Liver volume and hepatic adiposity in childhood: relations to body growth and visceral fat.

BACKGROUND AND OBJECTIVE: The sequence of prenatal growth restraint and postnatal catch-up growth may lead to hepato-visceral adiposity, insulin resistance and low-grade inflammation before the onset of puberty. In prepubertal children born appropriate for gestational age (AGA) or small for gestational age (SGA), we assessed potential relationships between the aforementioned sequence and liver volume. SUBJECTS/METHODS: The study population consisted of 86 children (41 AGA and 45 SGA with catch-up growth; age (mean±s.e.m.), 8.5±0.1 years), recruited into two prospective longitudinal studies. Anthropometry, endocrine-metabolic variables and inflammatory and hepatic markers were assessed, along with liver volume, hepatic adiposity and abdominal fat partitioning (by magnetic resonance imaging). RESULTS: AGA and SGA children differed in hepato-visceral adiposity, but had similar liver volumes. Boys had larger livers than girls, and higher sex hormone binding globulin and inflammation markers. Liver volume correlated with height Z-score, body mass index Z-score, HOMA-IR (homeostasis model assessment-insulin resistance) and with subcutaneous and visceral fat, but not with birth weight Z-score or with hepatic adiposity. Height, visceral fat, gender and HOMA-IR were major determinants of liver volume, together explaining 61% of its variance. CONCLUSIONS: The trajectory from prenatal restraint, via postnatal catch-up, to hepato-visceral adiposity and insulin resistance does not appear to be detectably influenced by prepubertal alterations of liver volume. Further follow-up will disclose the potential role of liver volume in the pubertal segment of this trajectory, and whether the augmented fat content and visceral adiposity in SGA subjects is followed by the development of metabolic syndrome and hepatic dysfunction in adulthood.

Perirenal fat is related to carotid intima-media thickness in children.

BACKGROUND/OBJECTIVES: It is well known that increased abdominal fat is associated with cardiovascular (CV) risk. Perirenal fat has been recently associated with CV risk in adults. However, studies with children are lacking. We investigated the relationship of perirenal fat and other abdominal fat depots (including preperitoneal, intra-abdominal and subcutaneous fat) with carotid intima-media thickness (cIMT-a surrogate marker of CV risk) in prepubertal children, so as to identify novel markers that can be easily assessed and used in the early prevention of cardiovascular disease. SUBJECTS/METHODS: Subjects were 702 asymptomatic prepubertal Caucasian children (418 lean, 142 overweight and 142 obese) who were recruited in a primary care setting. Ultrasound measurements (perirenal, preperitoneal, intra-abdominal and subcutaneous fat and cIMT), clinical (body mass index (BMI) and systolic blood pressure) and metabolic parameters (insulin resistance (HOMA-IR), high molecular weight (HMW) adiponectin and serum lipids) were assessed. RESULTS: Perirenal fat was associated with diverse metabolic and CV risk factors in all the studied subjects. However, in overweight and obese children, perirenal fat was mostly associated with cIMT (P<0.001) and was the only fat depot that showed independent associations with cIMT in multivariate analyses (overweight chidren: ß=0.250, P=0.003, r2=12.8%; obese children: ß=0.254, P=0.002, r2=15.5%) after adjusting for BMI, gender, age and metabolic parameters. Perirenal fat was also the only fat depot that showed independent associations with HMW-adiponectin in obese children (ß=-0.263, P=0.006, r2=22.8%). CONCLUSIONS: Perirenal fat is the main abdominal fat depot associated with cIMT, especially in overweight and obese children, and may thus represent a helpful parameter for assessing CV risk in the pediatric population.

α-Defensins and bacterial/permeability-increasing protein as new markers of childhood obesity.

