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Departments of Endocrinology and Metabolism (S.H., R.W.v.d.M., J.W.A.S., J.A.R.) and Radiology (S.H., R.W.v.d.M., H.J.L., A.d.R.), Leiden University Medical Center, 2300 RC Leiden, The Netherlands; Russell H. Morgan Department of Radiology and Radiological Science (M.S.), Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; and Philips Medical Systems (M.S.), Cleveland, Ohio 44106
Address all correspondence and requests for reprints to: S. Hammer, Department of Endocrinology and Metabolism (C4-R), Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail: S.Hammer{at}LUMC.nl.
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Abstract |
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Objective: Our objective was to assess the effects of progressive caloric restriction on myocardial and hepatic TG content and myocardial function.
Design: This was a prospective intervention study.
Participants: This study included 10 lean healthy men.
Interventions: Three-day partial (471 kcal/d) and complete starvation was performed.
Outcome Measures: Plasma levels of FFA, myocardial and hepatic TG content, and myocardial function were calculated.
Results: Plasma FFA increased from 0.6 ± 0.4 mmol/liter to 1.2 ± 0.4 and to 1.9 ± 0.7 mmol/liter, after partial and complete starvation, respectively (P < 0.001). Myocardial TG content increased from 0.35 ± 0.14% to 0.59 ± 0.27%, and 1.26 ± 0.49%, respectively (P < 0.01). The ratio between the early diastole and atrial contraction decreased from 2.2 ± 0.4 to 2.1 ± 0.4 (P = 0.7) and 1.8 ± 0.4, respectively (P < 0.01), and diastolic early deceleration from 3.4 ± 0.7 ml/sec2 x 10–3 to 2.9 ± 0.5 and 2.8 ± 0.9 ml/sec2 x 10–3, respectively (P < 0.05). Hepatic TG content decreased after partial starvation (from 2.23 ± 2.24% to 1.43 ± 1.33%; P < 0.05) but did not change upon complete starvation.
Conclusions: Progressive caloric restriction induces a dose-dependent increase in myocardial TG content and a dose-dependent decrease in diastolic function in lean healthy men. Hepatic TG content showed a differential response to progressive caloric restriction, indicating that redistribution of endogenous TG stores is tissue specific.
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Introduction |
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High plasma concentrations of free fatty acids (FFAs) may result in excessive FA uptake in nonadipose tissues, such as the liver and heart, which may affect normal organ function (7, 8). However, in humans the relation between myocardial TG accumulation and myocardial function was difficult to study by noninvasive methods because measurement of myocardial TG content is challenging due to artifacts induced by cardiac and respiratory motion. Recently, proton magnetic resonance spectroscopy (MRS) of the heart was developed that enables the measurement of myocardial TG content in humans in vivo (1, 10, 11, 12, 13). Using this method, Reingold et al. (14) documented that fasting for 48 h increases plasma FFA levels and myocardial TG content in healthy subjects, whereas myocardial TG content did not change after a single high fat meal. In another, cross-sectional study, Kankaanpää et al. (12) showed that increased levels of plasma FFA in obese subjects correlate positively with myocardial TG content and inversely with cardiac function. However, both studies did not address the relation between myocardial function in relation to myocardial TG content within the same subjects. In a recent study, we documented that the use of a very low calorie diet increases plasma FFA and myocardial TG content, associated with a decrease in myocardial diastolic function (15). Therefore, it appears that myocardial TG content is not fixed but varies within the same subject according to physiological conditions. It is yet unknown whether our recent findings of myocardial flexibility can be extrapolated when caloric restriction is progressively increased. Therefore, the aim of the present study was to extend the conditions of partial caloric restriction to complete caloric restriction, i.e. complete starvation. For this purpose we compared baseline observations with those obtained after 3-d partial starvation (471 kcal/d) and after 3-d complete starvation with respect to plasma levels of FFA, myocardial TG content, myocardial function, and hepatic TG content.
