Differential Pre-mRNA Splicing Regulates Nnat Isoforms in the Hypothalamus after Gastric Bypass Surgery in Mice

Background Neuronatin (NNAT) is an endoplasmic reticulum proteolipid implicated in intracellular signalling. Nnat is highly-expressed in the hypothalamus, where it is acutely regulated by nutrients and leptin. Nnat pre-mRNA is differentially spliced to create Nnat-α and -β isoforms. Genetic variation of NNAT is associated with severe obesity. Currently, little is known about the long-term regulation of Nnat. Methods Expression of Nnat isoforms were examined in the hypothalamus of mice in response to acute fast/feed, chronic caloric restriction, diet-induced obesity and modified gastric bypass surgery. Nnat expression was assessed in the central nervous system and gastrointestinal tissues. RTqPCR was used to determine isoform-specific expression of Nnat mRNA. Results Hypothalamic expression of both Nnat isoforms was comparably decreased by overnight and 24-h fasting. Nnat expression was unaltered in diet-induced obesity, or subsequent switch to a calorie restricted diet. Nnat isoforms showed differential expression in the hypothalamus but not brainstem after bypass surgery. Hypothalamic Nnat-β expression was significantly reduced after bypass compared with sham surgery (P = 0.003), and was positively correlated with post-operative weight-loss (R2 = 0.38, P = 0.01). In contrast, Nnat-α expression was not suppressed after bypass surgery (P = 0.19), and expression did not correlate with reduction in weight after surgery (R2 = 0.06, P = 0.34). Hypothalamic expression of Nnat-β correlated weakly with circulating leptin, but neither isoform correlated with fasting gut hormone levels post- surgery. Nnat expression was detected in brainstem, brown-adipose tissue, stomach and small intestine. Conclusions Nnat expression in hypothalamus is regulated by short-term nutrient availability, but unaltered by diet-induced obesity or calorie restriction. While Nnat isoforms in the hypothalamus are co-ordinately regulated by acute nutrient supply, after modified gastric bypass surgery Nnat isoforms show differential expression. These results raise the possibility that in the radically altered nutrient and hormonal milieu created by bypass surgery, resultant differential splicing of Nnat pre-mRNA may contribute to weight-loss

Serum leptin level and microvascular complications in type 2 diabetes

Background. Type 2 diabetes (T2DM) and its complications are highly prevalent in Egypt and are considered a major health problem. Insulin resistance arising from visceral obesity is the main pathological mechanism of T2DM. Leptin is an adipokine secreted from visceral adipose tissue and its level is proved to be higher in patients with T2DM, but its association with microvascular complications is not yet well-established, for this aim the present study was conducted. Methods. This cross-sectional study was conducted among 120 participants with T2DM recruited from the diabetes outpatient clinic of Alexandria Main Uni­versity Hospital, Alexandria, Egypt. Each participant was subjected to full history taking, complete physical examination and laboratory investigations. Results. Serum leptin level was significantly positively correlated with diabetes duration, BMI, WC, systolic and diastolic blood pressure, FPG, HbA1c, serum insu­lin level, HOMA-IR, total cholesterol, triglycerides and LDL-C. Regarding microvascular complications, serum leptin level was highly significantly positively correlated with UACR, peripheral neuropathy and retinopathy (P < 0.001) and significantly negatively correlated with e-GFR (P = 0.003). Conclusions. Serum leptin level is significantly correlated with microvascular complications in patients with T2DM in Alexandria, Egypt

Evaluation of the Genetic Association Between Adult Obesity and Neuropsychiatric Disease

Extreme obesity (EO, BMI>50) is frequently associated with neuropsychiatric disease (NPD). As both EO and NPD are heritable central nervous system disorders, we assessed the prevalence of protein truncating (PTV) and copy number variants (CNV) in genes/regions previously implicated in NPD, in adults with EO (n=149) referred for weight loss/bariatric surgery. We also assessed the prevalence of CNVs in patients referred to University College London Hospital (UCLH) with EO (n=218) and obesity (O, BMI 35-50, n=374) and a Swedish cohort of participants from the community with predominantly O (n=161). The prevalence of variants was compared to controls in ExAC/gnomAd database. In the discovery cohort (high NPD prevalence: 77%), the cumulative PTV/CNV allele frequency (AF) was 7.7 % vs 2.6% in controls (Odds Ratio (OR) 3.1, (95% CI 2-4.1, p<0.0001). In the UCLH EO cohort (intermediate NPD prevalence: 47%), CNV AF (1.8% vs 0.9% in controls, OR 1.95, 95% CI 0.96-3.93, p=0.06) was lower than the discovery cohort. CNV AF was not increased in the UCLH O cohort (0.8%). No CNVs were identified in the Swedish cohort with no NPD. These findings suggest PTV/CNVs, in genes/regions previously associated with NPD, may contribute to NPD in patients with EO

