Impact of estrogen receptor agonists and model of menopause on enzymes involved in brain metabolism, acetyl-CoA production and cholinergic function.

Our goal is to understand how loss of circulating estrogens and estrogen replacement affect brain physiology and function, particularly in brain regions involved in cognitive processes. We recently conducted a large metabolomics study characterizing the effects of rodent models of menopause and treatment with estrogen receptor (ER) agonists on neurochemical targets in hippocampus, frontal cortex, and striatum. Here we characterize effects on levels of several key enzymes involved in glucose utilization and energy production, specifically phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, and pyruvate dehydrogenase. We also evaluated effects on levels of ß-actin and α-tubulin, choline acetyltransferase (ChAT) activity, and levels of ATP citrate lyase. All experiments were conducted in young adult rats. Experiment 1 compared the effects of ovariectomy (OVX), a model of surgical menopause, and 4-vinylcyclohexene diepoxide (VCD)-treatments, a model of transitional menopause, with tissues collected at proestrus and at diestrus. Experiment 2 used a separate cohort of rats to evaluate the same targets in OVX and VCD-treated rats treated with estradiol or with selective ER agonists. Differences in the expression of metabolic enzymes between cycling animals and models of surgical and transitional menopause were detected. These differences were model-, region- and time- dependent, and were modulated by selective ER agonists. Collectively, the findings demonstrate that loss of ovarian function and ER agonist treatments have differing effects in OVX vs. VCD-treated rats. Differences may help to explain differences in the effects of estrogen treatments on brain function and cognition in women who have experienced surgical vs. transitional menopause.

Use of the REVERT® total protein stain as a loading control demonstrates significant benefits over the use of housekeeping proteins when analyzing brain homogenates by Western blot: An analysis of samples representing different gonadal hormone states.

Western blot is routinely used to quantify differences in the levels of target proteins in tissues. Standard methods typically use measurements of housekeeping proteins to control for variations in loading and protein transfer. This is problematic, however, when housekeeping proteins also are affected by experimental conditions such as injury, disease, and/or gonadal hormone manipulations. Our goal was to evaluate an alternative and perhaps superior method for conducting Western blot analysis of brain tissue homogenates from rats with distinct physiologically relevant gonadal hormone states. Tissues were collected from the hippocampus, frontal cortex, and striatum of young adult female rats that either were ovariectomized to model surgical menopause, or were treated with the ovatotoxin 4-vinylcyclohexene diepoxide (VCD) to model transitional menopause. Tissues also were collected from rats with a normal estrous cycle killed at proestrus when estradiol levels are high, and at diestrus when estradiol levels are low. Western blot detection of α-tubulin, ß-actin, and GAPDH was performed and were compared for sensitivity and reliability with a fluorescent total protein stain (REVERT®). Results show that the total protein stain was much less variable across samples and had a greater linear range than α-tubulin, ß-actin, or GAPDH. The stain was stable and easy to use, and did not interfere with the immunodetection or multiplexed detection of the housekeeping proteins. In addition, we show that normalization of our data to total protein, but not to GAPDH, revealed significant differences in α-tubulin expression in the hippocampus as a function of treatment, and that gel-to-gel consistency in measuring differences between paired samples run on multiple gels was significantly better when data were normalized to total protein than when normalized to GAPDH. These results demonstrate that the REVERT® total protein stain can be used in Western blot analysis of brain tissue homogenates to control for variations in loading and protein transfer, and provides significant advantages over the use of housekeeping proteins for quantifying changes in the levels of multiple target proteins.