Juan Pablo Méndez

Reproductive axis function and gonadotropin microheterogeneity in a male rat model of diet-induced obesity

Obesity causes complex metabolic and endocrine changes that may lead to adverse outcomes, including hypogonadism. We herein studied the reproductive axis function in male rats under a high-fat diet and analyzed the impact of changes in glycosylation of pituitary LH on the bioactivity of this gonadotropin. Rats were fed with a diet enriched in saturated fat (20% of total calories) and euthanized on days 90 or 180 of diet. Long-term (180 days), high-fat feeding rats exhibited a metabolic profile compatible with insulin resistance and metabolic syndrome; they concomitantly showed decreased intrapituitary and serum LH concentrations, low serum testosterone levels, and elevated serum 17beta-estradiol concentrations. A fall in biological to immunological ratio of intrapituitary LH was detected in 180 days control diet-treated rats but not in high-fat-fed animals, as assessed by a homologous in vitro bioassay. Chromatofocusing of pituitary extracts yielded multiple LH charge isoforms; a trend towards decreased abundance of more basic isoforms (pH 9.99-9.0) was apparent in rats fed with the control diet for 180 days but not in those that were fed the diet enriched in saturated fat. It is concluded that long-term high-fat feeding alters the function of the pituitary-testicular axis, resulting in hypogonadotropic hypogonadism. The alterations in LH function found in these animals might be subserved by changes in hypothalamic GnRH output and/or sustained gonadotrope exposure to an altered sex steroid hormone milieu, representing a distinctly different regulatory mechanism whereby the pituitary attempts to counterbalance the effects of long-term obesity on reproductive function.

Molecular Analysis of the SRD5A2 in 46,XY Subjects With Incomplete Virilization: The P212R Substitution of the Steroid 5α-Reductase 2 May Constitute an Ancestral Founder Mutation in Mexican Patients

Inactivating mutations of the SRD5A2 gene result in steroid 5α-reductase 2 deficiency, an autosomal recessive disorder expressed as a male-limited disorder of sex development. Herein, genomic DNA was isolated from 11 new patients with apparent steroid 5α-reductase 2 deficiency. Coding sequence abnormalities in SRD5A2 were assessed by exon-specific polymerase chain reaction, single-stranded conformation polymorphism, and direct sequencing. Likewise, enzymatic activity of the P212R gene variant of SRD5A2 was assessed. DNA analysis revealed mutations in all patients (G115D, R171S, N193S, E197D, G203S, P212R). Three individuals were compound heterozygotes, 6 were homozygotes, and 2 more were single heterozygotes for SRD5A2 mutations; remarkably, 40% of the mutant alleles (9/22) contained the gene variant P212R. The results described in this study represent, along with our previous reports, the largest number of patients with steroid 5α-reductase 2 deficiency belonging to nonrelated families. Regarding the frequency of the p.P212R mutation in our population and its presence throughout all of our country, it allows us to hypothesize that the presence of this mutation may constitute a founder gene effect.

Pituitary-testicular axis function, biological to immunological ratio and charge isoform distribution of pituitary LH in male rats with experimental diabetes.

Men with insulinopenic diabetes mellitus frequently present hypogonadism and exhibit circulating luteinizing hormone (LH) molecules with increased biological activity. To further study this latter issue, we analyzed the pattern of isoform distribution and the impact of changes in terminal glycosylation of pituitary LH on the bioactivity of this gonadotropin in experimental diabetes. Adult male rats were treated with streptozotocin or vehicle and euthanized on days 30, 60, or 90 posttreatment. All diabetic groups exhibited a significant decrease in serum insulin and testosterone levels as well as in sperm count; serum gonadotropins and 17beta-estradiol decreased only after 90 days of insulinopenia. Both the immunoreactive concentrations and the biological to immunological ratio of intrapituitary LH significantly increased in all experimental groups, as assessed by an in vitro homologous bioassay in HEK-293 cells expressing a recombinant LH receptor. Chromatofocusing of pituitary extracts revealed the presence of multiple LH charge isoforms; the pH distribution profile of LH in diabetic and control rats was indistinguishable on days 30 and 60 posttreatment. By contrast, the abundance of more basic isoforms (pH 9.99-9.0) decreased and that of isoforms with pH values 8.99-8.0 increased in rats with long-standing diabetes compared to controls. It is concluded that experimental diabetes alters the function of the pituitary-testicular axis, resulting in reduced sex steroids levels and hypogonadotropism. Long-standing insulinopenia leads to a paradoxical accumulation of intrapituitary LH molecules enriched in bioactivity with altered terminal glycosylation, which are apparently subserved by distinct mechanisms involving altered hypothalamic and/or gonadal inputs on the gonadotrope.

