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© 2008 American Society for Nutrition J. Nutr. 138:1047-1052, June 2008

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Dietary Calcium and Dairy Products Modulate Oxidative and Inflammatory Stress in Mice and Humans1,2

Michael B. Zemel* and Xiaocun Sun

Department of Nutrition, University of Tennessee, Knoxville, TN 37996

* To whom correspondence should be addressed. E-mail: mzemel{at}utk.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Methods
 Results
 Discussion
 LITERATURE CITED
 
We have recently shown 1{alpha},25-dihydroxycholecalciferol increased oxidative stress and inflammatory stress in vitro, whereas suppression of 1{alpha},25-dihydroxycholecalciferol with dietary calcium (Ca) decreased oxidative and inflammatory stress in vivo. However, dairy products contains additional factors, such as angiotensin-converting enzyme inhibitors, which may further suppress oxidative and inflammatory stress. Accordingly, this study was designed to study the effects of the short-term (3 wk) basal suboptimal Ca (0.4%), high-Ca (1.2% from CaCO3), and high-dairy (1.2% Ca from milk) obesigenic diets on oxidative and inflammatory stress in adipocyte fatty acid-binding protein-agouti transgenic mice. Adipose tissue reactive oxygen species (ROS) production and NADPH oxidase mRNA and plasma malondialdehyde (MDA) were reduced by the high-Ca diet (P < 0.001) compared with the basal diet and ROS and MDA were further decreased by the high-dairy diet (P < 0.001). The high-Ca and -dairy diets also resulted in suppression of adipose tissue tumor necrosis factor {alpha} and interleukin (IL)-6 mRNA (P = 0.001) compared with the basal diet, whereas an inverse pattern was noted for adiponectin and IL-15 mRNA (P = 0.002). Consequently, we conducted a follow-up evaluation of adiponectin and C-reactive protein (CRP) in archival samples from 2 previous clinical trials conducted in obese men and women. Twenty-four weeks of feeding a high-dairy eucaloric diet and hypocaloric diet resulted in an 11 (P < 0.03) and 29% (P < 0.01) decrease in CRP, respectively (post-test vs. pre-test), whereas there was no significant change in the low-dairy groups. Adiponectin decreased by 8% in subjects fed the eucaloric high-dairy diet (P = 0.003) and 18% in those fed the hypocaloric high-dairy diet (P < 0.05). These data demonstrate that dietary Ca suppresses adipose tissue oxidative and inflammatory stress.


   Introduction
TOP
 ABSTRACT
 Introduction
Methods
 Results
 Discussion
 LITERATURE CITED
 
Calcium (Ca)-rich diets have been demonstrated to exert an antiobesity effect that appears to be mediated in part by suppressing circulating 1{alpha},25-dihydroxycholecalciferol (1–4). Previous data from our laboratory demonstrate that 1{alpha},25-dihydroxycholecalciferol favors fatty acid synthesis and inhibits lipolysis via modulation of Ca2+ influx (5–11) and suppresses uncoupling protein 2 expression via the nuclear vitamin D receptor (12) and thereby decreases adipocyte apoptosis (13). In addition, 1{alpha},25-dihydroxycholecalciferol regulates adipose tissue fat depot location and expansion by promoting glucocorticoid production and release (14), resulting in a selectively greater effect in visceral than in subcutaneous depots. In support of this concept, dietary calcium (Ca) suppressed fat gain on eucaloric diets and accelerated fat loss on hypocaloric diets in both animals and humans (12,15–17).

Obesity is a principle causative factor in the development of metabolic disorders and increased oxidative stress in accumulated fat appears to be an important contributor to the pathogenesis of obesity-associated metabolic syndrome (18–21). Notably, visceral adiposity is characterized by low-grade systemic inflammation and obese subjects exhibit elevated production of inflammatory markers. We have recently shown that reactive oxygen species (ROS)3 production is modulated by mitochondrial uncoupling status and cytosolic Ca signaling and that 1{alpha},25-dihydroxycholecalciferol regulates ROS production in cultured murine and human adipocytes (22). Accordingly, it is possible that dietary Ca-induced suppression of 1{alpha},25-dihydroxycholecalciferol may reduce oxidative and inflammatory stress. In addition to Ca, dairy products contain additional bioactive compounds that appear to augment its antiobesity activity (23) and may also enhance its ability to suppress oxidative and inflammatory stress. Consequently, the objective of this study was to determine the effects of dietary Ca and dairy products on oxidative and inflammatory stress in a mouse model [adipocyte fatty acid-binding protein (aP2)-agouti transgenic mice].


