A Glucocorticoid Receptor Gene Marker Is Associated with Abdominal Obesity,Leptin, and Dysregulation of the Hypothalamic‐Pituitary‐Adrenal Axis

Objective: Abdominal obesity has a key role in the pathogenesis of prevalent and serious diseases and has been shown to be associated with an altered hypothalamic‐pituitary‐adrenal (HPA) axis function, which is regulated by endocrine feedback mediated via hippocampal glucocorticoid receptors (GR). Research Methods and Procedures: We examined the HPA axis function by repeated salivary samples for the assessment of cortisol, as well as other endocrine, anthropometric, metabolic, and circulatory variables in middle‐aged Swedish men (n = 284). With the restriction enzyme BclI, variants of the GR gene (GRL) locus were identified and two alleles with fragment lengths of 4.5 and 2.3 kilobases (kb) were detected. Results: The observed frequencies were 40.1% for the 2.3‐ and 2.3‐kb, 46.2% for the 4.5‐ and 2.3‐kb, and 13.7% for the 4.5‐ and 4.5‐kb genotypes. The larger allele (4.5 and 4.5 kb) was associated with elevated body mass index (BMI; p < 0.001), waist‐to‐hip circumference ratio (p = 0.015), abdominal sagittal diameter (p = 0.002), leptin (p < 0.001), and systolic blood pressure (borderline, p = 0.058). The 4.5‐ and 4.5‐kb allele was associated with leptin after adjustment for BMI. Moreover, salivary cortisol values, particularly after stimulation by a standardized lunch (p = 0.040 to 0.086), were elevated in the men with the larger allele. Discussion: These results indicate that there is an association between a deficient GR function, defined as a poor feedback regulation of the HPA axis activity, and a polymorphic restriction site at the GR gene locus. An abnormal control of HPA axis function due to genetic alterations may contribute to the pathogenesis of abdominal obesity.     

Effect of Smoking on the Paradox of High Waist‐to‐Hip Ratio and Low Body Mass Index

Objective: Research on diabetes mellitus (DM) indicates that people with a low body mass index (BMI) but a high waist‐to‐hip ratio (WHR) are in a particularly high‐risk group. The purpose of this study was to investigate the prevalence of and the effect of smoking on this paradoxical relationship. Research Methods and Procedures: Our study sample consisted of 3450 men and 4250 women who had participated in the Korean Nationwide Health Examination Survey. We divided the study sample into tertiles (low, medium, and high), according to the level of WHR and of BMI, which yielded nine different combinations. Individuals exhibiting so‐called paradox A had the highest WHR and the lowest BMI. Results: The prevalence of paradox A was 4.7% for men and 3.8% for women. The overall agreement of WHR and BMI groups was poor [for men: κ = 0.31 and 95% confidence interval (CI) = 0.29 to 0.34; for women: κ = 0.39 and 95% CI = 0.37 to 0.42 for women]. The odds ratios for subjects having paradox A were estimated using a logistic regression model after adjusting for age, age², height, education, smoking, use of alcohol, and exercise. The risk for paradox A among current smokers was 2.1‐fold (95% CI, 1.5 to 3.0) higher for men and 2.5‐fold (95% CI, 1.6 to 3.9) higher for women than for nonsmokers, after adjusting for age and covariates. Discussion: Cigarette smoking may increase the risk of paradox A. The findings of this study should be crossvalidated to different populations.     

Usefulness of Anthropometry and DXA in Predicting Intra‐abdominal Fat in Obese Men and Women

Objective: To investigate the usefulness of anthropometry and DXA in predicting intra‐abdominal fat (IAF) in obese men and women. Research Methods and Procedures: Observational, cross sectional study of 22 women and 18 men with a body mass index of 30 or above. IAF from 20 cm above and 10 cm below the L4 to L5 intervertebral disc was measured by magnetic resonance imaging (MRI) as a reference method. Central abdominal fat was measured from the upper border of L2 to the lower border of L4 by DXA. Waist and hip circumferences were also measured. Results: In obese women DXA, waist circumference and waist‐hip ratio were equally well correlated with IAF (r = 0.74, 0.75, and 0.70, respectively). In obese men DXA was moderately correlated with IAF measured by MRI (r = 0.46), whereas waist circumference and waist‐hip ratio were not significantly correlated with IAF. Discussion: The prediction of IAF in obese subjects was highly dependent on sex more than in non‐obese persons. Anthropometry and DXA were equally useful in obese women, whereas anthropometry had no predictive power and DXA was the only acceptable predictor of IAF in obese men.     

