MiR-27b Is Regulated By Glucocorticoid And Controls The Browning Effects Of White Adipose Via PRDM16

As we all known, glucocorticoid （GC） is the most important factor to regulatethe body’s metabolism. A large number of studies had confirmed that GC promotedadipocyte differentiation, increased triglyceride synthesis, inhibited lipolysis,promoted fat accumulation and caused metabolic syndrome. However, intracellularglucocorticoid concentrations can differ greatly from blood levels due to the action of11-hydroxysteroid dehydrogenase type1（11-HSD1）, an enzyme which canreactivate glucocorticoid by converting inert11-keto-glucocorticoids into activeforms （cortisol in humans, corticosterone in rodents）. The clinical study also foundthat serum GC levels of patients with metabolic syndrome were normal, but the highexpression of11-HSD1in liver and adipose tissue led to obesity, hyperglycemia anddyslipidemia. To find out molecular mechanisms of GC on regulating adipose canhelp us to elucidate causes of metabolic syndrome and lay the foundation for effectiveprevention and treatment of metabolic syndrome.Some studies found that browning of white fat could promote energyconsumption and improve metabolism. Enhancing the browning of white fat hasbecome a new target for the treatment of metabolic disorders. GC is a powerfulregulator of white adipocyte differentiation and recently has been reported to have anadditional role in brown adipose function. Our previous studies also found that theexpression of BAT-specific genes, including UCP1, Cidea, Cox7a1and Cox8b, weresignificantly decreased in the BAT of dexamethasone-treated mice. However, the roleof GC in the browning of white fat remains largely undetermined. Therefore, our study filtered out specific miRNAs to regulate browning of whitefat through treatment white adipocytes with GC, and then focused on the molecularmechanisms of GC in regulation on browning white adipose and body’s metabolism.It would provide a new strategy for preventing and treating of metabolic syndromethrough improving the body’s energy metabolism in clinic.Our research was divided into two parts.Part I: Essential Roles of glucocorticoid in regulating browning of white adiposePart II: Molecular mechanisms of glucocorticoid in regulating browning of whiteadiposePart I: Essential Roles of glucocorticoid in regulatingbrowning of white adiposeObjective. Glucocorticoid （GC） is a powerful regulator of white adipocytedifferentiation and recently has been reported to have an additional role in brownadipose function. Soumano et al. reported that in a brown adipose cell line, GCinhibited the transcriptional response of the uncoupling protein-1（UCP1） gene, aspecific mitochondrial protein involved in the regulation of thermogenesis and energyexpenditure in BAT. Strack et al. also found that corticosterone decreasednon-shivering thermogenesis and increased lipid storage in brown adipose tissue ofrats. Moreover, recently we observed that the expression of BAT-specific genes,including UCP1, Cidea, Cox7a1and Cox8b, were significantly decreased in the BATtissue of dexamethasone-treated mice. However, the role of GC in the browning ofwhite fat remained largely undetermined. The aim of the present study was toinvestigate regulation role of GC on white adipose browning. Methods. C57BL/6J mice were administrated with dexamethasone （dex） orvehicle （con）. To determine a role for GCs in browning of white fat, we measuredthe expression of brown fat functional genes in WAT of mice following treatmentwith dexamethasone, a synthetic long-term glucocorticoid. In vitro, UCP1geneexpression was investigated in glucocorticoid, including dexamethasone andoverexpression11-HSD1treatment in primary adipocytes isolated from SAT andVAT of mice, using real-time PCR.Results. We found that dexamethasone caused an enlargement of whiteadipocyte size and disturbance of glucose metabolism. The expression of UCP1inWAT, including both subcutaneous adipose tissue （SAT） and visceral adiposetissue （VAT）, was significantly decreased in dexamethasone-treated mice.Furthermore, in vitro experiments verified that UCP1expression was inhibited bydexamethasone treatment in primary adipocytes isolated from SAT and VAT.Consistent