# Identification of Pappalysin-2 (PAPP-A2), a modulator of Insulin-like Growth Factor-1 pathway, as a potential marker of teduglutide efficacy in patients with short bowel syndrome

Brune de Dreuille, Rémy Nicolle, Jérôme Cros, Dominique Cazals-Hatem, Anaïs Chassac, Nicolas Poté, Johanne Le Beyec-Le Bihan, André Bado, Maude Le Gall, Francisca Joly

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Brune de Dreuille, Rémy Nicolle, Jérôme Cros, Dominique Cazals-Hatem, Anaïs Chassac, et al. Identification of Pappalysin-2 (PAPP-A2), a modulator of Insulin-like Growth Factor-1 pathway, as a potential marker of teduglutide efficacy in patients with short bowel syndrome. Clinical Nutrition, 2025, 50, pp.128-136. 10.1016/j.clnu.2025.05.006. inserm-05337506

HAL Id: inserm-05337506

https://inserm.hal.science/inserm-05337506v1

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Original article

# Identification of Pappalysin-2 (PAPP-A2), a modulator of Insulin-like Growth Factor-1 pathway, as a potential marker of teduglutide efficacy in patients with short bowel syndrome

<figure><img src="image_2.png" /></figure>

Brune de Dreuille<sup>a, b</sup>, Rémy Nicolle<sup>a</sup>, Jérôme Cros<sup>a, c</sup>, Dominique Cazals-Hatem<sup>c</sup>, Anaïs Chassac<sup>d</sup>, Nicolas Poté<sup>d</sup>, Johanne Le Beyec-Le Bihan<sup>a, e</sup>, André Bado<sup>a</sup>, Maude Le Gall<sup>a</sup>, Francisca Joly<sup>a, b, *</sup>

## ARTICLE INFO

**Article history:**
Received 5 December 2024
Accepted 12 May 2025

**Keywords:**
Short bowel syndrome
Teduglutide
GLP-2
PAPP-A2

## SUMMARY

**Background:** Teduglutide, a glucagon-like peptide-2 (GLP-2) analog, is indicated to treat short bowel syndrome (SBS) since 2015. It has been shown to reduce parenteral support (PS) in SBS patients, although patients' response is quite heterogeneous. The exact mechanisms of action of GLP-2 on intestinal cells are still poorly understood. The aim of this study was to explore the intestinal action of teduglutide to identify molecular mechanisms underlying response heterogeneity.

**Methods:** A retrospective study was conducted in 39 SBS patients treated with teduglutide for at least 6 months. Intestinal biopsy specimens collected before and after treatment initiation were selected and analyzed by RNA sequencing to identify genes differentially expressed and pathways regulated following teduglutide treatment.

**Results:** Of the 39 patients included in the study, 29 (74%) had a colon in continuity. The overall response to teduglutide was a reduction by 75% (interquartile range: 42–100) in PS volume. Among the genes differentially expressed in the small bowel during teduglutide treatment, the most significantly upregulated gene was *PAPPA2* (q-value < 0.0001), encoding the metalloproteinase Pappalysin-2 (PAPPA-2) involved in Insulin-like Growth Factor (IGF) bioavailability. The best responders to teduglutide showed a lower *PAPPA2* expression at baseline (p < 0.01). *PAPPA2* expression at baseline also correlated positively with the percentage of remnant colon (p < 0.001).

**Conclusion:** We identified a new stakeholder, PAPPA-2, known to modulate IGF bioavailability, as playing a possible role in GLP-2 mechanism of action in human with SBS. We showed by association the existence of a greater spontaneous intestinal adaptation in SBS patients with a colon in continuity that could reduce sensitivity to teduglutide. Additionally, we suggest that initial *PAPPA2* expression could serve as a predictive biomarker for teduglutide efficacy.

© 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

Short Bowel Syndrome (SBS), which results from extensive intestinal resection, leads to severe malabsorption and is the leading cause of chronic intestinal failure. Patients need nutritional support to meet their energy and fluid requirements [1]. This parenteral support (PS) must be initiated after intestinal resection, and maintained over the long term in most patients. PS leads to several complications and significantly impacts patients'

**Abbreviations:** GLP-2, glucagon-like peptide-2; SBS, short bowel syndrome; PS, parenteral support; GLP-2R, glucagon-like peptide-2 receptor; CIC, colon in continuity; FFPE, formalin-fixed paraffin-embedded; IQR, interquartile range; IGF, Insulin-like Growth Factor; IGFBPs, Insulin-like Growth Factor Binding Proteins.

* Corresponding author. Gastroenterology and Nutritional Support Department, Hôpital Beaujon (AP-HP), Clichy, France.

