Recombinant Irisin Stimulates Browning Of White Adipocytes And Its Mechanism

Background:Obesity is the most common metabolic disorder in theworld. Obesity develops when energy intake exceedsenergy expenditure and is principally characterized byexcess adipose tissue. Obese subjects appear to havea high risk of developing cardiovascular diseases and type 2 diabetes. Increasing energy expenditure has emerged as a potential and attractive strategy to prevent obesity.Two different types of adipose tissue including white adipose tissue (WAT) and brown adipose tissue (BAT) have been widely studied. White adipocytes store energy (e.g., triglycerides), whereas brown adipocytes increase metabolic process and consume energy. Many studies have shown that the changes in BAT activity can profoundly affect adaptive thermogenesis and glucose homeostasis. Previous studies showed that some depots of white adipocytes could transfer into brown adipocytes after cold exposure and/or beta3-adrenergic signaling activation, which was called "browning". Browning of WAT depots appears to be protective against diet-induced metabolic disorders, including obesity and diabetes.The benefits of exercise in metabolic disease (including obesity and type 2 diabetes) prevention and progression have been well documented. In 2012, Harvard research team identified a new peptide "irisin", secreted by skeletal muscles and increased with exercise. It has been shown that PGC-1α and exercise upregulate the expression of fibronectin type III domain containing5 (Fndc5), a type I transmembrane protein of skeletal muscle. Fndc5 is then proteolysed at amino acid position 30 and 140 to give rise to irisin. Age and skeletal muscle mass are the primary predictors of the circulating level, with young male athletes having irisinlevels several fold higher than those in middle-aged obese women. The circulating irisin is significantly lower in individuals with type 2 diabetes compared with nondiabetic control subjects. Over-expression of irisin by adenoviral vector increases total body energy expenditure, induces modest weight loss, and improves glucose intolerance in high fat-fed mice. However, little is known about the molecular mechanism and signaling pathways mediating the induction of the functional "brown-like" adipocyte phenotype by irisin.Objective:The aim of this study was to establish an efficient system for the expression and purification of human recombinant irisin (r-irisin) and investigate the role of r-irisin in white adipocytes both in vitro and in vivo, as well as possible "browning" mechanisms by which irisin exerts its effects.Materials and methods:1. Expression and purification of human irisin from Pichia.2. Site-directed mutagenesis of irisin cDNA.3. Primary adipocyte culture.4. Differentiation of 3T3-L1 preadipocytes into mature adipocytes.5. Binding of r-irisin on the cell membrane of adipocytes.6. RNA isolation and Real-Time PCR to measure the expression of related genes.7. ICC to measure the protein level of UCP1 in 3T3L1-derived adipocytes with/without r-irisin.8. Western blot to measure the expression of UCP1 and signal pathway-related protein in primary adipocytes and 3T3L1-derived adipocytes after r-irisin treatment.9. Animal experiment to observe the browning effect of r-irisin in high fat diet-induced obese mice.10. Results are presented as mean±SE of at least three independent experiments, and each experiment was conducted in triplicate. Statistical significance among multiple groups was analyzed by one-way ANOVA followed by Student t-test for comparison of the resultsbetween two groups using the SPSS software package (SPSS 17.0). P<0.05 was considered to be statistically significant.Results:1. Expression and characterization of purified human recombinant irisin.(1). High quantity of r-irisin can be expressed in Pichia pastoris.(2). Coomassie brilliant blue stain showed that there were three protein bands present on the gel with a molecular weight of 25,22, and 15 kDa.(3). Treatment of the purifiedr-irisin with PNGase F can remove the bands owith molecular weight of 25 and 22 KDa.(4). Wemutated each ofthe two glycosylation sites at Asn7 or Asn52, individually. The mutant r-irisin protein from each mutant(N7H or N52H) showed only two bands with molecularweight 22 and 15 kDa.2. R-irisin can promote "browning" in primary rat adipocytes and 3T3L1-derived adipocytes.(1) Real-time PCR and western blot showed that the expression of UCP1 significantly increased in primarycultured rat adipocytes which were treated by irisin for 4 days, moreover, the increase is dose-dependent from 2 nmol/L to 100 nmol/L.(2) Gene expression studies showed thattreating 3T3L1-derived adipocytes with r-irisin (20 nmol/L) led to a rapid upregulation of several brown cellmarkers.(3) Western blot and ICC showed that UCP1 protein level was significantly increased in 3T3L1-derived adipocytes after r-irisin treatment.(4) The effects of upregulation of UCP1 and PGC-la by irisin was significantly reduced by the mutated irisin at either site.3. R-irisin promotes "browning" by activating ERK and p38MAPK signal pathway.(1). The phosphorylated p38 MAPK (P-p38) andphosphorylated ERK (P-ERK) were significantly increased at as early as 5 min, peaked between 10 and 20 min, and decreased at 30 min after the r-irisin treatment in both primary rat and 3T3-L1 adipocytes.(2). The irisin-induced UCP1 upregulationwas significantly reduced by either p38MAPK inhibitor (SB203580) or ERK inhibitor (U0126).4. The green fluorescence was observed clearly with increasing intensity along the membrane on luciferase-irisin-incubated cells, whereas no clear membrane signal was observed on luciferase-treated cells.5. R-irisin Affects the Body Weight and Glucose Homeostasis In Vivo.