Expression of genes related to ileal barrier function in heritage and modern broiler chickens.

1. An experiment was conducted to determine differences in the expression of genes encoding intestinal barrier proteins between fast, medium and slow-growing chickens. Chicken breeds Athens Canadian Random Bred (ACRB), Longenecker's Heritage (LHR), RedBro, Hubbard H1 (HH1), Cobb500 and Ross708 were raised from hatch for 35 d.2. Ileal samples were collected at embryonic day E19 (-2 days post-hatch), hatch and d 7, 14, 21, 28 and 35 post-hatch to assess the expression of genes encoding tight junction proteins (claudins, CLDN; occludin, OCLN; zonula occludens, ZO; and junctional adhesion molecules, JAM), mucin (Muc2), immunoglobulin A (IgA), polymeric immunoglobulin receptor (pIgR) and fatty acid binding protein (FABP2).3. Expression of CLDN-1 was increased (p < 0.0001) in LHR compared to Cobb500 while CLDN-5 was increased (p < 0.0001) in ACRB, HH1, RedBro and Ross708 compared to LHR as well as in ACRB compared to Cob500. Occludin was upregulated (p = 0.01) in ACRB and LHR compared to Ross708 at d 14 post-hatch. Expression of ZO-1 was upregulated (p = 0.001) in LHR compared to Ross708, HH1 and Cobb500. Tight junction genes, except CLDN-4, JAM-2 and JAM-3 were downregulated (p < 0.0001) at hatch and d 7 post-hatch. Expression of Muc2 was increased (p < 0.0001) in LHR compared to RedBro and from -2 d to d 7 post-hatch.4. Immunoglobulin A was increased (p = 0.001) in LHR compared to Ross708 and HH1 at -2 d post-hatch and in LHR compared to ACRB, Cobb500 and Ross708 at hatch. In addition, IgA expression was increased in all breeds at d 14 post-hatch while pIgR was upregulated (p = 0.02) in Cobb500 and Ross708 compared to ACRB, HH1, LHR and RedBro at hatch.5. The gene expression patterns suggest that selection for growth may have not induced changes in junctional complexes and immune defence genes. However, the results confirmed that the expression of these genes is age dependent.

Expression of genes associated with nutrient uptake in intestines of chickens with different growth potentials show temporal changes but are not correlated with growth.

1. The study was designed to compare the expression of genes that encode proteins located at either the brush border (BB) or basolateral (BL) of the gut epithelium among fast and slow-growing broilers.2. Six broiler breeds with different growth capacities were used: Ross 708, Hubbard H1 (HH1), Cobb 500, Longenecker's Heritage (LHR), Red-Bro, and the Athens Canadian Randombred Control (ACRB). Birds were sampled between embryonic day (ED) 19 and day 35 post-hatch (PH).3. Performance parameters indicated that Ross 708, HH1, and Cobb 500 had the highest body weights (BW) while ACRBs had the lowest.4. Quantitative RT-PCR was performed on 13 genes encoding proteins associated with nutrient processing and uptake. Statistical analysis was carried out (ANOVA) for eight BB genes: Aminopeptidase N (APN), four amino acid transporters, (ATBo,+, BoAT, bo,+AT, EAAT3) a di- and tri-peptide transporter (PepT1), and two sugar transporters (GLUT5 and SGLT1). Analysis of four amino acid transporters (CAT1, CAT2, LAT1, and γ+LAT1), and a single sugar transporter (GLUT2) associated with BL was carried out.5. Four BB associated genes (APN, EAAT3, BoAT, and b0,+AT) in the small intestine were negatively correlated with growth.6. In most cases, genes encoding BB proteins increased in expression over time (P < 0.05) in the small intestine, while, in the caeca, the expression decreased (P < 0.05). The mRNA of BL-associated proteins showed decreased (P < 0.05) expression over time in all gut segments, with exception of GLUT2, which increased in expression in the small intestine.7. The temporal changes in gene expression were consistent among bird lines and BB associated genes tended to increase over time, while BL associated genes tended to decrease over time. Correlation analysis indicated that mRNA expression of nutrient transporter genes may not be a good predictor of growth potential.

The effect of delayed feeding post-hatch on caeca development in broiler chickens.

1. Broiler chicks are frequently deprived of food up to 72 h due to uneven hatching rates, management procedures and transportation to farms. Little is known about the effect of delayed feeding due to extended hatching times on the early neonatal development of the caeca. Therefore, the objective of this study was to investigate the developmental changes and effects of a 48-h delay in feed access immediately post-hatch (PH) on the caeca.2. After hatch, birds (Ross 708) were randomly divided into two treatment groups (n = 6 battery pen/treatment). One group (early fed; EF) received feed and water immediately after hatch, while the second group (late fed; LF) had access to water but had delayed access to feed for 48 h. Contents averaging across all regions of the caeca were collected for mRNA expression as well as for histological analysis at -48, 0, 4 h PH and then at 1, 2, 3, 4, 6, 8, 10, 12 and 14 days PH.3. Expression of MCT-1 (a nutrient transporter), Cox7A2 (related to mitochondrial function) IgA, pIgR, and ChIL-8 (immune function) genes was affected by delayed access to feed that was dependent by the time PH. Expression of immune and gut barrier function-related genes (LEAP2 and MUC2, respectively) was increased in LF group. There was no effect of feed delay on expression of genes related to mitochondrial functions in the caeca, although developmental changes were observed (ATP5F1B, Cox4|1). Caecal mucus and muscle thickness were affected by delayed access to feed during caeca development.4. The data suggested a limited effect of delayed feed access PH on the developmental changes in caecal functions. However, the caeca seemed to be relatively resistant to delayed access to feed early PH, with only a few genes affected.

