Recent data from this laboratory demonstrate that increasing adipocyte intracellular Ca 2+ results in a coordinated stimulation of lipogenesis and inhibition of lipolysis. We have also noted that increasing dietary calcium of obese patients for 1 year resulted in a 4.9 kg loss of body fat ( P <0.01). Accordingly, we tested the possibility that calcitro‐phic hormones may act on adipocytes to increase Ca 2+ and lipid metabolism by measuring the effects of 1,25‐(OH) 2 ‐D in primary cultures of human adipocytes, and found significant, sustained increases in intracellular Ca 2+ and a corresponding marked inhibition of lipolysis (EC 50 ~50 pM; P<0.001), suggesting that dietary calcium could reduce adipocyte mass by suppressing 1,25‐(OH) 2 ‐D. To test this hypothesis, we placed transgenic mice expressing the agouti gene specifically in adipocytes on a low (0.4%) Ca/high fat/high sucrose diet either unsupplemented or with 25 or 50% of the protein replaced by non‐fat dry milk or supplemented to 1.2% Ca with CaCO 3 for 6 wk. Weight gain and fat pad mass were reduced by 26–39% by the three high calcium diets ( P <0.001). The high calcium diets exerted a corresponding 51% inhibition of adipocyte fatty acid synthase expression and activity ( P < 0.002) and stimulation of lipolysis by 3.4‐ to 5.2‐fold ( P < 0.015). This concept of calcium modulation of adiposity was further evaluated epi‐demiologically in the NHANES III data set. After controlling for energy intake, relative risk of being in the highest quartile of body fat was set to 1.00 for the lowest quartile of Ca intake and was reduced to 0.75, 0.40, and 0.16 for the second, third, and fourth quartiles, respectively, of calcium intake for women ( n =380;P≪ 0.0009); a similar inverse relationship was also noted in men ( n =7114; P< 0.0006). Thus, increasing dietary calcium suppresses adipocyte in‐tracellular Ca 2+ and thereby modulates energy metabolism and attenuates obesity risk.—Zemel, M. B., Shi, H., Greer, B., DiRienzo, D., Zemel, P. C. Regulation of adiposity by dietary calcium. FASEB J . 14, 1132–1138 (2000)

Intracellular Ca2+ plays a key role in the metabolic disorders associated with obesity and insulin resistance (1–3). Recombinant agouti protein, an obesity gene product, stimulates Ca2+ influx in a variety of cells (4, 5). Agouti also stimulates the expression and activity of fatty acid synthase, a key enzyme in de novo lipogenesis, and inhibits basal and agonist‐stimulated lipolysis in human and murine adipocytes via a Ca2+‐dependent mechanism (6, 7). These effects can be mimicked in the absence of agouti by either receptor or voltage‐mediated Ca2+ channel activation and inhibited by Ca2+ channel antagonism (6–8); agouti‐induced obesity in trans‐genic mice was markedly attenuated by 4 wk of Ca2+ channel antagonism (9). Thus, increasing adipocyte intracellular Ca2+ appears to promote triglyceride storage in human adipocytes by exerting a coordinated control of lipogenesis and lipolysis, serving to simultaneously stimulate the former and inhibit the latter. During the course of a previous, unrelated clinical trial investigating the antihypertensive effect of calcium in obese African‐Americans, we noted that increasing daily calcium intake from –400 to 1000 mg/day for 1 year resulted in a 4.9 kg reduction in body fat (Fig. 1). Although these data were inexplicable at the time, our recent data demonstrating regulation of adipocyte energy storage by intracellu‐lar Ca2+ lead to the proposal that increases in circulating calcitrophic hormones [1,25‐(OH) 2 ‐D and/or parathyroid hormone] secondary to low calcium diets stimulate adipocyte Ca2+ influx and thereby increase lipid storage. If this is correct, then increasing dietary calcium should suppress calcitro‐phic hormones and thereby reduce adipocyte intra‐cellular Ca2+ and lipid storage. The present study was conducted to address this concept. Figure 1 Open in figure viewer Effects of increasing dietary calcium for 12 months on body fat in obese African‐American males. Supplemental calcium was provided in the form of two cups of yogurt/day. Calcium intake was estimated from random prospective dietary records collected 1 day each week for each subject throughout the study. Baseline dietary calcium was estimated, duringa4wk lead‐in period, to be 447 ± 126 mg/day and increased to 1029 ± 74 mg/day during the 12 months of supplementation. Body fat was assessed using bioelectrical impedance (14). Data are mean ± standard deviation; n=11; ⋆P<0.01.