OBJECTIVES: The aim of this paper is to test whether α-defensins and bacterial/permeability-increasing protein were related to obesity and cardiovascular risk factors in prepubertal children. METHODS: Plasma α-defensins and bacterial/permeability-increasing protein, body mass index (BMI), waist circumference, systolic blood pressure (SBP), carotid intima media thickness (cIMT), HOMA-IR and HMW-adiponectin were assessed. RESULTS: In a cross-sectional study (N = 250), higher α-defensins concentrations were positively associated with BMI, waist, SBP, cIMT, HOMA-IR and negative correlated with HMW-adiponectin (all between r = 0.191 and r = 0.377, p ≤ 0.01 and p ≤ 0.0001). Conversely, plasma bacterial/permeability-increasing protein concentrations presented inversed associated with the same parameters (all between r = -0.124 and r = -0.329; p ≤ 0.05 and p ≤ 0.0001). In a longitudinal study (N = 91), α-defensins at age 7 were associated with BMI (ß = 0.189, p = 0.002; model R2 = 0.847) and waist (ß = 0.241, pthinsp;= 0.001; model R2 = 0.754) at age 10. CONCLUSIONS: α-Defensins and bacterial/permeability-increasing protein may be the markers of childhood obesity. Increased concentrations of α-defensins may predict BMI and abdominal fat deposition in children.

The macrophage activation product sCD163 is associated with a less favourable metabolic profile in prepubertal children.

OBJECTIVE: Macrophages are known to be involved in low-grade inflammatory processes such as obesity. soluble cluster of differentiation 163 (sCD163) is shed from the cell surface as specific macrophage activation marker. In prepubertal children, we studied if circulating sCD163 is associated with metabolic and cardiovascular risk markers. METHODS: A population of 236 school-aged Caucasian children (111 girls and 125 boys) aged 8 ± 1 year [81 normal weight (body mass index [BMI]-SDS < 1); 74 overweight (1 ≤ BMI-standard deviation score [SDS] < 2) and 81 with obesity (BMI-SDS ≥ 2)] were studied. BMI, waist circumference, fat mass and visceral fat were measured. Fasting serum sCD163, homeostatic model assessment of insulin resistance, high sensitivity C-reactive protein, gamma-glutamyl transpeptidase and lipids were quantified. RESULTS: Circulating sCD163 concentrations were higher in children with obesity (p < 0.0001). Associations were observed between circulating sCD163 and a less favourable metabolic profile as judged by higher waist circumference, fat mass, visceral fat, epicardial fat, homeostatic model assessment of insulin resistance, high sensitivity C-reactive protein, gamma-glutamyl transpeptidase and triglycerides (all between r = 0.173 and r = 0.363; p < 0.05 to p < 0.0001) and lower high-density lipoprotein-cholesterol (r = -0.285, p < 0.0001). In multiple regression analyses, circulating sCD163 was independently associated with HOMA-IR (ß = 0.162, p = 0.016; model R2 = 0.179) and high density lipoprotein-cholesterol/triglycerides ratio (ß = -0.167, p = 0.012; model R2 = 0.209). CONCLUSIONS: Childhood obesity may increase the risk of developing metabolic diseases later in life through chronic macrophage activation having deleterious effects on metabolism.

Uric acid, carotid intima-media thickness and body composition in prepubertal children.

BACKGROUND: Increased uric acid is an independent biomarker for cardiovascular disease in obese adolescents and adults. OBJECTIVE: We investigated whether uric acid relates to carotid intima-media thickness (cIMT) in prepubertal children, and whether body mass index (BMI) and preperitoneal fat modulate this association. METHODS: 359 asymptomatic prepubertal Caucasian children were stratified according to BMI categories (171 with BMI-SDS < 0; 188 with BMI-SDS ≥ 0) and according to preperitoneal fat levels (180 with preperitoneal fat <50th centile; 179 with preperitoneal fat >50th centile). Uric acid levels, insulin resistance (homeostasis model assessment insulin resistance; HOMA-IR), C-reactive protein (CRP), triacylglycerol (TG), systolic blood pressure (SBP), abdominal fat and cIMT (both by ultrasound) were assessed. RESULTS: Uric acid was associated with several cardiovascular risk factors, namely higher HOMA-IR, CRP, TG, BMI, waist, SBP, preperitoneal fat and cIMT (all P < 0.001 to P < 0.0001). Significant BMI and preperitoneal fat interactions were documented in the relationship between uric acid and cIMT (both P < 0.05), as uric acid was preferentially related to cIMT in heavier children (ß = 0.247, P < 0.001, r(2) = 9.1%) and in children with more preperitoneal fat (ß = 0.263, P < 0.0001, r(2) = 11.9%). CONCLUSIONS: Serum uric acid is associated with cIMT in asymptomatic prepubertal children. Both higher BMI and preperitoneal fat aggravate the potential risk of atherosclerotic disease imposed by higher concentrations of uric acid.