Endotelina-1, proteína C reactiva ultrasensible y actividad antioxidante en pacientes con hipotiroidismo subclínico y en eutiroideos con autoinmunidad tiroidea. Efectos del tratamiento con levotiroxina / Endothelin-1, high sensitivity C reactive protein and antioxidant activity in subclinical hypothyroid patients. Effects of levothyroxine treatment

RESUMEN La Endotelina-1 (ET1) y Proteína C Reactiva ultrasensible (PCRus) como marcadores de disfunción endotelial (DE) e inflamación vascular en hipotiroidismo subclínico (HS) han mostrado resultados controvertidos. El rol del estrés oxidativo y defensa antioxidante (TRAP) es motivo de discusión. Objetivos Establecer si el HS y la autoinmunidad tiroidea (AIT), excluyendo otros factores de riesgo cardiovascular, pueden causar DE e inflamación vascular, evaluadas a través de ET1 y PCRus, respectivamente. Establecer si TRAP juega algún rol. Evaluar cambios en ET1 y PCRus luego del tratamiento con levotiroxina (LT4). Material y métodos Se evaluaron prospectivamente 70 pacientes divididos en 3 grupos: HS: 41 pacientes (T4 normal,TSH >4,2 y <10 mUI/L), AIT: 10 pacientes eutiroideos (TSH <4,2 mUI/L) con aTPO y/o aTg (+) y Control: 19 pacientes eutiroideos sin AIT. Se excluyeron otros factores de riesgo cardiovascular. Se midió basalmente ET1, PCRus y TRAP plasmáticos, y en HS bajo LT4 (n = 24): ET1 y PCRus. Resultados No hubo diferencias significativas en edad, IMC, perfil lipídico y TRAP. ET1 y PCRus fueron significativamente mayores en pacientes con HS (media ± DS 1,77 ± 0,85 pg/ml y 1,5 ± 0,6 mg/l vs. controles (0,8 ± 0,3 pg/ml y 0,5 ± 0,2 mg/l) p <0,0001 y <0,008 respectivamente. Del mismo modo en AIT (1,4 ± 0.4 pg/ml y 2,3 ± 1,3 mg/l) vs controles p <0,0001 y <0,034, respectivamente. La TSH fue mayor en el grupo AIT vs. Control 2,57 ± 0,88 vs. 1,64 ± 0,5 mUI/L; p = 0,002. En HS bajo LT4 (8,7 ± 3,8 meses) se observó descenso de ET1 (p <0,001). ET1 correlacionó con TSH (r = 0,5 p <0,0001). El punto de corte de ET1 mediante curva ROC fue 1,32 pg/ml (Sensibilidad 81,6%-Especificidad 75%). Conclusiones ET1 y PCRus resultaron marcadores útiles para evaluar DE e inflamación vascular asociadas a HS. La defensa antioxidante no ejercería un rol en estos mecanismos. El tratamiento con LT4 produjo una significativa caída de ET1, pudiendo necesitarse un período más largo de eutiroidismo para normalizarla. En AIT, niveles de TSH >2,5 mUI/L podrían sugerir un mínimo grado de hipotiroidismo justificando la elevación en ET1 y PCR, sin descartar el rol de la AIT "per se".

