White adipose tissue (WAT) is a dynamic endocrine organ that can play a significant role in thermoregulation. WAT has the capacity to adopt structural and functional characteristics of the more metabolically active brown adipose tissue (BAT) and contribute to non-shivering thermogenesis under specific stimuli. Non-shivering thermogenesis was previously thought to be uncoupling protein 1 (UCP1)-dependent however, recent evidence suggests that UCP1-independent mechanisms of thermogenesis exist. Namely, futile creatine cycling has been identified as a contributor to WAT thermogenesis. The purpose of this study was to examine the efficacy of creatine supplementation to alter mitochondrial markers as well as adipocyte size and multilocularity in inguinal (iWAT), gonadal (gWAT), and BAT. Thirty-two male and female Sprague-Dawley rats were treated with varying doses (0 g/L, 2.5 g/L, 5 g/L, and 10 g/L) of creatine monohydrate for 8 weeks. We demonstrate that mitochondrial markers respond in a sex and depot specific manner. In iWAT, female rats displayed significant increases in COXIV, PDH-E1alpha, and cytochrome C protein content. Male rats exhibited gWAT specific increases in COXIV and PDH-E1alpha protein content. This study supports creatine supplementation as a potential method of UCP1-independant thermogenesis and highlights the importance of taking a sex-specific approach when examining the efficacy of browning therapeutics in future research.

Sprague-Dawley rats (n=32) underwent 8-weeks of creatine monohydrate (CM) supplementation (0, 2.5, 5, and 10 g/L). Total creatine concentrations (TCr) in female white fiber-dominant gastrocnemius (WGAS) and cardiac muscle (HRT) were significantly higher compared to males (p<0.05). CM supplementation increased TCr concentrations in female WGAS (p<0.05) and HRT (p<0.01) and in male red fiber-dominant gastrocnemius muscle (RGAS) (p<0.05). Future research should further investigate sex-differences in basal levels of TCr and the response to CM supplementation. Novelty – There is a sex- and tissue-dependant response to CM supplementation in rats.

Background Creatine is an essential compound that can store chemical energy and act as an energy buffer during times of metabolic stress in mammals. Creatine can be synthesized endogenously, however dietary supplementation with creatine monohydrate (CM) is a common medical intervention and is used as a common ergogenic aid in athletes. The aim of this study was to investigate the effect of increasing doses of dietary creatine on creatine uptake in brain, heart, skeletal muscle, and liver tissues in male and female rats in preparation for future dietary interventions. Methods 16 male (536g—670g) and 16 female (339g—487g) 12 week old Sprague‐Dawley rats were given access to drinking water with 0 g/L, 2.5 g/L, 5 g/L, or 10 g/L CM and 1% sucrose for 8 weeks. Free creatine and phosphocreatine tissue concentrations were measured using fluorometric assays and combined to report total creatine compound (TCr) concentrations in brain (BR), heart (HRT), liver (LIV), and skeletal muscle [soleus (SOL), extensor digitorum longus (EDL), and gastrocnemius (GAS)] tissues after the 8‐week dietary intervention. Results Males were significantly heavier than females (p<0.001) but there were no differences in body weight between groups of either sex. TCr concentrations in BR were higher with 2.5 g/L (p<0.01), 5 g/L (p<0.05), and 10 g/L (p<0.001) CM compared to control, with only the 2.5 g/L CM dose resulting in a higher TCr concentration in females (p<0.05) compared to males. TCr concentrations in SOL were higher with 5 g/L (p< 0.01) and 10 g/L (p<0.001) CM compared to controls. In EDL and GAS TCr concentrations were higher compared to controls with 2.5 g/L (p<0.01, p<0.05), 5 g/L (p<0.001, p<0.05), and 10 g/L (p<0.001, p<0.01) CM. TCr in GAS showed a trend with females (p<0.10) being higher than males. TCr concentrations in HRT of females were higher with 10 g/L (p<0.01) CM compared to controls and trending towards significance with 5 g/L (p<0.10) CM as compared to controls. TCr concentrations in HRT with 10 g/L CM were significantly higher in females (p<0.001) compared to males. No differences in TCr were observed between groups in LIV. Conclusions CM supplementation provided in water with 1% sucrose for 8 weeks leads to an increase in TCr in brain, heart, and skeletal muscle, but not in liver. Females appeared to be more responsive to CM supplementation in HRT, especially at the highest dose suggesting that dietary interventions involving CM may affect males and females differently. Support or Funding Information Supported by NSERC, Canada.

