MiR27a Regulates Adipocyte Reprogramming To Promote Obesity-Induced Insulin Resistance Through Repressing Of PPAR?

Adipose tissue chronic inflammation and metabolic imbalance play important roles in the development of obesity induced insulin resistance.Macrophage-like reprogramming of preadipocyte is an important part of adipose tissue immune abnormalities,while whitening of beige adipocytes is a major factor of metabolic reprogramming of adipose tissue.The nuclear transcription factor PPAR? is a key regulator of macrophages recruitment and the metabolic propensity in adipose tissue.Therefore,regulating the upstream signal molecule of PPAR? might be involved in the reprogramming of adipocytes.It has been confirmed that a great number of miRNA was released in obese adipocytes tissue and plasma,which may be due to the hypertrophy of adipocytes or secretion of diseased adipocytes.Previous studies showed that obese adipocytes can secret miR27 a that pulls the recruitment and polarization of macrophages in adipose tissue.Meanwhile,some researches demonstrated that miR27 a can induce the reprogramming of somatic cells and play a role in the regulation of preadipocyte to differentiate into white adipocytes or brown adipocytes.In addition,bioinformatics analysis and our previousstudy proved that PPAR? is a downstream target of miR27 a.But it is unclear whether miR27 a induces the reprogramming of adipocytes by inhibiting PPAR?.In view of our preliminary work,miR27 a can inhibit the expression of PPAR?,activate the NF-?B pathway,and is positively correlated with obesity-insulin resistance and chronic inflammation of adipose tissue.PPAR? participates in the white-beige metabolic reprogramming of adipocytes through mitochondrial dynamics,mitochondrial biosynthesis and mitochondrial autophagy.Thus,we speculate that miR27 a might be a key factors to cause obesity-induced insulin resistance.via regulating the downstream target gene PPAR? topromote the reprogramming of adipose precursor cells to macrophage-like cells to inhibit white adipocytes browning by interfering with mitochondrial dynamics and mitochondrial biosynthesis and induce mitochondrial autophagy in beige adipocytes,to promote whitening of beige adipocytes and participate in the regulation of metabolic reprogramming of adipocytes.The present study intends to interfere with miR27 a and/or PPAR? at the in vivo and in vitro levels.To investigate the role of miR27 a in inhibiting PPAR? expression,regulating the reprogramming of adipocytes and inducing insulin resistance.To prove the key role of miR27 a in the regulation of cellular and molecular network of metabolic imbalance in obesity,providing a new intervention target for obesity-induced insulin resistance.Aim 1: To explore the mechanism of miR27 a inducing macrophage-like reprogramming of preadipocytes by inhibiting PPAR?.1.To identify that miR27 a is a key regulator of macrophage-like reprogramming of preadipocyte.By establishing a high-fat diet and miR27 a intervention model in C57BL/6J mice.The results showed that miR27 a is involved in regulating serum biochemical parameters,glucose tolerance and insulin tolerance in model mice,and promoting the secretion of inflammatory factors in serum and adipose tissue of model mice,affecting the morphology of adipose tissue.In addition,transfection of 3T3-L1 preadipocytes with miR27 a mimics increased phagocytosis and migration and increased the number of cells expressing the macrophage makers F4/80 and MHC compared to controls,demonstrating that miR27 a promotes macrophage-like reprogramming of preadipocytes.2.To investigate the mechanism of miR27 a in regulating macrophage-like reprogramming in preadipocytes via restraining PPAR?.In this part,the expressions of PPAR? gene and protein in adipose tissue of C57BL/6J mice treated with miR27 a were examined.The results showed that the expression of PPAR? gene and protein level in miR27 a overexpression group and high-fat diet group had a significant decrease in adipose tissue compared with the low-fat diet group,compared with the high-fat diet group,the miR27 a knockdown group adipose tissue PPAR? gene and protein levels were significantly increased.We examined the expression of PPAR? and NF-?B inflammatory signaling pathway proteins in 3T3-L1 preadipocytes transfected with miR27 a mimics in the absence or presence with rosiglitazone(PPAR? activator)were used to detect the phagocytosis and migration and related inflammatory signaling pathway protein expression.The results showed that transfection of 3T3-L1 preadipocytes with miR27 a mimics reduced PPAR? expression,activated NF-?B inflammatory signaling pathway.In addition,the data indicate that PPAR? agonists may reverse the activation of NF-?B pathway mediated by miR27 a overexpression and inhibit the development of macrophage-like features in preadipocytes 3T3-L1.It is revealed that miR27 a induces macrophage-like reprogramming of adipose precursor cells by inhibiting PPAR? and thereby activating NF-?B pathway.Aim 2: To investigate the mechanism of miR27 a in regulating metabolic reprogramming via restraining PPAR?.1.Correlative studies about miR27 a and metabolic reprogramming of adipocyte.Obese C57BL/6J mice was induced by high-fat diet,combined with low temperature acclimation.The data indicated that compared with low-fat diet group,the level of miR27 a in both serum and adipose tissue in the high-fat diet group rose up significantly.After hypothermia domestication,the level of miR27 a in both serum and adipose tissue in high-fat diet group decreased notably.Compared with low-fat diet group,the adipocyte volume in the high-fat diet group increased,the organelles decreased and the expression level of UCP-1 and thermogenic unique genes declined significantly in high-fat diet group.There was no obvious difference in each index between low-fat diet group and low-fat diet group with hypothermia domestication.Compared to high fat diet group,weight,the imparied glucose regulation,and the other indexes were improved partly in high-fat diet group with hypothermia domestication.And the adipocyte volume decreased,organelles increased,intercellular space dilated and the expression level of UCP-1 and partial