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Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham NG7 2UH, United Kingdom
Address all correspondence and requests for reprints to: Francis B. Stephens, E Floor, School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom. E-mail: francis.stephens{at}nottingham.ac.uk.
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Abstract |
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Objective: The aim of the present study was to investigate the effect of an increase in skeletal muscle total carnitine (TC) content on muscle fuel metabolism.
Design: A 5-h iv infusion of saline (control) or L-carnitine was administered while serum insulin was maintained at a physiologically high concentration during two randomized visits.
Participants: Seven healthy, nonvegetarian young men (body mass index, 26.1 ± 1.6 kg/m2) participated in the present study at the University of Nottingham.
Main Outcome Measures: Skeletal muscle pyruvate dehydrogenase complex (PDC) activity and associated muscle metabolites were measured.
Results: The combination of hypercarnitinemia (600 µmol/liter) and hyperinsulinemia (160 mU/liter) increased muscle TC content by 15% (P < 0.01) and was associated with decreased pyruvate dehydrogenase complex activity (P < 0.05) and muscle lactate content (P < 0.05) by 30 and 40%, respectively, and an overnight increase in muscle glycogen (P < 0.01) and long-chain acyl-coenzyme A content (P < 0.05) by 30 and 40%, respectively, compared with control.
Conclusions: These results suggest that an acute increase in human skeletal muscle TC content results in an inhibition of carbohydrate oxidation in conditions of high carbohydrate availability, possibly due to a carnitine-mediated increase in fat oxidation. These novel findings may have important implications for our understanding of the regulation of muscle fat oxidation, particularly during exercise, when carnitine availability may limit fat oxidation, and in obesity and type 2 diabetes where it is known to be impaired.
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Introduction |
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It has been suggested (9, 10), and is the foundation of a multimillion dollar dietary supplement industry, that L-carnitine feeding can increase fat oxidation at rest and promote weight loss in humans. However, for this premise to be even considered as a viable hypothesis, an increase in skeletal muscle carnitine content is essential, and it is clear from the vast majority of the pertinent studies in healthy human volunteers presented in the literature that an increase in muscle carnitine content cannot be achieved via acute or chronic L-carnitine administration per se (11, 12, 13). For example, neither feeding L-carnitine daily for 3 months (12) nor iv infusing L-carnitine for 5 h in the fasted state (13) had an effect on muscle total carnitine (TC) content. On the other hand, we have recently demonstrated for the first time that maintaining hyperinsulinemia (
150 mU/liter) during 5 h of steady-state hypercarnitinemia (500 µmol/liter) increased skeletal muscle TC content by 13% in healthy human volunteers (13). We hypothesized that an increase in muscle carnitine content could alleviate the decline in fat oxidation rates routinely observed in healthy individuals during exercise requiring high glycolytic flux (a similar condition to that observed in obesity and type 2 diabetes) (14, 15). However, whether such an increase in skeletal muscle carnitine content would increase fat oxidation at rest warrants further investigation, particularly because insulin-resistant conditions, such as obesity and type 2 diabetes, are associated with an impaired ability of skeletal muscle to oxidize fatty acids, both at rest and during exercise (15, 16, 17, 18, 19, 20).
If an increase in muscle carnitine accumulation is associated with an increase in fat oxidation, then, because of the reciprocal relationship between fat and carbohydrate oxidation in skeletal muscle, one would expect a decrease in carbohydrate oxidation. With this in mind, the aim of the present study was to determine the effect of an insulin-mediated increase in muscle carnitine content on skeletal muscle PDC activity and muscle long-chain acyl-CoA, glycogen, glucose-6-phosphate, and lactate contents, thereby providing novel insight into the role of carnitine availability in the integration of fat and carbohydrate oxidation in human skeletal muscle.
