P40 The up-regulation role of H2S in 3T3L1 on adipogenesis

The endogenous gas, hydrogen sulfide (H 2 S), plays an important role in regulating cardiovascular function and inflammation. It also contributes to the pathogenesis of insulin resistance and glucose metabolism [1] , [2] , [3] . Recently, it has been reported that large amounts of H 2 S are formed from L-cysteine by cystathionine gamma lyase (CSE) in fat tissues and that H 2 S reduces glucose uptake in rat adipose tissue [3] . However, the biological roles of H 2 S in adipocytes and its contribution to adipogenesis are not yet clear. Here, we investigate whether H 2 S-synthesizing enzymes; cystathionine beta-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), are endogenously expressed in adipocytes and play a role in adipogenesis. In the present experiments, we stimulated the mouse fibroblast-like cell line, 3T3L1, to differentiate into adipocytes for up to one week as a model of adipogenesis in vitro . Over 7 days differentiation, whole cell lysate at day 0, 1, 3, 5 and 7 were collected for Western blotting of CBS, CSE and 3-MST. Endogenous CBS and 3-MST in adipocytes peaked at day 5 of differentiation whereas CSE protein expression was up-regulated in a time-dependent manner and maximally expressed at day 7 (n = 3, P  2 S on 3T3L1 cells was assessed after co-incubation of cells either with the slow-releasing H2S donor compound, GYY4137 (50 μ M), or the classical H 2 S donor, NaHS (50 μ M) for two days followed by harvesting, and real-time polymerase chain reaction (PCR) assay. Real-time PCR was performed for the following adipogenesis-related genes: fatty acids binding protein 4 (FABP4/aP2), peroxisome proliferator-activated receptor gamma (PPAR γ ), sterol regulatory element-binding protein 1 (SREBP1), CCAAT/enhancer binding protein alpha (CEBP α ), carbohydrate-responsive element-binding protein (ChREBP), fatty acid synthase (FAS), hormone-sensitive lipase (HSL), perilipin A and tail-interacting protein of 47 kDa (TIP47). Compared with the control group, NaHS and GYY4137- treated groups showed increased expression of PPAR γ , CEBP α , FAS and TIP47 (n = 6, P  μ M) inhibited lipolysis (5.79 ± 2.42 μ g/ml c.f. 45.9 ± 8.57 μ g/ml glycerol, n = 3, P  β 3 agonist, CL316,243 (10 nM). Taking all of these data into consideration, we suggest that H 2 S-synthesizing enzymes such as CBS, CSE and 3-MST are not only expressed in nerves and vascular tissues [4] , [5] , [6] , but that these enzymes are also present in large amounts in adipose tissue and that their up-regulation during adipogenesis promotes adipocyte maturation and prevent lipid degradation. Thus, it is possible that modulation of the H 2 S system may be useful for obesity treatment.