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. 2025 Aug 12;75(1):e334124. doi: 10.1136/gutjnl-2024-334124

Mucosal transcriptomic landscape along the small and large intestines in individuals with and without type 2 diabetes

Hannah Gilliam-Vigh 1,2,✉, Anne Marie Ellegaard 1,2, Martin Rønn Madsen 3, Asger Bach Lund 1,4,5, Benjamin Anderschou Holbech Jensen 2, Tina Vilsbøll 1,4,5, Kristoffer Rigbolt 3, Filip Krag Knop 1,4,6,*
PMCID: PMC12703299  PMID: 40803750

Abstract

Background

A detailed mapping of functional differences among intestinal regions in healthy individuals remains incomplete. Identifying regional alterations in individuals with type 2 diabetes (T2D) could enhance our understanding of disease-related intestinal changes.

Objective

To characterise the transcriptomic landscape along the entire intestinal tract in healthy individuals and those with T2D, and to create a publicly accessible database for future research.

Design

In this observational study, mucosal biopsies were obtained from 16 sites along the intestinal tract through anterograde and retrograde double-balloon endoscopy in 12 individuals with T2D and 12 normoglycaemic matched healthy individuals. Full transcriptomic analysis was performed. Genes with significantly different expressions between intestinal regions were analysed in terms of their biological mechanisms in healthy individuals, while regional expression profiles were compared between individuals with and without T2D.

Results

In healthy individuals, distinct gene clusters in the small and large intestines were associated with processes including immune response, mitochondrial activity and metabolism of organic substances. Individuals with T2D exhibited alterations in immune system activity and barrier permeability in the ileocaecal region and the large intestine.

Conclusion

Our study offers a detailed mapping of the transcriptomic landscape in the human intestinal tract, demonstrating regionalised gene expression profiles tied to critical biological processes. Notable alterations in immune system activity in the large intestine were observed in individuals with T2D. The publicly available database generated from this study (https://rnaseq.gubra.dk/) provides a valuable resource for exploring the mucosal transcriptome along the human intestinal tract.

Trial registration number

NCT03044860

Keywords: DIABETES MELLITUS, GENE EXPRESSION, GASTROINTESINAL ENDOSCOPY, GASTROINTESTINAL FUNCTION, GASTROINTESTINAL PHYSIOLOGY

WHAT IS ALREADY KNOWN ON THIS TOPIC

  • Previous studies have explored gene expression in specific regions of the human intestinal tract related to biological processes and type 2 diabetes (T2D), but lacked comprehensive, high-resolution mapping across the entire intestinal length.

WHAT THIS STUDY ADDS

  • This study presents the first detailed atlas of gene expression across 16 regions of the human intestine in individuals with and without T2D, identifying region-specific gene expression patterns and alterations associated with T2D.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

  • The publicly accessible database facilitates the identification of novel therapeutic targets and enhances our understanding of intestinal biology and T2D pathophysiology, potentially informing personalised treatment strategies and advancing metabolic disease research.

Introduction

The human intestinal tract is a complex system with distinct regions, each having unique anatomical and physiological characteristics.1 While the small intestine absorbs nutrients, the colon primarily absorbs water and electrolytes.2 3 Moreover, these regions exhibit varying degrees of permeability and immune function.4 The current functional classification of the intestines is based on the following six anatomical terms: duodenum, jejunum, ileum, caecum, colon and rectum. However, the functional heterogeneity within and between these regions remains to be fully described. This is particularly true for the many metres of jejunum, ileum and colon that may contain hitherto undescribed functional subregions.

Metabolic diseases, such as type 2 diabetes (T2D), have long been associated with intestinally derived pathophysiology.5,7 T2D has been linked to alterations to the gut microbiome,8,10 changes in mucosal permeability11 12 and potential malabsorption of micronutrients.13 Roux-en-Y gastric bypass surgery—altering the way nutrients travel through and, thus, stimulate the upper GI tract14,16—often leads to remission of T2D, further supporting the link between the intestines and T2D pathophysiology.17,19 Nevertheless, the region-specific involvement of the intestine in T2D pathophysiology remains poorly understood at an anatomical–functional level.

Here, we mapped the human intestinal mucosal transcriptome to compare gene expression profiles in individuals with and without T2D. Our objective was to create a freely available, searchable database of the human intestinal transcriptome. To achieve a high-resolution view of the transcriptomic landscape, we performed full messenger RNA sequencing on mucosal biopsies from seven well-defined anatomical regions in the small and large intestine.20 Additionally, we sampled every 30 cm along the jejunum and ileum during anterograde and retrograde double-balloon enteroscopy (DBE) in 12 healthy individuals and 12 sex, age and body mass index (BMI)-matched individuals with T2D.

