The Ketogenic Diet Reduces the Harmful Effects of Stress on Gut Mitochondrial Biogenesis in a Rat Model of Irritable Bowel Syndrome

Abstract

Functional alterations in irritable bowel syndrome have been associated with defects in bioenergetics and the mitochondrial network. Effects of high fat, adequate-protein, low carbohydrate ketogenic diet (KD) involve oxidative stress, inflammation, mitochondrial function, and biogenesis. The aim was to evaluate the KD efficacy in reducing the effects of stress on gut mitochondria. Newborn Wistar rats were exposed to maternal deprivation to induce IBS in adulthood. Intestinal inflammation (COX-2 and TRL-4); cellular redox status (SOD 1, SOD 2, PrxIII, mtDNA oxidatively modified purines); mitochondrial biogenesis (PPAR-γ, PGC-1α, COX-4, mtDNA content); and autophagy (Beclin-1, LC3 II) were evaluated in the colon of exposed rats fed with KD (IBD-KD) or standard diet (IBS-Std), and in unexposed controls (Ctrl). IBS-Std rats showed dysfunctional mitochondrial biogenesis (PPAR-γ, PGC-1α, COX-4, and mtDNA contents lower than in Ctrl) associated with inflammation and increased oxidative stress (higher levels of COX-2 and TLR-4, SOD 1, SOD 2, PrxIII, and oxidatively modified purines than in Ctrl). Loss of autophagy efficacy appeared from reduced levels of Beclin-1 and LC3 II. Feeding of animals with KD elicited compensatory mechanisms able to reduce inflammation, oxidative stress, restore mitochondrial function, and baseline autophagy, possibly via the upregulation of the PPAR-γ/PGC-1α axis.

Keywords: animal model; irritable bowel syndrome; ketogenic diet; mitochondria.

Figure 1

Western blot analysis of COX-2 (Panel (A)) and TLR-4 (Panel (B)) levels in colon samples of the control, IBS-Std, and IBS-KD rats, with each group consisting of four rats. Data were analyzed by Kruskal–Wallis analysis of variance and Dunn’s Multiple Comparison Test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

SOD 1 (Panel (A)) and SOD 2 (Panel (B)) levels, and western blot analysis of PrxIII (Panel (C)) in colon samples of the control, IBS-Std, and IBS-KD rats, with each group consisting of four rats. Data were analyzed by Kruskal–Wallis analysis of variance and Dunn’s Multiple Comparison Test.

Figure 3

Incidence of oxidatively modified purines at the D-loop in the colon samples of the control, IBS-Std, and IBS-KD rats, with each group consisting of four rats. Data obtained using the oxidized purines-sensitive enzyme formamidopyrimidine DNA glycosylase (Fpg). Panel (A): the graph represents the ratio between Fpg-treated and untreated band intensities expressed as the complement to 100%. Data were analyzed by Kruskal–Wallis analysis of variance and Dunn’s Multiple Comparison Test (** p < 0.01). Panel (B): Representative gels showing amplicons obtained from Fpg-treated and untreated total DNA.

Figure 4

Western blot analysis of PPAR-γ (Panel (A)), PGC-1α (Panel (B)), COX-4 (Panel (C)), and VDAC1 (Panel (D)) levels in colon samples of the control, IBS-Std, and IBS-KD rats, with each group consisting of four rats. Data were analyzed by Kruskal–Wallis analysis of variance and Dunn’s Multiple Comparison Test (* p < 0.05, ** p < 0.01).

Figure 5

mtDNA content relative to the β-actin nuclear gene in colon samples of the control, IBS-Std, and IBS-KD rats, with each group consisting of four rats. Data were obtained using qPCR. Data were analyzed by Kruskal–Wallis analysis of variance and Dunn’s Multiple Comparison Test (* p < 0.05).

Figure 6

Western blot analysis of Beclin-1 (Panel (A)) and LC3 II (Panel (B)) levels in colon samples of the control, IBS-Std, and IBS-KD rats, with each group consisting of four rats. Data were analyzed by Kruskal–Wallis analysis of variance and Dunn’s Multiple Comparison Test (* p < 0.05, ** p < 0.01).