LEPTIN-MELANOCORTIN PATHWAY VARIANTS AND GASTRIC EMPTYING IN PATIENTS WITH OBESITY

Increased intestinal bile acid (BA) absorption mediated by Apical sodium-dependent BA cotransporter (ASBT) is known to play a vital role in the onset and progression of obesity and associated disorders. Normally, ASBT is only present in the distal ileal villus cell brush border membrane (BBM). However, in Zucker rat model of obesity, ASBT is increased at three levels: villus cell BBM, along the crypt-villus axis and more proximally along the caudal oral length of the small intestine. This increased de novo expression of ASBT in the obese intestine can lead to increased transepithelial transport of BA and thus increased BA in the enterohepatic circulation during obesity. This was further substantiated by increased luminal BA pools in the OZR small intestine. However, the molecular mechanism of increased regional small intestinal expression of ASBT and its regulatory proteins to facilitate net increases in BA absorption in obesity is not known. Hypothesis: ASBT expression and expression of genes that are known to regulate ASBT will be induced/altered in proximal small intestine. Aim: Determine the expression of ASBT regulatory proteins along the proximal length of the obese small intestine. Methods: Starting from the end of the ileum going caudal-orally, five intestinal segments of equal length (S1-S5) were separated from LZR and OZR. Intestinal mucosa obtained from these segments were used for total RNA extraction and Real-time Quantitative PCR (RT-qPCR). Results: ASBT mRNA expression progressively decreased caudal-orally in both LZR and OZR, as expected, but was significantly higher in all the small intestinal segments in OZR. Moreover, FXR that is known to regulate ASBT at the level of the distal ileal cells in obesity, also increased significantly in all the proximal jejunal segments compared to its corresponding LZR intestinal segments. The expression of GATA4 that is known to repress ASBT expression, was absent in the distal ileum (S1) in both LZR and OZR. Moreover, its mRNA expression steadily increased along the proximal length of the LZR and OZR small intestine. However, this steady increase was significantly reduced in the comparative obese proximal intestinal segments S2, S3, S4 and S5. Finally, the expressions of BA activated G-protein-coupled BA receptor TGR5 (Gpbar1) and S1PR2 (Sphingosine-1-Phosphate Receptor 2) were unaltered in segments S1-S4 but were significantly increased in obese proximal small intestine (S5) (all experiments n=3, p<0.05). Conclusions: In the obese intestine, caudal oral increased ASBT expression is likely mediated by altered expressions of GATA4 and/or FXR and BA activated G-protein coupled receptors along the proximal length of obese small intestine. This increased expression of ASBT will contribute to the increase in the net absorption of BA, thus facilitating rapid onset and progression of obesity.

Background: Gastric emptying (GE) controls the rate and timing of food emptying and is a critical mediator of postprandial satiety and food intake regulation. Accelerated GE is a trait seen in human obesity. Mutations in the hypothalamic leptin-melanocortin 4 receptor (Leptin-MC4R) pathway have been linked to severe monogenic obesity. However, their influence on GE as a trait is unknown. We sought to investigate the association of leptin-MC4R pathway variants on GE in patients with obesity.
Methods: This is a cross-sectional study of patients with a history of severe obesity genotyped as part of the Mayo Clinic Biobank (n=3,000) study and had completed a GE test up to May 2022 (n=99) to assess differences in GE between carriers of a heterozygous variant in the leptin-MC4R pathway and non-carriers. Patients with bariatric surgery where excluded (n=4). GE was measured by scintigraphy after a standard 320kcal, 30% fat meal consisting of two 99mTc-radiolabeled eggs, toast, and 80 ml of milk. We evaluated GE% at 2 hours between both groups using ANCOVA with weight and age as covariates. We subdivided patients into carriers based on the location of the identified variants (i.e., upstream and downstream) and compared with non-carriers using ANOVA. Results are presented as mean and standard deviation (± SD). Statistical significance was set at 2-sided p < 0.05.
Results: A total of 95 subjects were included, 9 carriers (67% females; 39.78 + 12.33 years; BMI: 49.14 + 12.96 kg/m²) were compared with 86 non-carriers (87% female; 49.98 + 13.74 years; BMI: 40.75 + 6.29 kg/m²) (Table 1). At 2 hours, carriers had a delayed gastric emptying when compared to non-carriers (45.44 + 20.52% vs. 60.44% + 16.22%; p=0.047) (Fig 1A) with a significant effect of carrier status (β =7.11 + 3.21 min; p=0.02). At 4 hours, carriers persistently showed a delayed gastric emptying when compared to non-carriers (77.22 + 21.94% vs. 90% + 12.43%; p=0.046) (Fig 1A) with a significant effect of carrier status (β =7.42 + 2.63 min; p=0.005). In carriers, we identified variants in 4 genes upstream to the MC4R (LEPR, PCSK1, POMC, and SRC1), and 2 downstream (SIM1 and MC4R). When comparing upstream carriers vs. downstream carriers vs. non-carriers by location there was a significant difference among groups at 2 hours (50.16 + 23.11 vs. 33.33 + 8.08 vs. 60.44 + 16.22; p= 0.02) (Table 1, fig 1B), and, at 4 hours (80.50 + 23.3 vs. 69.33 + 21.03 vs. 90 + 12.43; p=0.01) (Table 1, fig 1B).
Conclusion: Carriers of heterozygous mutations in the leptin-melanocortin pathway had a delayed GE compared to non-carriers. These findings warrants further studies to determine the association between GE and leptin-melanocortin pathway variants in patients with obesity.