Conceived and designed the experiments: ER LMR WEK CBN VBK CFP. Performed the experiments: RDS MJM BRW JRB. Analyzed the data: KMH LRL CFP VBK WEK. Wrote the paper: KMH LMR LRL CFP RDS MJM BRW JRB VBK CBN ER WEK.
The authors have declared that no competing interests exist.
To determine if caloric restriction (CR) would cause changes in plasma metabolic intermediates in response to a mixed meal, suggestive of changes in the capacity to adapt fuel oxidation to fuel availability or metabolic flexibility, and to determine how any such changes relate to insulin sensitivity (SI).
Forty-six volunteers were randomized to a weight maintenance diet (Control), 25% CR, or 12.5% CR plus 12.5% energy deficit from structured aerobic exercise (CR+EX), or a liquid calorie diet (890 kcal/d until 15% reduction in body weight)for six months. Fasting and postprandial plasma samples were obtained at baseline, three, and six months. A targeted mass spectrometry-based platform was used to measure concentrations of individual free fatty acids (FFA), amino acids (AA), and acylcarnitines (AC). SI was measured with an intravenous glucose tolerance test.
Over three and six months, there were significantly larger differences in fasting-to-postprandial (FPP) concentrations of medium and long chain AC (byproducts of FA oxidation) in the CR relative to Control and a tendency for the same in CR+EX (CR-3 month P = 0.02; CR-6 month P = 0.002; CR+EX-3 month P = 0.09; CR+EX-6 month P = 0.08). After three months of CR, there was a trend towards a larger difference in FPP FFA concentrations (P = 0.07; CR-3 month P = 0.08). Time-varying differences in FPP concentrations of AC and AA were independently related to time-varying SI (P<0.05 for both).
Based on changes in intermediates of FA oxidation following a food challenge, CR imparted improvements in metabolic flexibility that correlated with improvements in SI.
ClinicalTrials.gov
Caloric restriction provides metabolic benefits in a variety of nonhuman animal species (reviewed in
Metabolic flexibility is an emerging indicator of (metabolic) health
Prior studies have examined the effects of type 2 diabetes, obesity, insulin resistance, and a family history of type 2 diabetes on metabolic flexibility, defined as the ability to shift substrate oxidation and measured by whole body and skeletal muscle respiratory quotient (RQ)
Thus far in human studies, metabolic flexibility has been measured as alterations in RQ, which serves as a surrogate for substrate oxidation and ranges from 1 for total carbohydrate oxidation to 0.7 for total fat oxidation3. Using changes in RQ as the primary variable, weight loss (with and without exercise training) has been associated with a trend towards increased metabolic flexibility, measured in the fasting state alone or in response to a hyperinsulinemic clamp
The main objective of the current study was to determine the effect of caloric restriction on the shift in substrate utilization in response to a mixed meal. A secondary aim was to evaluate the relationship of changes in metabolic flexibility, as measured by metabolic intermediate concentration changes from the fasted to fed states, to changes in insulin sensitivity over the course of a six-month intervention period. We hypothesized that caloric restriction, or the combination of caloric restriction and exercise, would cause a larger difference in the fasting to postprandial concentrations of fatty acids and acylcarnitines over the intervention period when compared to non-caloric restricted controls.
Forty-six healthy, non-smoking, overweight (25≤BMI<30), 25–50 year old men and 25–45 year old women were recruited to participate in and completed a six-month intervention (clinical trials number NCT00427193). The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
This Figure has been published previously
The study was approved by the Pennington Biomedical Research Center (PBRC, Baton Rouge, LA) Institutional Review Board and the CALERIE Data Safety Monitoring Board, and written informed consent was obtained for all participants.
Participants were randomized into one of four groups for 24 weeks: healthy weight maintenance diet (Control); caloric restriction (CR): 25% caloric restriction from baseline energy requirements; caloric restriction and exercise (CR+EX): 12.5% caloric restriction and 12.5% increase in energy expenditure through structured aerobic exercise; or, liquid calorie diet (LCD; 890 kcal/day) to rapidly achieve 15% weight loss followed by weight stabilization with a eucaloric diet designed to maintain body mass at this level. The group assignment was stratified to ensure equal distributions of sex and BMI in the four groups, and participants were analyzed as intention-to-treat.