Materials and methods

Biopsy collection

Mucosal biopsies were taken during two DBE procedures performed at Gentofte Hospital in Denmark. Mucosal biopsy retrieval, previously described in detail,21 involved propofol sedation and the use of a double-balloon enteroscope to collect biopsies from various parts of the intestinal tract. During the anterograde DBE, an ink mark was placed submucosally at maximum insertion to indicate depth of intubation, and during retraction, biopsies were taken from every 30 cm of the ileum and jejunum, Treitz area and the duodenum. During the retrograde procedure (aiming to reach the ink mark placed during the anterograde DBE), biopsies were taken from the ileum, the ileocaecal transition, caecum, ascending, transverse and descending colon, sigmoideum and rectum (figure 1 and online supplemental figure S1). Seven groups (regions 3–9) in the jejunum and proximal ileum were assigned 7–22 biopsies and processed together in case of multiple biopsies in the same region. Additional methods are provided in the online supplemental information.

Figure 1. Anatomical segments of biopsy sampling sites 1–16. Samples from the small intestine were collected at intervals of 30 cm and divided into seven groups.3,9 The other samples (duodenum, ligament of Treitz, ileocaecal, caecum, ascending colon, transverse colon, descending colon, sigmoideum and rectum) were collected from anatomically well-defined areas. Figure adapted from Rhee et al.21.

Figure 1

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Results

Participant characteristics

As previously reported,21 the group of healthy individuals comprised eight men and four women (with an age of 50±2 years; a BMI of 26.9±0.7 kg/m2; and a glycated haemoglobin (HbA1c) of 5.3%±0.1/34±1 mmol/mol). The group of participants with T2D comprised nine men and three women (with an age of 51±3 years; a BMI of 26.8±0.9 kg/m2; an HbA1c of 6.5%±0.4/48±4.1 mmol/mol; and a diabetes duration of 5.0±1.2 years). (All data here are presented as mean±SEM). All participants were of European descent.

Transcriptomic variance along the intestinal tract

We investigated gene expression variation across all samples along the intestinal tract in both groups. Biopsies were first visualised in a principal component analysis (PCA) plot containing both small and large intestinal samples (figure 2A). The samples segregated in location-specific clusters, explaining more than 75% of the observed variation, thus demonstrating a fundamental difference between small and large intestinal gene expression profiles (figure 2A). Notably, the small intestine demonstrated the highest degree of inter-regional variation, indicating significant transcriptional differences and potential functional heterogeneity along the intestinal landscape. We next separated large and small intestinal samples into independent PCAs to enhance the resolution of intrasectional differences.

Figure 2. Principal component analysis of gene expression data in mucosal biopsies sampled along the intestinal tract in individuals with and without T2D. Principal component analysis of mucosal biopsies from (A) the entire intestinal tract, (B) the small intestine and (C) the large intestine of individuals with and without T2D. Each dot represents a mucosal biopsy retrieved from healthy individuals (empty dots) and individuals with T2D (filled dots) and the colour indicates the intestinal segment from which it was obtained. PC, principal component; T2D, type 2 diabetes.

Figure 2

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The distribution of the small intestinal samples mirrored the biopsy retrieval with the duodenum next to the Treitz ligament, followed by biopsy 3–9 in numerical order and concluding with the most distal ileocaecal biopsy (figure 2B). Samples from individuals with T2D closely grouped with those from healthy individuals in most regions, corroborating that location is generally a stronger driver of mucosal gene expression differences than the presence or absence of T2D. For regions 7, 8 and 9, samples from individuals with T2D approximated the proximal small intestine (regions 4, 5 and 6), while similar samples from their normoglycaemic counterparts clustered closer to the ileocaecal sample (figure 2B). In the large intestine, the distribution of samples also mirrored biopsy retrieval (figure 2C). We did not observe a clear distinction between T2D and their healthy counterparts in any of the regions from the large intestine.

Clustering unveils spatial resolution of biological processes in the healthy intestines

To examine gene expression patterns throughout the healthy intestines, we generated heatmaps to visualise regionalised relative gene expression levels (figure 3). Genes that were differentially expressed in the small intestine (relative to the duodenum) and in the large intestine (relative to the rectum) were subjected to unsupervised hierarchical clustering (figure 3A and B). This exercise revealed six unique clusters in the small intestine and four in the large intestine (figures35, online supplemental Tables S1–S10).

