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1 From the Nutrition and Toxicology Research Institute Maastricht, University of Maastricht, Maastricht, Netherlands (NB, GBJH, and WHMS), and the Unite de Recherches sur les Obesites, Institut National de la Sante et de la Recherche Medicale (INSERM), Institut Louis Bugnard, Centre Hospitalier Universitaire de Toulouse, Universite Paul Sabatier, Toulouse, France (NV, AS, and DL)
2 Supported by a grant from the Dutch Dairy Association, Zoetermeer, Netherlands. 3 Address reprint requests to WHM Saris, Nutrition and Toxicology Research Institute Maastricht, University of Maastricht, PO Box 616, Maastricht, Netherlands. E-mail: w.saris{at}hb.unimaas.nl.
ABSTRACT
Background: Evidence from molecular and animal research and epidemiologic investigations indicates that calcium intake may be inversely related to body weight, possibly through alterations in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism.
Objective: We tested whether energy and substrate metabolism and adipose tissue enzyme messenger RNA (mRNA) expression can be altered by dietary calcium intake in healthy, nonobese, human volunteers consuming an isocaloric diet.
Design: Twelve healthy men [age: 28 ± 2 y; body mass index (BMI; in kg/m2): 25.2 ± 06] received 3 isocaloric diets [high calcium (1259 ± 9 mg/d), high dairy (high/high); high calcium (1259 ± 9 mg/d), low dairy (high/low); and low calcium (349 ± 8 mg/d), low dairy (low/low)] in a randomized crossover design. At the end of the 7-d dietary periods, 24-h energy expenditure and substrate metabolism were measured, and fat biopsy specimens were obtained to determine mRNA expression in genes involved in the lipolytic and lipogenic pathways.
Results: The 24-h energy expenditure was 11.8 ± 0.3, 11.6 ± 0.3, and 11.7 ± 0.3 MJ/24 h in the high/high, high/low, and low/low conditions, respectively. Fat oxidation in these conditions was 108 ± 7, 105 ± 9, and 100 ± 6 g/24 h. These differences were not statistically significant. mRNA concentrations of UCP2, FAS, GPDH2, HSL, and PPARG did not differ significantly. Serum 1,25(OH)2D3 concentrations changed from 175 ± 16 to 138 ± 15, 181 ± 23 to 159 ± 19, and 164 ± 13 to 198 ± 19 pmol/L in the high/high, high/low, and low/low conditions, respectively, and was significantly different between the high/high and low/low conditions (P < 0.05).
Conclusion: Altering the dietary calcium content for 7 d does not influence substrate metabolism, energy metabolism, or gene expression in proteins related to fat metabolism, despite significant changes in 1,25(OH)2D3 concentrations.
Key Words: Dietary calcium • energy expenditure • adipose tissue message RNA expression • body weight regulation • substrate metabolism • 1;25-dihydroxyvitamin D3 • 1;25(OH)2D3
INTRODUCTION
The prevalence of obesity has increased markedly during the past 2 decades, making obesity an important risk factor for the development of type 2 diabetes, various types of cancer, and cardiovascular complications. In recent years, an inverse relation between dietary calcium and body mass index (BMI; in kg/m2) was repeatedly observed (1-8). Some intervention studies also showed that dietary calcium may have weight-lowering effects, and, in addition, an even stronger effect was observed with dairy sources of calcium (9-12).
However, the mechanism is still unclear. A hypothesis to explain this relation was provided by Zemel et al (13). With an increased concentration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in cultures of human adipocytes and in transgenic mice, acute increases of the intracellular calcium concentration were observed (14, 15). This increased concentration of Ca2+ in the adipocytes lowered lipolysis and stimulated lipogenesis. Lowering the intake of dietary calcium leads to an increase in the serum concentration of 1,25(OH)2D3 within a few days (16). In this way, a low intake of dietary calcium may lead to a higher body weight by changing the balance of lipolysis and lipogenesis in adipose tissue through an increase in serum 1,25(OH)2D3 (1).
