Diana Cerjak

Hypothalamic oxytocin mediates adaptation mechanism against chronic stress in rats

Accumulation of continuous life stress (chronic stress) often causes gastric symptoms. Although central oxytocin has antistress effects, the role of central oxytocin in stress-induced gastric dysmotility remains unknown. Solid gastric emptying was measured in rats receiving acute restraint stress, 5 consecutive days of repeated restraint stress (chronic homotypic stress), and 7 consecutive days of varying types of stress (chronic heterotypic stress). Oxytocin and oxytocin receptor antagonist were administered intracerebroventricularly (icv). Expression of corticotropin-releasing factor (CRF) mRNA and oxytocin mRNA in the paraventricular nucleus (PVN) of the hypothalamus was evaluated by real-time RT-PCR. The changes of oxytocinergic neurons in the PVN were evaluated by immunohistochemistry. Acute stress delayed gastric emptying, and the delayed gastric emptying was completely restored after 5 consecutive days of chronic homotypic stress. In contrast, delayed gastric emptying persisted following chronic heterotypic stress. The restored gastric emptying following chronic homotypic stress was antagonized by icv injection of an oxytocin antagonist. Icv injection of oxytocin restored delayed gastric emptying induced by chronic heterotypic stress. CRF mRNA expression, which was significantly increased in response to acute stress and chronic heterotypic stress, returned to the basal levels following chronic homotypic stress. In contrast, oxytocin mRNA expression was significantly increased following chronic homotypic stress. The number of oxytocin-immunoreactive cells was increased following chronic homotypic stress at the magnocellular part of the PVN. Icv injection of oxytocin reduced CRF mRNA expression induced by acute stress and chronic heterotypic stress. It is suggested that the adaptation mechanism to chronic stress may involve the upregulation of oxytocin expression in the hypothalamus, which in turn attenuates CRF expression.

Hypothalamic oxytocin attenuates CRF expression via GABAA receptors in rats

Centrally released oxytocin (OXT) has anxiolytic and anti-stress effects. Delayed gastric emptying (GE) induced by acute restraint stress (ARS) for 90 min is completely restored following 5 consecutive days of chronic homotypic restraint stress (CHS), via up-regulating hypothalamic OXT expression in rats. However, the mechanism behind the restoration of delayed GE following CHS remains unclear. Gamma-aminobutyric acid (GABA)-projecting neurons in the paraventricular nucleus (PVN) have been shown to inhibit corticotropin releasing factor (CRF) synthesis via GABA(A) receptors. We hypothesized that GABA(A) receptors are involved in mediating the inhibitory effect of OXT on CRF expression in the PVN, which in turn restores delayed GE following CHS. OXT (0.5 μg) and selective GABA(A) receptor antagonist, bicuculline methiodide (BMI) (100 ng), were administered intracerebroventricularly (icv). Solid GE was measured under non-stressed (NS), ARS and CHS conditions. Expression of CRF mRNA in the PVN was evaluated by real time RT-PCR. Neither OXT nor BMI changed GE and CRF mRNA expression under NS conditions. Delayed GE and increased CRF mRNA expression induced by ARS were restored by icv-injection of OXT. The effects of OXT on delayed GE and increased CRF mRNA expression in ARS were abolished by icv-injection of BMI. Following CHS, delayed GE was completely restored in saline (icv)-injected rats, whereas daily injection of BMI (icv) attenuated the restoration of delayed GE. Daily injection of BMI (icv) significantly increased CRF mRNA expression following CHS. It is suggested that central OXT inhibits ARS-induced CRF mRNA expression via GABA(A) receptors in the PVN. GABAergic system is also involved in OXT-mediated adaptation response of delayed GE under CHS conditions.

Obesity, central adiposity and cardiometabolic risk factors in children and adolescents: A family-based study

The objective of this study was to assess genetic and phenotypic correlations of obesity‐related cardiometabolic risk factors in a family‐based cohort.

