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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 20, 2005 as doi:10.1096/fj.05-4985fje. |
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Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, NG7 2UH, UK
1 Correspondence: E Floor, School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, NG7 2UH, UK. E-mail: mbxfbs{at}nottingham.ac.uk
SPECIFIC AIMS
Increasing skeletal muscle carnitine content may alleviate the decline in muscle fat oxidation seen during intense exercise. Studies in humans, however, have failed to increase muscle carnitine content by dietary or intravenous L-carnitine administration. The aim of the present study was to determine whether insulin could augment Na+-dependent skeletal muscle carnitine uptake in healthy human subjects, secondary to increasing Na+/K+ ATPase pump activity.
PRINCIPAL FINDINGS
Eight healthy, non-vegetarian men (age 22.4±0.4 year, body mass 76.5±2.2 kg) reported to the laboratory on two occasions, separated by a 2 wk "washout" period. On each experimental visit, a 6 h euglycemic insulin clamp was performed while maintaining a fasting blood glucose concentration by the infusion of a 20% glucose solution. Each insulin clamp began at t = 0 and insulin was administered in a randomized manner at rates of 5 or 105 mIU•m–2•min–1, with the aim of achieving steady-state fasting (
5 mIU•L–1) or hyperinsulinemic (150 mIU•L–1) serum insulin concentrations, respectively, throughout each visit. After a 1 h equilibration period, a 5 h i.v. infusion of L-carnitine began in conjunction with the insulin clamp. At the onset of L-carnitine infusion, a bolus dose of 15 mg•kg–1 was administered over 10 min so as to rapidly reach a supraphysiological plasma concentration of 500 µmol•L–1. This was followed by a constant infusion at 10 mg•kg–1•h–1 for the next 290 min to maintain hypercarnitinemia. At t = 6 h, insulin and L-carnitine infusions were stopped and subjects were free to leave the laboratory once they had been fed and their blood glucose concentration was stable. During each experimental visit, 5 mL of arterialized venous (a-v) blood was obtained every hour for 6 h, and muscle biopsy samples were obtained from the vastus lateralis muscle immediately before and after each insulin clamp, using the percutaneous needle biopsy technique.
1. Intravenous L-carnitine infusion has no effect on skeletal muscle carnitine content in humans
A major finding from the present study was that maintaining plasma carnitine at a supraphysiological concentration (526.7±22.8 µmol·L–1) in healthy human volunteers by infusing L-carnitine intravenously for 5 h, during which serum insulin was maintained at a fasting concentration (7.1±0.4 mIU•L–1), did not measurably increase muscle TC accumulation (22.4±1.0 vs. 22.7±1.1 mmol•(kg dry muscle)–1) (Fig. 1
). In agreement with the vast majority of studies in which human skeletal muscle carnitine has content has been measured, our findings support the contention that human muscle carnitine content cannot be increased by L-carnitine supplementation alone, most likely due to the large concentration gradient between plasma and skeletal muscle carnitine compartments, the low Km (4.3 µmol·L–1) of the carnitine transporter protein (OCTN2) for carnitine and the rapid urinary clearance of L-carnitine.
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2. Insulin stimulates L-carnitine accumulation in human skeletal muscle
It would appear that if an increase in muscle carnitine content is to be achieved in humans, then an alternative strategy to simply increasing plasma carnitine concentration is required. We hypothesized that insulin could augment Na+-dependent skeletal muscle carnitine uptake secondary to its action of increasing sarcolemmal Na+/K+ ATPase pump activity and, thus, intracellular Na+ flux. In support of this hypothesis, Na+-dependent skeletal muscle uptake of other nutrients by skeletal muscle, such as amino acids and creatine, is augmented by insulin, and ouabain, a potent Na+/K+ ATPase pump inhibitor, has been shown to inhibit carnitine transport in isolated muscle cells.
We were able to demonstrate that maintaining hypercarnitinemia (476.9±15.2 µmol·L–1) in the presence of hyperinsulinemia (149.2±6.9 mIU•L–1) increased muscle TC content by 13% (22.0±0.9 vs. 24.7±1.3 mmol•(kg dm)–1, P<0.05) over 5 h (Fig. 1)
. Furthermore, we observed that steady-state plasma TC concentration during hyperinsulinemia (476.9±15.2 µmol·L–1) was lower compared with the control visit (526.7±22.8 µmol·L–1, P<0.01), while urinary TC excretion was unchanged (2.75±0.03 vs. 2.72 ± 0.05 g for the 5 h infusion periods). This leads us to conclude that, collectively, our observations demonstrate that insulin stimulates skeletal muscle TC accumulation in healthy humans during hypercarnitinemia.