Serum 1,25(OH)2D3 is an important regulator of a large number of genes (17). Of the genes regulated by 1,25(OH)2D3 are genes that are either related to fat breakdown and storage [HSL (hormone sensitive lipase), GPDH2 (glycerol phosphate dehydrogenase), and FAS (fatty acid synthase)] or adipocyte differentiation [PPARG (peroxisome-proliferator activated receptor )] (18-22). Furthermore, 1,25(OH)2D3 decreases the messenger RNA (mRNA) expression of UCP2 (23), which is correlated with basal metabolic rate (18). If changes in serum 1,25(OH)2D3 are paralleled by changes in the expression of these genes, this may provide more insight on how calcium intake affects body weight through changes in lipogenesis, lipolysis, and energy expenditure (EE).
The first well-controlled experimental trial to look at the effects of calcium intake on energy and fat metabolism in weight-stable subjects was published by Melanson et al (9), who observed a positive correlation between 24-h and sleeping fat oxidation and calcium intake, but they did not correct for protein intake. Furthermore, in most of the epidemiologic and dietary intervention studies performed in humans mentioned here, the investigators did not correct for protein intake (3, 4, 9, 24-26), which has been shown to have weight-lowering effects (27-31).
These data prompted us to investigate the effect of 3 isocaloric diets with a fixed macronutrient composition and different concentrations of calcium on 24-h EE and 24-h fat oxidation in vivo in humans. We hypothesized that a higher calcium intake would increase these indicators and that an additive effect may be observed of low-fat dairy. To study potential effects on a molecular level, mRNA expression of different genes related to fat metabolism (ie, UCP2, FAS, GPDH2, HSL, and PPARG) in adipose tissue was investigated as well.
SUBJECTS AND METHODS
Experimental subjects
Twelve untrained (did not perform any regular physical activity 3 h/wk), healthy men were recruited for participation in this study through advertisements in local newspapers. This study was approved by the local ethical committee of the University of Maastricht and the Academic Hospital of Maastricht. After the subjects had received a written and oral explanation of the procedures to be followed in this project, their informed consent was obtained. Furthermore, the habitual energy, micronutrient, and macronutrient intakes of the subjects was assessed by using 3-d food intake diaries, before the start of the experiment. Subjects were provided with a digital kitchen balance to allow them to make a more accurate estimation of their habitual food intake, and the macronutrient and micronutrient compositions of these habitual diets was calculated from these food records with the use of the Dutch food-composition table (32). Habitual physical activity of the subjects was also determined to assure that none of the subjects spent >3 h/wk in heavy physical exercise. Finally, before the start of the first dietary intervention period, body composition and maximal aerobic capacity were determined.
Maximal exercise test
At least 1 wk before the first stay in the respiration chamber, maximum oxygen uptake (
RESULTS
Subject characteristics
The average age of the subjects was 28 ± 8 y. The average BMI was 25.2 ± 0.6 (Table 1).
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TABLE 1. Physical characteristics of the male subjects1
Habitual dietary intake
The subjects had an average energy consumption of 12.6 ± 1.0 MJ/d, which was consumed as 14.8 ± 0.9% protein, 45.9 ± 2.8% from carbohydrate, and 35.7 ± 2.2% from fat. The habitual daily calcium intake of these subjects was 1027 ± 82 mg, and the dietary fiber intake of the subjects was 21.2 ± 1.9 g/d (Table 2).
