Somatostatin is key to explaining the long-term effect of anatomical rearrangements after sleeve gastrectomy on glucose metabolism

doi:10.21203/rs.3.rs-1975494/v1

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Somatostatin is key to explaining the long-term effect of anatomical rearrangements after sleeve gastrectomy on glucose metabolism

https://doi.org/10.21203/rs.3.rs-1975494/v1

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Background. Sleeve gastrectomy (SG) is one of the most commonly performed bariatric surgeries is able to treat diabetes (T2DM) better than many drugs. The mechanisms that underlie this phenomenon remains unclear. We propose that somatostatin (SST), isoforms, SST-14 and SST-28, are key to explaining the pathophysiological mechanisms behind T2DM improvement after SG.

Methods. We underwent surgeries on three groups of Wistar rats -fasting (FC), surgery control (Sham), and SG groups-. We measured plasma levels of glucose, insulin, SST-14 and SS-28 at several weeks after surgery, islet somatostatin receptor (SSTR) and cell populations at short and long-term after surgery. We performed a Pasireotide (SST-28 analogue) infusion assay in another group of rats to confirm the influence of SST-28 plasma levels on the delta-cell population.

Results. We found an elevation of the insulin response after surgery in SG animals but a decrease in insulin response in the long-term with a loss of beta cell mass. An increase in duodenal SST-28-producing cells in the duodenum and a loss of pancreatic SST-14-producing cells was measured after SG but not in controls. The expression of SSTR-5 in delta-cell populations from every group and the ability of the Pasireotide infusion assay to decrease the delta-cell population indicate the effect of SST-28 plasma levels on delta-cell maintenance.

Conclusion. After SG begins with a compensatory response in the duodenum, the depletion of beta cell mass after losing the brake that constitutes SST-14 at the paracrine level. This process may explain the percentage of T2DM relapse after SG.

Pancreas

Sleeve gastrectomy

Type 2 Diabetes mellitus

Bariatric surgeries

Somatostatin.

Sleeve gastrectomy surgery (SG) has become one of the most powerful tools for diabetes mellitus type 2 (T2DM) management, improving glycaemia and insulin resistance faster and better than pharmacological treatments ^1,2. The underlying mechanisms that produce these satisfactory outcomes remain unclear. Many articles have been published proposing ghrelin expression loss and low plasma ghrelin levels as the key to explaining fast glucose improvement in humans after SG ³. Most of the stomach ghrelin-producing cells in SG are remove. However, new studies have shown epsilon ghrelin-producing cell expansion in the rat pancreas after SG as a physiopathological response to stomach ghrelin-producing cell ablation, even showing increased plasma ghrelin levels ⁴. These findings could help to explain the 41.6% rate of diabetes relapse in patients five years after SG surgery ⁵. In addition, a recent article demonstrated long-term beta cell mass loss and insulin secretion failure after SG in Wistar rats ⁶. Contrary to most of the studies supporting the stimulation of insulin secretion and beta-cell mass maintenance by SG at least in the medium and short term ^7,8, this leads us to a more complex landscape where many other mechanisms appear to be involved.

Accordingly, we searched for an agent affected by SG surgery with a stronger effect on the beta cell population. Somatostatin (SST), a potent inhibitory hormone, appears to be a candidate. SST is secreted by several tissues, such as the duodenal mucosa, endocrine pancreas, hypothalamus, and gastric mucosa ^{9, 10, 11}. There are two active isoforms, SST-14 and SST-28, the last one prevailing in plasma. SST-14 is a strong paracrine inhibitory agent for beta-cell insulin secretion ^{12, 13}; it acts in human beta-cells mainly through somatostatin receptor type 2 (SSTR2) ¹⁴. Paradoxically, a high SST content was observed in the pancreas of diabetic Goto-Kakizaki (GK) rats ¹⁵, and new protective roles on beta-cell mass have been described for SST as a modulator of beta-cell response to stress ¹⁶. Interestingly, mice and human delta cells express a different and exclusive SST receptor, SSTR5. This receptor has a high affinity for SST-28, greater than for SST-14, and it is involved in the inhibition of cell growth processes ^17,18. SSTR5 is even being proposed as a therapeutic target in some neoplasms ¹⁹.