ABSTRACT The measurement of endothelin-1 (ET1) and high sensitivity C-reactive protein (hsCRP) as markers of endothelial dysfunction (ED) and vascular inflammation in subclinical hypothyroidism (SH) has shown controversial results. The role of oxidative stress and antioxidant defense (TRAP) is a matter of discussion. Objectives To establish if SH and thyroid autoimmunity (TAI), excluding other cardiovascular risk factors, may cause ED and vascular inflammation, evaluated through the measurement of ET1 and hsCRP respectively. To determine if TRAP could have some role. Additionally, changes in these parameters after treatment with levothyroxine (LT4) will be evaluated. Material and methods: 70 patients were prospectively evaluated. They were classified into: SH Group: 41 patients (normal T4, TSH> 4.2 and <10 mIU/L), TAI Group: 10 euthyroid patients (TSH <4.2 mUI/L) with positive aTPO and/or aTg and Control Group: 19 euthyroid patients without TAI. Other cardiovascular risk factors were excluded in patients and controls. Plasma ET1, hsCRP and TRAP were measured basally, and ET1 and hsCRP under LT4 therapy in the HS Group. Results There were no significant differences between the 3 groups in age, BMI, lipids and TRAP. ET1 and hsCRP were significantly higher in patients with SH (mean ± SD 1.77 ± 0.85 pg/ml and 1.5 ± 0.6 mg/l) vs. controls (0.8 ± 0.3 pg/ml y 0.5 ± 0.2 mg/l) p <0.0001 y <0.008 respectively. Similarly, in TAI patients (1.4 ± 0.4 pg/ml y 2.3 ± 1.3 mg/l) vs controls, p <0.0001 and <0.034, respectively. TSH was higher in the TAI patients versus control group (2.5 ± 0.88 versus 1.64 ± 0.5 mIU/L, p = 0.002). Twenty-four patients with SH showed a significant decrease in ET1 (p <0.001) under treatment with LT4 (8.7 ± 3.8 months). ET1 had a highly significant correlation (p <0.0001) with TSH (r = 0.5). The cut-off level of ET1 established by ROC curve was 1.32 pg/ml (Sensitivity 81.6%-Specificity 75%). Conclusions ET1 and hsCRP were useful markers to evaluate ED and vascular inflammation associated with SH. There were no differences in TRAP levels between patients and controls, so it does not appear that oxidative stress would have played any role. Treatment with LT4 produced a significant drop in ET1. Probably, a longer period of euthyroidism might be necessary to normalize ET1 levels. In TAI Group, TSH levels >2.5 mUI/L could suggest a "minimal degree" of hypothyroidism justifying the elevation in ET1 and hs CRP. The role of the TAI "per se" couldn't be completely ruled out.

Supplementation with n-3, n-6, n-9 fatty acids in an insulin-resistance animal model: does it improve VLDL quality?

Insulin-resistance (IR), of increased cardiovascular risk, is characterized by the production of altered VLDL with greater atherogenicity. Dietary fatty acids influence the type of circulating VLDL. But, it is not clear how dietary fatty acids impact VLDL characteristics in IR. AIM: to evaluate the effects of n-3, n-6 and n-9 fatty acid supplementation on preventing atherogenic alterations in VLDL, in a diet-induced IR rat model. Male Wistar rats (180-200 g) were fed: standard diet (control, n = 8) and a sucrose rich diet (30% sucrose in water/12 weeks, SRD; n = 24). Simultaneously, SRD was subdivided into SRD-C (standard diet), and three other groups supplemented (15% w/w) with: fish oil (SRD-n3), sunflower oil (SRD-n6) and high oleic sunflower oil (SRD-n9). Lipid profile, free fatty acids, glucose, and insulin were measured. Isolated VLDL (d < 1.006 g ml-1) was characterized by chemical composition and size (size exclusion-HPLC). In comparison with SRD-C: SRD-n3 showed an improved lipoprotein profile (p < 0.01), with lower levels of insulin and HOMA-IR (p < 0.05). SRD-n6 showed increased levels of HDL-cholesterol and lower insulin levels. SRD-n9 did not exhibit differences in lipid and IR profile, and even favored weight gain and visceral fat. Only SRD-n3 prevented the alterations in VLDL-TG% (54.2 ± 4.4% vs. 68.6 ± 8.2, p < 0.05) and showed lower large VLDL-% (22.5[19.7-35.6] vs. 49.1[15.5-82.0], p < 0.05), while SRD-n6 and SRD-n9 did not show effects. CONCLUSION: In IR, while n-3 PUFA showed expected favorable effects, supplementation with n-6 PUFA and n-9 MUFA did not prevent atherogenic alterations of VLDL. Thus, the recommendations of supplementation with these fatty acids in general diet should be revised.

Alteraciones glucémicas en los pacientes con enfermedad renal crónica / Glycaemic changes in patients with chronic kidney disease

En Argentina no se han realizado estudios destinados a establecer la prevalencia de disglucemias (glucemia alterada en ayunas [GAA], tolerancia alterada a la glucosa [TAG] y diabetes mellitus [DM]) en pacientes con enfermedad renal. Se decidió realizar un estudio observacional, evaluando la frecuencia con prueba de tolerancia oral a glucosa (PTOG) en pacientes con enfermedad renal crónica (ERC), sin registro de disglucemia en sus historias clínicas. Se realizó PTOG a 254 pacientes (60,62% masculinos), con ERC estadios 3, 4 y 5, en tratamiento conservador, hemodiálisis o trasplante. Los resultados mostraron pacientes con valores de DM: 10 pacientes según ayunas exclusivamente (3,94%; IC 95%: 1,35-6,53%); exclusivamente segunda hora, 11 pacientes (4,33%; IC 95%: 1,63-7,03%); por ambos criterios, 15 pacientes (5,91%; IC 95%: 2,81-9,00%); por al menos un criterio, 36 pacientes (14,17%; IC 95%: 9,69-18,66%). En análisis multivariado, la DM se asoció con valor de cintura (OR=1,033 por cm; IC 95%: 1,005-1,062; p=0,019) y con tratamiento sustitutivo vs. conservador (OR=0,41; IC 95%: 0,19-0,92; p=0,028). La GAA (criterio ADA) fue del 19,75% en tratamiento conservador vs. 9,24% en tratamiento sustitutivo, con diferencia estadísticamente significativa. No fue significativa la diferencia de TAG que evidenció 24,6 y 20,3% en tratamiento conservador y sustitutivo, respectivamente. Se propone la realización de PTOG en todo paciente con ERC, ya que permite la detección de todo el rango de disglucemias desconocidas, evitando el subdiagnóstico y favoreciendo la realización de tratamientos para evitar su progresión, en caso de estar ante la presencia de un grupo de riesgo para DM (GAA o TAG), así como la elección de la medicación más adecuada para el trasplante o el inicio del tratamiento de nuevos casos de DM no diagnosticada, para disminuir la morbimortalidad (AU)