with the reduced UCP1gene expression, dexamethasone led to a markeddecrease in the basal oxygen consumption rate （OCR）.Conclusions. These data indicated that dexamethasone inhibited the levels ofmitochondrial activity, thus altered the thermogenic program of WAT. This suggestedthat glucocorticoid played a negative role in the browning of white fat.Part II: Molecular mechanisms of glucocorticoid inregulating browning of white adiposeObjective. It is well known that WAT is the form of energy storage in the body, themore energy intake, the more volume and number of white adipose. Because of richin mitochondria, BAT dissipates energy for thermogenesis by intracellular oxidative phosphorylation to reduce the white fat storage. Recent studies found that, similar tothe brown fat, browning of white fat could promote energy consumption and improvemetabolism. So enhancing the browning of white fat had become a new target for thetreatment of metabolic syndrome. The first part of this study suggested that GCinhibited browning of white fat and over-expression of11beta-HSD1also suppressedbrowning of white adipocytes by increasing the activity of endogenous GC. Therefore,we further studied the molecular mechanisms of GC in regulating the browning ofwhite fat.Methods. To identify miRNAs which were regulated by dexamethasone, weused miRNA microarray analysis of human white adipocytes treated withdexamethasone for24hours. We further examined the expression of miR-27b byreal-time PCR to verificate the miRNA microarray results in both human and mouseadipocytes following dexamethasone treatment. To further characterize theassociation of GR with the miR-27b promoter, ChIP experiments were conducted inwhite adipocytes. To investigate the function of miR-27b in the browning of white fat,mimic-miR-27b or anti-miR-27b was transfected into primary cells from SAT andVAT to increase or inhibit miR-27b expression respectively. To identify potentialmiR-27b target genes, the miRNA target prediction web site TargetScan wasemployed. A putative miR-27b target site was identified at a highly evolutionarilyconserved octamer seed motif within the3’UTR of Prdm16. To determine whethermiR-27b could directly target Prdm16mRNA, we cloned the3’ UTR segment ofPrdm16containing the putative miR-27b target site （mPrdm16₃’ UTR-NM₀22113;3824-3831）, or a mutated seed site, into a pMiRGLO luciferase reporter construct. Toverify these findings at the protein level, miR-27b expression was modulated in SATadipocytes by transfecting mimic-miR-27b or VAT adipocytes by transfectinganti-miR-27b. Cells were simultaneously transfected with anti-miR-27b to test whether inhibition of miR-27b was effective at promoting browning effects in theabsence of Prdm16. Finally, we used the C57BL6/J mice with GC stimulation andmiR-27b was interfered for further observation the function of miR-27b in thebrowning of white fat in vivo.Results. miR-27b was one of the most upregulated miRNAs afterdexamethasone treatment. GC regulated the transcription of the miR-27b gene via thebinding of GR to the endogenous promoter region of miR-27b. miR-27b served as apotent negative regulator of the browning effects on white adipocyte. A putativemiR-27b target site was identified at a highly evolutionarily conserved octamer seedmotif within the3’UTR of Prdm16, a factor that controls both the development ofclassical brown fat and the browning of white fat. The effect of miR-27b on thebrowning of white adipocytes was largely mediated by Prdm16as its direct target.11-HSD1regulated miR-27b and decreased the browning effect of WAT, furthersuggesting miR-27b as a new target for prevent obesity. In vivo, experiments toverify the role of miR-27b in regulating the browning of white fat and affecting theoverall metabolism of the body were suggested that the anti-miR-27b-inducedbrowning effect of WAT is metabolically functional and had a favorable impact onglucose metabolism in mice.Conclusions. In summary, our data showed that miR-27b was activated by GCand pointed to a critical role for miR-27b in the control of the browning effect onWAT through PRDM16. It was suggested that miR-27b might represent a promisingtherapeutic target for the treatment of obesity and metabolic syndrome.