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Subjects and Methods |
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There were 10 nonsmoking, healthy men included in this study [age; mean ± SD: 23.7 ± 4.7 yr, range 20.8–36.0 yr; body mass index (BMI): 23.6 ± 0.9 kg/m2]. Women were excluded because the hormonal status or contraceptive use may affect lipid metabolism (16). The study population was partly based on a previous cohort (15). In each subject, medical history was obtained, and physical examination was performed. An electrocardiogram (ECG) was made during the first visit. Subjects with any aberrations on the ECG were excluded. In addition, a 2-h 75 g oral glucose tolerance test was performed in the fasted state, to exclude subjects suffering from diabetes mellitus (17). Other exclusion criteria were: obesity (BMI > 30 kg/m2); liver disease (increased plasma levels of alanine aminotransferase, aspartate aminotransferase, and/or 
-glutamyl transferase > 2 SD above the reference value of our institution); renal disease (defined by plasma creatinine levels > 2 SD above the reference value of our institution); use of any medication; and a history of (congenital) heart disease. Specifically, subjects with prior or present coronary artery disease (based on medical history) or hypertension (defined as sitting systolic blood pressure > 130 mm Hg and/or diastolic blood pressure > 85 mm Hg) were excluded. Written informed consent was obtained from all participants before the study. The local ethics committee approved the study.
Study design
The study consisted of three conditions. Baseline measurements were made, while subjects followed a normal diet but abstained from alcohol for 3 d (mean intake 2065 kcal/d). Subjects were admitted 4 h after the last meal for measurement of plasma concentrations of glucose, insulin, and lipids, and for evaluation by magnetic resonance imaging (MRI) and MRS. The second measurement was performed after a 3-d period of partial caloric restriction (471 kcal/d; Modifast Intensive, Nutrition & Santé Benelux, Breda, The Netherlands). The third measurement was performed after a 3-d period of complete starvation (0 kcal/d, only water was allowed), after which subjects were again admitted for blood sampling and MRI/MRS evaluation. Plasma concentrations of FFA and insulin were used to assess study compliance (18). Between all study occasions, a washout period with a minimum of 14 d was acquired (19), and the sequence of the second and third occasions was determined by balanced assignment.
1H-MRS of the liver and the heart
All MRI/MRS measurements were performed on a 1.5-Tesla Gyroscan ACS-NT MRI scanner (Philips Medical Systems, Best, The Netherlands) in the supine position. Localized single voxel (2 x 2 x 2 cm for the liver and 2 x 4 x 1 cm for the heart) spectra were recorded using a body coil for radiofrequency transmission and a surface coil (Ø 17 cm) for signal receiving. For the heart, the spectral volume was placed in the interventricular septum on four-chamber and short axis images at end systole, avoiding contamination with epicardial fat (Fig. 1
). Data collection was double triggered using ECG triggering and navigator echoes for compensation of respiratory motion as described earlier (13). For the liver, voxel sites were matched at both study occasions, carefully avoiding blood vessels and bile ducts. To detect weak lipid signals, water-suppressed spectra with 128 averages for the heart and 64 for the liver were collected. Spectral parameters were: a repetition time (TR) of 3000 msec, echo time (TE) of 26 msec, and 1024 data points over 1000 kHz spectral width. In the same voxel, using the same parameters except for a TR of 10,000 msec, unsuppressed spectra with four averages were collected. Spectra were analyzed in the time domain, using Java-based MR user interface software and prior knowledge files [version 2.2 (20)], as described earlier (13). Peak estimates of lipid resonances of myocardial TG at 1.3 parts per million (ppm) and 0.9 ppm were summed and calculated as a percentage of the unsuppressed water signal (TG content, TG/water x100).
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Imaging of the heart was performed using a body coil for radiofrequency transmission and a five-element synergy coil for signal receiving. To assess systolic function, the heart was imaged from apex to base with 12–14 imaging levels (dependent on the heart size) in short axis view using an ECG triggered, sensitivity encoding balanced steady-state free procession sequence. Imaging parameters were a field-of-view of 400 mm, a matrix size of 256 x 256, a slice thickness of 10 mm, a slice gap of 0 mm, a flip angle of 35°, a TE of 1.67 msec, and a TR of 3.34 msec. Temporal resolution was 25–39 msec. End diastolic and end systolic images were identified on all slices, and endocardial contours were drawn using MASS post processing software (Medis, Leiden, The Netherlands) as described previously (21). Left ventricular ejection fraction (LVEF) was calculated for the assessment of systolic function. Furthermore, an ECG-gated gradient-echo sequence with velocity encoding was performed to measure blood flow across the mitral valve for the determination of left ventricular diastolic function (22, 23). Imaging parameters included the following: a TE of 5 msec, a TR of 14 msec, a flip angle of 20°, a slice thickness of 8 mm, a field-of-view of 350 mm, a matrix size of 256 x 256, a velocity encoding of 100 cm/sec, and a scan percentage of 80%. Flow velocities in early diastole (E) and at atrial contraction (A) were measured, and their peak flow ratio was calculated (E/A ratio) using the FLOW analytical software package (Medis) by defining a region of interest on the modulus images in all cardiac phases. Furthermore, the mean deceleration of the E wave and an estimation of left ventricular filling pressures (E/Ea) (24) were measured. All spectroscopic and functional analyses were performed by an experienced observer, blinded to the interventions. During MRI, blood pressure and heart rate were measured twice with an automatic device (Dinamap DPC100X; Freiburg, Germany) and averaged for analysis.