   Methods
TOP
 ABSTRACT
 Introduction
 Methods
Results
 Discussion
 LITERATURE CITED
 
    Mice and diets. Thirty 6-wk-old male aP2-agouti transgenic mice from our colony were utilized. These animals express normal agouti protein specifically in adipose tissue under control of the aP2 promoter (24–27). The expression pattern of aP2-agouti transgenic mice mimics the normal adipose tissue selective expression pattern in obese and nonobese humans (25–27). These animals become overweight only when hyperinsulinemia is induced by daily subcutaneous insulin injections (24,27) or by chronic ingestion of a high-sucrose diet (28), making this model an appropriate tool for studying the development of diet-induced obesity and the resulting metabolic abnormalities.

Mice were randomly divided into 3 groups (10 mice/group) and fed a modified AIN 93 G diet (29), as described previously (3); the diet was modified to contain either suboptimal Ca (CaCO3, 0.4%, referred to as basal diet for comparison with high-Ca and -dairy treatments in Figs. 1–4), high Ca (Ca carbonate, 1.2%) or milk (Ca, 1.2%, with nonfat dry milk as the sole protein source). Sucrose was the sole carbohydrate source, providing 64% of energy, and fat was increased to 24% of energy with lard. Diet composition is described in Table 1. Mice were studied for 3 wk, during which food intake and spillage were measured daily and body weight and food consumption were assessed weekly. At the conclusion of the study, all mice were killed under isofluorane anesthesia and blood was collected via cardiac puncture; visceral fat pads (perirenal and abdominal), subcutaneous fat pads (subscapular), and soleus muscle were immediately excised, weighed, and used for further study, as described below.


Figure 1
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  		FIGURE 1  The effect of dietary Ca and milk on adipocyte intracellular ROS levels (A) and adipose NADPH oxidase gene expression ratio (B) in aP2-agouti transgenic mice. Values are means ± SEM, n = 10. Means without a common letter differ, P ≤ 0.001.

 

Figure 2
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  		FIGURE 2  The effect of dietary Ca and milk on plasma 1{alpha},25-dihydroxycholecalciferol (A) and MDA (B) in aP2-agouti transgenic mice. Values are means ± SEM, n = 10. Means without a common letter differ, P < 0.001.

 

Figure 3
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  		FIGURE 3  The effect of dietary Ca and milk on adipose tissue TNF{alpha} (A), IL-6 (B), MCP-1 (C), adiponectin (D), IL-15 (E), and muscle IL-15 gene expression ratio (F) in aP2-agouti transgenic mice. Values are means ± SEM, n = 10. Means without a common letter differ, P ≤ 0.002.

 

Figure 4
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  		FIGURE 4  The effect of dietary Ca and milk on plasma TNF{alpha} (A), IL-6 (B), and adiponectin (C) in aP2-agouti transgenic mice. Values are means ± SEM, n = 10. Means without a common letter differ, P < 0.03.

 

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  		TABLE 1 Mouse diet composition

 
This study was approved from an ethical standpoint by the Institutional Care and Use Committee of the University of Tennessee.

    Human study and diets. The samples used in this study were derived from frozen samples from 2 previously published studies. Details of experimental design and methods used are found in previously published literature (30,31). Briefly, in the hypocaloric diet study (30), obese subjects consumed balanced deficit diets (–500 kcal/d; –2092 kJ/d) and randomized to control (400–500 mg Ca/d; n = 16) or yogurt (1100 mg Ca/d; n = 16) treatments for 12 wk. In the eucaloric diet study (31), obese subjects were prescribed a low-Ca (500 mg/d)/low-dairy (<1 serving/d, where 1 serving = 245 g fluid milk, 227 g yogurt, or 42 g hard cheese) or high-dairy (1200 mg Ca/d diet including 3 servings of dairy) diet with no change in energy or macronutrient intake for 24 wk (n = 17/group). Plasma samples were obtained following an overnight fast via venipuncture collection into EDTA-treated tubes at the beginning and end of each study and frozen at –80°C for 24–30 mo. This study was approved from an ethical standpoint by the University of Tennessee-Knoxville Institutional Review Board.

