RESUMO
Growth hormone (GH) binding to GH receptor activates janus kinase 2 (JAK2)-signal transducer and activator of transcription 5b (STAT5b) pathway, which stimulates transcription of insulin-like growth factor-1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3) and insulin-like growth factor acid-labile subunit (IGFALS). Although STAT5B deficiency was established as an autosomal recessive disorder, heterozygous dominant-negative STAT5B variants have been reported in patients with less severe growth deficit and milder immune dysfunction. We developed an in vivo functional assay in zebrafish to characterize the pathogenicity of three human STAT5B variants (p.Ala630Pro, p.Gln474Arg and p.Lys632Asn). Overexpression of human wild-type (WT) STAT5B mRNA and its variants led to a significant reduction of body length together with developmental malformations in zebrafish embryos. Overexpression of p.Ala630Pro, p.Gln474Arg or p.Lys632Asn led to an increased number of embryos with pericardial edema, cyclopia and bent spine compared with WT STAT5B. Although co-injection of WT and p.Gln474Arg and WT and p.Lys632Asn STAT5B mRNA in zebrafish embryos partially or fully rescues the length and the developmental malformations in zebrafish embryos, co-injection of WT and p.Ala630Pro STAT5B mRNA leads to a greater number of embryos with developmental malformations and a reduction in body length of these embryos. These results suggest that these variants could interfere with endogenous stat5.1 signaling through different mechanisms. In situ hybridization of zebrafish embryos overexpressing p.Gln474Arg and p.Lys632Asn STAT5B mRNA shows a reduction in igf1 expression. In conclusion, our study reveals the pathogenicity of the STAT5B variants studied.
Assuntos
Fator de Transcrição STAT5 , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Hormônio do Crescimento , Transdução de Sinais/genética , RNA Mensageiro , Fator de Crescimento Insulin-Like I/genéticaRESUMO
Mammalian acid-labile subunit (ALS) is a serum protein that binds binary complexes between Insulin-like growth factors (IGFs) and Insulin-like growth factor-binding proteins (IGFBPs) extending their half-life and keeping them in the vasculature. Human ALS deficiency (ACLSD), due to homozygous or compound heterozygous mutations in IGFALS, leads to moderate short stature with reduced levels of IGF-I and IGFBP-3. There is only one corresponding zebrafish ortholog gene and it has not yet been studied. In this study we elucidate the role of igfals during zebrafish development. In zebrafish embryos igfals mRNA is expressed throughout development, mainly in the brain and subsequently also in the gut and swimbladder. To determine its role during development, we knocked down igfals gene product using morpholinos (MOs). Igfals morphant embryos displayed dorsalization in different degrees of severity, including a shortened trunk and loss of tail. Furthermore, co-injection of human IGFALS (hIGFALS) mRNA was able to rescue the MO-induced phenotype. Finally, overexpression of either hIGFALS or zebrafish igfals (zigfals) mRNA leads to ventralization of embryos including a reduced head and enlarged tail. These findings suggest that als plays an important role in dorso-ventral patterning during zebrafish development.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Animais , MutaçãoRESUMO
OBJECTIVE: Acid-labile subunit deficiency (ACLSD), caused by inactivating mutations in both IGFALS gene alleles, is characterized by marked reduction in IGF-I and IGFBP-3 levels associated with mild growth retardation. The aim of this study was to expand the known phenotype and genetic characteristics of ACLSD by reporting data from four index cases and their families. DESIGN: Auxological data, biochemical and genetic studies were performed in four children diagnosed with ACLSD and all available relatives. METHODS: Serum levels of IGF-I, IGFBP-3, acid-labile subunit (ALS), and in vitro ternary complex formation (ivTCF) were determined. After sequencing the IGFALS gene, pathogenicity of novel identified variants was evaluated by in vitro expression in transfected Chinese hamster ovarian (CHO) cells. ALS protein was detected in patients' sera and CHO cells conditioned media and lysates by Western immunoblot (WIB). RESULTS: Four index cases and four relatives were diagnosed with ACLSD. The following variants were found: p.Glu35Glyfs*17, p.Glu35Lysfs*87, p.Leu213Phe, p.Asn276Ser, p.Leu409Phe, p.Ala475Val and p.Ser490Trp. ACLSD patients presented low IGF-I and low or undetectable levels of IGFBP-3 and ALS. Seven out of 8 patients did not form ivTCF. CONCLUSIONS: This study confirms previous findings in ACLSD, such as the low IGF-I and a more severe reduction in IGFBP-3 levels, and a gene dosage effect observed in heterozygous carriers (HC). In addition, father-to-son transmission (father compound heterozygous and mother HC), preservation of male fertility, and marginal ALS expression with potential involvement in preserved responsiveness to rhGH treatment, are all novel aspects, not previously reported in this condition.
