The Leading Role of Peptide Tyrosine Tyrosine in Glycemic Control After Roux-en-Y Gastric Bypass in Rats
Roux-en-Y gastric bypass (RYGB) is one of the most effective surgical therapies for the rapid resolution of type 2 diabetes. However, the mechanisms underlying the entero-hormonal response after surgery and the role of peptide tyrosine tyrosine (PYY) in the restoration of normoglycemia are still not clear.
We reproduced the RYGB technique in Wistar and Goto–Kakizaki rats and performed serum hormonal, histological, and hormonal-infusion test.
Using the diabetic Goto–Kakizaki (GK) rat model, we demonstrated that PYY plasma levels showed a remarkable peak approximately 30 min earlier than GLP-1 or GIP after mixed-meal administration in RYGB-operated rats with PYY. The GLP-1 and GIP areas under the curve (AUCs) increased after RYGB in GK rats. Additionally, the findings suggested that PYY (3-36) infusion led to increased GLP-1 and GIP plasma levels close to those obtained after a meal. Finally, the number of GLP-1-positive cells appeared to increase in the three segments of the small intestine in GK-RYGB-operated rats beyond the early presence of nutrient stimulation in the ileum. Nevertheless, PYY-positive cell numbers appeared to increase only in the ileum.
At least in rats, these data demonstrate an earlier essential role for PYY in gut hormone regulation after RYGB. We understand that PYY contributes to GLP-1 and GIP release and there must be the existence of enteroendocrine communication routes between the distal and proximal small intestine.
KeywordsEnteroinsular axis Insulin secretion Peptide hormones Peptide tyrosine tyrosine Bariatric surgery Glucagon-like peptide-1
The effect of bariatric surgery on type 2 diabetes (T2DM) has generated considerable attention from the research community. Roux-en-Y gastric bypass (RYGB) is known as one of the most effective methods for maintaining body weight and improving glycemic control compared to medical therapy alone , but the underlying mechanism that leads to T2DM resolution remains controversial.
We now know that RYGB promotes substantial changes in the secretion of gut hormones, such as gastric inhibitory polypeptide (GIP) or mainly glucagon-like peptide-1 (GLP-1), an incretin hormone released postprandially by L cells from the ileum into the bloodstream. GLP-1 has been suggested to be an important contributor to insulin secretion and glucose tolerance and is key to explaining glycemic control improvement after RYGB [2, 3, 4].
However, some authors have reported enhanced glucose control after RYGB in a mouse model of functional GLP-1 and GLP-1 receptor deficiency, suggesting a GLP-1-independent mechanism for glycemic control improvement [5, 6]. In addition, notable increases in plasma peptide tyrosine tyrosine (PYY) levels have been reported after RYGB in rats and humans [7, 8]. Like GLP-1, PYY is an incretin hormone found in L cells in the mucosa of the ileum . Total plasma PYY is a mix of PYY (1-36) and the active endocrine isoform PYY (3-36), obtained by NH2-terminal PYY (1-36) residue cleavage by dipeptidyl peptidase-4 (DPP-4) . It was recently demonstrated that PYY (3-36) can modulate the insulin secretory response to glucose in the pancreas and restore glucose-induced suppression of glucagon secretion [7, 11]. Furthermore, PYY (3-36) can improve insulin sensitivity by increasing glucose uptake in adipose tissue and muscle in rodents  and slowing gastric emptying . It has also been reported to increase hepatoportal GLP-1 plasma levels after PYY (3-36) injection. This increase is probably due to neuropeptide Y2 receptor (NPY2R) activation in the small intestine . NPY2R activation has also shown marked postoperative glucose tolerance improvement and increased PYY plasma levels in wild-type mice after enterogastric anastomosis (EGA) but not in PYY-null mice .
