OBJECTIVE: In recent studies serum leptin levels were significantly higher in the luteal phase than in the follicular phase, but the mechanism of changing leptin levels are unknown. Several research lines indicate a potential role for tumor necrosis factor (TNF-alpha) in ovulation and reproductive events. As TNF-alpha appears to regulate leptin secretion, we speculated that TNF-alpha might be involved in leptin variations during the menstrual cycle. DESIGN AND METHODS: Nine healthy never obese and ten overweight normally cycling women were studied. TNF-alpha action - through the plasma levels of the soluble fraction of the tumor necrosis factor receptors 1 and 2 (sTNFR1 and sTNFR2) - and leptin concentrations were measured in the follicular (F), peri-ovulatory (PO) and luteal phases (L) of their menstrual cycles. RESULTS: Circulating leptin levels were significantly associated with the stage of the menstrual cycle (P<0.001), being higher in PO and L phases. However, only three of ten overweight subjects vs eight of nine lean women (Chi square P=0.014 after Fisher's exact test) showed significantly higher leptin levels in the PO and L than in the F phase (95% confidence interval (95% CI) of the differences, 3.7 to 10.2 ng/ml, paired t-test P=0.001). In these women (group 1), the changes in leptin levels parallelled the variations observed in plasma sTNFR1 (2.50+/-0.1 vs 2.11+/-0.05 ng/ml, P<0.0001, 95% CI, 0.21 to 0.56) and sTNFR2 levels (5.19+/-0.28 vs 4.55+/-0.25 ng/ml, P<0.0001, 95% CI, 0. 47 to 0.81). In the remaining women (group 2), leptin (95% CI, -1 to 9.2 ng/ml, P=not significant (NS)), sTNFR1 (95% CI, -0.3 to 0.14 ng/ml, P=NS) and sTNFR2 levels (95% CI, -0.95 to 0.39 ng/ml, P=NS) were essentially unaltered throughout the menstrual cycle. Group 2 women were similar in age (36.1+/-2.9 vs 37.3+/-1.4 years) and significantly overweight (body mass index 31+/-2.9 vs 23.9+/-1. 2 kg/m(2)) compared with group 1 women. A negative correlation was observed between leptin levels in the follicular phase and the change in plasma leptin from F to L phase in all subjects (r=-0.67, P=0.002). CONCLUSIONS: Circulating leptin and sTNFRs levels change significantly during the menstrual cycle of most lean women. In contrast, the levels of these molecules remain essentially unaltered during the F, PO and L phases in the majority of overweight women. Obesity might be associated not only with blunted diurnal excursions and dampened pulsatility, but also with blunted excursions during the menstrual cycle.
JM Fernandez-Real, C Gutierrez, J Vendrell, R Casamitjana and W Ricart
I Halperin, R Casamitjana, L Flores, M Fernandez-Balsells and E Vilardell
OBJECTIVE: The production of insulin-like growth factor binding protein-3 (IGFBP-3), the main IGF-I binding protein, is regulated by GH, and its serum levels are increased in acromegaly. We investigated its potential value as a parameter of acromegaly activity or remission in comparison with IGF-I, taking GH suppression below 2 microg/l after glucose load as the normal standard. METHODS: Data from 40 acromegalic patients (12 males and 28 females, aged 28 to 79 years) were obtained retrospectively from stored samples. From these, 145 pairs of IGF-I/IGFBP-3 values were collected; in 67 of them, simultaneous measurement of GH after glucose loading allowed their classification as active or inactive acromegaly. Relationships between IGF-I, IGFBP-3 and GH after glucose load were assessed, as well as differences between IGF-I and IGFBP-3 levels in active and inactive acromegaly. RESULTS: Significant positive correlation between IGF-I and IGFBP-3 in 145 samples was observed (r=0.49, P<0. 0001). As for the 67 samples in which activity or remission could be defined in terms of GH after glucose load, 50 were active and 17 inactive. Both IGF-I and IGFBP-3 significantly correlated with minimum GH (r=0.53, P<0.0001 and r=0.41, P<0.001 respectively). For both parameters, significant differences of means between active and inactive cases were observed (623+/-296 vs 300+/-108 ng/ml, P<0.0001 for IGF-I, and 4.1+/-1.3 vs 3.2+/-0.9 microg/ml, P<0.006 for IGFBP-3). Yet, when comparing in individual cases their classification as active or inactive with the finding of normal or increased IGF-I and IGFBP-3, among active cases 16% appeared as normal according to IGF-I, and 50% appeared as normal in terms of IGFBP-3. Among inactive cases, 23.5% appeared as active according to IGF-I, while 17.5% appeared as active in terms of IGFBP-3. CONCLUSION: Even though IGFBP-3 reflects GH secretion, it offers no advantage over IGF-I in the assessment of acromegaly, and it may underestimate disease activity in acromegalic patients.