MATERIALS AND METHODS Isolation and culture of human adipocytes Human subcutaneous adipose tissue was obtained from patients with no known history of metabolic disorders undergoing abdominal plastic surgery. Adipocytes were isolated by washing, mincing, collagenase digestion, and filtration as described previously (12, 17) and cultured in Dulbecco's modified Eagle's medium supplemented with 1% fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/ml), and gentamicin (50 μg/ml). Approval for this procedure was obtained from the Institutional Review Board for Human Subjects of the University of Tennessee. Cells were cultured in suspension and maintained in a thin layer at the top of the culture media, which was changed every day. Cells were studied –72 h after isolation and were serum‐starved prior to study. Animals and diets To evaluate the role of dietary calcium in regulating adiposity in vivo, transgenic mice expressing agouti specifically in adipocytes under the control of the aP2 promoter were studied. We have previously reported a characterization of these animals. Briefly, they exhibit a normal pattern of leptin expression and activity similar to that found in humans and exhibit a human pattern (adipocyte‐specific) of agouti expression (10). We have found these mice to be useful models for diet‐induced obesity in that they are not obese on a standard AIN‐93G diet, but become obese in response to hyperinsulin‐emia induced by either insulin administration (10) or high sucrose diets (11). Male aP2‐agouti transgenic animals from our colony were placed at 6 wk of age on a modified AIN 93‐G diet with suboptimal calcium (0.4%), sucrose as the sole carbohydrate source, and fat increased to 25% of energy with lard. They were randomized to four groups, as follows. The basal group continued this diet with no modifications; a high calcium group received the basal diet supplemented with CaCO 3 to increase dietary calcium by threefold to 1.2%; a medium dairy diet, in which 25% of the protein was replaced by non‐fat dry milk and dietary calcium was increased to 1.2%; and a high dairy group in which 50% of the protein was replaced by non‐fat dry milk, increasing calcium to 2.4%. Food intake and spillage was measured daily, and animals were weighed weekly. At the conclusion of the 6 wk feeding period, animals were killed by exsanguination under isoflu‐rane anesthesia, and blood was collected via cardiac puncture for glucose and insulin measurements. Fat pads (epididymal, perirenal, abdominal, and subscapular) were dissected, immediately weighed, frozen in liquid nitrogen, and stored at –80°C. Fatty acid synthase activity and mRNA levels were measured in abdominal fat as described below. Core temperature Core temperature was used as an indirect metabolic index to determine whether any reduction in efficiency of conversion of food energy to body weight was accompanied by increased thermogenesis (9). Temperature was measured via a thermocouple (Columbus Instruments, Columbus, Ohio). The probe was inserted a constant distance (1.8 cm) into the rectum of each animal. After stabilization (10 s), the temperature was recorded every 5 s for 30 s (9). All temperature measurements were made between 8:00 and 9:00 a.m. Intracellular calcium (human adipocytes) Intracellular Ca2+ was determined fluorometrically as described previously (17). Cells were washed with HEPES‐buffered salt solution loaded with Fura‐2‐acetoxymethyl ester (10 μΜ) for 45 min at 37°C in the dark with continuous shaking. Cells were then rinsed three times, resuspended, and intracellular Ca2+ was measured using dual excitation (340 and 380 nm)/single emission (510 nm) fluorometry. After the establishment of a stable baseline, the response to 1,25‐(OH) 2 ‐D or parathyroid hormone (10 pM‐100 nM) or their respective vehicles was determined. Digitonin (25 μΜ) and Tris/EGTA (100 mM, pH 8.7) were used to for calibration to calculate the final intracellular Ca2+ (18). Lipolysis Adipocytes were incubated for4hinthepresence or absence of forskolin (1 