Metabolic and molecular framework for the enhancement of endurance by intermittent food deprivation

Abstract

Evolutionary considerations suggest that the body has been optimized to perform at a high level in the food-deprived state when fatty acids and their ketone metabolites are a major fuel source for muscle cells. Because controlled food deprivation in laboratory animals and intermittent energy restriction in humans is a potent physiologic stimulus for ketosis, we designed a study to determine the impact of intermittent food deprivation during endurance training on performance and to elucidate the underlying cellular and molecular mechanisms. Male mice were randomly assigned to either ad libitum feeding or alternate-day food deprivation (ADF) groups, and half of the mice in each diet group were trained daily on a treadmill for 1 mo. A run to exhaustion endurance test performed at the end of the training period revealed superior performance in the mice maintained on ADF during training compared to mice fed ad libitum during training. Maximal O₂ consumption was increased similarly by treadmill training in mice on ADF or ad libitum diets, whereas respiratory exchange ratio was reduced in ADF mice on food-deprivation days and during running. Analyses of gene expression in liver and soleus tissues, and metabolomics analysis of blood suggest that the metabolic switch invoked by ADF and potentiated by exercise strongly modulates molecular pathways involved in mitochondrial biogenesis, metabolism, and cellular plasticity. Our findings demonstrate that ADF engages metabolic and cellular signaling pathways that result in increased metabolic efficiency and endurance capacity.-Marosi, K., Moehl, K., Navas-Enamorado, I., Mitchell, S. J., Zhang, Y., Lehrmann, E., Aon, M. A., Cortassa, S., Becker, K. G., Mattson, M. P. Metabolic and molecular framework for the enhancement of endurance by intermittent food deprivation.

Keywords: exercise; intermittent fasting; ketone; mitochondrial biogenesis; muscle.

Figure 1

Experimental design. Mice were randomly assigned to sedentary CTRL, sedentary ADF, daily running (EX), or combined EX and ADF groups.

Figure 2

Mice on ADF diet, with or without daily treadmill running, have reduced overall calorie intake, retain body mass, and exhibit improved glucose tolerance compared to mice fed ad libitum. A) Body weight. Body weights of mice in ADF, EX, and EXADF groups were not significantly different than mice in CTRL group at any time point. B) Fat mass as percentage of body weight. ADF mice had significantly more fat mass than EXADF group on food-deprivation day but not feeding day. There were no other significant differences in fat mass between groups. C) Lean mass as percentage of body weight. There were no significant differences in lean mass between groups. D) Food consumption during intervention wk 1–4. ADF and EXADF mice consumed fewer calories than CTRL and EX groups during wk 1, 2, 3, and 4. E) Blood glucose levels. Blood glucose levels were significantly lower in mice in ADF and EXADF groups compared to mice in CTRL and EX groups during wk 1–4. F) Results of glucose tolerance test. Blood glucose levels were measured 15, 30, 60, and 120 min after intraperitoneal injection of glucose (2 g/kg of 30% glucose solution). Two-way ANOVA was conducted on influence of ADF and exercise on glucose tolerance over course of 2 h. Mice in ADF and EXADF groups had significantly lower blood glucose levels compared to mice in CTRL group at 15 and 30 min time points. Group × Time interaction effect was not significant: F(12,125) = 1.59, P = 0.1. Values are means ± sem (n = 8 CTRL, 8 ADF, 9 EX, and 9 EXADF mice). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

ADF reduces RER and energy expenditure, and triggers ketogenesis, which is enhanced by exercise. A) Blood glucose levels on feeding and food-deprivation days before and after 45 min training on treadmill during fourth week of training. B). Blood ketone levels on feeding and food-deprivation days before and after 45 min training on treadmill during fourth week of training. C) RER during 60-h time period in mice in CTRL, ADF, EX, and EXADF groups. On food-deprivation days, RER was greatly reduced in mice in ADF and EXADF groups, while on feeding days mice in ADF and EXADF groups exhibited higher RERs compared to mice in CTRL and EX groups. D) Energy expenditure during 60 h time period in mice in CTRL, ADF, EX, and EXADF groups. Mice in ADF and EXADF groups exhibited lower energy expenditure on food-deprivation days compared to feeding days and compared to mice in CTRL and EX groups. V are means ± sem [n = 8 CTRL, n = 8 ADF, n = 9 EX, and n = 9 EXADF mice (A); n = 4 CTRL, n = 4 ADF, n = 4 EX, and n = 4 EXADF mice (B); and n = 7 CTRL, n = 8 ADF, n = 9 EX, and n = 8 EXADF mice (C, D)]. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

