A ketogenic diet alters mTOR activity, systemic metabolism and potentially prevents collagen degradation associated with chronic alcohol consumption in mice

Abstract

Introduction: A ketogenic diet (KD), which is a high fat, low carbohydrate diet has been shown to inhibit the mammalian target of rapamycin (mTOR) pathway and alter the redox state. Inhibition of the mTOR complex has been associated with the attenuation and alleviation of various metabolic and- inflammatory diseases such as neurodegeneration, diabetes, and metabolic syndrome. Various metabolic pathways and signalling mechanisms have been explored to assess the therapeutic potential of mTOR inhibition. However, chronic alcohol consumption has also been reported to alter mTOR activity, the cellular redox- and inflammatory state. Thus, a relevant question that remains is what effect chronic alcohol consumption would have on mTOR activity and overall metabolism during a KD-based intervention.

Objectives: The aim of this study was to evaluate the effect of alcohol and a KD on the phosphorylation of the mTORC1 target p70S6K, systemic metabolism as well as the redox- and inflammatory state in a mouse model.

Methods: Mice were fed either a control diet with/without alcohol or a KD with/without alcohol for three weeks. After the dietary intervention, samples were collected and subjected towards western blot analysis, multi-platform metabolomics analysis and flow cytometry.

Results: Mice fed a KD exhibited significant mTOR inhibition and reduction in growth rate. Alcohol consumption alone did not markedly alter mTOR activity or growth rate but moderately increased mTOR inhibition in mice fed a KD. In addition, metabolic profiling showed alteration of several metabolic pathways as well as the redox state following consumption of a KD and alcohol. A KD was also observed to potentially prevent bone loss and collagen degradation associated with chronic alcohol consumption, as indicated by hydroxyproline metabolism.

Conclusion: This study sheds light on the influence that a KD alongside alcohol intake can exert on not just mTOR, but also their effect on metabolic reprogramming and the redox state.

Keywords: Ketogenic diet; Metabolic reprogramming; Metabolomics; Mtor; Redox; Western blot.

Fig. 1

Consumption of a KD in mice leads to lower body weight gain. Growth curves for dietary groups on a normal diet without (ND) and with alcohol intake (ND + Alc.) and on a ketogenic diet without (KD) and with alcohol intake (KD + Alc.). Average weight ± standard deviation (n = 10) per group is given as a function of animal age. Mice were weighed three times a week. Asterisks (***p < = 0.001) indicate significant differences between ND vs KD and ND vs KD + Alc. groups as determined by post-hoc Tukey’s test

Fig. 2

A KD inhibits S6K1 phosphorylation in mice. A Representative Western blot image and B quantitative summary of pS6K1 and β-actin following separation by SDS-page. Average β-actin (~ 42 kDa) was used as control protein of pS6K1 (~ 75 kDa). Data represents the average relative pS6K1 intensity ± standard deviation (n = 4) per group. ND indicates normal diet; ND + Alc., normal diet and alcohol; KD, ketogenic diet; KD + Alc., ketogenic diet and alcohol. Asterisks (*p < 0.05) indicate significant differences between ND vs KD and ND vs KD + Alc. Groups, as determined by post-hoc Tukey’s test

Fig. 3

A KD and chronic alcohol consumption leads to metabolic alterations in mice. A PCA scores plot illustrates the natural separation of all significantly altered metabolites when the different groups (n = 6 to 10 per group) were compared. B Heatmap of main metabolites altered in different groups (n = 6 to 10 per group) following dietary intervention. Each metabolite was normalised to its respective internal standard (based on the relative metabolomics platform) and subsequently log transformed, as well as auto-scaled using MetaboAnalyst 5.0

Fig. 4

A KD lowers urinary amino acid levels irrespective of alcohol consumption. Bar graphs depict Cohen’s d-values (effect size) for each metabolite when relative abundance between the different mouse groups (n = 6 to 10 per group) were compared to the control group (ND). Leu indicates leucine; Ile, isoleucine; 2-KIC, 2-ketoisocaproic acid; 2-KMVA, 2-keto-3-methylvaleric acid; C4, butyryl-carnitine; Arg, arginine; Ala, alanine; Met, Methionine; HcyH, homocysteine; Ser, serine; ND, normal diet; ND + Alc., normal diet and alcohol; KD, ketogenic diet; KD + Alc., ketogenic diet and alcohol. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the control group, obtained via a post-hoc Tukey’s test

Fig. 5

A KD prevents increased urinary 4-hydroxyproline excretion observed during chronic alcohol intake. A Boxplots depict the relative 4-hydroxyproline intensity for dietary groups (n = 6–10 per group) on a normal diet without (ND) and with alcohol intake (ND + Alc.) and on a ketogenic diet without (KD) and with alcohol intake (KD + Alc.). For each boxplot the length of the box represents the 25th to 75th percentile inter-quartile range, the interior horizontal line represents the median, and the vertical lines stemming from the box extend to minimum and maximum values. B Bar graphs show the relative intensities for 4-hydroxyproline when the interaction of diet and alcohol was compared. Asterisks (*p < 0.05) indicate significant differences between the ND vs ND + Alc. groups as determined by post-hoc Tukey’’s test

Fig. 6

Alterations of TCA cycle intermediates are more prominent with dietary interventions than chronic alcohol consumption when the different mouse groups (n = 6–10 per group) were compared. Bar graphs reveal Cohen’s d-values (effect size; d > 0.8) for the significant metabolites when the relative abundance between the different mice groups were compared to the control group (ND). ND indicates normal diet; ND + Alc., normal diet and alcohol; KD, ketogenic diet; KD + Alc., ketogenic diet and alcohol. ***p < 0.001 vs. the control group, obtained via a post-hoc Tukey’s test

Fig. 7

A KD results in a more oxidative redox state in mice. Bar graphs (A–C) depict Cohen’s d-values (effect size) of alterations in metabolic indicators of NADH/NAD⁺ ratio, when the different dietary groups were compared to controls. Positive and negative effect size values respectively indicate increased and decreased levels in the experimental group vs the control group. An elevated NADH/NAD⁺ ratio drives the formation of amino acids from glutamic acid. Ala indicates alanine; Glu, glutamic acid; Ile, isoleucine; Leu, leucine; ND, normal diet; ND + Alc., normal diet and alcohol; KD, ketogenic diet; KD + Alc., ketogenic diet and alcohol. Asterisks (***p < = 0.001) indicate significant differences between the ND vs KD group as determined by post-hoc Tukey’s test

Fig. 8

The concentration levels (pg/mL) of pro-inflammatory biomarkers as measured in the serum of mice from all four dietary groups (n = 6 per group). The error bars represent the standard error. ND indicates normal diet; ND + Alc., normal diet and alcohol; KD, ketogenic diet; KD + Alc., ketogenic diet and alcohol. *p < 0.05 vs. the control group, obtained via a post-hoc Tukey’s test,