Intervention Effects Of Vitamin D On Simple Obesity In Rats

ObjectiveVitamin D besides have the function of bone metabolism, also have a function on maintaining the normal metabolism. There are many people with vitamin D deficiency in our country, and the incidence of simple obesity and metabolic syndrome increased significantly. The simple obesity and it’s closely related metabolic abnormalities, such as hypertension, type2diabetes and cardiovascular disease, are diseases with poor treatment effect. And these diseases could greatly reduce the patient’s quality of life, and take away a lot of social economic resources. Through observe the effects of vitamin D on metabolism index, our study want to provide experimental evidence for the intervention effects of Vitamin D on simple obesity in rats.Methods1Establishment of animal modelFifty two-month-old healthy male Wistar rats in the weight range of160-180g procured from the central animal house of Shandong University were maintained under controlled conditions (22℃±2℃,50%-60%relative humidity,12-/12-hour dark/light cycle). They were randomly divided into five groups:control group, fat group, low-dose group, middle-dose group and high-dose group. The control group were given common forage, and the fat group were given high-fat forage. The other three groups were all given high-fat forage and VD solution (5ml/kg) by ig once a day for nine weeks. The three groups were administrated with low, moderate and high dosages of0.6μg/ml,1.2μg/ml and2.4μg/ml. The body weight and body length of all rats were measured on the first day of this study and once a week thereafter. The fat group rats reached the standard of simple obesity after ig for nine weeks.2The collection of biological materialsPrior to killing the rats, they were fasting for12hours. The weight and body length were measured at0.1gram and0.1centimeters respectively. Blood was collected by decapitation and was treated with centrifugation (3500g,15min) to gain serum. Then the serum was refrigerated for later analysis of VD, TC, HDL-C, TG, Leptin and essential elements calcium concentration. Then the liver and epididymis fat pad were separated rapidly and weighted.3Lee’s index and fat mass indexThe epididymal fat pad was considered as the whole body fat, then the Lee’s index and fat mass index were calculated.4Morphological observation on adipose cell in ratsThe fat mass were dealted with paraffin section and HE staining after fixed by formalin. Then the morphology of fat cell was observed by microscope.5The determination of VD in serum5.1The pretreatment of serumAdd100μL serum and100μL methanol acetonitrile mixture (1:1) into centrifuge tube, then blend this mixed liquor. Add700μL n-hexane into the centrifuge tube, and the mixed liquor was treated with centrifugation (3000g,10min) to gain extracting solution after blended. Add extracting solution from the centrifuge tube into another centrifuge tube. The normal hexane extraction step was repeated twice to ensure complete extraction.Then the extracting solution was blowed dry by nitrogen, and the residue was dissolved by500μL methanol. Finally, the VD in serum was determined by HPLC-UVD.5.2Chromatographic conditionsChromatographic column was Agilent C18column (150mm X4.6mm,5μm), mobile phase were methanol, flow rate was1.0ml/min, detection wavelength was265nm, column temperature was21℃, and injection volume was20μl.6The determination of serum biochemical parameters The concentrations of TG, TC, HDL-TC and leptin in serum were determined by kit. And the concentrations of LDL-TC in serum were calculated by the formula of Friendward indirectly.7The determination of element Ca in serumThe serum was directly diluted with deionized water and Lanthanum oxide (20g/L) for analysis of Ca. Essential elements Ca in serum was determined by flame atomic absorption spectrophotometry.Results1The results of body weightDuring this study, the body weight of all rats in each group showed an obvious trend of rising. Compared with the fat group, the three different kinds of dose groups all showed a tardiness on rising on body weight. At the end of this study, the body weight of control group and high-dose group were obviously lower than that of fat group, low-dose group and middle-dose group (P<0.05). With the increase of intervention dose of VD, the body weight of rats was on a declining curve.2The results of body length and Lee’s indexThe body length of fat group and all the three different kinds of dose groups was obviously longer than that of control group (P<0.05). The Lee’s index of fat group, low-dose group and middle-dose group was obviously higher than that of control group and high-dose group (P<0.05). With the increase of intervention dose of VD, the body length of rats was on the rise, and the Lee’s index of rats was on a declining curve.3The results of liver weight, epididymis fat pad weight and fat mass indexThe liver weight of fat group was obviously higher than that of control group (P<0.05). The epididymis fat pad weight and fat mass index of fat group and three different kinds of dose groups were all obviously higher than that of control group (P<0.05). The epididymis fat pad weight of middle-dose group was obviously higher than that of fat group (P<0.05). The fat mass index of fat group and low-dose group was obviously higher than that of high-dose group (P<0.05). And with the