ADF switches fuel utilization from carbohydrates to fats during exercise, and enhances endurance running performance without affecting Vo_2max. A) VO₂ rates during Vo_2max exercise test 4 wk after initiation of dietary and exercise interventions. B) RER during Vo_2max exercise test 4 wk after initiation of dietary and exercise interventions. RER was significantly lower in EXADF group compared to other groups, indicating high reliance on fat and ketone metabolism during exercise. C) Vo_2max rates during exercise before initiation of diet and exercise interventions (basal) and 4 wk after initiation of interventions. D, E) Running endurance test of mice after 7 wk of diet and exercise interventions. Mice in EX and EXADF groups ran significantly further (D) and longer (E) than mice in CTRL and ADF groups. Mice in EXADF group ran significantly further and longer than mice in EX. Values are means ± sem [n = 8 CTRL, n = 8 ADF, n = 9 EX, and n = 9 EXADF mice (A, C); n = 7 CTRL, n = 8 ADF, n = 9 EX, and n = 8 EXADF mice (D, E)]. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Evidence that ADF induces mitochondrial biogenesis in soleus muscle. A) Mitochondrial DNA copy number analyzed by qPCR, normalized to actin, and displayed as relative fold change compared to CTRL group. Mitochondria DNA copy numbers were significantly greater in ADF and EXADF groups compared to CTRL and EX groups. B–E) NRF1, NRF2, TFAM, and SIRT1 mRNA levels were analyzed by qPCR, normalized to actin, and displayed as relative fold change compared to CTRL group. F) Immunoblot and densitometric quantification after actin normalization for oxphos of electron transport chain. Complex III was significantly increased in EXADF mice compared to CTRL and ADF mice, and complex IV was significantly increased in EXADF mice and EX mice compared to CTRL and ADF mice. Values are means ± sem [n = 7 CTRL, n = 8 ADF, n = 9 EX, and n = 8 EXADF mice (A, E); n = 7 CTRL, n = 8 ADF, n = 9 EX, and n = 7 EXADF mice (F)]. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

ADF affects major metabolic transcriptional pathways in soleus muscle. A) Heat map of significant GO pathways in soleus tissue. ADF and EXADF prominently enhanced expression of genes involved in fatty acid metabolism and mitochondrial activity while down-regulating genes involved in lipid synthesis and glycolysis. B–D) Volcano plots of GO pathways in soleus muscle of ADF, EX, and EXADF animals vs. CTRL. E–H) PPAR-α, hormone-sensitive lipase, ACADL, and PDK4 mRNA levels analyzed by qPCR, normalized to actin, and displayed as relative fold change compared to CTRL group. Values are means ± sem (n = 7 CTRL, n = 8 ADF, n = 9 EX, and n = 8 EXADF mice). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Impact of treadmill running and ADF on liver transcriptome. A) PCA of liver gene microarray data reveals that liver transcriptomes of mice in EX and EXADF groups closely overlap and are distinct from transcriptomes of mice in CTRL and ADF groups. B) Venn diagrams of up-regulated and down-regulated gene transcripts present in CTRL-ADF, CTRL-EX, and CTRL-EXADF pairwise comparisons. C–E) PGC-1α, FASN, and ACADL mRNA levels analyzed by qPCR, normalized to actin, and displayed as relative fold change compared to CTRL group. Values are means ± sem (n = 7 CTRL, n = 8 ADF, n = 9 EX, and n = 8 EXADF mice). *P < 0.05, **P < 0.01, ***P < 0.001. F–H) Top metabolic GO pathways exhibiting differences between mice in CTRL groups and mice in ADF, EX, and EXADF groups (ranked by z-score values).

Figure 8

Impact of treadmill running and ADF on plasma metabolome. A) PCA of plasma metabolomics data reveals that metabolomes of mice in ADF and EXADF groups closely overlap and are distinct from metabolomes of mice in CTRL and EX groups. B) Heat map showing relative levels of major metabolites in each of indicated pathways from each mouse in each group. C) Diagram showing metabolites feeding into TCA cycle that are either elevated (red) or reduced (green) in mice in ADF and EXADF groups compared to CTRL group. BCAA, branched chain amino acids; CoA, coenzyme A; IMP, inositol monophosphate; PNC, purine nucleotide cycle. D, E). Relative concentrations of ketogenic amino acids isoleucine and leucine in plasma of mice in 4 different groups. F–H) Relative concentrations of branched-chain α-keto acids plasma of mice in 4 different groups. I) Relative concentrations of ketone β-hydroxybutyrate in plasma of mice in 4 different groups. J–L) Relative concentrations of fatty acid metabolites glycerol, oleic acid, and palmitoleic acid in plasma of mice in 4 different groups. Values are means ± sem (n = 7 CTRL, n = 8 ADF, n = 9 EX, and n = 8 EXADF mice). *P < 0.05, **P < 0.01, ***P < 0.001.