Structural and Dynamic Features of Liver Mitochondria and Mitophagy in Rats with Hyperthyroidism

Abstract

This work investigated the effect of thyroxine on the biogenesis and quality control system of rat liver mitochondria. Chronic administration of thyroxine to experimental animals induced hyperthyroidism, which was confirmed by a severalfold increase in serum-free triiodothyronine and thyroxine concentrations. The uptake of oxygen was found to increase with a decrease in ADP phosphorylation efficiency and respiratory state ratio. Electron microscopy showed 36% of liver mitochondria to be swollen and approximately 18% to have a lysed matrix with a reduced number of cristae. Frequently encountered multilamellar bodies associated with defective mitochondria were located either at the edge of or inside the organelle. The number, area and perimeter of hyperthyroid rat mitochondria increased. Administration of thyroxine increased mitochondrial biogenesis and the quantity of mitochondrial DNA in liver tissue. Mitochondrial dynamics and mitophagy changed significantly. The data obtained indicate that excess thyroid hormones cause a disturbance of the mitochondrial quality control system and ultimately to the incorporation of potentially toxic material in the mitochondrial pool.

Keywords: energy metabolism; mitochondria; mitochondrial dysfunction; quality control system; thyroid hormones.

Figure 1

Typical electron micrographs of hepatic tissue in the control (A–C) and in experimental hyperthyroidism (D–I). N, nucleus. Scale bar, 2 µm. (J–N) Electron micrographs of mitochondrial morphological appearance: (J)—well-defined, intact, organized membranes and cristae; (K)—swollen mitochondria, slightly a decrease in the electron density of the matrix and irregular cristae; (L)—swollen mitochondria with MLBs; (M)—matrix vacuolization, cristae are almost not defined; (N)—damaged mitochondria with MLBs. (O–Q) Morphometric parameters of liver mitochondria from experimental rats. (O) The number of mitochondria per image (25 µm²). The number of examined images in each group was about 50. (P) Mitochondrial subpopulations occurring in the groups. The number of mitochondria analyzed in each group varied from 500 to 600. (Q) The ratio of the mitochondrial area to that of the total image area (25 µm²). CR, control; HR, hyperthyroidism; CRsw/HRsw, swollen mitochondria; HRsw + MLBs, swollen mitochondria with MLBs; CRdam, damaged mitochondria; HRdam + MLBs, damaged mitochondria with MLBs. *—multilamellar bodies (MLBs) (A–N); *—p < 0.05 (O–Q), ***—p < 0.001 as compared with the control data (n = 2–3 in each group).

Figure 1

Typical electron micrographs of hepatic tissue in the control (A–C) and in experimental hyperthyroidism (D–I). N, nucleus. Scale bar, 2 µm. (J–N) Electron micrographs of mitochondrial morphological appearance: (J)—well-defined, intact, organized membranes and cristae; (K)—swollen mitochondria, slightly a decrease in the electron density of the matrix and irregular cristae; (L)—swollen mitochondria with MLBs; (M)—matrix vacuolization, cristae are almost not defined; (N)—damaged mitochondria with MLBs. (O–Q) Morphometric parameters of liver mitochondria from experimental rats. (O) The number of mitochondria per image (25 µm²). The number of examined images in each group was about 50. (P) Mitochondrial subpopulations occurring in the groups. The number of mitochondria analyzed in each group varied from 500 to 600. (Q) The ratio of the mitochondrial area to that of the total image area (25 µm²). CR, control; HR, hyperthyroidism; CRsw/HRsw, swollen mitochondria; HRsw + MLBs, swollen mitochondria with MLBs; CRdam, damaged mitochondria; HRdam + MLBs, damaged mitochondria with MLBs. *—multilamellar bodies (MLBs) (A–N); *—p < 0.05 (O–Q), ***—p < 0.001 as compared with the control data (n = 2–3 in each group).

Figure 2

Morphometric parameters of liver mitochondria from experimental rats. (A) A histogram of distribution of the mitochondrial area in the groups. (B) A histogram of distribution of the mitochondrial perimeter in the groups. The number of mitochondria analyzed in each group varied from 500 to 600. CR, control; HR, hyperthyroidism. (n = 3 in each group).

Figure 3

Relative mtDNA levels in the liver of animals. A real-time qPCR was carried out to determine the mtDNA copy number, which is calculated as the ratio mtRNA to nuclear GADPH. CR-control, HR-hyperthyroidism. *—p < 0.05 compared with the control data (n = 8).

Figure 4

Fold changes of mRNA expression of mitochondrial dynamics, biogenesis and mitophagy genes in liver of experimental animals. CR, control; HR, hyperthyroidism.*—p < 0.05, **—p < 0.02, ***—p < 0.001 as compared with the control data (n = 10).

Figure 5

Immunoblotting analysis of mitochondrial dynamics and biogenesis proteins in liver tissue of control and hyperthyroid rats. (A) Representative Western blot of Drp1, OPA1, Mfn2, PGC1α, C₁-C₅-CR, T₁-T₅-HR. (B–E) Relative levels of appropriate proteins with respect to the loading control (GAPDH). CR, control; HR, hyperthyroidism. **—p < 0.02, ***—p < 0.001 as compared with the control data (n = 5).

Figure 6

Immunoblotting analysis of mitophagy proteins in liver tissue of control and hyperthyroid rats. (A) Representative Western blot of Parkin, PINK1, SQSTM1/p62, LC3A/B-I:II, BNIP3L, C₁-C₅-CR, T₁-T₅-HR. (B–F) Relative levels of appropriate proteins with respect to the loading control (GAPDH). CR, control; HR, hyperthyroidism. **—p < 0.02, ***—p < 0.001 as compared with the control data (n = 5–6).