Oral leptin supplementation throughout lactation in rats prevents later metabolic alterations caused by gestational calorie restriction.

OBJECTIVES: Calorie-restriction during gestation in rats has been seen to produce lasting detrimental effects in the offspring, affecting energy balance control and other related metabolic functions. Our aim was to assess whether leptin supplementation throughout lactation may prevent the dysmetabolic phenotype in adulthood associated with gestational calorie restriction. METHODS: Three groups of male Wistar rats were followed: the offspring of ad libitum fed dams (controls); the offspring of 20% calorie-restricted dams during gestation (CR); and CR rats supplemented with physiological doses of leptin throughout lactation (CR-Leptin). Pups were weaned with a standard diet (SD) until 4 months of age, and then half of the animals of each group were moved to a Western diet (WD) until 6 months of age. Body weight and food intake were recorded. Energy expenditure, locomotive activity, blood parameters, liver triglycerides (TG), and gene expression and specific proteins in liver and white adipose tissue (WAT) were measured in adulthood. RESULTS: Adult CR rats, but not CR-Leptin rats, displayed greater adiposity index and feed efficiency (both under SD) than controls, along with lower locomotive activity and energy expenditure, higher HOMA-IR index and greater circulating TG and leptin levels. CR animals also exhibited increased values of the respiratory exchange ratio and more severe signs of hepatic steatosis under WD than CR-Leptin animals. Gene expression analysis revealed that CR, but not CR-Leptin, animals displayed indicators of lower capacity for WAT expansion, along with decreased lipogenesis and lipolytic capacity under SD, and impaired lipogenic response of the liver to WD feeding, in accordance with diminished insulin sensitivity and WAT leptin signaling. CONCLUSIONS: Oral leptin supplementation in physiological doses throughout lactation in rats prevents most of the detrimental effects on energy homeostasis and metabolic alterations in adulthood caused by inadequate fetal nutrition.

Leptin intake in suckling rats restores altered T3 levels and markers of adipose tissue sympathetic drive and function caused by gestational calorie restriction.

BACKGROUND: Maternal calorie restriction during gestation in rats has been associated with altered white adipose tissue (WAT) sympathetic innervation and function in offspring. Here, we aimed to investigate whether supplementation with oral leptin (a breast milk component) throughout the lactation period may revert the aforementioned adverse programming effects. METHODS: Three groups of male and female rats were studied at the postnatal day 25: the offspring of control dams, the offspring of 20% calorie-restricted dams during pregnancy (CR) and CR rats supplemented with physiological doses of leptin throughout lactation (CR-Leptin). Tyrosine hydroxylase (TH) levels and its immunoreactive area, and mRNA expression levels of lipid metabolism-related genes and of deiodinase iodothyronine type II (Dio2) were determined in WAT. Triiodothyronine (T3) levels were determined in the blood. RESULTS: In CR males, leptin treatment restored the decreased TH levels and its immunoreactive area in WAT, and partially normalized expression levels of genes related to lipolysis and fatty acid oxidation (adipose triglyceride lipase, hormone-sensitive lipase, carnitine palmitoyltransferase 1b and peroxisome proliferator-activated receptor gamma coactivator 1-alpha). Leptin treatment also reverted the decreased T3 plasma levels and WAT lipoprotein lipase mRNA levels occurring in CR males and females, and the decreased Dio2 mRNA levels in CR females. CONCLUSIONS: Leptin supplementation throughout the lactation period reverts the malprogrammed effects on WAT structure and function induced by undernutrition during pregnancy. These findings support the relevance of the intake of leptin during lactation, bearing clear characteristics of essential nutrient, and provide a strategy to treat and/or prevent the programmed trend to obesity acquired by inadequate fetal nutrition.

Moderate calorie restriction during gestation programs offspring for lower BAT thermogenic capacity driven by thyroid and sympathetic signaling.

