Peripheral Blood miRome Identified miR-155 as Potential Biomarker of MetS and Cardiometabolic Risk in Obese Patients.

This study explored circulating miRNAs and target genes associated with metabolic syndrome (MetS) and cardiometabolic risk in obese patients. Small-RNA sequencing was used to assess the peripheral blood miRNome of 12 obese subjects (6 MetS and 6 non-MetS). Differentially expressed miRNAs and target genes were further analyzed by qPCR in a larger sample of obese patients (48 MetS and 32 non-MetS). miRNA:mRNA interactions were studied using in silico tools. miRNome analysis identified 10 downregulated miRNAs in MetS compared to non-Met patients (p < 0.05). In silico studies revealed three miRNAs (miR-155, miR-181a, and let-7a) and their predictive targets (CCAAT/enhancer-binding protein beta-CEBPB, KRAS proto-oncogene, GTPase-KRAS and suppressor of cytokine signaling 1-SOCS1) with a potential role in the insulin receptor signaling pathway. miR-155 expression was reduced and CEBPB mRNA levels were increased in MetS patients (p < 0.05), and these effects were correlated with the number of MetS diagnostic criteria (p < 0.05). Increased HOMA-IR (>7.6) was associated with low miR-155 levels, high CEBPB expression, and serum hsCRP (p < 0.05). miR-155 was negatively correlated with CEBPB, HOMA-IR, and plasma fibrinogen, and positively correlated with serum adiponectin (p < 0.05). Downregulation of circulating miR-155 is associated with insulin resistance, poor glycemic control, and increased MetS-related cardiometabolic risk, and these effects are potentially mediated by interaction with CEBPB.

Influence of polymorphisms in IRS1, IRS2, MC3R, and MC4R on metabolic and inflammatory status and food intake in Brazilian adults: An exploratory pilot study.

Polymorphisms in genes of leptin-melanocortin and insulin pathways have been associated with obesity and type 2 diabetes. We hypothesized that polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory markers and food intake composition in Brazilian subjects. This exploratory pilot study included 358 adult subjects. Clinical, anthropometric, and laboratory data were obtained through interview and access to medical records. The variants IRS1 rs2943634 A˃C, IRS2 rs1865434 C>T, MC3R rs3746619 C>A, and MC4R rs17782313 T>C were analyzed by real-time polymerase chain reaction. Food intake composition was assessed in a group of subjects with obesity (n = 84) before and after a short-term nutritional counseling program (9 weeks). MC4R rs17782313 was associated with increased risk of obesity (P = .034). Multivariate linear regression analysis adjusted by covariates indicated associations of IRS2 rs1865434 with reduced low-density lipoprotein cholesterol and resistin, MC3R rs3746619 with high glycated hemoglobin, and IRS1 rs2943634 and MC4R rs17782313 with increased high-sensitivity C-reactive protein (P < .05). Energy intake and carbohydrate and total fat intakes were reduced after the diet-oriented program (P < .05). Multivariate linear regression analysis showed associations of IRS2 rs1865434 with high basal fiber intake, IRS1 rs2943634 with low postprogram carbohydrate intake, and MC4R rs17782313 with low postprogram total fat and saturated fatty acid intakes (P < .05). Although significant associations did not survive correction for multiple comparisons using the Benjamini-Hochberg method in this exploratory study, polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory status in Brazilian adults. IRS1 and MC4R variants may influence carbohydrate, total fat, and saturated fatty acid intakes in response to a diet-oriented program in subjects with obesity.

Osteoporotic hip fractures in non-elderly patients: relevance of associated co-morbidities.