On the other hand, it has been known for a long time that the majority of circulating SST corresponds to the SST-28 fraction secreted by the upper digestive tract ^{20, 21, 22}. The plasticity of the duodenal SST-producing cell population under certain situations, such as some pharmacological treatments or ageing, is known ^{22, 23}. High duodenal SST plasma levels have been reported after total gastrectomy in animal models ²⁴.

These data lead us to hypothesize that SST is a key element in explaining SG outcomes. The objective of this study was to determine the role of the different somatostatin isoforms in the pathophysiological phenomena that lead to the resolution of diabetes after SG surgery in a healthy animal model. The study aim was also to give a mechanistic explanation to the episodes of hypoglycaemia and relapse in diabetes in the middle and long-term after surgery.

Animal procedures were approved by the Committee for Ethical Use and Care of experimental animals at Cadiz University. Thirty-six male Wistar rats and 15 additional 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 discarded to avoid cyclic variations in gonadotropins and their effect on glycaemic metabolism.

Experimental protocol:

Wistar rats were randomly divided into three groups: n = 12 in the Fasting control group (FG); n = 12 for the Sham-operated (Sham) group; and n = 12 for the Sleeve Gastrectomy-operated (SG). In each group, half of the animals (n = 6) were humanely killed 12 weeks after surgery, and the remaining animals in each group (n = 6) were humanely killed 24 weeks after surgery. The FG group animals (n = 12) underwent the same perioperative fasting periods, related to the surgical protocol. All the animals finished 12 h presurgical and 12 h postsurgical fasting periods. An intake readaptation period followed surgery to normalize fasting and a weight measurement every three days from surgery to the fourth week of survival were performed.

Fifteen additional rats were randomly divided into three groups, n = 5, infused with saline (control group); n = 5, infused with octreotide (octreotide group); and n = 5, SG-operated (SG group). These rats were humanely killed 12 weeks later for delta-cell population measurement.

Surgical procedures

Surgical procedures were performed in anaesthetized animals using continuous infusion of isoflurane 2–4% V/V (Abbott Columbus, OH, USA). The sham technique (Sham) (n = 12) reproduced the surgical injury over the stomach, but maintained its integrity. Sham was performed by a laparotomy of 5 cm in the upper third of the abdomen and an incision of approximately 3 cm in the middle area of the abdomen, sectioning the gastrosplenic ligament and exposing the stomach.

Sleeve gastrectomy (SG) (n = 12) was performed by a laparotomy of 5 cm in the upper third of the abdomen through sectioning of the gastrosplenic ligament and exposing the stomach, but this time, curved forceps were applied from the angle of Hiss to the antrum, performing a cylindrical stomach of approximately 0.5 cm in diameter. The stomach sectioned resected the most fundus, stomach-corpus at greater curvature and antrum. The pylorus was preserved. SG reduced the initial stomach volume by approximately 20%.

Oral glucose tolerance test (OGTT) and insulin measurement

Four, 11, and 23 weeks after surgery, an oral glucose tolerance test (OGTT) was performed in FG, sham, and SG rats after a 12 hour fast. A 2 g/kg, 20% w/v d-glucose solution was administered through gavage, and glycaemia was measured by a Glucocard G-Metre 1810 glucometer (Menarini Diagnostics, Italy) in blood samples obtained from the rat tails at 0, 30, 60, 90, and 120 minutes after glucose solution administration. The results are expressed as glucose mg/plasma dl.

Additionally, at four, 11, and 23 weeks after surgery, insulin measurement was performed in blood samples from the rat tails every 15 minutes for 60 minutes after glucose solution administration 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.