Assuntos
Glicoproteínas/deficiência , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like I/análise , Adolescente , Adulto , Idoso , Animais , Proteínas de Transporte/genética , Criança , Pré-Escolar , Cricetulus , Família , Feminino , Fertilidade , Variação Genética , Glicoproteínas/genética , Transtornos do Crescimento/genética , Heterozigoto , Humanos , Lactente , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/deficiência , Fator de Crescimento Insulin-Like I/deficiência , América Latina , Masculino , Pessoa de Meia-Idade , Mutação , Transfecção , Adulto JovemRESUMO
Allograft function and metabolic effects of four treatment regimens, namely, methylprednisone (MP) standard dose (MP-STD), deflazacort (DFZ), MP-late steroid withdrawal (MP-LSW), and MP-very low dose (MP-VLD), were evaluated in prepubertal patients. MP was decreased by month 4 post-transplantation to 0.2 mg/kg/day in MP-STD and DFZ patients and to <0.1 mg/kg/day in MP-LSW and MP-VLD patients. Starting in month 16 post-transplant, MP was switched to DFZ in the DFZ group and totally withdrawn in the MP-LSW group. Creatinine clearance diminished in the MP-STD and MP-LSW groups from 77 +/- 6 to 63 +/- 6 ml/min/1.73 m(2)and from 103 +/- 5 to 78 +/- 3 ml/min/1.73 m(2), respectively (p < 0.01 and p < 0.001, respectively). Height increased >0.5 SDS only in the MP-LSW and MP-VLD groups. The body mass index and fat body mass for height-age increased only in the MP-STD patients (p < 0.05 and p < 0.01, respectively). Fat body mass decreased in the DFZ group (p < 0.05), total cholesterol and LDL-cholesterol increased in the MP-STD group, while LDL-cholesterol and total cholesterol/HDL-cholesterol ratio decreased in the DFZ group (p < 0.01). Lumbar spine bone mineral density (BMD) for height-age showed an increase in the MP-LSW and MP-VLD groups (p < 0.01). Our data suggest that MP-LSW and MP-VLD strategies improve linear growth, BMD, the peripheral distribution of fat, and preservation of the bone-muscle unit and maintain the normal lipid profile. The MP-LSW patients had a concerning rate of acute rejections and graft function deterioration in prepubertal patients.
Assuntos
Composição Corporal/fisiologia , Transtornos do Crescimento/prevenção & controle , Crescimento/fisiologia , Transplante de Rim/efeitos adversos , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Rejeição de Enxerto/induzido quimicamente , Transtornos do Crescimento/fisiopatologia , Transtornos do Crescimento/reabilitação , Humanos , Imunossupressores/uso terapêutico , Transplante de Rim/reabilitação , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/metabolismo , Masculino , Metilprednisolona/uso terapêutico , Complicações Pós-Operatórias/prevenção & controle , Complicações Pós-Operatórias/reabilitação , Pregnenodionas/uso terapêuticoRESUMO
BACKGROUND: The most frequent monogenic causes of growth hormone insensitivity (GHI) include defects in genes encoding the GH receptor itself (GHR), the signal transducer and activator of transcription (STAT5B), the insulin like-growth factor type I (IGF1) and the acid-labile subunit (IGFALS). GHI is characterized by a continuum of mild to severe post-natal growth failure. OBJECTIVE: To characterize the molecular defect in a patient with short stature and partial GHI. PATIENT AND METHODS: The boy was born at term adequate for gestational age from non-consanguineous normal-stature parents. At 2.2â¯years, he presented proportionate short stature (height -2.77 SDS), wide forehead and normal mental development. Whole-exome analysis and functional characterization (site-directed mutagenesis, dual luciferase reporter assay, immunofluorescence and western immunoblot) were performed. RESULTS: Biochemical and endocrinological evaluation revealed partial GH insensitivity with normal stimulated GH peak (7.8â¯ng/mL), undetectable IGF1 and low IGFBP3 levels. Two heterozygous variants in the GH-signaling pathway were found: a novel heterozygous STAT5B variant (c.1896G>T, p.K632N) and a hypomorphic IGFALS variant (c.1642C>T, p.R548W). Functional in vitro characterization demonstrated that p.K632N-STAT5b is an inactivating variant that impairs STAT5b activity through abolished phosphorylation. Remarkably, the patient's immunological evaluation displayed only a mild hypogammaglobulinemia, while a major characteristic of STAT5b deficient patients is severe immunodeficiency. CONCLUSIONS: We reported a novel pathogenic inactivating STAT5b variant, which may be associated with partial GH insensitivity and can present without severe immunological complications in heterozygous state. Our results contribute to expand the spectrum of phenotypes associated to GHI.
