Triglyceride storage is altered across various chronic health conditions necessitating various techniques to visualize and quantify lipid droplets (LDs). Here, we describe the utilization of the BODIPY (493/503) dye in skeletal muscle as a means to analyze LDs. We found that the dye was a convenient and simple approach to visualize LDs in both sectioned skeletal muscle and cultured adult single fibers. Furthermore, the dye was effective in both fixed and nonfixed cells, and the staining seemed unaffected by permeabilization. We believe that the use of the BODIPY (493/503) dye is an acceptable alternative and, under certain conditions, a simpler method for visualizing LDs stored within skeletal muscle.

Adipocytes from post-menopausal females have higher basal lipolytic rates than pre-menopausal females, which contributes to increased risk of developing dyslipidemia following menopause. The purpose of this study was to delineate cellular mechanisms affecting adipose tissue function in the ovariectomized (OVX) mouse and also determine if physical activity or estrogen supplementation alter any detected changes. Female C57/Bl6 mice were placed into SHAM, OVX sedentary (OVX), OVX exercise (OVX-Ex), and OVX sedentary + 17beta-estradiol (OVX + E(2)) groups. Visceral fat mass, glycerol, and NEFA levels were significantly higher in OVX mice compared to SHAM animals, but were not elevated in the E(2)-treated animals. Voluntary running failed to change circulating levels of glycerol or NEFA in OVX mice, but did partially attenuate the increase in visceral fat mass. Adipose triglyceride lipase (ATGL) protein content was significantly elevated in visceral fat from OVX and OVX-Ex groups compared to SHAM, while ATGL-CGI-58 interaction was significantly higher in OVX than SHAM and OVX + E(2) mice. No significant differences in HSL phosphorylation were detected between groups, however, ERK1/2 phosphorylation was significantly elevated in the OVX mice. To determine if ERK1/2 function was critical for the increased glycerol levels, visceral fat was treated with MEK inhibitor PD98059, with no differences in glycerol release detected. Perilipin protein content was decreased significantly in OVX and OVX-Ex mice compared to SHAM. Thus, these data suggest that increased ATGL signaling and reduced perilipin protein content may contribute to increased NEFA and glycerol levels in OVX mice, which are attenuated with E(2) treatment, but not by exercise.

Decreases in female sex steroids enhance the accumulation of visceral fat mass, leading to a predisposition to developing metabolic diseases. The purpose of this study was to determine whether loss of ovarian function alters the amount and (or) the fatty acid (FA) composition of triacylglycerol (TAG) levels in the liver of ovary-intact (SHAM) or ovariectomized (OVX) mice. We also sought to determine whether voluntary wheel running could attenuate the associated changes in the liver. Twenty-two C57/BL6 female mice were divided into 2 groups (SHAM, OVX) and were then subdivided into sedentary and exercising groups (SHAM-Sed, SHAM-Ex, OVX-Sed, OVX-Ex). Visceral fat mass significantly increased in the OVX-Sed animals; however, the effect was attenuated in the OVX-Ex animals. Total hepatic TAG content did not significantly increase in the OVX-Sed animals; however, SHAM-Ex and OVX-Ex animals demonstrated significant decreases in TAG levels. A significant increase in the FA desaturase index (18:1/18:0 and 16:1/16:0) was detected in the OVX-Sed animals compared with all other groups, which corresponded to increases in stearoyl-CoA desaturase (SCD-1) content. These results indicate that loss of ovarian function alters FA composition of hepatic TAG mediated by increases in SCD-1. These data indicate that female sex steroids influence lipid metabolism in the liver and provide important insight concerning the influence of exercise on hepatic function.

Disruptions of ovarian function in women are associated with increased risk of metabolic disease due to dysregulation of peripheral glucose homeostasis in skeletal muscle. Our previous evidence suggests that alterations in skeletal muscle lipid metabolism coupled with altered mitochondrial function may also develop. The objective of this study was to use an integrative metabolic approach to identify potential areas of dysfunction that develop in skeletal muscle from ovariectomized (OVX) female mice compared with age-matched ovary-intact adult female mice (sham). The OVX mice exhibited significant increases in body weight, visceral, and inguinal fat mass compared with sham mice. OVX mice also had significant increases in skeletal muscle intramyocellular lipids (IMCL) compared with the sham animals, which corresponded to significant increases in the protein content of the fatty acid transporters CD36/FAT and FABPpm. A targeted metabolic profiling approach identified significantly lower levels of specific acyl carnitine species and various amino acids in skeletal muscle from OVX mice compared with the sham animals, suggesting a potential dysfunction in lipid and amino acid metabolism, respectively. Basal and maximal mitochondrial oxygen consumption rates were significantly impaired in skeletal muscle fibers from OVX mice compared with sham animals. Collectively, these data indicate that loss of ovarian function results in increased IMCL storage that is coupled with alterations in mitochondrial function and changes in the skeletal muscle metabolic profile.

Recent evidence suggests that ovarian hormones contribute to altered function of skeletal muscle, however the signaling processes thought to regulate muscle function remain undefined in females. Thus, the purpose of this investigation is to determine if ovarian hormone status is critical for contraction-induced activation of AMPK or MAPK in skeletal muscle. Female mice were divided into two groups, ovariectomy (OVX) and SHAM, which were then subjected to in situ isometric contractile protocols. AMPK, ERK 1/2, p38, and JNK phosphorylation were measured in the control and contracting limb. In the in situ protocol, OVX muscles were significantly more resistant to fatigue compared to the SHAM animals. In addition, the muscles from OVX mice demonstrated significantly lower levels of normalized AMPK phosphorylation at rest. AMPK phosphorylation was not increased in the muscles from SHAM mice after the in situ contractile protocol, while the OVX demonstrated significant increases in AMPK phosphorylation. After contraction, normalized ERK2 phosphorylation was significantly higher in the OVX group compared to the SHAM group. Both p38 and JNK phosphorylation increased in response to contraction; but no group differences were detected. A second set of SHAM and OVX animals were subjected to fatigue stimulated under in vitro conditions. Significant increases in AMPK and ERK2 phosphorylation were detected, but no differences were found between groups. In conclusion, removal of the ovaries results in different responses to contraction-induced changes in phosphorylation of AMPK and ERK2 in female mice and suggests hormones secreted from the ovaries significantly impacts cellular signaling in skeletal muscle.