| Objective:Investigate the weight-loss effect of electro-acupuncture (EA) treatment on Stat5 nestin-cre mice and its mechanisms; Obtain the genome-wide gene expression profiles in hypothalamus and Epi-WAT; Study the multiple pathway and targets about whole body regulating mechanism of weight-loss effect via acupuncture through STAT5-IGF1 pathway; Through this study, we aimed at to provide reliable and scientific evidences for the clinical application and promotion of acupuncture can resist obesity.Methods:12w male Stat5fl/fl and Stat5NKO mice were randomly divided into four groups: Stat5fl/fl group (only restriction), Stat5fl/fl+EA group (restriction+EA), Stat5NKO group (only restriction) and Stat5NKO+EA group (restriction+EA). After recorded the initial body weight, the EA groups were treated with acupuncture for two to four millimeter in Housanli and Neiting (left and right leg in turns) via 75% alcohol disinfection, connecting with the Hans electro-acupuncture device. Our parameter are dilatational wave (2/15 Hz) at a current intensity of 0.5 to 1 mA for 30 minutes, once a day, six times for one week, lasting for four weeks. During the remain day, we weighed food intake and body weight for each mouse.1.Evaluation of weight-loss effect via electro-acupuncture treatment on Stat5 NKO mice.Mice body weight and food intake were measured every week at the same time, TG, TC, LDL, glucose and leptin level were detected by Elisa combined colorimetric method. The morphology and size of adipocytes in epi-WAT were analyzed underlight microscope after HE staining, then we did cold room and metabolic cage experiments for assessing metabolic ability.2.Study the whole genome gene profiles of weight-loss effect via electro-treatment at Zusanli and Neiting points.We applied RNA-seq high-throughput sequencing technologies, combined with Tophat, Cufflinks and other software to calculate the level of Gene expression, following gene comparison on the UCSC genome browser, then we did GO (Gene ontology) and KEGG pathway to analysis genes classification. We also made comparison of gene expression profiles among Stat5fl/fl, Stat5fl/fl+EA group, Stat5NKO group and Stat5NKO+EA group, in order to detect target gene expression and signaling pathway for the weight-loss effect via electro-acupuncture.6-8w male C57BL6J wild type mice were randomly divided into different groups according to our experiment purpose. Mice were randomly divided into normal saline group and IGF1 group for gain of function study, mice were randomly divided into normal saline group and anti-IGF1 group for loss of function study; mice were randomly divided into AAV-empty group and AAV-Igfl group for over-express experiments after checking the over-expressed efficiency first.3.Study the effect on hypothalamus STAT5-IGF1 pathway in Stat5NKO obese mice induced by electro-treatment.We check IGF1 expression level in Stat5NKO mice and high fat diet induced wild type mice using WB at first. We measured body weight and food intake at different condition, then detected glucose and insulin level in blood, we also applied GTT and ITT test in mice for glucose and insulin tolerance at the same time. Proteins involved in insulin pathway were detected by WB in liver, including p-IRβ/t-IRβ, p-AKT/t-AKT etc. The TG and glycogen level in liver were measured by Elisa. Genes, like Pomc, Agrp, Npy, related to food intake in hypothalamus were detected by qPCR. Taking together, we explored the detail roles of EA on hypothalamus STAT5-IGF1 pathway.Results:1. The weight-loss effect of EA treatment on Stat5NKO obese mice.Compared to Stat5fl/fl group, body weight (P<0.01) and food intake (P<0.05) in the Stat5NKO group were significantly increased accompany with elevated ratio of epi-WAT/body weight (P<0.05) and increased size of adipocytes (P<0.01);Leptin, glucose, TG and TC levels in blood were abnormally elevated (P<0.01), the ability of cold resistance also decreased (P<0.01).Compared to Stat5NKO group, EA can significantly decreased body weight (P<0.01) and the ratio of epi-WAT/body weight (P<0.05), the adipocytes size, Leptin, glucose, TG and TC levels in