Tributyrin in Inflammation: Does White Adipose Tissue Affect Colorectal Cancer?

Abstract

Colorectal cancer affects the large intestine, leading to loss of white adipose tissue (WAT) and alterations in adipokine secretion. Lower incidence of colorectal cancer is associated with increased fibre intake. Fructooligosaccharides (FOS) are fibres that increase production of butyrate by the intestinal microbiota. Tributyrin, a prodrug of butyric acid, exerts beneficial anti-inflammatory effects on colorectal cancer. Our aim was to characterise the effects of diets rich in FOS and tributyrin within the context of a colon carcinogenesis model, and characterise possible support of tumorigenesis by WAT. C57/BL6 male mice were divided into four groups: a control group (CT) fed with chow diet and three colon carcinogenesis-induced groups fed either with chow diet (CA), tributyrin-supplemented diet (BUT), or with FOS-supplemented diet. Colon carcinogenesis decreased adipose mass in subcutaneous, epididymal, and retroperitoneal tissues, while also reducing serum glucose and leptin concentrations. However, it did not alter the concentrations of adiponectin, interleukin (IL)-6, IL-10, and tumour necrosis factor alpha (TNF)-α in WAT. Additionally, the supplements did not revert the colon cancer affected parameters. The BUT group exhibited even higher glucose tolerance and levels of IL-6, VEGF, and TNF-α in WAT. To conclude our study, FOS and butyrate supplements were not beneficial. In addition, butyrate worsened adipose tissue inflammation.

Keywords: butyrate; colon carcinogenesis; fructooligosaccharides; white adipose tissue.

Figure 1

Colon carcinogenesis experimental protocol: colon carcinogenesis was induced chemically by a single dose of azoxymethane (AOM), with 10 mg/kg body weight administered intraperitoneally on the 30th day, and subsequently three cycles of dextran sodium sulphate (DSS) consisted of 5 days drinking DSS and 15 days drinking water. The diet started on day 0 and finished on the 90th day.

Figure 2

Butyrate reduces body weight and leptin: evolution of body weight (A), weight gain (B), food consumption (C), percent survival (D), serum leptin (E), and water intake (F). Mice were divided into four groups: control group (CTRL—n = 9–10) fed with chow diet; and colon carcinogenesis-induced groups fed either with chow diet (CA—n = 9–10), tributyrin-supplemented diet (BUT—n = 5–6), or with FOS-supplemented diet (FOS—n = 9–10). Colon carcinogenesis was induced chemically by azoxymethane (AOM)/dextran sodium sulphate (DSS). Data are presented as means ± SEM. The groups were compared using one-way ANOVA followed by Bonferroni post hoc tests; for p < 0.05 (a), p < 0.01 (b), p < 0.001 (c), and p < 0.0001 (d); black circles (●), square (∎), triangles (▲), inverted triangles (▼) represents sample numbers in the corresponding group.

Figure 3

Butyrate reduces adipose tissue, gastrocnemius tissue mass, and liver weight: weights of subcutaneous (A), retroperitoneal (B), epididymal (C), mesenteric (D), brown (E), gastrocnemius skeletal muscle (F), and liver (G) tissue are presented below. Mice were divided into four groups: control group (CTRL—n = 9–10) fed with chow diet; and colon carcinogenesis-induced groups fed either with chow diet (CA—n = 9–10), tributyrin-supplemented diet (BUT—n = 5–6), or with FOS-supplemented diet (FOS—n = 9–10). Colon carcinogenesis was induced chemically by azoxymethane (AOM)/dextran sodium sulphate (DSS). Data are presented as means ± SEM. The groups were compared using one-way ANOVA followed by Bonferroni post hoc tests; for p < 0.05 (a), p < 0.01 (b), p < 0.001 (c), and p < 0.0001 (d).

Figure 4

Lipid profile: total cholesterol (A), triacylglycerol (B), and non-esterified fatty acid (C). Mice were divided into four groups: control group (CTRL—n = 9–10) fed with chow diet; and colon carcinogenesis-induced groups fed either with chow diet (CA—n = 9–10), TB-supplemented diet (BUT—n = 5–6), or with FOS-supplemented diet (FOS—n = 9–10). Colon carcinogenesis was induced chemically by azoxymethane (AOM)/dextran sodium sulphate (DSS). Data are presented as means ± SEM. After outlier exclusion, the groups were compared using one-way ANOVA followed by Bonferroni post hoc tests; black circles (●), square (∎), triangles (▲), inverted triangles (▼) represents sample numbers in the corresponding group.

Figure 5

Butyrate reduces glycemia: glucose tolerance test (A), area under the curve (B), and glycaemia (C). Mice were divided into four groups: control group (CTRL—n = 9–10) fed with chow diet; and colon carcinogenesis-induced groups fed either with chow diet (CA—n = 9–10), TB-supplemented diet (BUT—n = 5–6), or with FOS-supplemented diet (FOS—n = 9–10). Colon carcinogenesis was induced chemically by azoxymethane (AOM)/dextran sodium sulphate (DSS). Data are presented as means ± SEM. The groups were compared using one-way ANOVA followed by Bonferroni post hoc tests; for p < 0.01 (b), p < 0.001 (c), and p < 0.0001 (d); black circles (●), square (∎), triangles (▲), inverted triangles (▼) represents sample numbers in the corresponding group.

