The preprohormone expression profile of enteroendocrine cells following Roux-en-Y gastric bypass in rats
Graphical abstract
Introduction
Obesity and type 2 diabetes (T2D) have become a worldwide pandemic. A range of different interventions have been adopted [1] but none of these have shown to be perfect solutions. Lifestyle changes may provide long-term improvements on body-weight and T2D [2,3] but are often met with problems of adherence [4]. Pharmacological interventions for T2D are expensive and encompass life-long medication [5] whereas currently available anti-obesity drugs only lead to moderate weight-loss potentially accompanied by unwanted side-effects [6]. In contrast, bariatric surgeries constitute an effective way to achieve long-lasting, pronounced weight reductions with concomitant improvements in metabolic co-morbidities [7,8]. Accordingly, the development of novel medications that reflects the same clinical outcomes as bariatric surgeries is appealing [9].
Among bariatric surgery procedures [10], Roux-en-Y gastric bypass (RYGB) stands out due to its stable remission of T2D and pronounced, sustainable weight loss [11,12]. However, the underlying mechanisms of RYGB on body weight regulation and glucose homeostasis are not fully elucidated. With recent advances, it is likely that the mechanism is multifaceted, including stomach capacity restriction and intestinal malabsorption [13,14], adaptive enteroendocrine signaling [11,15,16], changes in bile acid secretion [17,18] and modulation of central neuronal circuits [19,20]. Among these mechanisms, the regulation of the enteroendocrine system is believed to play a pivotal role [21] with a large number of preclinical and clinical studies reporting RYGB induced effects on gut-derived hormones, such as glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK) and peptide YY (PYY) [[22], [23], [24], [25], [26]].
With the development of RNA sequencing (RNAseq) technologies [27] it is now possible to perform unbiased global transcriptome analysis enabling a comprehensive detection of transcribed genes with increased sensitivity, specificity and broad dynamic range. Here we used RNAseq to map changes in gut preprohormone gene expression following RYGB in a diet-induced obese (DIO) rat model [28]. The study describes the temporal changes in preprohormone gene expression signatures as compared to sham-operated (SHAM) and sham-operated weight-matched (SHAM W.M.) controls. To capture the diversity and potentially anatomically specific changes in preprohormone gene expression, laser capture microdissection (LCM) was applied to isolate chromogranin A positive enteroendocrine cells (EECs) along the whole rostro-caudal extension of the gastrointestinal (GI) tract.
Section snippets
Surgery
All animal experiments were conducted in accordance with internationally accepted principles for the care and use of laboratory animals. The described experiments were covered by the personal licenses (2013-15-2934-00784) issued by the Danish Committee for Animal Research.
Three (3) cohorts of thirty (30) male Sprague Dawley rats (Taconic, Ejby, Denmark and Janvier-Labs, Route du Genest, France) were included. They were offered ad libitum access to a two-choice diet consisting of a standard chow
Validation of RYGB model
Three cohorts of animals were used to characterize short (9 days), medium (22 days) and long-term (60 days) effects of RYGB surgery in DIO rats. The RYGB procedure led to an approximately 10% body weight loss within the first 9 days progressing to approximately 15% at both day 22 and 60 (Fig. 1A-C). The body weight reduction was mainly related to a marked 40% reduction in fat mass (Fig. 1F, G), whereas changes in lean mass was miniscule (Fig. 1D, E). Aside from a transient drop in energy intake
Discussion
Here we present a comprehensive characterization of enteroendocrine cell (EEC) mRNA expression along the gut following RYGB surgery in rats. Because EECs account for only 1% of all epithelial cells [30] it has historically been a challenge for researchers to get a clear overview of EEC specific transcriptomic changes. Using a combination of laser capture microdissection and immunohistochemistry we specifically isolated chromogranin A positive EECs and investigated preprohormone encoding gene
Conclusion
Taking advantage of LCM and NGS technologies, we present the first complete catalogue of RYGB induced preprohormone gene expression alterations as an important insight into the overall EECs response following RYGB in DIO rats. The data provides a spatial and temporal characterization of 54 regulated preprohormone encoding genes along the rat GI tract, including 16 that are not hitherto known. This comprehensive catalogue will support our understanding of hormone mediated effects of RYGB on
Acknowledgments
The authors would like to thank Farida Sahebzadeh and Lotte Handgaard Jørgensen for excellent technical assistance. The research was sponsored by Sanofi-Aventis Deutschland GmbH.
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