Effects Of Oxidative Stress Induced By A High Fat Diet On Calcium Metabolism And Related Molecular Mechanism

With growth in the living standard, the great changes in dietary patterns occur in urban and rural residents. Fat intakes are increasing in China, bringing about many health problems, such as osteoporosis. The increased energy intake can cause strong energy metabolic rate, which produces numerous ROS and consequently induces oxidative stress. Further, oxidative stress is one cause for pathogensis of many diseases. Although increasing evidences in postmenopausal models have demonstrated that oxidative stress plays a key role in abnormal calcium metabolism, previous studies have not addressed role of oxidative stress in high-fat diet (HFD)-induced bone mass loss. In contrast, antioxidant lipoic acid (LA), which not only scavenges ROS directly but also provides the reducing medium for the regeneration of the antioxidant from oxidized form, can prevent or at least attenuate oxidative injure. Therefore, the prevent study investigated the roles of HFD-induced oxidative stress in intestinal calcium absorption and bone metabolism and preventive effects of LA on them. Furthermore, their molecular mechamisms were also addressed.1. HFD-indcued oxidative stress inhibits intestinal calcium absorptionMale C57BL/6 mice were randomly assigned to one of four groups with eight mice in each group. The control group consumed an ordinary diet (4.9% fat, w/w). The other three groups were fed an HFD (21.2% fat), the HFD plus 0.1% lipoic acid (LA), or the HFD plus additional 0.9% calcium supplement (CS). After 9 wk, plasma and duodenal oxidative stress biomarkers including malondialdehyde, superoxide dismutase, catalase, total antioxidant capacity, reduced glutathione/oxidized glutathione ratio, and reactive oxygen species (ROS) were examined. The intestinal calcium absorption state was evaluated through examining the calcium balance, bone mineral density (BMD), and calcium metabolism biomarkers. Furthermore, quantitative RT-PCR was carried out to analyze the changes in expression of transcellular calcium absorption-related genes.The HFD induced marked decreases in intestinal calcium absorption and BMD of whole body, accompanied by redox imbalance and increased oxidative damage in duodenum; duodenal expression of calbindin-D9K, PMCA1b, and NCX was significantly down-regulated by 1.9-, 2.7-, and 1.5-fold, respectively. Furthermore, duodenal GSH/GSSG ratios were strongly positively correlated with the apparent calcium absorption rate a, ROS levels were negatively correlated with it, but dietary fat levels were not significantly correlated with it. Our results demonstrated that HFD-induced duodenal oxidation state could significantly down-regulate expression of calbindin-D9K, PMCA1b, and NCX, thus causing an inhibitory effect on intestinal calcium absorption. 2. HFD-induced abnormal bone metabolism associated with bone oxidative stessMale C57BL/6 mice (4 wk old) were fed with normal diet, high-fat diet (HFD), or HFD supplemented with 0.1% antioxidant LA. After 13-wk feeding, the markers of bone metabolism in plasma and in urine, and femora oxidative stress were measured. Moreover, IGF-1 and TNF-a in plasma were measured. The feeding dyslipidemic HFD induced both inhibitory bone formation reactions and enhancive bone resorption reactions, accompanied by impaired bone antioxidant system, low levels of IGF-1 in plasma, and high levels of TNF-a in plasma. In contrast, these alternatives were prevented completely or partially in mice fed LA supplement. Further, plasma propeptide of ? collagen C-propeptide as a marker of bone formation was positively correlated with both MDA (r=-0.687, P<0.001) and reduced glutathione/ oxidized glutathione (GSH/GSSG) ratio (r=0.565, P<0.003) of bone. Cross-linked N-telopeptides of bone type ? collagen as a marker of bone resorption was negatively correlated with both MDA (r=0.516, P<0.007) and GSH/GSSG ratio (r=-0.786, P<0.001). Dyslipidemia induces impaired bone antioxidant system. Oxidative stress could be an important mediator of hyperlipidemia-induced bone loss.3. Molecular mechanism for HFD-induced low bone massHigh-fat diet (HFD) leads to an increased risk of osteoporosis-related fracture, but the molecular mechanisms for its effects on bone metabolism have rarely been addressed. The present study investigated the possible molecular mechanisms for the dyslipidemic HFD-induced bone loss through comparing femoral gene expression profiles in the HFD-fed mice vs. the normal diet-fed mice during growth stage. We used Affymetrix 430A Gene Chips to identify the significant changes in genes expression involved in bone metabolism, lipids metabolism, and the related signal transduction pathways. Quantitative RT-PCR was carried out on some significant genes for corroboration of the microarray results. At the conclusion of the 13-wk feeding, the down-regulation of most of the genes related to bone formation and the up-regulation of most of the genes related to bone resorption were observed in the HFD-fed mice, according with the changes in plasma bone metabolic biomarkers. Together, the HFD induced a decrease in the majority of the adipogenesis-, lipid biosynthesis-, and fatty acid oxidation-related genes expression, such as PPARg and APOE. Furthermore, some genes engaged in the related signal transduction pathway were strongly regulated at transcript level, including IGFBP4, TGFbR1, IL17a, IL-4, and P53. These results indicate that the HFD may induce inhibitory bone formation and enhanced bone resorption, thus causing adverse bone status.Supplemental LA can attenuate HFD-induced negative effects on bone formation and prevent its enhancement effects on bone resorption associated with decreased oxidative stress. Furthermore, antioxidant LA can up-regulate genes involved in IGF-1 signaling pathway and down-regulate genes involved in P53 apoptotic pathway, thus contributing to attenuating inhibitory bone formation caused by HFD. Moreover, LA-induced the strong up-regulation of IL12a and down-regulation of PTHR1, TGFbR1, and IL17a may lead to depressing bone resorption.4. Effect of redox status onmatrix mineralizetion of osteoblast-like cell (MC3T3-E1)The role of redox status in matrix mineralization of MC3T3-E1 cells and preventive effect of antioxidant LA was studied. Our results suggested that LA prevented oxidative injure of MC3T3-E1 cells induced by hydrogen peroxide and down-regulated high NQO1 expression, which is involved in response to oxidative stress. The high NQO1 expression can increase stabilization of P53 expreesion, thus maybe induced osteoblastic apoptosis. This study demonstrated that LA can prevent P53-dependent MC3T3-E1 cells apoptosis induced by oxidative stress, associated with maintaing redox status. Moderate levels of LA (0.1-0.5mM) attenuate the impaired MC3T3-E1 cell proliferation and mineralization levels induced by hydrogen peroxide, but high levels of LA (2.5mM) can not prevent significantly its functions.