JM Fernandez-Real, M Vayreda, R Casamitjana, F Gonzalez-Huix and W Ricart
OBJECTIVE: Recent experimental work in mice has demonstrated that leptin is synthesized by muscle cells. As this latter tissue is the main target for insulin-estimulated glucose disposal, we hypothesized that the muscular and fat-free mass (FFM) compartments might influence serum leptin levels in humans through increased insulin resistance. DESIGN AND METHODS: We evaluated body composition (through bioelectric impedance and anthropometrical parameters), insulin resistance (using the fasting insulin resistance index (FIRI) and insulin sensitivity (S(I)) from the minimal model analysis) and leptin levels in 140 men and 114 women. RESULTS: Serum insulin, FIRI and leptin levels were significantly increased in men in the highest quintile of FFM. Leptin levels positively correlated with FFM in men (r=0.24, P=0004) but not in women (r=0.02, P=not significant). With weight gain, however, approximately 25% of the additional weight is lean mass, so that obese people have higher fat-free mass than lean people. Hence, we performed a multiple linear regression analysis in a stepwise manner to predict leptin levels, in which fat mass (FM), FFM, and FIRI, but not age or waist-to-hip ratio (WHR) independently contributed to 32%, 6% and 3% of the variance in serum leptin levels in men. In women, FM (49%), FIRI (3.6%) and WHR (2.4%), but not FFM or age explained this variance. In a sample of 40 subjects, S(I) and leptin correlated with mid-arm muscle circumference (r=-0.51, P=0.03 and r=0.53, P=0.02) and mid-arm muscle area (r=-0.52, P=0.03 and r=0.53, P=0.02) in men (n=17) but not in women (n=23). CONCLUSIONS: The fat-free mass compartment contributes to the variability of serum leptin levels in men. Whether insulin resistance at this level mediates an increased production of leptin merits further research.
F. Facchinetti, R. Perez-Fernandez, M. O. Toma, G.J. Gaudiero, M.J. Lechuga, J. Devesa and A. R. Genazzani
Abstract. The effect of chronic dopaminergic receptor blockade using domperidone (DOM) on the immature dog pituitary content of POMC-related peptides was evaluated. Six immature dogs were treated with DOM for 15 days, 3 times/day, po (3 mg/kg) together with DOM sc (0.6 mg/kg) at 21.00 h. Placebo was administered to six control animals with the same protocol. On the 16th day, the animals were killed, the whole pituitary removed, homogenized, and submitted to reverse-phase HPLC purification prior to radioimmunoassay (RIA) evaluation of β-endorphin, ACTH and α-MSH immunoreactivities (ir). DOM-treated dogs showed a pituitary concentration of β-EP and ACTH similar to the placebo-treated dogs. The total α-MSH ir was similar in both groups and distributed on two main peaks: one corresponding to α-MSH and another coeluting with des-acetyl-α-MSH [1-13(ACTH)NH2]. However, the percentage of α-MSH on total ir in DOMtreated dogs (15.4 ± 2.6%) was lower than in controls (37.5 ± 4.5%, P < 0.01); the corresponding percentage of 1-13(ACTH)NH2 content was 63.0 ± 3.8% vs 44.7 ± 3.7%, (P < 0.01). The α-MSH/1-13(ACTH)NH2 ratio was considerably decreased by the treatment (0.25 ± 0.06 vs 0.89 + 0.15, P < 0.01). < 0.01). Acetyl β-EP-like ir was also lower in treated (38.4 + 5.4 fmol/mg) vs control (86.6 + 19.2 fmol/mg, P < 0.05) animals. These data indicate that the dopaminergic system plays an important role in the control of acetylation processes of POMC-related peptides in the pituitary. This may be crucial as far as the biological activity of the peptides is concerned.
B K Rodiño-Janeiro, A Salgado-Somoza, E Teijeira-Fernández, J R González-Juanatey, E Álvarez and S Eiras
Obesity, a risk factor for coronary artery disease (CAD), is associated with inflammation and reactive oxygen species (ROS) production, while advanced glycation end-products, through their receptor (AGER or RAGE), play an important role on these processes. The aim of this study was to analyze the expression levels of RAGE, NADPH oxidase subunits, and catalase in adipose tissue in relation with CAD.
Design and methods
Patients undergoing heart surgery were included in two groups: with and without CAD. Epicardial adipose tissue (EAT) and subcutaneous adipose tissue (SAT) biopsies were analyzed for gene expression by RT-quantitative PCR, immunohistochemistry, or western blot.