Assays
Glucose, total cholesterol (TC), and TG were measured on a fully automated P800 analyzer (Roche, Almere, The Netherlands) and insulin on a Immulite 2500 random access analyzer with a chemoluminescence immunoassay (Diagnostic Products Corp., Los Angeles, CA). Coefficients of variation were less than 2% for glucose, TC, and TG, and less than 5% for insulin. Plasma FFAs were measured using a commercial kit (FFA-C; Wako Chemicals, Neuss, Germany).
Statistical analysis
All statistical analyses were performed using SPSS, version 12.01 (SPSS, Inc., Chicago, IL). Statistical comparisons among the three physiological conditions were made by repeated measures ANOVA. Pearson r values were used for correlation analysis. Data are shown as mean ± SD. P < 0.05 (two tailed) was considered significant. Based on a previous report, we expected a decrease in diastolic early deceleration. Therefore, P < 0.05 (one tailed) was considered significant for this parameter (15).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subject characteristics at baseline, after partial starvation, and after complete starvation are shown in Table 1. Postabsorptive plasma glucose levels decreased from 5.0 ± 0.3 mmol/liter at baseline to 4.3 ± 0.4 mmol/liter after partial (P = 0.001) and to 3.9 ± 0.5 mmol/liter after complete starvation (P < 0.001). This was associated with a dose-dependent decrease in plasma insulin levels. Simultaneously, plasma concentrations of FFA increased dose dependently from 0.6 ± 0.4 mmol/liter to 1.2 ± 0.4 mmol/liter after partial (P < 0.001) and to 1.9 ± 0.7 mmol/liter after complete starvation (P < 0.001). Plasma TG levels decreased after partial starvation (from 1.3 ± 0.4 mmol/liter to 0.9 ± 0.3 mmol/liter (P = 0.009) but did not change upon complete starvation (P = 0.677). TC increased from 5.0 ± 1.3 mmol/liter at baseline to 5.1 ± 1.4 mmol/liter after partial (P = 0.810) and to 5.9 ± 1.8 mmol/liter after complete starvation (P = 0.005).
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Myocardial TG content increased dose dependently from 0.35 ± 0.14% at baseline to 0.59 ± 0.27% after partial (P = 0.006) and to 1.26 ± 0.49% after complete starvation (P < 0.001; Fig. 2). Hepatic TG content correlated with BMI at baseline (r = 0.67; P = 0.033). Hepatic TG content significantly decreased after partial starvation (from 2.24 ± 2.24% to 1.43 ± 1.33%; P = 0.031), whereas it did not change after complete starvation (2.54 ± 2.53%; P = 0.378; Fig. 3).
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)Systolic and diastolic blood pressure, heart rate, and myocardial LVEF did not change significantly during/after partial and complete starvation, compared with baseline. Furthermore, estimated left ventricular filling pressures were unchanged after partial (8.8 ± 3.8; P = 0.742) and complete starvation (8.2 ± 2.5; P = 0.299) compared with baseline (9.3 ± 2.6). Diastolic E/A ratio decreased dose dependently from 2.2 ± 0.4 at baseline to 2.1 ± 0.4 after partial starvation (P = 0.687) and to 1.8 ± 0.4 after complete starvation (P = 0.005). E deceleration decreased dose dependently from 3.4 ± 0.7 ml/sec2 x 10–3 at baseline to 2.9 ± 0.5 x 10–3 ml/sec2 after partial (P = 0.036) and to 2.8 ± 0.9 after complete starvation (P = 0.032).