E-mail address: francisca.joly@aphp.fr (F. Joly).

<footnote><sup>a</sup> Centre de Recherche sur l'Inflammation (CRI), INSERM U1149, Université Paris Cité, 75018 Paris, France</footnote>

<footnote><sup>b</sup> Gastroenterology and Nutritional Support Department, Hôpital Beaujon (AP-HP), Clichy, France</footnote>

<footnote><sup>c</sup> Pathology Department, Hôpital Beaujon (AP-HP), Clichy, France</footnote>

<footnote><sup>d</sup> Pathology Department, Hôpital Bichat (AP-HP), Paris, France</footnote>

<footnote><sup>e</sup> Endocrine and Oncological Biochemistry Department, Hôpital Pitié-Salpêtrière (AP-HP)-Sorbonne University, Paris, France</footnote>
quality of life [2]. In this context, teduglutide, a glucagon-like peptide-2 (GLP-2) analog, was the first drug approved for the treatment of SBS-associated malabsorption.

GLP-2 is a proglucagon-derived hormone with trophic effects on the intestine, increasing villous height and crypt depth, and enhancing intestinal barrier, mesenteric blood flow and immune function [3,4]. GLP-2 is secreted by enteroendocrine L-cells in response to nutrient ingestion. In the gastrointestinal tract, these GLP-2 secreting cells are mainly located in the distal ileum and colon [5,6].

The expression of the GLP-2 receptor (GLP-2R) is very limited in cells located outside the proliferative compartment of the intestine. The GLP-2R is expressed by three main cell types: the scattered neurons of the enteric nervous system, the subepithelial myofibroblasts and some enteroendocrine cells [7–10]. GLP-2R has been shown to be mainly expressed in the proximal small bowel (duodenum and jejunum) and, to a lesser extent, in the ileum and colon [7,11].

The mechanism of action of GLP-2 in intestinal cells has been widely studied in rodent models [3]. Due to the cellular location of its receptor, the action of GLP-2 on epithelial cells is thought to be mediated by various factors, in particular growth factors. Several studies have shown that the intestinotrophic actions of GLP-2 are exerted indirectly through downstream signaling mediators arising from the niche, including IGF-1 and ErbB [12–14]. However, the mechanism of action of GLP-2 in human intestinal cells remains to be determined.

Teduglutide, which is now part of the therapeutic arsenal for SBS patients, allows increasing intestinal absorption to reduce PS dependence. After intestinal resection, several beneficial spontaneous adaptations occur in SBS patients, including remodeling of intestinal morphology and function, microbiota changes and the development of hyperphagia [15–18]. When these adaptations are not sufficient to achieve PS weaning, teduglutide is recommended in the ESPEN guidelines for the treatment of SBS in carefully selected patients [19]. In France, teduglutide has been marketed and used since 2015, and experts consider that it promotes intestinal adaptation in SBS patients [20].

The efficacy of teduglutide to reduce PS volume in SBS patients has been widely demonstrated in clinical trials as well as in real-life studies [21,22]. However, there is no consensus on the characteristics of the best responders versus non-responders. In particular, SBS patients show a range of intestinal anatomies, with or without a colon in continuity (CIC) with the small bowel, and the role of the colon in the response to teduglutide is ambivalent. Indeed, a greater reduction in PS volume under teduglutide has been shown in patients with jejunostomy compared to those with a CIC [23], however, the presence of a colon facilitates PS weaning during treatment [20,24].

To date, there is no clear criterion to predict the response to treatment. Moreover, there are no commercially available GLP-2 assays for measuring endogenous GLP-2 levels routinely, and no other predictive biomarker of efficacy has been identified to date. In the context of an expensive treatment like teduglutide, it could be useful to select patients and preferentially treat potential responders. The aim of this study was to explore potential mechanisms underlying response heterogeneity, by comparing the transcriptome of intestinal biopsy specimens from SBS patients taken before and after teduglutide initiation.

# 2. Materials and methods

## 2.1. Study design and patients

This was a single-center, observational, retrospective study conducted in the Department of Gastroenterology of Beaujon University Hospital (Clichy, France). The study was approved by

our institutional review board (IRB number 00006477). Patient consent was waived due to the non-interventional setting. All SBS patients treated with teduglutide for at least 6 months with intestinal biopsy specimens taken before and/or during treatment were included in the study. Teduglutide was administered once daily by subcutaneous injection at a dose of 0.05 mg/kg body weight. Intestinal biopsy specimens were collected during endoscopy performed at the time of treatment initiation and during the follow-up. Depending on the digestive anatomy of the patient and the access for endoscopy, the biopsies were taken in the duodenal or jejunal part of the small intestine. The endoscopy was systematically performed at treatment initiation, after approximately one year of treatment, then one or two years later depending on the results.

