Essential Role Of Visceral Adipose Browning In Obesity

With the development of social economy, obesity has become a global pandemic. Visceral obesity is the main risk factor for insulin resistance, type2diabetes and metabolic syndrome. The body’s energy intake increase and/or energy consumption reduction lead to obesity. Because of rich in mitochondria, brown adipose tissue makes energy dissipate in the form of heat which is produced through free fatty acid oxidation in lipid particles. Enhancing the function of brown adipose tissue can reduce fat accumulation that provides a way for the treatment of obesity and metabolic complications. Because it was found that white fat also has ability of browning under some condition, in recent years foreign scholars have turned their focus from how to strengthen the function of brown fat to white adipose browning.MicroRNAs (miRNAs) play important regulatory roles in a variety of biological processes including adipocytes differentiation, metabolic integration, insulin resistance and appetite regulation. The role of miRNA has tissue specificity and is more sophisticated on gene regulation. Mechanism of white adipose browning is still unclear and the process is under regulation of many transcription factors. Report about the regulation role of miRNA on white fat browning is very few so far.This study used diet induced obesity mice, obesity human, visceral primary preadipocytes culture model and miR-27b. We elucidated the ability of visceral adipose browning decreased in obesity and explained the molecular mechanism of miR-27b on regulation white adipose browning. MiR-27b played its role on adipose browning through regulating target genes Prdm16-a key transcription factor of browning adipocytes. Further we found that interference of miR-27b expression in vivo could enhance visceral fat browning in obesity and improve insulin sensitivity. These results would provide new strategies for treatment of visceral obesity and metabolic complications.Our research was divided into three parts.Part Ⅰ:Visceral adipose browning change in obese micePart Ⅱ:Molecular mechanism of visceral adipose browning change in obesityPart Ⅲ:Clinical study of visceral adipose browning change in obese humanPart Ⅰ:Visceral adipose browning change in obese miceObjective:It is well known that visceral obesity is closely related to the development of insulin resistance, type2diabetes, metabolic syndrome and cardiovascular disease. The body’s energy intake increase and/or energy consumption reduction lead to obesity. Enhancing the function of brown adipose tissue can reduce fat accumulation which provides a way for the treatment of obesity. Studies have found that function of brown adipose compensatory increase in obesity. Because mass of brown fat is limited, white adipose browning has become a research hotspot. How visceral adipose browning changes in obesity is unknown so far. The aim of the present study is to elucidate the change of visceral adipose browning in obesity.Methods:We used diet induced obesity mouse model and separated epididymal adipose tissue. The tissue was used to HE and Ucpl immunohistochemical staining. The expression of marker genes of visceral adipose browning, including Ucpl, Prdm16, Cidea, Cox8b and Pgcla was examined by Real-time fluorescent quantitative PCR and the protein expression of Ucpl and Prdm16was quantified by western blot. We detected the expression of miR-27b and Ucp1from different parts of white adipose tissue level in obese mice. MiR-27b was one kind of miRNAs found in our other research of white adipose browning.Results:The body and fat weight of DIO mouse was dramatically increased and glucose tolerance was impaired. We found that marker genes expression of visceral adipose browning, including Ucp1, Prdm16, Cidea, Cox8b and Pgc1α decreased significantly in visceral adipose of obese mice. The protein expression of Ucpl and Prdml6was also declined in obesity. However, level of miR-27b was elevated in visceral adipose tissue of obese mice. The expression of miR-27b was the highest in visceral fat compared to subcutaneous and perirenal fat.Conclusions:Visceral adipose browning of diet induced obesity mouse was declined. However, the expression of miR-27b increased. It suggested that miR-27b might have relationship with visceral adipose browning change in obesity.Part II:Molecular mechanism of visceral adipose browning change in obesityObjective:Importantly, the brown adipocyte-like cells in white adipose tissue can be generated by cold exposure or β3-adrenergic stimulation and regulated by transcription factor like Prdml6. The molecular mechanisms underlying this inducible brown adipogenesis have not been fully elucidated. In part Ⅰ, We have made it clear that visceral adipose browning declined in obesity, but its mechanisms have not yet been elucidated. Role of miRNA in white adipose browning is of little known and miR-27b is one kind of miRNAs found in our other research of white adipose browning. The present part focuses on regulation of white adipose browning and