Figure 6

Butyrate elevates adiponectin content in subcutaneous and retroperitoneal adipose tissue: subcutaneous (A), retroperitoneal (B), epididymal (C), and mesenteric (D) adipose tissues, and serum (E). Mice were divided into four groups: control group (CTRL—n = 7–8) fed with chow diet; and colon carcinogenesis-induced groups fed either with chow diet (CA—n = 7–8), TB-supplemented diet (BUT—n = 4–5), or with FOS-supplemented diet (FOS—n = 7–8). Colon carcinogenesis was induced chemically by azoxymethane (AOM)/dextran sodium sulphate (DSS). Data are presented as means ± SEM. After outlier exclusion, the groups were compared using one-way ANOVA followed by Bonferroni post hoc tests; for p < 0.05 (a); black circles (●), square (∎), triangles (▲), rhombus (◆) represents sample numbers in the corresponding group.

Figure 7

Butyrate promote hypertrophy of colonic crypts: Histological images of the colon (A) and colon crypt height (B). The tissues were subjected to haematoxylin and eosin (H&E) staining; the images represent 10× and 40× magnification; magnification bar: 100 µm. Mice were divided into four groups: control group (CTRL—n = 5) fed with chow diet; and colon carcinogenesis-induced groups fed either with chow diet (CA—n = 5), TB-supplemented diet (BUT—n = 5), or with FOS-supplemented diet (FOS—n = 5). Colon carcinogenesis was induced chemically by azoxymethane (AOM)/dextran sodium sulphate (DSS). Data are presented as means ± SEM. The groups were compared using one-way ANOVA followed by Bonferroni post hoc tests; for p < 0.05 (a).

Figure 8

Cell infiltration in the lamina propria of the colon mucosa: Histological images of the colon (A) percentage of eosinophils (blue arrows, (B)), plasma cells (green arrows, (C)), and fibroblasts (yellow arrows, (D)) in all lamina propria cells. The tissues were subjected to H&E staining; the images represent 100 × magnification; magnification bar: 50 µm. Mice were divided into four groups: control group (CTRL—n = 5) fed with chow diet; and colon carcinogenesis-induced groups fed either with chow diet (CA—n = 5), TB-supplemented diet (BUT—n = 5), or with FOS-supplemented diet (FOS—n = 5). Colon carcinogenesis was induced chemically by azoxymethane (AOM)/dextran sodium sulphate (DSS). Data are presented as means ± SEM. The groups were compared using one-way ANOVA followed by Bonferroni post hoc tests; for p < 0.05 (a), p < 0.01 (b); black circles (●), square (∎), triangles (▲), inverted triangles (▼) represents sample numbers in the corresponding group.

Figure 9

Colon carcinogenesis induction did not promote pro-inflammatory markers, but reduced IL-10 content in the colon: IL-6 (A), TNF-α (B), IL-10 (C), TNF-α/IL-10 ratio (D) in colon. Mice were divided into four groups: control group (CTRL—n = 5) fed with chow diet; and colon carcinogenesis-induced groups fed either with chow diet (CA—n = 5), TB-supplemented diet (BUT—n = 4), or with FOS-supplemented diet (FOS—n = 4). Colon carcinogenesis was induced chemically by azoxymethane (AOM)/dextran sodium sulphate (DSS). Data are presented as means ± SEM. The groups were compared using one-way ANOVA followed by Bonferroni post hoc tests; for p < 0.05 (a); black circles (●), square (∎), triangles (▲), inverted triangles (▼) represents sample numbers in the corresponding group.

Figure 10

Butyrate promoted pro-inflammatory markers (IL-6 and TNF-α) and IL-10 in different adipose depots: IL-6, TNF-α, and IL-10 concentrations in subcutaneous (A–C), retroperitoneal (D–F), epididymal (G–I), and mesenteric (J–L) adipose tissues. Mice were divided into four groups: control group (CTRL—n = 6–9) fed with chow diet; and colon carcinogenesis-induced groups fed either with chow diet (CA—n = 4–9), TB-supplemented diet (BUT—n = 3–4), or with FOS-supplemented diet (FOS—n = 5–9). Colon carcinogenesis was induced chemically by azoxymethane (AOM)/dextran sodium sulphate (DSS). Data are presented as means ± SEM. After outlier exclusion, the groups were compared using one-way ANOVA followed by Bonferroni post hoc tests; for p < 0.01 (b), p < 0.001 (c), and p < 0.0001 (d); black circles (●), square (∎), triangles (▲), rhombus (◆) represents sample numbers in the corresponding group.

Figure 11

Butyrate promoted elevation of VEGF in retroperitoneal adipose tissue: VEGF concentrations in subcutaneous (A), retroperitoneal (B), epididymal (C), and mesenteric (D) adipose tissue, serum (E), and the colon (F). Mice were divided into four groups: control group (CTRL—n = 6–9) fed with chow diet; and colon carcinogenesis-induced groups fed either with chow diet (CA—n = 4–9), TB-supplemented diet (BUT—n = 3–4), or with FOS-supplemented diet (FOS—n = 5–9). Colon carcinogenesis was induced chemically by azoxymethane (AOM)/dextran sodium sulphate (DSS). Data are presented as means ± SEM. After outlier exclusion, the groups were compared using one-way ANOVA followed by Bonferroni post hoc tests; for p < 0.05 (a), p < 0.01 (b); black circles (●), square (∎), triangles (▲), rhombus (◆) represents sample numbers in the corresponding group.