μΜ), and glycerol release into the culture medium was measured (18) to assess lipolysis. Glycerol release data was normalized for cellular protein. Fatty acid synthase activity and mRNA levels Immediately after death, adipose tissue was isolated and fatty acid synthase activity was measured in cytosolic extracts by measuring the oxidation rate of NADPH, as described previously (6, 9, 17). Enzyme activity was protein corrected using Coomassie blue dye. Total RNA was extracted by cesium chloride density gradient, electrophoresed, subjected to Northern blot analysis, and hybridized with a radiolabeled rat cDNA probe for fatty acid synthase using standard methods (6, 12, 17). Autoradio‐graphs were quantitated densitometrically, and all blots were stripped and reprobed with β‐actin as a loading control. Statistical analysis (in vitro and animal data) All data are expressed as mean ± sd. Data were evaluated for statistical significance by one‐way analysis of variance (ANOVA) or t test, depending on the number of comparisons made. All data sets with multiple comparisons were analyzed via ANOVA, followed by separation of significantly different group means via test the least significant difference using SPSS‐PC (v. 8.0). NHANES III analysis To determine whether the animal observations are relevant in defining a role for dietary calcium in modulating body composition at the population level, an analysis of the National Health and Nutrition Examination Survey (NHANES III) data set was conducted. This large cross‐sectional survey conducted between 1988 and 1994 followed a complex, four‐stage probability sampling scheme (13) designed to represent the entire U.S. civilian noninstitutionalized population over the age of 2 months. Only adults completing all three phases of the study (interview, physical examination, and laboratory examination) were included in this data analysis; respondents were excluded from this analysis if they could not provide complete, usable body composition/an‐thropometric data (e.g., amputees and individuals wearing casts), used insulin, or were pregnant, recently pregnant, or currently breast‐feeding. Body composition was assessed using the anthropometric and bioelectrical impedance data collected during the physical examination, with percent body fat calculated using the regression equations derived by Segal (14). Odds ratios for percent body fat and corresponding 95% confidence intervals were estimated by multiple logistic regression analysis with a robust variance estimation method using SUDAAN (15). Point estimates for all parameters were weighted to reflect the population distribution of each; variances were calculated using SUDAAN (15) to take the complex sampling design into account, as failure to account for the weighting and design effects of a complex sample design will result in distortion of estimates and underestimation of variance. Analyses were conducted separately for men and women, and all odds ratios were adjusted for age by including age in the model as a continuous variable. Other covariates included in the model were caloric intake, race/ ethnicity, and activity level. Characteristics of the study sample are shown in Tables 1 and 2. Table 1. Characteristics of NHANES III Study sample Women (n = 380) Men (n = 7114) Age (years) 28.7 ± 0.4 43.5 ± 0.44 Body mass index (kg/m2) 25.7 ± 0.4 26.6 ± 0.11 Body fat (%) 32.7 ± 0.6 25 ± 0.2 Calcium intake (mg/day) 720 ± 52 965 ± 15 Dairy product consumption (monthly frequency) 54.7 ± 3 51.4 ± 1 Energy intake (kcal/day) 1896 ± 68 2656 ± 28 Dietary fat (g/day) 74 ± 4 102 ± 2 Table 2. Quartiles of body fat for women and men in the NHANES III Study % Body fat Quartile Women Men 1 22.82 ± 0.45 15.47 ± 0.14 2 30.54 ± 0.17 22.83 ± 0.06 3 34.85 ± 0.18 27.55 ± 0.06 4 42.49 ± 0.44 34.08 ± 0.11