Figure 3. Heatmap of genes with differential expression along the intestines. Heatmap of genes with differential expression (Padj <0.05) along (A) the small intestine of healthy individuals with duodenum as reference and along (B) the large intestine of healthy individuals with rectum as reference. All genes in this plot were significantly differently expressed in at least one intestinal segment compared with the reference segment. Each row represents a specific gene, and each column represents an intestinal segment. P adj, adjusted p values.

Figure 3

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Figure 5. Cluster 1, 2, 3 and 4 of genes exhibiting altered expression in the healthy large intestine relative to the rectum. Distributions shared by all genes within the cluster and enriched pathways for Clusters 1 (A), 2 (B), 3 (C) and 4 (D). The x axes represent segments of the large intestine from the caecum on the left side to the rectum on the right side. The y axes represent the scaled normalisation of gene expression ranging from −2 to 2. Each grey line represents the expression of a single gene throughout the large intestine. The thick blue lines represent the calculated mean gene expression for each of the clusters. Detailed tables presenting the top 10 biological mechanisms enriched for genes within the cluster sorted by increasing P adj can be found in the online supplemental repository. Asc, ascending; Desc, descending; P adj, adjusted p values; Trans., transverse.

Figure 5

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Small intestine: Six clusters of genes (1, 2, 3, 4, 5 and 6) exhibiting distinct expression patterns along the small intestine comprised enough genes for gene ontology (GO) pathway enrichment analysis of biological functions (figure 4). The ontologies with the lowest adjusted p values (P adj) are listed (online supplemental Tables S1–S6). Cluster 1 comprised genes with increased expression from the duodenum to the ileocaecal location and was enriched with genes involved in cell responses to organic substances (P adj=0.025) (figure 4A, online supplemental Table S1). Cluster 2 included genes with a gradual increase in expression throughout the small intestine and a steep increase in expression at the very distal small intestine; this cluster displayed enrichment with genes associated with immune response and inflammation (P adj=7×10−13) (figure 4B, online supplemental Table S2). Cluster 3 was enriched by genes associated with RNA modification (P adj=5×10−03) and had the highest expression in the duodenum and ileocaecal region (figure 4C, online supplemental Table S3). Cluster 4 included genes with high expression in the duodenum and a gradual decline in expression through the small intestine until region 8, after which expression rose again towards the ileocaecal location. This cluster was enriched by genes associated with ribosomal biogenesis (P adj=3×10−44) (figure 4D, online supplemental Table S4). Cluster 5, enriched with genes related to cellular metabolic processes (P adj=1×10−10), decreased from the duodenum to region 9 (figure 4E, online supplemental Table S5). Finally, cluster 6 included genes with increasing expression from duodenum to region 5, and then, decreasing expression towards the ileocaecal region; this cluster was enriched with genes associated with the catabolism of lipids (P adj=2×10−10) (figure 4E, online supplemental Table S6).

Figure 4. Clusters 1, 2, 3, 4, 5 and 6 of genes exhibiting altered expression in the healthy small intestine relative to the duodenum. Distributions shared by all genes within the cluster and enriched pathways for Clusters 1 (A), 2 (B), 3 (C), 4 (D), 5 (E) and 6 (F). The x axes represent segments of the small intestine from the duodenum on the left side to the ileocaecal segment on the right side. The y axes represent the scaled normalisation of gene expression ranging from −2 to 2. Each grey line represents the expression of a single gene throughout the small intestine. The thick blue lines represent the calculated mean gene expression for each of the clusters. Detailed tables presenting the top 10 biological mechanisms enriched for genes within each cluster sorted by increasing P adj can be found in the online supplemental repository. P adj, adjusted p values.

Figure 4

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Large intestine: Cluster 1 in the large intestine comprised genes exhibiting the highest expression level in caecum and with decreasing expression levels along the remaining large intestine. Genes in cluster 1 were predominantly associated with the metabolism of organic substances and lipid metabolism (P adj=2×10−28) (figure 5A, online supplemental Table S7). Genes in cluster 2 were similarly expressed in all regions of the large intestine except for a very low level in the rectum; these genes were associated with mitosis (P adj=2×10−8) (figure 5B, online supplemental Table S8). The expression of cluster 3 and 4 genes rose from proximal to distal regions of the large intestine. Genes in cluster 3 were associated with glycoprotein metabolic processes (P adj=6×10−03) (figure 5C, online supplemental Table S9), while genes in cluster 4 related to intracellular organisation (P adj=0.026) (figure 5D, online supplemental Table S10).