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TABLE 2. Energy intake, macronutrient composition, and content of calcium and dietary fiber in the habitual and experimental diets during the 7-d intervention1
Experimental diets
All subjects completed the 3 different dietary regimens without any adverse effects of the diets, except for 1 subject who reported after his first 5.5-d dietary run-in (high/low diet) that he had not consumed all of the food items provided, because he felt he was overeating. Because the macronutrient composition and calcium content of the diet he had consumed were in line with the experimental diets, the data from this experiment were included in the final analysis. His EI during the 2 following dietary intervention periods was adjusted according to his EE during the first respiration chamber experiment. The average EI during the 7 days of the dietary interventions was 12.3 ± 0.3, 12.0 ± 0.6, and 12.3 ± 0.3 MJ/d for the high/high, high/low, and low/low diets, respectively. The experimental diets only differed in calcium concentration (P < 0.0001, low/low compared with high/high and high/low). All of the experimental diets provided significantly more dietary fiber and carbohydrates (P < 0.0001) and significantly less fat (P < 0.01) than the subjects' habitual diets (Table 2). The high-calcium diets provided significantly more calcium than the habitual diet (P < 0.01), and the low-calcium diet provided significantly less calcium (P < 0.0001). The content of energy and protein was not significantly different among any of the diets (Table 2). The dietary tool that was used in the present investigation does not allow for an accurate determination of vitamin D intake, but the average intake was 16 ± 8 IU/d during the 3 experimental diets, whereas the habitual intake was 28 ± 8 IU/d. This difference between the experimental and habitual diets was not statistically significant. To test whether the order in which the diets were given had an effect on the measurements of energy and substrate metabolism, all analyses were also performed with order of diet as a factor. No effects of order of the dietary intervention were observed.
Metabolic measurements
In all experiments, energy balance was achieved [(EI – EE) < 0.5 MJ]. On average, the absolute deviation from energy balance was 0.2 ± 0.0 MJ/d (high/high), 0.3 ± 0.1 MJ/d (high/low), and 0.2 ± 0.1 MJ/d (low/low) during the 24-h measurements in the respiration chamber. The total 24-h EE was 11.8 ± 0.3, 11.6 ± 0.3, and 11.7 ± 0.3 MJ/24 h in the high/high, high/low, and low/low conditions, respectively. These differences were not statistically different. The EE during the other periods (EESMR, EEsleep, EErest, and EEexercise) and the PAI were not statistically different either (Table 3).
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TABLE 3. Different components of energy expenditure (EE)1
The average 24-h RQ was 0.85 ± 0.01, 0.85 ± 0.01, and 0.86 ± 0.01, and the average 24-h fat oxidation in the 3 conditions was 108 ± 7, 105 ± 9, and 100 ± 6 g/d in the high/high, high/low, and low/low conditions, respectively (Figure 2). These differences were not statistically different. The average RQ during SMR in the 3 different dietary conditions was not statistically different either (0.82 ± 0.01, 0.81 ± 0.02, and 0.82 ± 0.01, respectively). No significant differences in RQ were observed during the other periods (sleep, rest, or exercise) (Table 3).
FIGURE 2.. Mean (±SEM) 24-h fat oxidation calculated from the indirect calorimetry values. The data were corrected for protein oxidation, which was calculated from 24-h nitrogen excretion in urine. n = 12. Differences between the 3 diets were tested with a 2-factor ANOVA; a Scheffé test was used for post hoc analysis. None of the differences were statistically significant.
Because of the relatively high intake of dietary protein, a positive 24-h protein balance (47.5 ± 4.6, 40.3 ± 4.4, and 38.7 ± 5.0 g in the high/high, high/low, and low/low conditions, respectively) was observed in all 3 experimental conditions. The 24-h fat balance was –16.1 ± 6.0, –10.3 ± 7.6, and –8.4 ± 5.2 g in the high/high, high/low, and low/low conditions, respectively, and 24-h carbohydrate balance was 11.6 ± 15.2, 29.6 ± 19.6, and 8.6 ± 14.1 g in the high/high, high/low, and low/low conditions, respectively. No significant differences in substrate balance were observed among the 3 diets (Table 3).
No differences among the 3 diets were observed in the concentrations of plasma calcium, FFA, glucose, insulin, or glycerol. All values were within the normal range. The serum 1,25(OH)2D3 concentrations at t = 0 were not significantly different among the 3 interventions. The serum concentration of 1,25(OH)2D3 decreased from 175 ± 16 to 138 ± 15 pmol/L during the high/high condition, decreased from 181 ± 23 to 159 ± 19 pmol/L in the high/low condition, and increased from 164 ± 13 to 198 ± 19 pmol/L in the low/low condition. A significant group x time interaction was observed (P < 0.05). The serum concentration of 1,25(OH)2D3 was significantly different between the high/high and low/low conditions at t = 7 (P < 0.05). Furthermore, the change in serum 1,25(OH)2D3 concentration was –39 ± 13, –22 ± 27, and 33 ± 15 pmol/L for the high/high, high/low, and low/low conditions, respectively. The difference in change was significantly different between the high/high and low/low conditions (P < 0.05). No differences were observed among the high/low and the other 2 conditions (Figure 3).
