Silvia I. García

Maternal pregestational BMI is associated with methylation of the PPARGC1A promoter in newborns.

We explored peroxisome proliferator‐activated receptor‐γ co‐activator 1α gene (PPARGC1A), peroxisome proliferator‐activated receptor‐γ gene (PPARG), and transcription factor A mitochondrial gene (Tfam) promoter DNA methylation in newborns between both extremes of abnormal fetal growth: Small (SGA) and large for gestational age (LGA) in relation to the mothers characteristics. We further sought for the association of rs9930506 variant at FTO gene and the promoter patterns of DNA methylation in the aforementioned genes, in relation to the offsprings birth weight. In a cross‐sectional study, 88 healthy pregnant women and their babies were included. According to the offspring birth weight, there were 57 newborns with appropriate weight for gestational age (AGA), 17 SGA, and 14 LGA. After bisulphite treatment of umbilical cord genomic DNA, a real‐time methylation‐specific PCR was used to determine the promoter methylation status in selected CpGs. Promoter methylated DNA/unmethylated DNA ratio, expressed as mean ± s.e., was 0.82 ± 0.15 (45% of alleles) for PPARGC1A, and 0.0044 ± 0.0006 (0.4% of alleles) for Tfam. PPARG promoter was almost 100% methylated in all samples. In univariate analysis, there was no association among characteristics of the newborn and gene promoter methylation. None of the maternal features were related with the status of promoter methylation, except for a positive correlation between maternal BMI and PPARGC1A promoter methylation in umbilical cord (Pearson correlation coefficient r = 0.41, P = 0.0007). Finally, FTO rs9930506 AA homozygous in the LGA group showed decreased levels of methylated PPARGC1A in comparison with AG + GG genotypes and AGA and SGA infants. In conclusion, our findings suggest a potential role of promoter PPARGC1A methylation in metabolic programming.

Short Allele of Serotonin Transporter Gene Promoter Is a Risk Factor for Obesity in Adolescents

Obesity and hypertension are increasing medical problems in adolescents. Serotonin transporter (5‐HTT) is involved in mood and eating disturbances. Encoded by the gene SLC6A4, the promoter shows functional insertion/deletion alleles: long (L) and short (S). Because individuals who are carriers for the short version are known to be at risk for higher levels of anxiety, we hypothesized that this variant may be associated with overweight. Data and blood samples were collected from 172 adolescents out of a cross‐sectional, population‐based study of 934 high school students. To replicate the findings, we also included 119 outpatients from the Nutrition and Diabetes Section of the Childrens County Hospital. We found that the S allele was associated with overweight (BMI > 85th percentile), being a risk factor for overweight independently of sex, age, and hypertension [odds ratio (OR): 1.85; 95% confidence interval (CI): 1.13, 3.05; p < 0.02]. Additionally, in the outpatient study, compared with the homozygous LL subjects, S allele carriers showed a higher BMI z‐score (1.47 ± 1.09 vs. 0.51 ± 1.4; p < 0.002) and were more frequent in overweight children. In conclusion, the S allele of the SLC6A4 promoter variant is associated with overweight being an independent genetic risk factor for obesity.

A Decreased Mitochondrial DNA Content Is Related to Insulin Resistance in Adolescents

The aim of this study was to investigate whether mitochondrial DNA (mtDNA) content is associated with insulin resistance (IR) in a sample of adolescents with features of metabolic syndrome. We further studied the link between polymorphisms in three genes involved in mitochondrial biogenesis and the presence of deleted mtDNA and mtDNA content. Data and blood samples were collected from 175 adolescents out of a cross‐sectional, population‐based study of 934 high school students. On the basis of the median value of homeostasis model assessment of IR (HOMA‐IR) of the whole sample (2.2), the population was divided into two groups: noninsulin resistance (NIR) and IR. mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out using a real‐time quantitative PCR method. Genotyping for peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) (pro12Ala), PPAR‐ γ coactivator‐1α (PGC‐1α) (Gly482Ser), and Tfam (rs1937 and rs12247015) polymorphisms was performed by PCR‐based restriction fragment length polymorphism. Long‐extension PCR was performed to amplify the whole mitochondrial genome. The mtDNA/nDNA ratio was significantly lower in the IR group (median: 9.08, range: 68.94) in comparison with the NIR group (12.24, 71.92) (P < 0.03). Besides, the mtDNA/nDNA ratio was inversely correlated with HOMA (R: −0.18, P < 0.02), glucose (R: −0.21, P < 0.008), and uric acid (R: −0.18, P < 0.03). Genotypes for the PPAR‐ γ, PGC‐1α, and Tfam variants were not associated with the mtDNA/nDNA ratio. Long‐extension PCR did not show significant levels of mtDNA deletions. In conclusion, our findings indicate that reduced mtDNA content in peripheral leukocytes is associated with IR. This result seems not to be related with the previously mentioned variants in genes involved in the regulation of mitochondrial biogenesis.