BACKGROUND: Maternal calorie restriction during pregnancy programs offspring for later overweight and metabolic disturbances. Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis and has recently emerged as a very likely target for human obesity therapy. OBJECTIVE: Here we aimed to assess whether the detrimental effects of undernutrition during gestation could be related to impaired thermogenic capacity in BAT and to investigate the potential mechanisms involved. METHODS: Offspring of control and 20% calorie-restricted rats (days 1-12 of pregnancy) (CR) were studied at the age of 25 days. Protein levels of uncoupling protein 1 (UCP1) and tyrosine hydroxylase (TyrOH); mRNA levels of lipoprotein lipase (LPL), carnitine palmitoyltransferase 1 (CPT1) and deiodinase iodothyronine type II (DIO2) in BAT; and blood parameters including thyroid hormones, were determined. The response to 24-h cold exposure was also studied by measuring body temperature changes over time, and final BAT UCP1 levels. RESULTS: Compared with controls, CR animals displayed in BAT lower UCP1 and TyrOH protein levels and lower LPL and CPT1 mRNA levels; they also showed lower triiodothyronine (T3) plasma levels. CR males, but not females, revealed lower DIO2 mRNA levels than controls. When exposed to cold, CR rats experienced a transient decline in body temperature, but the values were reestablished after 24 h, despite having lower UCP1 levels than controls. CONCLUSIONS: These results suggest that BAT thermogenic capacity is diminished in CR animals, involving impaired BAT sympathetic innervation and thyroid hormone signaling. These alterations make animals more sensitive to cold and may contribute to long-term outcomes of gestational calorie restriction in promoting obesity and related metabolic alterations.

Identification of early transcriptome-based biomarkers related to lipid metabolism in peripheral blood mononuclear cells of rats nutritionally programmed for improved metabolic health.

Moderate maternal calorie restriction during lactation protects rat offspring against obesity development in adulthood, due to an improved ability to handle and store excess dietary fuel. We used this model to identify early transcriptome-based biomarkers of metabolic health using peripheral blood mononuclear cells (PBMCs), an easily accessible surrogate tissue, by focusing on molecular markers of lipid handling. Male and female offspring of control and 20 % calorie-restricted lactating dams (CR) were studied. At weaning, a set of pups was killed, and PBMCs were isolated for whole-genome microarray analysis. The remaining pups were killed at 6 months of age. CR gave lower body weight, food intake and fat accumulation, and improved levels of insulin and leptin throughout life, particularly in females. Microarray analysis of weaned rat PBMCs identified 278 genes significantly differentially expressed between control and CR. Among lipid metabolism-related genes, expression of Cpt1a, Lipe and Star was increased and Fasn, Lrp1 and Rxrb decreased in CR versus control, with changes fully confirmed by qPCR. Among them, Cpt1a, Fasn and Star emerged as particularly interesting. Transcript levels of Cpt1a in PBMCs correlated with their levels in WAT and liver at both ages examined; Fasn expression levels in PBMCs at an early age correlated with their expression levels in WAT; and early changes in Star expression levels in PBMCs correlated with their expression levels in liver and were sustained in adulthood. These findings reveal the possibility of using transcript levels of lipid metabolism-related genes in PBMCs as early biomarkers of metabolic health status.

Protective effects of leptin during the suckling period against later obesity may be associated with changes in promoter methylation of the hypothalamic pro-opiomelanocortin gene.

Leptin supplementation of neonatal rats during the suckling period protects against being overweight in adulthood and ameliorates the control of food intake. This was associated with changes in the expression of hypothalamic genes involved in the central action of leptin: pro-opiomelanocortin (Pomc), leptin receptor (Lepr) and suppressor of cytokine signalling (Socs3). The purpose of the present study was to determine the methylation status within the promoter regions of these genes and to assess whether the observed changes in the expression levels of these genes could be explained by changes in their methylation status. Male rats were treated daily with an oral physiological dose of leptin or vehicle during the suckling period. After weaning, animals were fed with a normal-fat or a high-fat (HF) diet until aged 6 months. DNA was extracted from the hypothalamus and methylation within the promoter regions of the gene panel was measured by pyrosequencing. Pomc promoter methylation increased in control animals fed the HF diet but decreased in leptin-treated animals. In addition, there was a weak negative correlation between DNA methylation and POMC mRNA levels (P = 0·075). There were no changes in the methylation status of the CpG sites studied within the promoter regions of Lepr and Socs3 in response to leptin or HF treatments. This is the first demonstration that leptin treatment during lactation may programme methylation of an appetite-related gene in the hypothalamus of animals fed HF diets, with possible implications for gene expression and protection against the development of obesity.