These data and the revelation of enteroendocrine communication routes between the distal and proximal small intestine by a serial block-face scanning electron microscopy technique have indicated the presence of neuropods from L cells. These axon-like basal processes ended in synapse-like appositions to the enteric nervous system . These communication routes and the data reported previously led us to think about PYY as a possible new key to explaining glycemic control improvement after RYGB.
The aim of this study was to determine the contribution of PYY to glycemic control improvement after RYGB in a Goto–Kakizaki (GK) diabetic rat model by analyzing its ability to trigger changes in the gut hormone expression pattern.
Research Design and Methods
All animal procedures were approved by the Committee for Ethical Use and Care of Experimental Animals at Cadiz University. Thirty-six male GK rats and six male Wistar rats weighing 200–220 g, at an age of 10–11 weeks, were provided and kept at the Experimentation and Animal Production Service of University of Cadiz (SEPA). Female rats were not used to avoid the cyclic variations in gonadotropins and their effect on glycemic metabolism.
All animal procedures were performed with the approval of the University of Cadiz Committee for the Ethical Use and Care of Experimental Animals. This committee ensured that the procedures in all experiments were performed in accordance with international relevant guidelines and regulations of animal welfare. The internal Committee for the Ethical Use and Care of Experimental Animals followed the instructions marked for the Autonomous Andalusian Authority.
For hormonal and histological studies, 18 GK rats and 6 Wistar rats were randomly divided into four groups: n = 6 Wistar (Wistar); n = 6 Goto–Kakizaki (GK); n = 6 GK sham-operated (GK-sham); n = 6 GK RYGB-operated (GK-RYGB). Animals were kept for 12 weeks from surgery to sacrifice.
For the PYY (3-36) infusion assay, another 18 male GK rats were kept for 4 weeks after RYGB surgery. Rats were randomly divided into three groups: n = 6 GK saline-infused rats (RYGB-GK + vehicle); n = 6 GK PYY (3-36)-infused rats (RYGB-GK + PYY (3-36)); n = 6 GK mixed-meal-fed rats (RYGB-GK + meal). Mixed meal was prepared combining 0.5 g dextrose/ml 0.092 g vegetable oil/ml, and 0.125 nitrogen casein/ml in purified water .
The RYGB and sham groups were subjected to a pre- and postsurgical 12-hour fasting period. Briefly, to describe RYGB, animals were anesthetized with continuous infusion of 3% v/v isoflurane (Isoflo; Abbott 571329.8, Madrid). A laparotomy of 3 cm was made in the midline of the abdomen, and a gastric pouch with a volume of approximately 30% of the normal gastric volume was created. The remnant gastric fundus was anastomosed to the jejunum at 14 cm distal from the ligament of Treitz. The abdominal muscular and skin layers were closed in one layer using a continuous suturing technique [17, 18].
For the sham operation, animals were anesthetized, and a 3-cm incision was made in the midline of the abdomen. The jejunum was transected 40 cm distal to the angle of Treitz, and terminus–terminus anastomosis was performed. Abdominal layers were closed as above.
Sacrifice and Tissue Preparation
Animals were sacrificed 12 weeks after surgery by isoflurane inhalation overdose. Tissues were immediately removed, and 1 cm full-thickness segments of the duodenum, jejunum, and ileum were harvested and fixed in Bouin’s solution overnight at 4 °C. Later, the samples were dehydrated, embedded in paraffin, and cut into serial 10 μm microtome sections for immunostaining.
In rehydrated sections of intestine, GLP-1, PYY, and GIP release was analyzed by immunostaining using GLP-1 (1:100 rabbit Abcam ab22625, Cambridge, UK), PYY (1:500 rabbit Abcam ab22663, Cambridge, UK), and GIP (1:100 rabbit Abcam ab202792, Cambridge, UK) primary antibodies. The secondary antibody used was Alexa 488 anti-rabbit (1:250; Molecular Probes Inc. USA). DAPI was used to counterstain nuclei. To determine the positive cell fraction, the number of GLP-1-, PYY-, or GIP-positive cells and intestinal total areas were quantified in 10 fields per condition. The results were noted under randomized conditions by a single investigator and expressed as the number of GLP-1-, PYY-, or GIP-positive cells/mm2 of intestine.