Osteoporotic hip fractures (OHF) are not limited to elderly; however, studies in non-elderly are scarce. Thus, the aim of this study was to evaluate co-morbidities in non-elderly patients with OHF in a Community Teaching Hospital. All hospitalizations due to OHF during a 3-year period in a Community Teaching Hospital were retrospectively evaluated for co-morbidities, and patients 18-64 years old were compared with those ≥65 years old. Of all hospitalizations, 232 (0.73%) were due to hip fractures, and 120/232 (51.7%) patients had OHF. The comparison of the 13 (10.8%) OHF patients <65 years old (47.3 ± 9.7 years) with 107 (89.2%) ≥65 years old (80.4 ± 7.7 years) revealed a male predominance (61.5 vs. 27.1%, P = 0.022) and a distinct ethnic distribution with a lower proportion of Caucasians in the former (61.5 vs. 86.9%, P = 0.033). Moreover, non-elderly OHF patients had higher frequencies of insulin-dependent DM (38.5 vs. 3.7%, P = 0.001) and alcoholism (38.5 vs. 4.7%, P = 0.001) than aged patients. In contrast, rates of age-related co-morbidities such as stroke (7.7 vs. 18.7%, P = 0.461), heart failure (23.1 vs. 14.0%, P = 0.411), and dementia (7.7 vs. 15.9%, P = 0.689) were comparable in both groups. Logistic regression analysis demonstrated that insulin-dependent DM (OR = 25.4, 95% CI = 4.7-136.8, P < 0.001) and alcoholism (OR = 20.3, 95% CI = 3.9-103.3, P < 0.001) remained as independent risk factors for OHF in non-elderly patients. Osteoporosis is an important cause of HF in Community Hospital. Non-elderly patients with OHF have a peculiar demographic profile and associated co-morbidities. These findings reinforce the need of early osteoporosis diagnosis and rigorous fracture prevention in patients with DM and alcoholism.

Apolipoprotein E mRNA expression in mononuclear cells from normolipidemic and hypercholesterolemic individuals treated with atorvastatin.

BACKGROUND: Apolipoprotein E (apoE) is a key component of the lipid metabolism. Polymorphisms at the apoE gene (APOE) have been associated with cardiovascular disease, lipid levels and lipid-lowering response to statins. We evaluated the effects on APOE expression of hypercholesterolemia, APOE ε2/ε3/ε4 genotypes and atorvastatin treatment in Brazilian individuals. The relationship of APOE genotypes and plasma lipids and atorvastatin response was also tested in this population. METHODS: APOE ε2/ε3/ε4 and plasma lipids were evaluated in 181 normolipidemic (NL) and 181 hypercholesterolemic (HC) subjects. HC individuals with indication for lowering-cholesterol treatment (n = 141) were treated with atorvastatin (10 mg/day/4-weeks). APOE genotypes and APOE mRNA in peripheral blood mononuclear cells (PBMC) were analyzed by TaqMan real time PCR. RESULTS: HC had lower APOE expression than NL group (p < 0.05) and individuals with low APOE expression showed higher plasma total and LDL cholesterol and apoB, as well as higher apoAI (p < 0.05). Individuals carrying ε2 allele have reduced risk for hypercholesterolemia (OR: 0.27, 95% I.C.: 0.08-0.85, p < 0.05) and NL ε2 carriers had lower total and LDL cholesterol and apoB levels, and higher HDL cholesterol than non-carriers (p < 0.05). APOE genotypes did not affect APOE expression and atorvastatin response. Atorvastatin treatment do not modify APOE expression, however those individuals without LDL cholesterol goal achievement after atorvastatin treatment according to the IV Brazilian Guidelines for Dyslipidemia and Atherosclerosis Prevention had lower APOE expression than patients with desirable response after the treatment (p < 0.05). CONCLUSIONS: APOE expression in PBMC is modulated by hypercholesterolemia and the APOE mRNA level regulates the plasma lipid profile. Moreover the expression profile is not modulated neither by atorvastatin nor APOE genotypes. In our population, APOE ε2 allele confers protection against hypercholesterolemia and a less atherogenic lipid profile. Moreover, low APOE expression after treatment of patients with poor response suggests a possible role of APOE level in atorvastatin response.

Pharmacogenetics of OATP transporters reveals that SLCO1B1 c.388A>G variant is determinant of increased atorvastatin response.

