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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (8): 886-893.doi: 10.3736/jcim20120809

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Effects of ursolic acid on c-Cbl-associated protein expression in 3T3-L1 adipocytes with insulin resistance

Di Li, Guan-liang Wang, Meng-ya Shan, Jia-han Liu, Lin Wang, De-zeng Zhu()   

  1. Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
  • Received:2011-12-13 Accepted:2011-12-25 Online:2012-08-20 Published:2018-08-15
  • Contact: Zhu De-zeng E-mail:zdzzyx@163.com

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Objective: To observe the effects of ursolic acid (UA) on insulin resistance and cell differentiation in 3T3-L1 adipocytes and to explore the mechanisms.

Methods: 3T3-L1 adipocytes were cultured in Dulbecco‘s modified Eagle‘s medium (DMEM) supplemented with glucose (25 mmol/L) and insulin (10 -6 mol/L) to induce insulin resistance. After culture, glucose consumption of the adipocytes was detected by glucose oxidase method and glucose uptake was detected by using tritium-marked glucose. Drug concentration for following test was determined through detecting the effects of different concentrations of UA on the activity of 3T3-L1 adipocytes with insulin resistance by methyl thiazolyl tetrazolium (MTT) staining. 3T3-L1 adipocytes with insulin resistance were cultured with DMEM, rosiglitazone, and low- and high-dose UA, and then, glucose uptake and differentiation of 3T3-L1 adipocytes were detected. Finally, real-time fluorescence quantitative polymerase chain reaction and Western blot methods were used to detect the effects of UA on expressions of adipocyte lipid binding protein (aP2), c-Cbl-associated protein (CAP) and matrix metalloproteinase-1 (MMP-1) in 3T3-L1 cells with insulin resistance.

Results: After dealing with high glucose/hyperinsulin for 24 h, insulin resistance was induced successfully in the 3T3-L1 adipocytes. The concentrations of UA were defined to be 4 to 20 μmol/L. Compared with the model group, the glucose uptake was significantly increased in the rosiglitazone group and groups treated with low- and high-dose UA (P<0.01). The differentiation levels of 3T3-L1 adipocytes in the UA groups were lower than those in the control group and the rosiglitazone group. Effects of UA on the expressions of aP2 and MMP-1 were not obvious, but UA could up-regulate expression of CAP both in mRNA and protein levels (P<0.01).

Conclusion: Low- and high-dose UA can improve the glycometabolism and differentiation of 3T3-L1 adipocytes with insulin resistance by up-regulating the expression of CAP.

Key words: ursolic acid, diabetes mellitus, adipocytes, insulin resistance, cell differentiation, matrix metalloproteinase 1, proto-oncogene proteins c-cbl, 3T3-L1 cells

Figure 1

Effects of high glucose/hyperinsulin on glucose consumption in 3T3-L1 cellsData are expressed as $\bar{x}$±s, n=3. **P<0.01, vs normal cells; △△P<0.01, vs high glucose-treated 3T3-L1 cells. "

Figure 2

Effects of high glucose/hyperinsulin on glucose uptake in 3T3-L1 cells Data are expressed as $\bar{x}$±s, n=3. **P<0.01, vs normal cells; △△P<0.01, vs high glucose-treated 3T3-L1 cells."

Table 1

Effects of different concentrations of ursolic acid on viability of 3T3-L1 adipocytes ($\bar{x}$±s)"

Group n Survival rate of 3T3-L1 adipocytes
12 h 24 h 36 h 48 h
0 3 1 1 1 1
0.02 μmol/L ursolic acid 3 0.91±0.02 0.94±0.03 0.96±0.01 0.97±0.02
0.04 μmol/L ursolic acid 3 0.94±0.01 0.97±0.04 0.96±0.03 0.98±0.01
0.2 μmol/L ursolic acid 3 0.91±0.05 0.91±0.03 0.95±0.01 0.99±0.02
0.4 μmol/L ursolic acid 3 0.96±0.04 0.89±0.07 0.94±0.05 0.10±0.02
2 μmol/L ursolic acid 3 0.86±0.04 0.88±0.09 0.97±0.04 0.99±0.06
4 μmol/L ursolic acid 3 0.96±0.03 0.96±0.05 0.98±0.07 0.98±0.04
20 μmol/L ursolic acid 3 1.13±0.04 1.06±0.03 1.15±0.07 1.13±0.06
40 μmol/L ursolic acid 3 0.22±0.06 0.33±0.01 0.32±0.04 0.57±0.03
200 μmol/L ursolic acid 3 0.23±0.01 0.21±0.04 0.17±0.02 0.35±0.03

Table 2

Effects of ursolic acid on glucose uptake in 3T3-L1 adipocytes ($\bar{x}$±s)"

Group Concentration n Glucose uptake (d/min)
Normal 3 9 876±162▲▲
IR 3 7 288±128▲▲
Rosiglitazone 5 μmol/L 3 9 090±120**
Low-dose ursolic acid 10 μmol/L 3 8 111±875**▲▲
High-dose ursolic acid 20 μmol/L 3 8 497±117**▲▲

Table 3

Effects of ursolic acid on differentiation of 3T3-L1 adipocytes ($\bar{x}$±s)"

Group Concentration n Optical density570
Normal 12 0.54±0.03▲▲
IR 12 0.63±0.12
Rosiglitazone 5 μmol/L 12 0.77±0.04**
Low-dose ursolic acid 10 μmol/L 12 0.33±0.04**▲▲
High-dose ursolic acid 20 μmol/L 12 0.21±0.07**▲▲

Figure 3

Effects of ursolic acid on relative expressions of aP2, CAP and MMP-1 mRNAs in 3T3-L1 cells Data are expressed as mean ± standard deviation, n=3. **P<0.01, vs normal group; P<0.05, vs IR group. IR: insulin resistance; aP2: adipocyte lipid-binding protein; CAP: c-Cbl-associated protein; MMP-1: matrix metalloproteinase-1."

Figure 4

Effects of ursolic acid on expressions of aP2, CAP and MMP-1 proteins in 3T3-L1 cellsData are expressed as mean ± standard deviation, n=3. **P<0.01, vs normal group; **P<0.01, vs IR group. IR: insulin resistance; aP2: adipocyte lipid-binding protein; CAP: c-Cbl-associated protein; MMP-1: matrix metalloproteinase-1."

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