Isoform-specific <i>Nnat</i> expression in the hypothalamus after surgery in relation to weight-loss and circulating leptin.

<p>A–B) <i>Nnat</i>-α expression in the hypothalamus did not correlate with either change in body-weight or with circulating leptin after surgery; C–D) by contrast <i>Nnat</i>-β expression showed positive correlation with change in body-weight and weak positive correlation with circulating leptin after surgery; <i>key – Nnat-α expression shown with open squares, Nnat-β with filled circles, AU = arbitrary units where Nnat expression was standardised using ubiquitin (Ubc) as a reference gene</i>.</p

Change in body weight and leptin after chronic dietary switches.

<p>Control = standard dietary chow maintained, Control-HF = standard dietary chow switch to high-fat diet, Control-CR = standard dietary chow switch to caloric restriction; HF-Control = high-fat diet switch to standard dietary chow, HF = high-fat diet maintained, HF-CR = high-fat diet switch to caloric restriction; data presented as P value (post-hoc ANOVA).</p

Expression of <i>Nnat</i> isoforms in response to fasting versus <i>ad-libitum</i> feeding.

<p>A) Hypothalamic <i>Nnat</i>-α and -β showed a non-significant reduction in response to overnight fasting (n = 10) when compared to <i>ad-libitum</i> fed counterparts (n = 10); B) but a significant reduction after 24-h fasting (n = 10) compared to feeding (n = 11), equivalent for both isoforms (**<i>Nnat</i>-α P = 0.005, **<i>Nnat</i>-β P = 0.007); C–D) <i>Nnat</i> isoforms did not show a consistent or significant change after overnight fasting in the stomach or duodenum (n = 10 fasted, n = 10 <i>ad-libitum</i> fed in both tissues); <i>key – AU = arbitrary units where Nnat expression was standardised using ubiquitin (Ubc) as a reference gene</i>.</p

Hypothalamic and brainstem <i>Nnat</i> expression after modified gastric bypass <i>versus</i> sham surgery.

<p>A) <i>Nnat</i>-β showed a significant reduction in the hypothalamus (**P = 0.003) after modified gastric bypass (n = 8) compared to sham surgery (n = 7) whilst <i>Nnat</i>-α did not reduce significantly, consistent with a bypass-specific effect on <i>Nnat</i>-β expression; B) expression of <i>Nnat</i>-α and <i>Nnat</i>-β did not differ in the brainstem after modified gastric bypass (n = 8) compared to sham surgery (n = 7); <i>key – GBP = modified gastric bypass surgery, Sham = sham surgery, AU = arbitrary units where Nnat expression was standardised using an endogenous reference gene (ubiquitin (Ubc) for hypothalamus, hypoxanthine guanine phosphoriboribosyl transferase (Hprt) for brainstem).</i></p

Changes in body-weight, leptin and gut hormones after surgery.

<p>GBP = modified gastric bypass surgery; data presented as Mean (Standard Error of Mean) or P value (<i>t</i> test).</p

Results of our logistic regression analysis were compared with the GIANT-extremes results using combined data from obesity class 2 and 3 groups [11]; in terms of odds ratio (OR) with 95% confidence intervals (CI).

<p>There were no significant differences between the compared OR. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070735#pone-0070735-t002" target="_blank">Table 2</a> for allocated reference numbers of SNPs. The diagonal line represents the expected plotted values for our results, based on the GIANT-extremes results. The SNPs below the diagonal line are those which had a larger effect in our study compared to GIANT-extremes, whereas the SNPs above the diagonal line represent SNPs which had a larger effect in GIANT-extremes compared to our study.</p

Effect sizes (i.e. changes in BMI) within the bariatric cohort, calculated by using standardized BMI values were compared with the known effect sizes derived from inverse standardized BMI values in the GIANT-BMI meta-analysis [10] (A), and by using unstandardized BMI values (B).

<p>Of note, the <i>FTO</i> marker effect size plotted for the GIANT-BMI data relates to the SNP rs1558902 (SNP rs9939609 in our study). There were no statistically significant differences between the compared effect sizes. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070735#pone-0070735-t002" target="_blank">Table 2</a> for allocated reference numbers of SNPs.</p

Baseline demographic and clinical characteristics of bariatric patients.

*<p>Data are shown as mean ± SD.</p>**<p>Defined as presence of ≥1 major cardiovascular risk factor.</p

Association results with <i>FTO</i> SNP rs9939609 and <i>NEGR1</i> SNP rs2815752 in categories of BMI, compared with normal-weight controls.

<p>β, effect size; SE, standard error.</p