Fatty acid binding protein 3 (fabp3) is associated with insulin, lipids and cardiovascular phenotypes of the metabolic syndrome through epigenetic modifications in a northern european family population

BackgroundFatty acid-binding proteins (FABPs) play regulatory roles at the nexus of lipid metabolism and signaling. Dyslipidemia in clinical manifestation frequently co-occurs with obesity, insulin resistance and hypertension in the Metabolic Syndrome (MetS). Animal studies have suggested FABPs play regulatory roles in expressing MetS phenotypes. In our family cohort of Northern European descent, transcript levels in peripheral white blood cells (PWBCs) of a key FABPs, FABP3, is correlated with the MetS leading components. However, evidence supporting the functions of FABPs in humans using genetic approaches has been scarce, suggesting FABPs may be under epigenetic regulation. The objective of this study was to test the hypothesis that CpG methylation status of a key regulator of lipid homeostasis, FABP3, is a quantitative trait associated with status of MetS phenotypes in humans.MethodsWe used a mass-spec based quantitative method, EpiTYPER®, to profile a CpG island that extends from the promoter to the first exon of the FABP3 gene in our family-based cohort of Northern European descent (n=517). We then conducted statistical analysis of the quantitative relationship of CpG methylation and MetS measures following the variance-component association model. Heritability of each methylation and the effect of age and sex on CpG methylation were also assessed in our families.ResultsWe find that methylation levels of individual CpG units and the regional average are heritable and significantly influenced by age and sex. Regional methylation was strongly associated with plasma total cholesterol (p=0.00028) and suggestively associated with LDL-cholesterol (p=0.00495). Methylation at individual units was significantly associated with insulin sensitivity, lipid particle sizing and diastolic blood pressure (p<0.0028, corrected for multiple testing for each trait). Peripheral white blood cell (PWBC) expression of FABP3 in a separate group of subjects (n=128) negatively correlated with adverse profiles of metabolism (βWHR = −0.72; βLDL-c = −0.53) while positively correlated with plasma adiponectin (β=0.24). Further, we show that differential methylation of FABP3 affects binding activity with nuclear proteins from heart tissue. This region that we found under methylation regulation overlaps with a region actively modified by histone codes in the newly available ENCODE data.ConclusionsOur findings suggest that DNA methylation of FABP3 strongly influences MetS, and this may have important implications for cardiovascular disease.

Methylation of SOCS3 is inversely associated with metabolic syndrome in an epigenome-wide association study of obesity.

ABSTRACT Epigenetic mechanisms, including DNA methylation, mediate the interaction between gene and environment and may play an important role in the obesity epidemic. We assessed the relationship between DNA methylation and obesity in peripheral blood mononuclear cells (PBMCs) at 485,000 CpG sites across the genome in family members (8-90 y of age) using a discovery cohort (192 individuals) and a validation cohort (1,052 individuals) of Northern European ancestry. After Bonferroni-correction (Pα=0.05 = 1.31 × 10−7) for genome-wide significance, we identified 3 loci, cg18181703 (SOCS3), cg04502490 (ZNF771), and cg02988947 (LIMD2), where methylation status was associated with body mass index percentile (BMI%), a clinical index for obesity in children, adolescents, and adults. These sites were also associated with multiple metabolic syndrome (MetS) traits, including central obesity, fat depots, insulin responsiveness, and plasma lipids. The SOCS3 methylation locus was also associated with the clinical definition of MetS. In the validation cohort, SOCS3 methylation status was found to be inversely associated with BMI% (P = 1.75 × 10−6), waist to height ratio (P = 4.18 × 10−7), triglycerides (P = 4.01 × 10−4), and MetS (P = 4.01 × 10−7), and positively correlated with HDL-c (P = 4.57 × 10−8). Functional analysis in a sub cohort (333 individuals) demonstrated SOCS3 methylation and gene expression in PBMCs were inversely correlated (P = 2.93 × 10−4) and expression of SOCS3 was positively correlated with status of MetS (P = 0.012). We conclude that epigenetic modulation of SOCS3, a gene involved in leptin and insulin signaling, may play an important role in obesity and MetS.