As previously described, total energy expenditure measured using doubly labeled water was used for calculation of energy requirements
Individual exercise prescriptions were based on increasing energy expenditure 12.5% above resting energy expenditure as previously described
Plasma samples were obtained after an overnight fast and 60 and 90 minutes after a standard lunch mixed meal (postprandial and pooled) on an inpatient research unit at each of three time points: baseline (before initiation of the intervention), three months following initiation of the intervention and six months following initiation of the intervention (study exit). Samples were prepared and stored at −80°C for later analysis.
Targeted mass spectrometry was used to measure concentrations of eight free fatty acids (FFA), 15 amino acids (AA), and 45 acylcarnitines (AC) as described previously
As previously reported
Principal components analysis (PCA) was used as a data reduction technique. PCA is commonly used to reduce a large number of observed variables to a smaller number of constructed variables (principal components) that account for the variance in the observed variables
Specifically, we created a fasting-to-postprandial change score for each metabolite at each time point as follows: Change score = postprandial – fasting concentration. Then, PCA was performed using varimax rotation on the baseline change score for each metabolite class such that one PCA change score analysis was performed for each domain of FFAs, ACs, and AAs. The number of components retained for each model was selected to balance parsimony and the total percent of variance explained. Then, component loadings from baseline change PCAs were applied to the fasting to postprandial change scores for the three- and six-month time points normalized to the baseline values, and component scores were generated for each metabolic domain at each time point. This normalization provided a common metric for estimating change in components over time.
To determine whether the intervention affected change in fasting-to-postprandial differences (FPPD) over time, we used mixed models
Information on the main determinants from CALERIE is reported elsewhere. Briefly, CR and CR+EX improved two biomarkers associated with longevity, reduced core body temperature and fasting insulin concentrations as well as 24-hour energy expenditure, DNA damage, and cardiovascular risk profiles
Using PCA to analyze changes in the fasting-to-postprandial difference (FPPD), we identified single components for FPPD for each of the major analyte modules– FFA, AC, and AA– with eigenvalues of 6.37, 8.67, and 7.88 respectively, explaining 80%, 20% and 53% of the variance in the component (
Constituents | Loadings | Eigen-value | Cumulative Variance |
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Palmitic acid | 0.96 | 6.37 | 0.80 |
Linoleic acid | 0.95 | ||
Oleic acid | 0.95 | ||
Myristic acid | 0.93 | ||
Palmitoleic acid | 0.91 | ||
Stearic acid | 0.87 | ||
alpha-Linolenic acid | 0.86 | ||
Arachidonic acid | 0.66 | ||
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C12:1 | 0.77 | 8.67 | 0.20 |
C16 | 0.77 | ||
C14:1 | 0.75 | ||
C14:2 | 0.72 | ||
C16:2 | 0.67 | ||
C16:1 | 0.67 | ||
C12 | 0.69 | ||
C18:1 | 0.66 | ||
C6-DC | 0.62 | ||
C10:1 | 0.61 | ||
C10 | 0.56 | ||
C8:1 | 0.54 | ||
C8 | 0.53 | ||
C10-OH/C8-DC | 0.53 | ||
C18:2 | 0.53 | ||
