RESUMEN
A major challenge in the development of a gene therapy for hemophilia A (HA) is the selection of cell type- or tissue-specific promoters to ensure factor VIII (FVIII) expression without eliciting an immune response. As liver sinusoidal endothelial cells (LSECs) are the major FVIII source, understanding the transcriptional F8 regulation in these cells would help optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression. In silico analyses predicted several binding sites (BS) for the E26 transformation-specific (Ets) transcription factors Ets-1 and Ets-2 in the pF8. Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets-1/Est-2 combination, while Ets2 alone was ineffective. Moreover, Ets-1/Ets-2-DNA binding domain mutants (DBD) abolished promoter activation only when the Ets-1 DBD was removed, suggesting that pF8 up-regulation may occur through Ets-1/Ets-2 interaction with Ets-1 bound to DNA. pF8 carrying Ets-BS deletions unveiled two Ets-BS essential for pF8 activity and response to Ets overexpression. Lentivirus-mediated delivery of GFP or FVIII cassettes driven by the shortened promoters led to GFP expression mainly in endothelial cells in the liver and to long-term FVIII activity without inhibitor formation in HA mice. These data strongly support the potential application of these promoters in HA gene therapy.
Asunto(s)
Factor VIII , Hemofilia A , Animales , Células Endoteliales , Factor VIII/genética , Terapia Genética , Hemofilia A/genética , Hemofilia A/terapia , Lentivirus/genética , RatonesRESUMEN
BACKGROUND: Nutrition and growth in early postnatal life have a role in future diseases. Our aim was to investigate adiponectin oligomers in adequate-for-gestational-age obese children with respect to type and duration of feeding in the first year of life. METHODS: Adiponectin oligomers and cardiometabolic risk factors were measured in 113 adequate-for-gestational-age obese children, divided into group A (prolonged breast feeding, >6 mo), group B (short breast feeding, 1-6 mo), and group C (formula feeding from birth). RESULTS: All the parameters were similar among the groups. Adiponectin oligomers did not correlate with gestational age, months of breast feeding, and time of weaning. Total and high-molecular weight adiponectin were differently distributed across gender and pubertal stages (P < 0.02), being lower in males from the start of puberty. Prepregnancy BMI and at the end of the pregnancy were negatively associated (P < 0.04) with total and medium-molecular weight adiponectin in female and male offspring, respectively. CONCLUSIONS: Adiponectin oligomers and metabolic characteristics are similarly distributed in adequate-for-gestational-age obese children, irrespective of the type and duration of the feeding in the first year of life. Gender and mother's BMI in pregnancy are contributors to adiponectin regulation. Further studies will explain whether breastfeeding protects against metabolic impairment later in life.
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Adiponectina/metabolismo , Desarrollo Infantil/fisiología , Fenómenos Fisiológicos Nutricionales del Lactante , Obesidad/metabolismo , Adiponectina/genética , Índice de Masa Corporal , Estudios Transversales , Femenino , Edad Gestacional , Humanos , Lactante , Masculino , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Idiopathic Short Stature (ISS) defines a condition in which height is <-2SD compared to the mean of a reference population where systemic, endocrinological, nutritional or chromosomal disorders have not been identified and diagnosis is based on exclusion of any known causes of short stature. JAK/STAT pathway is triggered by GH binding to the GH receptor and promotes cellular growth through transcription of GH-responsive genes. In order to identify "candidate genes" differently expressed in ISS subjects with respect to control ones, we analyzed the expression of 84 genes related to JAK/STAT pathway by RT(2) Profiler PCR array approach in a total of 10 subjects. Then, we validated the observed data by Real Time PCR and ELISA assays in a major number of subjects. We found two genes that were differently expressed in ISS subjects with respect to the control group: CXCL9 and FCGR1A/CD64, both significantly up-regulated (fold change 2.17 and 1.70, respectively) and belonging to family of IFN-γ-inducible factors. Further, ISS subjects showed an increased gene expression of IFN-γ and IFI16, higher serum levels of IFN-γ but similar levels of CXCL9 when compared to healthy subjects. In addition, we showed a pubertal modulation of CXCL9 levels. These data suggest that inflammatory and regulatory factors of the cell cycle may be involved in the ISS condition, introducing a new perspective to its etiology.