Sustained acceleration of colonic transit following chronic homotypic stress in oxytocin knockout mice

Acute restraint stress delays gastric emptying and accelerates colonic transit via central corticotropin releasing factor (CRF) in rats. In contrast, central oxytocin has anxiolytic effects and attenuates the hypothalamus-pituitary-adrenal (HPA) axis in response to stress. Our recent study showed that up regulated oxytocin expression attenuates hypothalamic CRF expression and restores impaired gastric motility following chronic homotypic stress in mice. We studied the effects of acute and chronic homotypic stress on colonic transit and hypothalamic CRF mRNA expression in wild type (WT) and oxytocin knockout (OXT-KO) mice. Colonic transit was measured following acute restraint stress or chronic homotypic stress (repeated restraint stress for 5 consecutive days). (51)Cr was injected via a catheter into the proximal colon. Ninety minutes after restraint stress loading, the entire colon was removed. The geometric center (GC) was calculated to evaluate colonic transit. Expression of CRF mRNA in the supraoptic nucleus (SON) was measured by real time RT-PCR. Colonic transit was significantly accelerated following acute stress in WT (GC=8.1±0.8; n=7) and OXT KO mice (GC=9.4±0.3; n=7). The accelerated colonic transit was significantly attenuated in WT mice (GC=6.6±0.5; n=9) following chronic homotypic stress while it was still accelerated in OXT KO mice (GC=9.3±0.5; n=8). The increase in CRF mRNA expression at the SON was much greater in OXT-KO mice, compared to WT mice following chronic homotypic stress. It is suggested that oxytocin plays a pivotal role in mediating the adaptation mechanism following chronic homotypic stress in mice.

Sustained delayed gastric emptying during repeated restraint stress in oxytocin knockout mice.

We have recently shown that impaired gastric motility observed in acute restraint stress was restored following repeated restraint stress in mice. Repeated restraint stress up‐regulates oxytocin mRNA expression and down‐regulates corticotrophin‐releasing factor (CRF) mRNA expression at the hypothalamus. Oxytocin knockout mice (OXT‐KO) have been widely used to study the central oxytocin signalling pathways in response to various stressors. We studied the effects of acute and repeated restraint stress on solid gastric emptying and hypothalamic CRF mRNA expression in wild‐type (WT) and OXT‐KO mice. Heterozygous (HZ) parents (B6; 129S‐Oxttm1Wsy/J mice) were bred in our animal facility. Male OXT‐KO, WT and HZ littermates were used for the study. Solid gastric emptying was measured following acute restraint stress (for 90 min) or repeated restraint stress (for five consecutive days). Expression of CRF mRNA in the paraventricular nucleus (PVN) was measured by real‐time reverse transcriptase‐polymerase chain reaction. There were no significant differences of gastric emptying in WT (68.4 ± 4.1%, n = 6), HZ (71.8 ± 3.1%, n = 6) and OXT‐KO (70.6 ± 3.1%, n = 6) mice in nonstressed conditions. Acute stress significantly delayed gastric emptying in OXT‐KO mice (33.10 ± 2.5%, n = 6) WT (39.1 ± 1.1%, n = 6) and HZ mice (35.8 ± 1.2%, n = 6). Following repeated restraint stress loading, gastric emptying was significantly restored in WT (68.3 ± 4.5%, n = 6) and HZ mice (63.1 ± 2.6%, n = 6). By contrast, gastric emptying was still delayed in OXT‐KO mice (34.7 ± 1.3%, n = 6) following repeated restraint stress. The increase in CRF mRNA expression at the PVN was much pronounced in OXT‐KO mice compared to WT or HZ mice following repeated restraint stress. These findings suggest that central oxytocin plays a pivotal role in mediating the adaptation mechanism following repeated restraint stress in mice.

Impaired adaptation of gastrointestinal motility following chronic stress in maternally separated rats

Exposure to early life stress causes increased stress responsiveness and permanent changes in the central nervous system. We recently showed that delayed gastric emptying (GE) and accelerated colonic transit (CT) in response to acute restraint stress (ARS) were completely restored following chronic homotypic stress (CHS) in rats via upregulation of hypothalamic oxytocin (OXT) expression. However, it is unknown whether early life stress affects hypothalamic OXT circuits and gastrointestinal motor function. Neonatal rats were subjected to maternal separation (MS) for 180 min/day for 2 wk. Anxiety-like behaviors were evaluated by the elevated-plus-maze test. GE and CT were measured under nonstressed (NS), ARS, and CHS conditions. Expression of corticotropin-releasing factor (CRF) and OXT in the paraventricular nucleus (PVN) of the hypothalamus was evaluated by real time RT-PCR and immunohistochemistry. MS increased anxiety-like behaviors. ARS delayed GE and accelerated CT in control and MS rats. After CHS, delayed GE and accelerated CT were restored in control, but not MS, rats. CRF mRNA expression was significantly increased in response to ARS in control and MS rats. Increased CRF mRNA expression was still observed following CHS in MS, but not control, rats. In response to CHS, OXT mRNA expression was significantly increased in control, but not MS, rats. The number of OXT-immunoreactive cells was increased following CHS in the magnocellular part of the PVN in control, but not MS, rats. MS impairs the adaptation response of gastrointestinal motility following CHS. The mechanism of the impaired adaptation involves downregulation of OXT and upregulation of CRF in the hypothalamus in MS rats.