In Argentina, there have been no studies aimed at establishing the prevalence of dysglycaemia (impaired fasting glucose [IFG], impaired glucose tolerance [IGT] and diabetes mellitus [DM]) in patients with chronic kidney disease (CKD). Our group decided to conduct an observational study to evaluate the frequency with oral glucose tolerance test (OGTT) in CKD patients with no previous data for dysglycaemia in their medical records. OGTT was performed in 254 patients (60.62% male) with stage 3, 4 and 5 CKD under conservative treatment, haemodialysis or transplantation. Results for DM were found in 10 patients according to fasting glucose alone (3.94%; 95% CI: 1.35-6.53%), 11 patients with exclusively the second hour criterion (4.33%; 95% CI: 1.63-7.03%), 15 with both criteria (5.91%; 95% CI: 2.81-9.00%) and 36 patients with at least one criteria (14.17%; 95% CI: 9.69-18.66%). In a multivariate analysis, DM was associated with waist circumference (OR=1.033 per cm; 95% CI, 1.005 to 1.062; P=.019) and with conservative treatment vs. replacement therapy (OR=0.41; 95% CI: 0.19-0.92;P=.028). IGT was evident in 24.6% and 20.3 on conservative vs. replacement therapy, with no statistically significant difference. IFG (ADA criteria) was 19.75 vs. 9.24% in conservative vs. replacement therapy, with a statistically significant difference. OGTT is suggested for all CKD patients since it is able to detect the full range of unknown dysglycaemias, which avoids underdiagnoses and favours performing treatments to prevent progression in DM risk groups (IFG and/or IGT). It also aids in the selection of the most appropriate medication for transplantation or treatment initiation in new cases of undiagnosed DM to decrease morbidity and mortality (AU)

HemaSpot, a Novel Blood Storage Device for HIV-1 Drug Resistance Testing.

HemaSpot, a novel dried-blood storage filter device, was used for HIV-1 pol resistance testing in 30 fresh United States blood samples and 54 previously frozen Kenyan blood samples. Genotyping succeeded in 79% and 58% of samples, respectively, improved with shorter storage and higher viral load, and had good (86%) resistance mutation concordance to plasma.

Effect of insulin-resistance on circulating and adipose tissue MMP-2 and MMP-9 activity in rats fed a sucrose-rich diet.

BACKGROUND AND AIM: Adipose tissue produces different metalloproteinases (MMPs), involved in adipogenesis and angiogenesis. Different studies have shown that in obesity the behavior of different MMPs may be altered. However there are scarce data about the effect of insulin-resistance (IR) on MMP-2 and MMP-9 activity in adipose tissue. Our aim was to determine whether sucrose induced IR modifies MMP-2 and MMP-9 behavior in expanded visceral adipose tissue and the contribution of this tissue to circulating activity of these gelatinases. METHODS AND RESULTS: Male Wistar rats were fed with standard diet (Control) or standard diet plus 30% sucrose in the drinking water throughout 12 weeks (SRD). In epididymal adipose tissue vascular density, size and adipocyte density, PPARγ expression and MMP-2 and -9 were measured. Adipose tissue from SRD presented higher adipocyte size (6.32 ± 8.71 vs 4.33 ± 2.17 × 10(3) µm(2), p = 0.001) lower adipocyte density (164 (130-173) vs 190 (170-225) number/mm(2), p = 0.046) and lower vascular density (16.2 (12.8-23.5) vs 28.1 (22.3-46.5) blood vessels/mm(2), p = 0.002) than Control. MMP-2 and MMP-9 activity was decreased in SRD (1.93 ± 0.7 vs 3.92 ± 0.9 relative units, p = 0.048 and 1.80 ± 0.8 vs 5.13 ± 1.7 relative units, p = 0.004 respectively) in accordance with lower protein expression (0.35 ± 0.20 vs 2.71 ± 0.48 relative units, p = 0.004 and 1.12 ± 0.21 vs 1.52 ± 0.05 relative units, p = 0.036 respectively). There were no differences in PPARγ expression between groups. CONCLUSION: Insulin resistance induced by SRD decreases MMP-2 and MMP-9 activity in adipose tissue which would not represent an important source for circulating MMP-2 and -9. In this state of IR, PPARγ would not be involved in the negative regulation of adipose tissue gelatinases.