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Enanismo Hipofisario/metabolismo , Inflamación/metabolismo , Adolescente , Ciclo Celular/fisiología , Quimiocina CXCL9/metabolismo , Quimiocinas/metabolismo , Niño , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Reacción en Cadena de la Polimerasa , Receptores de IgG/genética , Receptores de IgG/metabolismoRESUMEN
Isolated GH deficiency (IGHD) is a rare disorder that occurs as an idiopathic form in most cases. The pathway JAK/STAT promotes cellular growth and it could be implicated in this condition. In order to characterize IGHD in the pediatric population and identify genes differently expressed before and after GH therapy, we performed a quantitative evaluation of 84 genes related to the JAK/STAT pathway which, by promoting cellular growth. RT(2) Profiler PCR Array and the other/subsequent evaluations were performed in three children with severe IGHD before and after 6 months of GH therapy and in three matched normal children. Gene profiling was modified by the IGHD status and the GH therapy, with a modulation of GHR and some inflammatory genes such as CRP. We found a heterozygous nonsense mutation R43X in the GHR gene in two out of three IGHD subjects, despite a good response to therapy. After therapy cardiovascular markers linked to genes as IL6, IL8 and TNF-α displayed a trend toward reduction. Pre- and post therapy status differently affects gene expression. Mutational screening of GHR may be useful in investigating IGHD's etiology. Genes linked to inflammation suggest to evaluate cardiovascular risks also in pediatric IGHD subjects.
Asunto(s)
Enanismo Hipofisario/tratamiento farmacológico , Enanismo Hipofisario/metabolismo , Hormona de Crecimiento Humana/uso terapéutico , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Adolescente , Proteínas Portadoras/genética , Niño , Enanismo Hipofisario/genética , Femenino , Terapia de Reemplazo de Hormonas , Hormona de Crecimiento Humana/deficiencia , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Quinasas Janus/genética , Masculino , Mutación , Factores de Transcripción STAT/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Trousseau sign was the first demonstration of a close relationship between cancer and thrombosis. Currently, venous thromboembolism (VTE) is five to six times more likely to occur in cancer patients, whereas there is a greater risk of cancer diagnoses following thromboses. In considering novel players, factor VIII (FVIII), an essential coagulation cofactor with emerging extracoagulative functions, has been identified as an independent VTE risk factor in cancer; however, the basis of this increase is unknown. OBJECTIVE: To investigate the possible direct expression and secretion of FVIII by cancer cells. METHODS: Bladder cancer, with a high VTE risk, and normal bladder tissue and epithelium, were used to investigate FVIII. Factor VIII protein and secretion were examined in bladder cancer cell lines. Expanding to other cancers, the Cancer Cell line Encyclopedia database was used to analyze FVIII, tissue factor, FV, FVII, FIX, FX, and von Willebrand factor (VWF) mRNA in 811 cell lines subdivided according to origin. Factor VIII protein synthesis, secretion, and bioactivity were investigated in a profile of cancer cell lines of differing origins. RESULTS AND CONCLUSIONS: Although expressed in the normal bladder epithelium, FVIII mRNA and protein were higher in matched bladder neoplasms, with synthesis and secretion of bioactive FVIII evident in bladder cancer cells. This can be extended to other cancer cell lines, with a pattern reflecting the tumor origin, and that is independent of VWF and other relevant players in the coagulation cascade. Here, evidence is provided of a possible independent role for FVIII in cancer-related pathophysiology.