    Total RNA extraction. A total cellular RNA isolation kit (Ambion) was used to extract total RNA from cells according to the manufacturer's instructions.

    Quantitative real-time PCR. Adipocyte and muscle 18s, NADPH oxidase, tumor necrosis factor (TNF){alpha}, monocyte chemotactic protein-1 (MCP-1), interleukin (IL)-6, adiponectin, and IL-15 were quantitatively measured using a Smart Cycler real-time PCR system (Cepheid) with a TaqMan 1000 Core Reagent kit (Applied Biosystems), as previously described (4,22). The primers and probe sets were obtained from Applied Biosystems TaqMan Assays-on-Demand Gene Expression primers and probe set collection according to the manufacturer's instructions.

    Plasma TNF{alpha}, IL-6, CRP, and 1{alpha},25-dihydroxycholecalciferol assay. Mouse TNF{alpha}, IL-6 (Assay Designs), and C-reactive protein (CRP) (LINCO) ELISA kits and a 1{alpha},25-dihydroxycholecalciferol ELISA kit (Alpco Diagnostics) were used to measure the concentration of these metabolites in plasma according to the manufacturer's instructions. Inter-assay CV were below 11% for all assays and intra-assay CV were below 8% for all assays.

    Adipocyte intracellular ROS assay. Adipose tissue digestion and adipocyte preparation were prepared as described previously (16). Intracellular ROS generation was assessed using 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate as described previously. Cells were loaded with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (2 µmol/L) for 30 min before the end of the incubation period (48 h). After washing twice with PBS, cells were scraped and disrupted by sonication on ice (20 s). Fluorescence (emission 543 or 527 nm) and DNA content were then measured as described previously. The intensity of fluorescence was expressed as arbitrary units/ng DNA.

    Lipid peroxidation. Plasma malondialdehyde (MDA) was used as an index of systemic lipid peroxidation and was determined using a TBARS Assay kit from Cayman Chemical according to the manufacturer's instructions.

    Statistical analysis. Data were evaluated for significance by 1-way ANOVA; differences in changes over time (i.e. body weight change in the mice and changes in CRP and adiponectin in the clinical trials) were analyzed as differences between pre- and post-test values for each variable. When ANOVA demonstrated treatment effects at P < 0.05, significantly different group means were separated by the least significant difference test using SPSS software. All data presented are expressed as means ± SEM.


   Results
TOP
 ABSTRACT
 Introduction
 Methods
 Results
Discussion
 LITERATURE CITED
 
All mice exhibited significant weight gain after consuming the high-sucrose, high-fat diets for 3 wk (Table 2); weight gain was significantly suppressed by the dairy diet but not by the high-Ca diet. Dietary Ca suppressed adipocyte intracellular ROS production (Fig. 1A) and corresponding NADPH oxidase gene expression (Fig. 1B). The dairy diet exerted a further inhibition of ROS generation compared with the high-Ca group, whereas NADPH oxidase was not further suppressed by the dairy diet compared with the Ca diet. Consistent with our previous observation, both dietary Ca and dairy induced a similar inhibition of circulating 1{alpha},25-dihydroxycholecalciferol (Fig. 2A), although there was a nonsignificant trend (0.05 < P < 0.10) toward greater suppression by the milk diet. The high-Ca diet also suppressed plasma MDA (Fig. 2B) and dairy exerted a significantly greater effect on MDA than dietary Ca per se.


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  		TABLE 2 Body weight, weight gain, and fat mass in ap2-agouti mice after consuming low-Ca, high-Ca, or milk dietary treatments for 3 wk1

 
Both the high-Ca and -dairy diets markedly decreased inflammatory cytokine gene expression in adipose tissue, with significant suppression of TNF{alpha} (Fig. 3A), IL-6 (Fig. 3B), and MCP-1 (Fig. 3C), whereas there was a corresponding diet-induced increase in adiponectin expression (Fig. 3D). Similarly, the high-Ca and milk diets markedly increased IL-15 expression in both adipose tissue (Fig. 3E) and skeletal muscle (Fig. 3F). These results were reflected in diet-induced changes in plasma cytokines, with significant decreases in plasma TNF{alpha} (Fig. 4A) and IL-6 (Fig. 4B) and a corresponding increase in plasma adiponectin in the mice fed the milk diet (Fig. 4C).