blood were also decreased, with the elevated ability of cold resistance.2.EA altered genome-wide gene expressions in hypothalamus and epi-WAT on Stat5NKO obese mice.RNA-seq results showed that compared to Stat5fl/fl group,336 genes (80% in 420) were up-regulated and 84 genes (20% in 420) were down-regulated in hypothalamus which indicated that Stat5 is a negative regulator. Compared to Stat5NKO group, EA down-regulated 370 genes (91.4% in 405), but only up-regulated 35 genes (8.6% in 405). In epi-WAT, compared to Stat5fl/fl group,1047 genes (48.6% in 2153) were up-regulated and 1106 genes (51.4% in 2153) were down-regulated at the same time. Compared to Stat5NKO group,361 genes (79.5% in 454) were up-regulated and 93 genes (20.5% in 454) were down-regulated at the same time. KEGG pathway analysis indicated that these genes (co-regulated in hypothalamus and epi-WAT) were involved in multiple pathways, including ECM, PPAR, cell adhesion, glycerophospholipid /glycerolipid metabolism, but EA also changed some uniquely pathways except these, such as adipocytokine signaling, fatty acid, cytokine-cytokine receptor, arachidonic acid metabolism, insulin signaling pathway and cell adhesion molecules (CAMs), systemic lupus erythematosus, vascular smooth muscle contraction. These changes might suggest possible mechanisms of acupuncture treatment at "Zusanli" and "Neting" acupoints.3.STAT5-IGF1 signaling was involved in the underlying mechanism of weight-loss effect induced by EA.Compared to Stat5fl/fl group, IGF1 expression level was increased in the Stat5NKO group, while EA treatment reversed the IGF1 expression.We also found an elevated IGF1 expression in the hypothalamus of high fat diet induced obese mice just the same as that in Stat5NKO mice. Next we applied gain and loss of function study to explore detail roles of IGF1 in the development of obesity. Compared to the normal saline group, the IGF 1-injected mice displayed a significant increase in food intake, while the anti-IGF1 injected mice showed reduced food intake. Neither treatment affected body weight during the 48-hour observation period. We found that the virus-treated mice displayed enhanced appetite but unchanged body weight, mimicking the effects of acute central IGF1 administration. Next we found that acute central IGF1 treatment or prolonged IGF1 over-expression in the ARC significantly increased serum insulin and reduced blood glucose levels. Glucose and insulin tolerance tests showed that both treatments led to improved glucose tolerance and enhanced insulin sensitivity, but the anti-IGF1 injected mice caused decreased serum insulin and elevated blood glucose levels. Western blotting studies demonstrated that the abundance of total ISR-beta and Akt did not differ between control and AAV-Igf1 mice, whereas the expression of the phosphorylated (active) forms of Akt and ISR-beta was significantly increased in the AAV-Igf1 mice. UCP1 expression in BAT from AAV-Igf1 mice was significantly increased, as compared with control mice. We also found that liver weight and hepatic triglyceride and glycogen levels were significantly reduced in AAV-Igf1 mice, as compared to control mice. Liver morphology did not differ between AAV-Igf1 and control mice. Acute IGF1 injection and long-term over-expression of IGF1 in the mouse brain did not affect Agrp and Npy expression, but significantly reduced Pomc expression, while central administration of the anti-IGF1 group increased Pomc expression, but did not change Agrp and Npy expression.Conclusion:1. EA treatment induced weight-loss effect on Stat5NKO obese mice.2. Weight-loss effect of EA treatment on Stat5NKO obese mice might be attributed to the alteration of genome-wide gene expression due to the loss of Stat5.3. IGF1 can promote food intake, improve glucose tolerance and enhance body insulin sensitivity in vivo, which served as downstream target gene of Stat5.4. IGF1 was not the directly target in the weight loss effect induced by EA, the increased expression of IGF1 in hypothalamus was an compensatory change after obesity formation. |
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