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Subjects and Methods |
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Seven healthy, nonsmoking, nonvegetarian young men (age, 22.4 ± 1.5 yr; body mass, 84.3 ± 5.0 kg; body mass index, 26.1 ± 1.6 kg/m2; fasting blood glucose concentration, 4.5 ± 0.1 mmol/liter; fasting serum insulin concentration. 5.2 ± 0.9 mU/liter) participated in the present study, which was approved by the University of Nottingham Medical School Ethics Committee in accordance with the Declaration of Helsinki. Before taking part in the study, all subjects underwent routine medical screening and completed a general health questionnaire. All gave their consent to take part in the study and were aware that they were free to withdraw from the experiment at any point.
Experimental protocol
Each subject reported to the laboratory at 0800 h on two occasions, separated by a 2-wk washout period, and voided their bladder. All subjects had abstained from carnitine-containing foods, alcohol, and strenuous exercise for the previous 24 h. On arrival, subjects were asked to rest in a supine position on a bed while a cannula was inserted retrogradely into a superficial vein on the dorsal surface of the nondominant hand. This hand was kept in a hand-warming unit (air temperature, 50–55 C) to arterialize the venous drainage of the hand (21), and a saline drip was attached to keep the cannula patent. A second cannula was placed in an antecubital vein in the nondominant forearm for the infusion of insulin and glucose, and a third cannula was inserted into an antecubital vein in the opposite arm for infusion of L-carnitine.
On each experimental visit, a 6-h euglycemic hyperinsulinemic insulin (human Actrapid; Novo Nordisk, Copenhagen, Denmark) clamp was performed (22) while maintaining a fasting blood glucose concentration of 4.47 ± 0.01 mmol/liter. The insulin clamp began at time 0. After a 10-min priming dose, insulin was infused at a rate of 105 mU/m2·min with the aim of achieving steady-state hyperinsulinemic serum insulin concentration throughout each visit to ensure that serum insulin concentration per se was not rate limiting to muscle carnitine transport. After a 1-h equilibration period, a 5-h iv infusion of 60 mM L-carnitine (Lonza Ltd., Basel, Switzerland) or the equivalent volume of saline (control) began, in randomized manner, in conjunction with the insulin clamp. At the onset of L-carnitine infusion a bolus dose of 15 mg/kg was administered over 10 min to rapidly reach a supraphysiological plasma concentration of approximately 500 µmol/liter. This was followed by a constant infusion at 10 mg/kg·h for the next 290 min to maintain hypercarnitinemia and to ensure that plasma carnitine concentration per se was not rate limiting to muscle carnitine transport. At time 6 h, the insulin and L-carnitine infusions were stopped, whereas the glucose infusion was continued for approximately 80 min to stabilize blood glucose concentration. During this time, on each visit, subjects were fed the same standardized, carnitine-free meal. The meal had an energy content of approximately 1500 kcal (55% carbohydrate, 35% fat, and 10% protein) and a total carbohydrate content of 220 g (35% of which were sugars). Thereafter, subjects were free to leave the laboratory once their blood glucose concentration was stable. Any food or drink from the meal that was not consumed before subjects left the laboratory was consumed that evening before 2200 h (usually consisting of a chocolate bar and a beverage high in carbohydrate), with the time and amount being noted and repeated on the following experimental visit. Subjects then returned to the laboratory at 0800 h the following morning in a fasted state from the previous evening.
Sample collection and analysis
During each experimental visit, 1 ml of arterialized venous blood was obtained every 5 min for monitoring blood glucose concentration (YSI 2300 STATplus; Yellow Springs Instruments, Yellow Springs, OH). In addition, 5 ml of arterialized venous blood was obtained every hour (and at 80 min) for 6 h and at 24 h the following morning. Two milliliters of this blood were collected into lithium heparin containers, and after centrifugation, the plasma was removed and immediately frozen in liquid nitrogen. These samples were then stored at –80 C and analyzed at a later date for free fatty acid (FFA) concentration, using an enzymatic-colorimetric assay kit (NEFA C kit; Wako Chemicals, Neuss, Germany), and TC concentration, using the radioenzymatic assay described previously by Cederblad et al. (23). The remaining blood was allowed to clot, and after centrifugation, the serum was stored frozen at –80 C. Insulin was measured in these samples at a later date with a RIA kit (Coat-a-Count Insulin; DPC, Los Angeles, CA).