AIMS: The relationship between variants in SLCO1B1 and SLCO2B1 genes and lipid-lowering response to atorvastatin was investigated. MATERIAL AND METHODS: One-hundred-thirty-six unrelated individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). They were genotyped with a panel of ancestry informative markers for individual African component of ancestry (ACA) estimation by SNaPshot(®) and SLCO1B1 (c.388A>G, c.463C>A and c.521T>C) and SLCO2B1 (-71T>C) gene polymorphisms were identified by TaqMan(®) Real-time PCR. RESULTS: Subjects carrying SLCO1B1 c.388GG genotype exhibited significantly high low-density lipoprotein (LDL) cholesterol reduction relative to c.388AA+c.388AG carriers (41 vs. 37%, p = 0.034). Haplotype analysis revealed that homozygous of SLCO1B1*15 (c.521C and c.388G) variant had similar response to statin relative to heterozygous and non-carriers. A multivariate logistic regression analysis confirmed that c.388GG genotype was associated with higher LDL cholesterol reduction in the study population (OR: 3.2, CI95%:1.3-8.0, p < 0.05). CONCLUSION: SLCO1B1 c.388A>G polymorphism causes significant increase in atorvastatin response and may be an important marker for predicting efficacy of lipid-lowering therapy.

Changes in plasma free fatty acid levels in septic patients are associated with cardiac damage and reduction in heart rate variability.

Free fatty acids (FFAs) have been shown to produce alteration of heart rate variability (HRV) in healthy and diabetic individuals. Changes in HRV have been described in septic patients and in those with hyperglycemia and elevated plasma FFA levels. We studied if sepsis-induced heart damage and HRV alteration are associated with plasma FFA levels in patients. Thirty-one patients with sepsis were included. The patients were divided into two groups: survivors(n = 12) and nonsurvivors (n = 19). The following associations were investigated: (a) troponin I elevation and HRV reduction and (b) clinical evolution and HRV index, plasma troponin, and plasma FFA levels. Initial measurements of C-reactive protein and gravity Acute Physiology and Chronic Health Evaluation scores were similar in both groups. Overall, an increase in plasma troponin level was related to increased mortality risk. From the first day of study, the nonsurvivor group presented a reduced left ventricular stroke work systolic index and a reduced low frequency (LF) that is one of HRV indexes. The correlation coefficient for LF values and troponin was r(2) = 0.75 (P < 0.05). All patients presented elevated plasma FFA levels on the first day of the study (5.11 +/- 0.53 mg/mL), and this elevation was even greater in the nonsurvivor group compared with the survivors (6.88 +/- 0.13 vs. 3.85 +/- 0.48 mg/mL, respectively; P < 0.05). Cardiac damage was confirmed by measurement of plasma troponin I and histological analysis. Heart dysfunction was determined by left ventricular stroke work systolic index and HRV index in nonsurvivor patients. A relationship was found between plasma FFA levels, LFnu index, troponin levels, and histological changes. Plasma FFA levels emerged as possible cause of heart damage in sepsis.

Atorvastatin effects on SREBF1a and SCAP gene expression in mononuclear cells and its relation with lowering-lipids response.

BACKGROUND: The transcription factors SREBP1 and SCAP are involved in intracellular cholesterol homeostasis. Polymorphisms of these genes have been associated with variations on serum lipid levels and response to statins that are potent cholesterol-lowering drugs. We evaluated the effects of atorvastatin on SREBF1a and SCAP mRNA expression in peripheral blood mononuclear cells (PBMC) and a possible association with gene polymorphisms and lowering-cholesterol response. METHODS: Fifty-nine hypercholesterolemic patients were treated with atorvastatin (10 mg/day for 4 weeks). Serum lipid profile and mRNA expression in PBMC were assessed before and after the treatment. Gene expression was quantified by real-time PCR using GAPD as endogenous reference and mRNA expression in HepG2 cells as calibrator. SREBF1 -36delG and SCAP A2386G polymorphisms were detected by PCR-RFLP. RESULTS: Our results showed that transcription of SREBF1a and SCAP was coordinately regulated by atorvastatin (r=0.595, p<0.001), and that reduction in SCAP transcription was associated with the 2386AA genotype (p=0.019). Individuals who responded to atorvastatin with a downregulation of SCAP had also a lower triglyceride compared to those who responded to atorvastatin with an upregulation of SCAP. CONCLUSION: Atorvastatin has differential effects on SREBF1a and SCAP mRNA expression in PBMC that are associated with baseline transcription levels, triglycerides response to atorvastatin and SCAP A2386G polymorphism.

CYP3A53A allele is associated with reduced lowering-lipid response to atorvastatin in individuals with hypercholesterolemia.