Plasma somatostatin measurement

At four and 23 weeks after surgery, a 4 ml/kg, 13.9 KJ/ml mixed-meal was administered to the 12-hour fasting rats by oral gavage. From FC, Sham and SG rats, blood samples obtained from the rat tails, every 15 minutes for 120 minutes. The samples were added to EDTA and centrifuged at 4000 x g for 15 minutes at 4°C; the plasma was then removed and stored at − 80°C. Total-SST, SST-14, and SST-28 plasma fractions were assessed by sandwich ELISA kits (MyBioSource Inc, San Diego CA, USA), (BMA-Biomedicals, CH-4302, Augst, Switzerland) according to the manufacturer’s instructions. SST values obtained for plasma SST are expressed as pg/ml. The total SST area under the curve (AUC) was calculated by the trapezoidal rule in the study.

Pasireotide assay

To perform the pasireotide infusion assay, an additional n = 18 rats were randomly divided into three groups. In the first group (Pasireotide), n = 6 rats were infused with 25 µg/kg body weight of Pasireotide (Signifor), an SST-28 analogue that preferentially binds to SSTR-5 rather than SSTR-2 (Novartis Europharm Limited. Elm Park, 4 Dublin, Ireland) every 12 hours for 30 days using an implanted catheter connected to an external miniature port (Instech Lab Inc. Plymouth Meeting, PA USA). In the second group, (Saline) n = 6 rats were infused with saline for the equivalent period using the same system. The last group (SG), n = 6 rats, was SG-operated as described above and animals were treated with the same protocol. Thirty days after surgery, the all groups animals were killed to study pancreatic delta-cell populations in the same way as in the other experimental groups described below.

Sacrifice and tissue preparations

The animals that were not involved in the pasireotide assay were killed 23 weeks after surgery by isoflurane inhalation overdose. The duodenum, hypothalamus, and pancreas were immediately removed and fixed in Bouin’s solution overnight at 4°C. The samples were dehydrated, embedded in paraffin and cut into serial 8 µm microtome sections.

Tissue immunostaining

In rehydrated sections of the duodenum, pancreas, and hypothalamus, SST-producing cell populations were analysed by immunostaining using rabbit anti-somatostatin IgG antibody (Abcam, Cambridge, CB4 OFL, UK) and mouse anti-insulin IgG antibody (Sigma Aldrich St Louis MO USA). The proliferation marker Ki67 and the differentiation markers Pax 6 and Nkx2.2 were also analysed using rabbit anti-ki67, rabbit anti-Pax6 and rabbit anti Nkx2.2 antibodies (Abcam, Cambridge, CB4 OFL, UK) and mouse anti-insulin IgG antibody (Sigma Aldrich, St Louis MO, USA).

The fluorescent secondary antibodies were Alexa 488 and 546 (Molecular Probes Inc. Eugene, OR USA). DAPI was used to counterstain the nuclei. The SST-positive cell number was measured in four slices of each type of tissue per condition and expressed as the number of SST-positive cells/mm² of tissue or islets in the pancreas.

The Ki67-, Pax6- and Nkx2.2-positive cell numbers were evaluated in four slices of the whole pancreas per condition and expressed as the number of ki67-, Pax6- or Nkx2.2-positive cells/mm² in the pancreas.

SSTR-2 and SSTR-5 expression in islets was analysed in four slices of whole pancreas per condition by immunostaining using rabbit anti-SSTR-2 IgG antibody (Invitrogen Corp. Carlsbad, CA, USA) and mouse anti-insulin IgG antibody (Sigma Aldrich, St Louis MO, USA); the secondary antibodies were Alexa 546 and 488, respectively (Molecular Probes Inc. Eugene, OR, USA). DAPI was used to counterstain the nuclei.

Every histological parameter was measured and noted by a single investigator using a fluorescence microscope with a digital camera and the image analysis Cell-D software (Olympus GmbH, Hamburg, Germany).

Statistical analysis

Data are presented as the means ± SEM. For secretion patterns, AUC and histological data analysis, one-way ANOVA followed by Tukey's/Bonferroni post hoc test was conducted using SPSS V21.0 software. The correlation between SSTR-2 expression and insulin AUC was determined by Pearson´s coefficient value. Statistical significance was accepted at P < 0.05 (*, #) or P < 0.01 (**, ##).