Moderate caloric restriction during gestation results in lower arcuate nucleus NPY- and alphaMSH-neurons and impairs hypothalamic response to fed/fasting conditions in weaned rats.

AIM: We aimed to characterize the developmental programming effects of moderate caloric restriction during early pregnancy on factors involved in hypothalamic control of energy balance. METHODS: Twenty-five-days-old offspring Wistar rats from 20% caloric restricted dams (from 1 to 12 days of pregnancy) (CR) and from control dams were studied under fed and 12 h fasting conditions. Morphometric studies on arcuate nucleus (ARC) and determinations of circulating parameters and hypothalamic levels of neuropeptide Y (NPY), proopiomelanocortin (POMC), long-form leptin receptor (ObRb), insulin receptor (InsR) and suppressor of cytokine signalling-3 (SOCS-3) mRNA were performed. RESULTS: CR animals did not show different body weight with respect to their controls, but presented higher food intake. They exhibited lower neuropeptide Y- and alpha-melanocyte-stimulating hormone-neurons (decreases of 18 and 13% in males, and 10 and 18% in females respectively) and lower total cells (decrease of 3% in males and 18% in females) in ARC. Under fed conditions, CR animals presented lower circulating leptin and ghrelin levels (decreases of 37 and 43% in males, and 15 and 34% in females respectively); furthermore, hypothalamic POMC, NPY (only in females), ObRb and InsR mRNA levels were reduced (39, 16 and 26% in males, and 112, 33, 61 and 56% in females), and those of SOCS-3 were increased (86% in males and 74% in females). Unlike control animals, under fasting conditions, ObRb, InsR and POMC mRNA levels did not decrease in CR females, and NPY mRNA decreased instead of increase in CR males. CONCLUSIONS: Moderate caloric restriction during gestation affects offspring hypothalamic structure and function, impairing its response to fed/fasting conditions, which suggests a predisposition to insulin and leptin resistance.

Comparison of murine fibroblast cell response to fluor-hydroxyapatite composite, fluorapatite and hydroxyapatite by eluate assay.

Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetic composite that contains the same molecular concentration of OH(-) groups and F(-) ions. The aim of this experiment was to evaluate the cellular responses of murine fibroblast NIH-3T3 cells in vitro to solid solutions of FHA and FA and to compare them with the effect of hydroxyapatite (HA). We studied 24, 48 and 72 h effects of biomaterials on cell morphology, proliferation and cell cycle of NIH-3T3 cells by eluate assay. Furthermore, we examined the ability of FHA, FA and HA to induce cell death and DNA damage. Our cytotoxic/antiproliferative studies indicated that any of tested biomaterials did not cause the total inhibition of cell division. Biomaterials induced different antiproliferative effects increasing in the order HA < FHA < FA which were time- and concentration-dependent. None of the tested biomaterials induced necrotic/apoptotic death of NIH-3T3 cells. On the other hand, after 72 h we found that FHA and FA induced G0/G1 arrest of NIH-3T3 cells, while HA did not affect any cell cycle phases. Comet assay showed that while HA demonstrated weaker genotoxicity, DNA damage induced by FHA and FA caused G0/G1 arrest of NIH-3T3 cells. Fluoridation of hydroxyapatite and different FHA and FA structure caused different cell response of NIH-3T3 cells to biomaterials.

Cytotoxicity of hydroxyapatite, fluorapatite and fluor-hydroxyapatite: a comparative in vitro study.

The purpose of this study was to evaluate the cytotoxicity of two formulations of hydroxyapatite (HA), namely fluorapatite (FA) and fluor-hydroxyapatite (FHA). HA is used as carrier material for antibiotics or anticancer drugs during treatment of bone metastasis. Negative control, represented by HA, was included for comparative purposes. Leukemia cells were used as a model cell line, and the effect of eluates of tested biomaterials on cell proliferation/viability and mechanism of antiproliferative activity were assessed. Study design attempted to reveal the toxicity of tested biomaterials with an emphasis to decide if tested biomaterials have promise for further studies in vivo. Results showed that eluates of FA and FHA inhibit the growth of leukemia cells and induce programmed cell death through mitochondrial/caspase-9/caspase-3-dependent pathway. Due to these differences compare to HA, it is concluded that FA and FHA have promise for evaluation of their behaviour in vivo.