Comparison of Thermogenic Sympathetic Response to Food Intake between Obese and Non‐obese Young Women

Objective: Sympathetic nervous system abnormality in humans is still a matter of debate. The present study was designed to examine diet‐induced autonomic nervous system activity and metabolic change in obese and non‐obese young women. Research Methods and Procedures: Sixteen age‐ and height‐matched obese and non‐obese young women participated in this study. Sympathovagal activities were assessed by means of our newly developed spectral analysis procedure of heart‐rate variability during the resting condition and after mixed‐food ingestion (480 kcal). Energy expenditure was also measured under these two conditions. Results: There was no significant difference in any of the parameters of the heart‐rate variability between the obese group and control group during the resting condition. In the control group, both absolute values (221.5 ± 54.5 vs. 363.8 ± 43.7 ms², p < 0.05) and relative values (0.23 ± 0.03 to 0.36 ± 0.02, p < 0.05) of a very‐low‐frequency component and global sympathetic nervous system index (1.46 ± 0.19 vs. 3.26 ± 0.61, p < 0.05) were significantly increased after mixed‐food ingestion compared with the values obtained after resting condition. However, no such sympathetic response was found in the obese group. Energy expenditure increased in the two groups after the meal, but the magnitude of the increase above the preprandial resting condition was significantly greater in the control group than in the obese group (11.2 ± 2.3 vs. 6.7 ± 0.8%, p < 0.05). Discussion: Our data suggest that despite identical sympathovagal activities at the resting condition, obese young women may possess a reduced sympathetic response to physiological perturbation such as mixed food intake, which might be related to lowered capacity of thermogenesis and the state of obesity.     

Autonomic Nervous System Activity and the State and Development of Obesity in Japanese School Children

Objective: The autonomic nervous system (ANS) plays an important role in regulating energy expenditure and body fat content; however, the extent to which the ANS contributes to pediatric obesity remains inconclusive. The aim of this study was to evaluate whether sympathetic and/or the parasympathetic nerve activities were altered in an obese pediatric population. We further examined a physiological association between the duration of obesity and the sympatho‐vagal activities to scrutinize the nature of ANS alteration as a possible etiologic factor of childhood obesity. Research Methods and Procedures: Forty‐two obese and 42 non‐obese healthy sedentary school children were carefully selected from 1080 participants initially recruited to this study. The two groups were matched in age, gender, and height. The clinical records of physical characteristics and development of the obese children were retrospectively reviewed to investigate the onset and progression of obesity. The ANS activities were assessed during a resting condition by means of heart rate variability power spectral analysis, which enables us to identify separate frequency components, i.e., total power (TP), low‐frequency (LF) power, and high‐frequency (HF) power. The spectral powers were then logarithmically transformed for statistical testing. Results: The obese children demonstrated a significantly lower TP (6.77 ± 0.12 vs. 7.11 ± 0.04 ln ms², p < 0.05), LF power (6.16 ± 0.12 vs. 6.42 ± 0.05 ln ms², p < 0.05), and HF power (5.84 ± 0.15 vs. 6.34 ± 0.07 ln ms², p < 0.01) compared with the non‐obese children. A partial correlation analysis revealed that the LF and HF powers among 42 obese children were negatively associated with the duration of obesity independent of age (LF: partial r = ?0.55, p < 0.001; HF: partial r = ?0.40, p < 0.01). The obese children were further subdivided into two groups based on the length of their obesity. All three spectral powers were significantly reduced in the obese group with obesity of >3 years (n = 18) compared to the group with obesity of <3 years. Discussion: Our data indicate that obese children possess reduced sympathetic as well as parasympathetic nerve activities. Such autonomic depression, which is associated with the duration of obesity, could be a physiological factor promoting the state and development of obesity. These findings further imply that preventing and treating obesity beginning in the childhood years could be an urgent and crucial pediatric public health issue.     