Insights into obesity from bariatric surgery & genetics

Bariatric surgery is the most effective treatment for the management of severe obesity. In Chapters 3 & 4 of this thesis, in two prospective parallel-group studies we compared the effects of the ‘gold-standard’ operation Roux-en-Y gastric bypass (RYGBP) vs. the newer, increasingly performed procedure, sleeve gastrectomy (SG). We showed that in adiposity-matched patients RYGB and SG resulted in comparable reductions in circulating leptin levels at 6 and 12 weeks post-surgery. However, RYGB and SG had differential effects on circulating gut hormones levels. Plasma acyl-ghrelin levels declined post-surgery, with superior decreases observed post-SG vs. post-RYGBP. Markedly increased meal-stimulated circulating levels of peptide YY3-36 (PYY3-36), active glucagon-like peptitde-1 (GLP-1) and glucagon were observed following RYGB and SG. However, these changes were significantly greater after RYGB compared to after SG. Both operations comparably enhanced circulating glucose-dependent insulinotropic peptide (GIP) early post-meal. Whereas late postmeal, GIP levels declined post-RYGBP, but were unchanged post-SG. Glucose, insulin and homeostasis model assessment for insulin resistance (HOMA-IR) changes were comparable following RYGBP and SG. Polymorphisms within the fat mass and obesity-associated gene (FTO) associate with adiposity. We have recently shown that normal-weight subjects homozygous for the rs9939609 FTO obesity-risk variant (A) display increased post-prandial appetite and attenuated reduction in plasma acyl-ghrelin levels. In Chapter 5 we extended this work to humans with severe obesity. We found no differences in acyl-ghrelin between obese AA and TT subjects (T being the protective allele of FTO rs9939609). Furthermore, in Chapter 6 we report that AA, AT and TT subjects exhibited comparable weight-loss post-RYGBP, with comparable weight-loss outcomes across the three genotypes seen post-SG. However, comparison of RYGBP vs. SG revealed superior weight-loss outcomes post-RYGBP in TT and AT subjects vs. SG, but comparable weight-loss post-RYGBP and post-SG in AA subjects. In Chapter 6 we report that overall RYGBP resulted in superior weight-loss vs. SG. Patients with type 2 diabetes (T2DM) exhibited comparable weight-loss vs. adipositymatched normoglycaemic patients following RYGBP. However, following SG weight-loss was greater in normoglycaemic patients compared to adiposity-matched T2DM patients. In addition, we report the prevalence of 32 common obesityassociated SNPs (single nucleotide polymorphisms) in our patient cohort, and show that FTO rs9939609 had the strongest effect on BMI. Collectively, this work further adds to the rapidly expanding field of bariatric research. Future research endeavors will bring us closer to developing less invasive surgical procedures and novel pharmacotherapies for the medical management of diabetes and obesity

Contribution of 32 GWAS-identified common variants to severe obesity in European adults referred for bariatric surgery

The prevalence of severe obesity, defined as body mass index (BMI) ≥35.0 kg/m2, is rising rapidly. Given the disproportionately high health burden and healthcare costs associated with this condition, understanding the underlying aetiology, including predisposing genetic factors, is a biomedical research priority. Previous studies have suggested that severe obesity represents an extreme tail of the population BMI variation, reflecting shared genetic factors operating across the spectrum. Here, we sought to determine whether a panel of 32 known common obesity-susceptibility variants contribute to severe obesity in patients (n = 1,003, mean BMI 48.4±8.1 kg/m2) attending bariatric surgery clinics in two European centres. We examined the effects of these 32 common variants on obesity risk and BMI, both as individual markers and in combination as a genetic risk score, in a comparison with normal-weight controls (n = 1,809, BMI 18.0–24.9 kg/m2); an approach which, to our knowledge, has not been previously undertaken in the setting of a bariatric clinic. We found strong associations with severe obesity for SNP rs9939609 within the FTO gene (P = 9.3×10−8) and SNP rs2815752 near the NEGR1 gene (P = 3.6×10−4), and directionally consistent nominal associations (P&lt;0.05) for 12 other SNPs. The genetic risk score associated with severe obesity (P = 8.3×10−11) but, within the bariatric cohort, this score did not associate with BMI itself (P = 0.264). Our results show significant effects of individual BMI-associated common variants within a relatively small sample size of bariatric patients. Furthermore, the burden of such low-penetrant risk alleles contributes to severe obesity in this population. Our findings support that severe obesity observed in bariatric patients represents an extreme tail of the population BMI variation. Moreover, future genetic studies focused on bariatric patients may provide valuable insights into the pathogenesis of obesity at a population level