Increase in MMP-2 activity in overweight and obese women is associated with menopausal status.

BACKGROUND: Metalloproteinases (MMPs) are synthesized in the subendothelium and are involved in the atherosclerosis and cardiovascular disease process because of their major significance in vascular remodeling and plaque rupture. MMPs are also synthesized in adipose tissue during angiogenesis; however, the role of these enzymes in obesity and insulin-resistant states is still controversial. OBJECTIVE: To evaluate MMP-2 activity in the circulation of overweight and obese women and in normal-weight controls, and to associate the levels of these factors with metabolic, adipose tissue and inflammation biomarkers. METHODS: Plasma MMP-2 activity, adiponectin and C-reactive protein concentration, lipoprotein profile and HOMA were determined in 39 healthy women (13 normal weight and 26 overweight/obese). RESULTS: Overweight/obese women were older (p < 0.001) than normal-weight women; 20/26 of overweight/obese women were postmenopausal compared with 4/13 of normal-weight women. Overweight/obese women had significantly higher plasma activity of MMP-2 than controls (mean relative area: 0.81 (range 0.4-1.92) vs. 1.33 (range 0.4-3.1); p < 0.005); this difference was lost after adjusting for menopausal status. MMP-2 activity positively correlated with waist circumference (p < 0.002), HOMA (p < 0.003), and high-sensitivity C-reactive protein (p < 0.05), apolipoprotein B (p = 0.006) and triglyceride/high density lipoprotein (HDL) cholesterol index (p < 0.001), and negatively with HDL cholesterol (p < 0.001), HDL2 cholesterol (p < 0.008), HDL3 cholesterol (p < 0.05) and adiponectin (p < 0.05). The association with HOMA and adiponectin persisted even after adjusting for menopausal status. CONCLUSION: Our finding of increased plasma activity of MMP-2 in overweight/obese women, associated with menopausal status, is important given that it fits in with an early stage of cardiovascular disease; the association of MMP-2 activity with obesity markers may be a link between adipose tissue and risk for cardiovascular disease.

Hepatic lipase activity is increased in non-alcoholic fatty liver disease beyond insulin resistance.

BACKGROUND AND OBJECTIVE: Hepatic lipase is a lipolytic enzyme mostly synthesized and localized at the surface of liver sinusoidal capillaries, which hydrolyses triglycerides and phospholipids of intermediate density, large low density (LDL) and high density lipoproteins. Hepatic lipase activity is increased in insulin resistant states. Non-alcoholic fatty liver disease (NAFLD) is characterized by insulin resistance. However, at present, no data are available regarding the behaviour of hepatic lipase with regard to the degree of hepatic steatosis. Our aim was to evaluate hepatic lipase activity in NAFLD patients and its relationship to the severity of hepatic steatosis. DESIGN AND PATIENTS: We studied 48 patients with NAFLD (diagnosed by ultrasonography and confirmed by liver biopsy) and 30 controls. Steatosis was semi-quantitatively assessed and considered as mild or grade 1, moderate or grade 2 and severe or grade 3. MEASUREMENTS: hepatic lipase activity, lipid and lipoprotein profile (including intermediate density lipoproteins and dense LDL), adiponectin, insulin, glucose and high sensitivity C-reactive protein were measured. Homeostasis model assessment for insulin resistance (HOMA) index was calculated. RESULTS: Patients with hepatic steatosis presented with higher hepatic lipase activity, HOMA and dense LDL and lower levels of adiponectin, high density lipoproteins, cholesterol and apoA-I. Hepatic lipase activity positively correlated significantly with the severity of hepatic steatosis. Hepatic lipase correlated with a more atherogenic profile and persisted higher in patients even after corrected for age, gender, body mass index, HOMA and adiponectin. CONCLUSION: The higher hepatic lipase activity in NAFLD patients contributes to a more atherogenic profile linked to increased cardiovascular risk, beyond the insulin resistance and the reduction in adiponectin.

HOMA-IR and non-HDL-C as predictors of high cholesteryl ester transfer protein activity in patients at risk for type 2 diabetes.

BACKGROUND AND AIMS: Metabolic syndrome (MS) and type 2 diabetes are highly associated with an abnormal lipoprotein profile, which may be generated and accentuated by high cholesteryl ester transfer protein (CETP) activity. Given the difficulty in measuring CETP activity, the aim was to identify simple biochemical predictors of high CETP activity. DESIGN AND METHODS: Eighty five subjects at risk for type 2 diabetes were classified according to the presence of MS. Lipoprotein profile, HOMA-IR and endogenous CETP activity were evaluated. RESULTS: As expected, MS patients presented higher concentration of glucose, insulin, triglycerides and non-HDL-C and lower HDL-C levels. Moreover, MS patients exhibited increased HOMA-IR and CETP activity. Employing a ROC curve for MS, high CETP activity was defined as >250%ml⁻¹ h⁻¹. The predictive variables of high CETP were non-HDL-C≥160mg/dl (OR=11.1;95%IC=3.3-38.2;p<0.001) and HOMA-IR>2.1 (OR=4.4;95%IC=1.3-14.8;p<0.05). CONCLUSIONS: High non-HDL-C and insulin resistance were predictors for increased CETP activity which measurement is not accessible for clinical laboratories.