FIGURE 3.. Mean (±SEM) serum concentrations of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] during the 7-d intervention period. n = 12. Differences over time between the 3 diets were tested for significance with a repeated-measures ANOVA. A Scheffé post hoc was used to determine differences between the 3 dietary conditions on different time points. A significant group x time interaction was observed (P < 0.05). #Significantly different from the low-calcium, low-dairy diet, P < 0.05.
The mRNA concentrations in adipose tissue were determined for 10 subjects only, because for 2 subjects, no complete sets of biopsies could be obtained because of their relatively low percentage of body fat. No differences were observed in the mRNA concentration of UCP2, FAS, GPDH2, HSL, or PPARG among the 3 diets (Figure 4).
FIGURE 4.. Mean (±SEM) changes in messenger RNA (mRNA) expression of adipose tissue UCP2 (uncoupling protein 2), FAS (fatty acid synthase), HSL (hormone sensitive lipase), PPARG (peroxisome-proliferator activated receptor ), and GPDH2 (glycerol phosphate dehydrogenase) genes relative to the prediet value, which was set at 1.00. n = 10. Differences between the 3 diets were tested with a 2-factor ANOVA; a Scheffé test was used for post hoc analysis. None of the differences were statistically significant.
DISCUSSION
In this carefully controlled dietary intervention study, no differences in energy or substrate metabolism were observed among 3 diets with varying contents of calcium and dairy protein, despite significant changes in the concentration of serum 1,25(OH)2D3. We did not see any differences in gene expression of 5 genes involved in the lipogenic and lipolytic pathways.
Because some epidemiologic investigations do show the long-term effects of an altered Ca2+ intake, it may be argued that the duration of the intervention period was not long enough to induce the necessary adaptations of the 1,25(OH)2D3 metabolism. However, we observed significant differences among the 3 diets for this hormone within 7 d, which is in line with previous research (43). This hormone was shown to have immediate effects on lipolysis (22, 23, 44, 45), so the significant changes in 1,25(OH)2D3 that were observed in this study should have induced the metabolic changes that we hypothesized. Nonetheless, other adaptations to a diet of high calcium and dairy may also occur that take longer than 7 d. The short duration of this intervention trial did not allow us to measure the effects of such longer-term adaptations.
It may also be argued that we studied the effects of calcium depletion rather than the effects of calcium supplementation because of the already high and possibly optimal calcium intake of our subjects. But this is contradicted by our data, because both the high/high and low/low conditions induced a significant change from the baseline values of the serum 1,25(OH)2D3 concentration in these subjects with a habitual intake of high calcium. Along with the results from 2 other recent investigations in which no relation between 1,25(OH)2D3 and body weight was observed (46, 47), our results indicate that the 1,25(OH)2D3 metabolism may not play a significant role in the relation of calcium with body weight, which is not in line with previous studies that investigated the relation among calcium intake, 1,25(OH)2D3, and substrate and energy metabolism (4, 10, 48-50).
However, part of this research that shows significant effects of altering calcium intake on body weight was performed in rodents, and the results may not carry to the human situation. The high-calcium diet induced a 1.2-fold increase in calcium intake, whereas increases of up to 2-fold were observed in the animal studies (11, 12). Furthermore, the results of the various experiments performed in cell cultures are not directly applicable to whole-body human metabolism because the intracellular calcium concentrations in these experiments (14, 22, 23, 45, 51, 52) were manipulated directly by calcium channel and vitamin D receptor agonists and not through an altered dietary intake of calcium. Therefore, they do not reflect the normal physiologic situation. Thus, it seems that the results from these cell culture and animal studies are not directly applicable to the human in vivo situation.