Assuntos
Agamaglobulinemia/genética , Síndrome de Laron/genética , Fator de Transcrição STAT5/genética , Agamaglobulinemia/imunologia , Pré-Escolar , Heterozigoto , Hormônio do Crescimento Humano/metabolismo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Síndrome de Laron/imunologia , Síndrome de Laron/metabolismo , Síndrome de Laron/fisiopatologia , Masculino , Testes de Função Hipofisária , Mutação Puntual , Índice de Gravidade de DoençaRESUMO
Germinal heterozygous activating STAT3 mutations represent a novel monogenic defect associated with multi-organ autoimmune disease and, in some cases, severe growth retardation. By using whole-exome sequencing, we identified two novel STAT3 mutations, p.E616del and p.C426R, in two unrelated pediatric patients with IGF-I deficiency and immune dysregulation. The functional analyses showed that both variants were gain-of-function (GOF), although they were not constitutively phosphorylated. They presented differences in their dephosphorylation kinetics and transcriptional activities under interleukin-6 stimulation. Both variants increased their transcriptional activities in response to growth hormone (GH) treatment. Nonetheless, STAT5b transcriptional activity was diminished in the presence of STAT3 GOF variants, suggesting a disruptive role of STAT3 GOF variants in the GH signaling pathway. This study highlights the broad clinical spectrum of patients presenting activating STAT3 mutations and explores the underlying molecular pathway responsible for this condition, suggesting that different mutations may drive increased activity by slightly different mechanisms.
Assuntos
Células Germinativas/metabolismo , Transtornos do Crescimento/genética , Perda Auditiva Neurossensorial/genética , Doenças do Sistema Imunitário/genética , Fator de Crescimento Insulin-Like I/deficiência , Mutação/genética , Fator de Transcrição STAT3/genética , Sequência de Aminoácidos , Pré-Escolar , Feminino , Células HEK293 , Hormônio do Crescimento Humano/farmacologia , Humanos , Lactente , Recém-Nascido , Fator de Crescimento Insulin-Like I/genética , Interleucina-5/metabolismo , Luciferases/metabolismo , Masculino , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Multimerização Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Transcrição Gênica/efeitos dos fármacos , Sequenciamento do ExomaRESUMO
Acid-labile subunit (ALS) is essential for stabilization of IGF-I and IGFBP-3 in ternary complexes within the vascular system. ALS deficient (ALS-D) patients and a subset of children with idiopathic short stature (ISS), presenting IGFALS gene variants, show variable degree of growth retardation associated to IGF-I and IGFBP-3 deficiencies. The aim of this study was to evaluate the potential pathogenicity of eleven IGFALS variants identified in ALS-D and ISS children using in silico and in vitro approaches. We were able to classify seven of these variants as pathogenic since they present impaired synthesis (p.Glu35Lysfs*87, p.Glu35Glyfs*17, p.Asn276Ser, p.Leu409Phe, p.Ser490Trp and p.Cys540Arg), or partial impairment of synthesis and lack of secretion (p.Leu213Phe). We also observed significant reduction of secreted protein for variants p.Ala330Asp, Ala475Val and p.Arg548Trp, while still retaining their ability to form ternary complexes. These findings provide an approach to test the pathogenicity of IGFALS gene variants.
Assuntos
Proteínas de Transporte/genética , Biologia Computacional/métodos , Simulação por Computador , Glicoproteínas/genética , Polimorfismo de Nucleotídeo Único/genética , Sequência de Aminoácidos , Animais , Células CHO , Proteínas de Transporte/química , Criança , Cricetinae , Cricetulus , Feminino , Glicoproteínas/química , Humanos , Masculino , Modelos Moleculares , Proteínas Mutantes/metabolismo , Alinhamento de Sequência , Software , TransfecçãoRESUMO
The present work examines the role of lipids in the development of the Type 1 diabetes induced by the administration of multiple low doses of streptozotocin (STZ) in C57BL/6J mice. The study was performed before and after the onset of clear hyperglycemia, and the results were as follows. First, 6 days after the first dose of STZ, while plasma glucose and insulin levels remained similar to those observed in the control mice, plasma free fatty acid (FFA) levels were significantly increased (P < 0.05). At that time, a marked increase of triglyceride content in gastronemius muscle was accompanied by a diminished activity of pyruvate dehydrogenase complex, suggesting an impaired glucose oxidation. Furthermore, a decrease of both triglyceride content and lipoprotein lipase activity was observed in the epididymal fat tissue. Second, 12 days after the first injection of STZ, hyperglycemia was accompanied by hypertriglyceridemia, a more pronounced increase of plasma FFA, and a significant (P < 0.05) reduction of insulinemia. At this time, both the adipose tissue and the gastrocnemius muscle showed a further deterioration of all parameters mentioned after 6 days. Moreover, in the gastrocnemius muscle, an impaired nonoxidative pathway of glucose metabolism was observed [significant reduction (P < 0.05) of glycogen mass, glucose-6-phosphate content, and glycogen synthase activities] at this time point. Finally, the data suggest for the first time that, in mice, Type 1 diabetes induced by multiple low doses of STZ and enhanced lipolysis of fat pads leads to an increase in the availability of plasma FFA, which seems to play a role in the early steps of diabetes evolution.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Ilhotas Pancreáticas/metabolismo , Metabolismo dos Lipídeos , Estreptozocina , Animais , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Tipo 1/induzido quimicamente , Progressão da Doença , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Índice de Gravidade de DoençaRESUMO
States of growth hormone (GH) resistance, such those observed in Laron dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF-1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF-1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH-resistant (GHR knockout [GHRKO]) mouse model with mice expressing the hepatic Igf-1 transgene (HIT) to generate the GHRKO-HIT mouse model. In GHRKO-HIT mice, serum IGF-1 levels were restored via transgenic expression of Igf-1, allowing us to study how endocrine IGF-1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH-resistant state, normalization of serum IGF-1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF-1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf-1 gene expression, blunted GH-mediated effects on the skeleton that are independent of serum or tissue IGF-1, and poor delivery of IGF-1 to the tissues. These findings are consistent with clinical data showing that IGF-I replacement therapy in patients with Laron syndrome does not achieve full skeletal growth.
