Effect of ketogenic diet on exercise tolerance and transcriptome of gastrocnemius in mice

Abstract

Ketogenic diet (KD) has been proven to be an optional avenue in weight control. However, the impacts of KD on muscle strength and exercise endurance remain unclear. In this study, mice were randomly allocated to normal diet and KD groups to assess their exercise tolerance and transcriptomic changes of the gastrocnemius. KD suppressed body-weight and glucose levels and augmented blood ketone levels of mice. The total cholesterol, free fatty acids, and β-hydroxybutyric acid levels were higher and triglycerides and aspartate aminotransferase levels were lower in KD group. There was no notable difference in running distance/time and weight-bearing swimming time between the two groups. Furthermore, KD alleviated the protein levels of PGC-1α, p62, TnI FS, p-AMPKα, and p-Smad3, while advancing the LC3 II and TnI SS protein levels in the gastrocnemius tissues. RNA-sequencing found that 387 differentially expressed genes were filtered, and Cpt1b, Acadl, Eci2, Mlycd, Pdk4, Ptprc, C1qa, Emr1, Fcgr3, and Ctss were considered to be the hub genes. Our findings suggest that KD effectively reduced body weight but did not affect skeletal muscle strength and exercise endurance via AMPK/PGC-1α, Smad3, and p62/LC3 signaling pathways and these hub genes could be potential targets for muscle function in KD-treated mice.

Keywords: exercise tolerance; high-throughput sequencing; ketogenic diet; lipid metabolism.

Figure 1

KD suppressed body weight and Glu level and augmented blood ketone level of C57BL/6 mice. (a) Body weight trajectories of mice on KD for 4 weeks. (b) Blood Glu level of mice on the KD for 4 weeks. (c) Blood ketone level of mice on the KD for 4 weeks. Data are expressed as mean ± SD (n = 10/group). Statistical significance was determined by one-way ANOVA. ^▲ P < 0.05, ^▲▲ P < 0.01 vs ND group. ND, normal diet; KD, ketogenic diet.

Figure 2

Effect of KD on lipid metabolism levels in C57BL/6 mice. Levels of (a) TC, (b) TG, (c) FFA, (d) β-HB, (e) AST, and (f) ALT in serum samples of mice treated with KD for 4 weeks. Data are expressed as mean ± SD. Differences of data in mice were assessed by two-tailed Student’s t-test. ^▲▲ P < 0.01 vs ND group. ND, normal diet; KD, ketogenic diet.

Figure 3

Effect of KD on exercise tolerance in C57BL/6 mice. (a) Running distance, (b) running time, (c) weight-bearing swimming time of mice treated with KD for 4 weeks. (d) Levels of blood Glu and (e) ketone in mice before and after swimming. Data are expressed as mean ± SD. Differences of data in mice were assessed by two-tailed Student’s t-test. ^▲▲ P < 0.01 vs ND group. ND, normal diet; KD, ketogenic diet.

Figure 4

Identification of DEGs in gastrocnemius tissues in KD treated mice. (a) Volcano plot: two black vertical lines represent 1.5 times upregulation and 1.5 times downregulation, green horizontal lines indicate P-value of 0.05. (b) Heatmap showing the expressive pattern of DEGs. Red represents upregulated genes and green represents downregulated genes.

Figure 5

GO analysis of DEGs in mice after KD treatment.

Figure 6

KEGG pathway analysis of DEGs in mice after KD treatment.

Figure 7

PPI network of upregulated DEGs and identification of hub genes. We mapped upregulated DEGs to the STRING database to construct a PPI network, and then re-imported these interactive network data into Cytoscape software. PPI network composed of 97 nodes and 210 edges. Hub genes obtained by the Degree algorithm, Cpt1b, Acadl, Eci2, Mlycd, and Pdk4 were considered to be key upregulated genes.

Figure 8

PPI network of downregulated DEGs and identification of hub genes. We also mapped downregulated DEGs to the STRING database to construct a PPI network and re-imported them into Cytoscape software. PPI network composed of 149 nodes and 684 edges. Hub genes obtained by Degree algorithm, Ptprc, C1qa, Emr1, Fcgr3, and Ctss were considered to be key downregulated genes.

Figure A1

Effect of KD on pathological changes, muscle fiber, and autophagy-related proteins of gastrocnemius tissues in mice. (a) HE staining of the gastrocnemius tissues showed that KD had no differential influence in mice. (b) Representative protein bands and (c) quantification data of PGC-1α, p62, LC3, TnI SS, TnI FS, p-AMPKα, AMPKα, p-Smad3, and Smad3 in gastrocnemius tissues of mice treated with KD for 4 weeks. Data are expressed as mean ± SD. Differences of data in mice were assessed by two-tailed Student’s t-test. ^▲ P < 0.05, ^▲▲ P < 0.01 vs ND group. ND, normal diet; KD, ketogenic diet.