Excess Dietary Sugar Alters Colonocyte Metabolism and Impairs the Proliferative Response to Damage

Abstract

Background & aims: The colonic epithelium requires continuous renewal by crypt resident intestinal stem cells (ISCs) and transit-amplifying (TA) cells to maintain barrier integrity, especially after inflammatory damage. The diet of high-income countries contains increasing amounts of sugar, such as sucrose. ISCs and TA cells are sensitive to dietary metabolites, but whether excess sugar affects their function directly is unknown.

Methods: Here, we used a combination of 3-dimensional colonoids and a mouse model of colon damage/repair (dextran sodium sulfate colitis) to show the direct effect of sugar on the transcriptional, metabolic, and regenerative functions of crypt ISCs and TA cells.

Results: We show that high-sugar conditions directly limit murine and human colonoid development, which is associated with a reduction in the expression of proliferative genes, adenosine triphosphate levels, and the accumulation of pyruvate. Treatment of colonoids with dichloroacetate, which forces pyruvate into the tricarboxylic acid cycle, restored their growth. In concert, dextran sodium sulfate treatment of mice fed a high-sugar diet led to massive irreparable damage that was independent of the colonic microbiota and its metabolites. Analyses on crypt cells from high-sucrose-fed mice showed a reduction in the expression of ISC genes, impeded proliferative potential, and increased glycolytic potential without a commensurate increase in aerobic respiration.

Conclusions: Taken together, our results indicate that short-term, excess dietary sucrose can directly modulate intestinal crypt cell metabolism and inhibit ISC/TA cell regenerative proliferation. This knowledge may inform diets that better support the treatment of acute intestinal injury.

Keywords: Colitis; DCA; Mitochondria; Renewal; Stemness.

Graphical abstract

Figure 1

Excess sugar impairs colonoid growth and reduces the expression of genes associated with Lgr5⁺ISCs. (A and B) Murine colonic crypts were cultured in increasing concentrations of sucrose, glucose, or fructose. (A) Representative images (magnification, 4×; scale bar: 200 μm), and (B) viability (percentage CellTiter-Glo luminescence of control), number, and size of colonoids after 5 days in culture are shown. (C and D) Human colonoids were dispersed to single cells and regrown with increasing concentrations of glucose. (C) Representative images (magnification, 4×; scale bar: 200 μm), and (D) average viability, number, and size of colonoids after 12 days in culture are shown. Data are representative of 2 experiments (n = 3) and data points are means ± SEM. One-way analysis of variance with multiple comparisons (with control), where ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (E and F) Murine colonoids cultured in 25 mmol/L (control) or 150 mmol/L of added glucose (Glucose) for 5 days were analyzed via RNAseq. (E) Volcano plot comparing control vs glucose-treated colonoids, red points in the volcano plot represent differentially expressed genes (-1.5 > FC >1.5; P < .05; FDR <0.3). (F) GSEA showing enriched gene sets (control vs glucose-treated). Concentrations of sugar represent the amount added in addition to what is present in the media (25 mmol/L). Avg, average; Conc., concentration; FDR, False Discovery Rate; NES, Normalized Enrichment Score.

Figure 2

Excess sugar is not toxic to fully developed colonoids. (A–C) Colonic crypts were isolated from mice and cultured for 5 days into fully developed 3-dimensional colonoids, which then were exposed to increased concentrations of added sucrose, glucose, or fructose for 2 days. (A) Representative images, (B) viability (percentage CellTiter-Glo [CTG] luminescence of control), and (C) number of organoids per well are shown. Images were taken at a magnification of 4× (scale bars: 200 μm). (D and E) After developing into mature human colonoids for 5 days, excess sugar was added for 2 days. (D) Average viability (percentage CTG luminescence of control) and (E) number of human colonoids are shown. (B–E) Data are representative of 2 experiments (n = 3) and data points are means ± SEM. Statistics represent (B) Kruskal–Wallis or (C and D) 1-way analysis of variance with multiple comparisons with control. ∗P < .05. Avg, average; Conc., concentration.

Figure 3

Excess glucose increases intracellular pyruvate but reduces total ATP levels in colonoids, and both metabolites are restored with DCA treatment. (A) Diagram of glycolysis and fatty acid oxidation pathways made with Biorender.com. (B–O) Isolated colonic crypts were cultured in 25 or 100 mmol/L of added glucose for 4 days, with and without 4 mmol/L DCA, and metabolite levels were measured via liquid chromatography–mass spectrometry. Quantified levels of metabolites as indicated. Data points represent means ± SEM and are representative of 2 experiments (n = 3–6). (C and F) Statistics represent 1-way analysis of variance or Kruskal–Wallis, where ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. ADP, Adenosine di-phosphate; AMP, Adenosine mono-phosphate; AU, arbitrary units; BP, bisphosphonate; CoA, Coenzyme-A; NAD, Nicotinamide Adenine Dinucleotide; NADH, Nicotinamide Adenine Dinucleotide Hydride.