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Discussion |
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Different degrees of starvation were associated with a considerable increase in plasma FFA levels, in accordance with previous observations (25, 26). These increased FFA levels reflect increased lipolysis of TG content in adipose tissue. Apparently, during starvation myocardial FA uptake exceeds the requirements of myocardial FA oxidation, resulting in increased TG stores. Moreover, progressive caloric restriction has dose-dependent effects on myocardial TG accumulation and myocardial function. However, a causal relationship between myocardial TG content and myocardial function cannot be derived from the present data.
Our data are supported by animal experiments. In those studies excessive exposure of the myocardium to plasma FA is accompanied by increased storage of myocardial TGs, resulting in the production of FA intermediates, and ultimately deteriorations in myocardial function (8, 27, 28). Accordingly, it has been suggested that in obese subjects, subclinical diastolic dysfunction is due to changes in myocardial metabolism (29, 30, 31, 32). Kankaanpää et al. (12) reported that alterations in left ventricular function in moderate obese subjects are associated with increased myocardial TG content, compared with lean subjects. Moreover, Szczepaniak et al. (1) showed increased myocardial TG content in overweight and obese subjects, which was accompanied by increased left ventricular mass. In accordance with our study, Reingold et al. (14) documented that short-term fasting leads to myocardial TG accumulation, although they did not document effects on myocardial function. The current results, documenting dose-dependent effects of caloric restriction on levels of plasma FFA, myocardial TG content, and diastolic function, extend these findings and support the general concept that increased myocardial TG content is associated with decreased myocardial function (33). Alternatively, starvation profoundly alters endogenous metabolic regulation and other, yet undefined, metabolic effects than merely increased levels of plasma FFA and myocardial TG content, which may be involved in explaining the reduction in myocardial diastolic function. For example, caloric restriction might change calcium homeostasis in the myocardium (34), which affects myocardial diastolic function (35).
Transmitral flow velocities are load dependent and can be affected by changes in intravascular volume. However, estimated left ventricular filling pressures were unchanged upon progressive caloric restriction. Therefore, we believe that the observed change in transmitral flow patterns results from a change in the relaxation of the left ventricle. Caloric restriction enhances adipose tissue lipolysis, reflected in increased levels of plasma FFA, due to reduced insulin levels. Similar to our results in the heart, others found corresponding results of increased TG content of skeletal muscle after fasting (19, 25, 26). Starvation affects more parameters of lipid metabolism because plasma FFAs stimulate the hepatic production of very low density lipoprotein (VLDL), which is an important supplier for TG to the heart (36, 37). Plasma FFA levels also increase during starvation and most likely will contribute to increased myocardial TG levels. However, the relative contribution of albumin-bound FAs vs. FAs derived from VLDL-TG to myocardial TG stores during caloric restriction cannot be derived from the present data.
We found a correlation between hepatic fat content and BMI, in accordance with previous observations (2, 38). However, despite the increase in the flux of plasma FFA to the liver, considering the increased plasma FFA levels, hepatic TG content was decreased after partial starvation but was unchanged after complete starvation. In line with our results, Westerbacka et al. (9) previously documented that a low fat diet in moderately obese women decreases hepatic TG content. Because hepatic TG content is tightly regulated by the balance of hepatic FA uptake, hepatic FA oxidation, and output of VLDL-TG particles, it is possible that this hepatic balance between FA uptake and TG output is differentially affected by partial and complete starvation. Nonetheless, our data indicate that progressive caloric restriction differentially affects tissue-specific stores of TG in heart and liver, and prove that myocardial TG content and myocardial function vary depending on nutritional conditions, at least with respect to progressive degrees of starvation. Additional studies are required to elucidate to which extent these results can be extrapolated to clinically relevant conditions like type 2 diabetes mellitus and obesity.
In conclusion, progressive caloric restriction induces a dose-dependent increase in myocardial TG content and a dose-dependent decrease in diastolic function in lean healthy men. Hepatic TG content showed a differential response to progressive caloric restriction, indicating that redistribution of endogenous TG stores is tissue specific, at least in lean healthy men.
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Footnotes |
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First Published Online November 20, 2007
1 S.H. and R.W.v.d.M. contributed equally. 
Abbreviations: A, Atrial contraction; BMI, body mass index; E, early diastole; ECG, electrocardiogram; FA, fatty acid; FFA, free FA; LVEF, left ventricular ejection fraction; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; ppm, parts per million; TC, total cholesterol; TE, echo time; TG, triglyceride; TR, repetition time; VLDL, very low density lipoprotein.
Received September 7, 2007.
Accepted November 13, 2007.
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