Oral Glucose Tolerance Test (OGTT) and Insulin Measurement
Three weeks after surgery, an oral glucose tolerance test (OGTT) was performed in Wistar, GK-RYGB, GK, and GK-sham 12-h fasting rats. A 20% w/vd-glucose solution (2 g/kg) was administered through gavage, and glycemia was measured by a Glucocard G-Meter 1810 glucometer (Menarini Diagnostics, Italy) in blood samples obtained from the rat tails at 0, 30, 60, 90, and 120 min after glucose solution administration .
Three weeks after surgery, after a 12-hour fasting period, insulin measurement was performed in blood samples from the rat tails every 10 min for 60 min after glucose solution administration using an ELISA kit (ALPCO Diagnostics, Salem, NH).
Hormonal Study and PYY Infusion Assay
Four weeks after surgery, a 4 ml/kg, 13.9 KJ/ml mixed meal was administered to the 12-h fasting rats by oral gavage. Blood samples obtained from the rat tails, every 15 min for 120 min, were added to EDTA tubes containing dipeptidyl peptidase-4 (DPP-4) inhibitor (10 μl/ml blood; Millipore) and centrifuged at 4000 × g for 15 min at 4 °C; the plasma was then removed and frozen at −80 °C. GLP-1 and GIP were assessed by sandwich ELISA kits (Cloud-Clone Corp, USA). PYY was measured using an ELISA kit (ALPCO Diagnostics, Salem, NH) according to the manufacturer’s instructions. The area under the curve (AUC) was calculated by the trapezoidal rule for every parameter in the study.
Three groups of 12-h fasting RYGB rats were included in the PYY intravenous infusions assay 4 weeks after surgery. Rat PYY (3-36) (Bachem, Bubendorf, Switzerland) was dissolved in saline (0.9% NaCl). A dose of 0.6 pmol kg−1 min−1 or vehicle (saline) was infused for 10 min in the first and second groups, and oral mixed meal was administered in the third group. GLP-1 and GIP plasma levels were quantified as above.
Data are presented as the means ± SEM. For AUC, histological and weight gain data analysis, one-way ANOVA followed by Tukey’s/Bonferroni’s post hoc test was conducted using SPSS v21.0 software. Statistical significance was accepted at P < 0.05.
OGTT and Insulin Secretion
Additionally, insulin secretion was analyzed in all the groups 3 weeks after surgery. Similar and higher insulin secretion patterns were shown by Wistar and GK-RYGB rats compared to GK or GK-sham rats (Fig. 2c). The GK-RYGB or Wistar AUCs were significantly higher (P < 0.05) than GK or GK-sham AUCs (Fig. 2d).
We also found intense differences regarding the GLP-1 secretion pattern between Wistar versus GK-RYGB rats and between GK versus GK-sham rats. The maximum GLP-1 plasma values occurred at 60 min after meal administration in Wistar, GK, and GK-sham groups but appeared 15 min later in GK-RYGB rats (Fig. 3c). GLP-1 AUCs were significantly increased in Wistar and GK-RYGB compared to GK and GK-sham groups (P < 0.05) (Fig. 3d).
Finally, GIP plasma levels were tested after mixed-meal administration. No differences were found between the groups in the first part of the assay, but higher GIP plasma levels appeared in the Wistar and GK-RYGB groups with respect to the GK and GK-sham groups in the second part of the assay (Fig. 3e). However, the AUC values of the Wistar and GK-RYGB groups were clearly different from the GK and GK-sham AUC values (P < 0.05) (Fig. 3f).
Twelve weeks after surgery, PYY, GLP-1, and GIP expression was studied in three intestinal segments—the duodenum, jejunum, and ileum of rats from each group—and the obtained values were expressed as the number of incretin-positive cells per square millimeter of the intestinal area.