The response to teduglutide was assessed as a reduction in PS (% and volume (mL/week)) achieved during treatment.

## 2.2. Data collection

Demographics and clinical data were collected from medical charts. Collected data included the age, gender, initial disease that led to intestinal resection, bowel anatomy features (remnant bowel length and presence of a CIC), and data on the response to teduglutide (changes in PS prescription during treatment). The length of colon in continuity was estimated from perioperative data records and expressed as a percentage of the usual length according to the Cummings classification [25].

## 2.3. RNA extraction and sequencing

Formalin-fixed paraffin-embedded (FFPE) blocks of intestinal biopsies performed before and during teduglutide treatment were identified in the Pathology Department of Beaujon Hospital. For all samples, the RNA was extracted using the Qiagen RNeasy FFPE kit following the manufacturer's instructions (Qiagen, Hilden, Germany). Overall, 10 ng of total RNA were used to generate a RNAseq sequencing library using the SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara) and sequenced by the Institut du Cerveau iGenseq plateform using a NovaSeq 6000 device with a minimum of 4 million reads sequenced. All RNAseq reads were aligned to the human genome reference sequence (Ensembl GRCH38) and Ensembl's reference transcriptome using STAR, quantified using FeatureCount and quantile-normalized.

## 2.4. RNAseq analysis

All analyses were performed using Rstudio (RStudio software, version 4.2.1.) Differential gene expression was analyzed based on the negative binomial distribution model using DESeq2 package. A q-value <0.05 was considered significant. Gene set enrichment analyses (GSEA) were performed using the algorithm implemented by Bioconductor for fast gene set enrichment analysis (FGSEA).

## 2.5. Statistics

Results are presented as a median (interquartile range [IQR]). Comparisons of continuous variables between ≥2 groups were performed using a non-parametric Mann-Whitney or Kruskal-Wallis test, and using a Dunn post-test for multi-parametric analyses. Correlations between quantitative parameters were assessed using the non-parametric Spearman test. A p-value <0.05 was considered significant.

All authors had access to the study data and have reviewed and approved the final manuscript.
# 3. Results

## 3.1. Patients' characteristics and intestinal biopsies

A total of 39 patients were included in the study. Their clinical characteristics are presented in Table 1. The median age was 50 years (IQR: 38–64) at the time of treatment initiation and 16 patients (41%) were women. The median remaining small bowel length was 70 cm (IQR: 27–100), and the colon was in continuity in 29 patients (74%). The overall response to teduglutide in the cohort was a reduction by 75% (IQR: 42–100) in PS volume during treatment, corresponding to a median reduction by 4500 mL per week (IQR: 2200–6325) in PS volume. Eighteen patients (46%) were weaned off PS during teduglutide treatment.

All intestinal biopsy specimens collected before and/or during treatment available for these patients were selected. Overall, 55 small bowel biopsy samples were analyzed: 29 collected before teduglutide initiation and 26 collected during treatment. Samples were collected from the duodenum of 17 patients and from the jejunum of 38 patients. For the samples taken during treatment, the median treatment duration before tissue collection was 24 months (IQR: 13–38; min-max: 6–64 months).

## 3.2. Overall changes in gene expression during teduglutide treatment

Differential gene expression in the small bowel between before and during teduglutide treatment is presented using a volcano plot in Fig. 1. Of the 10,146 genes studied, the expression of 1887 genes significantly changed during teduglutide treatment compared to before treatment, with a q-value <0.05. Among these genes, 963 were upregulated under teduglutide, and the most significantly upregulated gene was PAPPA2 (log2 Fold Change (FC) = 2.0, q-value = 1.01e-12).

