EGFP-tagged AdipoR1 protein expression and signaling function in MIN6 cells.

<p>(A) Representative fluorescence images of MIN6 cells transfected with empty vector (control), AdipoR1 or EGFP-tagged AdipoR1 (AdipoR1-EGFP) vectors. The scale bars represent the indicated dimensions. (B) Western blot analysis of cytosolic and membrane fractions of MIN6 cells transfected with control, AdipoR1or AdipoR1-EGFP vectors using anti-AdipoR1 antibody. Expected protein sizes are AdipoR1 (43 kDa) and AdipoR1-EGFP (70 kDa). (C) Akt phosphorylation in MIN6 cells transfected with control, AdipoR1 or AdipoR1-EGFP vectors. MIN6 cells were stimulated with 5μg/ml fAd in serum-free medium after transfections. Representative Western blots for Akt (60 kDa), phospho-Akt (Ser⁴⁷³) (60 kDa) and vinculin (124 kDa), as a loading control, are shown (left panel). Quantified values represent the ratios (compared with control) of phospho-Akt/Akt (right panel). Data are represented as mean ± SEM of four independent experiments. *p < 0.05, by ANOVA followed by Tukey’s honestly significant difference test compared with control.</p

Growth curves and blood glucose of AdipoR1/Akita, AdipoR1, Akita and WT mice.

<p>Growth curves and blood glucose levels of AdipoR1/Akita (n = 14), Akita (n = 11), AdipoR1 (n = 12), and WT (n = 10) mice. (A) Time course of body weight changes of mice between 4 and 10 weeks of age. Blood glucose after (B) an Ad lib feed and (C) a 16 h fast in mice between 4 and 10 weeks of age. Values are expressed as mean ± SEM. There were no significant differences between AdipoR1/Akita and Akita mice, nor between AdipoR1 and WT mice, whereas there were significant differences between AdipoR1/Akita vs AdipoR1 (* p<0.05), and Akita and WT (# p<0.05) mice by ANOVA followed by Tukey’s honestly significant difference test.</p

Pancreatic insulin contents and morphological characteristics of AdipoR1/Akita, AdipoR1, Akita and WT mice.

<p>Pancreatic tissue samples were obtained from AdipoR1/Akita, AdipoR1, Akita and WT mice at 15–20 weeks of age. (A) Pancreatic insulin contents of AdipoR1/Akita (n = 5), Akita (n = 5), AdipoR1 (n = 5) and WT (n = 5) mice. (B) Insulin positive area per islet area in AdipoR1/Akita (n = 5), Akita (n = 5), AdipoR1 (n = 5) and WT (n = 5) mice (upper panel). Quantification was performed on more than 20 islets from each mouse. Representative images of tissues/islet cells immunostained with anti-insulin antibodies (lower panel). Scale bars indicate 50 μm. (C) Representative images of electron micrographs of islet cells in AdipoR1/Akita, AdipoR1, Akita and WT mice. Similar results were obtained from three independent experiments. Scale bars indicate 2 μm. The small boxed areas have been enlarged and are shown in the corresponding large boxes. Arrows indicate insulin secretory granules. Data are shown as mean ± SEM. There were no significant differences between AdipoR1/Akita and Akita, or between AdipoR1 and WT, whereas there was significant differences between AdipoR1/Akita and AdipoR1 (* p<0.05), and between Akita and WT (# p<0.05) by ANOVA followed by Tukey’s honestly significant difference test. NS, Not significant.</p

β-cell specific AdipoR1-EGFP overexpression and signaling functions in AdipoR1 mice.

<p>(A) Pancreatic tissue samples were obtained from AdipoR1 and WT mice at 8 weeks of age. Pancreatic tissue sections were double immunostained for EGFP and insulin. DNA was stained by DAPI. Representative single-channel fluorescence and merged images are shown. Red, green and blue staining correspond to signals for AdipoR1-EGFP, insulin and DNA, respectively. Similar results were obtained from three independent experiments. The scale bars indicate 25 μm. (B) RT-PCR analysis of AdipoR1-EGFP transcripts (1910 bp) in pancreata of WT, AdipoR1 and AdipoR1/Akita mice. RT-PCR products were subjected to 1% agarose gel electrophoresis. β-actin (474 bp) was used as a loading control. (C) AMPK and Akt phosphorylation in WT and AdipoR1 mice islets stimulated with or without 5 μg/ml fAd. Representative Western blots for phospho-AMPK and AMPK (62 kDa), phospho-Akt and Akt (60 kDa), and vinculin (124 kDa) as a loading control, are shown (left panel). Quantified values represent the ratios (compared with control untreated WT mice) of phospho-AMPK/AMPK and phospho-Akt/Akt (right panel). Data are presented as mean ± SEM of three independent experiments. *p < 0.05, by ANOVA followed by Tukey’s honestly significant difference test compared with control mice.</p

Serum adiponectin levels.

<p>Serum adiponectin levels of AdipoR1/Akita (n = 8), AdipoR1 (n = 5), Akita (n = 6) and WT (n = 4) mice at 15–20 weeks of age. Values are expressed as mean ± SEM. *<i>P</i> < 0.05 by ANOVA followed by Tukey’s honestly significant difference test. NS, Not significant.</p

Supplemental material for Rare variants in <i>RNF213</i>, a susceptibility gene for moyamoya disease, are found in patients with pulmonary hypertension and aggravate hypoxia-induced pulmonary hypertension in mice

<p>Supplemental material for Rare variants in <i>RNF213</i>, a susceptibility gene for moyamoya disease, are found in patients with pulmonary hypertension and aggravate hypoxia-induced pulmonary hypertension in mice by Hatasu Kobayashi, Risako Kabata, Hideyuki Kinoshita, Takaaki Morimoto, Koh Ono, Midori Takeda, Jungmi Choi, Hiroko Okuda, Wanyang Liu, Kouji H. Harada, Takeshi Kimura, Shohab Youssefian and Akio Koizumi in Pulmonary Circulation</p

Amounts of the neonicotinoids excreted in urine during a 24 h period before (day 0) and after a single 2 μg dose was ingested (circles) and the model curves (red lines).

<p>The green bars are the means and the lower and upper blue whiskers are the standard deviations.</p

Amounts of the labeled compounds found to be excreted in the urine (μg d⁻¹) in a 24 h period after a single dose was ingested (circles) and the model curves (red lines).

<p>The green bars are the means and the lower and upper blue whiskers are the standard deviations.</p

Deuterium-labeled neonicotinoids used in the dosing study.

<p>Deuterium-labeled neonicotinoids used in the dosing study.</p

Distribution of neonicotinoid excretion rates in healthy adults (μg/d).

<p>The horizontal bars show the frequencies (the <i>x</i>-axis is the number of individual samples in each class). The black line boxes at the center of each graph show the first, second, and third quartiles. The lower whisker indicates the lowest value within the −1.5 interquartile range of the first quartile. The upper whisker indicates the highest value within the +1.5 interquartile range of the third quartile. Outlying values are shown as dots. The blue bars on the right-hand sides of the graphs are the means and standard deviations.</p