RAGE mRNA and protein expression in SAT from patients with CAD was lower than in patients without CAD. However, there was no change in EAT from patients with or without CAD. P22-PHOX and RAGE gene expression were higher in EAT than in SAT, whereas catalase mRNA levels were lower. NADPH oxidase subunits and catalase mRNA expression were not influenced by CAD. Whereas NADPH oxidase-dependent oxidative response of SAT and EAT to lipid circulating levels could be different; glycemic levels were not related with the analyzed genes expression.
This study demonstrates that RAGE expression in SAT, but not in EAT, is down-regulated in patients with CAD with respect to those without CAD. Although changes were not observed for NADPH oxidase subunits or catalase expression between CAD and non-CAD patients, a possible relationship between ROS production and RAGE expression in adipose tissues cannot be ruled out.
C Gil-Puig, S Seoane, M Blanco, M Macia, T Garcia-Caballero, C Segura and R Perez-Fernandez
Background: The transcription factor pituitary-1 (Pit-1) is mainly expressed in the pituitary gland, where it has critical roles in cell differentiation and as a transcriptional factor for GH and prolactin (PRL). It is also expressed in human extrapituitary tissues (placenta, lymphoid and haematopoietic tissues) and cell lines (human breast adenocarcinoma cells, MCF-7). Despite the widely suggested roles of GH and PRL in the progression of proliferative mammary disorders, Pit-1 expression in human mammary gland has not yet been reported.
Objective: To evaluate the expression of Pit-1 in human breast and, using the MCF-7 cell line, to investigate whether Pit-1 overexpression regulates GH expression and increases cell proliferation.
Methods: Using real-time RT-PCR, western blotting and immunohistochemistry, we evaluated the expression of Pit-1 mRNA and protein in seven normal human breasts and 14 invasive ductal mammary carcinomas. GH regulation by Pit-1 in MCF-7 cells was evaluated using RT-PCR, western blotting, ELISA and transfection assays. Cell proliferation was evaluated using bromodeoxyuridine.
Results: We found expression of Pit-1 mRNA and protein in both normal and tumorous human breast. We also found that Pit-1 mRNA levels were significantly increased in breast carcinoma compared with normal breast. In MCF-7 cells, Pit-1 overexpression increased GH mRNA and protein concentrations and significantly increased cell proliferation.
Conclusions: These findings indicate that Pit-1 is expressed in human breast, that it regulates endogenous human mammary GH secretion, and that it increases cell proliferation. This suggests that, depending on its level of expression, Pit-1 may be involved in normal mammary development, breast disorders, or both.
JM Fernandez-Real, P Botas-Cervero, A Lopez-Bermano, R Casamitjana, T Funahashi, E Delgado, S Kihara and W Ricart
BACKGROUND: In humans, adiponectin has been demonstrated to circulate in inverse proportion to the degree of insulin resistance. OBJECTIVE: To investigate the association between adiponectin and glycosylated haemoglobin (HbA1c) in a population-based study. DESIGN AND METHODS: Two hundred and ninety-seven individuals aged 30-75 years were enrolled in a cross-sectional study. They included patients with type 2 (non-insulin-dependent) diabetes mellitus and stable, good metabolic control (n=32) and individuals with glucose intolerance (n=54). Adiponectin was measured using a sandwich enzyme-linked immunosorbent assay (intra-assay and interassay coefficients of variation 3.3 and 7.4% respectively). RESULTS: Adiponectin correlated with age (r=0.161; P=0.006), body mass index (r=-0.197; P=0.001), diastolic blood pressure (r=-0.181; P=0.005), fasting glucose and HbA1c (r=-0.251 and r=-0.22 respectively; P<0.0001), high-density lipoprotein cholesterol (r=0.442; P<0.001) and serum triglycerides (r=-362; P<0.001). In multiple regression analysis, sex, age, fasting and post-load glucose, and adiponectin independently contributed to 40% of the variance in HbA1c. Among individuals with normal glucose tolerance, fasting glucose (P=0.0033), post-load glucose (P=0.0015), age (P=0.001) and adiponectin (P=0.0083) independently contributed to 21% of the variance in HbA1c. CONCLUSION: Adiponectin is significantly associated with altered glucose metabolism and independently contributes to the variance of HbA1c in a population-based manner.
R Pérez-Lobato, R Ramos, J P Arrebola, I Calvente, O Ocón-Hernández, C Dávila-Arias, M Pérez-García, N Olea and M F Fernández
Thyroid hormones (THs) are crucial for the correct maturation of the CNS and the neurodevelopment of the child. We aimed to investigate the association of TSH and free thyroxine (FT4) levels with cognitive functioning in children from the INMA-Granada cohort studied during their follow-up at the age of 9–11 years.
We evaluated 300 children from the original cohort, which comprised 668 eligible mother–son pairs recruited at birth from 2000 to 2002 in Granada (Spain).
