Effects of leucine on adipogenesis in 3T3-L1 preadipocytes during and after differentiation

Abstract

To observe the effects of leucine on adipogenesis in 3T3-L1 preadipocyte during and after differentiation, and to investigate possible mechanisms. Respectively, 0.0 (control), 0.5, 1.0 and 2.0 mmol/L leucine was added in 3T3-L1 cells and cell proliferation was measured by MTT. Then, 3T3-L1 preadipocyte was induced to differentiate. Leucine was added during whole differentiation period, or after differentiation for 4 days. The cells were stained with Oil Red O dye to observe lipid droplet. The culture media were collected and used to determine glycerol contents. Meanwhile, protein expressions related to lypolytic enzymes, leptin signaling pathway were determined by Western blot. MTT result showed that cell viabilities were (100.00±12.10)%, (102.73±12.38)%, (103.94±14.65)%, (108.70±5.05)% in 0.0, 0.5, 1.0 and 2.0 mmol/L leucine groups, respectively, there were no significant differences in cell proliferation among 4 groups (F=1.07, P=0.383). When 0.0, 0.5, 1.0 and 2.0 mmol/L leucine was added during differentiation, the relative number of lipid droplet was 1.00±0.06, 0.94±0.09, 0.82±0.08 and 0.79±0.04, respectively (F=11.74, P<0.001), and it was significantly lower in 1.0 and 2.0 mmol/L leucine groups than in control group (P=0.002 and P<0.001, respectively). There was no significant difference in lipid droplet when leucine was added after differentiation (F=0.16, P=0.924). When leucine was added during differentiation, the increment of glyceride contents in medium was (65.04 ± 11.75), (71.45 ± 23.71), (79.37 ± 17.63) and (110.32 ± 25.36) μmol/L, respectively (F=2.92, P=0.100). And it was significantly higher in 2.0 mmol/L leucine group (110.32 ± 25.36) μmol/L than in control group (65.04 ± 11.75) μmol/L (t=2.73, P=0.026). No significant difference of the increment of glyceride contents among 4 groups was observed when leucine was added after differentiation (F=0.80, P=0.528). Western blot results showed that leucine treatment during differentiation upregulated expression level of hormone-sensitive lipase phosphorylation (after 0.0 and 2.0 mmol/L leucine treatment,the protein levels were 1.00 ± 0.08 vs. 2.54 ± 0.27, P<0.001) , and downregulated the protein expression levels of perilipin A, leptin and leptin-related pathway, such as leptin receptor, Janus kinase 2 and suppressor of cytokine signaling-3 (after 0.0 and 2.0 mmol/L leucine was added, the protein levels were (1.00 ± 0.03) vs. (0.31 ± 0.07) , (1.00 ± 0.08) vs. (0.22±0.07) , (1.00±0.07) vs. (0.21 ± 0.04) , (1.00 ± 0.03) vs. (0.35 ± 0.05) , (1.00 ± 0.06) vs. (0.34 ± 0.05) , P<0.001). Leucine treatment after differentiation had no effects on these protein expressions (all P>0.05). Leucine inhibits adipogenesis during 3T3-L1 preadipocyte differentiation by the regulation of lypolytic enzymes and leptin signaling pathway; however, leucine has no effect on adipogenesis when differentiation completed.