<table><caption>**Table 1**
Clinical characteristics of the patients included in the study (n = 39).</caption><thead><tr><th></th><th>Patients included<br>N = 39</th></tr></thead><tbody><tr><td>Age, years, median (IQR)</td><td>50 (38–64)</td></tr><tr><td>Female gender, n (%)</td><td>16 (41)</td></tr><tr><td>Body weight, kg, median (IQR)</td><td>61.8 (53.0–67.5)</td></tr><tr><td>BMI, kg/m<sup>2</sup>, median (IQR)</td><td>21.2 (18.6–22.9)</td></tr><tr><td>Short bowel syndrome causes, n (%)</td><td></td></tr><tr><td>Mesenteric ischemia</td><td>12 (31)</td></tr><tr><td>Crohn's disease</td><td>9 (23)</td></tr><tr><td>CIPO</td><td>3 (8)</td></tr><tr><td>Radiation enteritis</td><td>3 (8)</td></tr><tr><td>Volvulus</td><td>5 (13)</td></tr><tr><td>Other</td><td>7 (18)</td></tr><tr><td>Bowel anatomy features</td><td></td></tr><tr><td>Remnant small bowel length (cm), median (IQR)</td><td>70 (27–100)</td></tr><tr><td>Residual colon (%), median (IQR)</td><td>70 (6–80)</td></tr><tr><td>Jejunostomy, n (%)</td><td>10 (26)</td></tr><tr><td>Colon in continuity, n (%)</td><td>29 (74)</td></tr><tr><td>≥ 50 % of colon remaining</td><td>27 (69)</td></tr><tr><td>&lt; 50 % of colon remaining</td><td>2 (5)</td></tr><tr><td>Parenteral support upon treatment initiation</td><td></td></tr><tr><td>PS Volume (mL/week), median (IQR)</td><td>7500 (4500–12,000)</td></tr><tr><td>PS Calories (kcal/week), median (IQR)</td><td>4820 (940–8680)</td></tr><tr><td>Number of days of infusion/week, median (IQR)</td><td>4.0 (2.7–5.0)</td></tr><tr><td>Response to teduglutide</td><td></td></tr><tr><td>Reduction in PS volume (% from baseline), median (IQR)</td><td>75 (42–100)</td></tr><tr><td>PS Volume change from baseline (mL/week), median (IQR)</td><td>4500 (2200–6325)</td></tr><tr><td>Weaned off PS, n (%)</td><td>18 (46)</td></tr></tbody></table>

Abbreviations: BMI = body mass index; CIPO = chronic intestinal pseudo-obstruction; IQR = interquartile range; PS = parenteral support.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Volcano plot of sequenced genes (n = 10,146). The x-axis indicates a log2 fold change after teduglutide treatment. The y-axis indicates the -log10 False Discovery Rate (FDR)-adjusted p-value. Each dot represents 1 gene. Red dots represent genes with a FDR-corrected p-value <0.05 (n = 1887), blue dots represent genes the expression of which did not change significantly. A differential gene expression analysis using the DESeq2 method adjusted for the biopsy site was used to generate the results.</figcaption></figure>

## 3.3. Changes in genes involved in the regulation of IGF transport and uptake by IGFBPs

PAPPA2 is known to be involved in the regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs). This specific pathway was
analyzed with GSEA, and Fig. 2 shows the evolution of the genes involved in IGF transport and uptake by IGFBPs between before and during the treatment with teduglutide. The tree on the left represents a hierarchical clustering of genes within the pathway, grouped based on similarities in their expression profiles across the samples. This clustering highlights patterns of co-expression among the genes within the pathway. Interestingly, other genes involved in the IGF transport and uptake pathway seemed to be upregulated in the jejunum of patients during teduglutide treatment, and two in particular were significantly upregulated: MMP1 (log2 FC = 2.5, q-value <0.001) and IGFBP5 (log2 FC = 0.9, q-value = 0.003).

### 3.4. Changes in PAPPA2 expression during teduglutide treatment

The overall changes in PAPPA2 expression were then analyzed in the different segments of the small bowel during teduglutide treatment. As shown in Fig. 3, PAPPA2 expression was strongly increased in the duodenum (log2 FC = 1.7, q-value <0.01), and the jejunum (log2 FC = 2.3, q-value <0.0001) during teduglutide treatment.

### 3.5. Jejunal PAPPA2 expression and response to treatment

The relationship between jejunal PAPPA2 expression and the response to teduglutide was then investigated (Fig. 4A-B). To this end, jejunal PAPPA2 expression before and during treatment with teduglutide was stratified according to patients' response to treatment (Fig. 4A). The response was assessed based on the percentage of PS volume reduction achieved during treatment. Patients were divided into three groups according to their response to treatment: non-responders (0-20% of PS volume reduction), partial responders (21-99% of PS volume reduction) and weaned

<figure><img src="image_4.png" /><figcaption>Fig. 3. Changes in PAPPA2 expression during teduglutide treatment in the duodenum and jejunum.</figcaption></figure>

Results are presented as medians, interquartile ranges, and minimum and maximum values. The y-axis is expressed in log₂ normalized RNAseq gene counts. False discovery rate-corrected p-values were used, **p < 0.01, ****p < 0.0001. NT: Not treated; T: Treated; TED: teduglutide.

off PS (100% of PS volume reduction). As shown in Fig. 4A, there was a significant difference in baseline PAPPA2 expression between the three response groups (p < 0.01). Indeed, the best responders to teduglutide showed the lowest PAPPA2 expression before treatment, and there was a negative correlation between PAPPA2 expression before teduglutide initiation and the PS volume

