| BackgroundEpidemiological studies have shown that intrauterine growth retardation (IUGR) is closely related to metabolic syndrome (MS) in adulthood. Reduced maternal protein consumption during pregnancy is the classic model to study the mechanism of MS in IUGR individuals. Although programming of hypertension, insulin resistance and altered glucose metabolism by maternal dietary restriction during pregnancy and lactation have been demonstrated in animal models, there were marked difference between reports of the effects on lipid metabolism. So, we would try to explore whether hyperlipidemia occurred in adulthood in IUGR rats. Liver and adipose tissue are important organs in lipid metabolism, whether the structure abnormalities of liver and adipose tissue occur in postnatal development period in IUGR models remains unclear. We would further confirm that hyperlipidemia occurred in adulthood in IUGR rats from the changes in tissue structure of organs.Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors belonging to the steroid, retinoid and thyroid hormone receptor superfamily of ligand-activated transcription factors. There are three distinct subtypes, PPARα, PPARβ and PPARγ. PPARα is mainly expressed in liver tissue and related to lipid metabolism. PPARy is mainly expressed in adipose tissue and possesses a variety of biological effects, which plays an important role in adipocyte differentiation, glucose and lipid metabolism and so on. It has been established that the abnormal expression of PPARs and PPARs-mediated fatty acid catabolism and adipocyte differentiation is closely related to metabolic syndrome. Till now, however, there is no report about the dynamic change of PPARs in the whole postnatal development period in IUGR individuals. And whether the expression of PPARs and PPARs-mediated fatty acid catabolism and adipocyte differentiation leads to hyperlipidemia in adulthood in IUGR rats remains unclear. The adipose tissue secretes several cytokines, such as leptin, adiponectin and adipocyte fatty acid binding protein (AFABP), which widely involve in the metabolic syndrome. In addition, some of them have been used to early predict the MS. The dynamic changes of circulating adipocytokines in IUGR rats need further investigation. It is meaningful to evaluate the possibility of early prediction for hyperlipidemia by monitoring the circulating adipocytokines in IUGR rats.PPARs are important therapeutic targets for MS. Conjugated linoleic acid (CLA), as one of essential fatty acids, takes part in the lipid metabolism, and meanwhile plays an important role in decreasing blood lipids by upregulate and activate PPARs. For IUGR individuals, whether intervention of CLA could early prevent hyperlipidemia in adulthood remain unclear. ObjectiveTo build and evaluate the rat models of IUGR. To observe the growth pattern after birth, the dynamic changes of blood lipids and the structure changes of liver and adipose tissue in development period in IUGR rats.MethodsSixteen male and sixteen female healthy3-month old SD rats were involved in this study. Pregnant rats were randomly divided into IUGR group and control group. The pregnant rats in IUGR group were fed with a10%low-protein diet during pregnancy, and21%normal protein diet was given to the pregnant rats during pregnancy in control group and the pregnant rats during lactation and the offspring rats after lactation in both groups. The offspring rats were weighed, and serum, liver and adipose tissue were collected at the age of1d,7d,21d,2m and3m. Eight rats entered the study at every time point for each group. Triglyceride (TG) in serum and liver were detected with the method of oxidase. Free fatty acid (FFA) in serum and liver were detected with copper color test. The structure of liver and adipose tissue was detected with paraffin section and hematoxylin-eosin staining, and the lipid deposition was detected with frozen section and stained with Sudan III.Results(1) The average birth weight of offspring was significantly lower in IUGR group (5.59±0.55g) compared to control group (6.95±0.64g)(P<0.001). The incidence of intrauterine growth retardation in IUGR group (76.09%) was significantly higher than control group (3.49%)(P<0.001). There was no difference in the average litter and perinatal mortality between the two groups.(2) The average body weight of offspring in IUGR group was significantly lower than control group at7d and2m (P=0.005, P=0.005), while there was no difference at21d and3m.(3) There was no difference in the serum TG and FFA level between the two groups at1d,7d,21d, and2m. But the serum TG and FFA level was significantly higher in the IUGR group compared to control group at3m (P<0.05).(4) The liver weight was significantly lower in IUGR group compared to control group at Id,7d and2m, while there was no difference at21d and3m. There was no difference in the ratios of liver weight to body weight between the two groups at any time points.(5) There was no difference in the hepatic TG and FFA content between the two groups at1d,7d,21d and2m. But the hepatic TG and FFA content was significantly higher in IUGR group compared to control group at3m (P<0.05).