The sequence of prenatal growth restraint and post-natal catch-up growth leads to a thicker intima-media and more pre-peritoneal and hepatic fat by age 3-6 years.

BACKGROUND: Infants born small-for-gestational-age (SGA) who develop post-natal weight catch-up are at risk for insulin resistance, central adiposity and cardiovascular disease in later life, even in the absence of overweight. OBJECTIVE: In young (age 3-6 years) non-obese SGA children, we assessed arterial health (as judged by intima-media thickness [IMT]) and abdominal fat distribution (subcutaneous, visceral, preperitoneal and hepatic components by magnetic resonance imaging [MRI] and/or ultrasound [US]) besides a selection of endocrine markers. METHODS: Comparisons of measures in SGA (n = 27) vs. appropriate-for-GA (AGA) children (n = 19) of similar height, weight and body mass index. Longitudinal outcomes (age 3-6 years) were carotid IMT (cIMT); fasting glucose, circulating insulin, IGF-I and high-molecular-weight (HMW) adiponectin; abdominal fat partitioning by US. Cross-sectional outcomes (age 6 years) were aortic IMT (aIMT) and abdominal fat partitioning by MRI. RESULTS: At 3 and 6 years, cIMT and IGF-I results were higher and HMW adiponectin lower in SGA than AGA children; at 6 years, SGA subjects had also a thicker aIMT and more pre-peritoneal and hepatic fat, and were less insulin sensitive (all P values between <0.05 and <0.0001). cIMT correlated positively with pre-peritoneal fat, particularly at 6 years. Post-SGA status and weight gain in early childhood (between 3 and 6 years) were independent predictors of cIMT at 6 years, explaining 48 % of its variance. CONCLUSION: SGA children aged 3-6 years were found to have a thicker intima- media and more pre-peritoneal and hepatic fat than AGA children of comparable size.

Circulating GLP-1 in infants born small-for-gestational-age: breast-feeding versus formula-feeding.

Prenatal growth restraint associates with the risk for later diabetes, particularly if such restraint is followed by postnatal formula-feeding (FOF) rather than breast-feeding (BRF). Circulating incretins can influence the neonatal programming of hypothalamic setpoints for appetite and energy expenditure, and are thus candidate mediators of the long-term effects exerted by early nutrition. We have tested this concept by measuring (at birth and at age 4 months) the circulating concentrations of glucagon-like peptide-1 (GLP-1) in BRF infants born appropriate-for-gestational-age (AGA; n=63) and in small-for-gestational-age (SGA) infants receiving either BRF (n=28) or FOF (n=26). At birth, concentrations of GLP-1 were similar in AGA and SGA infants. At 4 months, pre-feeding GLP-1 concentrations were higher than at birth; SGA-BRF infants had GLP-1 concentrations similar to those in AGA-BRF infants but SGA-FOF infants had higher concentrations. In conclusion, nutrition appears to influence the circulating GLP-1 concentrations in SGA infants and may thereby modulate long-term diabetes risk.

Increased serum IgG and IgA in overweight children relate to a less favourable metabolic phenotype.

BACKGROUND: The adaptive immune system has emerged as an unexpected modulator of insulin resistance. B lymphocytes accumulate in adipose tissue and produce pathogenic antibodies that cause insulin resistance. OBJECTIVE: We studied whether circulating immunoglobulins (IgG, IgA and IgM) were related to metabolic risk markers in pre-pubertal children with and without overweight. DESIGN AND METHODS: Subjects were 270 asymptomatic pre-pubertal Caucasian children (145 lean, 125 overweight) recruited in a primary care setting. Assessments included serum IgG, IgA and IgM concentrations (nephelometry), insulin resistance (HOMA-IR) and fasting lipids (triacylglycerol and high-density lipoprotein [HDL]-cholesterol). RESULTS: Overweight children had higher IgG and IgA serum levels than lean children (P ≤ 0.01). Increasing serum IgG and IgA, but not IgM, were associated with a less favourable metabolic phenotype, consisting of higher HOMA-IR and triacylglycerol and lower HDL-cholesterol, particularly in obese children, in whom serum IgG and IgA were both independently associated with HOMA-IR (ß = 0.308, P = 0.017, r2 = 9.5% and ß = 0.361, P = 0.005, r2 = 13.0%, respectively) and triacylglycerol (ß = 0.343, P = 0.006, r2 = 11.1% and ß = 0.354, P = 0.003, r2 = 12.2%, respectively). CONCLUSIONS: Increased circulating IgG and IgA in overweight children are associated with a less favourable metabolic phenotype, particularly in obese children. These results suggest a relationship between adaptive immunity and insulin resistance in childhood obesity.