Figure 4

Level of TCA cycle metabolites and phosphorylated PDH in glucose-treated colonoids. (A) Schema of TCA cycle. (B–I) Colonoids were cultured from single cells in 25 or 100 mmol/L of added glucose (totaling 50 and 125 mmol/L, respectively) for 4 days and metabolite levels from (B) media or (C–I) cells were measured via liquid chromatography–mass spectrometry. Data points represent means ± SEM and represent 2 experiments (n = 3–6). Statistics represent 1-way analysis of variance, where ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (J) Colonoids were cultured from single cells with or without 100 mmol/L added glucose (totaling 25 mmol/L for the control and 125 mmol/L for glucose-treated) for 6 days and protein expression of phospho-PDH (pPDH), PDH, and heat shock protein (HSP)90 were measured via Western blot. Representative blot of 4 experiments is shown. ADP, Adenosine di-phosphate; AU, arbitrary units; CoA, Coenzyme-A; FAD, Flavin adenine dinucleotide; FADH₂, Reduced Flavin adenine dinucleotide; HSP90, heat shock protein 90; NAD, Nicotinamide Adenine Dinucleotide; NADH, Nicotinamide Adenine Dinucleotide hydride.

Figure 5

Pyruvate dehydrogenase kinase inhibition with DCA rescues sugar-impaired colonoid development. (A and B) Murine colonoids were cultured in 150 mmol/L added sucrose, fructose, glucose (totaling 175 mmol/L), or no-sugar-added control, with or without DCA (dichloroacetate, 4 mmol/L). (A) Representative images after 4 days of culture in sugar and metabolic inhibitors (magnification, 4×; scale bars: 200 μm). (B) Quantification of panel A. Statistics represent Mann–Whitney comparing vehicle with DCA-treated, where ∗P < .05, ∗∗P < .01. (C) Colonoids grown from single cells in 150 mmol/L of added sucrose, fructose, or glucose (totaling 175 mmol/L) for 4 days with or without 7.8 mmol/L rotenone or 1 mmol/L 2-deoxyglucose (2-DG) for 4 days. Number of colonoids is shown. Data points represent means ± SEM and represent 2 experiments (n = 3). Stats represent 2-way analysis of variance with multiple comparisons (with vehicle control). (D–G) Colonoids with and without added glucose and/or DCA treatment were analyzed by RNAseq. (D) Principal component analysis (PCA) plot and (E) volcano plot comparing the transcriptome of glucose- and glucose/DCA-treated colonoids. (F) Transcript expression level of characteristic epithelial subset genes from various treated colonoids, as indicated. (G) GSEA of ISC signature enrichment (glucose/DCA-treated vs glucose-treated). Red points in volcano plot and dagger in heatmap represent differentially expressed genes (-1.5 > FC >1.5; P < .05; FDR <0.3). Avg, average; Ctrl, control; FDR, False Discovery Rate; NES, Normalized Enrichment Score.

Figure 6

The colonic epithelium can absorb luminal glucose, but do not express transporters to export glucose. (A and B) Lgr5^{IRES-GFP-cre-ERT2} mice were given a Cy5-glucose or anti-rat Cy5-secondary control enema for 30 minutes before being killed. (A) Representative images of colonic sections of individual and merged channels are shown. White arrowheads in magnified image show Cy5-glucose in Lgr5⁺ ISCs. Images were taken at a magnification of 40× (scale bar: 20 μm). (B) Percentage Cy5⁺ (glucose) colonic epithelial cells. Data are representative of 4 experiments and data points represent individual mice, error bars represent SEM. One-way analysis of variance was used to determine significance, where ∗∗P < .01 and ∗∗∗P < .001. (C–E) C57BL/6 or Rag1^-/- mice were fed HS or HF diets for 2 weeks and then treated with DSS. (C) Bulk colonic epithelium was isolated from Rag1^-/- female mice fed HS or HF diet for 2 weeks with or without 3 days of 3% DSS treatment, and transcriptome was assessed by RNAseq (n = 3–4). Glucose transporter expression level is shown. Data represent individual mice and error bars represent SEM. Dotted line represents no transcript. Blood glucose concentrations of HS- or HF-fed C57BL/6 mice: (D) postprandial and (E) fasted. Data are representative of 2–3 independent experiments (n = 3–4). Each data point represents individual mice, error bars represent SEM. (D) Mann–Whitney or (E) Student t test was used to determine significance. (F) FITC-dextran recovered from serum of HS- or HF-fed mice and from Std-fed mice treated with DSS for comparison. Data are representative of 2 independent experiments (n = 3), data points represent individual mice, error bars represent SEM. One-way analysis of variance was used to determine significance, where ∗P < .05. DAPI, 4′,6-diamidino-2-phenylindole; FPKM, fragments per kilobase exon per million mapped reads.