OGTT and insulin secretion measurement

At four, 11, and 23 weeks after surgery, OGTT was performed on the FC, sham and SG groups. No difference appeared between the glucose tolerance patterns of the FC, sham, and SG groups at four or 11 weeks (Fig. 1A, 1C). However, increased plasma glucose levels were observed in SG but not in control rats at 30 and 60 minutes after glucose administration at 23 weeks after surgery (Fig. 1E). The glucose area under the curve (AUC) was analysed for the three groups three times after surgery. Similar values were observed for FC, Sham, and SG AUC values at four or 11 weeks (Fig. 1B and 1D). In contrast, significantly higher AUC values appeared in SG rats with respect to FC rats and sham rats by 23 weeks of survival (Fig. 1F).

Insulin secretion was also tested after the OGTT at four, 11 and 23 weeks after surgery. Modified insulin secretion patterns appeared in SG rats with respect to controls at 11 and 23 weeks. An elevated insulin secretion pattern was shown by SG rats with respect to FC or Sham at eleventh week (Fig. 2C). However, it fell with respect to FC and Sham insulin secretion pattern at the end of the study 23 weeks after surgery (Fig. 2E). Insulin secretion AUCs were tested in each group. No differences were observed between the FC, Sham and SG groups at the fourth week (Fig. 2B), but very high AUC insulin values were found 11 weeks after surgery for SG rats. Decreased AUC insulin values were obtained for SG rats with respect to the controls at 23 weeks (Fig. 2F).

Hormonal study

To test changes in plasma somatostatin (SST) levels, total SST, SST-14 and SST-28 plasma fractions were assayed after oral mixed-meal administration in the three study groups four and 23 weeks after surgery. As Fig. 3A and 3D shows, a high total SST secretion pattern was observed after mixed-meal administration in SG rats but not in FC or sham groups at the two time points under study. Plasma total SST AUC values were increased in SG rats compared with controls at the fourth and twenty-third weeks (Figs. 3B and 3E).

SST-14 and SST-28 plasma fractions were analysed. As Figs. 3C and 3F show, the observed levels of circulating SST-14 were significantly lower than those of SST-28 and even lower in the SG group than in the controls at the twelfth and twenty-third weeks of the study.

Histological study. SST-producing cell expression. Endocrine-cell lineage marker study

Twelve and 24 weeks after surgery, SST expression was studied by immunostaining in three tissue samples from the duodenum, hypothalamus and pancreas of rats from each group. Expressing values were obtained as the number of SST-positive cells/mm² of each tissue area.

As Figs. 4B and 4E show, an increased number of SST-positive cells appeared in the duodenum from SG but not in FC or Sham rats, at weeks 12 and 24 of the study. In contrast, a low number of SST-positive cells were found in endocrine pancreas samples from SG at any time (Figs. 4C and 4F). No difference in SST-producing cell expression was observed between groups in the hypothalamus samples at any time (Figs. 4A and 4D).

Nkx2.2 and Pax6 identity marker delta-cell population expression was measured by immunostaining in pancreas samples from FC, sham and SG rats at 12 and 24 weeks after surgery. Significant and enhanced expression of Pax6 was observed in SG samples but not in control samples at all times (Fig. 4H). A significant high expression of the Nkx2.2 marker was registered in SG rats with respect to controls but only at the twenty-fourth week (Fig. 4I).

Relationship between SSTR-2 expression in the beta-cell population and insulin response

The AUC at 11 weeks postsurgery was correlated with the level of SSTR-2 receptor expression in the beta-cell population from animals at 12 weeks postsurgery. We observed a strong inverse correlation between the two parameters, as Figs. 5A, 5B and 5C show. Surprisingly, in the SG group, SSTR-2 expression almost doubled in 50% of the animals. This coincided with those with a lower insulin response as shown in figure and Table 5C. This did not occur in the sham or FC groups (Figures Tables 5A and 5B).

SSTR5 expression and pasireotide infusion assay

The expression of SSTR5 was tested in beta and delta-cell populations from FC, sham and SG rats at the two time points of study. In the delta cell population, a higher expression of the receptor for somatostatin type 5 was found than in beta cells (Figs. 5D, 5E and 5F). These differences were significant in the three groups at both 12 and 24 weeks after surgery P < 0.05 (*) or P < 0.01 (**).

