(M1130-02-08) Sex Hormone-Dependent Regulation of MCT1 on the Blood Brain Barrier

Purpose: Proton-dependent monocarboxylate transporters (MCTs) are involved in monocarboxylate uptake and efflux in a range of biologically important tissues and barriers, including the liver, kidney, and blood-brain barrier (BBB) (1). MCTs are involved in the transport of endogenous monocarboxylates including L-lactate, and monocarboxylate drugs, such as gamma-hydroxybutyrate and valproic acid (1). Among all the isoforms, MCT1 is expressed ubiquitously in the body, and is involved in drug absorption, distribution, and elimination. MCT1 can be found on the BBB, and is critical for brain distribution; increased BBB MCT1 may increase drug distribution to the brain (2). In our laboratory, we have published data demonstrating sex hormone-dependent regulation of hepatic MCT1, MCT4, SMCT1, and CD147 (3). The purpose of this project is to validate the BBB isolation technique and characterize MCT1 membrane protein expression localized on the BBB in animals treated with sex and cross-sex hormone therapy.

Methods: Male and female Sprague Dawley (SD) rats, ovariectomized (OVX) females and castrated (CST) males were obtained from Charles River at 8 weeks of age and were housed in a temperature-controlled room with a 12-hour day/night lighting cycle. At 10 weeks of age, OVX female and CST male SD rats were implanted subcutaneously with 60-day release pellets containing 1.5 mg estrogen and/or 50 mg progesterone or their corresponding control pellets (Innovative Research of America) (N = 6/treatment group). The BBB isolation technique was performed using a protocol developed by Brizca et al. (4). Following euthanasia, brains were harvested; cerebral hemispheres were isolated and homogenized with brain microvessel buffer. After four dextran centrifugation steps, pellets enriched with BBB were obtained. Membrane bound protein was extracted from BBB pellets using a ProteoExtract Native Membrane Extraction kit. Na⁺/K⁺ ATPase expression was evaluated by western blot to verify membrane isolation. BBB purity was assessed by western blot against platelet endothelial cell adhesion molecule-1 (PECAM-1) and synaptophysin. MCT1 protein expression levels were quantified by western blot and normalized to Na⁺/K⁺ ATPase expression.

Results: The purity of each BBB sample was validated by western blots against PECAM-1 and synaptophysin. Intact females showed the lowest MCT1 protein expression when compared with all other groups. OVX females treated with estrogen had significantly lower MCT1 protein expression than the corresponding placebo group. OVX with progesterone or both female sex hormones had lower MCT1 protein expression than their corresponding placebo groups, respectively. OVX females treated with estrogen showed similar MCT1 expression in comparison with intact females. CST males treated with estrogen had lower MCT1 protein expression than the corresponding placebo group. CST males with both female sex hormones showed significantly lower MCT1 expression when compared to CST estrogen or progesterone placebo group and progesterone group; however, there were also decreases in MCT1 expression in the corresponding placebo group. MCT1 protein expression in CST males treated with both female sex hormones was comparable to intact females.

Conclusion: MCT1 localized on the BBB can be regulated by sex hormone or cross-sex hormone treatment. OVX females treated with female sex hormones exhibited lower MCT1 protein expression, with estrogen significantly reducing protein expression of MCT1 on the BBB. These differences in BBB MCT1 expression may contribute to differences in brain distribution of MCT1 drug substrates.

References: 1. Morris ME, Felmlee MA. Overview of the proton-coupled MCT (SLC16A) family of transporters: characterization, function and role in the transport of the drug of abuse γ-hydroxybutyric acid. The AAPS journal. 2008;10(2):311-21.
2. Roiko SA, Felmlee MA, Morris ME. Brain uptake of the drug of abuse γ-hydroxybutyric acid in rats. Drug Metabolism and Disposition. 2012;40(1):212-8.
3. Cao J, Ng M, Felmlee MA. Sex Hormones Regulate Rat Hepatic Monocarboxylate Transporter Expression and Membrane Trafficking. J Pharm Pharm Sci. 2017;20(1):435-44.
4. Brzica H, Abdullahi W, Reilly BG, Ronaldson PT. A simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels. Journal of visualized experiments: JoVE. 2018(135).

Acknowledgments: This work is funded by NIDA/NIGMS DA052120.

Figure 1. Representative western blots results for (A) PECAM-1 (BBB marker protein); (B) synaptophysin (neuronal marker proteins); (C) Na+/K+ ATPase; (D) MCT1 in BBB isolated from Sprague–Dawley rats.



Figure 2. MCT1 membrane protein expression of (A) OVX and (B) CST implanted with estrogen, progesterone or both, their corresponding placebo, females (estrus) and males in BBB isolated from Sprague–Dawley rats. Expression was normalized to Na+/K+ ATPase . Results are presented as mean ± SD, N = 4 - 6. (A) *p < 0.05 compared to OVX estrogen placebo group; # p<0.05 compared to OVX progesterone placebo group. (B) *p < 0.05 compared to CST estrogen placebo group; # p<0.05 compared to CST progesterone placebo group; % p<0.05 compared to CST progesterone group.