Differentially expressed genes and functions between individuals with and without T2D

In individuals with T2D, the presence of residual faeces in the ileocaecal region impeded passage,19 thereby necessitating that biopsies from intestinal segments 8 and 9 were obtained exclusively via anterograde DBE. In contrast, segment 8 in healthy individuals was sampled via both anterograde and retrograde DBE, while segment 9 was obtained only via retrograde DBE (online supplemental Figure S1). Differences in access routes may affect the anatomical comparability of biopsy sites between groups, which should be considered when interpreting the results. In support of this, GO enrichment analysis of these segments revealed a strong signal for the ‘anterior/posterior pattern specification’ pathway (online supplemental Figures S2–S4), suggesting possible misalignment. Therefore, results from segments 7–9 were excluded from the comparison between disease states and transferred to the supplementary repository to avoid premature conclusions based on potentially inconsistent data.

Small intestinal regions 1–6: Differential gene expression analysis of the proximal small intestinal segments (duodenum, Treitz and segments 3–6) identified 387 genes (P adj <0.05). Enrichment analysis revealed changes in pathways relating to cholesterol synthesis and innate immune responses (figure 6A). A volcano plot showed differentially regulated genes related to immune activity, mitochondrial function and epithelial barrier maintenance in individuals with T2D (figure 6B). Among the top regulated genes, HLA-DQB1, a marker of antigen-presenting cells,22 was consistently upregulated in the small intestines of subjects with T2D (significance in the small intestinal region 1–6: P adj=3.01×10−04) (figure 6C). Similarly, APOBEC2, involved in cell turnover,23 was upregulated throughout the small intestine (P adj=5.89×10−04) (figure 6D), while GATD3A, a mitochondrial gene,24 was reduced throughout the small and large intestines (significance in the small intestinal region 1–6: P adj=1.3×10−03, in the large intestine: P adj=5.49×10−03) (figure 6E). We next referenced public single-cell RNA sequencing data from Abdulla et al25 to investigate which small intestinal cell types selectively express these genes of interest. Differentially expressed genes in the small intestine of individuals with T2D clustered into distinct expression patterns across cell types (figure 6F). A subset of genes, including CHRD2L, ADAM32, CCL23 and KCNK9, was predominately expressed in endothelial cells of the lymphatic vessel and the venule, pointing to altered immune cell traffic and vascular integrity. Another cluster, including HPCAL4, PRSS21 and UTS2, was selectively expressed in T cells and innate immune cells, potentially affecting immune homeostasis and vasoconstriction. Together, this indicates reduced vascular barrier integrity, increased immune activity, reduced mitochondrial activity and increased cell senescence in the small intestines of individuals with T2D.

Figure 6. Small intestinal differences between individuals with and without T2D. (A) Over-representation analysis of differentially expressed genes in the duodenum, ligament of Treitz and regions 3–6 of the small intestine. The 20 most significant pathways are shown in a descending order of significance, with darker green representing more significant pathways and lighter green representing lower significance (B) Volcano plot of gene expression differences. The x axis shows log2 fold change, and the y axis shows −log10 (P adj). Vertical dashed lines indicate thresholds at log2FC±1, and the horizontal line indicates a P adj of 0.05. Genes significantly downregulated (P adj <0.05, log2FC <−1) are marked in blue, and the 10 most significant are annotated. Upregulated genes (P adj <0.05, log2FC >1) are marked in red, with the top 10 annotated. (C–E) Expression of HLA-DQB1, APOBEC2 and GATD3A along the intestinal tract. Each plot shows gene expression (RPKM) on the y axis and anatomical sampling location from duodenum to rectum on the x axis. Grey shading denotes the large intestine. Points represent individual mucosal samples; lines represent mean expression per group. Grey and blue denote individuals without and with T2D, respectively. Arrows indicate the direction of significant differential expression from DESeq2 analyses performed separately for the small intestine, ileocaecal region and large intestine. (F) Relative expression (z-score) across cell types defined by small intestinal scRNAseq for genes differentially expressed in the small intestine. Row side colours represent direction of regulation in T2D relative to non-diabetic individuals. Genes significantly upregulated (red bar) or downregulated (blue bar) in individuals with T2D (P adj < 0.05, |log2FC| > 1) are shown. NS=not significant (P adj >0.05). Asc, ascending; Desc, descending; log2FC, log2 fold change; mRNA, messenger RNA; P adj, adjusted p values; RPKM, reads per kilobase per million mapped reads; scRNAseq, single-cell RNA sequencing; T2D, type 2 diabetes; Trans, transverse.