Decreased placental expression of pre-adipocyte factor-1 in children born small-for-gestational-age: association to early postnatal weight gain.

BACKGROUND: Preadipocyte factor-1 (Pref-1) is a key regulator of adipocyte differentiation acting as an inhibitor of adipogenesis; Pref-1 is highly expressed in embryonic tissues and placenta supporting a role in embryonic and fetal growth. The potential impact of placental Pref-1 expression in human pre- and postnatal development is unclear. OBJECTIVE AND HYPOTHESES: To assess the contribution of placental Pref-1 to fetal and postnatal growth. POPULATION AND METHODS: Placentas (N = 99) were collected at term delivery from singleton infants, who were born either appropriate (AGA; n = 59) or small-for-gestational-age (SGA; n = 40). Auxological data of all subjects were obtained at birth. In a subset of subjects (n = 31) we also obtained weight data at 4 mo and at 1 yr, together with body composition assessment (by DXA) at the age of 1 yr. Placental expression of Pref-1 was quantified by real-time PCR; the housekeeping gene GAPDH was used for comparisons. RESULTS: Pref-1 was significantly downregulated in the placentas from SGA babies as compared to AGA controls (P = 0.005). In SGA infants placental Pref-1 expression associated positively to body weight at 4 and 12 mo (r = 0.44, P = 0.05; r = 0.66, P = 0.001 respectively); at age 12 mo, placental Pref-1 expression was inversely related to total fat mass and positively correlated with total lean mass (r = -0.59, P = 0.01; and r = -0.59, P = 0.01,respectively). CONCLUSION: Placental Pref-1 expression in SGA fetuses was decreased and associated with postnatal body weight, suggesting a role of Pref-1 in the regulation of postnatal development.

Variations in the obesity genes FTO, TMEM18 and NRXN3 influence the vulnerability of children to weight gain induced by short sleep duration.

OBJECTIVE: Shorter sleep duration predisposes to obesity, but the mechanisms whereby sleep deprivation affects body weight are poorly understood. We tested whether this association is modulated by the obesity genes FTO, TMEM18 and NRXN3. SUBJECTS: Body mass index (BMI), waist circumference, visceral fat (abdominal ultrasound), homeostasis model assessment for insulin resistance (HOMA-IR), systolic blood pressure (SBP) and sleep time per 24 h were assessed in 297 asymptomatic children (151 boys, 146 girls; age range 5-9 years; BMI s.d. score range -2.0-4.0). Associations between sleep duration and the abovementioned outcomes were tested for three common single-nucleotide polymorphisms (SNPs), namely FTO (rs9939609), TMEM 18 (rs4854344) and NRXN3 (rs10146997), as well as for their combination. RESULTS: TT homozygotes (but not A(*) carriers) for the FTO SNP, exhibited nominal associations between decreasing sleep duration and increasing BMI, waist circumference, visceral fat and HOMA-IR (all P<0.05). Similar associations were observed in children with risk alleles (but not in those without risk alleles) for the TMEM18 and NRXN3 SNPs (P<0.05 to P<0.0001). The three SNPs had additive effects on the negative associations between sleep and, respectively, BMI (P<0.001), waist (P<0.005), visceral fat (P<0.001), HOMA-IR (P=0.010) and SBP (P<0.0005). The combined effects on obesity measures and SBP remained significant after correction for multiple testing. On average, 2 h of sleep less per night was associated with an increase in BMI of 1.0 s.d. (95% confidence interval 0.5-1.6 s.d.) and with 8.0 cm (95% confidence interval 3.6-12.2 cm) more waist circumference in genetically susceptible children. CONCLUSION: By age 7, common variations in FTO, TMEM18 and NRXN3 influence the vulnerability to metabolic complications of sleep deprivation. Further genetic studies are warranted to replicate these findings in other populations.