Assuntos
Desenvolvimento Ósseo , Osso e Ossos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Síndrome de Laron/sangue , Receptores da Somatotropina/metabolismo , Animais , Osso e Ossos/patologia , Modelos Animais de Doenças , Feminino , Terapia de Reposição Hormonal , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/uso terapêutico , Síndrome de Laron/tratamento farmacológico , Síndrome de Laron/genética , Masculino , Camundongos , Camundongos Mutantes , Receptores da Somatotropina/genética , TransgenesRESUMO
BACKGROUND: In acid-labile subunit (ALS)-deficient families, heterozygous carriers of IGFALS gene mutations are frequently shorter than their wild-type relatives, suggesting that IGFALS haploinsufficiency could result in short stature. We have characterized IGFALS gene variants in idiopathic short stature (ISS) and in normal children, determining their impact on height and the IGF system. PATIENTS AND METHODS: In 188 normal and 79 ISS children levels of IGF-1, IGFBP-3, ALS, ternary complex formation (TCF) and IGFALS gene sequence were determined. RESULTS: In sum, 9 nonsynonymous or frameshift IGFALS variants (E35Gfs*17, G83S, L97F, R277H, P287L, A330D, R493H, A546V and R548W) were found in 10 ISS children and 6 variants (G170S, V239M, N276S, R277H, G506R and R548W) were found in 7 normal children. If ISS children were classified according to the ability for TCF enhanced by the addition of rhIGFBP-3 (TCF+), carriers of pathogenic IGFALS gene variants were shorter and presented lower levels of IGF-1, IGFBP-3 and ALS in comparison to carriers of benign variants. In ISS families, subjects carrying pathogenic variants were shorter and presented lower IGF-1, IGFBP-3 and ALS levels than noncarriers. CONCLUSIONS: These findings suggest that heterozygous IGFALS gene variants could be responsible for short stature in a subset of ISS children with diminished levels of IGF-1, IGFBP-3 and ALS.
Assuntos
Estatura , Proteínas de Transporte/genética , Glicoproteínas/genética , Transtornos do Crescimento/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Proteínas de Transporte/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Mutação da Fase de Leitura/genética , Glicoproteínas/sangue , Heterozigoto , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Transdução de SinaisRESUMO
Understanding insulin-like growth factor-1 (IGF1) biology is of particular importance because, apart from its role in mediating growth, it plays key roles in cellular transformation, organ regeneration, immune function, development of the musculoskeletal system and aging. IGF1 bioactivity is modulated by its binding to IGF-binding proteins (IGFBPs) and the acid labile subunit (ALS), which are present in serum and tissues. To determine whether IGF1 binding to IGFBPs is necessary to facilitate normal growth and development, we used a gene-targeting approach and generated two novel knock-in mouse models of mutated IGF1, in which the native Igf1 gene was replaced by Des-Igf1 (KID mice) or R3-Igf1 (KIR mice). The KID and KIR mutant proteins have reduced affinity for the IGFBPs, and therefore present as unbound IGF1, or 'free IGF1'. We found that both KID and KIR mice have reduced serum IGF1 levels and a concomitant increase in serum growth hormone levels. Ternary complex formation of IGF1 with the IGFBPs and the ALS was markedly reduced in sera from KID and KIR mice compared with wild type. Both mutant mice showed increased body weight, body and bone lengths, and relative lean mass. We found selective organomegaly of the spleen, kidneys and uterus, enhanced mammary gland complexity, and increased skeletal acquisition. The KID and KIR models show unequivocally that IGF1-complex formation with the IGFBPs is fundamental for establishing normal body and organ size, and that uncontrolled IGF bioactivity could lead to pathological conditions.