The effect of intrauterine growth retardation on the expression of developmental factors in porcine placenta subsequent to the initiation of placentation.

Intrauterine growth retardation (IUGR) hinders fetal growth and postnatal development in swine; however the etiology of IUGR is essentially unknown. Expression of fourteen candidate genes associated with placental development or IUGR was examined in gestational day 50 (gd50) control and IUGR fetus whole placental tissue or areolae by real-time PCR. Endothelial nitric oxide synthase (ENOS) mRNA expression was elevated in gd50 IUGR placenta and areola compared to gd50 control. Since ENOS could modulate vascular tone and angiogenesis via nitric oxide production, data suggest that the increase in IUGR may be an adaptive response to poor perfusion to maintain pregnancy.

Circulating very-low-density lipoprotein characteristics resulting from fatty liver in an insulin resistance rat model.

The close association between nonalcoholic fatty liver and insulin resistance is now widely recognized. While the former is characterized by excessive intrahepatic triglyceride accumulation, the latter induces overproduction of very-low-density lipoprotein (VLDL) particles. It has not been well elucidated whether these apparently opposite mechanisms impact on VLDL characteristics or not. The aim of the present study was to evaluate the VLDL secretion and features resulting from insulin resistance and fatty liver in rats fed a sucrose-rich diet (SRD, i.e. addition of sucrose to drinking water during 12 weeks). No differences in calorie intake were observed in comparison to controls. Both groups showed similar weight gains throughout the treatment period. However, SRD rats showed an increased proportion of body fat as assessed by X-ray absorptiometry, increased visceral obesity, liver weight and fat accumulation in the liver (p < 0.04). Histological study revealed moderate micro- and macrovesicular steatosis. Fasting insulin, triglyceride and free fatty acid (FFA) levels increased while VLDLs decreased in SRD rats (p < 0.05). The chemical composition of VLDLs of SRD rats showed a higher percentage of triglycerides, and the VLDL triglyceride/protein ratio, an estimator of lipoprotein size, suggests that VLDL particles of SRD rats are larger than those of controls (p < 0.0005). FFA levels correlated with VLDL triglycerides (r = 0.49, p = 0.03) and liver fat content correlated with plasma triglycerides (r = 0.65), VLDL triglycerides (r = 0.55) and triglyceride/protein ratio (r = 0.52, p < 0.02). The VLDL secretion rate assay showed an increase in SRD rats (p < 0.02), confirming an overproduction despite liver fat accumulation. Our findings are consistent with an insulin resistance development model in which hepatic lipid content would constitute an important determinant of a triglyceride-rich, large-particle VLDL secretion; both features would increase its atherogenic potential.

Androgens in relationship to cardiovascular risk factors in the menopausal transition.

OBJECTIVE: To establish the relationship between androgens and cardiovascular disease (CVD) risk factors in the menopausal transition. METHODS: A total of 124 women were divided into four groups: 29 premenopausal (PreM), 35 women in the menopausal transition still menstruating (MTM), 29 women in the menopausal transition with 3-6 months amenorrhea (MTA), and 31 postmenopausal women (PostM). Levels of triglycerides, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, glucose and insulin were assayed in all samples and waist circumference was measured. In a subgroup of 83 women (19 PreM, 21 MTM, 28 MTA and 15 PostM), levels of total testosterone, androstenedione, dehydroepiandrosterone sulfate (DHEAS) and estradiol were determined. The free androgen index, Homeostasis Model Assessment (HOMA) index, Quantitative Insulin Sensitivity Check Index (QUICKI) and McAuley index, estradiol/total testosterone and triglyceride/HDL cholesterol ratios were calculated. RESULTS: Androstenedione was higher in MTA vs. PostM women (p < 0.05); DHEAS was higher in PreM women vs. the other three groups (p < 0.05). Sex hormone binding globulin (SHBG) in MTM women was higher than in MTA women (p < 0.05); the free androgen index was lower in MTM women than in MTA and PostM women. SHBG and the free androgen index showed negative and positive correlations, respectively with waist circumference, insulin resistance and lipids. In a multiple regression analysis, considering waist circumference, neither free androgen index nor SHBG showed significant differences between groups. The waist circumference correlated only with SHBG (p = 0.022) and correlations between SHBG and insulin resistance markers continued to be significant, but relationships between SHBG and lipoproteins and all correlations found with free androgen index were lost. CONCLUSIONS: An increment in the androgenic milieu that correlates with abdominal fat, insulin resistance and atherogenic lipoproteins becomes evident after the menopausal transition and suggests that evaluation of cardiovascular disease risk in these women should include androgens, considering that abdominal obesity is one of the main determinants of the relationship between androgenic parameters and cardiovascular risk factors.

Circulating small dense LDL, endothelial injuring factors and fibronectin in healthy postmenopausal women.