Sequential changes in the expression of genes involved in lipid metabolism in adipose tissue and liver in response to fasting.

The aim of this study was to provide a sequential analysis of the expression patterns of key genes involved in lipid metabolism in white adipose tissue (WAT) and liver and their relationship with blood parameters in response to fasting. Adult male rats were studied under different feeding conditions: feeding state, after 4, 8, or 24 h fasting, and after 3 h refeeding after 8 h fasting. Blood parameters and the expression of genes involved in lipogenesis and lipolysis in WAT and liver were analyzed. mRNA levels of genes involved in lipogenesis in liver (SREBP1c, FAS, and GPAT) had already decreased after 4 h fasting, as well as those of PPARgamma in WAT, whereas the decrease in SREBP1c, FAS, GPAT, and GLUT4 mRNA levels in WAT was observed after 8 h. Concerning lipolytic and fatty-acid-oxidation-related genes, liver PPARalpha, FGF21, CPT1, and PDK4 mRNA levels increased after 8 h fasting and those of ACOX1 after 24 h, and in WAT, ATGL, and CPT1 mRNA levels were greater after 24 h. Three hours refeeding increased the expression levels of PPARgamma in WAT, SREBP1c in both liver and WAT, and GPAT in liver, and decreased the expression levels of PPARalpha, CPT1, and PDK4 in liver. These results give new insight into the different adaptive time course response to fasting in the expression of genes involved in lipid metabolism, thus pointing out the very rapid response of lipogenic genes, particularly in liver, and the later response of lipolytic genes, particularly in WAT.

[De La Chapelle syndrome]. / Le syndrome de De La Chapelle.

OBJECTIVE: The De La Chapelle syndrome (XX male) is a peripheral hypogonadism concerning males with 46,XX karyotype. We conducted a retrospective study of 18 cases and report the main clinical biological and hormonal characteristics. PATIENTS AND METHODS: Clinical features (weight, height, aspect of the external genital organs, body hair, gynecomastia), hormone levels (testosterone, gonadotrophin, baseline and stimulated prolactin estradiol), and results of a Barr test and karyotype were recorded in all patients in addition to search for the SRY gene (in 8 of the 18 patients). Findings were compared with a matched male population and a Klinefelter syndrome population. RESULTS: Microrchidia was found in almost all the patients while the penis had a normal size. Signs of hypoandrogenism were frequent and gynecomastia was present in half the cases. De La Chapelle patients differed from Klinefelter patients by the absence of dysmorphism. DISCUSSION: Patients with De La Chapelle syndrome diagnosed around the age of 20 years do not have borderline disorders associating genitalia anomalies or sexual ambiguity. The majority of the patients bear the testis determining SRY gene on one of the X chromosomes, providing the rational explanation of the male phenotype, but 20% of the XX males doe not have this gene. The role of certain key genes that could be implicated in abnormal sexual differentiation is known, but the complexity and heterogeneous nature of this syndrome leaves many questions unanswered. Therapy is based on androgen replacement therapy given at an early stage.

Optimized transient insulin infusion in uncontrolled type 2 diabetes: evaluation of a pragmatic attitude.

Glucotoxicity generated by hyperglycemia creates a vicious circle worsening the imbalance of diabetes mellitus. A pump-optimized transient insulin treatment can be used to break this fate and restore some degree of insulin sensitivity in uncontrolled type 2 diabetes. The aim of this retrospective study was to evaluate type 2 diabetics with a secondary failure to a maximal oral antidiabetic therapy, treated with a transient subcutaneous insulin therapy during 3 days. The following criteria were analysed: delay before permanent insulin treatment, prognosis factors of evolution, weight evolution and glucose control in patients maintained under oral treatment. We studied 250 type 2 diabetics, and 515 insulin infusions. The average follow-up was 3.5 years. At the end of the follow-up 63 patients required insulin from the inception of the study (Group 1), 76 secondarily resumed insulin (Group 2), and 111 remained with oral treatment (Group 3). Patients in Group 1 were significantly older, with higher HbA1c and a lower body mass index (BMI). On average, the patients in Group 3 were submitted to less than 2 insulin infusions, their BMI from the beginning to the end of the follow-up remained stable, while HbA1c improved. We conclude that transient optimized insulin treatment during 3 consecutive days is effective. Thus, 45% of the initial global population remain under oral therapy after 3.5 years with a better glucose control and a stable weight.

