| AimsBased on a rat model of adult-onset insulin resistance (IR) induced by artificial nutrition control during suckling period, this study aimed to explore the effect and mechanisms of early postnatal nutrition environment on long-term metabolic reprogramming in offspring rat, and clarify the impact of early postnatal nutrition on adult-onset IR. The results will provide valuable evidences for optimal early postnatal feeding strategies to prevent and reduce the incidence of metabolic diseases such as IR.Research contentsThis study included 3 parts experiments. Part 1, Phenotype establishment of rat models. Three early postnatal nutrition (overnutrition, normo-nutrition and undernutrition) rat models were set up and IR status between groups were analyzed. Part 2, Mechanism exploration. The signaling pathway related to IR was screened and expressions for key gene signals in insulin targeted tissues were detected. The epigenetic regulation mechanism was also investigated. In addition, serum fatty acids and amino acids profiles were measured in this work. Part 3, Diet induction. The risks of developing IR and differential expression levels of key genes were compared between groups when exposed to high fat diet.MethodsOn postnatal day 2, Sprague-Dawley pups were assigned randomly to overnutrition (SL), normo-nutrition (NL) and undernutrition (LL) via artificially adjusted the number of pups nursed per dam. Litter size were adjusted to 3 pups/dam,10 pups/dam and 20 pups/dam for SL, NL and LL group, respectively. Only male rats were selected to be investigated in the current study. Rats were fed with normal diet after weaning (postnatal day 21). At 6 weeks after birth, each group was randomly divided into two subunits, and fed with normal diet or high fat diet till 16 weeks old. Physiological parameters, biochemical values and IR status were dynamically monitored, as well as morphological changes of insulin targeted organs, including skeletal muscle, visceral white adipose tissue and liver. Gene expression of IR related signaling pathway was detected by whole transcriptome sequencing. Real-time PCR and western blotting were employed to determine the expressions for mRNA and protein of important gene signals. In addition, DNA methylation of pivotal difference gene was detected via bisulfite sequencing PCR method. Moreover, gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were used to measure serum fatty acids and amino acids profiles.Results1. Comparing to rats in NL, the body weight of rats in SL group were 37.5% and 15.1% increased at 3 weeks and 16 weeks of age, respectively. Whereas, LL rats decreased 34.9% and 12.6% at week 3 and week 16, respectively. The weights of liver, skeletal muscle and visceral white adipose were shown the same pattern with body weight. However, we only investigated the significant differences of visceral adipose weight between SL and NL when expressed relative to body weight in all three groups of models. The difference of body weight and visceral white adipose between groups were preserved in rats when fed with high fat diet.2. At 16 weeks of age, SL rats exhibited increased IR index and glucose intolerance accompanied with elevated serum triglyceride and free fatty acid concentrations. However, the rats fasting serum glucose level was normal. There was no significant difference in IR status between LL and NL rats.3. The number of epididymal adipocyte increased at weaning and the adipocyte area increased at 16 weeks of age in SL rats. The epididymal adipocyte area persistently decreased in LL rats.4. The whole transcriptom study suggested that insulin signaling pathway was one of the key pathways that has been effected by different early postnatal nutrition. Insulin receptor(Insr), insulin receptor substrate 1(Irs1), protein kinase B(Akt2) and glucose transporter 4(Glut4) in IR related pathways were selected and to be investigated in the following study. Significantly decrease in mRNA levels for Glut4 and Irsl were observed in epididymal fat of SL rats compared to NL rats, accompanied by decreased protein levels for above all four components. In gastrocnemius, the mRNA levels for Akt2 and Glut4 significantly decreased in SL rats as well as protein levels of Insr and Glut4. For LL rats, we observed decreased expression of Glut4 mRNA in epididymal fat, whereas Akt2 mRNA and Irs1 protein levels were increased in gastrocnemius.5. In epididymal fat, gene Glut4 was characterized by hypomethylation associated with higher expression at 3 weeks of age, and hypermethylation associated with lower expression at 16 weeks of age in SL rats compared with NL rats.6. Significantly increased serum long chain saturated fatty acids, monounsaturated fatty acids and arachidonic acid were found in SL rats with lower serum concentration of taurine.7. Fed with high fat diet, SL rats developed more serious IR and glucose intolerance. Hyperleptinemia and hepatic steatosis were also observed. The gene expression levels of insulin pathway signals in gastrocnemius and epididymal fat further decreased.8. Exposed to high fat diet, LL rats exhibited glucose intolerance, adipocyte area dramatically expansion and severe hepatic steatosis, accompanied with significantly declined expressions for some insulin pathway signals in gastrocnemius and epididymal fat.Conclusions1. Early postnatal overnutrition induced persistent overweight and visceral white adipose accumulation in male rats while early postnatal undernutrition was shown the opposite effects, which not affected by dietary influence.2. Early postnatal overnutrition leaded to adult-onset IR in rats. Blunting insulin receptor signaling pathway in skeletal muscle and visceral white adipose, changing of Glut4 gene methyelation in visceral white adipose, elevating circulating free fatty acids levels and abnormal serum profiles of fatty acids and amino acids may contributed to the occurrence and development of IR in rats.3. High fat diet aggravated IR and the impaired insulin receptor signaling pathway in SL rats due to early postnatal nutrition overload.4. Although early postnatal undernutrition had no impact on adult-onset IR in rats, the risk for IR dramatically increased when exposed to high fat diet. |
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