BACKGROUND: In postmenopausal women (PMW), an adverse lipoprotein pattern and high risk of coronary artery disease has been described. Studies of the mechanisms promoting the higher atherogenic risk observed in healthy PMW are relevant. We evaluated the interactions among several circulating factors involved in the endothelial injury and inflammation in relation to LDL characteristics, beyond LDL cholesterol. METHODS: Lipoprotein profile, including apolipoproteins A-I and B, small dense LDL, hepatic lipase, cholesterol transfer protein (CETP), LDL composition and oxidability were assessed in PMW (n=30) in comparison to premenopausal (PreMW, n=28). The following emerging factors were measured: homocysteine, phospholipase A2, ferritin, hs-CRP and fibronectin from extracellular vascular matrix. Insulin-resistance was evaluated by waist circumference, HOMA and TG/HDL cholesterol ratios. RESULTS: The risk index apo B/apo A-I was significantly increased in PMW (p<0.0001), PMW showed higher proportion of small dense LDL which correlated with the increase in hepatic lipase activity (p<0.005) and with insulin-resistance markers (p<0.05), but not with CETP. Phospholipase A2 (p<0.05), homocysteine (p<0.005), hs-CRP (p<0.005), fibronectin (p<0.05) and ferritin (p<0.0001) were increased in PMW. LDL oxidability positively correlated with waist (p<0.02), homocysteine (p<0.05), fibronectin (p<0.05), hs-CRP (p<0.04), phospholipase A2 (p<0.05), and small dense LDL (p<0.01). After adjusting by menopausal condition, age and waist, LDL oxidability remained associated with waist (beta: 0.35, p=0.047), homocysteine (beta: 0,36 p<0,038), fibronectin (beta: 0,41 p=0.05), and small dense LDL (beta: 0.36, p=0.027). CONCLUSIONS: Evaluation of classic and non-traditional circulating risk factors in hypoestrogenism reflected endothelial and subendothelial inflammation and subclinical atherogenic processes.

Effects of storing pig ovaries at 4 or 20 degrees C for different periods of time on the morphology and viability of pre-antral follicles.

The purpose of this study was to evaluate the effect of cooling ovarian tissue on pig pre-antral follicles. Ovaries were maintained in saline solution (0.9%) at 4 or 20 degrees C for 6, 12 or 18 h. After storage, pre-antral follicles were morphologically evaluated. While primordial follicles were not affected by the storage, the percentage of morphologically normal growing follicles was significantly reduced in ovarian tissue stored at 20 degrees C for 12 or 18 h. To test the viability of stored follicles, growing follicles isolated from ovaries stored at 4 degrees C for 18 h and at 20 degrees C for 6 h were cultured for 3 days. Follicles stored in either condition presented the same growth pattern in vitro as fresh follicles. We conclude that storage of pig ovaries at 4 degrees C for up to 18 h or at 20 degrees C for up to 6 h does not affect the morphology of growing follicles or their ability to grow in vitro.

Metabolic syndrome throughout the menopausal transition: influence of age and menopausal status.

OBJECTIVE: To assess the relationship between the main components of both the metabolic syndrome and insulin resistance and menopausal status in the menopausal transition. METHODS: A total of 124 healthy women were divided into four groups according to their menstrual status: the first group consisted of 35 women in menopausal transition with menstrual bleeding (MTM) and with cycles between 35 and 80 days; the second group was composed of 29 women in menopausal transition with 3-6 months of amenorrhea (MTA). The third group consisted of 31 postmenopausal women (PostM) and the fourth group of 29 premenopausal women (PreM) with regular cycles. The metabolic syndrome was evaluated following the ATP III criteria. Evaluation of insulin resistance was made through the HOMA, QUICKI and McAuley indices and the triglycerides/high density lipoprotein (HDL) cholesterol ratio. RESULTS: The triglycerides/HDL cholesterol ratio increased in MTM, MTA and PostM women in comparison with PreM women. A slight decrease in the QUIKI index (p = 0.06) and a decrease in the McAuley index (p < 0.001) were observed in MTM, MTA and PostM women in comparison to PreM women. The relative frequencies of metabolic syndrome in the four groups were: PreM, 0%; MTM, 20%; MTA, 21%; and PostM, 22% (p = 0.0001). The most frequent markers of the metabolic syndrome were increased waist circumference, low HDL cholesterol levels and hypertension. Linear regression between menopausal status and metabolic syndrome was lost when age was added to the model. CONCLUSIONS: The frequency of metabolic syndrome increased from the time of the menopausal transition to the postmenopause. Abdominal obesity was the most frequent feature observed. Nevertheless, aging erased the effect of the menopause on the metabolic syndrome. In order to prevent cardiovascular disease, the metabolic syndrome must be evaluated from the time of the menopausal transition.

Endogenous hypertriglyceridemia intensifies the course of cerulein-induced pancreatitis in rat: relation with changes in the VLDL composition.

