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We have recently shown 1α,25-dihydroxycholecalciferol increased oxidative stress and inflammatory stress in vitro, whereas suppression of 1α,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 CaCO^sub 3^), and high-dairy 11.2% Ca from milk) obesigenic diets on oxidative and inflammatory stress in adipocyte fatty acid-binding protein-agrouti 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 α 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-reacttve 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.

Discussion

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 CaCO^sub 3^. 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.

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 eucaioric 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 lo:,25-dihydroxycholecaiciferol 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 1α,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, TNFα, 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 1α,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.