Asunto(s)
Factor VIII/metabolismo , Hemostáticos , Neoplasias , Coagulación Sanguínea , Factor VIII/genética , Humanos , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: We have identified a synonymous F8 variation in a severe hemophilia A (HA) patient who developed inhibitors following factor VIII (FVIII) prophylaxis. The unreported c.6273 G > A variant targets the consensus splicing site of exon 21. OBJECTIVES: To determine the impact of c.6273 G > A nucleotide substitution on F8 splicing and its translated protein. METHODS: Patient peripheral blood mononuclear cells were isolated and differentiated into monocyte-derived macrophages (MDMs). FVIII distribution in cell compartments was evaluated by immunofluorescence. The splicing of mutated exon 21 was assessed by exon trapping. Identified FVIII splicing variants were generated by site-directed mutagenesis, inserted into a lentiviral vector (LV) to transduce Chinese hamster ovary (CHO) cells, and inject into B6/129 HA-mice. FVIII activity was assessed by activated partial thromboplastin time, whereas anti-FVIII antibodies and FVIII antigen, by ELISA. RESULTS: HA-MDMs demonstrated a predominant retention of FVIII around the endoplasmic reticulum. Exon trapping revealed the production of two isoforms: one retaining part of intron 21 and the other skipping exon 21. These variants, predicted to truncate FVIII in the C1 domain, were detected in the patient. CHO cells transduced with the two FVIII transcripts confirmed protein retention and absence of the C2 domain. HA mice injected with LV carrying FVIII mutants, partially recovered FVIII activity without the appearance of anti-FVIII antibodies. CONCLUSIONS: Herein, we demonstrate the aberrant impact of a FVIII synonymous mutation on its transcription, activity, and pathological outcomes. Our data underline the importance of increasing the knowledge regarding the functional consequences of F8 mutations and their link to inhibitor development and an effective replacement therapy.
Asunto(s)
Hemofilia A , Animales , Células CHO , Cricetinae , Cricetulus , Factor VIII/genética , Factor VIII/metabolismo , Hemofilia A/genética , Humanos , Leucocitos Mononucleares/metabolismo , Ratones , Empalme del ARNRESUMEN
Several mutations in the melanocortin receptor 4 gene have been identified in humans and account for 3-6% of morbid obesity. In contrast, strong evidence of a causative role for melanocortin receptor 3 (MC3R) mutations are still lacking. In MC3R knockout mice, high feed efficiency rather than hyperphagia seems to contribute to increased fat mass. On the basis of this evidence, the objective of the present study was to investigate the presence of MC3R mutations in a group of 290 obese subjects (mean BMI 44.2+/-5.9 kg/m2). As a control, a group of 215 normal-weight subjects (mean BMI 22.4+/-2.7 kg/m2) was also screened. Three novel mutations in the MC3R gene (A293T, I335S and X361S) were identified among the obese patients. The mutations segregated with obesity in the members of the families studied. In vitro expression studies of each mutation demonstrated a loss of function of the I335S-mutated receptor. These findings suggest that, in humans, MC3R mutations may be a cause of a dominantly inherited form of obesity. However, this association as well as the specific phenotypic characteristics resulting from these mutations need to be further evaluated in larger series of obese subjects.
Asunto(s)
Mutación , Obesidad Mórbida/genética , Receptor de Melanocortina Tipo 3/genética , Adulto , Anciano , Animales , Células COS , Estudios de Casos y Controles , Chlorocebus aethiops , AMP Cíclico/metabolismo , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje , Receptor de Melanocortina Tipo 3/metabolismoRESUMEN
Vitamin D (VD) deficiency (VDD) correlates to obesity, with VD a recognized mediator of metabolic diseases. From a previous proteomic study identifying adiponectin as a link between VDD and pediatric obesity, herein we analysed another protein (SSP2301) increased with VDD. A focused 2D-electrophoretic analysis identified 4 corresponding plasma proteins, with one predicted to be fetuin B (FETUB). FETUB was studied due to its emerging role in metabolic diseases and cytogenetic location (3q27.3) with adiponectin. Results were confirmed in obese children, where plasma FETUB was higher with VDD. A direct effect by 1α,25-(OH)2D3 on hepatocellular FETUB synthesis was observed, with a time and dose dependent reduction. Further, we demonstrated the VD-receptor (VDR) is key, with FETUB "released" with VDR silencing. Finally, VD supplementation (6weeks) to juvenile mice fed a standard diet, reduced plasma FETUB. Only at 22weeks did liver FETUB correspond to plasma FETUB, highlighting the contribution of other VD-responsive tissues. Overall, FETUB is a key protein linking VDD to pediatric obesity. With an emerging role in metabolic diseases, we demonstrate that VD/VDR directly regulate FETUB.