AIMS: To study if the course of cerulein-induced pancreatitis in rats changes in a state of triglyceride-rich lipoprotein metabolism alteration. METHODS: Two groups of rats received control diet during a 90-day period (A) and sucrose-rich diet to induce endogenous hypertriglyceridemia (B). Subgroups A2 and B2 received i.p. 45 microg cerulein/kg body weight (to induce acute pancreatitis). Histological examination of pancreas tissue, serum pancreatic lipase, lipoprotein profile and VLDL chemical composition were assessed. Then, pancreatic lipase hydrolytic activity on VLDL-triglycerides was evaluated in vitro. RESULTS: Cellular vacuolization was observed in all of the cerulein-injected rats, but only in subgroup B2 fat necrosis was present. Serum triglycerides were higher in subgroup B1 than in subgroup A1 (mean +/- SEM, mg/dl 123,77 +/- 25.7 vs. 65.8 +/- 7, p < 0.01). Triglycerides from rats fed with sucrose-rich diet, decreased after cerulein-induced pancreatitis (80.38 +/- 11.3 vs. 123,77 +/- 25.7, p < 0.02). Moreover, the endogenous hypertriglyceridemic rats showed an increment of VLDL triglyceride content, which decreased when rats were injected with cerulein. A negative correlation was found between VLDL-triglyceride content and serum pancreatic lipase activity (r = 0.58, p < 0.02). The in vitro assay showed a decrease in VLDL-triglyceride content post incubation with pancreatic lipase enriched serum (mean +/- SD: 59.2 +/- 27.7%, p < 0.01). CONCLUSIONS: The endogenous hypertriglyceridemia intensifies the course of cerulein-induced pancreatitis and it could be related to the decrease in VLDL-triglycerides as a consequence of pancreatic lipase hydrolytic activity.

A new approach to the quantitative measurement of dense LDL subfractions.

OBJECTIVES: Small dense low-density lipoproteins (LDLs) should be considered a major risk factor for cardiovascular disease, but there is still no recommended method for measuring them or expressing clinical values. We measured the dense LDL portion relatively simply by isolating it using density ultracentrifugation and then giving it a relative, quantitative value. DESIGN AND METHODS: Dense LDLs (d=1.048-1.063 g/mL) were isolated from human plasma at the same time as total LDL (d=1.021-1.063 g/mL) by means of sequential ultracentrifugation, and the former was assessed as a percentage of the latter. A receiver operator characteristic (ROC) curve was used to compare the different LDL components as markers of dense LDLs. The proposed method was compared with non-denaturing gradient gel electrophoresis (NDGGE). In order to obtain clinical data, the dense LDL portion was measured in diabetic and postmenopausal subjects and healthy controls. RESULTS: The ROC curve showed that cholesterol level was a more accurate marker of dense LDLs. The within-run precision (CV) was 2.28%, and the between-run CV was 5.1%. Analytical recovery was 80.2+/-1.6%. The correlation between the proposed method and NDGGE was r=0.90, p<0.001. The dense LDL percentage significantly correlated with serum triglyceride (r=0.57, p<0.001) and high-density lipoprotein cholesterol levels (r=-0.33, p<0.01), but not with the LDL-cholesterol/apolipoprotein B ratio. The diabetic patients and postmenopausal women had higher dense LDL values than the healthy controls. CONCLUSIONS: The results obtained using this procedure are in line with those obtained using NDGGE, which is the conventional assay system for measuring LDL size. Determining the small dense LDL portion by means of its cholesterol content may be a better approach to characterising the risk of cardiovascular disease, even in the presence of relatively normal LDL-cholesterol levels.

Lipid and lipoprotein profile in menopausal transition. Effects of hormones, age and fat distribution.

The behavior of lipoproteins during the menopausal transition and their relationship with sex hormones and body fat distribution is still unclear. Our aim was to evaluate atherogenic IDL, LDL, Lp(a) and antiatherogenic HDL lipoproteins in four groups of women: premenopausal (n = 20), menopausal transition women with menstrual bleeding (n = 31), menopausal transition women with 3 to 6 months amenorrhea (n = 36), and postmenopausal women (n = 30). We also measured their FSH, LH and estradiol levels along with BMI and waist circumference. Menopausal transition and postmenopausal women showed higher values of waist circumference (p < 0.0032), LDL-cholesterol (p < 0.002), IDL-cholesterol (p < 0.002) and apoprotein B (p < 0.0001) than premenopausal women. Total-cholesterol (p < 0.0001), triglycerides (p < 0.004), IDL-cholesterol and Lp(a) were higher in menopausal transition women with amenorrhea and in postmenopausal women in comparison with premenopausal women. After adjustment according to age and waist circumference, multiple regression analysis showed the increase in total-cholesterol and LDL-cholesterol to be linearly associated to menopausal status and estradiol concentration, whereas Lp(a) was only related to menopausal status. Age was found to be an independent variable in relation to apoprotein B concentration changes. The effect of menopausal status on TG levels did not remain in the model when age, waist and BMI were included (beta = 0.05, p = 0.356). HDL-cholesterol levels were the same in all the groups. Menopause, age and the increase in abdominal fat distribution were three independent and significant factors impairing lipoprotein profiles from the beginning of the menopausal transition.