The 1-Week and 8-Month Effects of a Ketogenic Diet or Ketone Salt Supplementation on Multi-Organ Markers of Oxidative Stress and Mitochondrial Function in Rats

Abstract

We determined the short- and long-term effects of a ketogenic diet (KD) or ketone salt (KS) supplementation on multi-organ oxidative stress and mitochondrial markers. For short-term feedings, 4 month-old male rats were provided isocaloric amounts of KD (n = 10), standard chow (SC) (n = 10) or SC + KS (~1.2 g/day, n = 10). For long-term feedings, 4 month-old male rats were provided KD (n = 8), SC (n = 7) or SC + KS (n = 7) for 8 months and rotarod tested every 2 months. Blood, brain (whole cortex), liver and gastrocnemius muscle were harvested from all rats for biochemical analyses. Additionally, mitochondria from the brain, muscle and liver tissue of long-term-fed rats were analyzed for mitochondrial quantity (maximal citrate synthase activity), quality (state 3 and 4 respiration) and reactive oxygen species (ROS) assays. Liver antioxidant capacity trended higher in short-term KD- and SC + KS-fed versus SC-fed rats, and short-term KD-fed rats exhibited significantly greater serum ketones compared to SC + KS-fed rats indicating that the diet (not KS supplementation) induced ketonemia. In long term-fed rats: (a) serum ketones were significantly greater in KD- versus SC- and SC + KS-fed rats; (b) liver antioxidant capacity and glutathione peroxidase protein was significantly greater in KD- versus SC-fed rats, respectively, while liver protein carbonyls were lowest in KD-fed rats; and (c) gastrocnemius mitochondrial ROS production was significantly greater in KD-fed rats versus other groups, and this paralleled lower mitochondrial glutathione levels. Additionally, the gastrocnemius pyruvate-malate mitochondrial respiratory control ratio was significantly impaired in long-term KD-fed rats, and gastrocnemius mitochondrial quantity was lowest in these animals. Rotarod performance was greatest in KD-fed rats versus all other groups at 2, 4 and 8 months, although there was a significant age-related decline in performance existed in KD-fed rats which was not evident in the other two groups. In conclusion, short- and long-term KD improves select markers of liver oxidative stress compared to SC feeding, although long-term KD feeding may negatively affect skeletal muscle mitochondrial physiology.

Keywords: brain; ketogenic dieting; ketone salts; liver; mitochondria; oxidative stress; skeletal muscle.

Figure 1

Change in body mass, overall feed efficiency and serum beta-hydroxybutyrate (BHB) levels in 1 week-fed rats. Legend: Change in body mass is presented in panel (a). Feed efficiency is presented in panel (b). Serum BHB levels are presented in panel (c). Data in panel (a) are presented as mean ± standard error values (n = 10 per group). In panels (b,c), all bars are presented as mean ± standard error values and group means are indicated within each bar. For all panels, ANOVA p-values are presented below data, and different lettering (“a” versus “b”) indicates significant between-group differences whereas shared lettering (“a” as well as “b” or “a, b”) indicates lack of significant between-group differences. Abbreviations: SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate.

Figure 2

Oxidative stress and endogenous antioxidant-related markers in 1 week-fed rats. Legend: Oxidative stress and endogenous antioxidant related mRNAs are shown for gastrocnemius panel (a); brain panel (b); and liver panel (c). Tissue total antioxidant capacity levels are presented in panel (d). Fold-change for each mRNA are expressed relative to the SC group, all bars are presented as mean ± standard error values (n = 10 per group), group means are indicated within each bar, ANOVA p-values are presented below data, and different lettering in panel D (“a” versus “b”) indicates significant between-group differences whereas shared lettering (“a” as well as “b” or “a, b”) indicates lack of significant between-group differences. Abbreviations: Gclm, glutamate-cysteine ligase modulatory subunit; Gclc, glutamate-cysteine ligase catalytic subunit; Gsr, glutathione reductase; Gpx1, glutathione peroxidase; Cat, catalase; Sod2, supoeroxide dismutase 2; SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate.

Figure 3

Change in body mass, overall feed efficiency and serum BHB in 8 month-fed rats. Legend: Body mass changes over the intervention are presented in panel (a), feed efficiency is presented in panel (b) and serum BHB levels are presented in panel (c). Data in panel (a) are presented as mean ± standard error values (n = 7–8 per group). In panels (b,c), all bars are presented as mean ± standard error values and group means are indicated within each bar. In panels B and C ANOVA p-values are presented below data, and different lettering (“a” versus “b”) indicates significant between-group differences whereas shared lettering (“a” as well as “b” or “a, b”) indicates lack of significant between-group differences. Abbreviations: SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate.

Figure 4

Change in rotarod performance in 8 month-fed rats. Legend: Rotarod performance every 2 months are presented herein. All data are presented as mean ± standard error values (n = 7–8 per group). Given the significant group × tine interaction, post hoc analyses at each time point and within each group were performed. Specifically, different lettering (“a” versus “b”) indicates significant between-group differences whereas shared lettering (“a” as well as “b” or “a, b”) indicates lack of significant between-group differences, and asterisks indicate significant decreases within the KD rats at 6 and 8 months compared to 2 months values (p < 0.05). Abbreviations: SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate.