Asunto(s)
Fetuína-B/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Obesidad Infantil/complicaciones , Deficiencia de Vitamina D/complicaciones , Vitamina D/farmacología , Adolescente , Animales , Niño , Preescolar , Fetuína-B/genética , Células Hep G2 , Humanos , Ratones , Ratones Endogámicos C57BL , Obesidad Infantil/tratamiento farmacológico , Obesidad Infantil/metabolismo , Proteómica , Receptores de Calcitriol/metabolismo , Estudios Retrospectivos , Deficiencia de Vitamina D/tratamiento farmacológico , Deficiencia de Vitamina D/metabolismo , Vitaminas/farmacologíaRESUMEN
Obesity predisposes to vitamin D deficiency (VDD) and glucose abnormalities. It is currently debated if vitamin D administration may improve glucose homeostasis by interacting with modulators of insulin sensitivity, such as adiponectin and its oligomers. In a 4-week inpatient study on a metabolic rehabilitation program, consisting of individualized caloric restriction and aerobic physical exercise in obese subjects with VDD, we assessed the acute effects of 600,000 IU cholecalciferol given per os VD group, 12 subjects; body mass index (BMI) 42.7 ± 1.3 kg/m²) or placebo per os (PL group, 12 subjects, BMI 39.8 ± 0.9 kg/m²) on high (HWM-A), medium (MMW-A), and low molecular weight adiponectin (LMW-A), as quantified by western immunoblot (WIB) and ELISA. During the 4-week study, dieting promoted a similar magnitude of weight loss in VD and PL groups. Compared to the PL group, cholecalciferol administration increased 25(OH)Vit D levels (p < 0.001) and promoted a significant increase of HMW-A expression analyzed by WIB (p = 0.02). In parallel, a significant decrease of leptin/HMW-A ratio (p < 0.05), a biomarker of metabolic homeostasis, was observed. During the study, changes of MMW-A and LMW-A occurred independently of cholecalciferol administration, and were likely explained by weight loss. At odds with these findings, the ELISA assessment of adiponectin oligomers showed no modifications in the VD group or PL group. Current findings suggest that acute cholecalciferol administration selectively modifies HMW-A and the leptin/HMW-A ratio.
Asunto(s)
Adiponectina/sangre , Colecalciferol/administración & dosificación , Obesidad Mórbida/sangre , Obesidad Mórbida/tratamiento farmacológico , Adulto , Índice de Masa Corporal , Restricción Calórica , Colecalciferol/sangre , Ejercicio Físico , Femenino , Humanos , Resistencia a la Insulina , Leptina/sangre , Masculino , Peso Molecular , Método Simple Ciego , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/tratamiento farmacológico , Pérdida de PesoRESUMEN
Insulin-like growth factor-binding protein (IGFBP)-3 has been shown to potently inhibit cell proliferation in various cell systems. However, the specific mechanisms involved in the antiproliferative action of IGFBP-3 have yet to be elucidated. In the present study, we demonstrate that IGFBP-3 induces apoptosis in an insulin-like growth factor (IGF)-independent manner through the activation of caspases involved in a death receptor-mediated pathway in MCF-7 human breast cancer cells. Induction of IGFBP-3 using an ecdysone-inducible expression system inhibited DNA synthesis in an IGF-IGF receptor axis-independent fashion and resulted in the subsequent induction of apoptosis and an increase in caspase activity. Similar results were obtained when cells were transfected with GGG-IGFBP-3, an IGFBP-3 mutant unable to bind IGFs, corroborating the IGF-independent action of IGFBP-3. Additional caspase activity studies and immunoblot analyses using specific caspase substrates and/or caspase inhibitors revealed that the growth-inhibitory effect of IGFBP-3 results mainly from its induction of apoptosis (in particular, activation of caspase-8 and -7). Analyses of caspase-9 activity and release of cytochrome c into the cytosol confirmed that the mitochondria-mediated pathway is not involved. Taken together, these results show that IGFBP-3 expression leads to the induction of apoptosis through the activation of caspases involved in a death receptor-mediated pathway and that IGFBP-3 functions as a negative regulator of breast cancer cell growth, independent of the IGF-IGF receptor axis.