Secretin release after Roux-en-Y gastric bypass reveals a population of glucose-sensitive S cells in distal small intestine

Objectives Gastrointestinal hormones contribute to the beneficial effects of Roux-en-Y gastric bypass surgery (RYGB) on glycemic control. Secretin is secreted from duodenal S cells in response to low luminal pH, but it is unknown whether its secretion is altered after RYGB and if secretin contributes to the postoperative improvement in glycemic control. We hypothesized that secretin secretion increases after RYGB as a result of the diversion of nutrients to more distal parts of the small intestine, and thereby affects islet hormone release. Methods A specific secretin radioimmunoassay was developed, evaluated biochemically, and used to quantify plasma concentrations of secretin in 13 obese individuals before, 1 week after, and 3 months after RYGB. Distribution of secretin and its receptor was assessed by RNA sequencing, mass-spectrometry and in situ hybridization in human and rat tissues. Isolated, perfused rat intestine and pancreas were used to explore the molecular mechanism underlying glucose-induced secretin secretion and to study direct effects of secretin on glucagon, insulin, and somatostatin secretion. Secretin was administered alone or in combination with GLP-1 to non-sedated rats to evaluate effects on glucose regulation. Results Plasma postprandial secretin was more than doubled in humans after RYGB (P < 0.001). The distal small intestine harbored secretin expressing cells in both rats and humans. Glucose increased the secretion of secretin in a sodium-glucose cotransporter dependent manner when administered to the distal part but not into the proximal part of the rat small intestine. Secretin stimulated somatostatin secretion (fold change: 1.59, P < 0.05) from the perfused rat pancreas but affected neither insulin (P = 0.2) nor glucagon (P = 0.97) secretion. When administered to rats in vivo, insulin secretion was attenuated and glucagon secretion increased (P = 0.04), while blood glucose peak time was delayed (from 15 to 45 min) and gastric emptying time prolonged (P = 0.004). Conclusions Glucose-sensing secretin cells located in the distal part of the small intestine may contribute to increased plasma concentrations observed after RYGB. The metabolic role of the distal S cells warrants further studies.


Development and evaluation of a secretin radioimmunoassay
We obtained the following three secretin antisera: 1) ab103619 (Abcam, United Kingdom), 2) LS- Commercially available secretin ELISAs (LS-F27215 (LSBio) and CEB075Hu (Cloud Clone, Texas, USA) were included in our assay evaluation for comparison with our in-house developed assay.
Assays were evaluated according to the CLSI (Clinical and Laboratory Standards Institute) guidelines for Immunoassay (I/LA23-A, I/LA21-A2, and EP24-A) as previously described (2).
Recovery in percent was calculated as the measured concentration in the individual, spiked plasma sample minus the concentration in the corresponding non-spiked plasma sample divided by the theoretical spiked concentration of peptide and multiplied by 100.
Specificity was analyzed from recovery experiments. Known amounts of each peptide (5-300 pmol/L) were added to separate aliquots of healthy human plasma (a reserve plasma pool from a previous study (1) was used). One aliquot was measured in duplicate, using two separate assay runs or kits for each of the two commercial ELISAs.
Sensitivity was estimated by determining the lowest concentrations of added peptide that could be measured as being significantly different from zero (by paired analysis of three duplicate determinations) using a plasma pool spiked with 0, 1, 2, 5 pmol/L of human secretin. Precision was determined from multiple measurements of identical samples.
For all kits, the manufacturers' instructions were followed closely, including recommendations for sample preparation by extraction (purification columns and buffers).

Peptide extraction in rat tissue biopsies
The biopsies were cleaned in PBS and snap frozen on dry ice before storage at -80°C. Peptides were extracted using 1% trifluoroacetic acid (cat no. TS-28904, Thermo Fisher Scientific, USA) and homogenized with a bead mill (TissueLyzer, Qiagen Instruments AG, Switzerland) and a 5 mm steel bead. Peptides were purified using tc18 cartridges (cat no. 036810 Waters, USA). Before analysis of secretin and GLP-1 peptide content, samples were reconstituted in 1 ml assay buffer (80 mmol/L phosphate buffer, 10 mmol/L EDTA, 0.6 mM Thimerosal (cat no. T-5125, Sigma Aldrich, Denmark), 0.1% Human Serum Albumin (cat no. 12666, Merck KgaA, Germany), pH 7.5). Peptide content was normalized to tissue weight (pmol/g).

Immunohistochemistry
Tissue were paraffin-embedded on a HistoStar embedding workstation (Thermo Fisher Scientific, Massachusetts, USA), cut on a HM355S automatic microtome (Thermo Fisher Scientific, Massachusetts, USA) (4 μm) and mounted on cover slides. Next, the slides were heated at 60°C for 60 minutes to remove the paraffin and placed in a TissueClear bath for 10 min followed by several baths at decreasing ethanol concentrations, from 99-70%, to hydrate the samples. Heat-induced The venous effluent was collected for 1 min periods via the draining catheter using a fraction collector. The samples were immediately put on ice and stored at -20°C until analysis.

In situ hybridization
Pancreases were excised from non-fasted rats and transferred to fresh 4% paraformaldehyde (PFA), PBS for 24h at room temperature. Tissue was then transferred to 70% ethanol followed by infiltration (Shandon Excelsior, Thermo Fisher), and embedded in paraffin blocks. Sections of 4 μm were cut using a microtome (RM2125, Leica) and mounted on slides (superfrost Plus Slides, Thermo Scientific). The slides were baked at 60 °C for 1h and placed in xylene and ethanol for dewaxing. The in situ hybridization was performed using mRNA specific probes (Supplementary Table 3) following the manufacturers' manual of a commercially available in situ hybridization kit, RNAscope 2.5 HD detection (Cat.no. 322360, Advanced Cell Diagnostics, Milan, Italy) in combination with immunofluorescence. After the in situ hybridization procedure, the slides were incubated with blocking buffer (2 % BSA, PBS for 10 minutes) followed by incubation with primary insulin, glucagon or somatostatin antibody in blocking buffer (Supplementary Table 2) over night at 4 °C.
The day after the slides were washed and incubated with Alexafluor 488 conjugated secondary antibodies for 1 hour at room temperature (Supplementary Table 2). Coverslips were then mounted with ProLong Gold 32 Antifade Mountant with DAPI (P-3693 I, Thermofisher). The slides were analyzed using a Zeiss Widefield fluorescence microscope and AxioCam 506 mono camera or a Zeiss Axio Scan.Z1 Slide Scanner and AxioCam MRm camera.