Impact of genetic variants of IL-6, IL6R, LRP5, ESR1 and SP7 genes on bone mineral density in postmenopausal Mexican-Mestizo women with obesity.

BACKGROUND Since obesity and osteoporosis present a high genetic predisposition and polymorphisms of IL-6, IL6R, LRP5, ESR1 and SP7 may influence the risk of both diseases, the aim of this study was to analyze the possible association of polymorphisms in these genes, as well as their haplotypes, with BMD variations in postmenopausal Mexican-Mestizo women with grade 2 or grade 3 obesity. METHODS One hundred eighty unrelated postmenopausal women with grade 2 or grade 3 obesity were included. BMD was measured in total hip and lumbar spine by dual-energy X-ray absorptiometry. DNA was obtained from blood leukocytes. Rs1800795 of IL-6, rs2228145 of IL6R, rs3736228 of LRP5, rs9340799 (XbaI) and rs2234693 (PvuII), of ESR1, rs10876432 and rs2016266, of SP7 (and their haplotypes), were studied by real-time PCR allelic discrimination. Deviations from Hardy-Weinberg equilibrium were tested. Pairwise linkage disequilibrium between single nucleotide polymorphisms was calculated by direct correlation r(2), and haplotype analysis was conducted. RESULTS Using WHO criteria, 54.5% had grade 2 obesity, and 45.5% had grade 3 obesity. Regarding DXA results, 11.1% women had osteoporosis, 41.7% had osteopenia, and 47.2% had normal BMD. Genotype and haplotype analysis showed no significant differences with BMD variations at the lumbar spine, total hip or femoral neck. CONCLUSIONS We did not find a significant association between the polymorphisms analyzed or their haplotypes and BMD variations in postmenopausal women with obesity. The higher BMD observed in women with obesity could be the result of an adaptive response to the higher loading of the skeleton.

Genetic variants in ATP6 and ND3 mitochondrial genes are not associated with aggressive prostate cancer in Mexican–Mestizo men with overweight or obesity

Abstract Mitochondrial defects have been related to obesity and prostate cancer. We investigated if Mexican–Mestizo men presenting this type of cancer, exhibited somatic mutations of ATP6 and/or ND3.Body mass index (BMI) was determined; the degree of prostate cancer aggressiveness was demarcated by the Gleason score. DNA from tumor tissue and from blood leukocytes was amplified by the polymerase chain reaction and ATP6 and ND3 were sequenced. We included 77 men: 20 had normal BMI, 38 were overweight and 19 had obesity; ages ranged from 52 to 83. After sequencing ATP6 and ND3, from DNA obtained from leukocytes and tumor tissue, we did not find any somatic mutations. All changes observed, in both genes, were polymorphisms. In ATP6 we identified, in six patients, two non-synonymous nucleotide changes and in ND3 we observed that twelve patients presented non-synonymous polymorphisms. To our knowledge, this constitutes the first report where the complete sequences of the ATP6 and ND3 have been analyzed in Mexican–Mestizo men with prostate cancer and diverse BMI. Our results differ with those reported in Caucasian populations, possibly due to ethnic differences.

High Knowledge About Obesity and its Health Risks, with the Exception of Cancer, Among Mexican Individuals

Mexico has the second biggest prevalence in the world of obese adults (30%). We conducted a survey to determine knowledge concerning obesity co-morbidities. Three groups were surveyed with a questionnaire divided into three sections: demographic characteristics; knowledge and awareness in relation to obesity being a disease; causes of obesity and the health risks it represents; weight auto-perception and the subject’s personal experiences regarding weight. In all groups we found high knowledge regarding that obesity is a disease and the causes of its development, as well as that it greatly increases the risk of presenting type 2 diabetes, high blood pressure and knee osteoarthritis. However, in all groups, there was a gap in knowledge regarding the risk obesity poses for the development of breast and colon cancer. Aggressive health promotion campaigns concerning obesity, which have been implemented recently in Mexico, must emphasize cancer as a potential outcome for obese patients.

Triallelic digenic mutation in the prokineticin 2 and GNRH receptor genes in two brothers with normosmic congenital hypogonadotropic hypogonadism

Abstract Purpose/aim of the study: To date, different genes have been identified as responsible for the presence of normosmic congenital hypogonadotropic hypogonadism (nCHH). Herein, we report the molecular findings regarding the analysis of PROK2, in two brothers with nCHH. Subjects and methods: Two siblings with nCHH, in whom mutations in GNRHR, PROKR2 and FGFR1 had been investigated previously, as well as their family were studied. DNA was amplified by PCR and sequenced for the PROK2 gene. Controls were analyzed by restriction fragment-length polymorphism. The structure of PROK2 and its mutant protein were compared using a protein molecular model. Results: Both affected siblings exhibited a heterozygous p.R117W mutation in PROK2, while their mother was a heterozygous carrier and their father, an unaffected brother and their sister were homozygous wild type. Besides, both patients presented a homozygous p.E90K mutation in GNRHR that had been previously reported. Conclusions: We found a novel mutation in PROK2 in two siblings in whom a mutation in the GNRHR gene had been previously reported.