Angiotensin II enhances long-term memory in the crab Chasmagnathus

An opaque screen moving overhead provokes an escape response in the crab Chasmagnathus granulatus that habituates after a few presentations of the eliciting stimulus. Fifteen trials with a 180-s intertrial interval or 30 trials with a 90-s interval (strong training protocol) ensures long-term habituation (LTH) of the response for 24 h, whereas 10 trials (weak training protocol) fail to induce it. However, robust LTH is obtained when crabs are injected with human angiotensin (All; 50 pmol) immediately after a weak training protocol. This memory-enhancing effect of All is dose-dependent, reversible by saralasin (5 pmol), and vanishes either when the weak training protocol is reduced to only five trials, or when the peptide is given before training or 1 h after. LTH is impaired by saralasin (5 pmol) administered before or after the strong training protocol, but no amnestic effect is disclosed when the antagonist is given 1 h after. On the other hand, both All-like immunoreactivity and angiotensin-converting enzyme-like activity are described in diverse tissues of Chasmagnathus, namely, in gills and in both thoracic and supraesophageal ganglia. Results support the view that some components of the renin-angiotensin system and their influence on memory might have emerged early in evolution.

Methylation of TFAM gene promoter in peripheral white blood cells is associated with insulin resistance in adolescents

PURPOSE To explore whether DNA methylation of the mitochondrial transcription factor A (TFAM) promoter is associated with insulin resistance in a sample of adolescents with features of metabolic syndrome. METHODS The data and blood samples were collected from 122 adolescents out of a cross-sectional study of 934 high-school students. The population was divided into two groups: noninsulin resistance (NIR) and insulin resistance (IR). After bisulfite treatment of genomic DNA from peripheral leukocytes, we used methylation-specific polymerase chain reaction (PCR) to assess DNA methylation of three putative methylation target sites (CpG) in the TFAM promoter. RESULTS The ratio of the promoter methylated DNA/unmethylated DNA was 0.012+/-0.0009 (1.2% of alleles), and inversely correlated with the biochemical features of insulin resistance (plasma fasting insulin R: -0.26, p<0.004 and homeostasis model assessment (HOMA) index R: -0.27, p<0.002), and obesity (R: -0.27, p<0.002). Multiple regression analysis showed that the log-transformed HOMA index correlated with the status of promoter methylation of TFAM, independently of body mass index (BMI) Z score (beta: -0.33+/-0.094, p=0.00094). Finally, the TFAM promoter methylated DNA/unmethylated DNA ratio was found to be significantly associated with insulin resistance as dichotomous variable (NIR n=45, 0.014+/-0.002 and IR n=77, 0.011+/-0.001, respectively, p<0.016). CONCLUSION Our findings suggest a potential role of promoter TFAM methylation in the pathogenesis of insulin resistance in adolescents.

Mitochondrial DNA Depletion in Small‐ and Large‐for‐Gestational‐Age Newborns

Objective: To investigate whether mitochondrial DNA (mtDNA) content may be associated with clinical features, anthropometric variables, and laboratory findings in both extremes of abnormal fetal growth: small and large size for gestational age.

Antisense Inhibition of Thyrotropin-Releasing Hormone Reduces Arterial Blood Pressure in Spontaneously Hypertensive Rats

Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation. Recently, we described that the TRH precursor gene overexpression induces hypertension in the normal rat. In addition, we published that spontaneously hypertensive rats (SHR) have central extrahypothalamic TRH hyperactivity with increased TRH synthesis and release and an elevated TRH receptor number. In the present study, we report that intracerebroventricular antisense (AS) treatment with a phosphorothioate oligonucleotide against the TRH precursor gene significantly diminished up to 72 hours and in a dose-dependent manner the increased diencephalic TRH content, whereas normalized systolic blood pressure (SABP) was present in the SHR compared with Wistar-Kyoto (WKY) rats. Although basal thyrotropin was higher in SHR compared with WKY rats and this difference disappeared after antisense treatment, no differences were observed in plasma T4 or T3 between strains with or without AS treatment, indicating that the effect of the AS on SABP was independent of the thyroid status. Because the encephalic renin-angiotensin system seems to be crucial in the development and/or maintenance of hypertension in SHR, we investigated the effect of antisense inhibition of TRH on that system and found that TRH antisense treatment significantly diminished the elevated diencephalic angiotensin II (Ang II) content in the SHR without any effect in control animals, suggesting that the Ang II system is involved in the TRH cardiovascular effects. To summarize, the central TRH system seems to be involved in the etiopathogenesis of hypertension in this model of essential hypertension.