BACKGROUND: The cytochrome P450 isoenzyme 3A5 (CYP3A5) has an important role on biotransformation of xenobiotics. CYP3A5 SNPs have been associated with variations on enzyme activity that can modify the metabolism of several drugs. METHODS: In order to evaluate the influence of CYP3A5 variants on response to lowering-cholesterol drugs, 139 individuals with hypercholesterolemia were selected. After a wash-out period of 4 weeks, individuals were treated with atorvastatin (10 mg/day/4 weeks). Genomic DNA was extracted by a salting-out procedure. CYP3A5*3C, CYP3A5*6 and CYP3A5*1D were analyzed by PCR-RFLP and DNA sequencing. RESULTS: >Frequencies of the CYP3A5*3C and CYP3A5*1D alleles were lower in individuals of African descent (*3C: 47.8% and *1D: 55.2%) than in non-Africans (*3C: 84.9% and *1D 84.8%, p<0.01). Non-Africans carrying *3A allele (*3C and *1D combined alleles) had lower total and LDL-cholesterol response to atorvastatin than non-*3A allele carriers (p<0.05). CONCLUSION: CYP3A5*3A allele is associated with reduced cholesterol-lowering response to atorvastatin in non-African individuals.

Effects of ABCA1 SNPs, including the C-105T novel variant, on serum lipids of Brazilian individuals.

BACKGROUND: ABCA1 plays an important role in HDL metabolism. Single nucleotide polymorphisms (SNPs) in ABCA1 gene were associated with variation in plasma HDL-c. METHODS: The effect of the ABCA1 SNPs C-14T, R219K and of a novel variant C-105T on serum lipids was investigated in 367 unrelated Brazilian individuals (224 hypercholesterolemic and 143 normolipidemic). The relation between ABCA1 SNPs and the lipid-lowering response to atorvastatin (10 mg/day/4 weeks) was also evaluated in 141 hypercholesterolemic (HC) individuals. The polymorphisms were detected by PCR-RFLP and confirmed by DNA sequencing. RESULTS: Linkage disequilibrium was found between the SNPs C-105T and C-14T in the HC group. HC individuals carrying -105CT/TT genotypes had higher serum HDL-c and lower triglyceride and VLDL-c concentrations as well as lower TG/HDL-c ratio compared to the -105CC carriers (p<0.05). The R219K SNP was associated with reduced serum triglyceride, VLDL-c and TG/HDL-c ratio in the HC group (p<0.05), and with an increased serum apoAI in NL individuals. The effects of ABCA1 SNPs on basal serum lipids of HC individuals were not modified by atorvastatin treatment. CONCLUSIONS: The ABCA1 SNPs R219K and C-105T were associated with a less atherogenic lipid profile but not with the lowering-cholesterol response to atorvastatin in a Brazilian population.

Down-regulation of ABCB1 transporter by atorvastatin in a human hepatoma cell line and in human peripheral blood mononuclear cells.

PURPOSE: The effect of atorvastatin, an HMG-CoA reductase inhibitor, on expression and activity of the drug transporter ABCB1 in HepG2 cells and peripheral blood mononuclear cells (PBMCs) was examined. METHODS: Localization and expression of ABCB1 in hepatocytes was examined by indirect immunofluorescence. Expression of ABCB1 mRNA and ABCB1 activity were examined in atorvastatin-treated and control cells and PBMCs using real-time PCR and Rhodamine 123 efflux assay. RESULTS: Immunohistochemical analysis revealed that ABCB1 is located at the apical membrane of the bile canaliculi. Atorvastatin at 10 and 20 microM up-regulated ABCB1 expression resulting in a significant 1.4-fold increase of the protein levels. Treatment of HepG2 cells with 20 microM atorvastatin caused a 60% reduction on mRNA expression (p<0.05) and a 41% decrease in ABCB1-mediated efflux of Rhodamine123 (p<0.01) by flow cytometry. Correlation was found between ABCB1 mRNA levels and creatine kinase (r=0.30; p=0.014) and total cholesterol (r=-0.31; p=0.010). CONCLUSIONS. Atorvastatin leads to decreased ABCB1 function and modulates ABCB1 synthesis in HepG2 cells and in PBMCs. ABCB1 plays a role in cellular protection as well as in secretion and/or disposition, therefore, inhibition of ABCB1 synthesis may increase the atorvastatin efficacy, leading to a more pronounced reduction of plasma cholesterol.