Duplication of SOX9 associated with 46,XX ovotesticular disorder of sex development

RESEARCH QUESTION The purpose of the present study was to investigate whether ten unrelated SRY-negative individuals with this sex differentiation disorder presented a double dose of SOX9 as the cause of their disease. DESIGN Ten unrelated SRY-negative 46,XX ovotesticular disorder of sexual development (DSD) subjects were molecularly studied. Multiplex-ligation dependent probe amplification (MLPA) and quantitative real-time PCR analysis (qRT-PCR) for SOX9 were performed. RESULTS The MLPA analysis demonstrated that one patient presented a heterozygous duplication of the entire SOX9 coding region (above 1.3 value of peak ratio), as well as at least a ~ 483 kb upstream duplication. Moreover, no duplication of other SOX9 probes was observed corresponding to the region between -1007 and -1500 kb upstream. A qRT-PCR analysis showed a duplication of at least -581 kb upstream and ~1.63 kb of the coding region that encompasses exon 3. The limits of the duplication were mapped approximately from ~71539762 to 72122741 of Chr17. No molecular abnormalities were found in the remaining nine patients. CONCLUSION This study is thought to be the first report regarding a duplication of SOX9 that is associated with the presence of 46,XX ovotesticular DSD, encompassing at least -581 kb upstream, and the almost entire coding region of the gene.

Complete sequence of the ANKK1 gene in Mexican-Mestizo individuals with obesity, with or without binge eating disorder

BACKGROUND The aim of this study was to investigate if Mexican-Mestizo individuals with obesity, with or without binge eating disorder (BED), exhibited mutations or other type of genetic variants in the sequence of ANKK1. SUBJECTS AND METHODS Fifty unrelated individuals (21-53 years of age) with obesity, of Mexican-Mestizo ethnic origin were included; 25 of them had BED and 25 presented obesity without BED. The diagnosis of BED was based on criteria proposed in the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Besides, we also analyzed 100 individuals with normal body mass index. DNA from blood leukocytes was amplified by the polymerase chain reaction and all exons of ANKK1 were sequenced. RESULTS After ANKK1 sequencing we did not find any mutations; however, we observed various polymorphisms. One polymorphism, rs4938013 in exon 2 showed an association with obesity, whilst rs1800497 (also known as Taq1A) in exon 8, showed an association with BED (P = 0.020). Remarkable, for this study, the number of individuals for both polymorphisms for and additive model was sufficient to derive strong statistical power (80%, with a P < 0.05). CONCLUSIONS To our knowledge, this constitutes the first report where the complete sequences of ANKK1 has been analyzed in individuals with obesity, with or without BED. No mutations were found; however, one polymorphism was associated with obesity, with or without BED, and another one was associated with BED.

Serum concentrations of apelin-17 isoform vary in accordance to blood pressure categories in individuals with obesity class 3

ABSTRACT Background: The aim of this study was to investigate if serum concentrations of apelin-36, apelin-17, apelin-13 or apelin-12 were different in obesity class 3 individuals with hypertension, when compared to those without hypertension (normal or high-normal). Subjects and Methods: Twenty six individuals with obesity class 3-related hypertension and thirty three individuals without hypertension, who were divided in individuals with normal (n = 23) or with high-normal (n = 10) blood pressure (BP) were analyzed. All individuals presented obesity class 3, without diabetes mellitus. Measurements of all apelin isoforms were performed using enzyme-linked immunosorbent assay kits. Analysis of differences between groups of Apelin isoform concentrations was performed by a One-way ANOVA, with a Tukey test post hoc. Results: The individuals of the hypertensive group presented a slightly lower serum concentration of all apelin isoforms, but these differences were not statistically significant. These results were more evident when the group of patients without hypertension were divided based in normal and high-normal BP, observing that apelin-17 isoform were higher in individuals with high-normal BP in comparison to subjects with normal BP (P = 0.018); concentrations were also higher when compared to subjects with hypertension (P = 0.004). Conclusions: To our knowledge, this is the first study regarding the differences of apelin-17 isoform concentrations in individuals pertaining to different categories of BP, who presented obesity class 3. The group of patients that presented hypertension showed a lower concentration of all isoforms. This observation could be due to the fact that these patients were taking antihypertensive medication.