Figure 6

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Ileocaecal region: 442 genes were differentially expressed (P adj<0.05). Enrichment analysis indicated changes in mitochondrial function and glucose homeostasis (figure 7A). Upregulated genes were involved in ion transport, glycosylation and immune modulation, while the downregulated genes reflected impaired epithelial barrier protection and altered mitochondrial activity (figure 7B). Gene expression of LDHD, an enzyme involved in anaerobic glycolysis and a marker of tissue damage,26 was upregulated across the intestines, but especially in the large intestines, of individuals with T2D (small intestine 1–6: P adj=9.46×10−03, ileocaecal: P adj=1.6×10−10, large intestine: P adj=1.86×10−02) (figure 7C). CD24, important for gut leucocyte recruitment,27 was increased in the ileocaecal region (P adj=2.56×10−10) (figure 7D), while gene expression of LCN2, functioning as an antimicrobial peptide via iron-sequestering inhibiting, for example, E. coli growth,28 was increased in the ileocaecal region (P adj=2.04×10−²), suggesting increased mucosal activation towards luminal bacteria, hence pointing towards bacterial encroachment in subjects with T2D (figure 7E). Using single-cell RNA sequencing data from Abdulla et al,25 we examined the cell type-specific expression of genes differentially regulated in T2D. This revealed distinct expression profiles across intestinal cell types (figure 7F). MS4A12, HOXB13, SERPINA6, PPP1R14C, C6orf58 and IL20RA were prominently expressed in small intestinal enteroendocrine cells, pointing towards potential increase of enteroendocrine cell numbers or modifications in endocrine signalling, including increased capacity to bind corticosteroids and interleukins. TRIB3, CEACAM3 and DNAI4 were most prevalent in endothelial cells of the lymphatic vessels, indicating increased immune cell recruitment and changes in vessel tissue architecture. Together, this indicates increased immune responses, a decrease in mitochondrial function and changes in enteroendocrine signalling in the distal small intestine of individuals with T2D.

Figure 7. Ileocaecal differences between individuals with and without T2D. (A) Over-representation analysis of differentially expressed genes in the distal small intestine ileocaecal region. The 20 most significant pathways are shown in a descending order of significance, with darker green representing more significant pathways and lighter green representing lower significance. (B) Volcano plot of gene expression differences. The x axis shows log2 fold change, and the y axis shows -log10(P adj). Vertical dashed lines indicate thresholds at log2FC±1, and the horizontal line indicates a P adj of 0.05. Genes significantly downregulated (P adj <0.05, log2FC <−1) are marked in blue, and the 10 most significant are annotated. Upregulated genes (P adj <0.05, log2FC >1) are marked in red, with the top 10 annotated. (C–E) Expression of LDHD, CD24 and LCN2 along the intestinal tract. Each plot shows gene expression (RPKM) on the y axis and anatomical sampling location from duodenum to rectum on the x axis. Grey shading denotes the large intestine. Points represent individual mucosal samples; lines represent mean expression per group. Grey and blue denote individuals without and with T2D, respectively. Arrows indicate the direction of significant differential expression from DESeq2 analyses performed separately for the small intestine, ileocaecal region and large intestine. (F) Relative expression (z-score) across cell types defined by small intestinal scRNAseq for genes differentially expressed in the ileocaecal region. Row side colours represent direction of regulation in T2D relative to non-diabetic individuals. Genes significantly upregulated (red bar) or downregulated (blue bar) in individuals with T2D (P adj <0.05, |log2FC| > 1) are shown. NS=not significant (P adj >0.05). Asc, ascending; Desc, descending; log2FC, log2 fold change; P adj, adjusted p values; RPKM, reads per kilobase per million mapped reads; scRNAseq, single-cell RNA sequencing; T2D, type 2 diabetes; Trans, transverse.