Figure 5

Gastrocnemius oxidative stress-related proteins and markers in 8 month-fed rats. Legend: Gastrocnemius catalase (Cat), glutathione peroxidase (Gpx), and mitochondrial superoxide dismutase (Sod2) protein levels are presented with representative images in panel (a). In panel (b), tissue 4-hydroxynonenal (4-HNE) and protein carbonyls (Oxyblot) are presented with representative images. Fold-change for each protein are expressed relative to the SC group, all bars are presented as mean ± standard error values (n = 7–8 per group), group means are indicated within each bar and ANOVA p-values are presented below data. Abbreviations: SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate.

Figure 6

Brain oxidative stress-related proteins and markers in 8 month-fed rats. Legend: Brain catalase (Cat), glutathione peroxidase (Gpx), and mitochondrial superoxide dismutase (Sod2) protein levels are presented with representative images in panel (a). In panel (b), tissue 4-Hydroxynonenal (4-HNE), and protein carbonyls (Oxyblot) are presented with representative images. Fold-change for each protein are expressed relative to the SC group, all bars are presented as mean ± standard error values (n = 7–8 per group), group means are indicated within each bar and ANOVA p-values are presented below data. Abbreviations: SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate.

Figure 6

Brain oxidative stress-related proteins and markers in 8 month-fed rats. Legend: Brain catalase (Cat), glutathione peroxidase (Gpx), and mitochondrial superoxide dismutase (Sod2) protein levels are presented with representative images in panel (a). In panel (b), tissue 4-Hydroxynonenal (4-HNE), and protein carbonyls (Oxyblot) are presented with representative images. Fold-change for each protein are expressed relative to the SC group, all bars are presented as mean ± standard error values (n = 7–8 per group), group means are indicated within each bar and ANOVA p-values are presented below data. Abbreviations: SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate.

Figure 7

Liver oxidative stress-related proteins and markers in 8 month-fed rats. Legend: Liver catalase (Cat), glutathione peroxidase (Gpx), and mitochondrial superoxide dismutase (Sod2) protein levels are presented with representative images in panel (a). In panel (b), tissue 4-Hydroxynonenal (4-HNE), and protein carbonyls (Oxyblot) are presented with representative images. Fold-change for each protein are expressed relative to the SC group, all bars are presented as mean ± standard error values (n = 7–8 per group), group means are indicated within each bar, ANOVA p-values are presented below data and different lettering (“a” versus “b”) indicates significant between-group differences whereas shared lettering (“a” as well as “b” or “a, b”) indicates lack of significant between-group differences. Abbreviations: SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate.

Figure 8

Tissue mitochondrial glutathione, total antioxidant capacity, and ROS levels in 8 month-fed rats. Legend: Gastrocnemius mitochondrial oxidized (GSSG), reduced (GSH) and total glutathione are presented in panel (a); Brain mitochondrial oxidized (GSSG), reduced (GSH) and total glutathione are presented in panel (b); Liver mitochondrial oxidized (GSSG), reduced (GSH) and total glutathione are presented in panel (c); Tissue total antioxidant capacity levels are presented in panel (d); Tissue mitochondrial reactive oxygen species production levels are presented in panel (e). All bars are presented as mean ± standard error values (n = 7–8 per group), group means are indicated within each bar and ANOVA p-values are presented below data, and different lettering (“a” versus “b”) indicates significant between-group differences whereas shared lettering (“a” as well as “b” or “a, b”) indicates lack of significant between-group differences. Abbreviations: SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate.

Figure 9

Gastrocnemius mitochondrial function in 8 month-fed rats. Legend: State 3 pyruvate-malate is presented in panel (a); State 4 pyruvate-malate is presented in panel (b); Pyruvate-malate RCR is presented in panel (c); State 3 succinate is presented in panel (d); State 4 succinate is presented in panel (e); Succinate RCR is presented in panel (f). All bars are presented as mean ± standard error values (n = 7–8 per group), group means are indicated within each bar and ANOVA p-values are presented below data, and different lettering (“a” versus “b”) indicates significant between-group differences whereas shared lettering (“a” as well as “b” or “a, b”) indicates lack of significant between-group differences. Abbreviations: SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate; RCR, respiratory exchange ratio.

Figure 10

Brain and liver mitochondrial function in 8 month-fed rats. Legend: Brain state 3 and 4 pyruvate-malate and respiratory control ratio (RCR) values are presented in panel (a); Liver state 3 and 4 pyruvate-malate and RCR values are presented in panel (b); Liver state 3 and 4 succinate and RCR values are presented in panel (c). All bars are presented as mean ± standard error values (n = 7–8 per group), group means are indicated within each bar and ANOVA p-values are presented below data. Abbreviations: SC, standard chow-fed rats; KD, ketogenic diet-fed rats; SC + KS, standard chow-fed rats with supplemental sodium beta-hydroxybutyrate; RCR, respiratory exchange ratio.

Figure 11

Tissue citrate synthase activity in 8 month-fed rats. Legend: Tissue citrate synthase activity is presented. All bars are presented as mean ± standard error values (n = 7–8 per group), group means are indicated within each bar and ANOVA p-values are presented below data, and different lettering (“a” versus “b”) indicates significant between-group differences whereas shared lettering (“a” as well as “b” or “a, b”) indicates lack of significant between-group differences. Abbreviations: SC, standard control; KD, ketogenic diet; SC + KS, standard control with supplemental sodium beta-hydroxybutyrate.