Elsevier

Peptides

Volume 118, August 2019, 170100
Peptides

The preprohormone expression profile of enteroendocrine cells following Roux-en-Y gastric bypass in rats

https://doi.org/10.1016/j.peptides.2019.170100Get rights and content

Highlights

  • Enteroendocrine cell (EEC) transcriptome analysis throughout the rat gut.

  • RYGB markedly changes EEC-specific preprohormone gene expression in the obese rat.

  • 38 known and 16 predicted preprohormone genes were regulated by RYGB.

  • The regulated preprohormones may be targeted for treatment of obesity.

Abstract

Objective

Roux-en-Y gastric bypass (RYGB) leads to rapid remission of type 2 diabetes (T2D) and sustained body weight loss, but the underlying molecular mechanisms are still not fully understood. To further elucidate these mechanisms and identify potentially novel preprohormone encoding genes with anti-diabetic and/or anti-obesity properties, we performed a comprehensive analysis of gene expression changes in enteroendocrine cells after RYGB in diet-induced obese (DIO) rats.

Methods

The mRNA expression profiles of enteroendocrine cell enriched samples were characterized at 9, 22 and 60 days after RYGB surgery in a DIO rat model. Enteroendocrine cells were identified by chromogranin A immunohistochemistry and isolated by laser capture microdissection (LCM) from five regions covering the full rostro-caudal extension of the gastrointestinal (GI) tract. RNA sequencing and bioinformatic analyses were subsequently applied to identify differentially expressed preprohormone encoding genes.

Results

From the analysis of enteroendocrine cell mRNA expression profiles, a total of 54 preprohormones encoding genes were found to be differentially regulated at one or more time-points following RYGB. These included well-known RYGB associated preprohormone genes (e.g. Gcg, Cck, Gip, Pyy and Sct) and less characterized genes with putative metabolic effects (e.g. Nmu, Guca2a, Guca2b, Npw and Adm), but also 16 predicted novel preprohormone genes. Among the list of gene transcripts, Npw, Apln and Fam3d were further validated using in situ mRNA hybridization and corresponding peptides were characterized for acute effects on food intake and glucose tolerance in mice.

Conclusion

We present a comprehensive mRNA expression profile of chromogranin A positive enteroendocrine cells following RYGB in rats. The data provides a region-specific characterization of all regulated preprohormone encoding genes in the rat GI tract including 16 not hitherto known. The comprehensive catalogue of preprohormone expression changes may support our understanding of hormone mediated effects of RYGB on diabetes remission and body weight reduction.

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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