To further examine the effect of these diets in obese humans, we measured the plasma concentration of CRP, a key marker of low-grade inflammation, and the antiinflammatory cytokine adiponectin in archival samples derived from 2 previous clinical trials. The milk diet significantly suppressed plasma CRP levels in both eucaloric (Fig. 5A) and hypocaloric conditions (Fig. 5B) and increased the adiponectin concentration in both eucaloric (Fig. 5C) and hypocaloric conditions (Fig. 5D).


Figure 5
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  		FIGURE 5  Effects of dairy products on plasma CRP (A,B) and adiponectin (C,D) concentrations in obese subjects consuming a eucaloric diet for 24 wk (A,C) or a hypocaloric diet for 12 wk (B,D). Values are means ± SEM; n = 16 (A,C) or 17 (B,D). *Different from low dairy, P < 0.05. Low dairy: 400–500 mg/d Ca; high dairy: 1100–1200 mg/d Ca. Pre, Pretreatment; post, posttreatment.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Methods
 Results
 Discussion
LITERATURE CITED
 
Data from this study demonstrate a role for dietary Ca in modulating oxidative and inflammatory stress, with further effects on oxidative stress evident from dairy products when compared with CaCO3. Oxidative stress is augmented in obesity and a local increase in oxidative stress in accumulated fat causes dysregulated production of adipokines. Fat accumulation stimulates NADPH oxidase, a key factor in enzymatic cellular ROS production, expression in white adipose tissue (18). Consistent with this, the high-Ca diet in this study inhibited both NADPH oxidase expression and ROS production in mice; had the NADPH oxidase protein levels been similarly altered, we would anticipate a causative relationship between the suppression of this enzyme and of ROS production. Notably, dairy products exerted a greater inhibitory effect on ROS production in adipose tissue via an apparently NADPH oxidase-independent mechanism, as there was no further suppression of NADPH oxidase. These changes were reflected in significant decreases in systemic lipid peroxidation as demonstrated by decreased plasma MDA with the high-Ca diet, with a significantly greater effect on the milk diet. However, it should be noted that the milk diet contained 33% less sucrose than the other diets to compensate for the lactose contributed by the nonfat dry milk. This diet difference presents a limitation in interpreting the effects of the milk diet, as it is possible that the lower level of sucrose may contribute to reduced oxidative stress.

Previous data from our laboratory demonstrate that dietary Ca exerts an antiobesity effect that may be mediated in part via a 1{alpha},25-dihydroxycholecalciferol-mediated mechanism (32). Moreover, we have recently shown these genomic and nongenomic effects of 1{alpha},25-dihydroxycholecalciferol to directly regulate ROS production in adipocytes (16). In vivo data from the present study support these in vitro observations, with significant suppression of 1{alpha},25-dihydroxycholecalciferol on both the high-Ca and milk diets associated with decreases in both adipose tissue ROS and circulating MDA. However, the high-Ca and milk diets had identical Ca content and exerted comparable inhibition of 1{alpha},25-dihydroxycholecalciferol, whereas the milk diet resulted in further suppression of oxidative stress compared with the high-Ca diet. This suggests that the milk diet suppressed oxidative stress via an additional mechanism that could be attributed to either the difference in sucrose between the high-Ca and milk diets or to other components of the milk diet, as discussed below.