increase of intervention dose of VD, the liver weight, epididymis fat pad weight and fat mass index of rats were all on a declining curve.4Effect of VD on cell morphology of epididymis fat pad in rats4.1Effect of VD on cell sizeThe number of epididymis fat pad cells of fat group and low-dose group was obviously less than that of control group (P<0.01). The number of epididymis fat pad cells of middle-dose group and high-dose group was obviously more than that of fat group (P<0.05). And with the increase of intervention dose of VD, the cell size of epididymis fat pad in all rats was on a declining curve.4.2Effect of VD on cell morphologyThe volume of epididymis fat pad cells of control group was least in all groups, and the cells had a uniform size. The volume of epididymis fat pad cells of fat group was lagest in all groups, and the fat cells were very plentiful, but the shape of fat cells was irregular. The volume of epididymis fat pad cells of low-dose group was larger than control group and high-dose group, and the fat cells had a different size and polygon shape. The volume of epididymis fat pad cells of middle-dose group was among fat group and control group, and the fat cells had different size and polygon shape. The volume of epididymis fat pad cells of high-dose group was smaller than that of fat group. the fat cells of high-dose group had different size, and the cell membrane of fat cells was concave.5The serum concentrations of VD in ratsThe serum concentrations of VD of fat group and low-dose group was obviously lower than that of control group (P<0.01), and the serum concentrations of VD of high-dose group was obviously higher than that of control group (P<0.01). Compared with the fat group, the serum concentrations of all the three different kinds of dose group was obviously higher (P<0.01). And with the increase of intervention dose of VD, the serum concentrations of VD in rats were on rise.6The serum concentrations of serum biochemical parameters6.1The serum concentrations of TG, TC, HDL-TC and LDL-TC The serum concentrations of TC of fat group, low-dose group and middle-dose group were obviously higher than that of control group (P<0.05). Compared with fat group, the serum concentrations of TC in rats were on a declining curve with the increase of intervention dose of VD (P<0.05). The serum concentrations of TG of fat group, low-dose group and middle-dose group were obviously higher than that of control group (P<0.05), and the serum concentrations of TG in rats were on a declining curve with the increase of intervention dose of VD (P<0.05). The serum concentrations of HDL-C of fat group, low-dose group and middle-dose group was obviously lower than that of control group (P<0.05).And the serum concentrations of HDL-C in rats were on rise with the increase of intervention dose of VD. The serum concentrations of LDL-C of fat group, low-dose group and middle-dose group were obviously higher than that of control group (P<0.05). Compared with fat group, the serum concentrations of LDL-Cin rats were on a declining curve with the increase of intervention dose of VD (P<0.05).6.2The ratio of HDL-C/LDL-CThe ratio of HDL-C/LDL-C of control group was obviously higher than that of the other four groups (P<0.05). Compared with fat group, the ratio of HDL-C/LDL-C was on rise with the increase of intervention dose of VD, and the ratio of HDL-C/LDL-C of high-dose group was obviously higher than that of fat group (P<0.05).7The serum concentrations of leptinThe serum concentrations of leptin of control group was obviously lower than that of the other four groups (P<0.05). Compared with fat group, the serum concentrations of leptin in rats were on a declining curve with the increase of intervention dose of VD, and the serum concentrations of leptin of middle-dose group and high-dose group were lower than that of fat group (P<0.05).8The serum concentrations of Ca in ratsThe serum concentrations of Ca of fat group, low-dose group and middle-dose group were obviously lower than that of control group (P<0.01). Compared with fat group, the serum concentrations of Ca in rats were on rise with the increase of intervention dose of VD, and the serum concentrations of Ca of the three different kinds of dose groups were higher than that of fat group (P<0.01). And with the increase of intervention dose of VD, the serum concentrations of Ca in rats were on rise.9The results of correlation analysisThe fat mass index, Lee’s index, serum concentrations of TC, TG, LDL-C and leptin negatively correlated with the serum concentrations of vitamin D, and the serum concentrations of HDL-C and the ratio of HDL-C/LDL-C positively correlated with the serum concentrations of vitamin D. Multielement logistic regression analysis results showed that the serum concentrations of vitamin D was a protective factor for simple obesity in rats.Conclusions1The HPLC methods established in this study for detection of vitamin D in serum, was simple, rapid, sensitive, accurate and good repeatabilities.2The simple obesity can reduce the serum concentrations of vitamin D in rats.3Vitamin D supplements can improve the serum concentrations of vitamin D in rats.4Vitamin D have the intervention effects on the simple obesity in rats through reducing Lee’s index, fat mass index and serum concentrations of TG, TC, LDL-C, leptin and increasing serum concentrations of HDL-C and Ca.