The Study Of Effects On Aquaporin Adipose Expression In 3T3-L1 Adipocytes And The Relationship Between Body Mass And Expression Of Human Aquaporin Adipose

Objective:Aquaporin adipose(AQPap) is the physiological glycerol channel specific to adipocytes, and it is expressed predominantly in adipose tissue. In this study, lipid staining was used to evaluate the differentiation rate and the changes of function and morphology in 3T3-L1 adipocytes conversion process. We investigated the effects of hormone, nutrients and hypolipidemic drugs on the expression of AQPap in 3T3-L1 adipocytes, and discussed the relationship between body mass and expression of human aquaporin adipose(AQPap), in order to know the role of AQPap plays in obesity and diabetes mellitus.Methods:1 .Confluent preadipocytes were treated with luM dexamethasone ^ 0.5 mM IMX and 5ug/ml insulin for 48h, followed by standard medium DMEM culture with 5ug/ml insulin for another 48h, then refeeding with standard medium. Oil red O staining was used to evaluate the conversion process of 3T3-L1 adipocytes on dayO, 5, 7 and day9.2. 3T3-L1 cells on day 9 after differentiation were incubated with 10~6MU 10~7Mk 10'8M> 10~9M insulin for 6 hour, or incubated in DMEM with 10"8M insulin for 0, 3, 6h for the experiment on time course, total cellular RNA were extracted and used for RT-PCR. Such cells were also stimulated by epinephrine(10'6M> 10"7M> 10'8M> 10v?nIGF-1(10'M, 1Q-SM, 1(T9M, l(r10M), dexamethasone(10~6M> 1(T7VU 10"8M) or insulin(10"6M)+ dexamethasone(10"6M) respectively, total RNA was isolated for RT-PCR. After differentiation on day 9, 3T3-L1 cells were stimulated with different concentrations of glucose(5.6mM, ll.lmM, 16.8mM, 33.3mM) or glycerol(0.17mM, 1.7mM, 17 mM) for 48 hours, then total cellular RNA were extracted respectively, detecting the expression of AQPap mRNA by RT-PCR. After stimulation of insulin > epinephrine in 10~6M to the differentiated 3T3-L1 cells for 48h, or incubated with 16.8mM glucose for 48h, the crude membrane proteins were extracted, then investigated the effects of various factors on AQPap protein expression by Western blotting.The subjects were divided into three groups according to body mass index: the normal, the overweight, the obese. The expression of adipose AQPap were detected with RT-PCR and Western blotting.Results:1. After differentiation, the cells tured to be larger and round rather than of spindle shape, and containing large droplets of triglyceride. Over 95% of the cells appeared to be differentiated into mature adipocytes on day9. The droplets in the cytoplasm became red by staining with oil red O.2. There was no significant change of AQPap mRNA levels under the treatment with various concentrations of epinephrine(P>0.05) .Treatment with 10~9M insulin suppressed AQPap mRNA expression in differentiated 3T3-L1 adipocytes(P<0.05), and the results showed that the suppression of insulin on the expression of AQPap mRNA were dose-dependent and time-dependent in 3T3-L1 cells.IGF-1 could also decrease AQPap mRNA expression in differentiated 3T3-L1 adipocytes in 10"7M, but the suppression was weak compared to insulin with the same concentration.Desamethasone had no effect on the expression of AQPap mRNA in 3T3-L1 cells, but it could decrease the inhibition of insulin on the AQPap gene expression. After stimulation with high concentration of glucose, AQPap mRNA and protein expression were all augmented in 3T3-L1 cells (p<0.05). Incubation with high level of glycerol had no effect on AQPap mRNA expression although with the same osmotic pressure range as glucose stimulation. Hypolipidemic drugs such as fenofibrate^ lovastatin had no significance change on expression of AQPap mRNA in 3T3-L1 cells(p>0.05).3. There were significant differences between AQPap protein level of the obese and the normal (p<0.05 ) .RT-PCR showed that significant differences were found in the obese, the overweight and the normal subject ( p<0.05 ), but there was no significant change between the obese and the overweight ( p>0.05 ) .Conclusion:l.The differentiation process is essential and beneficial f...