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Caspasas/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Transducción de Señal/efectos de los fármacos , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Citocromos c/metabolismo , Citosol/metabolismo , ADN de Neoplasias/metabolismo , Ecdisona/farmacología , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor II del Crecimiento Similar a la Insulina/farmacología , Mitocondrias/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Ghrelin is a gastric hormone that exerts a stimulatory effect on appetite and fat accumulation. Ser(3) octanoylation is regarded as a prerequisite for ghrelin biological activity, although des-octanoylated forms may retain biological functions in vitro. Circulating ghrelin levels are usually low in obesity and in states of positive energy balance. Hence, the aim of our study was to analyze plasma active and serum total ghrelin levels in 20 obese (ages, 22-42 yr; body mass index, 41.3 +/- 1.1 kg/m(2)) and 20 lean subjects (ages, 22-43 yr; body mass index, 22.4 +/- 0.6 kg/m(2)) as well as their relationship to measures of glucose homeostasis, body fat, and resting energy expenditure (REE). The measured/predicted REE percentage ratio was calculated to subdivide groups into those with positive (> or = 100% ) and negative (<100%) ratio values. In obese patients, plasma active (180 +/- 18 vs. 411 +/- 57 pg/ml; P < 0.001) and serum total ghrelin levels (3650 +/- 408 vs. 5263 +/- 643 pg/ml; P < 0.05) were significantly lower when compared with lean subjects. Hence, ghrelin activity, defined as the proportion of active over total ghrelin levels, was similarly reduced in the obese state (6.1 +/- 0.9% vs. 8.4 +/- 1%; P < 0.05). There was a significant correlation between active and total ghrelin (r = 0.62; P < 0.001), and between total ghrelin and insulin (r = -0.53; P < 0.001) or insulin resistance using the homeostatis model of assessment-insulin resistance (r = -0.49; P < 0.001) approach. Significantly higher active ghrelin levels (214 +/- 22 vs. 159 +/- 30 pg/ml; P < 0.05) and ghrelin activity (8 +/- 1.7% vs. 4.9 +/- 0.9%; P < 0.05) were observed in patients with positive compared with negative measured/predicted REE ratio values. Our study shows that obesity is associated with an impairment of the entire ghrelin system. The observation that ghrelin is further decreased in cases of abnormal energy profit adds new evidence to the relationship between ghrelin activity and energy balance in obesity.
Asunto(s)
Metabolismo Energético , Obesidad/sangre , Hormonas Peptídicas/sangre , Adulto , Estudios de Casos y Controles , Femenino , Ghrelina , Homeostasis , Humanos , Insulina/sangre , Resistencia a la Insulina , Masculino , Obesidad/fisiopatología , DescansoRESUMEN
Obesity is currently the most important contributor to ill health and expenditure worldwide. More alarming is the fact that the pediatric population parallels adults, with obesity closely associated to type 2 diabetes mellitus (T2D), cardiovascular disease, hypertension, non-alcoholic fatty liver disease, vitamin D deficiency (VDD) and certain types of cancer. The observation in the early 1950s that android or truncal adipose tissue (AT) distribution compared to gynoid had a greater association with metabolic dysfunction, in particular T2D and cardiovascular disease risk, led to the hypothesis that obesity-associated complications are not associated with fat mass per se, but the pattern of fat distribution. This concept was further supported by groups of individuals with metabolic dysfunction despite a lean phenotype, and healthy obese people protected from metabolic dysfunction. It is now well recognized that an increase in visceral AT is an independent risk factor for the development of obesity-associated comorbidities with AT depot distribution, their anatomic, cellular and molecular features defining their role. The differences and the plasticity of subcutaneous, visceral and ectopic ATs to store and release fatty acids and to synthesize and secrete adipokines, defines the metabolic outcomes. The present review will examine the phenotypic and pathophysiological differences between the different AT depots, with a particular focus on the abdominal depots and their link to metabolic complications.