thermogenic unique genes increased.Using a hypertrophic white adipocyte model induced by palmitic acid,combined with cold stimulation.The results show that it was the same in vivo experiment: compared with control group,the expression level of miR27 a in hypertrophic white adipocyte increased significantly,and fell back after cold stimulation.Meanwhile,compared to control group,the hypertrophic white adipocyte lipid droplet volume increased,mitochondrial ATP activity and the expression level of thermogenic unique genes decreased.After cold stimulation,the above indexes all improved.Hence,in consideration that the level of miR27 a and beige change indexes are negative correlation,we speculate that miR27 a may inhibit the white adipocyte browning,correlative with adipocyte metabolic reprogramming induced by obesity probably.2.To clarify that miR27 a is the key regulatory factor for adipocytes metabolic reprogramming.By establishing the various mice models treated with high-fat diet or miR27 a intervention,knock down miR27 a by administering hypertrophic white fat cells,miR27 a overexpression in beige adipocyte and prepare the conditioned medium fromhypertrophic adipocyte to mimic in vivo condition,conditioned medium was co-incubated with brite.The changes in related indicators are detected in brite cells.The results indicated that compared with low-fat diet group,the indexes in both miR27 a overexpression and high-fat diet group were the same: the adipose tissue volume increased,organelles decreased,the expression level of UCP-1 and thermogenic unique genes declined.Compared with high-fat diet group,in miR27 a knockout group,adipocyte volume decreased,organelles increased,intercellular space dilated and the expression level of UCP-1 and thermogenic unique genes increased.After miR27 a knockout,the volume of intracellular lipid droplets decreased,mitochondrial ATP activity and thermogenic unique genes expression level increased significantly in the hypertrophic white adipocyte,after the brite overexpressed miR27 a or incubated with the conditioned medium containing miR27 a,lipid drpolets became larger,and mitochondrial activity and the expression level of UCP-1 protein and thermogenic unique genes decreased.These data indicated that miR27 a can inhibit the white adipocyte browning and promote the whitening of brite cells.This research indicated that miR27 a participate in transdifferentiation between white adipocyte and brite,which is the key factor to modulate adipocyte metabolic reprogramming.3.To elucidate the mechanism of miR27 a in affecting the metabolic reprogramming of adipocytes via PPAR?We established the model of C57BL/6J mice high fat diet and miR27 a intervention,as well as hypertrophic white adipocyte knockdowns miR27 a joint PPAR? inhibitor T0070907 intervention model.The models detect changes in the expression levels of mitochondrial dynamics-related signaling pathway proteins and mitochondrial biosynthesis-related pathway proteins.The results showed that high-fat diet and miR27 a overexpression inhibited PPAR?-Bnip3 signaling pathway of adipose tissue,inhibited the expression of Drp-1,therefore blocked mitochondrial division.However,they had no significant effect on mitochondrial fusion protein Opa-1 protein level.At the same time,overexpression of miR27 a reduced the protein expression levels of PGC-1?,Nrf-1 and Nrf-2,having inhibited the biogenesis of mitochondria.However,high-fat diet combined with a lentiviral knockdown miR27 a was in the opposite direction,having increased the expression of adipose tissue PPAR? and Bnip3 protein and promoted the mitochondrial division and biosynthesis-related protein expression in adipocytes.We examined mitochondrial dynamics and mitochondrial biosynthesis-related pathway proteins at the cellular level,and the results were consistent with the high-fat diet combined with lentiviral knockdown miR27 a levels in the body.The knockdown of miR27 a in hypertrophic white adipocyte increased the expression of PPAR?,Bnip3 and Drp-1,had no significant effects on the level of Opa-1 protein,and increased the protein expression levels of PGC-1?,Nrf-1 and Nrf-2 in the meanwhile.In addition,we observed that the interaction of T0070907,the PPAR? inhibitors,reversed the impacted of miR27 a knockdown,suggesting that miR27 might interfere mitochondrial fission-fusion balance and mitochondrial biogenesis by inhibiting PPAR?,so that it could inhibit the transdifferentiation of white adipocytes to brite.Then we established the intervention model of low-fat diet,low-fat diet C57BL/6J mice combined with a lentivirus overexpression of miR27 a and brite overexpress miR27 a in combination with PPAR? agonist rosiglitazone,detecting the expression of mitophagy-related protein.The results showed that after giving animals and brite the overexpression of miR27 a,the level of PPAR? dropped,and expression levels of FUNDC1,LC3?/II,ATG5 and ATG12 protein were significantly increased.The activation of PPAR? caused by PPAR? agonist rosiglitazone inhibited the development of brite to white adipocyte-like features via the FUNDC1 pathway.It is proved that miR27 a induces mitochondrial autophagy in brite by inhibiting PPAR? and promotes the conversion of brite to white adipocytes.In summary,the promising findings of present study are : 1.Adipocytes reprogramming plays an important role in obesity-induced insulin resistance;2.miR27 a is a key factor in macrophage-like reprogramming and metabolic reprogramming of adipocytes;3.The molecular mechanism of miR27 a inhibits PPAR?,thereby activating the NF-?B pathway,causing a macrophage-like reprogramming of preadipocytes;4.The molecular mechanism of miR27 a inhibits Bnip3 pathway and PGC-1? pathway,causing imbalance of mitochondrial dynamics and mitochondrial biosynthesis in white adipocytes,and inhibiting white adipocytes browning through inhibiting PPAR?;5.The molecular mechanism of miR27 a activates FUNDC1 pathway,causing mitophagy in brite,and promoting the whitening of beige adipocytes via inhibiting PPAR?.This experiment provides a new mechanism for obesity-induced insulin resistanceand provides a new target for obesity-induced insulin resistance treatment and drug intervention.