To Study The Mechanism On Irisin Transforming White Adipocyte Into Brown Or Beige Adipocyte

BackgroundObesity is the result of energy intake chronically exceeding energy expenditure.Classical treatments against obesity do not provide a satisfactory long-term outcome for the majority of patients.After the demonstration of functional brown adipose tissue exists in human adults,great effort has been devoted to develop therapies based on the adipose tissue itself through the conversion of fat-accumulating white adipose tissue into energy-dissipating brown adipose tissue.Studies focusing on the therapeutic potential of brown adipose tissue(BAT)against obesity have increased over the last decadeTwo types of adipose tissue have been previously identified:white adipose tissue(WAT)and brown adipose tissue(BAT).White fat stores energy(triglycerides)and brown adipose tissue produces heat and expends energy.The cells of white adipose tissue are larger,contain a larger single lipid droplet(the main chemical component is triglyceride),and have fewer mitochondria.The cells in brown adipose tissue are small,contain multiple lipid droplets,have a large number of mitochondria,and highly express uncoupled protein 1(UCP1).Therefore,brown adipose tissue can consume energy and generate heat.Later,it was found that white fat in humans and mice contains a different beige adipose tissue(bAT)from white fat and brown fat in terms of morphology,function,gene expression and response to external stimuli[5].The morphology and function of beige adipocytes are between white adipocytes and brown adipocytes,and the number of mitochondria is between white adipocytes and brown adipocytesMitochondrial biogenesis is a process in which the number of mitochondria in a cell increases.In the process,the specific mitochondria protein expression is very important,such as mitochondrial DNA polymerase polg(DNA polymerase gamma),etc.In addition,mitochondrial proliferation is regulated by a number of factors,such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha,which is a major regulator of mitochondrial proliferation.Mitochondrial DNA polymerase(polg)and pgc-1ahave been identified as key molecules for mitochondrial biogenesis.Of course,some other gene products also play important roles in the biological formation of mitochondria,such as a variety of protein molecules involved in the formation of mitochondrial structure.Irisin,secreted by skeletal muscles and increased with exercise,is a small polypeptide hormone containing 111 amino acids with an estimated molecular mass of 22 kDa.It becomes mature after the cleavage of fibronectin type III domain containing 5(Fndc5)at amino acid position 30 and 140.It was initially discovered as a PGC1-α-dependent myokine and as an endocrine molecule converting the white adipose tissue to the brown adipose tissue by stimulate UCP1 expression.When the Irisin molecule was discovered,it became a star molecule in the cream-colored transformation of white fat,offering hope in the fight against obesity.But the molecular mechanism by which Irisin causes the white fat to transform into beige remains unclear.The cream-colored white fat requires two conditions:one is the induced expression of UCP1,which is used to consume energy and fat.Another condition is the promotion of mitochondrial biogenesis or an increase in the number of mitochondria.We therefore hypothesized that irisin acting on skeletal muscle cells and then on adipocytes increase mitochondrial biogenesis or promote the mitochondria biogenesis,resulting in cream-colored white adipocytes.The main purpose of the present study is to explore whether exogenous irisin could increase the mitochondria numbers,namely,the mitochondria biogenesis during the browning process in differentiated 3T3L1 adipocytes.MethodsMouse 3T3-L1preadipocytes is a well-established cell line commonly used to Study adipocyte differentiation in vitro.1.In brief,preadipocytes were cultured with basic medium routinely used at 37℃ in a 5%CO2 incubator.After confluence,the cells were induced for the adipocyte differentiation with induction medium,two days after induction(day 2),the medium was changed to basic medium supplemented with insulin only for an additional 2 days.On day 4,the medium was switched back to basic medium for another 2 days.Irisin was added to the 3T3-L1 cells at day 4 in adipocyte differentiation for a total of 4 days’ treatment.On day 8,3T3-L1 mature adipocytes with or without irisin were processed for Oil Red O staining for confirming adipocyte differentiation,and western blotting to verify the expression of brown adipocyte-specific genes.2.RNA or proteins extraction and perform the reverse transcription-quantitative PCR(RT-qPCR)and Western blot to test the mRNA and protein levels of mitochondrial DNA polymerase[(DNA-directed)polymerase gamma,mitochondrial DNA polymerase),polg],PGC-1α(peroxisome proliferator-activated receptor gamma coactivator 1-alpha),and other mitochondrial genes.3.To determine whether irisin was able to increase mitochondria numbers with a specific staining.Results1.3T3-L1 preadipocytes were differentiated into mature adipocytes with differentiation medium(cocktail,reagents)and obvious lipid droplets were observed by oil red O staining2.The reverse transcription-quantitative PCR and western blot showed that irisin enhanced the expression of mitochondrial DNA polymerase(polg)and PGC-1α mRNA level and protein level in differentiated adipocytes and also irisin enhanced the expression of another mitochondria biogenesis related gene.3.mitochondria staining showed that irisin increased the mitochondria number in the cells treated with irisin compared to that treated with control buffer.Conclusion1.Irisin enhances significantly the expression of PGC-1α and polg(polymerase(DNA-directed)gamma),which are two molecule key for the mitochondria biogenesis.2.Irisin enhances the expression of other mitochondrial biogenesis related genes.3.Irisin treatment on 3T3 adipocytes increases the number of mitochondria in the cells.