A is expressed in both human and mouse islets and that levels are decreased in islets of T2DM donors as compared to islets from non-diabetic donors. In vitro studies in mouse and human islets demonstrated that glucose transiently increased HMG20A transcript levels, a result also observed in islets of gestating mice. In contrast, HMG20A expression was not altered in islets from diet-induced obese and pre-diabetic mice. The T2DM-associated rs7119 SNP, located in the 3' UTR of the HMG20A transcript reduced the luciferase activity of a reporter construct in the human beta 1.1E7 cell line. Depletion of Hmg20a in the rat INS-1E cell line resulted in decreased expression levels of its neuronal target gene NeuroD whereas Rest and Pax4 were increased. Chromatin immunoprecipitation confirmed the interaction of HMG20A with the Pax4 gene promoter. Expression levels of Mafa, Glucokinase, and Insulin were also inhibited. Furthermore, glucose-induced insulin secretion was blunted in HMG20A-depleted islets. In summary, our data demonstrate that HMG20A expression in islet is essential for metabolism-insulin secretion coupling via the coordinated regulation of key islet-enriched genes such as NeuroD and Mafa and that depletion induces expression of genes such as Pax4 and Rest implicated in beta cell de-differentiation. More importantly we assign to the T2DM-linked rs7119 SNP the functional consequence of reducing HMG20A expression likely translating to impaired beta cell mature function.


Assuntos
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Células Secretoras de Insulina/metabolismo , Polimorfismo de Nucleotídeo Único , Regiões 3' não Traduzidas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Glicemia/metabolismo , Linhagem Celular Tumoral , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Feminino , Predisposição Genética para Doença , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/patologia , Lipídeos/sangue , Masculino , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fenótipo , Ratos
14.
Cell Stress ; 2(6): 141-143, 2018 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31225479

RESUMO

Type 1 diabetes mellitus (T1DM) is defined as an autoimmune disease that targets the selective destruction of islet insulin-producing beta cells by infiltrating immune cells (insulitis). As a result, the organism is no longer able to produce insulin and develops hyperglycaemia and, if untreated, death. Despite advances in medical device technology and insulin analogues as well as strives in generating in vitro insulin-producing cells, there is still no robust therapy to substitute and protect beta cells that are lost in T1DM. Clinical trials aimed at blocking the immune-mediated beta cell destruction have had moderate success leaving a gap in our understanding of disease aetiology. Such breach in knowledge may stem from the oversight that inhibiting the immune attack likely impairs beta cell regeneration and emphasizes a fundamental paradigm in the approach to treat the disease: A non-mutually exclusive strategy in which the uncontrolled self-directed inflammatory immune response (and not the global immune system) as well as beta cell regeneration are exquisitely fine tuned in order to successfully regain immunological tolerance and restoration of a functional beta cell mass. As such, defining factors that can guide a pro-inflammatory immune cell destructive environment towards an anti-inflammatory environment facilitating beta cell survival and stimulate regeneration would define an unprecedented class of immune-regenerative therapeutic agents for T1DM. In our recent study we identify the liver receptor homolog 1 (LRH-1, also known as NR5A2) as a 'druggable' target that fulfills these criteria restoring glycemic control in various mouse models of T1DM as well as improving human islet survival and function both in vitro and in vivo (Nat Comms, 9:1488).

15.
Br J Pharmacol ; 174(21): 3795-3810, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28800677

RESUMO

BACKGROUND AND PURPOSE: Thyroid hormones induce several changes in whole body metabolism that are known to improve metabolic homeostasis. However, adverse side effects have prevented its use in the clinic. In view of the promising effects of thyroid hormones, we investigated the effects of levothyroxine supplementation on glucose homeostasis. EXPERIMENTAL APPROACH: C57BL/6 mice were treated with levothyroxine from birth to 24 weeks of age, when mice were killed. The effects of levothyroxine supplementation on metabolic health were determined. C57BL/6 mice treated with levothyroxine for 2 weeks and then challenged with streptozotocin to monitor survival. Mechanistic experiments were conducted in the pancreas, liver and skeletal muscle. RIP-B7.1 mice were treated with levothyroxine for 2 weeks and were subsequently immunized to trigger experimental autoimmune diabetes (EAD). Metabolic tests were performed. Mice were killed and metabolic tissues were extracted for immunohistological analyses. KEY RESULTS: Long-term levothyroxine supplementation enhanced glucose clearance and reduced circulating glucose in C57BL/6 mice. Levothyroxine increased simultaneously the proliferation and apoptosis of pancreatic beta cells, promoting the maintenance of a highly insulin-expressing beta cell population. Levothyroxine increased circulating insulin levels, inducing sustained activation of IRS1-AKT signalling in insulin-target tissues. Levothyroxine-treated C57BL/6 mice challenged with streptozotocin exhibited extended survival. Levothyroxine blunted the onset of EAD in RIP-B7.1 mice by inducing beta cell proliferation and preservation of insulin-expressing cells. CONCLUSIONS AND IMPLICATIONS: Interventions based on the use of thyroid hormones or thyromimetics could be explored to provide therapeutic benefit in patients with type 1 diabetes mellitus.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Glucose/metabolismo , Tiroxina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estreptozocina , Tiroxina/administração & dosagem
16.
Sci Rep ; 7(1): 3946, 2017 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-28638091