Assuntos
Crescimento e Desenvolvimento , Fator de Crescimento Insulin-Like I/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Disponibilidade Biológica , Tamanho Corporal/fisiologia , Peso Corporal/fisiologia , Feminino , Técnicas de Introdução de Genes , Hormônio do Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/genética , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Modelos Animais , Dados de Sequência Molecular , Complexos Multiproteicos/sangue , Tamanho do Órgão/fisiologia , Osteogênese , Ligação Proteica , Útero/citologia , Útero/crescimento & desenvolvimentoRESUMO
An increased availability of plasma free fatty acids (FFA) seems to play a role in the early stages of experimental type 1 diabetes mellitus induced in C57BL/6J mice by multiple low doses of streptozotoxin (mld-STZ). We analyzed the temporal changes of: (1) plasma and skeletal muscle lipids and their relationship with glucose metabolism; (2) triglyceride (Tg) concentration in isolated islets; (3) intraperitoneal glucose tolerance test; and (4) insulin secretion patterns when the three mutually interactive glucose signaling pathways were activated. Animals were killed by cervical dislocation at days 4, 6, 7, 8, 9 and 12 post first injection of mld-STZ. Compared with control mice, we observed: (1) at day 6, a significant increase of plasma FFA and both muscle and islet Tg content and a significant decrease of muscle pyruvate dehydrogenase activity. These parameters further deteriorated with time. (2) plasma Tg, glucose and insulin levels and glucose tolerance test were significantly different only after day 8. (3) an increase in both phases of the glucose plus palmitate-stimulated insulin secretion was observed at day 4. This effect progressively decreased since day 7 up to day 9. Moreover, an inhibitory action of cerulenin over glucose plus palmitate-stimulated insulin secretion was observed between days 6 and 9. Taken together these results suggest that early alteration in carbohydrate and lipid metabolism could represent a "metabolic window" which would develop between days 6 and 8. Afterwards, subsequent immunological alterations, apoptosis and necrosis induced the destruction of ß cells and would mask the results mentioned above.
Assuntos
Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Transtornos do Metabolismo dos Lipídeos/etiologia , Estreptozocina/administração & dosagem , Tecido Adiposo/imunologia , Tecido Adiposo/patologia , Animais , Autoimunidade/fisiologia , Peso Corporal/fisiologia , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/patologia , Relação Dose-Resposta a Droga , Epididimo , Insulina/sangue , Transtornos do Metabolismo dos Lipídeos/sangue , Transtornos do Metabolismo dos Lipídeos/induzido quimicamente , Transtornos do Metabolismo dos Lipídeos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Tamanho do Órgão , Fatores de TempoRESUMO
OBJECTIVE: Administration of multiple low doses of streptozotocin (mld-SZ) to mice results in the development of autoimmune diabetes. Hyperglycemia does not develop until a few days after the last injection. In this study, we explored immune-related alterations found in the very early stages of this diabetic syndrome and the capacity of mononuclear spleen cells (MSs) from mld-SZ mice to impair insulin secretion. METHODS: Mice injected with mld-SZ were used as an animal model of type 1 diabetes. MSs were isolated from control and mld-SZ mice at days 4, 6, 9, 12, and 16 after the first injection of the diabetogenic drug. MSs were transferred to normal syngeneic recipients or were cocultured with dispersed rat islet cells as an in vitro insulin secretion study. RESULTS: MSs from mld-SZ mice were able to diminish insulin secretion when transferred to normal syngeneic recipients and presented anti-beta-cell immune aggression when cocultured with dispersed rat islet cells as early as day 4 after mld-SZ administration. This capacity persisted throughout the experimental period. As early as 6 days after mld-SZ, islets showed insulitis followed by cell death with progressive severity. Hyperglycemia and diminished insulin secretion from perifused pancreatic islets only appeared at day 9 after mld-SZ. CONCLUSIONS: This study suggests that transferred or cocultured MSs from mld-SZ mice exert a functional immune aggression against beta cells at a very early stage, before donor mice develop impaired insulin secretion and hyperglycemia.