Figure 7

Sugar-supplemented water increases consumption and worsens DSS colitis. (A and B) Five-week-old female C57BL/6Tac mice were fed standard diet and water containing 10% sucrose, glucose, or fructose for 2 weeks and then treated with 3% DSS drinking water (dotted line) for 1 week. (A) Percentage initial weight and (B) survival are shown. (C–H) Five-week-old female C57BL/6Tac mice were fed HS, HF, or standard (Std) diet with 10% sucrose-supplemented water (SW) for 2 weeks. (C) Volume of water, (D) grams of sugar consumed per mouse, (E) weight change, (F) grams of food consumed per mouse, (G) kcal from food consumed per mouse, and (H) weights of mice on respective diets were measured. Analysis of variance was used to determine statistically significant differences in food or sugar intake between groups using body weight as a covariable, where ∗P < .05. (I) Mice were fed HF, HS, or Std diet with 10% mannitol water (MW) for 2 weeks and gavaged with carmine dye. The time to intestinally pass the carmine dye was measured. Data points represent means ± SEM and are representative of 2 experiments (n = 3–4). Multiple t tests performed comparing to Standard-fed mice were used to determine significance, where ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. (H) Five-week-old female C57BL/6Tac mice were fed diets with increasing concentrations of sucrose compared with fiber for 2 weeks. Weight change with different diets is shown. Kruskal–Wallis was used to determine significance.

Figure 8

Excess dietary sucrose leads to lethal DSS-induced colonic damage. (A–D) C57BL/6 mice were fed Std, HF, or HS diets for 2 weeks and then treated with 3% DSS drinking water for 1 week. (A) Percentage initial weight and (B) survival shown. (C) Representative H&E of colonic sections taken on day 6 of DSS (magnification, 4× and 20×; scale bar: 50 μm). (D) Histopathology score of blinded H&E sections, where scores of 1–5 indicate mild colitis, 6–10 indicate moderate colitis, and 11–17 indicate severe colitis. Kruskal–Wallis was used to determine significance, where ∗P < .05. (E and F) Mice fed diets with increasing concentrations of sucrose compared with fiber for 2 weeks and treated with DSS for 1 week. (E) Percentage initial weight and (F) survival shown. (G and H) Mice were fed 2 weeks of Std diet and switched to HS on the first day of DSS treatment (purple) or fed 2 weeks of HS then switched to Std diet on the first day of DSS (orange). (G) Percentage initial weight and (H) survival are shown. Data are representative of 3 experiments (n = 2–4) and data points represent means ± SEM. Multiple t tests were performed against HS per day using multiple comparisons and FDR <0.01, where ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001.

Figure 9

Effects of dietary sugar on the colonic microbiota. (A–D) C57BL/6 mice were fed HS or HF diets for 2 weeks and then treated with DSS, fecal samples were collected for 16S rRNA amplicon sequencing. (A) Schematic of diet/DSS treatment and days fecal samples were collected for 16S rRNA analysis. (Pre, day mice arrived at facility; Diet, 14 days of respective diet [sucrose-supplemented water (SW), Std with 10% sucrose in water]; DSS, collected during DSS treatment). (B) Relative abundances of the top 20 most abundant families. (C) Ordination plot based on the principle coordinate analysis (Bray–Curtis) shows taxonomic variations of microbial communities with various diet treatments (defined diets: HF and HS; standard diet: Std and SW). (D) Relative abundance of Akkermansia species as determined by 16S rRNA gene sequencing. (E and F) Mice fed HF, HS, or HS with SCFA supplementation in the water or tributyrin (TB)-supplemented HS diet for 2 weeks and then treated with DSS for 1 week. (E) Weight loss and (F) survival are shown. Data are representative of 2–3 independent experiments (n = 4). Data points represent means ± SEM. Multiple t tests were performed against HS per day, where ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. PC, Principal Coordinate.

