| Obesity is considered as the greatest public health threat of the 21 st century. The obese patients endure the lower quality of life and expectations, and gain the higher risk of developing type 2 diabetes,cardiovascular disease, liver steatosis and cancer. Obesity-related insulin resistance is an early event in the process of starting diabetes and other diseases, which is related to diabetes, dyslipidemia, hypertension, glucose intolerance and endothelium dysfunction. Numberous studies indicate that patients with obesity and insulin resistance have low levels of inflammation, and the chronic inflammation is an important cause of insulin resistance. Toll-like receptor 4(Toll-like receptor4, TLR4) is a pattern recognition receptor which play an important role in innate immune response and pathogen recognition. Recent studies showed that TLR4 involved in the innate immune response to invasion of pathogenic organisms, and many diseases induced by aseptic inflammation(including the occurrence, development and prognosis of obesity) were also related to the TLR4 signaling. Therefore, the role of TLR4 in obesity-related insulin resistance is worthy of further study.Micro RNAs are a class of highly evolutionarily conserved small single-stranded non-coding RNA molecules of 19 to 22 nucleotides. It negatively regulates gene expression in the post-transcriptional level by inhibiting the translation process of target genes. In recent years, many studied had reported that a variety of metabolic processes were affected by mi RNAs, including glucose and lipid metabolism, insulin secretion,cell differentiation, and inflammation. It has been identified that some mi RNAs play an important physiological role in the action of insulintarget tissues(liver, adipose tissue, skeletal muscle). It has been confirmed that mi RNAs not only exist in the cell, but also outside the cell.In different pathological conditions, plasma levels of certain miRNAs specific changes occur. It considers the possible use of plasma miRNAs as a new generation of biomarkers for the early diagnosis of the disease.However, It is still unknown whether the changes of plasma miRNAs expression profile is associated with TLR4 signaling when insulin resistance is induced by the high-fat diet. To this end, we established a high fat diet-induced insulin resistance in mouse models, and the mi RNAs of differential expression in the state of insulin resistance were screened; Further investigations on the mice with insulin resistance treated by TLR4 inhibitors TAK-242, the changes of plasma miRNAs expression profile were studied; the relationship among TLR4, miRNAs and high fat diet-induced insulin resistance was investigated.In this study, a high fat diet-induced insulin resistance in mouse models was established. 36 male C57BL/6 mice(born 21 days) were selected, and were randomly divided into two groups; the control group(12) was fed with general basic diet(low fat diet, LFD), the experimental group(24) was fed with high fat diet(high fat diet, HFD).When the two groups appeared to be significant differences in body weight, the glucose tolerance test(GTT) and insulin tolerance test(ITT)were performed, the occurrence of insulin resistance in mice was observed.After the mouse model of insulin resistance was established,the role of TLR4 inhibitors TAK-242 on insulin resistance was studied. LFD mice were still fed with general basic diet(namely, LFD control group); HFD mice were randomly divided into 2 groups, each group has 12 mice, and was fed with high fat diet. One group of HFD mice was injected intraperitoneally with TLR4 inhibitors TAK-242(0.5mg TAK-242/kg body weigh), 2 times a week(namely, HFD-T); Another group of HFDmice was designed as control group(namely, HFD-C). LFD control group and HFD-C group mice were only injected with an equal volume Dimethyl sulfoxide(DMSO). After been giving mice TLR4 inhibitors for GTT and ITT for 5 months, the mice were sacrificed. The blood samples were collected using EDTA-K2 anticoagulant tubes, and the peripheral blood mononuclear cells(PBMC) and plasma were isolated immediately.Western Blot technique was carried out to detect PBMC TLR4 protein levels. Automatic biochemical analyzer was used to detect plasma glucose(GLU), triglyceride(TG), total cholesterol(TC), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol(LDL-C), alanine aminotransferase(ALT) levels. The differential expressed miRNAs was screened by expression profiling of plasma mi RNAs, which was detected using mouse microRNA microarray.The target genes of differential