Intrauterine hyperglycaemia during late gestation caused mitochondrial dysfunction in skeletal muscle of male offspring through CREB/PGC1A signaling
Authors: Yan Yi-Shang , Mo Jia-Ying , Huang Yu-Tong , Zhu Hong , Wu Hai-Yan , Lin Zhong-Liang , Liu Rui , Liu Xuan-Qi , Lv Ping-Ping , Feng Chun , Sheng Jian-Zhong , Jin Min , Huang He-Feng 
Published: July 2024

Abstract
Background
Maternal diabetes mellitus can influence the development of offspring. Gestational diabetes mellitus (GDM) creates a short-term intrauterine hyperglycaemic environment in offspring, leading to glucose intolerance in later life, but the long-term effects and specific mechanism involved in skeletal muscle dysfunction in offspring remain to be clarified.

Methods
Pregnant mice were divided into two groups: The GDM group was intraperitoneally injected with 100 mg/kg streptozotocin on gestational days (GDs) 6.5 and 12.5, while the control (CTR) group was treated with vehicle buffer. Only pregnant mice whose random blood glucose level was higher than 16.8 mmol/L beginning on GD13.5 were regarded as the GDM group. The growth of the offspring was monitored, and the glucose tolerance test was performed at different time points. Body composition analysis and immunohistochemical methods were used to evaluate the development of lean mass at 8 weeks. The exercise capacity and grip strength of the male mouse offspring were assessed at the same period. Transmission electron microscopy was used to observe the morphology inside skeletal muscle at 8 weeks and as a foetus. The genes and proteins associated with mitochondrial biogenesis and oxidative metabolism were investigated. We also coanalyzed RNA sequencing and proteomics data to explore the underlying mechanism. Chromatin immunoprecipitation and bisulfite-converted DNA methylation detection were performed to evaluate this phenomenon.

Results
Short-term intrauterine hyperglycaemia inhibited the growth and reduced the lean mass of male offspring, leading to decreased endurance exercise capacity. The myofiber composition of the tibialis anterior muscle of GDM male offspring became more glycolytic and less oxidative. The morphology and function of mitochondria in the skeletal muscle of GDM male offspring were destroyed, and coanalysis of RNA sequencing and proteomics of foetal skeletal muscle showed that mitochondrial elements and lipid oxidation were consistently impaired. In vivo and in vitro myoblast experiments also demonstrated that high glucose concentrations impeded mitochondrial organisation and function. Importantly, the transcription of genes associated with mitochondrial biogenesis and oxidative metabolism decreased at 8 weeks and during the foetal period. We predicted Ppargc1α as a key upstream regulator with the help of IPA software. The proteins and mRNA levels of Ppargc1α in the skeletal muscle of GDM male offspring were decreased as a foetus (CTR vs. GDM, 1.004 vs. 0.665, p = 0.002), at 6 weeks (1.018 vs. 0.511, p = 0.023) and 8 weeks (1.006 vs. 0.596, p = 0.018). In addition, CREB phosphorylation was inhibited in GDM group, with fewer activated pCREB proteins binding to the CRE element of Ppargc1α (1.042 vs. 0.681, p = 0.037), Pck1 (1.091 vs. 0.432, p = 0.014) and G6pc (1.118 vs. 0.472, p = 0.027), resulting in their decreased transcription. Interestingly, we found that sarcopenia and mitochondrial dysfunction could even be inherited by the next generation.

Conclusions
Short-term intrauterine hyperglycaemia significantly reduced lean mass in male offspring at 8 weeks, resulting in decreased exercise endurance and metabolic disorders. Disrupted organisation and function of the mitochondria in skeletal muscle were also observed among them. Foetal exposure to hyperglycaemia decreased the ratio of phosphorylated CREB and reduced the transcription of Ppargc1α, which inhibited the transcription of downstream genes involving in mitochondrial biogenesis and oxidative metabolism. Abnormal mitochondria, which might be transmitted through aberrant gametes, were also observed in the F2 generation.