Streptozotocin-Induced Adaptive Modification of Mitochondrial Supercomplexes in Liver of Wistar Rats and the Protective Effect of Moringa oleifera Lam

Abstract

The increasing prevalence of diabetes continues to be a major health issue worldwide. Alteration of mitochondrial electron transport chain is a recognized hallmark of the diabetic-associated decline in liver bioenergetics; however, the molecular events involved are only poorly understood. Moringa oleifera is used for the treatment of diabetes. However, its role on mitochondrial functionality is not yet established. This study was aimed to evaluate the effect of M. oleifera extract on supercomplex formation, ATPase activity, ROS production, GSH levels, lipid peroxidation, and protein carbonylation. The levels of lipid peroxidation and protein carbonylation were increased in diabetic group. However, the levels were decreased in Moringa-treated diabetic rats. Analysis of in-gel activity showed an increase in all complex activities in the diabetic group, but spectrophotometric determinations of complex II and IV activities were unaffected in this treatment. However, we found an oxygen consumption abolition through complex I-III-IV pathway in the diabetic group treated with Moringa. While respiration with succinate feeding into complex II-III-IV was increased in the diabetic group. These findings suggest that hyperglycemia modifies oxygen consumption, supercomplexes formation, and increases ROS levels in mitochondria from the liver of STZ-diabetic rats, whereas M. oleifera may have a protective role against some alterations.

Figure 1

Moringa oleifera effect on mitochondrial respiratory chain. (a) Oxygen consumption (^∗ P < 0.05 versus D; ^# P < 0.05 versus C); (b) enzymatic activity of complex I and II (^∗ P < 0.05 versus C and M); (c) F₁/F₀ ATPase activity (^∗ P < 0.05 versus C and M; ∗^∗ P < 0.05 versus M + oligo) from liver mitochondria. C: control; D: diabetic; M: Moringa-treated diabetic groups.

Figure 2

Characterization of OXPHOS proteins expressed in liver mitochondria during diabetes. (a) OXPHOS cocktail specificity demonstrated by a Western blot from liver-isolated mitochondria of diabetic rats and treated with Moringa extract. Relative expression of (b) MTC01 subunit of CIV, (c) SDHB subunit of CII, (d) UQCRC2 subunit of CIII, (e) NDUFB8 subunit of CI, and (f) ATP5A subunit of CV was performed by densitometric analysis from gel. Molecular mass standards are shown on the left panel of the gel. C: control; D: diabetic; M: Moringa-treated group. Data are shown as mean band density normalized relative to UQCR2. Significant differences are represented by ^∗ P < 0.05 versus C; ^# P < 0.05 versus D.

Figure 3

Electrophoretic representative pattern of liver mitochondrial solubilized of the different groups (5 g de digitonin/g protein). (a) Blue native polyacrylamide gel electrophoresis (BN-PAGE) stained with Coomassie blue; (b) complex I in-gel activity; (c) complex II in-gel activity; (d) complex IV in-gel activity; (e) complex IV in-gel activity; (f) high resolution clear native polyacrylamide gel electrophoresis (hrCN-PAGE) of complex V. B: bovine heart solubilized mitochondria (positive control); C: control; D: diabetic; M: Moringa-treated diabetic groups.

Figure 4

H₂O₂ production by mitochondrial respiratory chain measured by Amplex Red (UA) in liver mitochondria from different treatments (C: control; D: diabetic; M: Moringa-treated diabetic groups) oxidizing (a) pyruvate plus malate or (b) succinate as substrates and the effects of respiratory chain inhibitors (R: rotenone; A: antimycin A; Ml: malonate). Mitochondria were studied during state 4 respiration. To correct for the increase in background fluorescence of the Amplex Red/HRP detection system overtime, fluorescence was monitored for a period of ten minutes. This background was subtracted from resorufin trace. Data are means ± SEM (n = 5).