Animal models suggest that acetylcarnitine production is essential for maintaining metabolic flexibility and insulin sensitivity. Because current methods to detect acetylcarnitine involve biopsy of the tissue of interest, noninvasive alternatives to measure acetylcarnitine concentrations could facilitate our understanding of its physiological relevance in humans. Here, we investigated the use of long–echo time (TE) proton magnetic resonance spectroscopy (1H-MRS) to measure skeletal muscle acetylcarnitine concentrations on a clinical 3T scanner. We applied long-TE 1H-MRS to measure acetylcarnitine in endurance-trained athletes, lean and obese sedentary subjects, and type 2 diabetes mellitus (T2DM) patients to cover a wide spectrum in insulin sensitivity. A long-TE 1H-MRS protocol was implemented for successful detection of skeletal muscle acetylcarnitine in these individuals. There were pronounced differences in insulin sensitivity, as measured by hyperinsulinemic-euglycemic clamp, and skeletal muscle mitochondrial function, as measured by phosphorus-MRS (31P-MRS), across groups. Insulin sensitivity and mitochondrial function were highest in trained athletes and lowest in T2DM patients. Skeletal muscle acetylcarnitine concentration showed a reciprocal distribution, with mean acetylcarnitine concentration correlating with mean insulin sensitivity in each group. These results demonstrate that measuring acetylcarnitine concentrations with 1H-MRS is feasible on clinical MR scanners and support the hypothesis that T2DM patients are characterized by a decreased formation of acetylcarnitine, possibly underlying decreased insulin sensitivity.
Lucas Lindeboom, Christine I. Nabuurs, Joris Hoeks, Bram Brouwers, Esther Phielix, M. Eline Kooi, Matthijs K.C. Hesselink, Joachim E. Wildberger, Robert D. Stevens, Timothy Koves, Deborah M. Muoio, Patrick Schrauwen, Vera B. Schrauwen-Hinderling
Submitter: Francis B. Stephens | email@example.com
Authors: Francis B. Stephens, Dumitru Constantin-Teodosiu, and Paul L. Greenhaff
MRC/ARUK Centre for Musculoskeletal Ageing Research, School of Life Sciences, University of Nottingham, UK
Published November 10, 2014
Insulin resistance is characterized by a ‘metabolic inflexibility’ in the reciprocal relationship between fatty acid and glucose oxidation within skeletal muscle (1). In keeping with Muoio et al (2), the recent JCI article of Lindboom and colleagues (3) asserts that “the transformation of excessive acetyl-CoA into acetylcarnitine is important to maintain metabolic flexibility”, and that “compromised capacity to generate acetylcarnitine, either due to reduced carnitine acetyl transferase (CRAT) activity or low carnitine concentration, may reduce pyruvate dehydrogenase (PDH) activity, hence reducing oxidative degradation of glucose”. Contrary to this opinion, biochemically determined human muscle acetylcarnitine content is reduced under insulin-stimulated, high glucose conditions (4), and increased in the insulin resistant state (5, 6). What’s more, compelling evidence has demonstrated a positive, constant linear relationship exists between muscle acetyl-CoA formation and acetylcarnitine accumulation over low to maximum rates of mitochondrial flux (7, 8, 9, 10). Furthermore, Lindeboom et al (3) themselves report maximal ex vivo CRAT activity in participants with type 2 diabetes (T2D) of 52.6 nmol/mg protein/min, which is far in excess of PDH flux rates even during high intensity exercise (15 nmol/mg protein/min; 10). This clearly demonstrates that CRAT is not limiting the equilibrium between acetyl-CoA and acetylcarnitine, making it highly unlikely that acetylcarnitine accumulation could be compromised for reasons other than rates of acetyl-CoA formation from PDH or β-oxidation reactions. Illustrated another way, if CRAT activity was indeed limiting in insulin resistant individuals, then mitochondrial ATP production would be markedly suppressed, as the rapid sequestering of mitochondrial free CoA as acetyl-CoA would immediately compromise PDH and TCA cycle flux. This clearly doesn’t happen, rather it’s likely that group differences in ex vivo maximal CRAT activity and in vivo mitochondrial function reported by Lindboom et al. would be dissipated if corrected for mitochondrial content, particularly given the apparent impairment of muscle mitochondrial function in patients with T2D is accounted for by differences in mitochondrial content (11). Finally, the resting muscle acetylcarnitine content of participants with T2D in Lindboom et al. was 12% of the measured total carnitine pool, making it highly improbable that carnitine availability was limiting to PDH flux and acetylcarnitine formation.
Nearly half a century of research has elucidated that muscle acetylcarnitine content reflects the balance of PDH flux, fatty acid oxidation, acetyl-CoA availability and the prevailing muscle ATP demand. The mechanistic control of the relative contribution from each, and its dysregulation in disease, remains to be determined.