Figure 7

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Large intestine: 2046 genes were differentially expressed in the large intestine. Enrichment analysis pointed to augmented lymphocyte activation and antigen-driven immune cell recruitment (figure 8A). Downregulated genes in individuals with T2D were important for immune regulation, T cell signalling and gut barrier function, while upregulated genes included transcription factors, oncogenes and enteroendocrine hormones (figure 8B). Gene expression of LAMC3, a part of the basement membrane and binding sites for integrin receptors, was downregulated throughout the small and the large intestines in T2D (P adj=2.52×10−⁴; P adj=0.19; P adj=6.21×10−¹⁴) suggesting decreased mucosal structure (figure 8C). The gene CLDN8, encoding a tight junction protein,29 was upregulated in the large intestine of individuals with T2D (P adj=6.77×10−⁷) (figure 8D). This may reflect compensatory tight junction remodelling in response to epithelial stress. ACTL8, an oncogene commonly associated with colorectal cancer,30 was upregulated (P adj=2.92×10−⁴) (figure 8E), possibly indicating increased regenerative pressure or altered cytoskeletal organisation in the diabetic gut. Next, single-cell RNA sequencing data25 was used to examine the cell type-specific expression of genes differentially regulated in T2D. Several gene expression patterns were detected across cell types. In the large intestinal T cells, genes like HSPB3, TMEM105, PDZK1, PNMA8C, ATP6V1B1, CYP24A1, RGPD3 and FAM153A were most prevalent, which indicates reduced resilience to stress and reduced T cell activation, while genes including PPY, GREB1 and INSL5 were most prevalent in enteroendocrine cells, suggesting altered enteroendocrine hormone signalling. Immune surveillance genes such as HLA-DQB2, HLA-DQA1 and HLA-DQA2 were unsurprisingly most prevalent in B cells, macrophages and dendritic cells. Their downregulation in the large intestines of individuals with T2D indicates less antigen presenting cells or reduced antigen presentation capacity potentially increasing the risk for systemic infection or bacterial translocation fuelling T2D progression.

Figure 8. Large intestinal differences between individuals with and without T2D. (A) Over-representation analysis of differentially expressed genes in the large intestine. The 20 most significant pathways are shown in a descending order of significance, with darker green representing more significant pathways and lighter green representing lower significance. (B) Volcano plot of gene expression differences. The x axis shows log2 fold change, and the y axis shows −log10(P adj). Vertical dashed lines indicate thresholds at log2FC±1, and the horizontal line indicates a P adj of 0.05. Genes significantly downregulated (-1 > log2FC, P adj <0.05) are marked in blue, and the 10 most significant are annotated. Upregulated genes (log2FC >1, P adj <0.05) are marked in red, with the top 10 annotated. (C–E) Expression of LAMC3, CLDN8 and ACTL8 along the intestinal tract. Each plot shows gene expression (RPKM) on the y axis and anatomical sampling location from duodenum to rectum on the x axis. Grey shading denotes the large intestine. Points represent individual mucosal samples; lines represent mean expression per group. Grey and blue denote individuals without and with T2D, respectively. Arrows indicate the direction of significant differential expression from DESeq2 analyses performed separately for the small intestine, ileocaecal region and large intestine. (F) Relative expression (z-score) across cell types defined by large intestinal scRNAseq for genes differentially expressed in the large intestine. Row side colours represent direction of regulation in T2D relative to non-diabetic individuals. Genes significantly upregulated (red bar) or downregulated (blue bar) in individuals with T2D (P adj <0.05, |log2FC| > 1) are shown. NS=not significant (P adj >0.05). Asc, ascending; Desc, descending; log2FC, log2 fold change; P adj, adjusted p values; RPKM, reads per kilobase per million mapped reads; scRNAseq, single-cell RNA sequencing; T2D, type 2 diabetes; Trans, transverse.

Figure 8

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Discussion

This study provides a comprehensive transcriptomic overview of the human intestine, revealing region-specific functions, cellular composition and gene expression differences between healthy individuals and those with T2D, based on mucosal biopsies collected throughout the entire intestinal tract using DBE. In healthy individuals, clustering revealed distinct patterns of gene expression in the small and large intestines. In the small intestine, clusters with distinct and region-specific gene expression profiles were enriched for genes linked to cellular response to organic substances, immune response, inflammation and ribosomal biogenesis (figure 4). Moreover, in the large intestine, gene expression related to mitosis and lipid metabolism was found to differ between regions (figure 5).

The immune system plays a crucial role in maintaining gut homeostasis. Importantly, our study reveals new knowledge on the biological mechanisms involved along the GI tract. Cluster analysis of the small intestine corroborates that genes most abundantly expressed in the distal small intestine (cluster 2) were enriched for biological processes related to the immune system with the GO terms ‘Lymphocyte Differentiation’ and ‘Mononuclear Cell Differentiation’ most significantly enriched and regulatory T cells, monocytes and myeloid dendritic cells presumably increased in this part of the intestines compared with the proximal small intestine (figure 2B, online supplemental Table S2). Both intestinal lymphocytes and monocytes are an integral part of maintaining gut barrier function and intestinal tolerance.31 32 Our results align well with histological mapping of lymphocytes showing a high abundance in gut-associated lymphoid tissue, macroscopically visible as Peyer’s patches.33 While the distribution of mononuclear cells in human small intestinal mucosa remains unreported, previous research has shown an elevated presence of these cells in the distal small intestine of mice.34 In the large intestine, immune functions were more uniformly distributed across segments, resulting in only a modest over-representation of immune pathways in cluster 3 (figure 5C, online supplemental figure S9)

