Progesterone and estrogen regulate oxidative metabolism in brain mitochondria

Abstract

The ovarian hormones progesterone and estrogen have well-established neurotrophic and neuroprotective effects supporting both reproductive function and cognitive health. More recently, it has been recognized that these steroids also regulate metabolic functions sustaining the energetic demands of this neuronal activation. Underlying this metabolic control is an interpretation of signals from diverse environmental sources integrated by receptor-mediated responses converging upon mitochondrial function. In this study, to determine the effects of progesterone (P4) and 17beta-estradiol (E2) on metabolic control via mitochondrial function, ovariectomized rats were treated with P4, E2, or E2 plus P4, and whole-brain mitochondria were isolated for functional assessment. Brain mitochondria from hormone-treated rats displayed enhanced functional efficiency and increased metabolic rates. The hormone-treated mitochondria exhibited increased respiratory function coupled to increased expression and activity of the electron transport chain complex IV (cytochrome c oxidase). This increased respiratory activity was coupled with a decreased rate of reactive oxygen leak and reduced lipid peroxidation representing a systematic enhancement of brain mitochondrial efficiency. As such, ovarian hormone replacement induces mitochondrial alterations in the central nervous system supporting efficient and balanced bioenergetics reducing oxidative stress and attenuating endogenous oxidative damage.

Figure 1

P4 and E2 enhance mitochondrial respiratory activity. Oxygen electrode measurements of respiration using isolated rat brain mitochondria. A, Representative traces of mitochondrial oxygen consumption with or without in vivo E2 (30 μg/kg), P4 (30 μg/kg), E2/P4, or sesame oil vehicle control treatment (Ct) in the presence of l-malate (Mal; 5 mm), l-glutamate (Glut; 5 mm), and ADP (410 μm) to initiate state 3 respiration and atractyloside to induce state 4_o respiration. The traces are representative of six to eight separate experiments. B, Percent changes in mitochondrial RCR (state 3/state 4_o). The data represent mean ± sem of eight separate experiments. *, P < 0.05 compared with control; n = 8.

Figure 2

P4 and E2 enhance COX activity and expression. A, Relative rate of COX activity of isolated whole-brain mitochondria with or without in vivo E2 (30 μg/kg), P4 (30 μg/kg), E2/P4, or sesame oil vehicle control treatment. The bars represent mean ± sem from four separate experiments with two animals per group for each experiment. *, P < 0.05 compared with control; **, P < 0.01 compared with control; n = 8. B, Total RNA was isolated from brain after 24 h exposure to E2 (30 μg/kg), P4 (30 μg/kg), E2/P4, or sesame oil vehicle control. Expression of COXI, COXII, COXIII, and COXIV mRNA was assessed by real-time RT-PCR. Bars represent mean relative expression ± sem from eight animals per group. *, P < 0.05 compared with control; n = 8.

Figure 3

P4 and E2 increase expression of complex V, subunit α protein. Total protein was isolated from brain after 24 h exposure to E2 (30 μg/kg), P4 (30 μg/kg), E2/P4, or sesame oil vehicle control. Expression of complex V, subunit α was determined by Western blot analysis. Bars represent mean relative expression ± sem from four animals per group. *, P < 0.05 compared with control; n = 4.

Figure 4

P4 and E2 do not alter mitochondrial biogenesis. Total DNA was isolated from brain after 24 h exposure to E2 (30 μg/kg), P4 (30 μg/kg), E2/P4, or sesame oil vehicle control. The relative copy number of COXII (mitochondrial) and β-actin (nuclear) DNA was assessed by real-time PCR, and the ratio of COXII/ β-actin was used as a marker of relative mitochondrial number. Bars represent mean ± sem from eight animals per group. *, P < 0.05 compared with control; n = 8.

Figure 5

Ovarian hormones reduce rate of peroxide production and free radical leak in brain mitochondria. A, Fluorescent Amplex Red measurements of H₂O₂ production in isolated rat brain mitochondria exposed in vivo to ovarian hormones. Spectrofluorometer measurements were made of H₂O₂ production by isolated brain mitochondria in the presence of malate (5 mm) and glutamate (5 mm) plus ADP (410 μm) to initiate state 3 respiration. Values represent relative mean H₂O₂ production rates ± sem of eight separate experiments. *, P < 0.05 compared with control; n = 8. B, The percent free radical leak was calculated for isolated whole-brain mitochondria after in vivo E2 (30 μg/kg), P4 (30 μg/kg), E2/P4, or sesame oil vehicle control treatment. Free radical leak was calculated as the percentage of H₂O₂ production to twice the rate of O₂ consumption. The bars represent mean ± sem from eight animals per group. *, P < 0.05 compared with control; **, P < 0.01 compared with control; n = 8.

Figure 6

P4 and E2 reduce lipid peroxidation of brain mitochondria. Whole-brain mitochondria were isolated 24 h after in vivo exposure to E2 (30 μg/kg), P4 (30 μg/kg), E2/P4, or sesame oil vehicle control treatment. Lipid peroxides in brain mitochondria were measured using the leucomethylene blue assay (A) or by measuring TBARS (B). The bars represent mean ± sem from eight animals per group. *, P < 0.05 compared with control; **, P < 0.01 compared with control; n = 8.

Figure 7

P4 and E2 alter the antioxidant profile of brain mitochondria. Whole-brain mitochondria were isolated 24 h after in vivo exposure to E2 (30 μg/kg), P4 (30 μg/kg), E2/P4, or sesame oil vehicle control treatment. Expression of the mitochondrial antioxidant proteins MnSOD (A) and peroxiredoxin V (B) were measured using Western blot analysis. The bars represent mean ± sem from four animals per group. *, P < 0.05 compared with control; **, P < 0.01 compared with control; n = 4.