Muscle biopsy samples were obtained from the vastus lateralis muscle immediately before and after each insulin clamp and the following morning, using the percutaneous needle biopsy technique (24), and were snap-frozen in liquid nitrogen less than 5 sec after removal from the limb. One portion of the sample was subsequently freeze-dried and stored at –80 C, and the remainder was stored wet in liquid nitrogen. After removal of visible blood and connective tissue, the freeze-dried muscle samples were powdered, and FC, acetylcarnitine (AC), long-chain acylcarnitine, and long-chain acyl-CoA (LCA-CoA) contents were determined radioenzymatically using a modified version of the radioenzymatic method of Cederblad et al. (25). Values were subsequently summed to calculate muscle TC. To reduce the variance in nonmuscle constituents, muscle carnitine content was adjusted for the highest total creatine content from each pair of samples. Total creatine was calculated as the sum of free creatine and phosphocreatine content determined spectrophotometrically using the method of Harris et al. (26). Muscle glycogen, glucose-6-phosphate, and lactate content was also determined using a modified version of the spectrophotometric method of Harris et al. (26).
The remainder of the frozen muscle was used to determine PDC activity as previously described (6). Briefly, the activity of PDC in its dephosphorylated active form (PDCa) was assayed in a buffer containing NaF and dichloroacetate and was expressed as rate of acetyl-CoA formation (millimoles per minute per kilogram wet muscle) at 37 C.
Statistical analysis
A two-way ANOVA (time and treatment effects) (GraphPad Prism 4.02; GraphPad Software Inc., San Diego, CA) was performed to locate differences in serum insulin, plasma carnitine, plasma FFA, and blood glucose concentrations as well as muscle PDC activity and carnitine, long-chain acyl-CoA, glycogen, lactate, and glucose-6-phosphate content. When a significant main effect was detected, data were further analyzed with Student’s paired t tests using the Bonferroni correction. Statistical significance was declared at P < 0.05, and all the values presented in text, tables, and figures are means ± SEM.
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Results |
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After the 60-min equilibration period, the 105 mU/m·min euglycemic hyperinsulinemic clamps produced similar steady-state (1–6 h) serum insulin concentrations of 160.1 ± 1.9 and 155.8 ± 3.9 mU/liter, during the saline (control) and L-carnitine infusions, resulting in glucose disposal rates of 9.9 ± 0.9 and 10.5 ± 0.7 mg/kg·min, respectively, and whole-body glucose disposal values of 300.2 ± 19.3 and 316.8 ± 15.0 g over the 6 h, respectively (please note that these data refer to serum insulin concentration, which is routinely lower than plasma insulin concentration). Whole-body glucose disposal was also similar between the 80-min recovery periods after the two clamps of 32.5 ± 4.1 and 37.2 ± 3.5 g for control and carnitine, respectively.
Plasma carnitine
The plasma TC profiles over the course of the control and carnitine infusion visits during the two hyperinsulinemic clamps are illustrated in Fig. 1
. From similar basal plasma TC concentrations of 53.8 ± 4.3 and 50.5 ± 4.6 µmol/liter, the saline infusion had no effect on plasma TC concentration, whereas the bolus 15 mg/kg L-carnitine infusion (indicated by the arrow) produced a mean peak plasma TC concentration of 723.0 ± 63.9 µmol/liter, which remained elevated above 600 µmol/liter throughout the carnitine visit and was greater than during control at every time point (P < 0.001). Plasma TC concentration during carnitine was also greater than control the following morning after 24 h (47.7 ± 3.8 vs. 72.9 ± 5.3 µmol/liter, for control and carnitine, respectively; P < 0.001).