It is possible that this 1{alpha},25-dihydroxycholecalciferol-independent component may be explained in part by the suppression of the adipocyte renin-angiotensin system. Dairy contains angiotensin-converting enzyme inhibitory peptides (23) and adipose tissue expresses all components of the renin-angiotensin system (33–37); stimulation of this system promotes oxidative and inflammatory responses (38–43), whereas antagonism of this system suppresses oxidative stress (44,45). The high concentration of leucine in milk may also indirectly contribute to suppression of oxidative stress. The provision of leucine to skeletal muscle promotes protein synthesis and inhibits protein degradation (46,47); recent data suggest that leucine alters energy partitioning between adipose tissue and muscle (48), resulting in reduced energy storage in adipose tissue and increased fat oxidation and energy utilization (presumably for protein synthesis) in skeletal muscle. This shift in metabolic flexibility may cause acceleration of energy utilization and result in less oxidative and inflammatory stress resulting from nutrient overload. Finally, it is possible that the significant decrease in adiposity in the milk diet may have contributed to the additional effects of the milk diet compared with the high-Ca diet.

Oxidative stress also appears to play a role in regulating inflammatory status in adipose tissue (4,18). The high-Ca diet also resulted in significant suppression of inflammatory cytokines and promotion of antiinflammatory cytokines in mice; however, although there was a trend toward a stronger effect of the milk diet, this trend was significant only for circulating adiponectin. Similar effects were also found in the retrospective analysis of archival clinical samples from obese subjects, with high dairy suppressing circulating CRP and increasing adiponectin under both eucaloric and hypocaloric conditions. However, because these clinical studies compared high- to low-dairy diets and did not include a high-Ca/low-dairy group, we cannot distinguish between Ca and noncalcium contributions to these effects.

Although adipocytes directly generate inflammatory mediators, adipose tissue-derived cytokines also originate substantially from these nonfat cells, among which the infiltrated macrophages appear to play a prominent role (49). Infiltration and differentiation of adipose tissue-resident macrophages are under the local control of chemokines, many of which are produced by adipocytes. Adipocyte-derived MCP-1 plays a crucial role in the recruitment of monocytes and T lymphocytes into adipose tissue and obesity is associated with increased expression of MCP-1 in adipose tissue in both rodents and humans (50–52). We recently found 1{alpha},25-dihydroxycholecalciferol stimulated MCP-1 expression in 3T3-L1 adipocytes and markedly stimulated inflammatory cytokine production from both adipocytes and macrophages in coculture (53). Consistent with this, data from the present study demonstrate that circulating 1{alpha},25-dihydroxycholecalciferol was reduced with increased Ca intake, concurrent with reductions in proinflammatory indices, suggesting a role for dietary Ca in attenuating the cytokine dysregulation associated with diet-induced obesity.

Excess adiposity per se is insufficient to explain the increased oxidative and inflammatory burden found in metabolic syndrome compared with uncomplicated obesity. Indeed, Van Guilder et al. (54) recently reported significantly higher oxidative stress (oxidized LDL) and inflammatory burden such as CRP, TNF{alpha}, and IL-6 in otherwise healthy obese subjects with metabolic syndrome compared with BMI-matched obese subjects without metabolic syndrome. Our sample analysis from previously conducted clinical trials suggest that dairy foods appear to exert a beneficial effect on circulating CRP and adiponectin levels independently of changes in body weight, because one of the studies was conducted under eucaloric conditions with no significant changes in body weight (31). However, it is not possible to fully preclude the effects of reduced adiposity, because subjects had reduced adiposity despite no change in body weight (31).

In summary, this study provides further in vivo evidence that dietary Ca and dairy inhibit oxidative and inflammatory stress in a mouse model of diet-induced obesity and oxidative stress as well as in obese adult humans. Dietary Ca-induced suppression of circulating 1{alpha},25-dihydroxycholecalciferol may be responsible for Ca-induced suppression of oxidative and inflammatory stress, although further effects of dairy foods on oxidative stress appear to be mediated by additional mechanisms.


   FOOTNOTES
 
1 Supported by a grant from the National Dairy Council. Back

2 Author disclosures: X. Sun, no conflicts of interest; M. Zemel has grants from the National Dairy Council and has been compensated as a speaker by the National Dairy Council. Back

3 Abbreviations used: aP2, adipocyte fatty acid-binding protein; CRP, C-reactive protein; IL, interleukin; MDA, malondialdehyde; MCP-1, monocyte chemotactic protein-1; ROS, reactive oxygen species; TNF, tumor necrosis factor. Back

Manuscript received 18 December 2007. Initial review completed 11 February 2008. Revision accepted 24 March 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Methods
 Results
 Discussion
 LITERATURE CITED
 

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