17β-Estradiol Directly Lowers Mitochondrial Membrane Microviscosity and Improves Bioenergetic Function in Skeletal Muscle

Abstract

Menopause results in a progressive decline in 17β-estradiol (E2) levels, increased adiposity, decreased insulin sensitivity, and a higher risk for type 2 diabetes. Estrogen therapies can help reverse these effects, but the mechanism(s) by which E2 modulates susceptibility to metabolic disease is not well understood. In young C57BL/6N mice, short-term ovariectomy decreased-whereas E2 therapy restored-mitochondrial respiratory function, cellular redox state (GSH/GSSG), and insulin sensitivity in skeletal muscle. E2 was detected by liquid chromatography-mass spectrometry in mitochondrial membranes and varied according to whole-body E2 status independently of ERα. Loss of E2 increased mitochondrial membrane microviscosity and H₂O₂ emitting potential, whereas E2 administration in vivo and in vitro restored membrane E2 content, microviscosity, complex I and I + III activities, H₂O₂ emitting potential, and submaximal OXPHOS responsiveness. These findings demonstrate that E2 directly modulates membrane biophysical properties and bioenergetic function in mitochondria, offering a direct mechanism by which E2 status broadly influences energy homeostasis.

Keywords: estrogen; hormone replacement therapy; hydrogen peroxide; insulin resistance; membrane viscosity; menopause; mitochondria; ovariectomy.

Figure 1. 2 week-OVX decreases mitochondrial function and induces an oxidative shift in SM cellular redox environment

(A) Citrate synthase activity in RG. (B) JO₂ measured in PmFbs from RG. (C) Fatty acid-supported JO₂. Abbreviations: glutamate/malate (G/M), succinate (Succ), rotenone (Rot), antimycin A (AmA), ascorbate (Asc), N,N,N′,N′-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD), palmitoyl-carnitine (PC), almitoyl-CoA (P-CoA), L-carnitine (L-Carn). (D–F) Mitochondrial respiratory kinetics. Pyruvate titrations in the presence of malate and ADP (D), and ADP titrations in the presence of G/M (E) or pyruvate/malate (F). Kinetic parameters K_M and Vmax were determined from fitting to Michaelis Menten functions. Changes in Vmax were significant (*p<0.05). (G) Total GSH and GSSG concentration and resulting GSH/GSSG ratios (H) in RG. (I) JH₂O₂ measured in PmFbs pre-incubated with or without 1-chloro-2,4-dinitrobenzene (CDNB) for GSH depletion, then added pyruvate. Values are means ± SEM; * p<0.05; ** p<0.005; *** p<0.0005, N = 8-15 mice/group.

Figure 2. E2 therapy reverses the OVX-induced pro-diabetogenic state

(A) Study design. (B) Uterine mass at sacrifice. (C) Body Composition. (D) Fasting blood glucose. ! p<0.05, !! p<0.005 vs. pre-OVX for each group, and # p<0.05 vs. OVX-4w[ctl]. (E) Glucose tolerance test. Inset: Area under the curve (AUC). (F) Insulin tolerance test. Inset: slope of the least-squared-regression line calculated from the first 20 min. (G) HOMA-IR scores calculated as in Figure S2E. (H–I) Ex-vivo basal and insulin-stimulated ³H-2-Deoxy-glucose uptake in whole EDL (H) and soleus (I) muscles. Values are means ± SEM, * p<0.05, ** p<0.005 vs. NC-Pro and # p<0.05, ## p<0.005 vs. OVX-4w[ctl], N = 6 mice/group.