Soluble fatty acid synthase relates to bone biomarkers in prepubertal children.

SUMMARY: Circulating soluble fatty acid synthase (FASN, a key enzyme in de novo biosynthesis of fatty acids, expressed in both adipocytes and osteoblasts) is clinically related to a less favorable bone profile in healthy prepubertal children. Soluble FASN may participate in the reciprocal regulation between fat and bone metabolism. INTRODUCTION: Fatty acid synthase (FASN), a key enzyme in de novo biosynthesis of fatty acids, is expressed in adipocytes and osteoblasts. We hypothesized that FASN may participate in the crosstalk between fat and bone. To this aim, we studied the relation between circulating soluble FASN (an extracellular FASN that reflects previously intracellular enzymatic activity) and adipose tissue and bone biomarkers in prepubertal children. METHODS: Circulating soluble FASN, total and high molecular weight (HMW) adiponectin, bone biomarkers [osteocalcin (OC), uncarboxylated osteocalcin (ucOC), C-terminal cross-linked telopeptide of type I collagen (CTX), bone-specific alkaline phosphatase (BSAP)], and a profile of energy metabolism [body fat, insulin resistance and secretion (HOMA), serum lipids] were assessed in 84 asymptomatic prepubertal children (44 girls, 40 boys, age 6.8 ± 0.1 year). Serum 25-OH Vitamin D (Vit D) was additionally measured. RESULTS: Circulating soluble FASN increased with increasing HMW adiponectin (r = 0.29, p = 0.01) and decreasing serum Vit D (r = -0.21, p < 0.05), and was related to a less favorable bone profile, showing negative associations with bone-derived metabolic parameters [total OC (r = -0.33, p = 0.002) and ucOC (r = -0.37, p < 0.0001)] and a positive association with the CTX-to-BSAP ratio (r = 0.31, p < 0.01). These correlations were not explained by age, gender, body fat, insulin resistance or secretion or serum lipids; however, they were predominant in those subjects with Vit D levels below the population median. CONCLUSIONS: Circulating soluble FASN relates to both adipose tissue and bone biomarkers in prepubertal children, with associations that are dependent on Vit D concentrations. These findings suggest that FASN may participate in the crosstalk between fat and bone metabolism.

Placental FTO expression relates to fetal growth.

OBJECTIVE: The fat mass and obesity-associated gene (FTO) participates in the control of postnatal weight gain. We assessed whether FTO is expressed in human placenta and whether such expression relates to prenatal weight gain and to the rs9939609 single nucleotide polymorphism (SNP) in FTO. DESIGN AND SUBJECTS: In a birth cohort study, placentas from women (n = 147) with an uncomplicated, singleton, term pregnancy were weighed at delivery. Real-time PCR was used to study, in placental tissue, the expression of FTO and of housekeeping genes (TATA box binding protein and succinate dehydrogenase complex, subunit A) and to genotype the rs9939609 SNP in FTO. Weights and lengths of the newborns were measured; circulating insulin and insulin-like growth factor-I (IGF-I) were quantified in cord blood. RESULTS: FTO was highly expressed in placenta and was associated with increased fetal weight and length (P<0.001 to P<0.0001). Maternal parity showed an interaction (P<0.001) in the association between placental FTO expression and placental weight. Placental FTO mRNA expression was associated with increased fetal-to-placental weight ratio (P<0.005) in infants from primiparous women, and was associated with increased fetal weight and length and placental weight (P<0.001 to P<0.0001) in infants from nonprimiparous women. These associations were not explained by either cord insulin or IGF-I. Placental FTO expression was unrelated to placental FTO rs9939609 SNP. CONCLUSION: FTO is expressed in the human placenta. In a maternal parity-dependent manner, placental FTO may participate either in the control of fetal weight gain or in the partitioning between placental and fetal growth.

LIGHT is associated with hypertriglyceridemia in obese subjects and increased cytokine secretion from cultured human adipocytes.