discussed the potential mechanisms.Methods:In vitro, we cultured primary epididymal preadipocytes of C57BL/6J mice aged3weeks by collagenase digestion method and transfected with LNA-anti-miR-27b or Pre-miRTM miR-27b Precursor. Marker genes expression of adipose tissue browning, including Ucpl, Prdml6, Cidea, Cox8b and Pgcla was detected by qRT-PCR. Protein expression of Ucpl and Prdm16was also examined by western blot. Oxygen consumption rate was measured by Seahorse XF24Extracellular Flux Analyzer. Luciferase report assays were used to confirm target genes of miR-27b.In vivo, on the basis of constructing diet induced obesity mice model, the mice were injected with lentivirus packaging miR-27b interference plasmid through tail intravenous one time, with a total dose of5x107TU/ml. The body and fat weight were measured and marker genes expression of visceral adipose browning, including Ucp1, Prdm16, Cidea, Cox8b and PGC1α were examined by Real-time fluorescent quantitative PCR and the protein expression of Ucpl and Prdml6was detected by western blot. The whole oxygen consumption of mice was examined using a set of16OxyMax(?) Metabolic Activity Monitoring chambers (Columbus, OH, USA).Results:In vitro, the expression of adipose browning marker genes, including Ucp1, Prdm16, Cidea, Cox8b and PGC1α was elevated in the primary visceral preadipocytes transfected with LNA-anti-miR-27b and oxygen consumption rate was also increased. On the contrary, the level of marker genes decreased in the preadipocytes transfected with Pre-miRTM miR-27b Precursor and oxygen consumption rate was also decreased. Luciferase reporter assays showed that miR-27b mimics reduced the activity of the reporter construct harboring a wild-type3’UTR but not the one with mutations in the seed sequences. This demonstrated that miR-27b interacts with the predicted target sites in the Prdm16transcript.In vivo, when miR-27b was interfered, marker genes expression of visceral adipose tissue browning, including Ucpl, Prdm16, Cidea, Cox8b and Pgc1α was elevated and whole oxygen consumption in mice was also increased. Insulin sensitivity of the mouse was improved compared with the control group. But there was no change in the body and fat weight, which might be associated with the duration of the effect of virus. Further research should be considered with the interference role of miR-27b for a long time.Conclusions:MiR-27b regulated the function of white adipose browning through inhibiting protein expression of the target gene Prdm16. Enhancing visceral adipose browning in obese mice through interfering miR-27b expression in vivo could reduce visceral fat drop and improve insulin sensitivity. Although there was no obvious change in body weight and fat mass, miR-27b was still a target for treating obesity and metabolic complication.Part Ⅲ:Visceral adipose browning change in obese humanObjective:Seeking visceral obesity treatments has been the hotspot in the study of scholars at home and abroad. Improving the function of white adipose browning gradually becomes a new way to treat obesity and metabolic complications. In part II, we have confirmed that miR-27b could regulate visceral adipose browning in vitro and in vivo. The present part aims to elucidate the change of visceral adipose browning in obese human and the role of miR-27b in regulation of primary human visceral adipocytes browning. It will clarify clinical significance of miR-27b in regulation human visceral fat browning and provide a new path to treat obesity and metabolic complications in clinic.Methods:Collecting greater omental adipose tissue of patients operated elective cholecystectomy (because of cholecystitis and gall bladder calculi and eliminate tumor and severe kidney disease) and recording patients’ basic information. Subjects were divided by BMI<25kg/m2and BMI>25kg/m2in the two groups. Using real-time fluorescent quantitative PCR to detect the expression of visceral adipokine like Adiponectin, Leptin, and MCP-1. The expression of miR-27b and marker genes of fat browning such as UCP1, PRDM16, CIDEA and PGC1α was detected by qRT-PCR. Primary human visceral preadipocytes were cultured by enzyme digestion method and then cells were transfected with mimic-miR-27b or LNA-anti-miR-27b. Marker genes of fat browning such as UCP1, PRDM16, CIDEA and PGC1α were detected by qRT-PCR.Results:Compared with BMI<25kg/m2group, the expression of marker genes of visceral fat browning like UCP1, PRDM16, CIDEA and PGCla decreased in BMI>25kg/m2group. MiR-27b expression was also elevated in obese human visceral fat. The gene expression of UCP1, PRDM16, CIDEA and PGC1α was decreased in primary human visceral preadipocytes which miR-27b was interfered. On the contrary, overexpression of miR-27b caused decline of above genes expression in primary human visceral preadipocytes.Conclusions:The function of visceral adipose tissue browning in obese human was impaired compared with normal weight people and miR-27b expression increased. The browning function of human visceral fat adipocytes was also regulated by miR-27b.