Indole-3-propionic acid links gut dysfunction to diabetic retinopathy: a biomarker and novel therapeutic approach

Abstract

Background: Both host and microbe metabolism of tryptophan (Trp) is altered in diabetes; however, the molecular mechanisms are incompletely understood.

Objective: We used strategies to increase either angiotensin converting enzyme-2 (ACE-2) dependent or independent Trp absorption in a model of type 2 diabetes, db/db mice, and tested whether the strategies could prevent development of diabetic retinopathy (DR), the most common microvascular complication of diabetes. Additionally, we investigated levels of Trp metabolites in humans with and without DR.

Design: Enhanced ACE-2 dependent Trp absorption was achieved with gavage of genetically modified bacteria that preserved intestinal ACE2:sodium coupled neutral amino acid transporter expression. ACE-2 independent Trp absorption was achieved by gavage of the Trp dipeptide (Isoleucine-Trp; IW) absorbed via solute carrier family 15 member 1. Both strategies were used either as a prevention (6 months treatment) or intervention (3 months treatment) and at the conclusion, intestinal, metabolic and retinal studies were performed including spatial mass spectroscopy (MS). Plasma Trp metabolites and gut permeability markers were measured in individuals with T2D with (n=30) and without (n=40) DR and compared with healthy controls (n=35).

Results: Lactobacillus paracasei-ACE2 or IW treatment prevented DR, corrected dysbiosis, enriched Trp-metabolising bacteria, improved gut barrier integrity, boosted incretin secretion and restored glucose homeostasis in db/db mice. Spatial MS identified indole propionic acid (IPA) as a metabolite in the retinal pigment epithelial layer protecting the posterior blood retinal barrier. T2D individuals with DR demonstrated elevated serum markers of endotoxemia and intestinal barrier disruption while showing reduced levels of the beneficial metabolite IPA and elevated levels of the toxic metabolite indole sulfate.

Conclusion: Nutraceutical strategies that restore Trp metabolism or IPA serve as both a biomarker and a treatment for DR.

Keywords: BACTERIAL TRANSLOCATION; GUT INFLAMMATION; INTESTINAL BARRIER FUNCTION; INTESTINAL PERMEABILITY; METABOLOMICS.

Figure 1:. Oral administration of LP-ACE2 and IW prevents the development of DR in db/db mice.

Schematic of experimental design shows the preventive treatment regimen in db/db mice. Starting at two months of age, immediately after the establishment of diabetes, mice were gavaged with LP-ACE2 (three times per week) or IW (daily) for six months. At study completion (eight months of age), tissues were collected for analysis. Saline-treated db/db mice and non-diabetic WT mice served as controls (A). Retinal function was assessed using ERGs. Photopic a- wave (n=3–5/group) (B), Photopic b-wave (n=4–12/group) (C), Scotopic a-wave (n=4–9/group) (D), and Scotopic b-wave (n=4–8/group) (E) Amplitudes were significantly improved in LP-ACE2- and IW-treated mice compared to saline-treated db/db controls. Visual acuity, measured by OKN, showed increased spatial frequency thresholds in treated groups (n=10–11/group) (F). DR was evaluated by quantifying acellular capillaries in retinal flat mounts. Representative brightfield images and quantification revealed a significant reduction in acellular capillaries in LP-ACE2- and IW-treated mice, indicating reduced retinal vascular pathology (n=4–6/group) (G, H). Each black data point represents an individual experimental animal. The schematic of experimental design was created using BioRender, an online scientific illustration software. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s HSD correction for multiple comparisons. Scale bar = 20 μm.

Figure 2:. LP-ACE2 and IW treatment ameliorates gut dysbiosis, structural alterations, and goblet cell dysfunction in db/db mice.