Besides exploring the functional differences between intestinal regions in the healthy intestine, our aim was to understand the differences in gene expression between matched individuals with and without T2D. Our PCA plot revealed substantial location-specific segregation of samples indicating pronounced differences between small and large intestinal gene expression profiles. Thus, the differential analysis was performed separately for the small and large intestines to enhance resolution of each region. Further, the segments of the small intestine were separated for the analysis in a proximal part (consisting of segments duodenum, ligament of Treitz and 3–6) and the ileocaecal region, while the segments 7–9 were disregarded as explained below. We observed region-specific transcriptional changes along the intestine in individuals with T2D. Broadly, our results indicate an increased immune activation throughout the intestines, reduced epithelial barrier function and increased epithelial senescence in subjects with T2D compared with healthy matched controls. While we detected differences in both the small and large intestines, the most pronounced changes were extensive immune and epithelial remodelling in the large intestines.

Immunological changes were further observed in the proximal small intestines with pathways related to interferon-beta production being over-represented and several genes indicating increased immune surveillance, among others: HLA-DQB1,35 CCL23,36 USP6,37 TLR4 (not shown).38 Several of the most significantly upregulated genes in the ileocaecal region, LCN2,39 CD2427 and IL20RA,40 suggest enhanced immune activity in this transitional zone between the less populated small intestine and the densely colonised large intestine, where the epithelium and associated immune cells must balance defence against pathogenic translocation with tolerance to commensal microbiota.1 4 41 Our results indicate that the immune activity is increased in the proximal to middle of the small intestine and in the transitional zone of the ileocaecal region in the individuals with T2D, pointing towards the small intestine as a focal point for T2D-related immune regulation.

The results from the large intestine indicate a marked shift in immune activity with 2046 differentially expressed genes, which were enriched in pathways related to both the adaptive and innate immune system activation. Relevant genes involved in immune activity include HLA-DQB2,35 FCRL6,42 EGR143 and FOS.44 Among these are the latter two transcriptional factors, suggesting persistent reconfiguration of the large intestinal mucosa. These results align well with numerous studies advocating a strong connection between intestinal inflammation and impaired glucose homeostasis. To this end, animal and human data suggest that low-grade inflammation of the intestinal mucosa, driven by either immune cell activation or microbial dysbiosis, contributes to the development and progression of T2D.45,48 Transfer of microbiota from lean, metabolically healthy individuals to individuals with metabolic dysfunction increases the recipients’ glucose tolerance,49 while elevated levels of circulating lipopolysaccharide (LPS), a marker of microbial translocation have been observed in individuals with T2D.50 51 LPS52 and circulating inflammation markers CRP53 and MCP-154 are all correlated with increased HbA1c levels. Notably, genetic55 or pharmacological suppression56 of inflammatory mediators, including TNF-α, improves insulin sensitivity in both mice and humans. We found increased immune activation in the intestines of individuals with T2D. In the large intestine, pathways related to extracellular matrix reorganisation are significantly regulated (P adj=3.35×10−5), while genes related to barrier function like MUC3, LAMC3, CLDN8 and CLDN2 (not shown) were among the most differentially expressed in the ileocaecal region and large intestine. These changes indicate, together with changes in fibroblasts, goblet cells and endothelial cells, a reorganisation of the gut barrier function, possibly to accommodate immune recruitment following bacterial encroachment into the mucosal layers. Blood bacterial DNA concentrations have been shown to be higher for individuals who later developed T2D, while adipose tissue from individuals with T2D has been shown to contain bacteria reminiscent of the individual’s gut microbiota,57 thus indicating bacterial translocation as a contributor to the low-grade inflammation, a key part of the pathophysiology behind T2D. Furthermore, glucose homeostasis is a trait that can be transplanted with the gut microbiome,58 while hyperglycaemia per se also drives gut barrier dysfunction.59 Thus, more research into intestinal inflammation and gut permeability in T2D is urgently warranted.

