Ana Mercedes Cavaleiro

Association of polymorphisms of glutamate‐cystein ligase and microsomal triglyceride transfer protein genes in non‐alcoholic fatty liver disease

Background and Aims:  Although the metabolic risk factors for non‐alcoholic fatty liver disease (NAFLD) progression have been recognized, the role of genetic susceptibility remains a field to be explored. The aim of this study was to examine the frequency of two polymorphisms in Brazilian patients with biopsy‐proven simple steatosis or non‐alcoholic steatohepatitis (NASH): −493 G/T in the MTP gene, which codes the protein responsible for transferring triglycerides to nascent apolipoprotein B, and −129 C/T in the GCLC gene, which codes the catalytic subunit of glutamate‐cystein ligase in the formation of glutathione.

Analysis of gonadotropin pulsatility in hirsute women with normal menstrual cycles and in women with polycystic ovary syndrome

OBJECTIVE To study the relation between plasma gonadotropin pulsatility, androgen levels, and estrogen levels in patients with polycystic ovary syndrome (PCOS), in hirsute women with normal menstrual cycles, and in healthy women. DESIGN Prospective study. SETTING University medical center-based cellular and molecular endocrinology laboratory. PATIENT(S) Eight healthy women (group 1), 9 hirsute women with normal menstrual cycles (group 2), and 19 women with PCOS (group 3). INTERVENTION(S) Plasma concentrations of LH and FSH were measured by RIA every 15 minutes for 12 hours. MAIN OUTCOME MEASURE(S) Rhythmic parameters of 12-hour LH and FSH secretion. RESULT(S) Rhythmic parameters of 12-hour LH secretion were significantly higher in patients with PCOS (group 3) than in controls (group 1) or in hirsute women with normal menstrual cycles (group 2). The frequency of LH pulses was statistically higher in patients with PCOS (group 3) than in controls (group 1). Statistically significant correlations were found when the frequency of LH pulses was plotted against basal LH concentrations and rhythmic parameters of 12-hour LH secretion. CONCLUSION(S) Luteinizing hormone pulse amplitude was higher in patients with PCOS than in hirsute women with normal menstrual cycles or in healthy women. The LH pulse frequency was increased only in patients with PCOS compared with healthy women and not in hirsute women with normal menstrual cycles.

MTP -493G/T gene polymorphism is associated with steatosis in hepatitis C-infected patients

The reduction of hepatic microsomal transfer protein (MTP) activity results in fatty liver, worsening hepatic steatosis and fibrosis in chronic hepatitis C (CHC). The G allele of the MTP gene promoter, -493G/T, has been associated with lower transcriptional activity than the T allele. We investigated this association with metabolic and histological variables in patients with CHC. A total of 174 untreated patients with CHC were genotyped for MTP -493G/T by direct sequencing using PCR. All patients were negative for markers of Wilsons disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake lower than 100 g/week. The sample distribution was in Hardy-Weinberg equilibrium. Among subjects with genotype 1, 56.8% of the patients with fibrosis grade 3+4 presented at least one G allele versus 34.3% of the patients with fibrosis grade 1+2 (OR = 1.8; 95%CI = 1.3-2.3). Logistic regression analysis with steatosis as the dependent variable identified genotypes GG+GT as independent protective factors against steatosis (OR = 0.4, 95%CI = 0.2-0.8; P = 0.01). The results suggest that the presence of the G allele of MTP -493G/T associated with lower hepatic MTP expression protects against steatosis in our CHC patients.