To evaluate the impact of LP-ACE2 and IW on diabetes-induced gut dysbiosis, 16S rRNA sequencing and metatranscriptomic analyses were performed. Microbial diversity was assessed by measuring α-diversity using observed species counts (n=4–9/group) (A), β-diversity via partial least squares discriminant analysis (PLS-DA) (n=4–9/group) (B), and phylogenetic differences (n=4–9/group) (C). To assess structural changes in the gut, immunohistochemistry was performed on small intestinal sections. Representative brightfield images show H&E staining, Ki67-positive proliferating cells, and Alcian blue staining for mucin-producing goblet cells (D). Quantitative analysis included villus-to-crypt length ratio (n=6–10/group) (E), Ki67 proliferative index (n=8/group) (F), and goblet cell counts (n=13/group) (G). Both LP-ACE2 and IW treatments significantly improved gut architecture, enhanced epithelial proliferation, and increased mucin production, indicating restoration of gut barrier integrity. Each black data point represents an individual experimental animal. All statistically significant changes were normally distributed and determined using one-way ANOVA with Tukey’s HSD correction for multiple comparisons. Scale bar = 50 μm.

Figure 3:. IPA treatment mitigates gut barrier defects and improves glucose metabolism in db/db mice.

LP-ACE2 and IW treatment restores diabetes-induced depletion of indole levels measured by ELISA (n=4–8/group) and IPA levels presented as fold change and measured by LC/MS (n=4–5/group) (A-B). The presence of IPA in the WT retina was detected by MALDI imaging (n=5) (C). Schematic of the experimental design for IPA treatment regimen in db/db mice. Starting at two months of age, immediately after diabetes onset, mice were gavaged with IPA (daily) for six months. At study completion (eight months of age), tissues were collected for analysis. Saline-treated db/db mice and non-diabetic WT mice served as controls (D). The effect of IPA on gut epithelial and endothelial barrier dysfunction were evaluated using immunofluorescence staining. Representative images of intestinal sections showing the expression of epithelial junctional proteins ZO-1 (green) and p120-catenin (red) and endothelial barrier markers PV1 (green) and VE-cadherin (red) (E). The quantification of immunostaining of ZO-1(n=8/group) (F), p120-catenin (n=8/group) (G), PV-1 (n=6/group) (H), and VE-cadherin (n=6/group) (I). To assess the effect of IPA on glucose metabolism, the levels of incretins, GIP (n=6–7/group) (J) and GLP-1 (n=6–7/group) (K) were measured in plasma samples. Each black data point represents an individual experimental animal. The schematic of experimental design was created using BioRender, an online scientific illustration software. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with HSD correction for multiple comparisons. Scale bar = 50 μm (20X magnification).

Figure 4:. IPA treatment preserves canonical signaling and mitigates diabetes induced inflammation.

To assess the impact of IPA on canonical signaling, the expression of TLR-4 and its downstream effectors MyD88, Caspase-1, and NLRP3 were detected in the small intestine tissue lysates by Western Blots and quantified (n=3/group) (A-E). The effect of IPA on TLR-4, MyD88, and IL-1β expression in RPE cells was detected using immunostaining and flow cytometry (F-K). Representative images of TLR4 (red) expression and its quantification (n=7–8/group) (F,G), the number of MyD88 positive cells measured by flow cytometry (n=3–4/group) (H, I), and IL-1β (green) staining and its quantification (n=7/group) (J,K). Each black data point represents an independent biological replicate. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s HSD correction for multiple comparisons. In Western Blot images, each lane represents a sample from an individual experimental mouse. Scale bar = 50 μm (20X magnification).

Figure 5:. IPA treatment modulates intestinal immune cell populations and reduces retinal immune cell infiltration in db/db mice.

A schematic illustrates the experimental approach involving photoconversion of intestinal immune cells and their migration to the retina (A). Immunostaining of retinas from KIK::GR mice revealed a higher number of pathogenic Th17 cells in diabetic mice (lower panel) compared to controls (upper panel) (B). Red Th17 cells represent cells that migrated from the photoactivated gut region to the retina. The effect of IPA treatment on hematopoiesis in diabetic mice was assessed by detecting long-term hematopoietic stem cells (LT-HSC⁺) (n=3–4/group) and granulocyte-monocyte progenitors (GMP⁺) (n=3–4/group) in the bone marrow (C,D), peripheral monocytes(n=3–4/group) and classical monocytes(n=3–4/group) (E,F) and Th17 cells in the gut (n=3–4/group) and retina (n=2–3/group) (G, H) using flow cytometry. Each black data point represents an independent biological replicate. The schematic of experimental design was created using BioRender, an online scientific illustration software. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s HSD correction for multiple comparisons. Scale bar = 20 μm (20X magnification).

Figure 6:. LP-ACE2, IW and IPA treatments attenuates diabetes-induced reduction of AhR and PXR expression in intestine and HIEC under diabetic mimicking conditions.