RESUMO

LH-21 is a triazol derivative that has been described as a low-permeant neutral CB1 antagonist, though its pharmacology is still unclear. It has been associated with anti-obesity actions in obese rats. However, its role in preventing type 2 diabetes (T2D) onset have not been studied yet. Given CB1 receptors remain as potential pharmacological targets to fight against obesity and T2D, we wanted to explore the metabolic impact of this compound in an animal model of obesity and pre-diabetes as well as the lack of relevant actions in related central processes such as anxiety. C57BL/6J mice were rendered obese and pre-diabetic by feeding a high-fat diet for 15 weeks and then treated with LH-21 or vehicle for two weeks. Food intake, body weight and glucose handling were assessed, together with other relevant parameters. Behavioural performance was evaluated by the open field test and the elevated plus maze. LH-21 did not affect food intake nor body weight but it improved glucose handling, displaying tissue-specific beneficial actions. Unexpectedly, LH-21 induced anxiolysis and reverted obesity-induced anxiety, apparently through GPR55 receptor. These results suggest that LH-21 can be a new candidate to fight against diabetes onset. Indeed, this compound shows potential in counteracting obesity-related anxiety.


Assuntos
Ansiedade/prevenção & controle , Glicemia/metabolismo , Obesidade/metabolismo , Estado Pré-Diabético/metabolismo , Receptor CB1 de Canabinoide/antagonistas & inibidores , Triazóis/administração & dosagem , Animais , Comportamento Animal , Dieta Hiperlipídica , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Obesidade/prevenção & controle , Estado Pré-Diabético/prevenção & controle
17.
Genes (Basel) ; 8(3)2017 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-28282933

RESUMO

Paired box 4 (PAX4) is a key factor in the generation of insulin producing ß-cells during embryonic development. In adult islets, PAX4 expression is sequestered to a subset of ß-cells that are prone to proliferation and more resistant to stress-induced apoptosis. The importance of this transcription factor for adequate pancreatic islets functionality has been manifested by the association of mutations in PAX4 with the development of diabetes, independently of its etiology. Overexpression of this factor in adult islets stimulates ß-cell proliferation and increases their resistance to apoptosis. Additionally, in an experimental model of autoimmune diabetes, a novel immunomodulatory function for this factor has been suggested. Altogether these data pinpoint at PAX4 as an important target for novel regenerative therapies for diabetes treatment, aiming at the preservation of the remaining ß-cells in parallel to the stimulation of their proliferation to replenish the ß-cell mass lost during the progression of the disease. However, the adequate development of such therapies requires the knowledge of the molecular mechanisms controlling the expression of PAX4 as well as the downstream effectors that could account for PAX4 action.

18.
Diabetologia ; 59(4): 755-65, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26813254

RESUMO

AIMS/HYPOTHESIS: A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM. METHODS: Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements. RESULTS: PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death. CONCLUSIONS/INTERPRETATION: The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Mutantes
19.
Sci Rep ; 5: 15672, 2015 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-26503027

RESUMO

PAX4 is a key regulator of pancreatic islet development whilst in adult acute overexpression protects ß-cells against stress-induced apoptosis and stimulates proliferation. Nonetheless, sustained PAX4 expression promotes ß-cell dedifferentiation and hyperglycemia, mimicking ß-cell failure in diabetic patients. Herein, we study mechanisms that allow stringent PAX4 regulation endowing favorable ß-cell adaptation in response to changing environment without loss of identity. To this end, PAX4 expression was monitored using a mouse bearing the enhanced green fluorescent protein (GFP) and cre recombinase construct under the control of the islet specific pax4 promoter. GFP was detected in 30% of islet cells predominantly composed of PAX4-enriched ß-cells that responded to glucose-induced insulin secretion. Lineage tracing demonstrated that all islet cells were derived from PAX4(+) progenitor cells but that GFP expression was confined to a subpopulation at birth which declined with age correlating with reduced replication. However, this GFP(+) subpopulation expanded during pregnancy, a state of active ß-cell replication. Accordingly, enhanced proliferation was exclusively detected in GFP(+) cells consistent with cell cycle genes being stimulated in PAX4-overexpressing islets. Under stress conditions, GFP(+) cells were more resistant to apoptosis than their GFP(-) counterparts. Our data suggest PAX4 defines an expandable ß-cell sub population within adult islets.


Assuntos
Apoptose/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/citologia , Fatores de Transcrição Box Pareados/metabolismo , Animais , Desdiferenciação Celular/fisiologia , Linhagem da Célula , Proliferação de Células/fisiologia , Diabetes Mellitus/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/genética , Hiperglicemia/patologia , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/classificação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Transcrição Box Pareados/genética , Regiões Promotoras Genéticas/genética
20.
Curr Gene Ther ; 15(4): 436-46, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26122098

RESUMO

Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional ß-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes.


Assuntos
Vetores Genéticos , Ilhotas Pancreáticas/fisiologia , Lentivirus/genética , Transdução Genética/métodos , Animais , Células Cultivadas , Citometria de Fluxo , Glucagon/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Masculino , Camundongos Endogâmicos C57BL
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