Effects of short-term add-on ezetimibe to statin treatment on expression of adipokines and inflammatory markers in diabetic and dyslipidemic patients.

AIM: The influence of short-term add-on ezetimibe to simvastatin treatment on expression of adipokines and inflammatory markers was investigated in diabetic and nondiabetic patients with hypercholesterolemia. METHOD: Hypercholesterolemic nondiabetic (HC, n = 37) and diabetic (DM, n = 47) patients were treated with simvastatin (SV, 10 or 20 mg/d/8-wk) and then SV plus ezetimibe (SV + EZ, 10 mg each/d/4 wk). Serum lipids, glycemic profile, and inflammatory markers (hsCRP, adiponectin, resistin, VCAM-1, and ICAM-1) were evaluated before and after the add-on ezetimibe therapy. mRNA expression of ADIPOR1, ADIPOR2, RETN, VCAM1, and ICAM1 was measured by real-time PCR in peripheral blood mononuclear cells (PBMC). RESULTS: Serum concentrations of LDL and HDL cholesterol, and adiponectin were higher in HC than DM patients (P < .05). The add-on ezetimibe therapy reduced total and LDL cholesterol, apoB and adiponectin serum levels in HC and DM groups, and resistin in HC subjects (P < .05). DM patients showed higher expression of ADIPOR1, ADIPOR2, RETN, and VCAM1 in PBMC than subjects in HC group, before and after add-on ezetimibe therapy (P < .05). PBMC RETN mRNA expression was reduced by add-on ezetimibe therapy in HC individuals (P < .05), but not in DM subjects. CONCLUSION: Short-term add-on ezetimibe to simvastatin treatment suppressing effects on hypercholesterolemia and adiponectinemia is independent of the diabetes status. Resistin serum levels and leukocyte mRNA expression are influenced by add-on ezetimibe to statin treatment.

Implementing a clinical assessment protocol for sensory and skeletal function in diabetic neuropathy patients at a university hospital in Brazil.

CONTEXT AND OBJECTIVE: Physiotherapy can contribute towards recovering or preventing physical and sensory alterations in diabetic neuropathy patients. Our objective was to create and apply a protocol for functional assessment of diabetic neuropathy patients' lower limbs, to guide future physiotherapy. DESIGN AND SETTING: Clinical study at the University Hospital and teaching/research center of Universidade de São Paulo. METHODS: An intentional sample of diabetic neuropathy patients was utilized. The protocol was divided into: (1) preliminary investigation with identification of relevant clinical diabetes and neuropathy characteristics; (2) thermal, tactile and proprioceptive sensitivity tests on the feet; (3) evaluations of muscle function, range of motion, lower limb function, foot anthropometry. RESULTS: The patients' mean age was 57 years, and they had had the diagnosis for 13 years on average. Distal numbness and tingling/prickling were present in 62% and 67%, respectively. There were tactile sensitivity alterations above the heel in 50%, with thermal sensitivity in 40% to 60%. The worst muscle function test responses were at the triceps surae and foot intrinsic muscles. Longitudinal plantar arches were lowered in 50%. Decreased thermal and tactile sensitivity of the heels was found. There was a general reduction in range of motion. CONCLUSIONS: The results provided detailed characterization of the patients. This protocol may be easily applied in healthcare services, since it requires little equipment, at low cost, and it is well understood by patients.

ADIPOQ and IL6 variants are associated with a pro-inflammatory status in obeses with cardiometabolic dysfunction.

