Sex differences in cardiovascular disease (CVD), including aortic stenosis, atherosclerosis and cardiovascular calcification, are well documented. High levels of testosterone, the primary male sex hormone, are associated with increased risk of cardiovascular calcification, whilst estrogen, the primary female sex hormone, is considered cardioprotective. Current understanding of sexual dimorphism in cardiovascular calcification is still very limited. This review assesses the evidence that the actions of sex hormones influence the development of cardiovascular calcification. We address the current question of whether sex hormones could play a role in the sexual dimorphism seen in cardiovascular calcification, by discussing potential mechanisms of actions of sex hormones and evidence in pre-clinical research. More advanced investigations and understanding of sex hormones in calcification could provide a better translational outcome for those suffering with cardiovascular calcification.

Caloric restriction (CR) is a nutritional intervention that reduces the risk of age-related diseases in numerous species, including humans. CR's metabolic effects, including decreased fat mass and improved insulin sensitivity, play an important role in its broader health benefits. However, the extent and basis of sex differences in CR's health benefits are unknown. We found that 30% CR in young (3-month-old) male mice decreased fat mass and improved glucose tolerance and insulin sensitivity, whereas these effects were blunted or absent in young female mice. Females' resistance to fat and weight loss was associated with decreased lipolysis, lower systemic energy expenditure and fatty acid oxidation, and increased postprandial lipogenesis compared to males. Positron emission tomography-computed tomography (PET/CT) with 18F-fluorodeoxyglucose (18F-FDG) showed that peripheral glucose uptake was comparable between sexes. Instead, the sex differences in glucose homeostasis were associated with altered hepatic ceramide content and substrate metabolism: compared to CR males, CR females had lower TCA cycle activity but higher blood ketone concentrations, a marker of hepatic acetyl-CoA content. This suggests that males use hepatic acetyl-CoA for the TCA cycle whereas in females it accumulates, thereby stimulating gluconeogenesis and limiting hypoglycaemia during CR. In aged mice (18-months old), when females are anoestrus, CR decreased fat mass and improved glucose homeostasis to a similar extent in both sexes. Finally, in a cohort of overweight and obese humans CR-induced fat loss was also sex- and age-dependent: younger females (<45 years) resisted fat loss compared to younger males while in older subjects (>45 years) this sex difference was absent. Collectively, these studies identify age-dependent sex differences in the metabolic effects of CR and highlight adipose tissue, the liver and oestrogen as key determinants of CR's metabolic benefits. These findings have important implications for understanding the interplay between diet and health and for maximising the benefits of CR in humans.

Caloric restriction (CR) is a nutritional intervention that reduces the risk of age-related diseases in numerous species, including humans. CR's metabolic effects, including decreased fat mass and improved insulin sensitivity, play an important role in its broader health benefits. However, the extent and basis of sex differences in CR's health benefits are unknown. We found that 30% CR in young (3-month-old) male mice decreased fat mass and improved glucose tolerance and insulin sensitivity, whereas these effects were blunted or absent in young female mice. Resistance to fat and weight loss in females was not explained by decreased energy expenditure compared to males. Positron emission tomography-computed tomography (PET/CT) with 18F-fluorodeoxyglucose (18F-FDG) showed that peripheral glucose uptake was comparable between sexes. Instead, CR caused a pronounced suppression of hepatic gluconeogenesis and altered liver ceramide content in males but not in females. In aged mice (18-months old), when females are anoestrus, CR decreased fat mass and improved glucose homeostasis to a similar extent in both sexes. CR-induced fat loss in humans was also sex- and age-dependent: younger females (<45 years) resisted fat loss compared to younger males while in older subjects (>45 years) this sex difference was absent. Collectively, these studies identify age-dependent sex differences in the metabolic effects of CR and highlight adipose tissue, the liver and oestrogen as key determinants of CR's metabolic benefits. This has important implications for understanding the interplay between diet and health and for maximising the benefits of CR in humans.

Cholesterol has important functions in the organization of membrane structure and this may be mediated via the formation of cholesterol-rich, liquid-ordered membrane microdomains often referred to as lipid rafts. Methyl-beta-cyclodextrin (cyclodextrin) is commonly used in cell biology studies to extract cholesterol and therefore disrupt lipid rafts. However, in this study we reassessed this experimental strategy and investigated the effects of cyclodextrin on the physical properties of sonicated and carbonate-treated intracellular membrane vesicles isolated from Cos-7 fibroblasts. We treated these membranes, which mainly originate from the trans-Golgi network and endosomes, with cyclodextrin and measured the effects on their equilibrium buoyant density, protein content, represented by the palmitoylated protein phosphatidylinositol 4-kinase type IIα, and cholesterol. Despite the reduction in mass stemming from cholesterol removal, the vesicles became denser, indicating a possible large volumetric decrease, and this was confirmed by measurements of hydrodynamic vesicle size. Subsequent mathematical analyses demonstrated that only half of this change in membrane size was attributable to cholesterol loss. Hence, the non-selective desorption properties of cyclodextrin are also involved in membrane size and density changes. These findings may have implications for preceding studies that interpreted cyclodextrin-induced changes to membrane biochemistry in the context of lipid raft disruption without taking into account our finding that cyclodextrin treatment also reduces membrane size.