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From similar basal concentrations of 0.33 ± 0.05 and 0.30 ± 0.04 mmol/liter, plasma FFA concentration decreased after the commencement of the insulin clamp, and was maintained at 0.039 ± 0.002 and 0.046 ± 0.01 mmol/liter during the control and carnitine visit, respectively. By 24 h, plasma FFA concentration was 0.22 ± 0.02 and 0.19 ± 0.02 mmol/liter during the control and carnitine visit, respectively. There were no significant differences between each visit.
Muscle carnitine
Skeletal muscle TC data during the control visit are presented in Table 1. Skeletal muscle TC content was unchanged after 5 h of saline infusion in conjunction with hyperinsulinemia (
0.9 ± 0.8 mmol/kg dry muscle; P > 0.05) and was the same as the basal state the following morning. However, 5 h of L-carnitine infusion during hyperinsulinemia (carnitine visit) increased skeletal muscle TC by 15% (4.1 ± 1.0 mmol/kg dry muscle; P < 0.01), which remained elevated overnight, although it was not significantly different from the preinfusion value.
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. There was a trend for an increase (12%) in muscle FC content after the control infusion visit, which was paralleled by a corresponding decrease in AC (P < 0.05). Muscle FC increased by 17% during the carnitine infusion visit (P < 0.05), retuning to basal overnight, whereas no change was observed in AC. Skeletal muscle long-chain acylcarnitine was unchanged after both infusion visits. PDC activity
Muscle PDC activity increased during the euglycemic hyperinsulinemic clamp from 0.49 ± 0.04 to 1.07 ± 0.09 mmol/min·kg wet muscle (P < 0.01) during the control visit and from 0.43 ± 0.07 to 0.74 ± 0.06 mmol/min·kg wet muscle (P < 0.01) during the carnitine visit (Fig. 2A). However, the increase in PDC activity after the carnitine infusion visit was not as pronounced as after the control infusion visit, such that postinfusion PDC activity was 31% less than control (P < 0.05). Twenty-four hours after the commencement of the insulin infusion, PDC activity had returned to its basal value of 0.46 ± 0.09 (P < 0.01) and 0.47 ± 0.12 mmol/min·kg wet muscle for the control and carnitine visit, respectively.
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Muscle glycogen content increased during each euglycemic hyperinsulinemic clamp, from a basal content of 506 ± 25 and 487 ± 23 mmol/kg dry muscle, to postinfusion values of 642 ± 31 (P < 0.05) and 651 ± 37 mmol/kg dry muscle (P < 0.01) during control and carnitine visits, respectively (Fig. 2B). Twenty-four hours after the commencement of insulin infusion in the control visit, muscle glycogen had not changed further (567 ± 22 mmol/kg dry muscle), whereas after the carnitine visit, muscle glycogen content increased again to 736 ± 24 mmol/kg dry muscle, which was significantly greater than the preinfusion value (P < 0.001) and the corresponding 24-h control value (P < 0.01). The difference in muscle glycogen content between control and carnitine at 24 h was approximately 170 mmol/kg dry muscle or 40 mmolkg wet muscle (assuming 1 kg dry muscle for every 4.3 kg wet muscle) (26). Thus, assuming skeletal muscle comprises 40% of total body mass, corresponding to an average of approximately 35 kg for the subjects in this study, the whole-body difference in skeletal muscle glycogen content (glucosyl units) between control and carnitine at 24 h was approximately 1.4 mol or 250 g.