Several limitations should be considered when interpreting the present data set. While the depth of anterograde DBE was similar in the two groups,21 allowing for a uniform biopsy sampling, the retrograde DBE presented challenges. Specifically, in the majority of participants with T2D, obstruction of the passage into the ileum (often due to residual faeces21) prevented a comprehensive sampling at 30 cm intervals. Therefore, far from all participants in the two groups had the entire small intestine sampled.21 Furthermore, the small intestinal length is reported to be increased in individuals with T2D.60 Together, these notions may explain variances in gene expression. This claim is strengthened by pathways linked to ‘anterior/posterior pattern specification’ and ‘regionalisation’ being downregulated in individuals with T2D in regions 7 and 8 (online supplemental figures S1-S3). This disparity in biopsy locations provides a plausible explanation for some of the observed differences in gene expression but cannot rule out potential changes in gene expression due to the pathophysiology of T2D. Taken together, when using our data set, it is important to consider this potential bias in sampling locations in the distal small intestine between groups (that might involve a larger ‘uncharted’ area in the participants with T2D), which have been accounted for in all analyses included here. Additionally, our data set is limited by a small sample size (12 individuals in each group) and the limited demographic diversity of the participants (all of European descent). Due to a relatively small sample size, we did not further evaluate the impact of sex, age and BMI on gene expression levels. It is worth noting that for most genes, we observed considerable interindividual variability in expression, underscoring the importance for future studies aimed at understanding transcriptional responses along the intestinal tract. Lastly, it is important to note that the mucosal biopsies were retrieved after a 12-hour fast in sedated subjects due to the nature of the endoscopic procedures. This means that the gene expression in the biopsies reflects the fasting state, and thus, relevant differences between intestinal regions and between individuals with and without T2D in response to stimuli, such as a meal, are inherently beyond the scope of this data set.

This study represents the first map of the transcriptomic landscape of the human intestinal tract across 16 regions through frequent biopsies using DBE in living subjects. While the resulting data provide deep insights into mucosal molecular dynamics and T2D pathophysiology, we focus here on immune-related gene expression exemplifying how this data set can be used. We encourage readers to explore the database we made publicly accessible at https://rnaseq.gubra.dk/. There, researchers can explore the full extent of our findings and potentially uncover further associations and patterns that extend beyond the scope of this manuscript.

In conclusion, based on a unique DBE-based sampling procedure in 24 participants,21 we here provide a comprehensive database describing the transcriptomic landscape spanning the entire length of the human intestinal tract in individuals with and without T2D. The database represents a searchable atlas of the expression of 19.739 genes in the intestinal mucosa from 16 sites along the intestinal tract elucidating the anatomical distribution of GO enrichment analysis-derived biological functions in the human intestines as well as gene expression alterations in both cohorts. This publicly accessible, searchable database, housed at https://rnaseq.gubra.dk,20 provides a valuable resource for researchers investigating gene expression patterns that will catalyse further advances in our understanding of intestinal biology and T2D pathophysiology. In the present paper, we have used the database to demonstrate (1) regionalised gene expression patterns that may link anatomically well-defined areas of the intestines with the activity of processes such as immune response, inflammation, mitochondrial respiration, lipid metabolism and mitosis; and (2) regionalised gene expression changes in the intestines of individuals with T2D, highlighting cell-specific differences in immune system activity and barrier permeability, particularly within the ileocaecal region and across the large intestine. We highlight that the results discussed represent only the tip of the iceberg and hope that it will encourage other researchers to use this database for further exploration of gut biology in health and disease.

Supplementary material

online supplemental file 1
gutjnl-75-1-s001.docx (14.3MB, docx)
DOI: 10.1136/gutjnl-2024-334124
online supplemental file 2
gutjnl-75-1-s002.pptx (2.7MB, pptx)
DOI: 10.1136/gutjnl-2024-334124

Footnotes

Funding: This study was funded by Gentofte Hospital (Not Applicable), Novo Nordisk Fonden (Not Applicable).

Provenance and peer review: Not commissioned; externally peer reviewed.

Patient consent for publication: Not applicable.

Ethics approval: The study was approved by the Scientific-Ethical Committee of the Capital Region of Denmark (journal number H-3-2010-115) and the Danish Data Protection Agency and registered with ClinicalTrials.gov (NCT03044860). The study adhered to the latest revision of the Declaration of Helsinki and both oral and written consents were obtained from all participants before enrolment.

Data availability free text: Anonymous mRNA data are available indefinitely at https://rnaseq.gubra.dk/. The study protocol is included as a data supplement available with the online version of this article.

Patient and public involvement: Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

Data availability statement

Data are available in a public, open access repository.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

online supplemental file 1
gutjnl-75-1-s001.docx (14.3MB, docx)
DOI: 10.1136/gutjnl-2024-334124
online supplemental file 2
gutjnl-75-1-s002.pptx (2.7MB, pptx)
DOI: 10.1136/gutjnl-2024-334124

Data Availability Statement

Data are available in a public, open access repository.

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