Investigating the role of hexose-6-phosphate dehydrogenase in mouse metabolism and muscle function

Hexose-6-phosphate dehydrogenase (H6PD) is an endoplasmic reticulum (ER)-luminal enzyme involved in the microsomal pentose phosphate pathway (PPP). It is ubiquitously expressed and plays an important role in the metabolism of glucocorticoids. However, in a mouse model, the deficiency of H6PD leads to a phenotype in skeletal muscle which is at least partially independent of glucocorticoids. The mechanisms by which this progressively deteriorating myopathy develops are not well understood to date. Therefore, a better characterization of this phenotype and the investigation of its development are important to understand the functions of H6PD and the ER-PPP.
The first part of this thesis consists of an extensive review on H6PD and its role in health and disease. Various different aspects of H6PD and of the ER-PPP in mice and humans have been explored within the past 30 years, however, these studies led to a variety of conflicting findings. Here, we aimed to summarize and discuss the progress made in this field as well as to identify areas where our current understanding is lacking. To this end, we reviewed the available literature, specifically concentrating on the involvement of H6PD in the development of metabolic disorders, myopathy, and cancer. We aimed to evaluate the potential of H6PD as a target for therapeutic interventions in these diseases.
In the second part of this thesis we aimed to investigate the hypothesis that mice deficient in H6PD (H6PD KO) may be more susceptible to cardio-, hepato-, and nephrotoxicity caused by xenobiotics. Recent publications indicated a role of H6PD in the mitigation of ER stress. Doxorubicin, a chemotherapeutic drug known to cause both acute and chronic cardiomyopathy was suggested to cause ER stress both in mice as well as a rat cell model of cardiomyocytes. Furthermore, doxorubicin was found to increase the activity of H6PD in mouse hearts. Therefore, we hypothesized that H6PD KO mice might be more susceptible to the adverse effects of doxorubicin. We compared the effects of two different dosages of doxorubicin in female wildtype (WT) and H6PD KO mice after two time intervals. For this purpose, we measured markers of ER stress after a single dose of doxorubicin. We were not able to confirm our hypothesis, as we did not find a significant difference in the effect of doxorubicin in mouse hearts, livers, or kidneys.
In the third and final part of this thesis, we present a detailed analysis of the skeletal muscle phenotype in H6PD KO mice. In order to understand the mechanisms by which myopathy in H6PD KO mice develops, we systematically characterized the muscle structure, function, and metabolic pathways in these animals. To this end, we analyzed the muscle architecture using Hematoxylin/Eosin staining as well as transmission electron microscopy. Moreover, we measured muscle force generation in vivo by grip strength and running to exhaustion on a treadmill. To further investigate metabolic alterations in H6PD KO mice, we compared the respiratory quotient and the energy exchange rate of mice at rest and during treadmill exercise. Additionally, we measured mitochondrial respiration of soleus and extensor digitorum longus muscle fibers ex vivo. To examine the biological pathways involved, we performed gene and protein expression studies of enzymes involved in muscle physiology, glucose metabolism, as well as lipid binding and transport. In order to identify potential sex-specific differences in the effect of H6PD KO, we employed both male and female mice.
In conclusion, this thesis emphasizes the importance of H6PD and its role in ER homeostasis for the proper function and survival of a variety of cells and tissues. A more complete understanding of the role of the ER-PPP, and the enzymes involved in it, may provide new therapeutic options in various disorders, including metabolic disorders, myopathy, and cancer.