Waist Circumference as the Best Predictor of Noninsulin Dependent Diabetes Mellitus (NIDDM) Compared to Body Mass Index,Waist/hip Ratio and Other Anthropometric Measurements in Mexican Americans—A 7-Year Prospective Study

Although a number of obesity-related variables are recognized risk factors for NIDDM, few studies have addressed which one is the best predictor. A cohort of 721 Mexican Americans aged 25–64 years who were free of NIDDM at baseline were followed for an average of 7.2 years; 105 new cases of NIDDM were diagnosed. Body weight, body mass index (BMI), waist and hip circumferences, waist/hip ratio (WHR), triceps and subscapular skinfolds were all positively predictive of NIDDM independent of age and sex. There were modest to strong correlations between these anthropometric variables, however, waist circumference was the strongest predictor of NIDDM. The predictive power of a single measurement of waist circumference was at least equal to that of WHR and BMI combined. The risk of NIDDM for those in the highest quartile of waist circumference was 11 times greater than for those in the lowest quartile (95% confidence interval: 4.2–28.8). The waist-NIDDM relation was stronger in subjects with BMI ≤ 27 kg/m² (OR: 6.0 for a 1 SD difference) than in subjects with BMI > 27 kg/m2 (OR: 1.7 for a 1 SD difference). In multivariate analysis, waist circumference was the only significant predictor of NIDDM in models that included other anthropometric variables either separately or simultaneously. WHR and BMI were independent predictors of NIDDM after adjustment for each other, however, their predictive abilities disappeared after adjustment for waist circumference. The data indicate that waist is the best obesity-related predictor of NIDDM. This finding suggests that the distribution of body fat, especially abdominal localization, is a more important determinant than the total amount of body fat of the development of NIDDM in Mexican Americans.     

Relation of Nodal expression to the specification of the dorsal‐ventral axis and tissue patterning in the starfish Patiria pectinifera

In this study, we attempted to reveal fundamental aspects of starfish embryogenesis, particularly embryonic axis specification or determination, in Patiria pectinifera. We first cloned PpNodal, which is known to play an important role in the specification of the embryonic axis in a wide range of animals, and studied its expression profile. PpNodal expression was first detected at the mid‐blastula stage and showed a single peak around the onset of gastrulation. These features of Nodal expression were shifted to later stages by several hours, compared with those of sea urchin embryos. After the gastrulation started, the expression level became gradually lowered up to the early bipinnaria stage, while the expression level became drastically lowered in sea urchin embryos during gastrulation. The localized Nodal expression in the presumptive oral region was not observed in starfish embryos, unlike in sea urchin embryos. Furthermore, SB431542, an inhibitor of Nodal receptor, did not affect the formation of the DV axis, although it caused the loss of left‐right asymmetry. In contrast to this, SB525334, a specific inhibitor of TGF‐beta receptor, caused the complete loss of the DV axis. Thus, the usage of signaling molecules during early embryogenesis likely varies among echinoderm classes.     

The influence of starvation and natural refeeding on the rate of triacylglycerol/fatty acid substrate cycling in brown adipose tissue and different white adipose sites of the ratin vivo. The role of insulin and the sympathetic nervous system

Triacylglycerol/fatty acid substrate cycling was measuredin vivo in brown adipose tissue (BAT) and white adipose tissue (WAT) of fed, starved and refed rats. Starvation (24 h) significantly decreased the rate of cycling in BAT, and refeeding chow diet led to a rapid, 6-fold increase in cycling. Cycling rate in WAT was much lower than in BAT, and was not influenced by fasting or refeeding. Similar rates of cycling were found in epididymal, mesenteric, subcutaneous, and scapular WAT depots. Sympathetic denervation of interscapular BAT abolished the response of the tissue to refeeding, as did acute suppression of insulin secretion. Similarly, rats fasted for 3 days showed no acute increase in the activity of the cycle following refeeding.     

The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

Simple embryo injection of long single‐stranded donor templates with the CRISPR/Cas9 system leads to homology‐directed repair in Xenopus tropicalis and Xenopus laevis

We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology‐directed repair (HDR)‐mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single‐stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte‐specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell‐specific manner of slc45a2.L with sfGFP (X. laevis).     

High‐efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system

The base‐editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum‐Base Editor 3 (GhBE3) base‐editing system has been developed to create single‐base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32‐GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an ‘editing window’, approximately six‐nucleotide windows of ?17 to ?12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off‐target sites predicted by CRISPR‐P and Cas‐OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole‐genome sequencing analyses on two GhCLA‐edited and one wild‐type plants with about 100× depth showed that no bona fide off‐target mutations were detectable from 1500 predicted potential off‐target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.