Alisporivir Treatment Alleviates Mitochondrial Dysfunction in the Skeletal Muscles of C57BL/6NCrl Mice with High-Fat Diet/Streptozotocin-Induced Diabetes Mellitus

Abstract

Diabetes mellitus is a systemic metabolic disorder associated with mitochondrial dysfunction, with mitochondrial permeability transition (MPT) pore opening being recognized as one of its pathogenic mechanisms. Alisporivir has been recently identified as a non-immunosuppressive analogue of the MPT pore blocker cyclosporin A and has broad therapeutic potential. The purpose of the present work was to study the effect of alisporivir (2.5 mg/kg/day i.p.) on the ultrastructure and functions of the skeletal muscle mitochondria of mice with diabetes mellitus induced by a high-fat diet combined with streptozotocin injections. The glucose tolerance tests indicated that alisporivir increased the rate of glucose utilization in diabetic mice. An electron microscopy analysis showed that alisporivir prevented diabetes-induced changes in the ultrastructure and content of the mitochondria in myocytes. In diabetes, the ADP-stimulated respiration, respiratory control, and ADP/O ratios and the level of ATP synthase in the mitochondria decreased, whereas alisporivir treatment restored these indicators. Alisporivir eliminated diabetes-induced increases in mitochondrial lipid peroxidation products. Diabetic mice showed decreased mRNA levels of Atp5f1a, Ant1, and Ppif and increased levels of Ant2 in the skeletal muscles. The skeletal muscle mitochondria of diabetic animals were sensitized to the MPT pore opening. Alisporivir normalized the expression level of Ant2 and mitochondrial susceptibility to the MPT pore opening. In parallel, the levels of Mfn2 and Drp1 also returned to control values, suggesting a normalization of mitochondrial dynamics. These findings suggest that the targeting of the MPT pore opening by alisporivir is a therapeutic approach to prevent the development of mitochondrial dysfunction and associated oxidative stress in the skeletal muscles in diabetes.

Keywords: alisporivir; diabetes mellitus; lipid peroxidation; mitochondria; mitochondrial dysfunction; mitochondrial permeability transition pore.

Figure 1

Induction scheme of diabetes mellitus (a), body weight gain (b), intraperitoneal glucose tolerance test, IPGTT (c), and intraperitoneal insulin sensitivity test, IPIST (e) in control (CTR), alisporivir-treated control (CTR+Ali), diabetic (DM), and alisporivir-treated diabetic (DM+Ali) mice. The total areas under the curve (AUC) of the IPGTT (d) and IPIST (f) are shown. The tests were conducted on the 60th day from the beginning of the experiment. ** In subfigure b, the difference between the CTR and DM+Ali groups is significant at p < 0.01. **** differences between the CTR and DM groups are significant at p < 0.0001. All data are presented as mean ± SEM (n = 5). Data in subfigures (a,c,d) are from Belosludtseva et al. Biology 2021, 10, 839, doi: 10.3390/biology10090839.

Figure 2

Representative electron micrographs of mouse skeletal muscle mitochondria in the experimental groups: CTR (a), CTR + Ali (b), DM (c), and DM + Ali (d). Samples from two skeletal muscles (M. quadriceps femoris) were analyzed in each experimental group. The number of the examined fields of view (25 μm²) in the groups was 30–50. Red arrows indicate individual mitochondria. Scale bar: 1 μm.

Figure 3

The number of mitochondria per field of view (25 μm²). The number of examined fields of view was 30–50 in each group.

Figure 4

Levels of the proteins of mitochondrial respiratory chain complexes. (a) Data from the Western blot analysis. The letter of “M” indicates a positive control (rat heart tissue lysate—mitochondrial extract). Quantification of complex I/tubulin ratio (b), complex II/tubulin ratio (c), complex III/tubulin ratio (d), complex IV/tubulin ratio (e), and complex V/tubulin ratio (ATP synthase, f). The data are presented as mean ± SEM (n = 6).

Figure 5

The relative mRNA levels of Atp5f1a (a), Ppif (b), Ant1 (c), and Ant2 (d) in the skeletal muscles of experimental animals. The values are given as mean ± SEM (n = 9).

Figure 6

Changes in the external (Ca²⁺) upon the successive addition of Ca²⁺ pulses (10 μM) to the suspension of the skeletal muscle mitochondria of the experimental animals (a). Ca²⁺ retention capacity of skeletal muscle mitochondria of experimental animals (b). The values are given as means ± SEM (n = 6).

Figure 7

Alisporivir treatment suppresses the DM-induced lipid peroxidation in mouse skeletal muscle mitochondria. Lipid peroxidation was assessed by the level of TBARS in the skeletal muscle mitochondria of the experimental animals. All data are mean ± SEM (n = 5).

Figure 8

The relative mRNA levels of Drp1 (a), Mfn2 (b), Opa1 (c), and Ppargc1a (d) in the skeletal muscle of experimental animals. The values are given as mean ± SEM (n = 6–9).