Thyrotropin-releasing hormone receptor (TRHR) gene is associated with essential hypertension

In essential hypertension, a polygenic and multifactorial syndrome, several genes interact with the environment to produce high blood pressure. Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation. We have described that TRH overexpression induces hypertension in a normal rat, which was reversed by TRH antisense treatment. This treatment also reduces the central TRH hyperactivity in spontaneously hypertensive rats and normalizes blood pressure. Human TRH receptor (TRHR) belongs to the G protein-coupled seven-transmembrane domain receptor superfamily. Mutations of these receptors may result in constitutive activation. As it has been demonstrated that hypertensive patients have a blunted TSH response to TRH injection, suggesting a defect in the TRHR, we postulate that the TRHR gene is involved in human hypertension. We studied 2 independent populations from different geographic regions of our country: a sample of adult subjects from a referral clinic and a population-based sample of high school students. In search of molecular variants of TRHR, we disclosed that a polymorphic TG dinucleotide repeat (STR) at −68 bp and a novel single nucleotide polymorphism, a G→C conversion at −221 located in the promoter of the TRHR are associated with essential hypertension. As STRs detected in gene promoters are potential Z-DNA-forming sequences and seem to affect gene expression, we studied the potentially different transcriptional activity of these TRHR promoter variants and found that the S/−221C allele has a higher affinity than does the L/G−221 allele to nuclear protein factor(s). Our findings support the hypothesis that the TRHR gene participates in the etiopathogenesis of essential hypertension.

Increased Expression of Magnocellular Vasopressin mRNA in Rats with Deoxycorticosterone-Acetate Induced Salt Appetite

The neuropeptides arginine vasopressin (AVP) and oxytocin (OT) have been implicated in the genesis of hypertension due to deoxycorticosterone acetate (DOCA)-salt treatment of uninephrectomized rats. In this work, we studied if DOCA treatment of intact rats in doses arousing a salt appetite (a prehypertensive state), modulated mRNA for AVP and OT in the hypothalamus. Male Sprague-Dawley rats were offered both tap water and 3% NaCl in separate bottles and received vehicle or subcutaneous injections of 10 mg DOCA on alternate days for 7 days (4 injections) or 17 days (9 injections). They developed a preference for 3% NaCl solutions 24–48 h after treatment. Brain slices from rats killed on the 8th or 18th day were exposed to 35S-labeled probes encoding prepro-AVP mRNA or OT mRNA, respectively. Expression of these mRNAs was measured in the magnocellular and parvocellular divisions of the paraventricular nucleus (PVN) and magnocellular cells of the supraoptic nucleus (SON). No changes were obtained in neuropeptide mRNA levels in the parvocellular division of the PVN between control and the two groups of DOCA-treated rats. However, DOCA-treated animals presented an increased number of grains per cell for AVP mRNA in the magnocellular division of the PVN and in magnocellular cells of the SON, as shown by group mean comparisons and frequency histograms. No changes were detected for OT mRNA. In a second series of studies, control or DOCA-treated rats were offered 3% NaCl or water as the only choice. Animals drinking 3% NaCl showed increased AVP and OT mRNA levels, whether they received DOCA or not. However, AVP mRNA levels in both nuclei were higher in DOCA-treated rats drinking 3% NaCl than in controls drinking salt solution. In comparison, control and DOCA-treated rats drinking water showed lower levels of AVP mRNA. OT mRNA levels in the SON remained unchanged in the same groups. The results suggest that in the magnocellular cells of the PVN and SON, increments in AVP mRNA are obtained following increments in salt intake produced by either mineralocorticoid treatment or exclusive salt drinking. In rats offered salt solution and water to drink, DOCA effects on AVP mRNA developed before changes occurred in serum sodium levels. Because combined DOCA + salt treatment induced a higher response in terms of AVP mRNA expression, we suggest that AVP could be a target of the central effects of the mineralocorticoid.

Angiotensin-(1–7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

J. Neurochem. (2012) 120, 46–55.