Figure 10

HS diet induces lethal colitis independent of changes to the microbiota. (A and B) Germ-free mice were fed HS or Std diet for 2 weeks, then treated with 1% DSS for 1 week. (A) Percentage initial weight and (B) survival curve are shown. (C–E) Germ-free female C57BL/6 mice were gavaged with FMT from mice fed HS or HF diet for 2 weeks. After 3 days of intestinal colonization, mice were treated with 3% DSS drinking water for 1 week. (C) Schematic of FMT and DSS treatment. (D) Weight loss and (E) survival were measured. Data points represent means ± SEM from 2 independent experiments (n = 4) and ∗P < .05, ∗∗P < .01. GF, germ-free.

Figure 11

High-sucrose diet increases spare respiratory capacity of colonic crypt cells. (A–J) Colonic crypts isolated from mice fed HS or HF diet for 2 weeks and plated on a Matrigel-coated Seahorse XF analyzer plate. (A) Representative OCR trace and (B) quantification of basal OCR (average OCR before oligomycin addition). (C) Representative ECAR trace after and (D) quantification of basal ECAR (average ECAR before oligomycin addition). (E) OCR to ECAR ratio, using basal rates from panels B and D. (F) Schema of how SRC, ATP-associated OCR, and basal OCR were measured. (G) Quantification of SRC (difference between basal and maximal oxidative rates, achieved after FCCP injection). (H) Representative ECAR trace after 3-hour glucose deprivation and (I) glycolytic rate measured by subtracting basal rate after 2-deoxyglucose (2-DG) injection from maximum response after glucose injection. (J) Tabulated ATP-associated OCR (difference between basal OCR and OCR after oligomycin injection). (K) Five-week-old C57Bl/6 female mice were fed HS or HF diet for 2 weeks, the colonic epithelium was isolated, and ATP concentration was measured. (L–N) Lgr5^{eGFP-Cre-ERT2} reporter mice were fed HS or HF diet for 2 weeks and colonic crypts were dispersed to single cells or fixed for microscopy and fluorescently stained. (L) Mean fluorescent intensity of MitoTracker Deep Red (MTDR MFI) and (M) MitoSox for all EPCAM⁺ cells. (N) Ratio of MitoTracker Deep Red MFI to MitoSox MFI in EPCAM⁺ cells. Data are representative of 2–4 experiments (n = 2–4) and data points represent means ± SEM. Tabulated bar charts represent means ± SEM, with each point representing 1 mouse. (E, J, and K) Significance was determined by Student t test or Mann–Whitney, where ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. Metabolic inhibitors used were oligomycin (oligo), FCCP, 2-DG, and rotenone with antimycin (rot/aa).

Figure 12

Metabolic changes in colonic ISCs with increased dietary sucrose. (A–I) Lgr5^{IRES-GFP-cre-ERT2} mice were fed HS or HF diet for 2 weeks, and colonic epithelium was isolated and stained for flow cytometry. (A–F) Lgr5⁺ ISCs were sorted for metabolic analysis on a Seahorse XF analyzer. (A) Representative trace of OCR, (B) tabulated basal OCR, (C) representative trace of ECAR, (D) tabulated basal ECAR, and (E) ratio of basal OCR to ECAR (taken from panels B and D). (F) Tabulated SRC (difference between basal and maximal oxidative rates, achieved after FCCP injection). (G) Ratio of MitoTracker Deep Red mean fluorescence intensity (MFI) to MitoSox MFI in Lgr5⁺ cells. (H) MFI of MitoTracker Deep Red (MTDR) and (I) MitoSox for Lgr5⁺ cells. Data points represent individual mice, means ± SEM from 4 independent experiments (n = 2–4) are shown. (E) Student t test or Mann–Whitney test used to determine significance. Metabolic inhibitors used were oligomycin (oligo), FCCP, 2-deoxyglucose (2-DG), and rotenone with antimycin (Rot/AA). (J) Representative images of p-PDH staining (±HS diet, ±DSS; 3 days) and (K) quantification of the level of p-PDH MFI in crypt base cells (magnification, 40×; scale bars: 50 μm). Data are representative of 2 independent experiments (n = 2–3). Data points represent means ± SEM. Kruskal–Wallis was used to determine statistical significance, where ∗P < .05. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 13

HS diet reduces expression of proliferation-related genes in Lgr5⁺ISCs. (A–D) Lgr5⁺ ISCs were isolated by flow cytometry from Lgr5^{eGFP-Cre-ERT2} HS- or HF-fed mice and analyzed by RNAseq. (A) Transcript expression level of epithelial subset gene signatures and (B) Principal Component Analysis (PCA) plot showing variance with percentages on axes representing the percentage variance explained by each principle component. (C) Volcano plot for genes comparing HS- or HF-treated samples, where red points represent differentially expressed genes (-1.5 > FC >1.5; ∗P < .05; FDR <0.3). (D) GSEA of Lgr5⁺ ISC RNAseq data showing enrichment of genes in HF-fed or HS-fed mice for gene sets indicated. For heatmap, dagger represents DEG in HS vs HF Lgr5⁺ ISCs (-1.5 > FC >1.5; ∗P < .05; FDR <0.3). FDR, False Discovery Rate; max, maximum; min, minimum; NES, Normalized Enrichment Score.