Assuntos
Autoimunidade/efeitos dos fármacos , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Hiperglicemia/imunologia , Células Secretoras de Insulina/imunologia , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Baço/citologia , Baço/imunologia , Estreptozocina/farmacologiaRESUMO
La talla baja idiopática (TBI) incluye a un grupo heterogéneo de pacientes con fallas en su crecimiento. Una causa probable de TBI puede ser la insensibilidad a la GH (IGH). La proteína de unión de GH de alta afinidad (GHBP) se genera por el clivaje proteolítico de la porción extracelular del receptor de GH (GHR) y su determinación se propone como un marcador periférico del nivel de GHR en los tejidos. El objetivo de este trabajo fue evaluar los niveles de GHBP circulantes y su asociación con factores de crecimiento y el polimorfismo del exón 3 del gen GHR en niños con TBI. Los niños con TBI presentaron talla, IMC, IGF-I, IGFBP-3, ALS y niveles de GHBP significativamente más bajos que un grupo de niños de edad comparable (p<0.001). El genotipo del exón 3 del GHR no fue un factor determinante de las diferencias observadas. La máxima respuesta de GH de los tests de estímulo de secreción correlacionó negativa y significativamente con los niveles de GHBP (r= -0.28, p= 0.012). Los perfiles de distribución de la concentración de GHBP, IGF-I, ALS y BP3 expresadas en score de desvío estándar (SDE) en la TBI, mostraron un sesgo hacia niveles bajos. En conclusión, los marcadores de acción de GH y los niveles de GHBP fueron bajos en la TBI, independientemente del genotipo del exón 3 del gen GHR. En un subgrupo de niños con TBI, niveles disminuidos de GHBP y de componentes del sistema de los IGFs, colaborarían en la evaluación de la IGH sugiriendo la búsqueda de defectos en el GHR.
Idiopathic Short Stature (ISS) includes a heterogeneous group of children with growth failure. One possible explanation for the growth failure is a reduced responsiveness to growth hormone (GH). Human circulating GH is partially bound to a highaffinity binding protein (GHBP) which is derived from proteolytical cleavage of the extracellular domain of the GH receptor. Many reports have demonstrated a close relationship between GHBP and liver GH receptor status in physiological conditions and diseases. Moreover, serum GHBP measurement has been proposed as an useful peripheral index of GH receptor abundance. Our objective was the evaluation of serum GHBP levels and its probable association with serum growth factors (IGF-I, IGFBP-3 and ALS) and the exon 3 polymorphism of the extracellular domain of the GHR gene in ISS children. Children with ISS presented significantly lower height SDS, BMI SDS, serum components of the IGFs system and GHBP concentration as compared to an age-matched control group of normal children (p<0.001). Interestingly, exon 3 genotype did not influence the differences observed in these parameters. The maximal GH response obtained after two GH provocative tests inversely and significantly correlated to GHBP serum levels (r= -0.28, p= 0.012). A frequency study showed a deviation to low SDS values of serum GHBP, IGF-I, IGFBP-3 and ALS. Conclusion: 1- in children with ISS the exon 3 genotype of the GHR gene is not a factor that could explain the lower levels observed in circulating GHBP concentration and components of the IGFs system; 2- low serum GHBP together with low IGF-I, IGFBP-3 or ALS levels would help pointing to GH insensitivity due to GH receptor gene abnormalities in ISS.
Assuntos
Humanos , Masculino , Feminino , Pré-Escolar , Criança , Adolescente , Hormônio do Crescimento Humano/biossíntese , Insuficiência de Crescimento/etiologia , Biomarcadores/metabolismo , Hormônio do Crescimento Humano/genética , Peptídeos e Proteínas de Sinalização Intercelular/genéticaRESUMO
Rats fed a sucrose-rich diet (SRD) develop hypertriglyceridemia and a marked decline in beta cell function. The purpose of this study was to determine whether changes in triglyceride concentration and/or altered pyruvate dehydrogenase complex (PDHc) activity contribute to the beta cell dysfunction, and to analyze the effect of dietary fish oil on the altered patterns of insulin secretion and peripheral insulin resistance. Rats were fed an SRD for 210 d. One-half of the rats continued consuming the SRD until d 270. The other half received an SRD in which fish oil (FO) was partially substituted for corn oil until d 270. A group of rats was fed a control diet (CD) throughout the experiment. The islets of rats fed the SRD had a greater triglyceride concentration and lower PDHc activity than those fed the CD. Insulin secretion patterns under the stimulus of glucose, palmitate or L-arginine were impaired in SRD-fed compared with CD-fed rats. This was accompanied by peripheral insulin resistance, mild hyperglycemia, a sharp increase of plasma triglyceride and free fatty acid levels and greater epididymal and retroperitoneal fat weights. FO normalized and/or improved these variables. Our results indicate that the increased fat storage and decreased PDHc activity in the beta cells play a key role in the abnormal insulin secretion of rats chronically fed an SRD. This is consistent with the reversion of these alterations by dietary FO.