3. Hypercarnitinemia during hyperinsulinemia increases OCTN2 transcription
The Na+-dependent, active transport of carnitine into human skeletal muscle is mediated via a high-affinity, novel organic cation transporter OCTN2. The mechanisms regulating carnitine transporter protein content in human skeletal muscle have not been elucidated or characterized in vivo. An additional novel finding from the present study was that the combination of hypercarnitinemia and hyperinsulinemia increased skeletal muscle OCTN2 mRNA expression by 2.3 ± 0.3-fold, which was significantly greater (P<0.05) than the 1.4 ± 0.2-fold increase observed when hypercarnitinemia was combined with an insulin clamp that maintained a fasting serum insulin concentration (Fig. 2
). The findings suggest that OCTN2 is regulated at the transcriptional level, which presents another possible therapeutic target for increasing skeletal muscle carnitine stores.
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CONCLUSIONS AND SIGNIFICANCE
More than 95% of the body’s total carnitine store exists within skeletal muscle tissue as either free (80%) or acyl (20%) carnitine where, as a substrate for the carnitine acyltransferase enzymes, it plays essential roles in the translocation of long chain fatty acids into the mitochondrial matrix for subsequent ß-oxidation and in regulating the mitochondrial acetyl-CoA/CoASH ratio. For example, during intense exercise carnitine buffers excess acetyl-CoA production, in the form of acetylcarnitine, when its rate of condensation with oxaloacetate is less than its rate of formation by pyruvate oxidation (i.e., pyruvate dehydrogenase complex flux is in excess of the rate of acetyl-CoA utilization by the TCA cycle). However, this acetylation depletes the free carnitine pool, and it has been suggested that the resulting reduction in the availability of free carnitine in the reaction catalyzed by carnitine palmitoyltransferase 1 (CPT-1), the rate-limiting step in long-chain acyl-CoA entry into mitochondria, might be limiting to fat oxidation under these conditions. In this respect, previous research has demonstrated that as exercise intensity increases to >75% Wmax, muscle free carnitine content falls by 65% (to 5.6 mmol•(kg dm)–1) and is paralleled by a 35% decrease in the rate of fat oxidation (measured using a [U-13C]palmitate tracer). Based on this evidence it was hypothesized that muscle free carnitine availability becomes limiting to fat oxidation at a concentration of 5 mmol•(kg dm)–1 (or 1.52 mM intracellular water). This seems plausible given the reported Km of CPT-1 for free carnitine in human skeletal muscle is 0.48 mM. Furthermore, the catalytic site of CPT-1 for carnitine is located within the contact sites of the outer and inner mitochondrial membranes, which will limit its exposure to the predominantly cytosolic store of free carnitine. It is our premise, therefore, that the 2.7 mmol•(kg dm)–1 (0.82 mM intracellular water) increase in total carnitine content observed in the present study could alleviate the decline in fat oxidation rates routinely observed at exercise intensities above 75% Wmax. It is possible that such an increase could positively affect fat oxidation rates during exercise in metabolic diseases such as obesity and type 2 diabetes. Both conditions are associated with an impaired ability of skeletal muscle to oxidize fatty acids during exercise, occurring perhaps as a result of high glycolytic flux and, consequently, reduced muscle free carnitine availability limiting long-chain acyl-CoA translocation.
In conclusion, the present study demonstrates that 5 h of L-carnitine infusion in the presence of hyperinsulinemia increases OCTN2 mRNA expression by 2.3-fold and muscle total carnitine content by 13%. The latter finding could have a significant effect on the integration of fat and carbohydrate oxidation in contracting skeletal muscle and therefore be of clinical importance in treating obesity and type 2 diabetes, particularly as it appears that exercise combined with weight loss, rather than weight loss alone, is required to enhance fasting skeletal muscle fat oxidation rates and improve insulin sensitivity in obese and type 2 diabetic patients.
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FOOTNOTES
2 Current address: Centre for Integrated Systems, Biology and Medicine, School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, UK. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4985fje;
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) with supplementation is presented numerically (n=8). 
P < 0.05, significantly greater than pre infusion value.
