Effects And Mechanisms Of Intrauterine Malnutrition And Early Growth Hormone Intervention On AMPK-α1/SREBP-1c/ACC-1Pathway In Rats

Background:Epidemiological studies have shown that small for gestational age (SGA) is closely related to dyslipidemia, which plays a central role in the pathogenesis of adult metabolic syndrome. Thus, it is necessary to find the potential targets for lipid dysregulation in SGA. AMP-activated protein kinase (AMPK), one of the serine/threonine kinase protein kinase and a heterotrimer of three subunits, ie, α, β, and γ, is widely existing in eukaryotic cells, playing an important role in regulating the hepatic lipid homeostasis. It has been established that abnormal expression of AMPK-al participates in dyslipidemia. Up to now, however, there is no report about the dynamic change of AMPK-al in the whole postnatal development period in SGA individuals. And whether the expression of AMPK-al and AMPK-al-mediated fatty acid catabolism leads to dyslipidemia in adulthood in SGA rats remains unclear. Therefore, we aim to explore the role of AMPK-α1and its targeted genes in the occurrence of dyslipidemia in adult SGA rats. In addition, epigenetic modification has been demonstrated to participate in developmental programming of metabolic disease in SGA individuals, but so far little attention has been paid to the association between epigenetic modification of AMPK-α1gene and hepatic lipid metabolism in SGA rats. Recently, the role of microRNAs (miRNAs) in lipid metabolism arouses more concern. Researchers found that miR-370could affect AMPK-α1、sterol regulatory element-binding protein-1c (SREBP-1c) and acetyl CoA carboxylase1(ACC-1) expression via modulation of miR-122to regulate lipid metabolism. Whether these two miRNAs are also involved in the regulation of AMPK-α1、SREBP-1c and ACC-1expression in SGA rats needs to be further discussed.At present, AMPK has been considered as an important target for hepatic lipid dysregulation. Many studies have shown that growth hormone (GH) therapy in SGA children would be beneficial for various metabolic parameters. It was reported that exogenous GH therapy could improve the total and phosphorylated protein levies of AMPK-α, resulting in improvement of lipid metabolism. For SGA individuals, whether GH could early prevent dyslipidemia in adulthood by activating AMPK-α1remain unclear. The results of the study were expected to provide theoretical basis for GH therapy in SGA children.Part Ⅰ Effects and mechanisms of intrauterine malnutrition on AMPK-α1/SREBP-1c/ACC-1pathway in ratsObjective:To explore the molecular mechanism of dyslipidemia by observing the dynamic change of AMPK-α1/SREBP-1c/ACC-1pathway, pathway associated miRNA and histone acetylation in the whole postnatal development period in hepatic tissue of SGA rats.Methods:SGA rat model was established by using semi-starvation treatment during the whole pregnancy. At1,21and70days of age, the serum and liver tissue of offspring rats were collected after measuring the body weight and body length. Serum triglycerides (TG) levels and hepatic TG contents were measured using commercial kits by employing enzymatic assay. The expression of AMPK-α1and nSREBP-1c protein in liver tissue was assessed by western blot. The expression of AMPK-α1, SREBP-lc and ACC-1mRNA in liver tissue was detected by real-time RT-PCR. The acetylation status surrounding the promoter region of AMPK-al in liver tissue was investigated by CHIP-PCR. The different miRNA expression profiles in liver tissue between SGA and AGA rats were examined by miRNA microarray. Real-time RT-PCR was performed to validate the results.Results:1. Comparison of body weight and body length between SGA and AGA rats:At1day of age, the body weight and body length of SGA rats were significantly lower than AGA rats (all P<0.01). From1to21days of age, SGA rats showed catch-up growth, however, the body weight and body length of which were still lower than AGA rats at21days of age (P<0.05and P<0.01). At70days of age, SGA rats still showed lower body weight and body length when compared to AGA rats (all P<0.05).2. Comparison of serum TG levels between SGA and AGA rats:There were no differences of serum TG levies between SGA and AGA rats at1or21days of age. At70days of age, compared to AGA rats, SGA rats exhibited massive elevation of serum TG levles (P<0.05).3. Comparison of hepatic TG contents between SGA and AGA rats:At1and21days of age, the hepatic TG contents of SGA rats were similar with AGA rats. At70days of age, the hepatic TG contents of SGA rats was significantly higher than AGA rats (P<0.05). 4. Comparison of protein expression between SGA and AGA rats:Hepatic protein expression of AMPK-al and nSREBP-lc were similar between SGA and AGA rats at1day of age. At21and70days of age, SGA rats showed significantly decreased expression of AMPK-al (all P<0.05) and increased expression of nSREBP-lc (all P<0.05) at protein levels in liver tissue when compared to AGA rats.5. Comparison of gene mRNA expression between SGA and AGA rats:Hepatic mRNA expression of AMPK-al, SREBP-lc and ACC-1were similar between SGA and AGA rats at1day of age. At21and70days of age, SGA rats showed significantly decreased expression of AMPK-α1(P<0.05and P<0.01) and increased expression of SREBP-lc (all P<0.01) and ACC-1(P<0.05and P<0.01) at mRNA levels in liver tissue when compared to AGA rats.6. Comparison of histone H3acetylation surrounding the promoters of AMPK-al between SGA and AGA rats:The levels of acetylated histone H3surrounding the promoters of AMPK-al in liver tissue were similar between SGA and AGA rats at1day of age. At21and70days of age, the levels of acetylated histone H3surrounding