BACKGROUND: LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells) is a member of the tumor necrosis factor (TNF) family, primarily expressed in lymphocytes, which was associated with the induction of pro-inflammatory cytokines and alterations of lipid homeostasis in animal models. We aimed to analyze whether LIGHT has a role in the human obesity-associated inflammatory status. METHODS: The association between circulating LIGHT concentrations and clinical variables was studied in 190 subjects with different degrees of obesity and glucose tolerance. The expression and release of 21 different cytokines, and the expression of genes involved in lipid metabolism were also evaluated after stimulation with LIGHT in cultured human differentiated adipocytes. RESULTS: Serum LIGHT concentrations positively associated with body mass index (BMI), fat mass, glycated hemoglobin and fasting triglycerides, and negatively with high-density lipoprotein cholesterol. Circulating LIGHT concentrations were significantly increased in morbidly obese subjects and in patients with type 2 diabetes. LIGHT induced the secretion of several cytokines and upregulated the expression and secretion of interleukin-6 (IL-6), IL-8, Growth Regulated Oncogene (GRO) and monocyte chemotactic protein-1 (MCP-1). These observations were concomitant with the activation of nuclear factor (NF)-kappaB signalling in human differentiated adipocytes. LIGHT also upregulated the expression and synthesis of its own receptor (herpesvirus entry mediator (HVEM)) and decreased the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and fatty acid synthase. CONCLUSION: These data suggest that LIGHT may have a role in mediating chronic inflammation and alterations of lipid metabolism in obese subjects.

Thyroid hormone responsive Spot 14 increases during differentiation of human adipocytes and its expression is down-regulated in obese subjects.

CONTEXT: Very limited information is available regarding the function of human thyroid hormone responsive Spot 14 (human S14, hS14) in adipogenesis and human adiposity. OBJECTIVE: To evaluate hS14 levels during differentiation of human pre-adipocytes, in human fat depots and isolated fat cells. DESIGN: This was a cross-sectional study. SUBJECTS: A total of 161 omental (OM) and 87 subcutaneous (SC) adipose tissue samples obtained during elective surgical procedures from a population who varied widely in terms of obesity. MEASUREMENTS: hS14 gene expression and protein levels during adipogenesis were assessed by RT-PCR, western blot, and using an automated confocal imaging approach. RESULTS: hS14 gene expression levels were decreased in OM adipose tissue from overweight (-42.0%) and obese subjects (-56.5%) compared with lean subjects (P<0.05 and P<0.0001, respectively). hS14 mRNA (but not hS14-related) was inversely associated with obesity measures such as body mass index (P=0.001), percent fat mass (P=0.001), waist-to-hip ratio (P=0.020), and systolic blood pressure (P=0.031). hS14 gene expression and protein levels were up-regulated at the early stages of differentiation of human pre-adipocytes as well as for 3T3-L1 cells. That observation was most prominent in those individual cells exhibiting the more marked differentiation features. hS14 gene expression levels increased by approximately 45 000-fold in mature adipocytes. Increased hS14 levels were also found in stromal-vascular cells/pre-adipocytes (3.8-fold, P<0.05) and in adipose tissue samples (1.9-fold, P<0.0001) from SC compared with OM fat depots. CONCLUSIONS: These results suggest that hS14 is involved in human adipogenesis, but inversely related to obesity and OM fat accumulation.

Decreased circulating lactoferrin in insulin resistance and altered glucose tolerance as a possible marker of neutrophil dysfunction in type 2 diabetes.

CONTEXT: Lactoferrin is an innate immune system protein with multiple beneficial health activities. OBJECTIVE: To gain insight in the interaction between innate immune system and metabolic disturbances (obesity and insulin resistance), we investigated the relationship between circulating lactoferrin and chronic inflammation-associated insulin resistance according glucose tolerance status in Caucasian population. DESIGN, SETTING, PARTICIPANTS, AND MAIN OUTCOME MEASURES: Circulating nonstressed lactoferrin (ELISA), metabolic variables, and inflammatory markers were measured in 229 men, 94 with normal (NGT) and 135 with altered glucose tolerance (AGT). Lactoferrin secretion by neutrophil was investigated in whole-blood culture (four young NGT subjects, four older NGT subjects, and four patients with type 2 diabetes) under microbial lipopolysaccharide (LPS) with IL-6 and rosiglitazone treatment. We also tested the lactoferrin action in THP-1 cells under LPS stimulus. RESULTS: Circulating lactoferrin was significantly decreased in patients with AGT (431.5 +/- 187.5 vs. 493.5 +/- 238.9 ng/ml, P = 0.02). In addition, circulating lactoferrin was negatively associated with hyperglycemia and obesity measures and positively with insulin sensitivity. Lactoferrin was negatively related to inflammatory markers, especially in AGT subjects. In ex vivo experiments, we found a significant decrease in LPS-induced lactoferrin release from neutrophils in subjects with type 2 diabetes. IL-6 coincubation decreased LPS-induced lactoferrin release in NGT subjects (P < 0.001). Finally, rosiglitazone treatment led to increased lactoferrin secretion (398 +/- 193 vs. 280.1 +/- 104.9 ng/ml, P < 0.0001). Lactoferrin decreased nuclear factor-kappabeta activation and IL-6, IL-8, and macrophage chemoattractant protein-1 expression under LPS challenge. CONCLUSIONS: Decreased circulating lactoferrin levels may play a role in chronic low level inflammation-associated insulin resistance.