<figure><img src="image_5.png" /><figcaption>Fig. 2. Heatmap of the expression of genes involved in the regulation of Insulin-like Growth Factor (IGF) transport and uptake in the jejunum before and during teduglutide treatment.</figcaption></figure>

Row Z-Score = (value of gene expression - mean)/standard deviation. n = 17 before treatment, n = 21 during treatment; TED: teduglutide. False discovery rate-corrected p-values were used, **p < 0.01, ***p < 0.001, ****p < 0.0001.
reduction achieved during treatment (r = -0.67, p = 0.006, Fig. 4B). On the contrary, after treatment initiation, there was no difference in PAPPA2 expression between the three response groups (p = 0.5, Fig. 4A).

### 3.6. Jejunal PAPPA2 expression and intestinal anatomy

Jejunal PAPPA2 expression before and during teduglutide treatment was then analyzed according to the proportion of

remaining colon (Fig. 4C-D). Before treatment, PAPPA2 expression was related to the percentage of remnant colon (p < 0.001, Fig. 4C): patients with jejunostomy or a short colon length (0–50%) showed a significantly lower PAPPA2 expression at baseline than patients with a large portion of remaining colon (51–89%) or the whole CIC (90–100%). This result was confirmed by the strong positive correlation between baseline jejunal PAPPA2 expression and the proportion of remaining colon (r = 0.80, p = 0.0002, Fig. 4D). These

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Jejunal PAPPA2 expression stratified according to response to treatment with teduglutide and remaining colon in continuity.</figcaption></figure>

A. Jejunal PAPPA2 expression before and during treatment with teduglutide, stratified according to the level of response to treatment. Results are presented as medians, interquartile ranges, and minimum and maximum values. An ANOVA was used to generate p-values, **p < 0.01, ns: not significant. TED: teduglutide; NR: Non-responders (0–20 % of PS reduction); PR: Partial Responders (21–99 % of PS reduction), W: Weaned off PS (100 % of PS reduction). B. Correlation between jejunal PAPPA2 expression before treatment initiation and the response to treatment (in % of PS reduction), n = 17. Spearman correlation test. C. Jejunal PAPPA2 expression in the jejunum before and during teduglutide treatment, stratified according to the proportion of remaining colon. Results are presented as medians, interquartile ranges, and minimum and maximum values. An ANOVA was used to generate p-values, ***p < 0.001, ns: not significant. TED: teduglutide. D. Correlation between jejunal PAPPA2 expression before treatment initiation and the proportion of remaining colon (%), n = 17. Spearman correlation test.

The y-axis (panels A, C) and the x-axis (panels B, D) are expressed in log₂ normalized RNAseq gene counts.
differences were no longer significant after teduglutide initiation (p = 0.4, Fig. 4C).

### 3.7. Response to teduglutide according to the clinical characteristics

The response to teduglutide according to the intestinal anatomy is shown in Fig. 5. There was a negative correlation between the percentage of PS reduction achieved during treatment and the percentage of remnant colon ($r = -0.48, p < 0.01$, Fig. 5A). Regarding the response to treatment according to the absolute reduction in PS volume (mL/week), the reduction was significantly higher in patients with end-jejunostomy compared to patients with a CIC ($p < 0.05$, Fig. 5B).

Regarding the complete weaning off PS during teduglutide treatment, the characteristics and comparison of weaned off and non-weaned off patients are presented in Table 2. Weaned off patients have significantly longer bowel length ($p = 0.001$), smaller proportion of remnant colon ($p = 0.02$) and lower jejunal *PAPPA2* expression before treatment initiation ($p = 0.02$). A bivariate analysis between remaining colon and *PAPPA2* expression showed no significance for these variables ($p = 0.33$ for both factors), which may be explained by the high correlation between these two variables. The same comparison between the characteristics of responders and non-responders has been performed (Supplementary Table 1). The response was here defined as in teduglutide clinical trials, as a reduction of PS volume higher than 20% compared to treatment initiation.

## 4. Discussion

In this study, we identified *PAPPA2* as a gene that was strongly upregulated during teduglutide treatment, and which appeared to play an important role in the response to treatment. This gene encodes a metalloproteinase called Pregnancy-Associated Plasma Protein-A2 (PAPP-A2). PAPP-A2 shares 45% of its amino acid sequence with PAPP-A, a better-known metalloproteinase [26]. Both are regulatory proteins of the IGF axis. Through their proteolytic activity against the IGFBPs, they can release IGF from its circulating complex [27]. In particular, PAPP-A2 regulates IGF-1 bioavailability by specifically cleaving IGFBP3 and IGFBP5 [28]. In addition, the expression of IGFBP5 was also significantly increased in the jejunum of our patients during teduglutide treatment. Our