the promoters of AMPK-al in liver tissue were significantly lower in SGA rats than in AGA rats (all P<0.01).7. Comparison of miRNA expression profiles between SGA and AGA rats:At1day of age,8upregulated miRNAs with more than1.3changes and22downregulated miRNAs with less than0.75changes were discovered in liver tissue of SGA rats.8. Comparison of miR-370and miR-122expression between SGA and AGA rats: At1day of age, the hepatic levels of miR-122were similar between SGA and AGA rats, while the hepatic levels of miR-370were significantly lower in SGA rats than in AGA rats (P<0.05), which were consistent with the results obtained by microarray analysis. At21and70days of age, the hepatic levels of miR-122were similar between SGA and AGA rats, while the hepatic levels of miR-370could not be detected at the two time points.Conclusion:1. The growth restriction in offspring subjected to maternal undernutrition during pregnancy observed at birth, persisted to adult.2. Dylipidemia observed in adult SGA rats might be a result of abnormal expression of AMPK-α1/Srebp-1c/ACC-1pathway in liver tissue during childhood and adulthood.3. The levels of acetylated histone H3surrounding the promoters of AMPK-α1were significantly lower in liver tissue of SGA rats than in AGA rats during childhood and adulthood.4. The expression of miRNA-370was significantly lower in SGA rats than in AGA rats, which may participated in metabolic regulation in embryonic and neonatal period of SGA rats.Part Ⅱ Effects and mechanisms of early growth hormone intervention on AMPK-α1/SREBP-lc/ACC-1pathway in SGA ratsObjective:To explore whether growth hormone intervention could improve lipid metabolism via accommodating AMPK-α1/SREBP-1c/ACC-1pathway and histone acetylation in liver tissue of SGA rats.Methods:SGA rat model was established by previous method. Daily subcutaneous injections of rhGH or saline were given from21to35days of age in AGA or SGA rats, then the rats were randomly divided into four groups:SGA+rhGH group, SGA+NS group, AGA+rhGH group and AGA+NS group.. The serum and liver tissue of offspring rats were collected after measuring the body weight and body length. Serum insulin-like growth factor1(IGF-1) levels were accessed by enzyme-linked immunosorbent assay. Serum TG levels and hepatic TG contents were measured using commercial kits by employing enzymatic assay. The expression of AMPK-α1and nSREBP-1c protein in liver tissue was assessed by western blot. The expression of AMPK-al, SREBP-lc and ACC-1mRNA in liver tissue was detected by real-time RT-PCR. The acetylation status surrounding the promoter region of AMPK-al in liver tissue was investigated by CHIP-PCR. The expression of miR-122in liver tissue was performed by real-time RT-PCR.Results:1. Comparison of body weight and body length among groups:At70days of age, the body weight and body length of SGA+NS group were still significantly lower than AGA+NS and SGA+rhGH groups (P<0.01and P<0.05); the body weight and body length were similar among SGA+rhGH, AGA+NS and AGA+rhGH groups.2. Comparison of serum IGF-1levels among groups:In order to explore the effectiveness of intraperitoneal injection of rhGH, we measured the serum IGF-1levels at21,35and70days of age by employing enzyme-linked immunosorbent assay. At21days of age, compared to AGA group, SGA group showed lower serum IGF-1levles (P<0.05). At35days of age, SGA+rhGH group showed higher serum IGF-1levels than SGA+NS group (P<0.05). At70days of age, there were no differences of serum IGF-1levies among SGA+rhGH, SGA+NS, AGA+NS and AGA+rhGH groups.3. Comparison of serum TG levels among groups:At70days of age, compared to AGA+NS and SGA+rhGH groups, SGA+NS group exhibited massive elevation of TG levies (all P<0.05); there were no differences of serum TG levles among SGA+rhGH, AGA+NS and AGA+rhGH groups.4. Comparison of hepatic TG contents among groups:At70days of age, compared to AGA+NS and SGA+rhGH groups, SGA+NS group showed increased hepatic TG contents (all P<0.05); there were no differences of hepatic TG contents among SGA+rhGH, AGA+NS and AGA+rhGH groups5. Comparison of protein expression among groups:At70days of age, SGA+NS group showed significantly decreased expression of AMPK-Ω1(all P<0.05) and increased expression of nSREBP-1c (all P<0.05) at protein levels when compared to AGA+NS and SGA+rhGH groups; there were no differences of AMPK-al and nSREBP-lc expression expression at protein levels among SGA+rhGH, AGA+NS and AGA+rhGH groups.6. Comparison of gene mRNA expression among groups:At70days of age, SGA+NS group showed significantly decreased expression of AMPK-al (all P<0.05) and increased expression of SREBP-lc (all P<0.05) and ACC-1(all P<0.05) at mRNA levels when compared to AGA+NS and SGA+rhGH groups; there were no differences of AMPK-α1、SREBP-lc and ACC-1expression at mRNA levels among SGA+rhGH, AGA+NS and AGA+rhGH groups.7. Comparison of histone H3acetylation surrounding the promoters of AMPK-al among groups:At70days of age, the levels of acetylated histone H3surrounding the promoters of AMPK-al were significantly lower in SGA+NS group when compared to AGA+NS and SGA+rhGH groups (all P<0.05); which were similar among SGA+rhGH, AGA+NS and AGA+rhGH groups.8. Comparison of miR-122expression among groups:There were no differences of miR-122expression among SGA+NS、SGA+rhGH、AGA+NS and AGA+rhGH groups at70days of age.Conclusion:1. Early postnatal administration of GH could improve the growth retardation in SGA rats. 2. Early postnatal administration of GH could also improve lipid metabolism in SGA rats, which might be associated with the expression change in AMPK-α1/SREBP-1c/ACC-1pathway.3. GH could induce changes of histone acetylation.