The effect of LP-ACE2 or IW treatment on AhR and PXR protein expression was determined by Western Blots and quantified (n=3/group) (A-C). After 24 hrs of DIZE, IW and IPA treatment, AhR and PXR expression in HIEC was determined under diabetic mimicking conditions. HIECs were exposed to LPS and high glucose (HG) for 24 hours to mimic diabetes in vitro. Representative images of AhR (green) (n=10–19/group) and PXR (red) (n=8–18/group) expression in all the cohorts and their quantification (D-F). Each black data point represents an independent biological replicate. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s HSD correction for multiple comparisons. In Western Blot images, each lane represents a sample from an individual experimental mouse. Scale bar = 50 μm (20X magnification).

Figure 7:. IW restores diabetes-induced retinal PXR downregulation in db/db mice and DIZE, IW, and IPA enhance PXR expression in RPE and HRECs.

Representative images of human retina from healthy controls or diabetics showing PXR expression (red) and its quantification (n=5–6/group) (A,B). Representative images of PXR expression in the retinal tissue sections of all three experimental cohorts, WT, db/db treated with saline and db/db treated with IW and its quantification (n=3–4/group) (C,D). After 24 hours of exposure to DIZE, IW, or IPA, PXR expression was determined in RPE (green; E, G; n=7–8/group) and HRECs (red; F, H; n=6–8/group) under diabetic mimicking conditions. Each black data point represents an independent biological replicate. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s HSD correction for multiple comparisons. In Western Blot images, each lane represents a sample from an individual experimental mouse. Scale bar = 50 μm (20X magnification).

Figure 8:. Intervention with LP-ACE2, IPA and IW in db/db mice significantly enhanced gut barrier integrity, preserved retinal structure and function, and modulated immune responses.

Schematic of experimental design shows the intervention regimen in db/db mice. Starting at five months of age, three months after the establishment of diabetes, db/db mice were gavaged with LP-ACE2 (three times per week), IW and IPA (daily) for another 3 months. At study completion (6 months of diabetes), tissues were collected for analysis. Saline-treated db/db mice and non-diabetic WT mice served as controls (A). The levels of blood glucose (n=3–7/group) (B) and HgA1c (n=3–7/group) (C) were measured in all five cohorts. The effects of LP-ACE2, IW and IPA on diabetes-induced gut epithelial and endothelial barrier dysfunction were evaluated using immunofluorescence staining. Representative images show expression of epithelial junctional proteins p120-catenin (red), ZO-1 (green) and endothelial barrier integrity markers PV1 (green) and VE-cadherin (red) (D). The quantification of epithelial markers (n=8–10/group) (E-F) and endothelial markers (n=6–10/group) (G-H) confirmed changes in these markers in saline-treated db/db mice with restoration in all three treatment cohorts. Plasma levels of gut permeability markers were measured by FABP2 ELISA (n=3–6/group) (I) and LPS (n=3–6/group) (J). Each black data point represents an individual experimental replicate. The schematic of experimental design was created using BioRender, an online scientific illustration software. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s HSD correction for multiple comparisons. Scale bar = 50 μm (20X magnification).

Figure 9:. Impaired tryptophan metabolism and barrier function in T2D subjects with DR.

Targeted metabolomic analysis was performed to assess L-Trp and its downstream metabolites in the plasma of T2D individuals and age- and sex-matched healthy controls. Plasma levels of L-Trp (n=18–29/group) (A), indole (n=15–24/group) (B), IPA (n=16–25/group) (C), IA (n=8–12/group) (D), ICAld (n=11–20/group) (E), ILA (n=12–25/group) (F), IGA (n=15–19/group) (G), and IAA (n=14–17/group) (H) in T2D subjects with and without DR. Plasma levels of deleterious indole metabolite IS in T2D subjects with and without DR (n=17–23/group) (I). Gut barrier integrity was evaluated by quantifying plasma levels of gut permeability markers: FABP2 (n=18–28/group) (J), PGN (n=18–29/group) (K), and LBP (n=16–28/group) (L) using ELISA. All markers were significantly altered in T2D subjects compared to controls with further changes correlating with DR severity. Each black dot represents an individual human subject. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s HSD correction for multiple comparisons.