Figure 14

The transcriptome of the colonic epithelium with increased dietary sucrose at steady-state and after DSS-induced damage. (A and B) Rag1^-/- mice were fed HF or HS diet for 2 weeks, and then exposed to 3% DSS drinking water for 7 days. (A) Percentage age initial weight and (B) survival shown (n = 3–4, means ± SEM). (C–H) Bulk colonic epithelium was isolated from Rag1^-/- female mice fed HS or HF diet for 2 weeks with or without 3 days of 3% DSS treatment and the transcriptome was measured via RNAseq (n = 3–4). (C) Expression level of epithelial subset gene signatures. (D) Principal Component Analysis (PCA) plot showing variance between samples from differentially treated mice (as indicated) with percentages on axes representing percentage variance explained by each principle component. (E) Volcano plot comparing genes isolated from colon epithelium of HS- or HF-treated mice and (F) volcano plot comparing genes isolated from colon epithelium of HS/DSS- or HF/DSS-treated mice. Red points represent differentially expressed genes (-1.5 > FC >1.5, ∗P < .05, FDR <0.3). Specific genes are called out with arrowheads. (G) GSEA of colonic epithelium RNAseq data showing enrichment of genes in HF/DSS-treated or HS/DSS-treated mice for gene sets as indicated. (H) Heatmap of regulatory enzymes of glycolysis and TCA cycle where red and blue represent high or low expression level, respectively, normalized across rows. For heatmaps, differentially expressed genes are denoted with a dagger, comparing HS/DSS vs HF/DSS epithelium (-1.5 > FC >1.5, ∗P < .05, FDR <0.3). Epi-Mes, Epithelial-Mesenchymal; max, maximum; min, minimum; Reg., regulatory.

Figure 15

After DSS-induced damage, HS diet reduces colonic crypt cell proliferation but does not increase epithelial cell death. (A and B) Colons were isolated from HS- or HF-fed mice, treated 3 days with or without DSS. (A) Representative images of colonic sections stained for Ki67 in green (magnification, 20×; scale bars: 50 μm) and (B) percentage of EPCAM⁺ cells that are Ki67⁺ from flow cytometric analysis. (C) Representative TUNEL of colonic sections after 3 days of 3% DSS treatment in mice fed HS or HF diet for 2 weeks. Images were taken at a magnification of ×10 (scale bars: 100 μm). (D) Percentage of cells that are TUNEL⁺. (E) Mice were fed HS or HF diet and treated 3 days with 3% DSS, colonic epithelium was isolated and stained with activated caspase-3 for flow cytometric analysis. Data represent the percentage of EPCAM⁺ cells that are activated caspase-3⁺. (F) Number of EPCAM⁺ colonocytes after 4 days of DSS from flow cytometric analysis. (G–J) Lgr5^{eGFP-Cre-ERT2}Rosa^{LSL-TandemDimerTomato} mice fed HS or HF diet for 2 weeks and injected with tamoxifen to induce Tomato expression from Lgr5-expressing ISCs on first day of DSS treatment. (G) Representative images of colonic crypts with Lgr5^eGFP (green) and Tomato⁺ progeny (red) (magnification, 60×; scale bars: 10 μm), (H) number of TdT⁺ cells per crypt, (I) height of most distant Tomato⁺ progeny from bottom of crypt (averaged per GFP⁺ crypt), and (J) percentage of GFP⁺ crypts containing Tomato⁺ progeny at the specified position along crypts are shown after 3 days of DSS. (K) Lgr5^{eGFP-Cre-ERT2}Rosa^LSL-TdTomato mice were fed HS or HF diet for 2 weeks, injected with tamoxifen to induce Tomato expression on the first day of DSS treatment, and injected with EdU on day 3 of DSS, 4 hours before being killed. Percentage of EdU⁺ cells, as measured by microscopy, are shown. Data are representative of 2 experiments (n = 2–5) and data points represent means ± SEM. (F and K) One-way analysis of variance or Kruskal–Wallis test were used to determine significance, where ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. avg, average; DAPI, 4′,6-diamidino-2-phenylindole; Max. maximum.