C8:1-DC | 0.51 | ||
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Leucine/Isoleucine | 0.91 | 7.88 | 0.53 |
Phenylalanine | 0.91 | ||
Methionine | 0.87 | ||
Histidine | 0.83 | ||
Valine | 0.81 | ||
Tyrosine | 0.81 | ||
Aspartate/Asparagine | 0.81 | ||
Serine | 0.79 | ||
Proline | 0.71 | ||
Ornithine | 0.66 | ||
Arginine | 0.65 | ||
Glycine | 0.56 | ||
Alanine | 0.56 |
*Changes were computed as postprandial metabolite concentration minus preprandial metabolite concentration. For these differences, PCA was performed separately for each metabolite class: fatty acids, acylcarnitines, and amino acids. Key metabolites within each component (
Metabolite Changes | Preprandial Median (IQR) | Postprandial Median (IQR) |
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Acetyl carnitine, C2 (nM) | 6941.3 (3053.4) | 4169.4 (1929.1) |
Propionyl carnitine, C3 (nM) | 386.6 (243.2) | 471.9 (273.9) |
Butyryl/Isobutyryl carnitine, C4/Ci4 (nM) | 136.0 (112.8) | 160.3 (64.5) |
Tiglyl carnitine, C5:1 (nM) | 52.0 (21.6) | 48.1 (25.1) |
Isovaleryl/3-Methylbutyryl/2-Methylbutyryl carnitine, C5's (nM) | 102.0 (54.8) | 110.6 (61.0) |
β-Hydroxy butyryl carnitine, C4OH (nM) | 21.0 (18.0) | 16.9 (19.6) |
Hexanoyl carnitine, C6 (nM) | 0 (0) | 21.3 (69.2) |
3-Hydroxy-isovaleryl/Malonyl carnitine, C5OH/C3DC (nM) | 92.8 (88.0) | 104.1 (111.2) |
Methylmalonyl/Succinyl carnitine, Ci4DC/C4DC (nM) | 21.8 (14.1) | 22.6 (13.7) |
Octenoyl carnitine, C8:1 (nM) | 151.4 (76.3) | 122.8 (68.4) |
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Glutaryl carnitine, C5DC (nM) | 28.9 (17.7) | 23.0 (13.7) |
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Decatrienoyl carnitine, C10:3 (nM) | 96.8 (47.7) | 71.0 (41.0) |
Decadienoyl carnitine, C10:2 (nM) | 28.5 (11.7) | 21.1 (15.2) |
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Myristoyl carnitine, C14 (nM) | 12.7 (7.4) | 8.5 (4.4) |
3-Hydroxy-tetradecenoyl carnitine, C14:1-OH (nM) | 8.7 (5.3) | 6.0 (5.1) |
3-Hydroxy-tetradecanoyl/Dodecanedioyl carnitine, C14-OH/C12-DC (nM) | 5.6 (3.9) | 3.5 (3.2) |
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3-Hydroxy-hexadecanoyl/Tetradecanedioyl carnitine, C16-OH/C14-DC (nM) | 2.1 (1.9) | 2.1 (1.6) |
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Stearoyl carnitine, C18 (nM) | 26.2 (9.0) | 23.7 (11.4) |
3-Hydroxy-octadecenoyl carnitine, C18:1-OH (nM) | 3.9 (1.9) | 2.3 (1.8) |
3-Hydroxy-octadecanoyl/Hexadecanedioyl carnitine, C18-OH/C16-DC (nM) | 3.2 (2.8) | 3.0 (1.8) |
Arachidoyl carnitine, C20 (nM) | 3.5 (2.6) | 3.2 (2.5) |
Octadecenedioyl carnitine, C18:1-DC (nM) | 4.3 (2.3) | 4.0 (2.4) |
3-Hydroxy-eicosanoyl/Octadecanedioyl carnitine, C20-OH/C18-DC (nM) | 4.7 (3.5) | 4.1 (3.7) |
Docosanoyl carnitine, C22 (nM) | 2.2 (1.8) | 1.9 (1.6) |
3-Hydroxy- |
22.1 (12.2) | 16.0 (8.0) |
Heptanedioyl carnitine, C7-DC (nM) | 0 (6.2) | 0 (4.9) |
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3-Hydroxy-palmitoleoyl/ |
5.3 (1.9) | 3.2 (2.2) |
3-Hydroxy-linoleyl carnitine, C18:2-OH (nM) | 3.7 (4.3) | 2.9 (4.9) |
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Glutamate/Glutamine (µM) | 102.9 (36.9) | 105.6 (40.5) |
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Citrulline (µM) | 30.7 (11.8) | 28.4 (10.7) |
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*Those loading most heavily (component load ≥|0.5|) in principal component analyses are identified in bold.