(1) The mRNA levels of brown adipocyte marker genes were significantly elevated in the adipose tissues in both normal and high fat diet induced fat mice, which were injected r-irisin for 14 days.(2) IHC showed that the expression of UCP1 significantly increased in irisin-injected mice.(3) The body weight of irisin-injected mice decreased.(4) IPGTT showed that r-irisin treatment of fat mice significantly improved glucose tolerance.(5) Radioimmunoassay showed that that r-irisin treatment of fat mice significantly reduced levels of fasting insulin.Conclusion:1. Pichiapastoris can express high quantity of r-irisin, and r-irisin has two glycosylation sites.2. R-irisin can promote "browning" in primary rat adipocytes and 3T3L1-derived adipocytes.3. R-irisin promotes "browning" by activating ERK and p38MAPK signal pathway.4.3T3L1-derived adipocytes may have yet to be identified irisin receptor on the cell membrane.5. R-irisin can decrease body weight, promote the expression of brown-specific genes, and improve high fat diet induced insulin resistance.Background:Obesity-associated metabolic complications, including metabolic syndrome and type 2 diabetes (T2DM), are growing health problems worldwide. Obesity results from excessive energy intake compared to energy expenditure, which leads to increased adipose tissue mass and ectopic fat accumulation. Brown adipose tissue (BAT), a thermogenic tissue, consumes the energy as heat, which is otherwise stored by white adipose tissue (WAT). The thermogenic capability of BAT is mainly mediated by the mitochondria uncoupling protein 1 (UCP1), which uncouples the electron transport chain from energy production, resulting in the heat production. Thus, increasing BAT mass and UCP1 expression, thereby "burning" more fat, becomes an inspiring therapeutic strategy for preventing and managing obesity and its associated diseases. Irisin, an exercise-induced myokine identified in both mice and humans, leads to the "browning" of subcutaneous (sc) white adipocytes by increasing the expression of mitochondrial UCP1 in mice. The precursor of irisin is full-length fibronectin type Ⅲ domain containing 5 (FNDC5) whose overexpression protects high-fat diet-induced obesity in mice through the formation of brite adipocytes. Our and others’ results in animal studies illuminate irisin’s potential as an attractive therapeutic target to treat metabolic disorders, such as obesity and diabetes. Despite numerous clear-cut experimental data of irisin’s beneficial effects on animals, the physiological role and thermogenic effect of irisin in human remains controversial.Objective:The aim of this study was to investigate the role of irisin on human WAT, as well as possible signaling mechanisms by which irisin exerts its effects.Materials and methods:1. Production of recombinant irisin (r-irisin).2. Harvest of human subcutaneous white adipose tissue (scWAT).3. Isolation and expansion of human primary preadipocytes.4. Differentiation of human preadipocytes into mature adipocytes.5. UCP1 expression was detected by Immunofluorescent (IF) or immonocytochemistry (ICC) staining.6. mRNA expression was detected by quantitative real-time PCR (qRT-PCR).7. Human fat tissue culture and r-irisin treatment.8. Statistics:The data are expressed as the mean±standard deviation (SD). All of the experiments were repeated at least three times. Comparisons among values for all groups were performed by one-way analysis of variance (ANOVA). Student’s t-test was used for analysis of differences between different groups. Differences were considered to be significant at P<0.05.Results:1. Isolation of cultured human mature adipocytes.2. R-irisin has browning effects on human mature adipocytesderived from scWAT. (1) Q-PCR showed that the expression of brown adipose gene (UCP1, PGC-la and PRDM16) was significantly up-regulated by 4-day of r-irisin treatment.(2) The results of ICC and IF showed that UCP1 protein level significantly increased after 4-day of r-irisin treatment.3. Effects of r-irisin on adipogenic differentiation. After treatment with r-irisin for 18 days, the percentage of mature adipocytes in the r-irisin treated group was reduced. Furthermore, r-irisin treatment resulted down-regulation of adipogenic-related genes (C/EBP-β and Adipoq) and browning-related genes (UCP1 and PRDM16) expression during adipogenic differentiation of preadipocytes from most donors.4. Irisin upregulates UCP1 expression in fresh scWAT fragment by activating ERK/p38 MAPK signaling.(1) R-irisin treatment increased the levels of UCP1 proteinin some but not all human scWAT fragments.(2) The levels of phosphorylated p38 MAPK (p-p38) and phosphorylated ERK (p-ERK) in fresh fat tissue were significantly enhanced by r-irisin treatment for 60 min.(3) ScWAT fragments were treated with p-p38 (SB203580) or p-ERK (U0126) inhibitor for 30 min followed by r-irisin. Both inhibitors abolished the irisin-induced up-regulationof UCP1 protein.5. Positive correlation of basal levels of beige gene expression with irisin-induced browning.(1) The transcription levels of two browning related genes (UCP1 and PRDM16) in scWAT varied among donors after r-irisin treatment for 4 days.(2) ScWAT from different donors showed highly variable basal levels of three beige cell-related genes (UCP1, CD137 and TMEM26).(3) The relative expression changes of both UCP1 and PRDM16 levels in scWAT after irisin treatment positively correlated with basallevels of UCP1, CD137 and TMEM26.Conclusion:1. Irisin up-regulated the expression of browning-associated genes in humans.2. Irisin inhibited adipogenesis, thereby reducingthe formation of new adipocytes.3. Irisin activated the p38/ERK MAPK signaling pathway.4. The abundance of brite adipocytes in scWATscorrelated positively with responsiveness to r-irisin treatment.