| Objective: Docosahexaenoic acid (DHA) is the main n-3polyunsaturated fatty acids in the body. A recent study reported that DHAimproving insulin resistance (IR) as well as inhibiting inflammation, therefore,is used for the prevention and improvement of type2diabetes mellitus (T2D).However, the exact mechanism of this action is not entirely clear.There is a very close relationship between chronic low-gradeinflammation and insulin resistance induced by obesity. Many studies haveconfirmed the crosstalk between insulin signaling system andpro-inflammatory cytokines signal conduction pathway such as TNFα, IL-1β,IL-6and so on. Consequently pro-inflammatory pathway inhibits the insulinsignaling pathway leading to insulin resistance. Recent studies have found thatDHA has a strong anti-inflammatory role. Further more, some studies showedthat DHA increases adipose tissue insulin sensitivity in the high-fatdiet-induced obese mice by inhibiting the inflammatory response.The liver is the vital organ involved in glucose and lipid metabolism ofbody, as well as the important target organ for insulin resistance. Compared tothe adipose tissue, when inflammation occurs, except the invasion ofinflammatory cells, hepatic inherent macrophages (Kupffer cells) can beactivated to exacerbate the inflammatory response. How DHA inhibitinflammation and improve the mechanism of hepatic insulin resistance in type2diabetes?In this research, a high-fat diet combined with a small dose STZ-inducedobesity-related type2diabetic rats model was used as subject, before and afterdietary supplementation with DHA, to observe the improvement of ratshepatic insulin resistance and inflammatory response. This research is aim toinvestigate the anti-inflammation mechanism of DHA improving hepatic insulin resistance and to provide new experimental evidence for the preventionand treatment of type2diabetes and its complications.Methods:1Animal studiesThe livers of type2diabetic rats and type2diabetic rats fed with DHAprepared by Hou Lianguo et al were used as subjects. The experimentalanimals were divided randomly into Con, T2D and T2D+DHA group.2Biochemical analysesFasting blood-glucose was determined by Abbott Antuo fast bloodglucose meter. Fasting serum insulin was detected by radioimmunoassay.Insulin resistance of type2diabetic rats was evaluated by calculating theHOMA-IR and HOMA-ISI levels.3Determination of liver glycogen contentThe glycogen content of the liver tissue assay kit (Nanjing Jiancheng)was used to detect glycogen content in the liver tissue by anthrone method.4Gene expression profiling by quantitative RT-PCRPromega total RNA extraction kit extracted total RNA from liver tissue ofrats. G6Pase, PEPCK, TNFα, IL-1β, IL-6, IL-10, Arg-1mRNA levels wereassayed by quantitative RT-PCR with18S rRNA as an internal.5Detection of the target gene protein levels in liver of rats by Western blotTotal protein was extracted from liver tissue of rats. TAK1, P-TAK1, JNK,P-JNK, AKT, P-AKT, GSK3β, P-GSK3β and the PEPCK protein levels inliver of rats were assayed by Western blot.6Hematoxylin-eosin staining of liver tissueHematoxylin-eosin staining of paraffin sections of the liver tissue wasperformed to observe the pathological changes of the liver tissue.7Statistical analysisAll statistical analyses were conducted using SPSS16.0software. Thedata are expressed as means±SEM. Data were evaluated using One-wayANOVA when significance applied (P<0.05). Results:1DHA improved systemic insulin resistance in type2diabetic rats.The results showed obvious insulin resistance in type2diabetic rats.Dietary supplementation with DHA decreased the extent of insulin resistanceand increased insulin sensitivity, but could reduce neither blood glucose norserum insulin levels in type2diabetic rats.2DHA alleviated hepatic inflammatory response in type2diabetic rats.2.1Effect of DHA on hepatic inflammatory cell infiltration in type2diabeticrats.The results of hematoxylin-eosin staining of paraffin sections of the livertissue showed that dietary supplementation with DHA significantly reducedinfiltration of hepatic inflammatory cells in type2diabetic rats.2.2Effect of DHA on pro-inflammatory cytokines TNFα, IL-1β, IL-6mRNAexpression in the liver of rats.The results of quantitative RT-PCR showed that pro-inflammatory factorsTNFα, IL-1β, IL-6mRNA expression levels were significantly increased inthe liver tissue of type2diabetic rats, and that dietary supplementation withDHA obviously decreased these genes expression.2.3Effect of DHA on TAK1and JNK active forms in inflammatory signalingpathways.The results showed that TAK1and JNK are activated in liver tissue oftype2diabetic rats, meanwhile dietary supplementation with DHA can inhibitTAK1and JNK activation in liver tissue of type2diabetic rats. These resultsindicated that DHA can inhibit TNFα-mediated TNFα-TAK1-JNK signalingpathway.2.4Effect of DHA on anti-inflammatory factors IL-10, Arg-1mRNAexpression in the liver of type2diabetic rats.The results showed that the mRNA expression of anti-inflammatorycytokines IL-10, Arg-1were significantly reduced in the liver of type2diabeticrats and that dietary supplementation with DHA increased the expression ofthese anti-inflammatory cytokines.3DHA improved hepatic insulin resistance in type2diabetic rats. 3.1Effect of DHA on glycogen metabolism liver of rats.The results showed that GSK3β is activated in the liver tissue of type2diabetic rats, followed by the glycogen synthase phosphorylation inactivation,as a result the liver tissue can not be normal glycogen synthesis, manifested asdecreased glycogen content. Dietary supplementation with DHA can inhibitthe activation of GSK3β so that the glycogen synthesis increase.3.2Effect of DHA on the expression of gluconeogenesis-related genes in theliver of type2diabetic rats.The results showed that the mRNA expression of PEPCK and G6Pase,key rate-limiting enzymes in gluconeogenesis, were significantly increasedresulting in enhanced gluconeogenesis in the liver tissue of type2diabetic rats.Dietary supplementation with DHA could only decrease the mRNAexpression and protein expression of PEPCK.3.3Effect of DHA on AKT active form in insulin signaling pathways.The results suggested that AKT was inhibited in liver tissue of type2diabetic rats, however, dietary supplementation with DHA can made AKTactivate in liver tissue of type2diabetic rats. These results indicated that DHAcan inhibit TNFα-mediated TNFα-TAK1-JNK signaling pathways by reducingthe expression of TNFα, thereby improving hepatic insulin resistance of type2diabetic rats.Conclusion:1DHA improved systemic insulin resistance in type2diabetic rats.2DHA improved hepatic insulin resistance and alleviated inflammatoryresponse in type2diabetic rats.3DHA can relieve the inhibition of the insulin signal Ins/IRS-PI3K-AKT,by downregulating TNFα-mediated TNFα-TAK1-JNK inflammatory signalingpathway, to improve the liver of obese type2diabetic insulin resistance. |
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