J Nutr. 2002 Apr.
Acetyl-L-carnitine supplementation differently influences nutrient partitioning, serum leptin concentration and skeletal muscle mitochondrial respiration in young and old rats.
Iossa S, Mollica MP, Lionetti L, Crescenzo R, Botta M, Barletta A, Liverini G.
Department of General and Environmental Physiology, University of Naples Federico II, Italy.
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Abstract: Variations in energy balance, body composition, and nutrient partitioning induced by acetyl-L-carnitine (ALCAR) supplementation were studied in young (2 mo) and old (24 mo) Wistar rats. Changes in skeletal muscle metabolism as well as in serum free triiodothyronine and leptin levels were also evaluated. Rats were administered 0 (control) or 15 g/L ALCAR in their drinking water for 1 mo. ALCAR treatment significantly decreased body lipid percentage in young rats and significantly increased body protein percentage in old rats. The percentage of metabolizable energy (ME) intake stored as lipid was lower in ALCAR-treated young rats, whereas the percentage of ME intake stored as protein was greater in ALCAR-treated old rats compared with their age-matched controls. In addition, ALCAR supplementation significantly decreased serum leptin levels in old rats. Elevated skeletal muscle respiration was found in old rats treated with ALCAR, due to an increase in mitochondrial protein mass. In conclusion, ALCAR supplementation decreases efficiency of lipid deposition in young rats and increases efficiency of protein deposition in old rats. In addition, ALCAR supplementation partly reduces the leptin resistance that occurs in old rats, and improves ATP production in skeletal muscle mitochondria through an increase in mitochondrial protein content.

Excerpts from the full text article:

Introduction

Both humans and rodents tend to gain weight as they age (1, 2, 3); therefore, rats are considered the preferred animal model for studying changes in energy partitioning with age. In previous work, we found that the partitioning of energy from incoming nutrients is differently regulated with age (4). In fact, young rats were characterized by storage of lipid and protein, whereas in middle-aged rats, protein deposition approached zero and the excess of the ingested energy over energy expenditure was stored entirely as fat, so that age-associated obesity began to develop (4). Interestingly, a number of studies in a variety of species have shown that L-carnitine supplementation influences nutrient partitioning and thus body composition (5, 6, 7, 8, 9). Because carnitine, a cosubstrate of carnitine acyl-transferase, is essential for translocation of fatty acids into the mitochondrial matrix (10), it is conceivable that carnitine level influences fatty acid utilization. In addition, we have shown that dietary supplementation with a carnitine derivative, acetyl-L-carnitine (ALCAR), slowed some hepatic metabolic dysfunction due to age, probably through maintenance of mitochondrial function (11).

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Materials and methods

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One group of young and old rats served as control, whereas the other two groups received water supplemented with a 15 g/L (pH adjusted to 6.0) solution of ALCAR. Young and old rats drank about the same amount of water per day (data not shown), which would provide a daily ALCAR dose of 1.0 g/(kg body · d). The treatment period lasted 1 mo.

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Results

Final body weight and the body water percentage did not differ in either young or old rats due to ALCAR treatment (Table 1). Body lipid percentage increased significantly with age and was lower in ALCAR-treated young rats compared with their controls (Table 1). Body protein percentage was lower in old rats than in young rats and was greater due to ALCAR only in old rats (Table 1). Body energy content was greater in old than in young rats and ALCAR treatment increased it only in old rats (Table 1).

ME intake, energy expenditure and net energy expenditure (energy expenditure excluding the total cost of storage) decreased with age, and ALCAR supplementation decreased energy expenditure and net energy expenditure only in old rats (Table 2). Body energy, protein and fat gain decreased with age. ALCAR treatment increased body protein gain in old rats and decreased fat gain in young rats, so that body energy gain increased in old rats and decreased in young rats (Table 2).

The percentage of ME intake stored as fat decreased with age and was lower than controls in ALCAR-treated young rats (Fig. 1A). The percentage of ME intake stored as protein decreased with age in controls but was greater in ALCAR-treated old rats than in the other three groups (Fig. 1B).

The percentage of ME intake utilized for energy maintenance (net energy expenditure/ME intake) was greatest in control old rats and least in old rats treated with ALCAR (Fig. 2A). The percentage of ME intake stored as body energy, an index of metabolic efficiency, was lower in ALCAR-treated young rats and greater in ALCAR-treated old rats than in their respective controls (Fig. 2B). The percentage of ME intake utilized for lipid and protein storage was lower in old than in young control rats and it significantly increased due to ALCAR treatment only in old rats (Fig. 2C).