FT4 and TSH concentrations were measured, and cognitive development was assessed using neuropsychological tests (n=187). Children with chronic disease related to thyroid function and/or cognitive development were excluded.
Median TSH and FT4 levels were 3.1 μIU/ml and 1.2 ng/dl respectively. In multivariable regression analyses adjusted for maternal and child characteristics, children with TSH levels in the top tertile had worse verbal comprehension and immediate and long-term recall. Children with FT4 levels in the top tertile had better attention and lower impulsivity and were at a lower risk of scoring below the 20th percentile in intelligence quotient (OR=0.24; 95% CI=0.08–0.74; P=0.013) and in abstract reasoning ability (OR=0.28; 95% CI=0.09–0.88; P=0.029).
Our findings indicate that circulating THs and TSH may in the top tertile have an impact on cognitive functions; thus, higher TSH slightly but significantly increased the risk of a lower score in certain neuropsychological tests.
José Manuel Fernández-Real, Marek Straczkowski, Begoña Lainez, Matilde R Chacón, Irina Kowalska, Abel López-Bermejo, Antonio García-España, Agnieszka Nikolajuk, Ida Kinalska and Wifredo Ricart
Objective: Serum concentrations of soluble tumor necrosis factor-α (TNF-α) receptor 2 (sTNFR2) are associated with insulin resistance. In a recent study, we provided evidence for the existence of a biologically active form of sTNFR2 produced by alternative splicing (DS-TNFR2). We aimed to evaluate whether this circulating DS-TNFR2 is associated with insulin action in humans.
Design and methods: Real time PCR (light cycler technology) evaluated DS-TNFR2 expression in monocytes. DS-TNFR2 was measured using a monoclonal antibody against an epitope present in TNFR2 (first 14 residues of the juxtamembrane region) but predicted to be absent in soluble proteolytic cleavage-produced TNFR2. Insulin sensitivity was measured using euglycemic hyperinsulinemic clamp (n = 76) and homeostatic model of assessment (HOMA) value in a replication study of 223 subjects.
Results: Real time PCR confirmed gene expression of DS-TNFR2 in monocytes from healthy subjects. A significant and positive association was found between serum DS-TNFR2 concentration and insulin sensitivity (P = 0.032, n = 76). This association was most significant in subjects with normal glucose tolerance (r = 0.44, P = 0.002). The subjects in whom DS-TNFR2 was detectable were more insulin sensitive than those with undetectable DS-TNFR2 (42.12±22.08 vs 31.71± 16.95 μmol × kg−1 × min−1, P = 0.039). DS-TNFR2 was inversely associated with body mass index, waist-to-hip ratio, systolic and diastolic blood pressure, fasting serum glucose, serum triglycerides and serum uric acid concentration and with the HOMA value (P = 0.03) in the replication study. Circulating DS-TNFR2 declined with increased number of components of the metabolic syndrome.
Conclusion: Native sTNFR2 and DS-TNFR2 show opposite associations with insulin action. DS-TNFR2 might play a role as a counterpart of the proinflammatory environment associated with insulin resistance.
R Martínez, C Fernández-Ramos, A Vela, T Velayos, A Aguayo, I Urrutia, I Rica, L Castaño and on behalf of the Spanish Congenital Hyperinsulinism Group
Congenital hyperinsulinism (CHI) is a clinically and genetically heterogeneous disease characterized by severe hypoglycemia caused by inappropriate insulin secretion by pancreatic β-cells.
To characterize clinically and genetically CHI patients in Spain.
Design and methods
We included 50 patients with CHI from Spain. Clinical information was provided by the referring clinicians. Mutational analysis was carried out for KCNJ11, ABCC8, and GCK genes. The GLUD1, HNF4A, HNF1A, UCP2, and HADH genes were sequenced depending on the clinical phenotype.
We identified the genetic etiology in 28 of the 50 CHI patients tested: 21 had a mutation in KATP channel genes (42%), three in GLUD1 (6%), and four in GCK (8%). Most mutations were found in ABCC8 (20/50). Half of these patients (10/20) were homozygous or compound heterozygous, with nine being unresponsive to diazoxide treatment. The other half had heterozygous mutations in ABCC8, six of them being unresponsive to diazoxide treatment and four being responsive to diazoxide treatment. We identified 22 different mutations in the KATP channel genes, of which ten were novel. Notably, patients with ABCC8 mutations were diagnosed earlier, with lower blood glucose levels and required higher doses of diazoxide than those without a genetic diagnosis.
Genetic analysis revealed mutations in 56% of the CHI patients. ABCC8 mutations are the most frequent cause of CHI in Spain. We found ten novel mutations in the KATP channel genes. The genetic diagnosis is more likely to be achieved in patients with onset within the first week of life and in those who fail to respond to diazoxide treatment.