findings are consistent with the fact that IGF-1 has been identified as a mediator of GLP-2 trophic action [29,30]. Moreover, we observed an upregulation of *MMP1* expression during teduglutide treatment. This gene encodes the matrix metalloproteinase-1 (MMP-1), which has been shown to cleave the complex between IGF2 and IGFBP2, thus releasing IGF-2 [31]. The upregulation of *MMP1* during teduglutide treatment thus suggests a complementary role of IGF-2 in the trophic action of GLP-2. This result is consistent with the fact that IGF-2 null mice have been shown to have a partially impaired response to GLP-2 treatment [29]. Previous studies demonstrated that GLP-2 exerts its intestinotrophic effect through an indirect mechanism that requires both IGF-1 and the intestinal epithelial IGF-1 receptor [12,29]. The biological activity of IGF-1 is modulated by IGFBPs which are highly expressed in the intestine. In this regard, Austin et al. [32] suggested a role of PAPP-A in GLP-2 action in mice, this protease specifically cleaving IGFBP4, that exerts complex effects on the intestine. These findings are supported by a study conducted by Shawe-Taylor et al. [33] on human cells that showed an increase degradation of IGFBP-4, -5, -7 in the medium, probably associated with an increase in IGF bioavailability. Consistently, our study is the first to suggest a role of PAPP-A2 in the IGF-mediated intestinotrophic action of GLP-2 in human SBS, being consistent with the fact that the intestinotrophic effect of IGF-1 is mainly dependent on IGFBP-3 and -5 [34]. Little is known about the expression and regulation of this protease, but mutations in the *PAPPA2* gene have been associated with growth retardation and short stature in children due to low IGF-1 availability [35]. Furthermore, a high expression of *PAPPA2* has been associated with lower survival rates in lung cancer [36] and gastric cancer [37]. Upon teduglutide treatment, during which we have previously reported the development of abnormal intestinal polyps [38], it could be important to study *PAPPA2* expression in the intestinal mucosa of patients who developed polypoid lesions, and investigate whether this protease plays a role in lesion development.

We also found that *PAPPA2* expression was lower before treatment initiation in the best responders, and increased significantly during treatment, whereas its expression was already high before treatment initiation in non-responders, and remained stable during teduglutide treatment. Since GLP-2 increases *PAPPA2* expression, we could assume that teduglutide non-responders could have higher endogenous GLP-2 levels at baseline, and high activation of IGF signaling cascades. The correlation between *PAPPA2*

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Response to teduglutide according to the intestinal anatomy.</figcaption></figure>

**A.** Correlation between PS reduction during treatment and the presence of a remaining colon, n = 39. Spearman correlation test. **B.** Comparison of PS reduction between patients with jejunostomy (n = 10) and patients with a colon in continuity (n = 29). A Mann-Whitney test was used to generate p-values, \*p < 0.05, line passing at the median. TED: teduglutide.
<table><caption>**Table 2**
Comparison of the clinical characteristics of the weaned off and non-weaned off patients.</caption><thead><tr><th></th><th>Weaned n = 18</th><th>Non-weaned n = 21</th><th>p-value <sup>a</sup></th></tr></thead><tbody><tr><td>Age, years, median (IQR)</td><td>56 (41-64)</td><td>46 (36-60)</td><td>0.27</td></tr><tr><td>Female, n (%)</td><td>4 (22)</td><td>12 (57)</td><td>0.06</td></tr><tr><td>Body weight, kg, median (IQR)</td><td>64.5 (59.0-72.0)</td><td>57.0 (51.0-66.0)</td><td>0.06</td></tr><tr><td>BMI, kg/m2, median (IQR)</td><td>21.8 (18.7-23.8)</td><td>20.7 (18.6-22.0)</td><td>0.33</td></tr><tr><td>Bowel anatomy features</td><td></td><td></td><td></td></tr><tr><td>Jejunostomy, n (%)</td><td>6 (33)</td><td>4 (19)</td><td>0.46</td></tr><tr><td>Colon in continuity, n (%)</td><td>12 (66)</td><td>17 (81)</td><td>0.46</td></tr><tr><td>≥50 % colon remaining, n (%)</td><td>10 (55)</td><td>17 (81)</td><td>0.17</td></tr><tr><td>&lt;50 % colon remaining, n (%)</td><td>2 (11)</td><td>0 (0)</td><td>0.17</td></tr><tr><td>Remnant small bowel length (cm), median (IQR)</td><td>80 (78-133)</td><td>30 (20-70)</td><td>0.001</td></tr><tr><td>Remnant colon (%), median (IQR)</td><td>55 (1-79)</td><td>80 (50-100)</td><td>0.02</td></tr><tr><td>Short bowel syndrome causes, n (%)</td><td></td><td></td><td></td></tr><tr><td>Mesenteric ischemia</td><td>6 (33)</td><td>6 (29)</td><td>1</td></tr><tr><td>Crohn's disease</td><td>4 (22)</td><td>5 (24)</td><td>1</td></tr><tr><td>Radiation enteritis</td><td>2 (11)</td><td>1 (5)</td><td>0.59</td></tr><tr><td>CIPO</td><td>2 (11)</td><td>1 (5)</td><td>0.59</td></tr><tr><td>Volvulus</td><td>2 (11)</td><td>3 (14)</td><td>1</td></tr><tr><td>Other</td><td>2 (11)</td><td>5 (24)</td><td>0.42</td></tr><tr><td>PAPPA2 expression in the jejunum before treatment, median (IQR)</td><td>7.7 (7.1-8.0)</td><td>9.0 (8.2-9.5)</td><td>0.02</td></tr></tbody></table>