Serum free T3 levels significantly decreased with age and ALCAR treatment had no effect (Fig. 3A). Serum leptin levels were greater in old than in young control rats and were lower due to ALCAR supplementation only in old rats (Fig. 3A). RMR decreased significantly with age whether rats were fed or food-deprived. ALCAR-treatment had no effect in either condition (Fig. 3B).

In skeletal muscle homogenates, no effect of age was found on state 3 respiration, and ALCAR supplementation increased it only in old rats (Table 3). On the other hand, ALCAR supplementation did not influence state 4 respiration, which increased significantly with age (Table 3). In addition, we measured state 3 and state 4 respiration in isolated muscle mitochondria to take into account changes in respiratory specific activity due to ALCAR supplementation (Table 4). Isolated mitochondria were pure and intact. In agreement with others (22, 23), we found that the contamination of isolated mitochondria by other ATPase-containing membranes was <10% in all groups. The RCR indicated the high quality of the mitochondrial preparations (Table 4). There were no effects of age or ALCAR supplementation on state 3 respiration. State 4 respiration, in contrast, increased with age and was lower in ALCAR-treated old rats than in controls. We calculated the mitochondrial protein mass (mg/g wet tissue) by dividing the SDH activity in muscle homogenates (Table 3) by the SDH activity in isolated muscle mitochondria (Table 4). This calculation could be done because our mitochondrial preparations were minimally contaminated as discussed above. Mitochondrial protein mass was greater in ALCAR-treated old rats than in controls (Fig. 4).

Significant inverse correlations were found between serum leptin levels and ME intake in each group of rats (Fig. 5). In addition, slopes of the regression lines for the correlation between leptin and ME intake were different in old rats compared with young rats and in old rats after ALCAR supplementation compared with control old rats (Fig. 5).

Discussion

We studied the effect of ALCAR supplementation on body composition, nutrient partitioning, and skeletal muscle metabolism in young and old Wistar rats. ALCAR is used largely as a nutritional supplement because of its importance in cellular energy production. Its use could be equivalent to that of L-carnitine because ALCAR is deacetylated during or immediately after uptake into intestinal cells and a portion of the newly formed intracellular free carnitine is apparently reacetylated (24). However, at the cellular level, ALCAR seems to be the active molecule in some metabolic pathways (25, 26). Carnitine and ALCAR are both involved in the transport of fatty acids and acetyl groups into the mitochondria. ALCAR is also an intracellular energy reservoir of acetyl groups (10).

Old rats had greater body energy and fat content, but lower body protein content than young rats (Table 1). These changes could be due to a decrease in the ability to use fat as a metabolic fuel with age, so that more amino acids would be utilized for energy production rather than for body protein synthesis. This suggestion is in agreement with our previous work that showed an age-related decline in fatty acid oxygen consumption in perfused liver (11). Taking into account the lower tissue carnitine levels found in old rats by Kalaiselvi and Panneerselvam (27), the impairment in fat utilization could be due in part to low levels of carnitine.

The present results also showed that long-term ALCAR dietary treatment differently influenced body composition and nutrient partitioning in young and old rats. In fact, we found that ALCAR supplementation significantly decreased fat gain and body fat content in young rats and significantly increased protein gain and body protein content in old rats (Tables 1, 2).

Decreased fat gain in young rats supplemented with ALCAR is consistent with other studies conducted in a variety of species. In fact, Ji et al. (7) found that carnitine-fed salmon had lower lipid concentrations in white muscle and visceral organs. Rabie and Szilagyi (9) showed that in broilers supplemented with carnitine, the quantity and percentage of abdominal fat were reduced. Finally, in rats treated with caffeine, carnitine and choline, there was a significant loss of adipose fat mass (6). Lower fat gain of young rats treated with ALCAR (Table 2) could be due to increased fat utilization as metabolic fuel. This suggestion is in agreement with our previous work that showed increased fatty acid oxygen consumption in perfused liver from rats treated with ALCAR (11) and with the results of Penn et al. (8), who showed a decrease in fatty acid oxidative rates in liver homogenates from carnitine-deprived piglets. However, the greater fat utilization found in young rats treated with ALCAR was not associated with increased energy expenditure (Table 2). A possible explanation could be the trend for a lower ME intake (P = 0.17) found in these rats (Table 2), so that carbohydrate and protein oxidation was insufficient to meet body energy needs and a greater amount of fat would be oxidized. Interestingly, after ALCAR treatment, a substantial decrease in body fat without changes in protein balance could be induced through only minor changes in ME intake.