RESULTS Figure 2 demonstrates that both 1,25‐(OH) 2 ‐D and parathyroid hormone (PTH) stimulate significant, sustained increases in intracellular Ca2+ in primary cultures of human adipocytes (P<0.001; EC 50 ~ 50 pM for 1,25‐(OH) 2 ‐D and ‐10 nM for PTH). 1,25‐(OH) 2 ‐D treatment also resulted in marked (83%) inhibition of forskolin‐stimulated lipolysis (P<0.001) in human adipocytes (Fig. 3). PTH treatment exerted little effect on lipolysis (data not shown) despite its stimulation of an intracellular Ca2+ response, most likely as a result of an accompanying activation of adenylate cyclase. Figure 2 Open in figure viewer Effects of 1,25‐(OH) 2 ‐D (left panel) and parathyroid hormone (right panel) on intracellular calcium in human adipocytes. Figure 3 Open in figure viewer Effects of 1,25‐(OH) 2 ‐D on forskolin‐stimulated lipolysis in human adipocytes. Forskolin treatment resulted in a ~ twofold increase in glycerol release; data shown are normalized to 100% for the forskolin‐treated group. (n=6/group; P<0.001). Treatment of aP2‐transgenic‐agouti mice with the high fat/high sucrose basal diet resulted in a weight gain of 24%, which was reduced by 26 and 29% by the high calcium and medium dairy diets, respectively (P<0.04), and further reduced by 39% by the high dairy diet (P<0.04; Fig. 4). These differences occurred despite the lack of any difference in food intake. Measurement of core temperature, an indirect metabolic index, reflected these observations, with –0.5°C increases in core temperature in response to all three high calcium diets (P<0.03; Fig. 5). This increase, coupled with the lack of difference in food intake, is indicative of a shift in efficiency of energy metabolism from energy storage to thermo‐genesis. Figure 4 Open in figure viewer Effects of calcium and dairy products on 6 wk weight gain in transgenic mice expressing agouti in adipose tissue under the control of the aP2 promoter. Figure 5 Open in figure viewer Effects of calcium and dairy products on core temperature in transgenic mice expressing agouti in adipose tissue under the control of the aP2 promoter. This shift in energy metabolism was evident in studies of fatty acid synthase, a key enzyme in de novo lipogenesis that is highly sensitive to regulation by nutrients and hormones (12). The basal diet caused a 2.6‐fold increase in fatty acid synthase activity, and this effect was markedly attenuated by all three high calcium diets (P< 0.002; Fig. 6A). The diets caused corresponding decreases in adipocyte fatty acid syn‐thase mRNA, with a 27% reduction on the high calcium diet and a 51% reduction on the medium and high dairy diets (P<0.01; Fig. 6B). Adipocyte lipolysis responded to dietary manipulations in an inverse fashion to the fatty acid synthase responses. The basal diet caused a marked (67%) suppression of lipolysis (P<0.0001); however, lipolysis was stimulated 3.4‐ to 5.2‐fold by the high calcium diets (P<0.015; Fig. 6C), with greater effects from the high dairy diets than from the high calcium diet. Assessment of fat pad mass after 6 wk of dietary treatment provides further support for these findings. Table 3 demonstrates that all three high calcium diets caused a 36% reduction in mass of the epididymal, abdominal, perirenal, and subscapular adipose tissue compartments (P<0.001). Epidymal and subscapular fat pad mass was reduced by –50% by all three diets, whereas the abdominal fat pads exhibited greater decreases on the medium and high dairy diets than on the high calcium diet (P<0.001; Table 3). Table 3. Effects of calcium and dairy products on fat pad mass in transgenic mice expressing Agouti in adipose tissue under control of the aP2 prromoter Basal High calcium Medium dairy High dairy Abdominal (g) 2.239 ± 0.109 1.807 ± 0.082⋆ 1.661 ± 0.127⋆⋆ 1.680 ± 0.113⋆⋆ Perirenal (g) 1.675 ± 0.124 1.271 ± 0.098⋆ 1.172 ± 0.123⋆ 1.052 ± 0.094⋆ Epididymal (g) 0.198 ± 0.036 0.110 ± 0.017⋆ 0.110 ± 0.014⋆ 0.097 ± 0.015⋆ Subscapular (g) 1.592 ± 0.318 0.680 ± 0.069⋆ 0.663 ± 0.068⋆ 0.639 ± 0.087⋆ Sumb (g) 5.703 ± 0.548 3.649 ± 0.238⋆ 3.705 ± 0.276⋆ 3.787 ± 0.251⋆ Figure 6 Open in figure viewer Effects of calcium and dairy products on adipocyte fatty acid synthase activity (upper panel), fatty acid synthase mRNA (middle panel, data are expressed as fatty acid syn‐thase (FAS):actin ratio.) and lipolysis in transgenic mice expressing agouti in adipose tissue under the control of the aP2 promoter. Data are expressed as mean ± standard deviation (ra=10/group). Statistical significance is as indicated in each panel. Serial measurements of plasma glucose and insulin demonstrate a diabetogenic effect of the basal high fat/high sucrose/low calcium diet, with an increase in fasting glucose from 98 ± 10 to 130 ± 11 mg/dl (P<0.02) and a corresponding degree of compensatory hyperinsulinemia. These increases were attenuated by the high calcium and medium dairy diets and prevented by the high dairy diet (Fig. 7). Figure 7 Open in figure viewer Effects of calcium and dairy products on fasting plasma glucose and insulin levels in transgenic mice expressing agouti in adipose tissue under the control of the aP2 promoter. Top panel depicts insulin and the bottom panel depicts glucose. Table 4 summarizes the NHANES III data analysis. After controlling for energy intake, activity level, age, race, and ethnicity, the odds ratio of being in the highest quartile of body fat was markedly reduced from 1.00 for the first quartile of calcium intake to 0.75, 0.40, and 0.16 for the second, third, and fourth quartiles, respectively (multiple R2 = 0.20; P= 0.0009), in adult women. Similarly, the regression model for males demonstrated an inverse relationship between calcium and dairy intakes and body fat (multiple R2=0.40; P=0.0006), although a comparable dose‐responsive reduction in relative risk (odds ratio) by quartile of calcium intake was not evident from the model. Table 4. Effects of dietary calcium, and dairy intake on the risk of being in the highest quartile of body fat for women Quartile of calcium and dairy consumption Calcium intake (mg/day; mean ± sem ) Dairy consumption (servings/month) mean ± sem Odds ratio of being in the highest body fat quartile 1 255 + 20 14.4 + 1.9 1.00 2 484 + 13 38 + 1.3 0.75 (0.13, 4.22)b 3 773 + 28 57.2 + 1.0 0.40 (0.04, 3.90)b 4 1346 + 113 102.8 + 3.6 0.16 (0.03, 0.88)b

DISCUSSION We have previously shown that agouti, an obesity gene expressed in human adipocytes, stimulates Ca2+ influx (4, 5) and promotes energy storage in human adipocytes by coordinately stimulating the expression and activity of fatty acid synthase, a key enzyme in de novo lipogenesis and inhibiting lipolysis inaCa2+‐dependent fashion (6, 7). The carboxyl terminus of agouti protein, which retains functional activity in an in vitro assay system (20), bears a striking spatial homology in number and spacing of cysteine residues to spider and snail venoms (ω‐conotoxins, plectoxins), which target Ca2+ channels (21). Moreover, this agouti modulation of adipocyte lipid metabolism is fully mimicked by Ca2+ channel agonists and inhibited by Ca2+ channel antagonists (6–8). In addition, treating transgenic mice overex‐pressing agouti with a Ca2+ channel antagonist (nifedipine) for 4 wk resulted in significant decreases in adipocyte lipogenesis and an 18% reduction in adipose tissue mass (9). Thus, adipocyte Ca2+ modulates energy storage, suggesting that adipocyte Ca2+ is a logical target for pharmacological and/or nutritional regulation of adiposity. Dietary calcium modulation of intracellular calcium, mediated by suppression of calcitrophic hormones, has previously been demonstrated to attenuate the risk of hypertension, and possibly type II diabetes as well (23, 24). Intracellular calcium plays a key role in multiple related metabolic disorders, including hypertension, cardiac hypertrophy, insulin resistance, and hyperinsulinemia, all of which are commonly associated with obesity. It is now well recognized that these are not merely co‐morbid factors that occur secondary to obesity, but rather are part of an integrated metabolic syndrome referred to as ‘syndrome X’ (25), ‘plurimetabolic syndrome’, the ‘deadly quartet’ (obesity, hypertri‐glyceridemia, hypertension and insulin resistance/ hyperinsulinemia), or ‘generalized cardiovascular and metabolic disease’ (23, 24). Regardless