Abbreviations: BMI = body mass index; CIPO = chronic intestinal pseudo-obstruction; IQR = interquartile range; PS = parenteral support.

expression before treatment initiation and PS volume reduction achieved during treatment could make PAPPA2 level a predictive factor for response to teduglutide.

Given the limited sample size and the imbalance between response groups in our cohort, performance indices could not be calculated to evaluate the performance of PAPPA2 in this context. A larger prospective cohort would be needed to confirm the potential of initial PAPPA2 expression as a predictive biomarker for teduglutide efficacy.

Our results also showed a positive correlation between the percentage of remaining colon and the expression of PAPPA2 before treatment initiation, but this correlation disappeared during treatment. GLP-2 being secreted mainly in the colon, this result confirmed our assumption: in patients with a CIC, increased levels of endogenous GLP-2 could result in significant spontaneous intestinal adaptation and thus, in a lower sensitivity to treatment with GLP-2 analogs. The relationship between the endogenous GLP-2 levels and the response to GLP-2 analogs had never been investigated, and these results highlighted the interest of measuring GLP-2 levels before initiating teduglutide as they may correlate with treatment response.

Nonetheless, the precise location of the enteroendocrine cells that secrete GLP-2 is still under investigation. Recent studies have shown that enteroendocrine cells are not necessarily specific to the type of hormones they secrete, and that the hormones they secrete may depend on their environment [39,40], thus challenging the usual classification of enteroendocrine cells. However, several studies have shown that GLP-2-secreting cells would be mainly located in the distal ileum and colon [5,6]. In SBS patients who have undergone ileum and colon resection, GLP-2 secretion is not increased after a meal, in contrast to healthy controls [41]. On the other hand, in a cohort of SBS patients with a CIC, GLP-2 secretion during fasting and postprandial phases is significantly higher than in control subjects [42], with no difference between patients with and without ileum [18]. These results indicate an adaptive process of hormone secretion by enteroendocrine cells after intestinal resection, and higher GLP-2 levels in patients with a CIC before teduglutide initiation. Measurement of plasma GLP-2 levels in SBS patients before treatment initiation could allow to correlate GLP-2 levels and post-resection intestinal anatomy (length of small intestine, presence of ileum, remaining portions of colon, etc.), and thus

help to better describe the sites of GLP-2 secretion in SBS patients.

Our results showed that the response to teduglutide correlated negatively with the proportion of colon remaining. In particular, 4 non-responder patients had to increase their PS volume during treatment and these patients all had more than 50% of remaining colon. Three of them presented hyperphagia but the remnant small bowel length of these 4 patients was less than 40 cm.

However, several studies have reported that the presence of a CIC is a predictive factor for achieving PS weaning under teduglutide [22], i.e. the best response to treatment. This could suggest that the treatment still provides a benefit for patients with a CIC, despite the presence of spontaneous intestinal adaptation induced by endogenous GLP-2. This observation could be explained by the fact that patients with a CIC have significantly lower PS needs before treatment, and that small initial PS volumes favor PS weaning. However, the decrease in PS volume during treatment is lower in these patients than in patients with jejunostomy, even though some of them might be weaned off PS. This raises the question of the real benefit of treatment in terms of patients' quality of life. It would be interesting to assess the benefit of teduglutide in patients with jejunostomy who will gain several days without PS infusion, compared to patients with a CIC who will sometimes gain one day but will achieve PS weaning.