Asunto(s)
Grasa Abdominal/patología , Grasa Abdominal/fisiopatología , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/patología , Enfermedades Cardiovasculares/fisiopatología , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/fisiopatología , Humanos , Resistencia a la Insulina , Obesidad/complicaciones , Obesidad/patología , Obesidad/fisiopatología , Factores de RiesgoRESUMEN
Key circulating molecules that link vitamin D (VD) to pediatric obesity and its co-morbidities remain unclear. Using a proteomic approach, our objective was to identify key molecules in obese children dichotomized according to 25OH-vitamin D (25OHD) levels. A total of 42 obese children (M/Fâ=â18/24) were divided according to their 25OHD3 levels into 25OHD3 deficient (VDD; nâ=â18; 25OHD<15 ng/ml) or normal subjects (NVD; nâ=â24; >30 ng/ml). Plasma proteomic analyses by two dimensional (2D)-electrophoresis were performed at baseline in all subjects. VDD subjects underwent a 12mo treatment with 3000 IU vitamin D3 once a week to confirm the proteomic analyses. The proteomic analyses identified 53 "spots" that differed between VDD and NVD (p<0.05), amongst which adiponectin was identified. Adiponectin was selected for confirmational studies due to its tight association with obesity and diabetes mellitus. Western Immunoblot (WIB) analyses of 2D-gels demonstrated a downregulation of adiponectin in VDD subjects, which was confirmed in the plasma from VDD with respect to NVD subjects (p<0.035) and increased following 12mo vitamin D3 supplementation in VDD subjects (p<0.02). High molecular weight (HMW) adiponectin, a surrogate indicator of insulin sensitivity, was significantly lower in VDD subjects (p<0.02) and improved with vitamin D3 supplementation (p<0.042). A direct effect in vitro of 1α,25-(OH)2D3 on adipocyte adiponectin synthesis was demonstrated, with adiponectin and its multimeric forms upregulated, even at low pharmacological doses (10(-9) M) of 1α,25-(OH)2D3. This upregulation was paralleled by the adiponectin interactive protein, DsbA-L, suggesting that the VD regulation of adiponectin involves post-transciptional events. Using a proteomic approach, multimeric adiponectin has been identified as a key plasma protein that links VDD to pediatric obesity.
Asunto(s)
Adiponectina/metabolismo , Obesidad Infantil/metabolismo , Deficiencia de Vitamina D/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adiponectina/química , Adolescente , Animales , Biomarcadores , Niño , Preescolar , Colecalciferol/uso terapéutico , Femenino , Humanos , Masculino , Ratones , Multimerización de Proteína , Proteómica/métodos , Deficiencia de Vitamina D/tratamiento farmacológicoRESUMEN
While visceral adipose tissue (VAT) associates to obesity, there is debate for subcutaneous adipose tissue (SAT). One explanation may be SAT subcompartments, superficial-SAT (sSAT) and deep-SAT (dSAT), recently recognized as independent depots. Our aim was to establish roles for sSAT/dSAT with obesity by examining the expression of proteins key to adipocyte metabolism. Paired biopsies from sSAT and dSAT of 10 normal-weight (BMI 21.8 ± 0.8 kg/m(2)) and 11 obese subjects (BMI 44 ± 2.1 kg/m(2)) were analyzed for differences in insulin sensitivity using adiponectin, GLUT4 and resistin, glucocorticoid metabolism by 11ßHSD1 and alterations of the adipokines leptin and TNFα. Between lean and obese subjects, sSAT and dSAT changes for GLUT4, resistin and TNFα were equivalent. Resistin and TNFα increased in both obese SAT sub-compartments; 33-fold (sSAT; P < 0.006) and 18.5-fold (dSAT; P < 0.003) higher resistin, with undetectable in leans to significant TNFα levels in obese. In contrast, GLUT4 showed 5.5-fold (sSAT; P < 0.03) and 7-fold (dSAT; P < 0.03) lower levels in obese, correlating to BMI (r = -0.6423, P = 0.007) and HOMA-IR (r = -0.5882, P = 0.017). Exclusive sSAT-specific differences were observed for adiponectin, leptin, and 11ßHSD1. Both sSAT 11ßHSD1 and leptin increased in obese, with 11ßHSD1 2.5-fold (P = 0.052) and leptin 3.3-fold (P < 0.008) higher, with 11ßHSD1 correlating to HOMA-IR (r = 0.5203, P = 0.0323) and leptin to BMI (r = 0.5810, P = 0.01). In contrast, obese had 7-fold (P < 0.02) lower sSAT adiponectin, correlating to BMI (r = -0.5178, P = 0.027) and HOMA-IR (r = -0.4570, P = 0.049). Overall, sSAT and dSAT are distinct abdominal adipose tissue depots with independent metabolic functions. Between the two, sSAT shows clear independent effects that associate to obesity and its metabolic complications.