Association of single nucleotide polymorphisms in the gene encoding GLUT1 and diabetic nephropathy in Brazilian patients with type 1 diabetes mellitus

Mesangial cells subject to high extracellular glucose concentrations, as occur in hyperglycaemic states, are unable to down regulate glucose influx, resulting in intracellular activation of deleterious biochemical pathways. A high expression of GLUT1 participates in the development of diabetic glomerulopathy. Variants in the gene encoding GLUT1 (SLC2A1) have been associated to this diabetic complication. The aim of this study was to test whether polymorphisms in SLC2A1 confer susceptibility to diabetic nephropathy (DN) in Brazilian type 1 diabetes patients. Four polymorphisms (rs3820589, rs1385129, rs841847 and rs841848) were genotyped in a Brazilian cohort comprised of 452 patients. A prospective analysis was performed in 155 patients. Mean duration of follow-up was 5.6 ± 2.4 years and the incidence of renal events was 18.0%. The rs3820589 presented an inverse association with the prevalence of incipient DN (OR: 0.36, 95% CI: 0.16 - 0.80, p=0.01) and with progression to renal events (HR: 0.20; 95% CI: 0.03 - 0.70; p=0.009). AGGT and AGAC haplotypes were associated with the prevalence of incipient DN and the AGAC haplotype was also associated with the prevalence of established/advanced DN. In conclusion, rs3820589 in the SLC2A1 gene modulates the risk to DN in Brazilian patients with inadequate type 1 diabetes control.

Hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) and guanylate kinase 1 (GUK1) are differentially expressed in GH-secreting adenomas

Pituitary tumors, adenomas in their vast majority, represent around 10–15% of the intracranial neoplasms. Pituitary carcinomas are exceedingly rare. Clinically, these neoplasms cause hormonal dysfunctions, and mass effect symptoms as headache and visual disorders in the case of macroadenomas. Pituitary tumorigenesis is still poorly understood. In order to investigate the expression of cancer-related genes in pituitary tumors, we employed a human cancer cDNA macroarray membrane with 1176 well-characterized human genes related to cancer and tumor biology. We were able to identify several differentially expressed genes, among them hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) and guanylate kinase 1 (GUK1) which were over expressed in a pool of clinically nonfunctioning pituitary adenomas, compared with a spinal cord metastasis of a nonfunctioning pituitary carcinoma. HGS and GUK1 mRNA expression were chosen to be validated by quantitative RT-qPCR, however, only GUK1 had the differential expression confirmed between the adenomas and the metastasis of a pituitary carcinoma. We have also investigated HGS and GUK1 mRNA expressions in a series of 46 pituitary adenomas (18 nonfunctioning, 12 GH-secreting, nine PRL-secreting, and seven ACTH-secreting adenomas). HGS and GUK1 were significantly over expressed in GH-secreting adenomas, compared with ACTH-secreting adenomas and nonfunctioning tumors, and with PRL-secreting adenomas, respectively. We have shown that these genes, involved in tumorigenesis in other tissues, are as well over expressed in the pituitary tumors, however, their role in the oncogenesis of these tumors need to be further investigated.

Hyperinsulinism/hyperammonemia (HI/HA) syndrome due to a mutation in the glutamate dehydrogenase gene

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a rare autosomal dominant disease manifested by hypoglycemic symptoms triggered by fasting or high-protein meals, and by elevated serum ammonia. HI/HA is the second most common cause of hyperinsulinemic hypoglycemia of infancy, and it is caused by activating mutations in GLUD1, the gene that encodes mitochondrial enzyme glutamate dehydrogenase (GDH). Biochemical evaluation, as well as direct sequencing of exons and exon-intron boundary regions of the GLUD1 gene, were performed in a 6-year old female patient presenting fasting hypoglycemia and hyperammonemia. The patient was found to be heterozygous for one de novo missense mutation (c.1491A>G; p.Il497Met) previously reported in a Japanese patient. Treatment with diazoxide 100 mg/day promoted complete resolution of the hypoglycemic episodes.

Dietary advanced glycated end-products and medicines influence the expression of SIRT1 and DDOST in peripheral mononuclear cells from long-term type 1 diabetes patients:

