Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress

Abstract

Mitochondrial-supported bioenergetics decline and oxidative stress increases during aging. To address whether the dietary addition of acetyl-l-carnitine [ALCAR, 1.5% (wt/vol) in the drinking water] and/or (R)-alpha-lipoic acid [LA, 0.5% (wt/wt) in the chow] improved these endpoints, young (2-4 mo) and old (24-28 mo) F344 rats were supplemented for up to 1 mo before death and hepatocyte isolation. ALCAR+LA partially reversed the age-related decline in average mitochondrial membrane potential and significantly increased (P = 0.02) hepatocellular O(2) consumption, indicating that mitochondrial-supported cellular metabolism was markedly improved by this feeding regimen. ALCAR+LA also increased ambulatory activity in both young and old rats; moreover, the improvement was significantly greater (P = 0.03) in old versus young animals and also greater when compared with old rats fed ALCAR or LA alone. To determine whether ALCAR+LA also affected indices of oxidative stress, ascorbic acid and markers of lipid peroxidation (malondialdehyde) were monitored. The hepatocellular ascorbate level markedly declined with age (P = 0.003) but was restored to the level seen in young rats when ALCAR+LA was given. The level of malondialdehyde, which was significantly higher (P = 0.0001) in old versus young rats, also declined after ALCAR+LA supplementation and was not significantly different from that of young unsupplemented rats. Feeding ALCAR in combination with LA increased metabolism and lowered oxidative stress more than either compound alone.

Figure 1

Dietary supplementation of ALCAR+LA partially reverses the age-related decline in average mitochondrial ΔΨ. Rats were fed ALCAR+LA as described, and average hepatocellular ΔΨ was assessed by using R123. Results showed that average ΔΨ was significantly lower in cells from old animals relative to young. However, ALCAR+LA supplementation significantly increased average ΔΨ over that seen in unsupplemented old rats. Columns denoted by the same letter are statistically different (P ≤ 0.05) from each other.

Figure 2

LA supplementation reverses the ALCAR-induced and age-related decline in hepatocellular ascorbate levels. Hepatocytes (1.0 × 10⁶ cells) were hydrolyzed in 10% (wt/vol) metaphosphoric acid, and the ascorbate content in the supernatant was analyzed by HPLC with electrochemical detection (31). Results show that old rats had significantly lower hepatocellular ascorbate concentrations than cells isolated from young animals. For rats fed 1.5% (wt/vol) ALCAR, ascorbate levels in hepatocytes from both young and old rats were significantly lower than corresponding controls (not shown). Cosupplementation of LA with ALCAR negated both the age-related and ALCAR-induced decline in hepatocellular ascorbate concentrations. Columns denoted by the same letter are statistically different (P ≤ 0.05) from each other.

Figure 3

ALCAR+LA supplementation lowers 2,′7′-dichlorofluorescin (DCF) appearance. Young and old rats were treated with or without ALCAR+LA, and hepatocytes were isolated as described. Cells (40,000) were incubated with dichlorofluorescin diacetate (20 μM, final concentration), and the rate of fluorescence appearance was measured by using a Cytofluor fluorescent plate reader (as described in Materials and Methods). Because of the difference in oxygen consumption characteristics, all rates of fluorescence were expressed in relation to oxygen consumption. Results show that ALCAR+LA supplementation significantly lowered the age-related increase in DCF appearance, indicating lower ROS and/or reactive nitrogen species in hepatocytes under this feeding regimen. Columns denoted by the same letter are statistically different (P ≤ 0.05) from each other.

Figure 4

ALCAR+LA supplementation significantly lowers the steady-state age-related accumulation of lipid peroxidation. MDA, a marker of lipid peroxidation, was measured by gas chromatography/MS as described (29, 30). Results show no statistical increase (P ≥ 0.05) in MDA levels with ALCAR, LA, or both supplements in liver tissue from young animals. MDA levels were significantly increased in liver from old rats relative to young controls, and ALCAR supplementation alone further increased hepatic MDA content over that of nonsupplemented old rats. LA alone and ALCAR+LA supplementation significantly decreased hepatic MDA content in livers from old rats. Columns denoted by the same letter are statistically different (P ≤ 0.05) from each other.