Differential Effects of Exercise Timing on Skeletal Growth in Mice

Introduction

Skeletal growth is crucial for adult height and bone health. Studies have shown that exercise can effectively increase bone density, but the optimal exercise timing remains unclear. This study aimed to investigate the optimal exercise timing by comparing the effects of exercise at different time periods on skeletal growth in mice.

The research was jointly conducted by the School of Stomatology, Tongji Medical College, Huazhong University of Science and Technology, and several other research institutions, and published in Nature Metabolism.

Background and Objectives

Existing studies have confirmed that exercise can increase bone mass and strength, but the differential effects of exercise at different time periods on skeletal growth remain unclear. This study aimed to explore the effects of exercise at different times of the day on skeletal growth in mice and to unravel the underlying molecular mechanisms.

Research Methods

Experimental Subjects and Groups

The experimental subjects were 3-week-old C57BL/6J mice, including 890 males and 98 females, provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology. The experiment was divided into six groups: ZT1 (early rest period), ZT13 (early active period), ZT17, ZT21, and a control group. The mice exercised on a treadmill for 30 minutes per day, five days a week, for five weeks.

Experimental Procedures

1. Exercise Protocol

   • Days 1-5: Mice gradually adapted to the treadmill, starting at 6 m/min and gradually increasing to 16 m/min.
   • Following 4 weeks: Mice exercised for 30 minutes at 16 m/min, five days a week.

2. RNA Sequencing

   • Total RNA was extracted from the skeletal tissues of sedentary and exercised mice for RNA sequencing analysis.
   • The JTK_CYCLE algorithm was used to analyze the circadian expression of genes in skeletal tissues.

3. Micro-Computed Tomography (Micro-CT)

   • A micro-computed tomography scanner was used to measure the total femur length, bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), and other parameters in exercised and sedentary mice.

4. Metabolomics and Proteomics Analyses

   • The effects of exercise at different time periods on metabolic pathways in skeletal tissues, including the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), were investigated.

Main Results

Skeletal Growth Effects

The study found that mice exercising during the early active period (ZT13) exhibited a 5% increase in femur length, a 26% increase in BV/TV, and a 54% increase in Tb.Th. Compared to exercise at other time periods, ZT13 exercise had a more significant promoting effect on skeletal growth.

Gene Expression Analysis

ZT13 exercise significantly upregulated the expression of genes related to skeletal growth, including genes involved in bone growth, matrix synthesis, and energy metabolism. The ZT13 exercise group had 10.1% of genes upregulated and 10.3% downregulated, significantly higher than the percentages of changes in other time periods.

Metabolic Pathway Analysis

In the ZT13 exercise group, genes related to the TCA cycle and OXPHOS were significantly upregulated, while the ZT1 exercise group primarily showed high expression of genes involved in the glycolysis pathway. ZT13 exercise promoted bone cell proliferation and differentiation by increasing ATP production and the NAD+/NADH ratio.

OXPHOS Activation and Skeletal Growth

Through real-time micro-positron emission tomography (PET) observation of oxygen metabolism in the skeletal tissues of exercised mice, it was found that ZT13 exercise significantly increased oxidative phosphorylation activity and led to a slight accumulation of intermediate products in energy metabolism.

The use of the oxidative phosphorylation activator AICAR further demonstrated that the significant promotion of skeletal growth during the early active period was mainly achieved through the enhancement of OXPHOS activity in skeletal tissues.

Conclusion

This study showed that the optimal exercise timing for promoting skeletal growth is the early active period (ZT13), as exercise during this time can significantly promote skeletal growth. This effect is achieved through significantly increasing the expression of genes related to skeletal growth and energy metabolism pathways.

Research Significance

The results indicate that timely exercise strategies can significantly improve bone health, especially during childhood and adolescence, helping to maximize skeletal growth potential. By identifying the appropriate exercise timing, more scientific exercise programs can be designed to prevent the occurrence of bone diseases.

This study not only theoretically proposed the differential effects of “optimal exercise timing” on skeletal growth but also provided new perspectives and methods for non-pharmacological interventions in skeletal growth.

Research Highlights

• This study provided in-depth insights into the significant effects of exercise timing on skeletal growth and its molecular mechanisms.
• Through high-throughput transcriptomics and metabolomics analyses, it was the first to confirm the regulatory effects of exercise at different time periods on energy metabolism pathways in skeletal tissues.
• The potential importance of exercise during the early active period for bone health was discovered, providing a theoretical basis for designing optimized bone health intervention strategies.