Effects of filtration of semen doses from subfertile boars through neuter Sephadex columns.

This study was designed to develop a method of improving the quality of sperm obtained from subfertile Piétrain boars. Seminal doses were filtered through neuter Sephadex columns (G-25 Medium, G-50 Fine, G-50 Medium and G-75, length 10 +/- 0.5 cm, flow rate 1 ml/20 s). Doses were prepared by pooling 10 ml semen samples collected from 58 asthenoteratospermic boars and diluted the sperm-cell rich fraction 1 : 6 in Betsville thawing solution extender. Sperm quality was determined before and after the filtering process. Sperm morphology and motility were assessed using the computer program SCA 2002 production, and sperm vitality was evaluated by fluorescence multistaining. ORT and HRT tests were used to determine the osmotic resistance of spermatozoa, and metabolic performance was assessed by measuring l-lactate production. Results indicate that the filtration process rendered increased proportions of mature spermatozoa and of viable spermatozoa with an intact acrosome, nucleus and mitochondrial sheath. Sperm filtration led to decreased percentages of spermatozoa with proximal and distal droplets and of agglutinated spermatozoa, along with slightly diminished ORT values. HRT scores and L-lactate production were unaffected. Our findings indicate that filtering through a Sephadex column improves the sperm morphology and vitality of seminal doses obtained from subfertile boars, but produces no functional changes in the spermatozoa. All four column types yielded similar results.

Hyaluronic acid delays boar sperm capacitation after 3 days of storage at 15 degrees C.

The present study was undertaken to determine the effects of the addition of hyaluronic acid (HA), ranged from 12.5 to 200 microg/ml, on boar sperm capacitation status during a storage time (up to 3 days) at 15 degrees C in Beltsville thawing solution (BTS). The raw extender was the negative control whereas different concentrations of caffeine (CAF), ranged from 0.25 to 8mM, served as positive controls. Sperm viability, motility, morphology, and osmotic resistance were also determined before and after assessing the treatments. Samples were obtained from 28 healthy and post-pubertal Piétrain boars and sperm parameters were tested immediately after the addition of treatments and after 1, 2 and 3 days of refrigeration at 15 degrees C. Sperm capacitation status was determined by chlortetracycline (CTC) staining and sperm viability by means of a multiple fluorochrome-staining test. Sperm motility and morphology were assessed using phase-contrast microscopy accompanied by a computer assisted sperm analysis system (CASA). Whereas HA delayed sperm capacitation, CAF increased the frequency of capacitated spermatozoa after 2 days of cooling. Moreover, HA did not modify other sperm parameters, such as sperm velocity, whereas CAF increased progressive motility during the first 2 days of cooling and then decreased. It can be concluded that the addition of HA at 50 and 100 microg/ml to the BTS extender may delay sperm capacitation after 3 days of cooling.

Boar spermatozoa and prostaglandin F2alpha. Quality of boar sperm after the addition of prostaglandin F2alpha to the short-term extender over cooling time.