Submitter: Patrick Schrauwen | firstname.lastname@example.org
Authors: Deborah M. Muoio, Patrick Schrauwen, Vera Schrauwen-Hinderling
Department of Human Biology, Maastricht University Medical Center, Maastricht, The Netherlands
Published November 10, 2014
Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that interconverts acetyl-CoA and its membrane permeant carnitine conjugate, acetylcarnitine. Although the biochemistry of this enzyme has been studied for over fifty years, its physiological function remains poorly understood. As recently discussed in (1-3), several lines of evidence suggest CrAT plays a key role in mitigating lipid-induced inhibition of pyruvate dehydrogenase (PDH), and that CrAT and/or carnitine insufficiency might contribute to obesity-related impairments in glucose homeostasis. First, provision of L-carnitine in the context of mixed substrate availability increases carbon flux through the PDH complex. This is evident in intact isolated mitochondria, cultured myocytes, perfused working hearts, and at the whole body level during acute L-carnitine infusions (2-7). Moreover, muscle-specific deletion of CrAT in mice disrupts glucose homeostasis and pyruvate flux, providing direct evidence of a strong functional link between the two enzymes (2). Additionally, in obese rodents and humans, dietary supplementation with L-carnitine has been shown to enhance systemic glucose tolerance in association with increases in plasma acetylcarnitine levels, whole body glucose oxidation and muscle PDH activity (2,8). Notably, the anti-diabetic actions of L-carnitine are more pronounced in the setting of metabolic disease (3), suggesting that carnitine and/or CrAT activity might not limit PDH flux in circumstances of normal health and energy balance. To this point, studies by Greenhaff and colleagues showed that a prolonged euglycemichyperinsulinemic clamp lowers muscle acetylcarnitine levels in healthy individuals (9), which may not be surprising given that insulin potently suppresses lipolysis, both systemically and locally, thereby reducing substrate competition and the potential for acetyl-CoA accumulation. Importantly, this experimental paradigm does not mimic the nutrient milieu of a mixed meal, or the lipid-enriched state of an obese and/or type 2 diabetic individual.
Stephens et al. question the idea that CrAT activity and/or L-carnitine availability could limit pyruvate oxidation based on flux capacity of the enzyme measured in a homogenate system after membrane permeabilization in the presence of saturating carnitine concentrations. One can argue that these in vitro conditions are unlikely to recapitulate the regulatory constraints imposed by intact mitochondria functioning in a physiologic context. For example, L-carnitine and acetylcarnitine are present in multiple cellular compartments. Thus, mitochondrial import and export of L-carnitine and acetylcarnitine, as well as enzyme inhibitors such palmitoyl-CoA (10), could affect the rate of the CrAT reaction in vivo. Additionally, total CrAT activity measured in whole cell lysates does not necessarily reflect enzyme abundance and function in the microenvironment surrounding PDH. Lastly, contrary to the prediction that CrAT insufficiency would result in rapid sequestering of mitochondrial free CoA, bringing PDH and the TCA cycle to a halt, ablation of CrAT in a mouse model did not produce either of these outcomes (2). Taken together, these findings underscore the need for further work to elucidate the physiological function of CrAT in the context of health and disease. The non-invasive MR measurement reported in the Journal (1) will now enable further study of the importance of this pathway in the etiology and treatment of type 2 diabetes.
1. Lindeboom, L., Nabuurs, C. I., Hoeks, J., Brouwers, B., Phielix, E., Kooi, M. E., Hesselink, M. K., Wildberger, J. E., Stevens, R. D., Koves, T., Muoio, D. M., Schrauwen, P., and Schrauwen-Hinderling, V. B. (2014) Long-echo time MR spectroscopy for skeletal muscle acetylcarnitine detection. J Clin Invest 124, 4915-4925
2. Muoio, D. M., Noland, R. C., Kovalik, J. P., Seiler, S. E., Davies, M. N., DeBalsi, K. L., Ilkayeva, O. R., Stevens, R. D., Kheterpal, I., Zhang, J., Covington, J. D., Bajpeyi, S., Ravussin, E., Kraus, W., Koves, T. R., and Mynatt, R. L. (2012) Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility. Cell Metab 15, 764-777
3. Ringseis, R., Keller, J., and Eder, K. (2012) Role of carnitine in the regulation of glucose homeostasis and insulin sensitivity: evidence from in vivo and in vitro studies with carnitine supplementation and carnitine deficiency. Eur.J.Nutr. 51, 1-18
4. Noland, R. C., Koves, T. R., Seiler, S. E., Lum, H., Lust, R. M., Ilkayeva, O., Stevens, R. D., Hegardt, F. G., and Muoio, D. M. (2009) Carnitine insufficiency caused by aging and overnutrition compromises mitochondrial performance and metabolic control. J Biol.Chem. 284, 22840-22852
5. Mingrone, G. (2004) Carnitine in Type 2 Diabetes. Annals of the New York Academy of Sciences 1033, 99-107
6. Mingrone, G., Greco, A. V., Capristo, E., Benedetti, G., Giancaterini, A., De Gaetano, A., and Gasbarrini, G. (1999) L-carnitine improves glucose disposal in type 2 diabetic patients. Journal of the American College of Nutrition 18, 77-82
7. Broderick, T. L., Quinney, H. A., and Lopaschuk, G. D. (1992) Carnitine stimulation of glucose oxidation in the fatty acid perfused isolated working rat heart. J Biol Chem 267, 3758-3763
8. Power, R. A., Hulver, M. W., Zhang, J. Y., Dubois, J., Marchand, R. M., Ilkayeva, O. R., Muoio, D. M., and Mynatt, R. (2007) Carnitine revisited: potential use as adjunctive treatment in diabetes. Diabetologia 50, 824-832
9. Stephens, F. B., Constantin-Teodosiu, D., Laithwaite, D., Simpson, E. J., and Greenhaff, P. L. (2006) An acute increase in skeletal muscle carnitine content alters fuel metabolism in resting human skeletal muscle. J.Clin.Endocrinol.Metab 91, 5013-5018
10. Seiler, S. E., Martin, O. J., Noland, R. C., Slentz, D. H., Debalsi, K. L., Ilkayeva, O. R., An, J., Newgard, C. B., Koves, T. R., and Muoio, D. M. (2014) Obesity and Lipid Stress Inhibit Carnitine Acetyltransferase Activity. J Lipid Res 55, 635-44