Carnitine supplementation to obese Zucker rats prevents obesity-induced type I to type II muscle fiber transition and favors an oxidative phenotype of skeletal muscle

The different contractile and metabolic functions of individual skeletal muscles in the body are caused by a different muscle fiber distribution. Two major types of muscle fibers, which differ in their myosin heavy-chain (MHC) isoforms and their enzymatic capacity [1], can be distinguished. Type I fibers are mitochondria-rich, have a high oxidative capacity utilizing mostly oxidative phosphorylation [2, 3], and are myoglobin-rich with red appearance. Type II fibers (subdivided in IIa, IIb and IId/x) have a lower oxidative capacity due to fewer mitochondria content than type I muscle fibers and thus depend on glycolytic metabolism to generate ATP.

Interestingly, muscle fiber distribution is altered by genetic and/or high fat diet-induced obesity due to induction of fiber transition from type I to type II thereby leading to a change of the muscle’s metabolic phenotype [4‐7]. The resulting reduced type I fiber content is associated with mitochondrial dysfunction characterized by an impaired mitochondrial oxidative enzyme capacity of skeletal muscle [8]. Noteworthy, genetic and/or high fat diet-induced obesity and diabetes cause an impairment of carnitine status [9‐11], whereas normalization of carnitine status due to carnitine supplementation reverses mitochondrial dysfunction under these conditions [4, 9, 10, 12, 13]. At large, these findings indicate that carnitine supplementation through normalizing carnitine status is able to prevent type I to type II fiber transition and thereby the metabolic shift from oxidative to glycolytic of skeletal muscle under unloading conditions or metabolic stress.

Obesity- and diabetes-induced type I to type II fiber transition and decreases of oxidative capacity of skeletal muscle has been explained by down-regulation of peroxisome proliferator-activated receptor γ, coactivator-1α (PGC-1α) and peroxisome proliferator-activated receptor δ (PPARδ) [4, 9, 14]. Both, PGC-1α and PPARδ are critical regulators of genes involved in type II to type I fiber transition, mitochondrial biogenesis, cellular and mitochondrial fatty acid uptake, β-oxidation, carnitine uptake, tricarboxylic acid cycle, respiratory chain, and angiogenesis [15‐20]. Due to these functions PPARδ and PGC-1α are typically higher expressed in oxidative type I muscle fibers than in glycolytic type II muscle fibers [15, 21]. Interestingly, carnitine supplementation was reported to increase expression of PGC-1α and PPARδ in rodent models of unloading [4], and genetic and diet-induced obesity and diabetes [10]. Based on these observations we hypothesized that carnitine supplementation through inducing PGC-1α and PPARδ in skeletal muscle counteracts obesity and/or diabetes-induced muscle fiber transition from type I to type II and restores the muscle fiber distribution and the muscle oxidative metabolic phenotype observed during non-obese and non-diabetic states. As a model object we used obese Zucker rats, an established genetic model of obesity, insulin resistance, and metabolic syndrome, which were fed either a carnitine supplemented or a control diet with a low native carnitine concentration for 4 wk. Lean Zucker rats served as healthy non-obese and non-diabetic controls.

In the present study we tested the hypothesis that carnitine supplementation to obese Zucker rats counteracts the obesity-induced muscle fiber transition from type I to type II and, thereby, improves fatty acid utilization in skeletal muscle. The dietary carnitine dosage (3 g/kg diet) fed to the rats related to 156 to 216 mg/kg body weight based on an average daily feed consumption of 26 g and a body weight of 360 (initial) to 500 (final) g. This carnitine dosage is slightly higher when compared to that used in clinical studies with human subjects with different metabolic disorders in which carnitine dosages of up to 4 g/d corresponding to 60 mg/kg body weight for an individual weighing 70 kg were found to be effective [28]. A key finding of the present study is that carnitine supplementation to obese rats resulted in an increased number of type I fibers and a decreased number of type II fibers in rectus femoris muscle when compared to non-supplemented obese rats. This indicates that carnitine induces type II to type I fiber transition in femoris muscle of obese rats which was also confirmed by the finding that the type I fiber specific MYH7 mRNA level in rectus femoris muscle was markedly elevated in the obese carnitine group. Interestingly, the fiber type distribution of rectus femoris muscle was similar between the obese carnitine and the lean control group, whereas rectus femoris muscle of the obese control group had a lower number of type I fibers and a greater number of type II fibers compared to that of the lean control group. A reduction of type I fibers and a lower oxidative enzyme activity in muscle of obese and diabetic rodent models compared to lean models has been well documented [6, 7]. In addition, several studies reported that obese subjects have a decreased proportion of type I muscle fibers and an overall decrease in mitochondrial enzymes indicating that muscle oxidative capacity is impaired in obese subjects [29‐32], which likely contributes to the impaired whole body fatty acid utilization and the elevated blood lipid levels in these subjects. Moreover, it was shown that insulin sensitivity correlates positively with the proportion of type I muscle fibers and negatively with the proportion of type II muscle fibers [29‐31]. There is a large body of evidence that carnitine supplementation improves glucose tolerance in insulin resistant and/or diabetic humans (reviewed by [28]). The observed up-regulation of genes in involved in glycolysis (HK2) by carnitine in muscle of the obese rats is also supportive of a beneficial effect of carnitine on glucose homeostasis. Insulin resistant and/or diabetic subjects are likely particularly sensitive to carnitine supplementation because diabetic subjects were reported to have diminished plasma free carnitine concentrations, even though the levels were still within or only slightly below the physiological range (25–50 μmol/l) reported for healthy subjects [33‐35]. Given the observed increase in type I muscle fiber proportion in obese rats and the relationship between type I muscle fiber proportion and insulin sensitivity it is not unlikely that the improvement of glucose tolerance and insulin sensitivity by carnitine supplementation in obese and diabetic subjects is due to the effect of carnitine on muscle fiber distribution. Even though a very recent study reported that carnitine supplementation at a non-physiologically high dosage promoted intestinal formation of the proatherogenic trimethylamine-N-oxide and accelerated atherosclerosis development in mice [36], the fact that none of the carnitine supplementation studies in humans reported any adverse effects even at very high dosages (e.g., 4 g oral carnitine) indicates that safety concerns with carnitine supplementation are unfounded. Collectively, the present findings corroborate our hypothesis that carnitine supplementation to obese rats counteracts the obesity-induced muscle fiber transition from type I to type II and is able to restore the muscle fiber distribution and the oxidative metabolic phenotype observed in lean animals.