An explanation for the discrepancy with the observational research and our results may be that because of the larger statistical power in epidemiologic investigations, the differences in body weight and body composition caused by subtle differences in energy and substrate metabolism that are expected from changes in calcium intake are more likely to be detected (3, 4, 25, 26, 53-58). Recently, Jacobsen et al (59) did not observe any differences in substrate and energy metabolism either, using a comparable study design. From a post hoc power analysis, it was calculated that to reach significant differences in EE with the variance and differences observed between groups in the present investigation, we should include at least 43 subjects. This shows that 7-d changes in dietary calcium content may not have a robust effect on either EE or fat oxidation, which is in line with recent work from Shapses et al (60). However, if the observed differences would sustain for a prolonged period, they may contribute to a significant effect on body weight. For example, if the effects on fat oxidation observed in this project would last for 1 y, they may account theoretically for a difference in body weight of 3.0 kg (assuming an additional fat oxidation of 8 g/d and with the assumption that an additional 38000 kJ needs to be expended to lose 1 kg body fat). An impairment of fat oxidation is suggested as an important prerequisite to become obese (61). This may be the link with the observed correlations between calcium intake and obesity in the epidemiologic studies.
Nonetheless, data from 2 interventional studies indicate a possible role for calcium in body weight regulation. In one human intervention trial, an additive effect of increasing dietary calcium intake on weight loss was observed (50). However, the endpoint of that experiment was body weight and not substrate metabolism. Furthermore, the subjects were obese, and it was previously shown that obese subjects show a larger increase in serum 1,25(OH)2D3 in response to a low-calcium diet (16). In another human intervention study, Melanson et al (9) observed a relation between acute Ca2+ intake and fat oxidation in normal weight subjects who consumed an energy-balanced diet. The correlation between acute Ca2+ intake and fat oxidation was rather weak with r2 ranging from 0.32 to 0.38 (P = 0.03–0.07), and food intake was not as well controlled as in the present randomized clinical trial. Furthermore, in the interventional study by Melanson et al (9) and also some other previously published observational studies (3, 4, 24-26), an increased protein intake that is related to an increase in calcium intake may have confounded the outcome measures. Although those 2 intervention studies may point at an effect of calcium intake on energy and substrate metabolism, differences in methods and subjects make it difficult to compare them with the present study.
An alternative mechanism that may be responsible for the observed epidemiologic relation between dairy and calcium intake and body weight is a reduced fat absorption from the gut induced by an increased calcium intake. In a recent investigation Jacobsen et al (59) observed that increasing the dietary calcium intake from 675 to 1850 mg/d induced no differences in energy or substrate metabolism but a rather large increase of the fecal fat excretion of 8.2 g/d (from 6.0 to 14.2 g/d). Papakonstantinou et al (62) also showed in rats that increasing the dietary calcium intake decreased the energy that was available from the diet from 94% to 90%. Although the magnitude of these results is not in line with previous research showing more modest effects of dietary calcium on fat absorption (63, 64), it is clear that fat absorption may be an important mechanism by which calcium intake can influence body composition.
Summarizing the results from this investigation, we have shown that a 7-d dietary intervention with either a high- or a low-calcium diet induces significant changes in the concentration of serum 1,25(OH)2D3. However, these changes were not accompanied by differences in energy and substrate metabolism or alterations in the mRNA concentration of 5 genes involved in fat and substrate metabolism, which is in contrast with previous data. Possible explanations for this discrepancy are first that we have successfully corrected for the confounding effects of protein intake. Second, the power of the present investigation may be too small to detect differences in energy and substrate metabolism induced by different concentrations of dietary calcium.
Finally, the results from other investigations (59, 63-65) also seem to indicate that the relation between intake of dairy and calcium and body weight that was observed in epidemiologic reports may not be mediated by 1,25(OH)2D3 but by other mechanisms, for example, the decreased fat absorption that is related to an increased calcium intake. This mechanism indeed warrants further investigation.
ACKNOWLEDGMENTS
NB was the principal investigator, GBJH provided practical assistance during the experiment, AS performed the mRNA analyses, NV assisted in the mRNA analyses and in writing the manuscript, DL supervised the mRNA analyses and assisted in writing the manuscript,. WHMS supervised the study and the writing of the manuscript. None of the authors had a financial conflict related to this work.
REFERENCES