BACKGROUND: Polymorphisms in genes encoding adiponectin (ADIPOQ) and interleukin-6 (IL6) have been associated with adiposity and obese-related phenotypes. This study investigated the relationship of ADIPOQ and IL6 gene polymorphisms with pro-inflammatory and cardiometabolic markers in obese patients. METHODS: Anthropometric and body composition parameters were measured in 249 Brazilian subjects (30 to 68 yr). Metabolic and inflammatory markers and adipokines were analyzed in blood samples. ADIPOQ rs2241766 (45 T > G) and IL6 rs1800795 (-174G > C) polymorphisms were analyzed by real-time PCR and PCR-RFLP, respectively. RESULTS: Type 2 diabetes, hypertension, dyslipidemia and increased values of waist circumference, body fat, leptin, fibrinogen, IL-1ß, hsCRP and TNFα were related to obesity (p < 0.05). Multiple linear regression analysis showed a positive correlation between BMI and waist circumference, body fat, leptin, fibrinogen, PAI-1, IL-1ß, hsCRP and TNFα values (p < 0.001) but not with adiponectin. Obese group had altered metabolic status, resistance to leptin and insulin, and atherogenic and pro-inflammatory profiles. ADIPOQ and IL6 variants were not directely related to obesity, leptin resistance or alterations in cardiometabolic markers. Individuals carrying ADIPOQ 45G allele (TG + GG genotype) had higher IL-6, IL-1ß and TNFα levels than TT genotype carriers (p < 0.05). IL6 -174GG genotype was associated with increased IL-1ß levels (p = 0.033). CONCLUSION: Obesity is associated with leptin resistance, cardiometabolic alterations and a pro-inflammatory status. Our results are suggestive that ADIPOQ and IL6 polymorphisms contribute to cardiometabolic risk in obese individuals.

[Food phytosterol ester efficiency on the plasma lipid reduction in moderate hypercholesterolemic subjects]. / Eficiência dos Esteres de Fitoesteróis Alimentares na Redução dos Lípides Plasmáticos em Hipercolesterolêmicos Moderados.

OBJECTIVE: This study aimed at relating the pattern of response to dietary plant sterol ester (PSE) treatment of plasma lipids concentrations and apo E polymorphisms. METHODS: Patients (20-60y old: 50 women; 10 men) with primary moderate hypercholesterolemia were fed margarine (20g/d), received no treatment (placebo), and were fed PSE (2.8g/d = 1.68 phytosterols), during 3 periods of 4 weeks each, in a crossover, double-blind study. DNA was extracted from white blood cells for the apo E polymorphisms. RESULTS: PSE treatment significantly lowered TC and LDL-C 10% and 12%, respectively, in relation to the baseline, and 6% and 8% in relation to the placebo phase, but HDL-C and TG levels were not modified. In regard to the apo E genotyping, no significant difference occurred between apo E 3/3 and apo E (3/4). CONCLUSION: Dietary plant sterol ester (PSE) treatment reduced cholesterolemia, and the reduction of LDL-C in absolute values was more pronounced when the initial LDL - C concentration were elevated.

Influence of PCSK9 polymorphisms on plasma lipids and response to atorvastatin treatment in Brazilian subjects.

BACKGROUND: The proprotein convertase subtilisin/kexin type 9 (PCSK9) has a key role in the regulation of plasma low-density lipoprotein (LDL) cholesterol by enhancing the degradation of LDL receptor. Functional variants in PCSK9 have been associated with differences in plasma lipids and may contribute to the variability of the response to cholesterol-lowering drugs. OBJECTIVE: To investigate the influence of PCSK9 variants on plasma lipid profile and response to atorvastatin in Brazilian subjects. METHODS: PCSK9 E670G, I474V, and R46L single nucleotide polymorphisms (SNPs) and plasma lipids were evaluated in 163 hypercholesterolemics (HC) and 171 normolipidemics (NL). HC patients with indication for cholesterol-lowering drug therapy (n = 128) were treated with atorvastatin (10 mg/d/4 wk). PCSK9 SNPs were analyzed by real time polymerase chain reaction. RESULTS: Frequencies of the PCSK9 SNPs were similar between the HC and NL groups. Logistic regression analysis showed a trend of association between PCSK9 E670G and hypercholesterolemia after adjustment for covariates (P = .059). The 670G allele was associated with high basal levels of LDL cholesterol (P = .03) in HC patients using the extreme discordant phenotype method. No association tests were performed for R46L variant because of its very low frequency, whereas the I474V polymorphism and PCSK9 haplotypes were not related to hypercholesterolemia or variability on plasma lipids in both NL and HC groups (P > .05). LDL cholesterol reduction in response to atorvastatin was not influenced by PCSK9 genotypes or haplotypes. CONCLUSIONS: PCSK9 E670G polymorphism but not I474V contributes to the variability on plasma LDL cholesterol levels in hypercholesterolemic subjects. Both PCSK9 variants have no influence on cholesterol-lowering response to atorvastatin.

Leptin receptor gene polymorphisms are associated with adiposity and metabolic alterations in Brazilian individuals.