Quantitative polymerase chain reaction was employed to quantify expression of two genes coding for advanced glycation end-product receptors [RAGE (AGER) and AGER1 (DDOST)] and of the gene coding the deacetylase SIRT1 (SIRT1) in peripheral blood mononuclear cells from type 1 diabetes patients without [Group A, n = 35; 28.5 (24–39) years old; median (interquartile interval)] or with at least one microvascular complication [Group B, n = 117; 34.5 (30–42) years old]; 31 healthy controls were also included. In a subgroup of 48 patients, daily advanced glycation end-products intake before blood collection was assessed. Lower expression of DDOST was found in patients than in controls after adjustment for sex, age, use of statins, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. Higher expressions of AGER, DDOST and SIRT1 were observed in Group A. Stratifying by complications, AGER and DDOST expressions were higher in those without retinopathy and without diabetic kidney disease, respectively, compared to patients with these complications. Patients using statins or angiotensin receptor blockers presented higher expression of DDOST. Expression of SIRT1 was higher in patients consuming ≥12,872 KU daily of advanced glycation end-products. Although AGER, DDOST and SIRT1 are differently expressed in peripheral blood mononuclear cells from type 1 diabetes patients with and without microvascular complications, they are also influenced by dietary advanced glycation end-products and by statins and angiotensin receptor blockers.

Hormetic modulation of hepatic insulin sensitivity by advanced glycation end products

Because of the paucity of information regarding metabolic effects of advanced glycation end products (AGEs) on liver, we evaluated effects of AGEs chronic administration in (1) insulin sensitivity; (2) hepatic expression of genes involved in AGEs, glucose and fat metabolism, oxidative stress and inflammation and; (3) hepatic morphology and glycogen content. Rats received intraperitoneally albumin modified (AlbAGE) or not by advanced glycation for 12 weeks. AlbAGE induced whole-body insulin resistance concomitantly with increased hepatic insulin sensitivity, evidenced by activation of AKT, inactivation of GSK3, increased hepatic glycogen content, and decreased expression of gluconeogenesis genes. Additionally there was reduction in hepatic fat content, in expression of lipogenic, pro-inflamatory and pro-oxidative genes and increase in reactive oxygen species and in nuclear expression of NRF2, a transcription factor essential to cytoprotective response. Although considered toxic, AGEs become protective when administered chronically, stimulating AKT signaling, which is involved in cellular defense and insulin sensitivity.

Association between the CYBA and NOX4 genes of NADPH oxidase and its relationship with metabolic syndrome in non-alcoholic fatty liver disease in Brazilian population

BACKGROUND Oxidative stress has been implicated in the progression of severe forms of non-alcoholic fatty liver disease (NAFLD). NADPH oxidase produces reactive oxygen species. In the present study, we investigated for the first time two single nucleotide polymorphisms (SNPs) in the regulatory region of genes encoding NADPH oxidase 4 (NOX4) and p22phox (CYBA) in NAFLD. METHODS A total of 207 biopsy-proven NAFLD patients [simple steatosis (n = 27); nonalcoholic steatohepatitis (NASH) (n = 180)] were evaluated. Genomic DNA was extracted from peripheral blood cells, and polymorphisms in CYBA (unregistered) and NOX4 (rs3017887) were determined by direct sequencing of PCR. RESULTS Associations of CYBA-675 T/A with high-density lipoprotein (HDL) (TT vs TA vs AA; P < 0.01) and triglycerides (TGL) (TT vs XA; P < 0.01) were observed only in NASH patients. For polymorphisms in the NOX4 gene, NOX4 (rs3017887) CA + AA genotypes was significant associated with alanine aminotransferase (ALT) (CA + AA vs CC; P = 0.02). However, there was no association of SNPs in the CYBA and NOX4 genes encoding the NADPH oxidase system proteins and the presence of NASH. Regarding the clinical results, it was observed that the most advanced degrees of fibrosis occurred in patients diagnosed with type 2 diabetes mellitus (66.9% vs 37.5%, P < 0.01) and those who were more obese (32.2 vs 29.0 kg/m2, P < 0.01). In addition, serum glucose and insulin levels increased significantly in the presence of NASH. CONCLUSIONS There were associations between the presence of the allele A in the NOX4 SNP and a higher concentration of ALT in the NAFLD population; between the presence of the AA genotype in the polymorphism of the CYBA-675 T/A CYBA gene and a higher level of TGL and lower HDL in NASH patients. The presence of metabolic syndrome was associated with advanced degrees of fibrosis in NAFLD patients.