Skeletal muscle lactate content, presented in Table 1, was increased 56% (P < 0.05) after the control infusion visit but was unchanged after the carnitine infusion visit. Twenty-four hours after the commencement of the saline infusion, muscle lactate content returned to basal. No significant differences were observed in skeletal muscle glucose-6-phosphate content during each visit (Table 1). Skeletal muscle LCA-CoA content tended to decrease after the control and carnitine infusion visits (Table 1). However, although muscle LCA-CoA was maintained at this lower value (P = 0.08), the following morning after the cessation of the hyperinsulinemic clamp in the control visit, muscle LCA-CoA content had returned to basal in the carnitine visit, such that it was 39% greater than control (P < 0.05).
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Discussion |
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), achieved via iv L-carnitine infusion during a 6-h euglycemic hyperinsulinemic clamp, resulted in a 30% decrease in muscle PDC activity (Fig. 2A) and a 40% decrease in muscle lactate content (Table 1). Furthermore, after an overnight fast, muscle glycogen (Fig. 2B) and LCA-CoA (Table 1) content had increased by 30 and 40%, respectively, in the carnitine group compared with control. The total amount of glucose infused during the control and carnitine visits in the present study was approximately 330 and 350 g, respectively, and the subjects consumed the same diet after each visit, consisting of approximately 220 g of carbohydrate. Thus, the 250-g difference in whole-body muscle glycogen content (assuming skeletal muscle contributes to 40% of total body mass; see Results) between the control and carnitine visits was not attributable to a difference in the amount of carbohydrate administered. Taken together, these findings lead us to conclude that the increase in muscle carnitine content observed in the present study inhibited glycolytic flux (decrease in lactate) and carbohydrate oxidation at the level of the PDC, thereby diverting muscle glucose uptake toward glycogen storage (nonoxidative glucose disposal).
An increase in nonoxidative glucose disposal, calculated indirectly as the difference between whole-body glucose disposal and oxidation (measured by indirect calorimetry), during steady-state L-carnitine infusion in the presence of an elevated serum insulin concentration (75 mU/liter), has been previously reported in other human studies (27, 28, 29). However, the fate of the glucose was not determined in these experiments, nor were the mechanisms involved elucidated. The key question, therefore, is what was the mechanism responsible for the apparent carnitine-mediated decrease in carbohydrate oxidation observed during the carnitine visit of the present study?
Because of carnitine’s role in long-chain fatty acid translocation into the mitochondrial matrix (2, 3), it is entirely plausible that the reduction in PDC activation could have been caused by a carnitine-mediated increase in skeletal muscle long-chain fatty acid oxidation via CPT1 (possibly from im stores, as arterialized venous plasma FFA concentration was not different between visits). Indeed, according to Randle’s glucose-fatty acid cycle (30, 31, 32, 33), increasing ß-oxidation would result in an increase in muscle acetyl-CoA and, therefore, an inhibition of PDC activity and thereby carbohydrate flux. The decrease in PDC activity after the insulin clamp in the carnitine trial of the present study was paralleled by a reduction in muscle lactate content and resulted in an accumulation of muscle glycogen overnight, conditions that are both consistent with the premise that carbohydrate oxidation was inhibited. In support of this theory, muscle LCA-CoA content returned to basal overnight during the carnitine visit (whereas it remained suppressed during the control visit), which suggests that fat oxidation was indeed increased. However, it should be noted that we have previously hypothesized that muscle carnitine availability becomes limiting to CPT1 at a value of around 6 mmol/kg dry muscle (7, 13), a content routinely observed in healthy humans during intense exercise. This raises the question as to why an increase in muscle carnitine content would increase fat oxidation at rest, where free carnitine content is around 20 mmol/kg dry muscle, well above the reported Km of CPT1 for carnitine (0.5 mM) (34). At present, this point requires further investigation but may be related to the compartmentalization of carnitine in skeletal muscle, which is not evident from measurements made on homogenized muscle biopsy samples.