ALCAR supplementation increased protein deposition (Table 2) and hence metabolic efficiency (Fig. 2B) only in old rats. This increase can be the result of enhanced protein synthesis and/or reduced protein catabolism. The increased metabolic efficiency found in old rats after ALCAR treatment means a higher efficiency of oxidative phosphorylation at the cellular level (see also below). Consequently, more ATP could be produced during fatty acid oxidation and more amino acids could be channeled through protein synthesis. The information regarding carnitine’s effects on protein metabolism in the literature is controversial. Owen et al. (28) found that carcass proteins were unaffected by L-carnitine supplementation in weaning pigs. In contrast, Tao et al. (29) found an increased nitrogen balance in intravenously fed growing rats supplemented with carnitine. In addition, Heo et al. (5) found that carnitine reduced urinary nitrogen excretion and increased protein accretion in growing pigs fed a low energy diet. The above different effects of ALCAR administration could be explained by the species studied, dietary treatment as well as by the age of the animals used in the various studies.

Our results also showed that RMR, measured in fed and food-deprived rats (Fig. 3B), as well as energy expenditure and net energy expenditure (energy expenditure excluding the cost of fuel storage) (Table 2), decreased significantly with age. Therefore, the reduction in energy expenditure found in old animals is due in part to a decrease in RMR. ALCAR supplementation did not restore the age-dependent decline in RMR and energy expenditure.

To gain insight into the hormonal modifications induced by ALCAR supplementation, we measured serum levels of free T3 and leptin, which are involved in the regulation of nutrient metabolism (12, 13, 14). ALCAR treatment neither affected serum free T3 levels in young rats nor restored the age-dependent decline in old rats (Fig. 3A). A similar finding was obtained by Janssens et al. (30) in exercising pigeons in which decreased thyroid hormone levels were not influenced by orally supplemented carnitine. Our serum leptin results (Fig. 3A) confirm previous observations that serum leptin levels increase with age (4, 31). Interestingly, ALCAR treatment decreased serum leptin levels in old rats, but not in young rats. The latter result is different from that of Hongu and Sachan (6), who supplemented a diet with caffeine, choline and carnitine. A significant positive correlation (r2 = 0.97, P < 0.0001) between serum leptin levels and body fat mass was always found, in agreement with the observation that leptin circulates in proportion to body fat mass in rodents (32). In addition, we found significant inverse correlations between serum leptin levels and ME intakes (Fig. 5). This result is consistent with the inhibitory action of leptin on food intake (33). Interestingly, the slope of the regression line obtained for old rats (-5.3 ± 0.8) was significantly higher than that obtained for young rats (-43.2 ± 6.3) and for old rats treated with ALCAR (-40.4 ± 1.8). This finding suggests that the responsiveness to the anorexic effect of leptin is reduced in old rats but can be restored by ALCAR supplementation. The reduced sensibility to the anorexic effect of leptin in old rats is in agreement with previous studies showing reduced response to systemic (34) or intracerebroventricular injection of leptin in old rats (35).

In the present work, mitochondrial oxidative capacities together with mitochondrial protein content were assessed in skeletal muscle, a tissue that contributes greatly to whole-body energy expenditure and lipid utilization. Measurements of mitochondrial respiratory activities were performed on both homogenates and isolated mitochondria. ALCAR treatment increased maximal oxidative capacity (state 3 respiration) in homogenates (Table 3) but not in isolated mitochondria (Table 4) in old rats, due to increased mitochondrial protein content, which occurred in old rats supplemented with ALCAR. Moreover, we observed a significant increase in state 4 respiration with age, in both homogenates and isolated mitochondria. With the limitation that state 4 respiratory rate in the presence of saturating amounts of oligomycin can give only a rough indication of the mitochondrial proton leak, the increase in state 4 respiratory rate may indicate that the proton leak pathway increases with age. In agreement with this suggestion, Lal et al. (36) found that the proton leak was lower in skeletal muscle mitochondria from young than from old rats. An implication of this finding could be that the inefficiency of the oxidative phosphorylation system is greater in old than in young rats, so that mitochondria from old rats could not completely satisfy cellular energy requirements, with a possible impairment in the functional ability of the muscle cell. Interestingly, ALCAR treatment decreased state 4 respiratory rate only in old rats and therefore could restore the capacity of muscle cells to produce energy.

In conclusion, the results of this work indicate that ALCAR supplementation differently influences nutrient partitioning in young and old rats. In fact, lipid deposition is lower in young rats, whereas protein deposition is enhanced in old rats. In addition, ALCAR supplementation in old rats can reduce leptin resistance and improve ATP production in skeletal muscle mitochondria through an increase in mitochondrial protein content and, perhaps, in the efficiency of the oxidative phosphorylation system.