To determine the role of SST-28 in the pancreatic delta-cell population, animals were infused with saline (control group), pasireotide (Signifor, an SST-28 analogue -pasireotide group-) or SG-operated (SG group). The delta-cell population showed a decreased delta-cell population in pasireotide-infused rats compared with saline-infused rats (P < 0.05) but less than that caused by SG (Fig. 5G).

Many publications have been written about the mechanisms underlying the improvement of T2DM after SG. We offer a new explanation for the changes in carbohydrate metabolism that lead to the resolution of T2DM after SG. We performed SG on healthy Wistar rats and explored the short- and long-term effects of SG on glucose metabolism and the SST-producing cell population of the enteropancreatic axis. We found significant changes in the plasma total SST profile after mixed meal administration at four and 23 weeks after surgery in SG rats, as shown in Figs. 3A and 3B. However, the source of the plasma SST elevation was a mystery due to the presence of somatostatin-producing cells in several tissues, such as the hypothalamus, pancreas or gut ^{9, 10, 11}.

We investigated circulating SST composition and an elevated range of SST-28 was found but not of SST-14 (Figs. 3C and 3F). Additionally, we analysed SST tissue expression by evaluating the number of SST-producing cells. Surprisingly, an elevated number of SST-producing cells was observed in the duodenum with respect to the hypothalamus and pancreas from SG rats (Figs. 4 and 4E). In addition, a low number of SST-producing cells were located in pancreas samples from SG rats with respect to the control groups (Figs. 4C and 4F). These data are consistent with those published who proposed the main participation of SST-28 from the duodenum in total postprandial SST circulating levels ^{20, 21}, presenting a compensatory mechanism once gastric SST-producing cells are removed after SG.

More interesting was the low number of SST-producing cells located in the pancreas if we attended to SST-14 paracrine beta-cell inhibition ^12,13. This situation could lead to an increase or alteration in the insulin secretion pattern. Any one significant increase in insulin secretion was found, and normal plasma glucose levels were observed four weeks after SG, but a high plasma insulin level and elevated plasma insulin AUC were observed at 11 weeks. A low secretion was found at 23 weeks (Figs. 2C-2F) according to our previous experiences, probably due to a beta-cell mass loss and an alpha-cell mass increase ⁶.

These data suggest a possible progressive elevation of insulin secretion due to a lack of paracrine inhibition by SST-14-producing delta-cells and a possible mechanism explaining the improvement in blood glucose levels after surgery. This suggestion would agree with observations in GK diabetic rats in which there is a hyperplasia of the delta-cell population as diabetes develops ¹⁵. However, this situation, if sustained, would lead to a long-term depletion of beta-cell mass and a loss of insulin production, as Figs. 2E and 2F show. This hypothesis is supported by the long-term effect of insulin hypersecretion on beta cell mass that leads to exhaustion ²⁵, the positive modulatory effects of low SST concentrations on alpha-cell population expansion through the expression of miRNA-132-3p observed in vitro ²⁶ and the protective paracrine inhibitory effect of SST-14 on insulin production by beta cells ¹⁶. Thus, the lack of the SST-14 brake would be behind the short-term improvement in glycaemia but also the long-term depletion of beta-cell mass and the relapses in T2DM reported five after surgery in almost 41.6% of SG in humans ⁵.

This seems to be a reasonable explanation, but what happens when there is no long-term relapse after surgery? SSTR-2 is the receptor for SST-14 present in the beta cell population ¹⁴. Surprisingly, animals with better long-term insulin values showed higher expression of the receptor in the beta-cell mass (Figs. 5A-5C), what appears to be an adaptive situation to the low levels of SST-14 in the islet as described in other endocrine-lineage cells in response to drops in hormone levels ²⁷. However, it would also be interesting to analyse the functionality of these receptors.