In contrast to the above suggestion that the increase in muscle carnitine content augmented muscle glycogen accumulation by increasing fat oxidation and decreasing carbohydrate flux, the observations of the present study could also suggest a direct effect of intracellular carnitine on glycogen synthase activity and, therefore, rates of glycogen synthesis. For example, an increase in glycogen synthesis would reduce the amount of glucose entering glycolysis under insulin clamp conditions and, therefore, pyruvate and lactate accumulation and PDC activity. However, at present, we do not know of any reports in the literature as to the effects of carnitine per se on skeletal muscle glycogen synthase activity. Also, the observed decrease in muscle lactate content and PDC activity was evident before the increase in muscle glycogen content (although this could be because an increase in muscle carnitine content to a degree capable of eliciting an intracellular response might have only occurred toward the end of the L-carnitine infusion).
The reciprocal relationship between carbohydrate and fat oxidation in skeletal muscle would suggest that the apparent decrease in carbohydrate flux observed was the result of, or resulted in, an increase in fat oxidation. Thus, these findings could be of major importance in the treatment of insulin-resistant states, such as obesity and type 2 diabetes, because both conditions are associated with an impaired ability of skeletal muscle to oxidize fatty acids, both at rest and during exercise (15, 16, 17, 18, 19, 20). Furthermore, reducing or preventing im lipid accumulation increases insulin sensitivity (35, 36, 37, 38). In addition, a carnitine-mediated inhibition of carbohydrate oxidation (at the level of PDC) and an increase in muscle glycogen storage could also be of relevance in these conditions, because the inability of insulin to activate glycogen synthase in obese individuals appears to precede the development of type 2 diabetes (39, 40). Increasing skeletal muscle fat oxidation in obesity and type 2 diabetes is important, particularly during exercise, because exercise combined with weight loss, rather than weight loss alone, enhances fasting skeletal muscle fat oxidation rates and improves insulin sensitivity in obese patients (15, 18). However, whether free carnitine availability is responsible for the impairment of fat oxidation observed during exercise in obesity/type 2 diabetes has not been investigated. There is enhanced glucose use during exercise in obesity and type 2 diabetes (14, 15), and it is feasible therefore that this could reduce muscle free carnitine content, because of PDC flux being in excess of the rate of acetyl-CoA use by the tricarboxylic acid cycle, which would decrease fat oxidation. Accordingly, if skeletal muscle free carnitine availability were to be increased, then the inhibition of fat oxidation observed during exercise in these conditions could possibly be alleviated. However, these views should be tempered by the understanding that subject numbers in the study were relatively low and restricted to healthy young male volunteers. Whether an increase in muscle carnitine content will affect muscle fuel metabolism in insulin-resistant individuals, as observed in the present study in healthy male volunteers, remains to be determined and should ideally be quantified using stable isotope technologies in combination with muscle biopsy samples.
In conclusion, increasing skeletal muscle carnitine content decreases muscle lactate content and PDC activity and increases glycogen storage in conditions of high carbohydrate availability, possibly via a carnitine-mediated increase in muscle fat oxidation, as muscle LCA-CoA was increased. Additional investigation is required to directly identify whether an increase in fat oxidation occurred. Nevertheless, these novel findings have important implications for our understanding of the regulation of muscle fat oxidation, particularly during exercise, when carnitine availability may limit fat oxidation, and in obesity and type 2 diabetes where fat oxidation is known to be impaired.
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Footnotes |
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First Published Online September 19, 2006
Abbreviations: AC, Acetylcarnitine; CoA, coenzyme A; CPT1, carnitine palmitoyltransferase 1; FC, free carnitine; FFA, free fatty acids; LCA-CoA, long-chain acyl-CoA; PDC, pyruvate dehydrogenase complex; TC, total carnitine.
Received July 21, 2006.
Accepted September 11, 2006.
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) and L-carnitine (•) infusion combined with 6 h iv insulin infusion at a rate of 105 mU/m2·min. Values are means ± SEM (n = 7). ***, P < 0.001, significantly greater than the corresponding control value.