This study showed that carnitine supplementation in obese rodents resulted in a higher type I muscle fiber content compared to obese rats without carnitine supplementation. The observed decrease of carnitine levels in plasma and muscle in the obese control group is consistent with observations from recent studies showing that whole body carnitine status is strongly compromised in rodent models of genetic and diet-induced obesity and diabetes [10]. As the main reason for this phenomenon an impaired hepatic carnitine biosynthesis has been identified [10, 11]. In contrast, carnitine supplementation to obese and/or diabetic rodent models is able to restore the carnitine status to normal [10] or even supraphysiological levels as shown herein. Although our observations do not proof a causal link between carnitine status and muscle fiber distribution, our results suggest that the carnitine-induced change in muscle fiber distribution is due to an improvement of carnitine status.

In addition, the present study shows that the carnitine-induced change in the contractile phenotype of skeletal muscle of obese rats is also accompanied by a change in the metabolic phenotype. In agreement with the high content of mitochondria and the preferential use of fatty acids for energy production of type I fibers, genes involved in fatty acid transport and uptake (FABP3, CD36, LPL), β-oxidation (ACADL, ACADM), carnitine shuttle (SLC25A20), carnitine uptake (SLC22A5) and TCA cycle and respiratory chain (SDHA) in femoris muscle were strongly up-regulated in the obese carnitine compared to the obese control group. In line with our findings in skeletal muscle, carnitine supplementation was also shown to stimulate TCA activity, mitochondrial respiration and ATP production in the diabetic rat heart [37, 38]. Thus, these carnitine-induced effects on gene expression of rectus femoris muscle are indicative of an improved capacity of the muscle for oxidative utilization of fatty acids. Since skeletal muscle significantly contributes to whole-body fatty acid utilization, the improved oxidative metabolic phenotype of skeletal muscle at least partially explains the pronounced NEFA- and TG-lowering effects of carnitine supplementation observed in the present rat model of obesity and insulin resistance but also in other studies [39].

To gain insight into the mechanisms underlying muscle fiber transition we determined the mRNA and/or protein levels of the main regulators of fiber composition. The two main regulators of type II to type I fiber transition, mitochondrial biogenesis, and oxidative enzyme expression, PPARδ and PGC-1α, but also PGC-1β, which induces similar effects as PGC-1α on the contractile and metabolic phenotype of skeletal muscle [40, 41], were clearly stronger expressed in rectus femoris muscle of the obese carnitine group than in the obese control group. Regarding that PGC-1α and PGC-1β also regulate angiogenesis through inducing the expression of angiogenic factors like VEGFs, we also determined transcript levels of VEGFA (encoding VEGFa) and VEGFB (encoding VEGFb). VEGFb particularly favors the utilization of fatty acids not only by increasing capillary density and thereby blood perfusion but also by inducing the expression of fatty acid transport proteins (FATPs, CD36) [42]. Correspondingly, VEGFb is mainly expressed in tissues with high mitochondria content and with preferential use of fatty acids as energy source like oxidative skeletal muscle, heart, and brown adipose tissue [43]. The observation that the relative mRNA level of VEGFB was markedly elevated in the obese carnitine group is therefore in line with the induction of PGC-1α and PGC-1β and provides a further indication for the improved oxidative phenotype of rectus femoris muscle in rats of the obese carnitine group. Collectively, our findings strongly suggest that the up-regulation of genes encoding PGC-1α, PGC-1β, and PPARδ in rectus femoris muscle by L-carnitine supplementation is responsible for the observed type II to type I fiber transition and the oxidative metabolic phenotype of skeletal muscle of obese Zucker rats.

In conclusion, the results of this study demonstrate that carnitine supplementation to obese Zucker rats significantly improves carnitine status, counteracts the obesity-induced muscle fiber transition from type I to type II and favors an oxidative metabolic phenotype of skeletal muscle which preferentially uses fatty acids as energy source. The enhanced capacity of skeletal muscle to utilize fatty acids was demonstrated by the carnitine-induced up-regulation of genes involved in fatty acid uptake and transport, carnitine uptake, fatty acid β-oxidation, mitochondrial fatty acid uptake (carnitine shuttle system) and tricarboxylic acid cycle. These metabolic changes in skeletal muscle are likely to contribute to the pronounced NEFA- and TG-lowering effects of carnitine supplementation in obese Zucker rats. Therefore, carnitine supplementation is supposed to be beneficial for the treatment of elevated levels of metabolic fuels (e.g., fatty acids) which are frequently found in subjects with obesity, insulin resistance, diabetes or metabolic syndrome.