Prostaglandin F2alpha (PGF2alpha) has been used to improve reproductive performance in swine. The goal of the present work was to determine how the addition of PGF2alpha affects boar sperm quality. Eleven different treatments were evaluated: eight with only PGF2alpha (0.625, 1.25, 2.50, 5, 10, 12.50, 25 and 50mg PGF2alpha/100ml) and three binary treatments (0.625mg PGF2alpha/100ml+200microg/ml hyaluronic acid (HA), 1.25mg PGF2alpha/100ml+200microg/ml HA, 0.625mg PGF2alpha/100ml+7.5microM caffeine (Caf)). All these substances were added to 16 ejaculates from 16 healthy and sexually mature boars (n=16), and each ejaculate was considered as a replicate. Our study also assessed the effects of these 11 treatments over different periods of preservation. Sperm quality was tested immediately after the addition of treatments (time 0), and after 1, 3, 6 and 10 days of cooling at 15 degrees C. To evaluate sperm quality, five parameters were analysed: (1) sperm viability, acrosome and mitochondrial sheath integrity (using a multiple fluorochrome-staining test), (2) sperm motility, (3) sperm morphology and (4) agglutination (using a computer assisted system) and (5) osmotic resistance (using the ORT). Parametric (analysis of variance for repeated measures) and non-parametric tests (Friedman test) were used as statistical analyses. Treatments with PGF2alpha concentrations higher than 12.5mg/100ml were cytotoxic while the others did not damage boar spermatozoa. Thus, the other treatments may be used to produce profitable effects without adverse effects. Moreover, the addition of PGF2alpha at 5mg/100ml to sperm diluted in BTS may maintain sperm viability and motility better after 6 days of cooling, because significant differences were observed (P<0.05) compared with control at the same time.

Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs.

This study evaluated the effects of cooling, freezing and thawing on the plasma membrane integrity, kinetics and expression of two sugar transporters glucose transporter-3 and -5 (GLUT-3 and GLUT-5) in spermatozoa from Iberian boars. Semen samples were collected twice weekly from eight young, fertile Iberian boars of the 'Entrepelado' and 'Lampiño' breeds. The samples were suspended in a commercial extender and refrigerated to 17 degrees C for transport to the laboratory (step A), where they were further extended with a lactose-egg yolk-based extender and chilled to 5 degrees C (step B) prior to freezing in the presence of glycerol (3%). Spermatozoa were assessed for plasma membrane integrity and sperm motility at each of the steps, including post-thaw (step C). Aliquots were also prepared for immunocytochemical localisation of the sugar transporters (fixed and thin smears for transmission and scanning electron microscopy levels respectively) and for SDS-PAGE electrophoresis and subsequent western blotting, using the same antibodies (rabbit anti-GLUT-3 and anti-GLUT-5 polyclonal antibodies). The results showed lower percentages of progressively motile spermatozoa at step C in both breeds, while the percentage of live spermatozoa was significantly lower only in the 'Entrepelado' breed. The results obtained from electron microscopy clearly showed that Iberian boar spermatozoa expressed the hexose transporters, GLUT-3 and GLUT-5. The pattern of expression, in terms of location and concentration, was characteristic in each case but, in the case of isoform GLUT-5, it remained constant during the different steps of freezing-thawing protocol. These results indicate that cryopreservation affects the status of sperm cells of Iberian boars by altering the distribution of some membrane receptors and decreasing the percentage values of parameters linked to sperm quality.

Structural and ultrastructural features of boar seminal vesicles.

The morphological features of boar seminal vesicles were examined by light and transmission microscopy. Boar seminal vesicles consist of glandular tissue arranged in multiple lobules containing a system of ramified secretory tubules. The secretory tubules are composed of a mucosa formed by an epithelium and an underlying lamina propria and, are surrounded by a muscular layer. The epithelium is made up of columnar cells and occasional basal cells. Mast cells are frequently found among epithelial cells. Three types of columnar cells, considered different stages of the secretory cell cycle, are present: principal cells, clear cells and dense cells. Principal cells are functionally differentiated cells characterised by abundant mitochondria, great development of the rough endoplasmic reticulum and presence of secretory granules in their cytoplasm. The apical surface of many principal cells shows apical blebs filled with PAS-positive material. No acid mucosubstances are detected. Microvilli cover the apical surface except in the apical blebs. Dense cells, arranged between principal cells, are also functional differentiated cells but with signs of cellular degeneration. Clear cells are an initial differentiated stage of columnar cells and are characterised by the presence of a poorly developed rough endoplasmic reticulum and by the absence of secretory granules. Proliferating cells are present among columnar cells. Basal cells contain scarce cytoplasm, few organelles and no secretory granules. The lack of mitotic activity in these cells suggests that they do not act as precursors of columnar cells.