An epigenetic map of age-associated autosomal loci in northern European families at high risk for the metabolic syndrome

BackgroundThe prevalence of chronic diseases such as cancer, type 2 diabetes, metabolic syndrome (MetS), and cardiovascular disease increases with age in all populations. Epigenetic features are hypothesized to play important roles in the pathophysiology of age-associated diseases, but a map of these markers is lacking. We searched for genome-wide age-associated methylation signatures in peripheral blood of individuals at high risks for MetS by profiling 485,000 CpG sites in 192 individuals of Northern European ancestry using the Illumina HM450 array. Subjects (ages 6–85 years) were part of seven extended families, and 73% of adults and 32% of children were overweight or obese.ResultsWe found 22,122 genome-wide significant age-associated CpG sites (Pα=0.05 = 3.65 × 10−7 after correction for multiple testing) of which 14,155 are positively associated with age while 7,967 are negatively associated. By applying a positional density-based clustering algorithm, we generated a map of epigenetic ‘hot-spots’ of age-associated genomic segments, which include 290 age-associated differentially methylated CpG clusters (aDMCs), of which 207 are positively associated with age. Gene/pathway enrichment analyses were performed on these clusters using FatiGO. Genes localized to both the positively (n = 241) and negatively (n = 16) age-associated clusters are significantly enriched in specific KEGG pathways and GO terms. The most significantly enriched pathways are the hedgehog signaling pathway (adjusted P = 3.96 × 10−3) and maturity-onset diabetes of the young (MODY) (adjusted P = 6.26 × 10−3) in the positive aDMCs and type I diabetes mellitus (adjusted P = 3.69 × 10−7) in the negative aDMCs. We also identified several epigenetic loci whose age-associated change rates differ between subjects diagnosed with MetS and those without.ConclusionWe conclude that in a family cohort at high risk for MetS, age-associated epigenetic features enrich in biological pathways important for determining the fate of fat cells and for insulin production. We also observe that several genes known to be related to MetS show differential epigenetic response to age in individuals with and without MetS.

Hypothalamic circuit regulating colonic transit following chronic stress in rats

Although acute stress accelerates colonic transit, the effect of chronic stress on colonic transit remains unclear. In this study, rats received repeated restraint stress (chronic homotypic stress) or various types of stress (chronic heterotypic stress) for 5 and 7 days, respectively. Vehicle saline, oxytocin (OXT), OXT receptor antagonist or corticotropin-releasing factor (CRF) receptor antagonists were administered by intracerebroventricular (ICV) injection prior to restraint stress for 90 min. Immediately after the stress exposure, the entire colon was removed and the geometric center (GC) of Na51CrO4 (a nonabsorbable radioactive marker; 0.5 μCi) distribution was calculated to measure the transit. Gene expression of OXT and CRF in the paraventricular nucleus (PVN) was evaluated by in situ hybridization. Accelerated colonic transit with the acute stressor was no longer observed following chronic homotypic stress. This restored colonic transit was reversed by ICV injection of an OXT antagonist. In contrast, chronic heterotypic stress significantly accelerated colonic transit, which was attenuated by ICV injection of OXT and by a CRF receptor 1 antagonist. OXT mRNA expression in the PVN was significantly increased following chronic homotypic stress, but not chronic heterotypic stress. However, CRF mRNA expression in the PVN was significantly increased following acute and chronic heterotypic stress, but not chronic homotypic stress. These results indicate that central OXT and CRF play a pivotal role in mediating the colonic dysmotility following chronic stress in rats.