Asunto(s)
Biomarcadores/metabolismo , Obesidad/metabolismo , Grasa Subcutánea Abdominal/metabolismo , Adipocitos/metabolismo , Adipocitos/fisiología , Adipoquinas/metabolismo , Adulto , Biopsia , Femenino , Glucocorticoides/metabolismo , Glucosa/metabolismo , Humanos , Masculino , Persona de Mediana Edad , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The proinflammatory state of metabolic disorders encompasses the alterations in leukocyte counts and acute-phase reactants, and thus, predisposes to acute and chronic cardiovascular events linked to fat accumulation. Leptin is a marker of adiposity and also yields regulatory effects on innate and adaptive immunity; however, its role on the immune function of obese subjects remains to be elucidated. The aim of this study is to determine the influence of obesity and the role of leptin concentrations on lymphocyte counts and immunoglobulin levels as broad markers of immune function. Cross-sectional analysis in 147 obese (64 M, BMI 43 ± 8.1 kg/m(2)) and 111 age- and sex-matched controls (36 M, BMI 22.5 ± 2.6 kg/m(2)) by assessment of peripheral leukocyte counts, immunoglobulin (Ig) A, G, M levels, leptin, glucose and lipid homeostasis, and acute-phase reactants. Compared to controls, all the leukocyte components were significantly increased in obesity (p < 0.0001 for all) except for basophils and eosinophils. While IgA and IgG levels were similar between groups, IgM levels were lower (p < 0.001) in obese individuals. A significant relationship was evident between leptin and leukocyte counts (p < 0.001), with this latter being correlated to insulin resistance, adiposity, and lipid profile. At the stepwise multiple regression analysis, leukocytes were best predicted by leptin (ß = 0.43, p < 0.0001) and male gender (ß = 0.15, p < 0.05), yet when obesity entered the equation, it acted as an independent predictor of leukocytes (ß = 0.51, p < 0.0001). Leptin also acted as a predictor of IgA levels (ß = 0.20, p < 0.01). Current results show that IgM levels are significantly decreased in patients with obesity in association to significant increments in leukocyte counts. These latter are markedly correlated to leptin levels, insulin resistance, lipid profile, and adiposity. This circumstance, and the significant correlation seen between leptin and IgA levels, may suggest an indirect intervention of leptin in the immunologic alterations consequent to obesity and related to its cardiovascular risk.
Asunto(s)
Inmunoglobulinas/sangre , Leptina/sangre , Linfocitos , Obesidad/sangre , Obesidad/inmunología , Adulto , Biomarcadores/sangre , Biomarcadores/metabolismo , Femenino , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Recuento de Linfocitos , Linfocitos/citología , Masculino , Persona de Mediana Edad , Obesidad Mórbida/sangre , Obesidad Mórbida/inmunologíaRESUMEN
High molecular weight (HMW-A) adiponectin levels mirror alterations in glucose homeostasis better than medium (MMW-A) and low molecular weight (LMW-A) components. In 25 patients with wide-range extreme obesity (BMI 40-77 kg/m(2)), we aimed to explore if improvements of multimeric adiponectin following 4-wk weight loss reflect baseline OGTT-derived insulin sensitivity (ISIOGTT) and disposition index (DIOGTT). Compared to 40 lean controls, adiponectin oligomers were lower in extreme obesity (p < 0.001) and, within this group, HMW-A levels were higher in insulin-sensitive (p < 0.05) than -resistant patients. In obese patients, short-term weight loss did not change total adiponectin levels and insulin resistance, while the distribution pattern of adiponectin oligomers changed due to significant increment of HMW-A (p < 0.01) and reduction of MMW-A (p < 0.05). By multivariate analysis, final HMW-A levels were significantly related to baseline ISIOGTT and final body weight (adjusted R(2) = 0.41). Our data suggest that HMW adiponectin may reflect baseline insulin sensitivity appropriately in the context of extreme obesity. Especially, we documented that HMW-A is promptly responsive to short-term weight loss prior to changes in insulin resistance, by a magnitude that is proportioned to whole body insulin sensitivity. This may suggest an insulin sensitivity-dependent control operated by HMW-A on metabolic dynamics of patients with extreme obesity.