OBJECTIVE: The aim of the study was to investigate whether adiposity and metabolic markers, such as leptin, glucose, and lipids, are influenced by leptin (LEP) and leptin receptor (LEPR) gene polymorphisms in a sample of our population. SUBJECTS AND METHODS: A group of 326 individuals of Caucasian-European descent, aged 30 to 80 years, 87 men and 239 women, 148 obese and 178 non-obese, was randomly selected at two clinical hospitals in the city of Sao Paulo, Brazil. All individuals declared their ethnic group as white during the initial interview. Anthropometric measurements, body mass index (BMI), and fat mass were evaluated. Blood samples were drawn for DNA extraction and measurements of leptin, soluble leptin receptor, glucose, and lipids. LEP -2548G>A and LEPR Lys109Arg (c.326A>G), Gln233Arg (c.668A>G) and Lys656Asn (c.1968G>C) polymorphisms were detected by PCR-RFLP. RESULTS: Increased leptin and serum lipids, and LEPR Arg223Arg (GG genotype) were associated with higher risk for obesity (p < 0.05), while reduced risk was found in LEPR Arg109Arg (GG genotype) carriers (OR: 0.38, 95%CI: 0.19-0.77, p = 0.007). Multiple linear regression analysis showed a relationship between LEPR 223Arg, increased waist circumference, and leptinemia (p < 0.05), while LEPR 109Arg was associated with high total cholesterol and triglycerides (p < 0.05). LEPR haplotype 3 (AGG: 109Lys/233Arg/656Lys) carriers have increased risk for obesity (OR: 2.56, 95% CI: 1.19-5.49, p = 0.017). Moreover, this haplotype was associated with increased BMI, waist circumference, and leptinemia (p < 0.05). CONCLUSIONS: LEPR polymorphisms are associated with obesity, hyperleptinemia, and atherogenic lipid profile, suggesting their potential role for leptin resistance and cardiovascular risk. Moreover, LEPR haplotype 3 confers susceptibility to adiposity and hyperleptinemia in our population.

Effects of atorvastatin on CYP3A4 and CYP3A5 mRNA expression in mononuclear cells and CYP3A activity in hypercholeresterolemic patients.

BACKGROUND: Variability of response to statins has been related to polymorphisms in genes involved in cholesterol homeostasis and statin metabolism, such as CYP3A4 and CYP3A5. We investigated the effects of atorvastatin on CYP3A4 and CYP3A5 mRNA expression in mononuclear cells and on CYP3A activity and their interactions with common variants. METHODS: Unrelated individuals (n=121) with hypercholesterolemia (HC) were treated with atorvastatin (10 mg/day/4 weeks). Ninety-two normolipidemic (NL) subjects were selected as a control group. Genotype analysis of CYP3A4*1B (rs2740574), CYP3A4*22 (rs35599367), CYP3A5*3C (rs776746), and CYP3A5*1D (rs15524) and mRNA levels in peripheral blood mononuclear cells (PBMCs) were estimated. CYP3A activity was phenotyped by the urinary cortisol to 6-beta-hydroxy-cortisol ratio. RESULTS: LDL cholesterol reduction in response to atorvastatin was positively correlated with change in CYP3A4 (R(2)=0.039, p=0.037) and CYP3A5 (R(2)=0.047, p=0.019) mRNA levels and negatively correlated with CYP3A activity (R(2)=0.071, p=0.022). CYP3A5*3C (AGT haplotype) was associated to lower basal CYP3A5 mRNA expression in HC (p<0.045), however none of the haplotype groups impacted treatment. CONCLUSION: It is likely that cholesterolemia status changes promoted by atorvastatin play a role in regulating CYP3A4 and CYP3A5 mRNA expression in PBMCs, as well as CYP3A activity. CYP3A5*3C (AGT haplotype) also contributes for the variability of CYP3A5 mRNA levels in PBMCs.

Differentiation of African components of ancestry to stratify groups in a case-control study of a Brazilian urban population.