[The impact of dipper and non-dipper characteristics in the fluctuation of arterial blood pressure. A study of a population of 484 diabetic patients]. / Les conséquences du caractère dipper et non dipper de la courbe de pression artérielle. Etude d'une population de 484 patients diabétiques.

Abnormal pattern of circadian blood pressure variations carries a high risk of cardiovascular complications. The aim of this study was to assess the frequency of abnormal blood pressure rhythm in diabetes and its consequences on micro and macrovascular complications. 484 diabetes mellitus patients were submitted to 24-h ambulatory blood pressure monitoring. They were divided into two groups according to the absence (non-dipper: group 1; n = 167) or presence (dipper: group 2; n = 317) of nocturnal BP reduction = 10% of daytime BP. Following data were collected and compared between these two groups: body mass index, glycated haemoglobin, urinary albumin excretion, research of retinopathy by fundoscopy, tests for presence of a macrovascular disease. There were no significant differences among the two groups in sex, body mass index, type and duration of diabetes and glycemic control. Clinical SBP and DBP did not differ from significant manner between non-dipper and dipper (140 +/- 18/81 +/- 1 versus 138 +/- 19/81 +/- 10 mmHg). Non-dipper 24-h SBP and 24-h DBP were higher than those of dipper (129 +/- 16/76 +/- 9 versus 122 +/- 15/73 +/- 8 mmHg; p < 0.001). Non-dipper were older than dipper (59.9 +/- 13 versus 55.8 +/- 15 years; p < 0.001) and there was more hypertensive patients in group 1 than in group 2 (50% versus 39%; p < 0.01). Macro- and microvascular diabetes complications were more common in non-dipper. In conclusion high blood pressure is frequently observed in diabetic patients. Its association with a diminished nocturnal BP fall could explain a higher risk of complications, especially retinopathy, nephropathy and cardiac events.

[Chronic complications of type 2 diabetes mellitus. Clinical course after 5 years of follow-up]. / Complicaciones crónicas de la diabetes mellitus tipo 2. Evolución tras 5 años de seguimiento.

OBJECTIVE: To evaluate the modifications in the prevalence of complications and the incidence of end-points in diabetic patients observed for five years, and the effectiveness of a diabetes care protocol on the process indicators. DESIGN: Prospective observational study between 1991 and 1996. SETTING: Primary care centre. PARTICIPANTS: Diabetic patients monitored between 1991 and 1996. MEASUREMENTS AND MAIN RESULTS: Social and demographic variables, DM epidemiology variables, cardiovascular risk factors, complications and end-points were measured. 318 of the 352 patients selected in 1991 were followed. Average age was 68.6 (SD 11.2) and 39% were male. Mean observance was for 53 months (SD 10). There was an increase of insulin use (19%) and self-analysis (34.7%) both in men (chi 2 = 14.7, p < 0.001) and in women (chi 2 = 40.5, p < 0.001), independently of age (chi 2 = 37.77, p < 0.001). Complications increased: microvascular ones from 33.4% to 42.1%, macrovascular ones from 22.3% to 37.2%. The most common end-points were CVA (7.8%) and angor (3.6%). 22 patients died (6.9%), with ischaemic cardiopathy (30%) and neoplasm (30%) the most common causes. Hypertension increased from 51.6% to 59.8% and hypercholesterolaemia from 42.6% to 47%. Obesity (42%) and tobacco dependency (28%) remained stable. Systolic blood pressure went down by 4.7 mmHg and diastolic pressure by 3.76 mmHg, and in women, over-65s and those who had had the illness longest (> 10 years). 6.5% (CI 1-12.9%) of patients improved their blood pressure (< 135/85 mmHg). HbA1c worsened independently of sex, age or years of evolution of diabetes.

[Anemia in primary care: etiology and morphological characteristics]. / Anemias en atención primaria: etiología y características morfológicas.