expressed miRNAs were predicted in TLR4 signaling pathway using bioinformatics methods, and the GO and KEGG database molecular annotation system were used to investigate the main effects of the mi RNAs targeted genes on the biological functions or signal pathway.The results showed that during the period of developing insulin resistance in mice model, compared with the control group, the weight of high fat diet mice gained more rapidly, that significant difference in body weight appeared after feeding to 20 weeks(P<0.05). After having been fed by high fat diet for 7 months, GTT and ITT results showed that the ability to regulate glucose in mice fed a high fat diet was impaired, and the hypoglycemic effect of insulin was reduced, which suggested that insulin resistance appeared in high fat diet group. After the mice with insulin resistance(keeping a high fat diet)had been treated by TLR4 inhibitors TAK-242 for five months, the glucose tolerance and insulin sensitivity were improved. The plasma biochemical variables results showed that, compared with LFD control group, plasma TG, TC, LDL-Clevels were significantly increased(P<0.05) in HFD-T and HFD-C groups; GLU, HDL-C and ALT levels were also increased, but no statistical significance(P>0.05). There was no significant difference in GLU, TG, TC, LDL-C, HDL-C, ALT levels between HFD-T and HFD-C groups(P>0.05). Western Blot results showed that, PBMC TLR4 protein expression was not found in LFD control group; a high level of TLR4 protein expression was detected in HFD-C group; compared with HFD-C group, the expression level of TLR4 protein was significantly lower in the HFD-T group. The results suggested that PBMC TLR4 protein was partly inhibited by TAK-242 in mice fed a high fat diet. The above results revealed that insulin resistance was affected by TLR4 in mice fed a high fat diet, and the insulin resistance was improved after inhibited expression of TLR4 protein.The screening results of micro RNA microarray chip showed that,comparing miRNAs expression between HFD group and LFD control group, 185 miRNAs were significant in the high fat diet group, including6 up-upregulated and 179 down-regulated miRANs, which suggested that the miRNAs expression profile of plasma was changed in mice with high fat diet-induced insulin resistance. A significant difference of miRANs was also found between HFD-T group and LFD control group, the total number of differential expression miRNAs was 171, and all of them were down-regulated, which suggested that there were also differences for plasma miRNA expression profile between LFD control group and HFD-T group. Comparing miRNAs expression between HFD-C group and HFD-T group, 13 miRNAs were significant in HFD-T group, all of them were down-regulated, which suggested that the changes of 13 mi RNAs expression levels may be associated with the partial inhibition of TLR4 protein expression.Bioinformatics analysis results showed that a total of 10 interaction proteins with TLR4 were predicted; the difference of mmu-miR-3095-3p,mmu-miR-5113, mmu-miR-709 and mmu-miR-335-3p expression levels was more than 1000 times between HFD-C group and HFD-T group,and their target genes can be found in TLR4-interaction protein or Toll like receptor signaling pathway; GO and KEGG analyses showed 74% of these target genes belonged to the biological processes genes, and the transcription factors accounted for 82%. Thus, the inhibitor of TLR4,TAK-242, can affect the miRNAs expression whose target genes were TLR4 signaling pathways downstream genes and TLR4-interaction protein genes.In conclusion, insulin resistance is successfully induced by a high fat diet in mice model; the plasma miRANs expression profile is different in mice with insulin resistance; inhibiting the expression of TLR4 protein may alleviate insulin resistance in mice, and with a change in the expression profile of plasma mi RNAs; the target genes of the significant differential expression mi RNAs can exist in TLR4-interaction protein or Toll like receptor signaling pathway. Results suggest that when insulin resistance occurs, there is a change in plasma miRNAs expression profile,this change is associated with TLR4 and its signaling pathways.The finding enrichs the possible mechanisms of insulin resistance,and provides experimental and theoretical foundations for further studies on the role of TLR4 and mi RNAs in insulin resistance. It also provides a basis for finding miRNAs diagnostic markers for early diagnosis of insulin resistance. |
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