of terminology, a growing body of evidence suggests that these conditions are all characterized by an underlying impairment in intracellular Ca2+ (1–3, 23, 24, 26–28). Indeed, Resnick (23) has proposed a unifying ‘ionic hypothesis’ in which the varying metabolic abnormalities associated with syndrome X represent different tissue‐specific manifestations of a cellular lesion characterized, in part, by elevations in steady‐state intracellular Ca2+ levels. Consistent with this concept, correcting elevations in intracellular Ca2+ results in clinical improvements in blood pressure, insulin resistance, platelet aggregation, and left ventricular hypertrophy (23). Our previous studies of the mechanisms of agouti‐induced obesity indicate that obesity may also be partly a manifestation of a ‘lesion’ in intracellular Ca2+ regulation (4–7, 9); data from the present study lend further support for this hypothesis. For adipocyte Ca2+ to serve as a logical target for nutritional regulation, human adipocytes would need to exhibit responsiveness to calcitrophic hormones. Our observation in the present study that human adipocytes respond to both parathyroid hormone and 1,25‐(OH) 2 ‐D with dose‐responsive increases in intra‐cellular Ca2+ suggests that low Ca2+ diets, by virtue of stimulating a calcitrophic hormone response, will increase adipocyte intracellular Ca2+ whereas higher calcium diets will suppress this response. Accordingly, a coordinated down‐regulation of lipogenesis and up‐regulation of lipolysis would be predicted to result from increasing dietary calcium. Data from the present study of transgenic mice overexpressing agouti in adipose tissue to mimic the human pattern of expression support this prediction, as the obesity‐promoting effects of the lard/sucrose‐based diet were significantly attenuated on the high calcium and high dairy diets. These data are further supported by the measurements of fatty acid synthase mRNA and activity, as well as the lipolysis data. Thus, these data demonstrate that increasing dietary calcium attenuates diet‐induced adiposity by modulating adipocyte intracellular Ca2+ and thereby coordinately regulating lipogenesis and lipoly‐sis (6–9). Dairy and elemental sources of calcium exerted qualitatively comparable effects; however, calcium in the form of dairy exerted a greater effect on attenuating fat deposition than a comparable quantity of elemental calcium. Consistent with this, a recent randomized clinical trial demonstrated a markedly greater weight loss (7.0 vs. 1.7 kg) in patients maintained on a milk‐based diet for 16 wk vs. those maintained on conventional hypocaloric diet at the same level of energy intake (16). Although this difference was attributed to the novelty of the milk‐based diet possibly contributing to a greater level of compliance, data presented herein suggest that this effect may also be attributable to suppression of 1,25‐(OH) 2 ‐D and adipocyte Ca2+, with a consequent reduction in the efficiency of energy utilization. This concept is supported by our population‐based observations in NHANES III. These data demonstrate a profound reduction in the odds of being in the highest quartile of adiposity associated with increases in calcium and dairy product intake. This analysis was controlled for both energy intake and physical activity. Thus, these data indicate that, for any given level of energy intake and expenditure, a low calcium diet favors increased adipose tissue energy storage, but the converse was true for higher calcium diets. Accordingly, dietary calcium appears to modulate the efficiency of energy utilization, with low calcium diets favoring increased efficiency of energy storage and higher calcium diets reducing energy efficiency and instead favoring increased thermogenesis. This concept is further supported by our observation of reduced energy efficiency and increased core temperature in the transgenic mice fed the higher calcium diets.

ACKNOWLEDGMENTS Funding for this work was provided by the National Dairy Council, Rosemont, Illinois.