Comprehensive investigations of key mitochondrial metabolic changes in senescent human fibroblasts

Abstract

There is a paucity of detailed data related to the effect of senescence on the mitochondrial antioxidant capacity and redox state of senescent human cells. Activities of TCA cycle enzymes, respiratory chain complexes, hydrogen peroxide (H₂O₂), superoxide anions (SA), lipid peroxides (LPO), protein carbonyl content (PCC), thioredoxin reductase 2 (TrxR2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), reduced glutathione (GSH), and oxidized glutathione (GSSG), along with levels of nicotinamide cofactors and ATP content were measured in young and senescent human foreskin fibroblasts. Primary and senescent cultures were biochemically identified by monitoring the augmented cellular activities of key glycolytic enzymes including phosphofructokinase, lactate dehydrogenase, and glycogen phosphorylase, and accumulation of H₂O₂, SA, LPO, PCC, and GSSG. Citrate synthase, aconitase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and complex I-III, IIIII, and IV activities were significantly diminished in P25 and P35 cells compared to P5 cells. This was accompanied by significant accumulation of mitochondrial H₂O₂, SA, LPO, and PCC, along with increased transcriptional and enzymatic activities of TrxR2, SOD2, GPx1, and GR. Notably, the GSH/GSSG ratio was significantly reduced whereas NAD⁺/NADH and NADP⁺/NADPH ratios were significantly elevated. Metabolic exhaustion was also evident in senescent cells underscored by the severely diminished ATP/ADP ratio. Profound oxidative stress may contribute, at least in part, to senescence pointing at a potential protective role of antioxidants in aging-associated disease.

Keywords: Aging; Antioxidants; Mitochondria; Oxidative stress; Senescence.

Fig. 1. Identification of senescent fibroblasts by monitoring total cellular glucose and glycogen degradative enzyme activities and oxidative stress markers in confluent serial subcultures.

Enzyme activities of (A) phosphofructokinase (PFK), (B) lactate dehydrogenase (LDH), and (C) glycogen phosphorylase (GP), along with levels of (D) hydrogen peroxide (H₂O₂) and (E) superoxide anions (SA), (F) lipid peroxides (LPO), (G) protein carbonyl content (PCC), and (H) oxidized glutathione (GSSG) in P5-P35 confluent cells. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate significant difference from control P5 values (n = 6).

Fig. 2. Mitochondrial specific activities of TCA cycle enzymes in primary P5 and senescent P25 and P35 fibroblasts.

Enzyme activities of (A) aconitase (can), (B) α-ketoglutarate dehydrogenase (α-KGDH), (C) succinate dehydrogenase (SDH), (D) malate dehydrogenase (MDH), (E) NAD⁺- isocitrate dehydrogenase (ICD), and (F) NADP⁺-ICD in young (P5) and senescent (P25 and P35) confluent cells. ***p < 0.001 and ****p < 0.0001 indicate significant difference from control P5 values (n = 6).

Fig. 3. Mitochondrial specific activities of electron transport chain (ETC) enzymes in primary P5 and senescent P25 and P35 fibroblasts.

Enzyme activities of (A) complex I-III, (B) complex II-III, (C) complex IV, and (D) citrate synthase (CS) in young (P5) and senescent (P25 and P35) confluent cells. ***p < 0.001 and ****p < 0.0001 indicate significant difference from control P5 values (n = 6).

Fig. 4. Mitochondrial oxidative stress markers in primary P5 and senescent P25 and P35 fibroblasts.

Generation levels of (A) hydrogen peroxide (H₂O₂), (B) superoxide anions (SA), (C) lipid peroxides (LPO), and (D) protein carbonyl content (PCC), and transcriptional and enzyme activities of thioredoxin reductase 2 (TrxR2) (E, F) and superoxide dismutase 2 (SOD2) (G, H) in young (P5) and senescent (P25 and P35) confluent cells. **p < 0.01), ***p < 0.001, and ****p < 0.0001 indicate significant difference from control P5 values (n = 6).

Fig. 5. Mitochondrial glutathione status in primary P5 and senescent P25 and P35 fibroblasts.

Transcriptional activities of (A) glutathione peroxidase 1 (GPx1) and (B) glutathione reductase (GR), and enzyme activities of (C) GPx1 and (D) GR, along with levels of (E) reduced glutathione (GSH), (F) oxidized glutathione (GSSG), and (G) GSH/GSSG ratio in young (P5) and senescent (P25 and P35) confluent cells. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate significant difference from control P5 values (n = 6).

Fig. 6. Mitochondrial NAD+, NADH, NADP+, and NADPH levels in primary P5 and senescent P25 and P35 fibroblasts.

Levels of (A) NAD⁺, (B) NADH, (C) NAD⁺/NADH ratio, (D) NADP⁺, (E) NADPH, and (F) NADP⁺/NADPH ratio in young (P5) and senescent (P25 and P35) confluent cells. **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate significant difference from control P5 values (n = 6).

Fig. 7. Cellular ATP and ADP levels in primary P5 and senescent P25 and P35 fibroblasts.

Levels of (A) ATP, (B) ADP, and (C) ATP/ADP ratio in young (P5) and senescent (P25 and P35) confluent cells. ****p < 0.0001 indicates significant difference from control P5 values (n = 6).

Fig. 8. A working model of mitochondrial senescence [65].

Replicative senescence induces metabolic changes in mitochondria evident as accumulation of ROS, shutdown of TCA and ETC pathways, glutathione depletion, and energy exhaustion. ROS, reactive oxygen species; ETC, electron transport chain; PFK, phosphofructokinase; LDH, lactate dehydrogenase; GP, glycogen phosphorylase; H₂O₂, hydrogen peroxide; SA, superoxide anions; LPO, lipid peroxides; PCC, protein carbonyl content; GSSG, oxidized glutathione; TrxR2, thioredoxin reductase 2; SOD2, superoxide dismutase 2; GPx1, glutathione peroxidase 1; GR, glutathione reductase; GSH, reduced glutathione.