The mechanism that causes the loss of islet delta-cell mass after surgery remains unclear, but the high plasma level of SST found after SG in our model could play a key role. The anti-proliferative effect of SST analogues by inducing cell cycle arrest or apoptosis through protein tyrosine phosphatases in neuroendocrine tumours is well known ²⁸. We tested the delta-cell mass proliferation ratio after SG, but no difference was found between the SG and control groups in our model (Fig. 4G). Regarding the delta-cell population plasticity, we also consider the possibility that sustained high plasma SST-28 levels inhibit delta-cell mass identity. In this sense, SSTR-5, a high affinity receptor for SST-28 ^17,18, was observed on delta cells from SG and control rats (Fig. 5D). Moreover, the ability of delta cells to transdifferentiate into beta cells under stress conditions has been tested ²⁹; we consider sustained hyperinsulinism as an important stress situation ^{25, 30}. Additionally, high expression of beta-cell markers, such as Pax 6 and Nkx2.2, was found in the delta-cell population from the SG group at four and 24 weeks but not in the control groups (Figs. 4H and 4I).

To confirm these findings, three groups of five rats were used. We used pasireotide, an SST-28 analogue with high selective binding to SSTR-5, an SST-28 high affinity receptor present on delta-cells (Fig. 5D). We administered saline or pasireotide to each group or performed SG. The pasireotide group showed a significant delta-cell population decrease with respect to the saline group (control) and was close to that obtained after SG (Fig. 5E).

Thus, a paradoxical situation would arise where high plasma levels of SST-28 would induce a decrease in the islet delta-cell population and paracrine and endocrine levels of SST-14 limiting the SST-14 brake on beta-cell insulin secretion. This theory is supported by the inverse correlation between insulin AUC and SSTR-2 islet expression at 23 weeks after surgery observed in the SG group. This allows a situation of hyperinsulinemia that leads to long-term beta-cell mass exhaustion and depletion after SG and also implies a higher affinity of SSTR-2 present in the beta cell population for SST-14 than for SST-28.

The beta-cell-enhanced SSTR-2 expression seems to be a compensatory mechanism that could allow for reasonable levels of insulin secretion preventing beta-cell depletion and diabetes relapse after surgery sometimes, as we can observe if we analyse Table 5C. This would also explain why a relapse in diabetes does not always appear after SG and the number of hypoglycaemia episodes after SG in those patients without this adaptation.

We are aware that many questions remain to be answered, but this could be a good approach forward to determine the underlying mechanisms causing the effects of SG on glucose metabolism.

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. As a prior communication, 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.

Data availability: All the data supporting the results and critical resources will be available at the institutional repository of the University of Cadiz (http://hdl.handle.net/10498/26564).

Statement of Animal Welfare: The authors declare that 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.

Funding sources: The authors declare the research project was supported by University of Cadiz and Institute for Biomedical Science Research and Innovation (INIBICA).

Acknowledgment: The authors desire to thank Ms. Sofie Winderick for the technical support at the Department of anatomy laboratory.

Authors ´contributions: A Camacho, G Pérez-Arana and JA Prada designed the project and followed the financially the project. D Almorza and J Bancalero analysed the procedures for statistical analysis. J Bancalero, A Camacho, A Ribelles, performed the surgical techniques, followed the animal survival period and performed the functional test. G Pérez-Arana directed and measured the histological techniques. J Bancalero, FJ Visiedo and A Gonzalez analyses the samples with ELISA and histological techniques. All the authors participated in preparing the manuscript.

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No competing interests reported.

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Somatostatin is key to explaining the long-term effect of anatomical rearrangements after sleeve gastrectomy on glucose metabolism

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Abstract

Figures

Background

Research Design And Methods

Experimental protocol:

Surgical procedures

Oral glucose tolerance test (OGTT) and insulin measurement

Plasma somatostatin measurement

Pasireotide assay

Sacrifice and tissue preparations

Tissue immunostaining

Statistical analysis

Results

OGTT and insulin secretion measurement

Hormonal study

Histological study. SST-producing cell expression. Endocrine-cell lineage marker study

Relationship between SSTR-2 expression in the beta-cell population and insulin response

SSTR5 expression and pasireotide infusion assay

Discussion

Conclussion

Declarations

References

Additional Declarations

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