Assuntos
Sacarose Alimentar/administração & dosagem , Óleos de Peixe/farmacologia , Insulina/metabolismo , Metabolismo dos Lipídeos , Pâncreas/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ingestão de Energia , Técnica Clamp de Glucose , Hiperinsulinismo/metabolismo , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Concentração Osmolar , Perfusão , Ratos , Ratos Wistar , Triglicerídeos/metabolismoRESUMO
Feeding rats a sucrose rich diet (SRD) induces hypertriglyceridemia and insulin resistance. The purposes of this study were to determine the time course of changes in lipid and glucose metabolism in the gastrocnemius muscle, both in the basal state and after the euglycemic hyperinsulinemic clamp, in rats fed a SRD for 3, 15 or 30 wk, and to analyze the changes in glucose-stimulated insulin secretion from perifused isolated islets from SRD-fed rats and their relationships to peripheral insulin insensitivity. A control group of rats was fed a control diet (CD) for the same period of time. After 3 wk of consuming the SRD, long-chain acyl CoA (LCACoA) levels in muscle were greater than in rats fed the CD, an early indication of the disturbance of lipid metabolism. Neither glycogen storage nor glucose oxidation were impaired at this time. Moreover, the biphasic patterns of glucose-stimulated insulin secretion showed a marked increase in the first peak, which helped maintain normoglycemia in SRD-fed rats. After 15 or 30 wk of consuming the SRD, triglyceride and LCACoA levels in muscles were greater than in rats fed the CD. Glucose oxidation as well as insulin-stimulated glycogen synthase activity and glycogen storage were lower than in rats fed the CD. Moreover, the altered pattern of insulin secretion further deteriorated. This was accompanied by peripheral insulin resistance and moderate hyperglycemia. Our results indicate that the dyslipemia present in rats chronically fed a SRD may play an important role in the progressive deterioration of insulin secretion and sensitivity in this animal model.
Assuntos
Sacarose Alimentar/administração & dosagem , Glucose/metabolismo , Insulina/metabolismo , Metabolismo dos Lipídeos , Músculo Esquelético/metabolismo , Animais , Sacarose Alimentar/farmacologia , Resistência à Insulina , Secreção de Insulina , Masculino , Ratos , Ratos Wistar , Triglicerídeos/sangueRESUMO
En estudios previos en ratones C57BL/ KsJ mdb/mdb, observamos alteraciones en la secreción de insulina inducida por glucosa, y un menor efecto inhibitorio de la somatostatina sobre la secreción de insulina. En el presente trabajo se estudiaron los patrones de secreción de glucagon bajo estimulación con arginina-glucosa, en perfusión de cortes de páncreas de ratones con diabetes genética de 20 a 90 días de vida. También se investigó si las células alfa presentaban una disminuida sensibilidad a la somatostatina. Los resultados muestran que: a) en ratones mdb/mdb de 20 a 90 días, los patrones de secreción de glucagon presentan hipersecreción basal y un primer pico disminuido; b) la somatostatina inhibe la secreción estimulada de glucagon por debajo de los valores basales en ratones mdb/mdb de 20 a 30 días de edad. En estadios posteriores (40 a 90 días de vida), la somatostatina ejerce un menor efecto inhibitorio, ya que los niveles de glucagon permanecen por en cima de los valores basales. Esto puede indicar un progresivo deterioro en la sensibilidad de las células alfa a la somatostatina, hecho que fue previamente observado para las células beta
Assuntos
Camundongos , Animais , Masculino , Feminino , Diabetes Mellitus Experimental/fisiopatologia , Glucagon/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Somatostatina/farmacologia , Fatores Etários , Camundongos Endogâmicos C57BL , Pâncreas/metabolismo , PerfusãoRESUMO
En estudios previos en ratones C57BL/ KsJ mdb/mdb, observamos alteraciones en la secreción de insulina inducida por glucosa, y un menor efecto inhibitorio de la somatostatina sobre la secreción de insulina. En el presente trabajo se estudiaron los patrones de secreción de glucagon bajo estimulación con arginina-glucosa, en perfusión de cortes de páncreas de ratones con diabetes genética de 20 a 90 días de vida. También se investigó si las células alfa presentaban una disminuida sensibilidad a la somatostatina. Los resultados muestran que: a) en ratones mdb/mdb de 20 a 90 días, los patrones de secreción de glucagon presentan hipersecreción basal y un primer pico disminuido; b) la somatostatina inhibe la secreción estimulada de glucagon por debajo de los valores basales en ratones mdb/mdb de 20 a 30 días de edad. En estadios posteriores (40 a 90 días de vida), la somatostatina ejerce un menor efecto inhibitorio, ya que los niveles de glucagon permanecen por en cima de los valores basales. Esto puede indicar un progresivo deterioro en la sensibilidad de las células alfa a la somatostatina, hecho que fue previamente observado para las células beta (AU)