Asunto(s)
Adiponectina/sangre , Resistencia a la Insulina , Obesidad Mórbida/sangre , Adiponectina/química , Adulto , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/sangre , Femenino , Homeostasis , Humanos , Insulina/sangre , Masculino , Persona de Mediana Edad , Obesidad Mórbida/dietoterapia , Estructura Cuaternaria de Proteína , Pérdida de PesoRESUMEN
Mutations affecting exon 3 splicing are the main cause of autosomal dominant Isolated GH Deficiency II (IGHDII) by increasing the level of exon 3-skipped mRNA encoding the functionally inactive dominant-negative 17.5-kDa isoform. The exons and introns of the gene encoding GH (GH1) were screened for the presence of mutations in 103 sporadic isolated GH deficiency cases. Four different variations within exon 3 were identified in 3 patients. One carried c.261C>T (p.Pro87Pro) and c.272A>T (p.Glu91Val), the second c.255G>A (p.Pro85Pro) and c.261 C>T, and the third c.246G>C (p.Glu82Asp). All the variants were likely generated by gene conversion from an homologous gene in the GH1 cluster. In silico analysis predicted that positions c.255 and c.272 were included within 2 putative novel exon splicing enhancers (ESEs). Their effect on splicing was confirmed in vitro. Constructs bearing these 2 variants induced consistently higher levels both of transcript and protein corresponding to the 17.5-kDa isoform. When c.255 and c.272 were combined in cis with the c.261 variant, as in our patients, their effect was weaker. In conclusion, we identified 2 variations, c.255G>A and c.272A>T, located in 2 novel putative exon splicing enhancers and affecting GH1 splicing in vitro by increasing the production of alternatively spliced isoforms. The amount of aberrant isoforms is further regulated by the presence in cis of the c.261 variant. Thus, our results evidenced novel putative splicing regulatory elements within exon 3, confirming the crucial role of this exon in mRNA processing.
Asunto(s)
Empalme Alternativo , Enanismo Hipofisario/genética , Hormona de Crecimiento Humana/genética , Mutación , Elementos de Respuesta , Adolescente , Sustitución de Aminoácidos , Línea Celular , Niño , Biología Computacional , Enanismo Hipofisario/metabolismo , Exones , Sistemas Especialistas , Conversión Génica , Hormona de Crecimiento Humana/química , Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/metabolismo , Humanos , Masculino , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Hipófisis/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Abdominal visceral tissue (VAT) and subcutaneous adipose tissue (SAT), comprised of superficial-SAT (sSAT) and deep-SAT (dSAT), are metabolically distinct. The antidiabetic agents thiazolidinediones (TZDs), in addition to their insulin-sensitizing effects, redistribute SAT suggesting that TZD action involves adipose tissue depot-specific regulation. We investigated the expression of proteins key to adipocyte metabolism on differentiated first passage (P1) preadipocytes treated with rosiglitazone, to establish a role for the diverse depots of abdominal adipose tissue in the insulin-sensitizing effects of TZDs. Adipocytes and preadipocytes were isolated from sSAT, dSAT, and VAT samples obtained from eight normal subjects. Preadipocytes (P1) left untreated (U) or treated with a classic differentiation cocktail (DI) including rosiglitazone (DIR) for 9 days were evaluated for strata-specific differences in differentiation including peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and lipoprotein lipase (LPL) expression, insulin sensitivity via adiponectin and glucose transport-4 (GLUT4), glucocorticoid metabolism with 11 beta-hydroxysteroid dehydrogenase type-1 (11 beta HSD1), and alterations in the adipokine leptin. While depot-specific differences were absent with the classic differentiation cocktail, with rosiglitazone sSAT had the most potent response followed by dSAT, whereas VAT was resistant to differentiation. With rosiglitazone, universal strata effects were observed for PPAR-gamma, LPL, and leptin, with VAT in all cases expressing significantly lower basal expression levels. Clear dSAT-specific changes were observed with decreased intracellular GLUT4. Specific sSAT alterations included decreased 11 beta HSD1 whereas secreted adiponectin was potently upregulated in sSAT with respect to dSAT and VAT. Overall, the subcompartments of SAT, sSAT, and dSAT, appear to participate in the metabolic changes that arise with rosiglitazone administration.