In response to CR, the AC FPPD was amplified (more negative FPPD) over time relative to Control (P = 0.02 at 3 mo., P = 0.002 at 6 mo., overall P<0.001) (
Fasting to postprandial difference (FPPD) scores were computed as postprandial minus fasting concentration. Difference scores were used in principal component analyses and single component solutions were retained as described in
As previously reported
Each bar represents insulin sensitivity improvements for participating individuals. A. By intervention group. CR = Caloric restriction; CR+EX = Caloric restriction with exercise; Control = Healthy weight maintenance diet; LCD = Liquid calorie diet B. Intervention groups combined.
As described in
In order to determine whether larger FPPD corresponded to changes in fasting concentrations, postprandial concentrations or both, we evaluated raw data for individual metabolites loading most heavily on each component. By evaluating raw data, the intervention-induced amplifications in FFPD were attributable to changes in the fasting concentrations, as opposed to the postprandial concentration of the metabolites; that is, increased fasting AC and decreased fasting AA over time. As seen in
Baseline and three month acylcarnitine concentrations are shown for both fasting (preprandial) and postprandial assessments. The six acylcarnitines that had the largest loadings on the acylcarnitine factor (see
Baseline and three month amino acids concentrations are shown for both fasting (preprandial) and postprandial assessments. The five amino acids that had the largest loadings on the amino acid factor (see
In this randomized controlled trial of three healthy lifestyle interventions (CR, CR+EX, and a healthy weight maintenance diet) and a very low calorie diet, we observed that the CR intervention significantly increased the fasting-to-postprandial difference (FPPD) in circulating acylcarnitines (AC) and free fatty acids (FFA). Moreover, we observed that increased FPPD for both AC and AA were related to greater insulin sensitivity. Thus, this study expands prior investigations of metabolic flexibility from 1) acute responses to an infusion or meal to responses to a prolonged intervention, 2) responses to glucose/insulin infusions to more clinically-relevant mixed meals, 3) assessments made by increases in RQ to changes in a broad panel of metabolic intermediates that includes both substrates and products of key energy producing pathways.
The term metabolic flexibility is used to describe the efficient transition between substrate utilization in response to changes in substrate supply or energy demand. One example is the shift from the use of fatty acids as the main energy source during fasting conditions towards glucose utilization in a fed state
To our knowledge, this is the first report of a relation between an increased FPPD for AA and greater insulin sensitivity. While in prior investigations, amplified FPPD gradients in fatty acid and glucose oxidation have been recognized as signs of improved metabolic flexibility
Here, we showed that CR (at a level of −25% from basal energy requirements) improved the ability to shift energy substrates with feeding, and that caloric restriction plus exercise (CR+EX), with an identical relative energy deficit, imparted roughly half of the ability to shift substrate seen with CR alone. In contrast, in a study by Kelley and Goodpaster, a combination of weight loss and exercise training increased the rate of fatty acid relative to glucose oxidization in the fasting state (lower fasting respiratory quotient [RQ]) as compared to before the intervention
Our observations add metabolic flexibility, as measured by changes in metabolic intermediates in response to a mixed meal, to the list of improvements that occur in the setting of CR elucidated through the CALERIE study. In addition to this effect, CR or CR+EX reduced core body temperature, 24-hour energy expenditure, fasting insulin concentrations, DNA damage, and cardiovascular risk profiles
The careful control of the caloric restriction and exercise interventions is a great strength of this analysis. To minimize type I error rates, we used PCA to reduce the dimensionality of the data. We performed these analyses cognizant that using PCA in small samples can result in ‘overfitting,’ where findings are sample-specific rather than representative of the population of interest
In summary, CR improved metabolic flexibility evidenced by higher fasting AC and FFA concentrations and widened FPPD gradients for these metabolites. Furthermore, the change in the FPPD gradient of AC and AA concentration was related to improvements in insulin sensitivity.
CONSORT Checklist.
(DOC)
Trial Protocol.
(DOC)
We appreciate the contributions from the remaining members of the Pennington CALERIE Research Team, The Duke Pepper Center, and the Sarah W. Stedman Center Nutrition and Metabolism Center.