Assuntos
Camundongos , Animais , Masculino , Feminino , Diabetes Mellitus Experimental/fisiopatologia , Glucagon/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Somatostatina/farmacologia , Pâncreas/metabolismo , Perfusão , Fatores Etários , Camundongos Endogâmicos C57BLRESUMO
En este trabajo se hace una introducción sobre aspectos de la fisiopatología de la DMID, cuyos conocimientos son el fundamento para comprender y aplicar estrategias de predicción y prevención [ptes.1 y 2]. Se comentan luego, en forma breve, conceptos generales sobre predicción y prevención [ptes.4 y 5]. En la parte 6 se describen y discuten los resultados obtenidos en nuestro laboratorio. Los mismos fueron divididos en 3 subgrupos: a) Sobre modelos experimentales aplicables en estudios sobre predicción y prevención: el modelo de diabetes por transferencia de células mononucleares [CMN] de ratón diabético por múltiples dosis bajas de SZ [mdb-SZ] a ratón singéneico normal ha sido de gran utilidad en estudios posteriores de los puntos B) y C). Por otra parte creemos que el otro modelo descripto [diabetes por transferencia de CMN de niños con DMID a ratón atímico normal] tiene una gran importancia por el hecho de usar CMN humanas y poder concentrar dichas células CMN [agresoras y/o protectoras] en forma selectiva en el páncreas del ratón receptor. b) Sobre predicción: más allá del diagnóstico preclínico individual o en grupos de riesgo ya conocidos, nuestros estudios en este punto se centraron en la búsqueda de nuevos grupos de riesgo de padecer DMID. En ese aspecto se describió un nuevo grupo, niños no diabéticos con intolerancia esporádica a la glucosa sin antecedentes familiares de diabetes. También se observó que pacientes con diabetes no insulino dependiente con fallo secundario a los hipoglucemiantes orales, presentan, en alrededor de un 30 por ciento de los casos, agresión inmune celular anti-célula beta pancreática [AIC]. c) Sobre prevención: aquí se utilizaron 3 estrategías diferentes: c.1- la administración de CMN diabéticas modificadas, previo a la transferencia de células diabéticas agresoras, protegió contra las alteraciones en la tolerancia a la glucosa y la secreción de insulina [SI]. Sin embargo creemos que el resultado más significativo es la protección por medio de este mecanismo de vacuna cuando la agresión anti-célula beta es mucho más importante como en el modelo de diabetes por inyección de mdb-SZ... (TRUNCADO)(AU)
Assuntos
Humanos , Animais , Animais de Laboratório , Diabetes Mellitus Tipo 1 , Dieta para Diabéticos , Ilhotas Pancreáticas , Teste de Tolerância a Glucose , InsulinaRESUMO
En este trabajo se hace una introducción sobre aspectos de la fisiopatología de la DMID, cuyos conocimientos son el fundamento para comprender y aplicar estrategias de predicción y prevención [ptes.1 y 2]. Se comentan luego, en forma breve, conceptos generales sobre predicción y prevención [ptes.4 y 5]. En la parte 6 se describen y discuten los resultados obtenidos en nuestro laboratorio. Los mismos fueron divididos en 3 subgrupos: a) Sobre modelos experimentales aplicables en estudios sobre predicción y prevención: el modelo de diabetes por transferencia de células mononucleares [CMN] de ratón diabético por múltiples dosis bajas de SZ [mdb-SZ] a ratón singéneico normal ha sido de gran utilidad en estudios posteriores de los puntos B) y C). Por otra parte creemos que el otro modelo descripto [diabetes por transferencia de CMN de niños con DMID a ratón atímico normal] tiene una gran importancia por el hecho de usar CMN humanas y poder concentrar dichas células CMN [agresoras y/o protectoras] en forma selectiva en el páncreas del ratón receptor. b) Sobre predicción: más allá del diagnóstico preclínico individual o en grupos de riesgo ya conocidos, nuestros estudios en este punto se centraron en la búsqueda de nuevos grupos de riesgo de padecer DMID. En ese aspecto se describió un nuevo grupo, niños no diabéticos con intolerancia esporádica a la glucosa sin antecedentes familiares de diabetes. También se observó que pacientes con diabetes no insulino dependiente con fallo secundario a los hipoglucemiantes orales, presentan, en alrededor de un 30 por ciento de los casos, agresión inmune celular anti-célula beta pancreática [AIC]. c) Sobre prevención: aquí se utilizaron 3 estrategías diferentes: c.1- la administración de CMN diabéticas modificadas, previo a la transferencia de células diabéticas agresoras, protegió contra las alteraciones en la tolerancia a la glucosa y la secreción de insulina [SI]. Sin embargo creemos que el resultado más significativo es la protección por medio de este mecanismo de vacuna cuando la agresión anti-célula beta es mucho más importante como en el modelo de diabetes por inyección de mdb-SZ... (TRUNCADO)