GLP-1 expression appeared to be increased in the three segments of the small intestine in GK-RYGB rats with respect to the Wistar, GK, and GK-sham groups (P < 0.05) (Fig. 4d–f). A remarkable decrease in GLP-1-positive cells in the ileum was observed in the GK and GK-sham groups of rats (P < 0.01) (Fig. 4f).
Low expression values of GIP-positive cells were found in the duodenum of the GK and GK-sham groups with respect to the Wistar and GK-RYGB groups (P < 0.05) (Fig. 4 g–i). However, no differences were found in the number of GIP-positive cells in the jejunum or ileum between any groups.
PYY (3-36) Infusion Assay
To test the role of PYY in stimulating GIP and GLP-1 secretion, a pool of GK-RYGB rats was randomly divided into three groups 4 weeks after surgery and fasted for 12 h. In this oral test, grouped animals were fed with mixed meal, infused with PYY (3-36), or infused with saline solution. The data showed a normal GLP-1 secretion pattern with a peak 75 min after meal administration in the GK-RYGB+meal group. A slight elevation in the GLP-1 plasma level, not as high as the GK-RYGB+meal group, appeared in the PYY (3-36)-infused rats. No increase in GLP-1 plasma levels was found in the saline-infused rats (Fig. 6a).
Significant differences were found between the GK-RYGB+vehicle AUC versus GK-RYGB+PYY (3-36) AUC (P < 0.05) and GK-RYGB+meal AUC (P < 0.01) (Fig. 6b).
A GIP secretion pattern similar to that described for the GK-RYGB group was found in the GK-RYGB+meal group. The described curve was similar to that described in Fig. 3e for the GK-RYGB group. A similar elevation in GIP plasma level was detected in the second part of the assay (from 60 min after infusion to the end of the test) in the GK-RYGB+PYY group (3-36), but no GIP plasma elevation was found in the GK-RYGB+vehicle rat group (Fig. 6c).
Statistical differences were determined between the AUC of the GK-RYGB+meal group versus the AUC of the GK-RYGB+vehicle group; no differences were found versus the AUC of the GK-RYGB+PYY (3-36) group (P < 0.05) (Fig. 6d).
As presented in the data, a positive effect on glucose homeostasis was observed following RYGB in our model (Fig. 2a, b). This improvement can be attributed to increased insulin secretion (Fig. 2c, d), independent of body weight reduction (Fig. 1). A large number of authors reported variations in incretins GLP-1 and GIP secretion patterns after RYBG. They tried to explain this phenomenon, such as increased GLP-1 secretion, due to rapid delivery of nutrients to the distal bowel and L-cell stimulation [20, 21, 22] or/and suppression of GIP secretion by K cells due to a restricted delivery of nutrients in the duodenum [2, 23]. However, recently, a new actor may play an important role in glycemic regulation. Postprandial increased plasma levels of PYY have been reported in patients after RYGB . PYY may indirectly stimulate postprandial insulin secretion in mice .
We investigated changes in plasma profiles of GLP-1, PYY, and GIP after meal administration in the RYGB group and controls 4 weeks after surgery. Our data revealed that PYY, GLP-1, and GIP AUC after a mixed-nutrient meal were increased postoperatively in the RYGB group, with values higher than or similar to those of the nondiabetic controls (Fig. 3d–f). These findings were consistent with several studies that reported increased postprandial PYY and GLP-1 plasma levels after RYGB [25, 26]. We also observed one peak in RYGB on PYY secretion 15 min after meal administration, while the peaks of GLP-1 and GIP secretion appeared 30 and 25 min later (Fig. 3a, b). As above, the data on GLP-1 and GIP secretion profiles have been supported for a long time by previous studies in rodents and patients, with similar responses to OGTT [27, 28]. However, the early PYY secretion profile in only the RYGB group was notably interesting. Specifically, attending to the effect of PYY (3-36) on neuropeptide Y2 receptor (NPY2R) leads to increased hepatoportal GLP-1 plasma levels and improved glucose tolerance .