Figure 3. E2 therapy restores mitochondrial function and cellular redox balance in SM

(A) JO₂ measured in PmFbs from RG. C I-supported JO₂ measured by the sequential addition of pyruvate/malate (Pyr/Mal) and ADP (left panel); or glutamate/malate (G/M) and ADP (middle panel), and C II-supported JO₂ measured in the presence of rotenone (Rot), succinate (Succ), and ADP (right panel). (B) Fatty acid-supported JO₂ in RG PmFbs, as in Figure 1C. (C) Total GSH and GSSG levels in whole gastrocnemius. (D) Resulting 2GSH/GSSG ratios. (E) Left: JH₂O₂ measured after the addition of Pyr, followed by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). !! p<0.005 vs Pyr. Right: PmFBs were pre-incubated with 1-chloro-2,4-dinitrobenzene (CDNB), then added pyruvate. (F) JH₂O₂ measured after the addition of succinate, auranofin. (G) Mitochondrial free radical leak (JH₂O₂/JO₂*2) under succinate-supported respiration. Values are means ± SEM, * p<0.05; ** p<0.005; *** p<0.0005 vs. NC-Pro, and # p<0.05, ## p<0.005 vs. OVX-4w[ctl], N = 6 mice/group.

Figure 4. E2 therapy restores OVX-induced loss of C I, C I/II+III activities

(A) Western blot analysis of OXPHOS complexes and (B) citrate synthase activity in isolated mitochondria. (C) Relative specific activity of respiratory complexes normalized to CS activity (1 CSU = 1 µmol/min/mg prot). See Figure S4. Values are means ± SEM, * p<0.05, ** p<0.005 vs. NC-Pro, and # p<0.05, ## p<0.005 vs. OVX-4w[ctl], N = 4-5 mice/group.

Figure 5. E2 decreases microviscosity in biomimetic mitochondrial membranes

(A) Sample fluorescence MC-540 emission spectra from LUVs made with PC:PE:PS and increasing [E2], or cholesterol (negative control). (B) Peak MC-540 F values from (A). Values are means ± SEM, from 3 independent LUVs preparations. * p<0.05; ** p<0.005 vs [E2] 0%. (C) Representative pressure-area (π-A) isotherms of DPPC monolayers containing 5 mol% E2 or cholesterol. (D) Average mean molecular area (Mma) at relevant surface pressures extrapolated from 3 independent π-A isotherms. (E) The surface elasticity moduli (Cs⁻¹) as a function of Mma, corresponding to the respective π-A isotherms shown in panel C. (F) Cs⁻¹ calculated from 3 independent π-A isotherms (dotted lines). Values are means +/− SEM.

Figure 6. E2 localizes to mitochondrial membranes and decreases microviscosity independent of ER α

(A) Representative extracted ion chromatograms for E2. (B) E2 content in mitochondrial membrane extracts measured by LC/MS. (C) Fluorescence MC-540 emission spectra in intact SM mitochondria. (D) Peak MC-540 F values from C. Data are means +/− SEM, * p<0.05, ** p<0.005, *** p<0.0005 vs NC-Pro, and # p<0.05, ## p<0.005 vs. OVX-4w[ctl], n=6-8 mice/group. (E) E2 content and respective CI relative specific activity (F). Values are means +/− SEM, * p<0.05 vs. NC-skmERαKO, and # p<0.05 vs. OVX-skmERαKO, N = 3-4 mice/group.

Figure 7. In-vitro E2 exposure of OVX mitochondria restores membrane microviscosity JH₂O₂ emitting potential and OXPHOS steady-state flux

(A) Experimental design. (B) E2 content in mitochondrial membranes. Values are means +/− SEM, N = 7-14 mice/group. (C) Peak F values from MC-540 emission spectra in fresh intact SM mitochondria pre-incubated +/− 3 nM E2. Values are means +/− SEM of three replicate measures, N = 8 mice/group. (D) Specific activities of complexes, measured as in Figure 4. N = 7 mice/group. (E) JH₂O₂ and (F) maximal JO₂ capacity measured in isolated mitochondria as in Figure 1B. (G) JO₂, (H) JATP, and (I) resulting ATP/O ratios (JATP/JO₂), in the presence of G/M/Pyr/Succ and ADP. Values are means ± SEM. For all panels, * p<0.05, ** p<0.005 vs. NC-Pro, and ! p<0.05, !! p<0.005 vs. OVX-4w. N = 7 mice/group.