(6) At all time points, the hepatic cord and some hepatocytes of offspring in IUGR group had an irregular arrangement. Compared to control group, the hepatocytes of offspring in IUGR group were mild enlarged, some with fatty degeneration, at3m.(7) The lipid deposition in white adipose cells and brown adipose cells increased with individual development in the two groups, and partial brown adipose cells were replaced by white ones. And this change was more obvious in IUGR group than control group at3m. There was fewer small fat cells in WAT in IUGR group.Conclusions(1) It was an effective method to make animal models of IUGR by feeding rat with a10%low-protein diet during pregnancy.(2) The body weight and liver weight of offspring rats in IUGR group exhibited stage catch-up growth.(3) Hyperlipidemia occurred in adulthood in IUGR rats.(4) There were structure changes in liver and adipose tissue in postnatal development period in IUGR rats, with higher lipid content in liver tissue and fatty degeneration in some hepatocytes. Part Ⅱ Mechanism of Intrauterine Growth Retardation on Hyperlipidemia in the RatsObjective(1) To explore the role of PPARα and PPARα-mediated fatty acid β-oxidation in the occurrence of hyperlipidemia in IUGR rats.(2) To explore the role of PPARγ and PPARγ-mediated adipocyte differentiation in the occurrence of hyperlipidemia in IUGR rats.(3) To evaluate the possibility of early prediction for hyperlipidemia by monitoring the circulating adipocytokines in IUGR ratsMethodsIUGR rats and control ones were obtained by previous method. The offspring rats were killed, and serum, liver tissue and white adipose tissue (WAT) were collected at the age of1d,7d,21d,2m and3m. Eight rats entered the study at every time point for each group. The expression of PPARα protein in the liver tissue was detected by immunohistochemical staining and western blot. The expression of PPARγ protein in white adipose tissue (WAT) was detected by western blot. The expression of PPARα, Cpt1a, Acox1and Acox3mRNA in liver tissue and PPARγ, LPL and AFABP mRNA in WAT was detected by real-time RT-PCR. The serum leptin, adiponectin and AFABP were measured by enzyme-linked immunosorbent assay (ELISA). Results(1) The expression of PPARa protein in liver tissue in IUGR group was significantly lower than control group at Id,7d,21d,2m and3m. Compared to control group, hepatic PPARa mRNA expression in IUGR group decreased by35%(P=0.014) at1d,30%(P=0.012) at7d,49%(P=0.032) at21d,54%(P=0.002) at2m and62%(P<0.001) at3m, respectively.(2) Compared to control group, hepatic Cpt1a mRNA expression in IUGR group decreased by55%(P<0.001) at Id,47%(P=0.008) at7d,51%(P=0.014) at21d,37%(P=0.011) at2m and55%(P<0.001) at3m, respectively.(3) There was no difference in hepatic Acoxl mRNA expression between the two groups at Id,7d and2m. The hepatic Acox1mRNA expression was significantly lower in IUGR group than control group at21d and3m (P=0.036, P=0.009).(4) There was no difference in hepatic Acox3mRNA expression between the two groups at1d. Compared to control group, hepatic Acox3mRNA expression in IUGR group decreased by31%(P=0.018) at7d,50%(P=0.022) at21d,37%(P=0.018) at2m and46%(P=0.012) at3m, respectively.(5) The expression of PPARy protein in WAT in IUGR group was significantly lower than control group at7d,21d,2m and3m. Compared to control group, the PPARy mRNA expression in WAT in IUGR group decreased by30%(P=0.027) at7d,48%(P=0.001) at21d,45%(P=0.002) at2m and56%(P<0.001) at3m, respectively.(6) There was no difference in LPL mRNA expression in WAT between the two groups at7d,2m and3m. The LPL mRNA expression in WAT was significantly lower in IUGR group than control group at21d (P=0.038).(7) There was no difference in AFABP mRNA expression in WAT between the two groups at7d. Compared to control group, the AFABP mRNA expression in WAT in IUGR group increased by43%(P=0.015) at21d,34%(P=0.033) at2m and73%(P=0.006) at3m, respectively.(8) There was no difference in serum leptin level between the two groups at Id. The serum level was significantly higher in IUGR group than control group at7d,21d,2m and3m.(9) There was no difference in serum adiponectin level between the two groups at1d and7d. The serum adiponectin level was significantly lower in IUGR group than control group at21d,2m and3m.(10) There was no difference in serum AFABP level between the two groups at Id and7d. The serum AFABP level was significantly higher in IUGR group than control group at21d,2m and3m.Conclusion(1) The lower hepatic PPARa expression in postnatal development period downregulated the expression of key genes (Cpt1a, Acox1, Acox3) involved in fatty acid metabolism, and then leaded to reduced ability of mitochondrial and peroxisomal fatty acid β-oxidation, which might involved in the occurrence of hyperlipidemia in IUGR rats.(2) The lower expression of PPARγ in WAT in postnatal development period lead to abnormality of adipocyte differentiation, which might involved in the occurrence of hyperlipidemia in IUGR rats.