Acylcarnitines are carnitine esters derived from fatty acids or amino acids transferred into the mitochondria [1
]. Elevated AC production can occur when β-oxidation rates are in excess of complete oxidation to CO2
through the TCA cycle [1
]. Previous studies have shown that AC concentrations increase with duration of fasting in humans [8
]. By contrast, glucose-stimulated insulin secretion during an OGTT [10
] or hyperinsulinemia during a euglycemic clamp significantly reduce AC concentrations [4
]. The acute postprandial pattern of AC species was unknown - as past studies were designed to understand the impact of glucose/insulin challenge and not to mimic the physiology of a mixed meal.
As a result of a growing body of data in the literature, plasma AC levels are proposed as biomarkers in subjects with insulin resistance [3
]. Indeed, AC concentrations are higher in the fasting state in insulin resistant populations and decrease less during glucose challenge tests [4
]. This latter observation may be due to a continued release of adipose FFA as a result of adipose insulin resistance, or could be due to use of IMCL [33
]. With respect to the role of dietary fatty acids, very little is known. AC composition has been shown in one study to resemble longer-term, dietary fatty acid composition [6
] and also in another study, to increase acutely after a a single oral bolus of oil [34
]. Thus, both endogenous and dietary fatty acids could impact AC composition and concentration. Given the multiple sources of metabolic substrates that could serve as precursors of plasma AC, we sought to determine how AC concentration would change under physiologic conditions which would elicit an insulin response suppressing adipose fatty acid release, while at the same time leading to dietary fat absorption. Our key finding was that the responses of different AC species varied after the meal. The concentrations of the saturates C14:0, C16:0, and C18:0 remained steady, while the mono- and polyunsaturates fell significantly. This finding is in line with data supporting greater oxidation of long-chain unsaturated fatty acids (18:1, 18:2) compared to saturates in humans [35
] and in rodents [37
]. Using an isotopic dilution method in vivo, Kanaley et al measured a fatty acid label present in the same AC species [5
]. In that study, as in the present project, isotope administration did not result in labeling of downstream AC products - most likely due to administration at tracer amounts used for turnover measurements. The seminal finding of Kanaley et al was that the plasma FFA is an original precursor of muscle AC, but that intramyocellular-TG serves as a key intermediate compartment between these two pools in fasted individuals [5
]. The present results echo these findings, but in the fed state here, by demonstrating a postprandial pattern of reductions in plasma FFA, which precede reductions in long-chain and medium-chain AC concentrations.
The transition from the fasted to fed states is characterized by changes in the metabolism of long-chain fatty acids which is determined by a balance between adipose fatty acid release, availability of tissue lipid stores, mitochondrial fatty acid transfer, release of dietary-fatty acids by intravascular lipolysis, tissue fatty acid uptake, and mitochondrial fatty acid oxidation rates. As shown in the supplementary data, from 0 to 2h postprandially LCAC (C16, C18:1, C18:2) were constant against a background of significant plasma FFA reduction. One interpretation of this finding is that from 0–2h, the source of the AC was IMCL. Concurrently, the intermediate fatty acid beta-oxidation byproducts (C8, C10, C10:1, C12, C12:1, C14:1, C14:2, and C16:1) fell from the onset of eating (0–2h). This fall in by-products could have occurred due to their accelerated use, or a decline in their production. Our data are consistent with the latter, since whole-body fat oxidation remained unchanged. At 4h, C16, C18:1 and C18:2 finally reached a nadir and then began to rise in the late postprandial phase when dietary fatty acids were being liberated in the plasma compartment, and at the same time adipose fatty acid release resumed (). Altogether, these data suggest that the use of IMCL serves as a ~3h buffer for the dramatic loss of plasma FFA after the onset of eating. If this interpretation of events is accurate, adipose insulin resistance in the fed state
could result in significant overload of muscle beta-oxidation, leading to negative effects such as inflammation and defects in insulin signaling [38
Our secondary findings include a strong influence of LBM to increase the production of short-chain AC in both the fasted and fed states. Mihalik and colleagues found significant positive correlations between BMI and multiple fasting AC species [4
]. Our data suggest that for at least the short-chain AC, these relationships were due to a direct provision of substrate (branch-chain amino acids) mediated through elevated LBM. Many of the subjects described in the literature as having the highest fasting AC concentrations were overweight, obese and diabetic, and had a higher LBM either because of male gender [40
], ethnicity (African American [3
]) or obesity [42
]. The composite of increased FFA substrate and availability of muscle mass rich in mitochondria suggests that overweight and obese individuals have both the source and the means to form AC species.
We also observed that those individuals with the lowest LCAC concentrations in both the fasted and fed states were those whose glucose oxidation was the highest at those times. Conversely, positive and significant associations were found between fasting LCAC and fasting fat oxidation. In the fed state, the influence of fatty acid flux was evident by the positive relationships between adipose fatty acid release, post-meal FFA concentrations, and postprandial AC concentrations. Consistent with past observations of higher acetyl-carnitine in fasted insulin resistant subjects [3
], during the fed state, a high insulin sensitivity index was associated with lower postprandial LCAC, specifically C14 and C14:1. Interestingly, these two AC, frequently used as biomarkers of inborn errors in metabolism [2
], were repeatedly predictive of whole body substrate oxidation in the fasting and fed states. Lastly, dietary fatty acid spillover is found in individuals who have appropriately suppressed fatty acid turnover in the fed state. These are the same people who exhibited high insulin sensitivity, low postprandial nadir FFA concentrations, and a switch to glucose oxidation in the fed state. If this scenario is correct, then dietary fatty acid spillover is a natural result of active adipose liberation of dietary fatty acids through lipolysis but slow turnover of FFA in the blood.
The present study had a number of limitations, including the small sample size of postprandial studies compared to larger fasting metabolomic analyses, the restricted ethnicity of the subjects, and the assessment of AC after only one meal. We were surprised at the very low level of variability between the subjects in their patterns of postprandial AC change at each time point (suppl. fig. 1 and 2
). The strong correlations found between fasting (or nadir) AC concentrations and various subject characteristics were present for multiple similar AC species (either long or short-chain AC), which provides support for these findings. The rigor of our standardization of the meal protocol, particularly amongst this group of subjects with a wide range of insulin sensitivities, likely facilitated the testing of our hypothesis. Given the present data, future study designs can now be devised to determine meal labeling parameters that will result in non-steady state tracking of fatty acids into the mitochondria.
In summary, the present study is the first to demonstrate the temporal pattern of change in plasma AC in overweight subjects consuming a mixed meal and we have demonstrated that conditions that impact fatty acid flux contribute to the control of AC concentrations. Elevated adipose fatty acid release postprandially led to increased FFA, which was associated with elevations in products of incomplete beta-oxidation. Insulin sensitivity at muscle and adipose can both lead to lower post-meal AC. Future studies investigating AC as biomarkers of metabolic function should include a focus on the precursor molecules used for AC production. In subjects with elevated body weights, precursors of short-chain AC could be derived from muscle stores of branch-chain amino acids and/or from plasma sources (e.g., diet). Moreover, medium- and long-chain AC can be derived from IMCL in the short term, and diet and peripheral stores over the long-term. A better definition of the role of AC during the transition from the fasted to fed states will aid in the understanding of how substrate overload contributes to metabolic dysfunction postprandially.