OBJECTIVE: To find the morphological characteristics and causes of the types of anaemia seen at a primary care centre. DESIGN: Descriptive, observational study. SETTING: Urban health centre. PATIENTS: People attending for a year who had an anaemia defined by haemoglobin figures below 13 g/dl in males and 12 g/dl in women. MEASUREMENTS AND MAIN RESULTS: 152 patients with anaemia were identified. The most common types of anaemia were iron-deficiency anaemia (IDA), anaemia due to chronic illness (ACI) and post-haemorrhage anaemia (48%, 26.3% and 6.6%, respectively). Anaemia due to vitamin B12 deficit was detected in four patients, Thalassaemia minor in two, haemolytic anaemia in two, and a refractory anaemia in one patient. The most common cause of IDA was gynaecological in origin; and the commonest cause of ACI was neoplasm. The main findings of digestive origin in IDA were oesophagitis in two patients, duodenal ulcer in one, erosive gastritis in one, gastric neoplasm in one, colonic neoplasm in two and Crohn's disease in one. 13.7% of the anaemia studied in PC required hospital referral. CONCLUSIONS: Anaemia is a common health problem in primary care (PC), with a rough incidence of one case per month per doctor. Its main types are iron-deficiency anaemia and anaemia due to chronic illness. Most cases were detected in PC and most can be studied properly at this care level.

[Analysis of hypercholesterolemia care costs in primary health care]. / Análisis de los costes de la atención a la hipercolesterolemia en un equipo de atención primaria.

OBJECTIVES: 1) To calculate the care costs of Hypercholesterolaemia in Primary Care in terms of the number of visits to the doctor, analyses and drugs. 2) To calculate the effectiveness of the intervention. DESIGN: A retrospective study. SETTING: Primary Care Centre. PATIENTS: 97 histories of patients diagnosed as having Hypercholesterolaemia, obtained by systematic sampling from the Centre's morbidity register, were analysed. A control with the same age, sex and risk factors was chosen for each one of them. MEASUREMENTS: The study period was 12 months. For those patients diagnosed as having Hypercholesterolaemia after the opening of the centre, the initial figures and those at the end of the study for total Cholesterol, LDL Cholesterol (cLDL) and HDL cholesterol (cHDL) were analysed. RESULTS: There were no differences between the demographic characteristics and the risk factors of the cases and controls. An excess of attendances (average of 9.0 against 6.0, p = 0.01), of hypolipaemic and non-hypolipaemic drugs (average of 4.0 against 3.0, p = 0.006) and of analyses (average of 2.0 against 0.0, p = 0.0001) were detected for the cases. The following variations were found between the initial and final figures: for total Cholesterol (-7.2%, p = 0.001), for cLDL (-11.6%, p = 0.006) and cHDL (+18.2%, p < 0.0001). CONCLUSIONS: 1) Hypercholesterolaemia care in a mainly selective manner for people at high risk was shown to be reasonably effective and affordable. 2) Before starting other strategies for detecting Hypercholesterolaemia, adequate prioritisation and evaluation of their impact on health-care delivery are required.

Nucleotide sequence of the 85B-protein gene of Mycobacterium bovis BCG and Mycobacterium tuberculosis.

We have cloned and sequenced the genes coding for the 85B antigen from M. bovis BCG and M. tuberculosis. Within this gene, the only difference in sequence between M. bovis BCG and M. tuberculosis corresponds, respectively, to a C-->T yielding a Leu-->Phe replacement at position 100 of the mature 85B protein. Therefore as we described previously for the 85A gene, there is also very little variation between these two species within the 85B gene.

The use of ultrasound for the determination of periodontal bone morphology.

The purpose of this study was to assess the value of ultrasound in the measurement of periodontal bone morphology. Four patients scheduled for periodontal surgery were selected for the study. The Ocu-Scan 400 machine was used and adjusted according to the manufacturer's instructions. Teeth involved in the study were measured on the facial aspect at three different points: mesial, midfacial and distal (except one tooth measured only at the mesial point). Two different presurgical measurements were made with the use of ultrasound. The distance between the gingival margin and the crest of alveolar bone was measured during the surgical procedure. No correlation could be made between the different measurements obtained. From the results the following was concluded: measurement of alveolar bone topography with the presently available ultrasound probe is not accurate, and ultrasound measurement is a fast and painless procedure.