| Mobilizing BAT(Brown adipose tissue)and browning WAT(white adipose tissue)into beige adipocytes,have emerged as a promising target to combat obesity and other metabolic disorders due to their common thermogenic ability of energy expenditure and heat production through mitochondria.In general,browning of white adipose tissues is characterized by the certain external factor stimulation such as chronic cold exposure and hormonal interaction,which lead to the formation of multilocular lipid droplets,increased expression of UCP1 and mitochondrial number,which results in beige adipocyte development.Despite the major thermoregulatory role of classical BAT,excess amount of WAT in humans and their capability of trans-differentiation,makes the induction of browning within WAT more interesting strategy to counteract weight gain.Mitochondrial biogenesis has substantial influence during beige adipocyte differentiation in WAT and its dysfunction ends up in detrimental effects on energy metabolism in adipocytes.Mitochondrial transcription and replication are regulated by nucleus-encoded genes,and the product is then translocated to mitochondria.In addition to the induced expression of UCP1 upon external physiological stimuli,PGC1?(PPAR?coactivator1-?),Polg(mtDNA polymerase ?)are activated to initiate internal metabolic response.These PGC1? and Polg along with Nrf1,2(Nuclear respiratory factor 1,2),TFAM(transcription factor A,mitochondria)and mTERF1(mitochondrial transcription termination factor 1)controls the transcription and replication of mtDNA,and fallout in enhanced mitochondrial biogenesis and function.Many pharmacological and natural agents have shown significant increase in WAT browning by activation of these transcription factors and signaling pathways of mitochondrial biogenesis.GH(Growth hormone)not only play central role in growth and development but also regulates the adipocyte proliferation and differentiation.It has been reported that GH inhibits adipocyte differentiation,reduces triglyceride accumulation,increases lipolysis,lipid deposition,and the reduction in its secretion has been linked to obesity.Generally,GH upon binding to GHR,can activate PI3K,MAPK and STAT5 signaling pathways.However,STAT5b has been reported to be specifically activated by GH.In addition to this,various studies have shown that,STAT5 can increase the expression of UCP1 and hence directly or indirectly involved in browning of WAT.However,proper molecular mechanism of GH in beige fat formation/formation o is still unknown.In this study,we hypothesized that,GH can transform WAT into beige adipocytes by increasing mitochondrial biogenesis and by changing UCP1 expression.Method1.3t3-L1 preadipocytes were cultured and differentiation was induced in the presence and absence(control)of Growth hormone(GH)after which lipid content was measured by Oil red O staining.2.To elucidate the effect of GH on adipocytes browning,mitotracker green fluorescent dye was used to evaluate the mitochondrial content and distribution after GH treatment.3.To further investigate the browning effect,protein expression of browning markers(UCP1 and Cidea)was checked by Western Blot.4.To determine the effect of GH on mitochondrial biogenesis,Protein and gene expression of key regulators of mitochondrial regulators(Polg,PGC1?,Tfam,PPARy)were determined by Western Blot and Real time PCR.5.Three inhibitors PD98059(MAPK inhibitor),LY294002(PI3K inhibitor)and Lestaurtinib(JAK/STAT inhibitor)were used to treat 3T3-L1 white adipocytes along with and without Growth hormone to figure out GH signaling pathway closely related to mitochondria changes.6.The luciferase reporters of Polg,PGC1?(Mitochondrial biogenesis markers)and Cidea(thermogenesis marker)were constructed,each reporter was transfected into Hepg2 cells for 24 hours.Cells were then treated with GH and luciferase assay was carried out by Dual luciferase assay kit.7.C57BL/6J mice were fed with high fat diet for 12 weeks and divided into 2 groups.One group was injected with GH every day for 1 month according to their bodyweight(ng/g.bw)while another group received PBS.Mice body weight was measured weekly during the treatment.8.Mice were sacrificed,whole body weight as well as organ weights were measured.Subcutaneous and visceral adipose tissues were separated and a small portion was extracted as total proteins sample,thereafter protein level was measured by Western blot.Results1.3T3-L1 adipocytes treated with GH showed the less lipid content but increased mitochondrial content by Oil red O staining and Mitochondrial fluorescent dye respectively.2.3T3-L1 adipocytes treated with GH showed increased expression of UCP1 and Cidea as compared to control group by western blot.3.Western blot and PCR analysis showed that GH also increased the expression of Polg,PGC1?,Tfam and other important proteins related to mitochondrial activity(PPARy,Cyc,PDK2).4.3T3-L1 adipocytes treated with Lestaurtinib showed the reduced levels of Polg and PGC1?,the cells treated with the PD98059 and LY2940 also showed the reduced expression of Polg and PGC1? though the reduction was not as strong as with Lestaurtinib.5.Increased expression of PPARy;Tfam and UCP1 were also diminished in the cells treated with Lestaurtinib.6.Dual luciferase assay showed that GH treatment somewhat enhanced the transactivation of Polg and PGC1? through STAT5 pathway.7.Dual luciferase assay also showed that GH treatment enhanced the transactivation of Cidea through STAT5 pathway.8.Growth hormone treatment in obese mice resulted in decrease in overall body weight as compared to control group.9.Western blot analysis showed increased levels of UCP1,Cidea,Polg,PGC1?,Tfam in ssubcutaneous and visceral adipose tissues from GH treated mice.Conclusion1.These results indicates that the growth hormone has the potential to transform 3T3-L1 adipocytes into beige/brown-like adipocytes.2.Growth hormone treatment increased the mitochondrial number in 3T3-L1 adipocytes3.Growth hormone has a possible role in to enhance mitochondrial biogenesis and activity in 3T3-L1 adipocytes.4.Growth hormone impacts the expression of key regulators of mitochondria mainly through JAK/STAT pathway,other GH signaling pathways also have some effect.5.STAT5 has possible role in the transactivation of PGC1? or Polg to promote browning of 3T3-L1 white adipocytes by mitochondrial biogenesis.6.STAT5 also increases the transactivation of Cidea which shows that GH is also capable of enhancing thermogenesis which is another important factor in browning of white adipocytes.7.GH decreases body weight and increases level of key regulators of mitochondrial biogenesis proteins in obese mice via mitochondrial biogenesis. |
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