In this way, the widespread distribution of NPY2R in the small intestine of rats  and its potential to increase hepatoportal GLP-1 plasma levels  are also acknowledged. Therefore, we analyzed GLP-1, GIP, and PYY expression in the small intestine of our model and found increased PYY expression in the ileum of the RYGB group similar to the nondiabetic controls, but not in the duodenum or jejunum, and increased expression of GIP in the jejunum. Nevertheless, GLP-1 expression appeared to increase in the three areas of the small intestine in the RYGB group (Fig. 4a–i). These data suggested widespread and increased expression of GLP-1, but not GIP, beyond the early presence of nutrient stimulation in the ileum of the RYGB group, which is probably due to other stimulation mechanisms. Some of these mechanisms could be explained by the presence of neuropods found in mice . These neuropods respond as an axon-like basal process containing peptide-secreting vesicles from the PYY-secreting enteroendocrine cells. One possibility is that neuropods contribute to trigger GLP-1 release in the jejunum and even GLP-1 and GIP release in the duodenum, linking the distal and proximal small intestine.
In summary, these data confirm a central role for PYY in glucose homeostasis improvement after RYGB in GK rats. Our experiments revealed a high peak in PYY plasma levels earlier than any other enterohormone after mixed-meal administration. This suggests the possibility that PYY is the first enterohormone to be secreted due to meal presence in the distal intestine. Recently, PYY was shown to trigger GLP-1 and GIP secretion. The PYY local immunohistochemical expression in the ileum and widespread expression of GLP-1 in the small intestine—beyond the place where the meal arrives early—support this thought. Additionally, the test of PYY (3-36) infusion in our model reports similar plasma profiles of GLP-1 and GIP after a meal or PYY (3-36) infusion and confirms our theory.
These findings allow us to think about PYY not only as an incretin that may mediate direct effect on insulin sensitivity  or gastric emptying . As seen in our model, we propose that PYY is the trigger that allows secretion of other important enterohormones, such as GLP-1 or GIP.
Despite numerous efforts, further research is required to answer many more questions regarding PYY endocrine signals and function in a physiological context or after RYGB in animal models and humans.
Acknowledgments and Grant Support
The authors desire to thank the Asociacion Gaditana de Apoyo al Investigador (AGAI) and Sustainable Social Development Research Institute (INDESS) University of Cadiz for economic support.
Authors’ contribution statement
All authors (A.C., G.M.P.A., and J.A.P.) designed the project. G.P.A. and D.A.G. analyzed the statistical procedures. A.C. and J.A.P. performed surgical techniques and followed the animal survival period. G.M.P.A. and A.R.G. performed the histological techniques. All authors participated in manuscript elaboration.
Compliance with ethical standards
Disclosure and Conflict Statement
The authors declare no conflict of interest. All the authors declare to have read and signed the disclosure and conflict statement. We did not receive payment or services from a third party (government, commercial, private foundation, etc.) for any aspect of the submitted work (limited to the grant). We have no financial relationships (regardless of the amount of compensation) with entities that could be related to the aim of the study. We have no patents or manuscripts, pending or issued, broadly relevant to this work. There are no other relationships or activities that readers could perceive to have influenced, or that give the appearance of, or potentially influence, what we have written in the submitted work.
We, the authors, state that the article is original, it has not been submitted for publication in another journal, and it has not yet been published either wholly or in part.
Statement of Animal Welfare
The authors declare that an ethical approval was obtained for our study. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted and ethical approval was obtained from the University of Cadiz Committee for the Ethical Use and Care of Experimental Animals. This committee ensured that the procedures in all experiments were performed according to international guidelines and regulations for the welfare of the animals. The internal Committee for the Ethical Use and Care of Experimental Animals followed the instructions marked for the Autonomous Andalusian Authority.
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