Overall, these findings raise the question of the benefits of teduglutide in patients with a CIC, in whom high endogenous GLP-2 levels lead to significant spontaneous intestinal adaptation. To go further, the relevance of modifying the dose and/or timing of teduglutide administration in SBS patients with a CIC should perhaps be discussed. Indeed, if endogenous GLP-2 levels are elevated in these patients, a higher dose of teduglutide could be needed to observe an effect on the intestinal mucosa. On the contrary, GLP-2R saturation could also occur so that a higher dose would have no additional effect, but this has never been demonstrated yet. Moreover, a higher dose could increase the risk of developing intestinal polyps during treatment. Regarding the timing of treatment initiation, earlier treatment could be beneficial in these patients, to combine the effects of GLP-2 analogs with spontaneous adaptation to reinforce natural mucosa adaptations. Further studies are needed to confirm these assumptions.

This study has several limitations. First, the intestinal biopsy specimens collected during teduglutide treatment were collected

<footnote><sup>a</sup> Wilcoxon-Mann-Whitney test for continuous data. Fischer's exact test (n ≤ 5) or Pearson's Chi2 test (n > 5) for categorical data.</footnote>
at different timepoints and anatomic location for each patient, due to the retrospective design of the study, and the fact that we used the biopsies available to perform the analyses. Still, the changes in gene expression observed in our cohort did not correlate with treatment duration. Second, our population of SBS patients was heterogeneous in terms of etiology and intestinal anatomy, and PS duration before teduglutide initiation varied greatly from one patient to another, which could have affected the response to treatment. Finally, endogenous GLP-2 levels were not reported in this study, as these dosages are not performed routinely. In a future study, it would be important to assess the endogenous GLP-2 levels in patients to confirm our hypothesis of a link between teduglutide response and spontaneous intestinal adaptation. Moreover, a larger prospective study with standardized evaluation of treatment response is needed to confirm the predictive value of PAPPA2 expression as a biomarker for teduglutide efficacy.

## 5. Conclusion

We identified a potential mediator involved in GLP-2 mechanism of action, PAPP-A2, a possible key actor for IGF modulation in the intestinotrophic action of GLP-2 in human cells. This study also allowed identifying mechanisms of greater spontaneous intestinal adaptation in SBS patients with a CIC, probably due to endogenous GLP-2 secretion, resulting in lower sensitivity to teduglutide. These results suggest that PAPPA2 expression at teduglutide initiation could serve as a predictive biomarker of treatment efficacy.

## Author contributions

Brune de Dreuille (Conceptualization: Supporting; Formal analysis: Lead; Funding acquisition: Equal; Investigation: Equal; Writing – original draft: Lead).

Rémy Nicolle (Conceptualization: Equal; Formal analysis: Lead; Investigation: Equal; Methodology: Lead; Writing – review & editing: Equal).

Jérôme Cros (Conceptualization: Lead; Methodology: Lead; Resources: Equal; Writing – review & editing: Supporting).

Dominique Cazals-Hatem (Conceptualization: Equal; Investigation: Equal; Resources: Lead; Writing – review & editing: Supporting).

Anaïs Chassac (Investigation: Equal; Methodology: Supporting; Resources: Lead; Writing – review & editing: Supporting).

Nicolas Poté (Investigation: Equal; Methodology: Equal; Resources: Lead; Validation: Equal; Writing – review & editing: Equal).

Johanne Le Beyec-Le Bihan (Conceptualization: Supporting; Formal analysis: Supporting; Methodology: Equal; Writing – review & editing: Lead).

André Bado (Conceptualization: Supporting; Formal analysis: Equal; Methodology: Equal; Resources: Lead; Writing – review & editing: Lead).

Maude Le Gall (Conceptualization: Supporting; Formal analysis: Supporting; Methodology: Equal; Writing – review & editing: Lead).

Francisca Joly (Conceptualization: Lead; Formal analysis: Supporting; Funding acquisition: Lead; Investigation: Lead; Resources: Equal; Supervision: Lead; Writing – original draft: Supporting; Writing – review & editing: Lead).

## Writing assistance

Sophie Pégorier.

Source of funds: research association (Association pour l'Étude des Fonctions Digestives).

## Funding statement

This work was supported by a research grant (Prix NCM-Elsevier) awarded by SFNCM (Société Francophone Nutrition Clinique et Métabolisme).

## Conflict of interest

BD received a grant from Takeda for the implementation of a clinical study.

Fj has participated in NPS Pharmaceuticals' Scientific, Shire's Advisory Board, Takeda, Zealand and Vectibio advisory boards and in randomized controlled clinical trials of teduglutide supported by NPS and takeda, and randomized controlled clinical trials for Zealand (for Glepaglutide) and Vectivbio (Apraglutide).

All other authors report no conflicts of interest.

## Acknowledgments

Institut du Cerveau iGenseq plateform for the sequencing.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.clnu.2025.05.006.

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