Background Supplementation with dietary creatine has been reported to improve markers of bone structure in animals and humans. Trabecular bone is one of two types of bone responsible for strength and structural support in the body. It serves to support areas that experience mechanical stress. The purpose of this study was to investigate the effect of varying doses of dietary creatine on the trabecular bone morphology of male and female rats. Methods 32 Sprague‐Dawley rats (16 male, 16 female) were randomized into one of four groups: 0g/L, 2.5g/L, 5g/L, or 10g/L of creatine monohydrate (CM) in drinking water with 1% sucrose (for palatability). Dietary interventions began at 12 weeks and lasted for 8 weeks. Ex vivo scanning and analysis was conducted on the proximal region of left femurs using micro‐computed tomography (mCT). Standard measures of trabecular bone morphology were measured at the proximal femur: trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), percent bone volume (BV/TV), connectivity (Conn) and connectivity density (Conn.Dn). Results There were no significant differences in Tb.Th, Tb.Sp, Tb.N, BV/TV, Conn, or Conn.Dn between creatine dose groups for males or females. Males had greater Tb.Th than females (p<0.01). Greater Tb.Sp was also observed in males as compared to females (p<0.01). Females demonstrated significantly higher BV/TV (p<0.01) as well as a greater Tb.N (p<0.01) than males. Conn was significantly different between sexes (p<0.05) with a higher value in females compared to males. Conn.Dn was also significantly higher in females (p<0.01) compared to males. Conclusions Eight weeks of different dietary intakes of creatine does not appear to have an effect on proximal femur Tb.Th, Tb.Sp, Tb.N, BV/TV, Conn, or Conn.Dn. However, differences in sex were observed for each dependent variable which is suggestive of a difference in the trabecular structure between males and females. Support or Funding Information Supported by NSERC (Canada).

Activation of white adipose tissue (WAT) thermogenesis, known as WAT browning, has emerged as an attractive approach to treat obesity and obesity‐related diseases. Traditionally BAT or browning WAT has been defined by the expression of uncoupling protein 1 (UCP1), a mitochondrial protein that uncouples respiration from ATP production. However, recent work has highlighted creatine metabolism or futile cycling as a potential thermogenic pathway. Creatine‐driven futile cycling is a catabolic process which results in ATP oxidation by mitochondrial creatine kinase, resulting in the phosphorylation of creatine and increases in ADP concentrations that drive thermogenic respiration. The purpose of the present study was two‐fold: 1) to determine the effects of creatine supplementation on markers of WAT browning, and 2) to determine if creatine supplementation exhibits sex differences. Thirty‐two Sprague‐Dawley rats (16 male, 16 female) were randomly assigned to one of four experimental groups: control, 2.5g/L, 5g/L and 10g/L of creatine monohydrate (CM) and 1% sucrose via drinking water. Rats had ad libitum access to their drinking water and a standard chow diet (AIN‐93G) throughout the study. Following the 8‐week intervention, brown interscapular adipose (BAT) and white subcutaneous inguinal (iWAT) fat depots were collected. Western blotting analysis demonstrated no significant changes in BAT samples for both males and females. Males had lower cytochrome C than females with 10g/L of creatine monohydrate (p<0.05). In iWAT, mitochondrial markers (COXIV, PDH, citrate synthase, and cytochrome C) and GAMT exhibit no changes in males, however, a decrease in UCP‐1 at 5g/L compared to control was observed (p<0.05). Females exhibit a lower control PDH content than males (p<0.01) in iWAT, accompanied by an increase in PDH levels at all doses of CM compared to control (p<0.05). Furthermore, in females COXIV and cytochrome C presented significant increases with 5g/L and 10g/L, respectively (p<0.05). Both males and females did not present with body weight differences despite dose differences. This study presents novel work demonstrating that female WAT exhibits less mitochondrial markers than males under control conditions, however it demonstrates the potential for CM supplementation to increase female adipose thermogenicity to a similar level observed in males. Support or Funding Information Supported by NSERC, Canada