(3) Hyperleptinemia, hypoadiponectinemia and high serum AFABP level arose in postnatal development period in IUGR rats, and changes of these adipocytokines occurred earlier than blood lipid. Early prediction for hyperlipidemia might be carried out by monitoring the circulating leptin, adiponectin and AFABP in IUGR rats. Part Ⅲ Effect of Conjugated Linoleic Acid Intervention on Hyperlipidemia in IUGR ratsObjectiveTo evaluate the possibility of prevention of hyperlipidemia in IUGR rats by early nutrition intervention of conjugated linoleic acid.MethodsIUGR rats and control ones were obtained by previous method. The offspring in IUGR group were fed a21%normal protein diet with2%CLA (IUGR+CLA group) or a21%normal protein diet (IUGR group) after lactation at21d till3m. The offspring in control group were fed a21%normal protein diet with2%CLA (CON+CLA group) or a21%normal protein diet (control group) after lactation at21d till3m. The offspring rats were killed, and serum, liver tissue white adipose tissue was collected at the age of2m and3m. Eight rats entered the study at every time point for each group. Triglyceride (TG) in serum and liver were detected with the method of oxidase. Free fatty acid (FFA) in serum and liver were detected with copper color test. Hepatic PPARα protein and adipose PPARγ protein expression was detected by western blot. The expression of PPARα, Cpt1a, Acox1and Acox3mRNA in the liver tissue and PPARγ and LPL mRNA in the WAT was detected by real-time RT-PCR. The serum leptin, adiponectin and AFABP were measured by enzyme-linked immunosorbent assay (ELISA).Results(1) The average body weight of offspring was significantly lower in IUGR+CLA group and IUGR group compared to control group at2m (P<0.01), without difference between IUGR+CLA group and IUGR group. At3m, the average body weight of offspring was significantly lower in IUGR+CLA group compared to IUGR group and control group (P<0.05).(2) There was no difference in the serum TG level between the four groups at2m. At3m, the serum TG level in the IUGR+CLA group was significantly lower than IUGR group (P<0.05), no difference between IUGR+CLA group and control group. At2m and3m, there was no difference in serum FFA level between IUGR+CLA group and IUGR group or control group.(3) At2m, there was no difference in hepatic TG and FFA content between the four groups. At3m, the hepatic TG and FFA content in IUGR+CLA group was significantly lower than IUGR group (P<0.05), no difference between IUGR+CLA group and control group.(4) At2m and3m, the hepatic PPAR a protein expression in IUGR+CLA group was significantly higher than IUGR group (P<0.05). At2m, the hepatic PPAR a mRNA expression was significantly increased compared to control group (P<0.05), no difference between IUGR+CLA group and IUGR group. At2m, there was no difference in hepatic Cptla, Acoxl and Acox3mRNA expression between IUGR+CLA group and IUGR group or control group. At3m, compared to IUGR group, the hepatic PPAR a, Cptla, Acoxl and Acox3mRNA expression in IUGR+CLA group upregulated37%(P<0.01),43%(P<0.05),44%(P<0.01) and36%(P<0.01), respectively.(5) At2m and3m, the adipose PPAR Y protein expression in IUGR+CLA group was significantly higher than IUGR group (P<0.01), but lower than control group (P<0.01). At2m, there was no difference in the adipose PPAR γY mRNA expression between IUGR+CLA group and IUGR group or control group. At3m, the adipose PPAR γ mRNA expression in IUGR+CLA group downregulated than control group (P<0.05), but upregulated30%(P<0.05) than IUGR group. At2m and3m, there was no difference in adipose LPL mRNA expression between the four groups.(6) At2m and3m, the serum leptin level in IUGR+CLA group was significantly lower than IUGR group (P<0.05). At2m, there was no difference in serum adiponectin level between the four groups. At3m, the serum adiponectin level in IUGR+CLA group was significantly higher than IUGR group (P<0.01), no difference between IUGR+CLA group and control group. At2m, the serum AFABP level in IUGR+CLA group was significantly lower than IUGR group (P<0.01), but higher than control group (P<0.05). At3m, the serum AFABP level in IUGR+CLA group was significantly lower than IUGR group (P<0.01), no difference between IUGR+CLA group and control group.(7) There was no effect of CLA on normal rats, except that the serum leptin level in CON+CLA group was significantly lower than control group at2m and the serum AFABP level in CON+CLA group was significantly lower than control group at3m.Conclusion(1) CLA reduced the body weight, lowered serum TG and decreased hepatic lipid content in adulthood in IUGR rats.(2) CLA might induce hepatic PPAR a expression, which repaired the ability of mitochondrial and peroxisomal fatty acid β-oxidation, then play a role in anti-hyperlipidemia and decreasing hepatic lipid content in IUGR rats.(3) CLA might induce adipose PPAR Y expression, which promoted adipocyte differentiation, then play a role in anti-hyperlipidemia in IUGR rats.(4) CLA exerted an influence on circulating adipocytokines in IUGR rats and normal ones. |
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