BACKGROUND: Balancing the subject composition of case and control groups to create homogenous ancestries between each group is essential for medical association studies. METHODS: We explored the applicability of single-tube 34-plex ancestry informative markers (AIM) single nucleotide polymorphisms (SNPs) to estimate the African Component of Ancestry (ACA) to design a future case-control association study of a Brazilian urban sample. RESULTS: One hundred eighty individuals (107 case group; 73 control group) self-described as white, brown-intermediate or black were selected. The proportions of the relative contribution of a variable number of ancestral population components were similar between case and control groups. Moreover, the case and control groups demonstrated similar distributions for ACA <0.25 and >0.50 categories. Notably a high number of outlier values (23 samples) were observed among individuals with ACA <0.25. These individuals presented a high probability of Native American and East Asian ancestral components; however, no individuals originally giving these self-described ancestries were observed in this study. CONCLUSIONS: The strategy proposed for the assessment of ancestry and adjustment of case and control groups for an association study is an important step for the proper construction of the study, particularly when subjects are taken from a complex urban population. This can be achieved using a straight forward multiplexed AIM-SNPs assay of highly discriminatory ancestry markers.

Influence of polymorphisms and cholesterol-lowering treatment on SCARB1 mRNA expression.

AIM: This study evaluated the influence of polymorphisms and cholesterol-lowering treatments on SCARB1 mRNA expression in peripheral blood mononuclear cells and in HepG2 and Caco-2 cells. METHODS: Blood samples were drawn from normolipidemic (NL, n = 166) and hypercholesterolemic (HC, n = 123) individuals to extract DNA and total RNA and to analyze the lipid profile. After a 4-week washout period, 98 HC individuals were treated with atorvastatin (10 mg/day/4 weeks) whereas 25 were treated with ezetimibe (10 mg/day/4 weeks), followed by simvastatin (10 mg/day/8 weeks) and simvastatin plus ezetimibe (10 mg each/day/4 weeks). HepG2 and Caco-2 cells were treated with atorvastatin, simvastatin and ezetimibe at various concentrations for 12 and 24 h and collected for RNA extraction. SCARB1 mRNA expression was measured by TaqMan® assay and SCARB1 c.4G> A, c.726 + 54C> T and c.1080C> T polymorphisms were detected by PCR-RFLP. RESULTS: High LDL cholesterol (> 160 mg/dL) values were associated with low baseline SCARB1 mRNA expression in PBMC. Allele T carriers for SCARB1 c.726+54C> T had lower basal SCARB1 transcription in PBMC (p < 0.05). Simvastatin, atorvastatin and ezetimibe treatments did not modify the SCARB1 mRNA level in PBMC from HC patients. Similarly, these cholesterol-lowering drugs did not modulate the SCARB1 expression in HepG2 and Caco-2 cells in spite of the concentration and time of exposure (p > 0.05). CONCLUSION: LDL cholesterol levels and SCARB1 c.726 + 54C> T are associated with low mRNA expression in mononuclear cells. Cholesterol-lowering drugs do not modulate SCARB1 expression in PBMC from HC subjects or in HepG2 and Caco-2 cells.

Influence of SCARB1 polymorphisms on serum lipids of hypercholesterolemic individuals treated with atorvastatin.

BACKGROUND: The SR-BI is a key component on the cholesterol metabolism. Polymorphisms in the SR-BI gene (SCARB1) were related with variations on plasma lipoprotein profile and other risk factors for cardiovascular disease. We tested the relationship of 3 SCARB1 single nucleotide polymorphisms (SNPs) with hypercholesterolemia in a Brazilian population and whether these variants can influence lipid-lowering response to atorvastatin. METHODS: c.4G>A, c.726+54C>T and c.1050C>T SNPs and serum concentrations of lipid and apolipoproteins were evaluated in 147 hypercholesterolemic (HC) and 185 normolipidemic (NL) unrelated Brazilian subjects. HC patients were treated with atorvastatin (10 mg/day/4 weeks). RESULTS: Frequencies of SCARB1 polymorphisms were similar between the HC and NL groups (p>0.05). The T allele for c.726+54C>T was associated with higher LDL-c in NL and with higher apoB and apoB/apoAI in HC (p<0.05). HC individuals carrying c.1050C allele carriers (CC and CT genotypes) had lower change of total cholesterol, LDL-c, apoB and apoB/apoAI ratio (p<0.05) than the TT genotype carriers in response to atorvastatin. CONCLUSION: The SCARB1 polymorphisms are related with variations in serum lipids in the Brazilian population and c.1050C>T SNP is associated with lipid-lowering atorvastatin response.