Effects Of BRL37344, A Kind Of Selective Beta 3 Adrenergic Receptor Agonists, On Expression Of UCP1 And GLUT4 In Primary Human Omental Adipocytes

Objectives:Obesity is one kind of pathological state, which caused by too much energy accumulation when energy intake exceeding expenditure in body. It is a most important risk factor of several diseases, such as insulin resistance, type 2 diabetes, arteriosclerosis, metabolic syndrome and so on. In recent years, researches on obesity and adipocytes metabolism have been received more attentions and selective beta 3-adrenergic receptor agonists have been the hotspot in this field for its potential anti-obesity and anti-diabetic effect. Our experiment was based on human preadipocyte from omental adipose tissue, approaching to the primary culture method of human preadipocyte and set up a model for omental adipocyte. To investigate the effect of BRL37344, a kind of beta 3-adrenergic receptor agonists, on expression of uncoupling protein 1 (UCP1) as well as glucose transporter 4 (GLUT4) in primary adipocytes from human omental adipose tissue.Methods:This study consisted of two groups of subjects undergoing elective open abdominal surgery, for a total of 26 subjects. One group included 16 subjects for the study of primary omental preadipocyte culture model. The other group included 10 subjects for the study of drugs intervention. All the adipose tissue specimens were obtained from the abdominal omental region freshly, about 10-20g. The adipose tissue samples were immediately transported to the laboratory in sterile centrifuge tubes containing basal medium and arrived in the laboratory within 0.5h after removing the tissue from the patient. Usually, the preadipocyte isolation experiment was started immediately according to the collagenase digestion procedure. After being cultured with differentiation medium for several days, the preadipocyte would change into differentiated cells. Morphology and extracting stained intracytoplasmic lipid with oil red 0 all verified their preadipocyte identity. These cells were incubated for 24 hours in the presence of selectiveβ3-adrenergic receptor agonist BRL37344 at different concentration (10-9mol/L,10-7mol/L). After the interference of BRL37344, extracted the total RNA and protein, then investigated the mRNA protein expression of UCP1 and GLUT4 using RT-PCR and Western-blot separately. Data in the text and figures were expressed as means±SE. Statistical analysis was undertaken using a one-way ANOVA for comparison of control versus treatments groops. The threshold for significance was P<0.05.Resluts:1. Human omental preadipocytes were primary cultured successfully and then differentiated in vitro into mature adipocytes. The cultured preadipocyte adherenced after incubation for 24h, small size, shaped mostly fusiform, proliferated about 2-3 days, then could reach 70%-80% confluence. Cultured with differentiation medium, we could observe small lipid droplet at day 8-10, these droplets augmented gradually and confluenced into a few big ones, like grapes, the nuclear was squeezed into one side of the cell. After day 15, cells were completely filled with multiple lipid droplets and had a decreased production of matrix proteins. During the period of lipid accumulation, the differentiated preadipocytes detach very easily from the well bottom and they became vulnerable to vibration or other mechanical stress.2. The adipocytes were stained by oil red 0 and the purity could reach 95%.3. The mRNA and protein expression of UCP1 and GLUT4 were higher than those in control group, when the cells were incubated with BRL37344(10-9mol/L, 10-7mol/L) for 24h, present a dose-response effect.Conclusions:1. Human omental preadipocytes were primary cultured and differentiated into adipocytes successfully in this experiment according to the research methods of scholars domestic and overseas. The adipocytes were stained by oil red 0 and the purity could reach 95%. These cells had three typical characteristics which could demonstrate preadipocytes.①From adipose tissue, fusiform shape, no lipid droplet in the intracytoplasm.②Muliplicated rapidly, close to the doubling time with collagenoblast.③After forming monolayer confluens and cultured with differention medium, the cells could chang into mature adipocytes, according to the measuration of enzyme as well as the obeservation of lipid accumulation. This culture model offered a much better platform for the study of development, differentiation and endocrine functions of preadipocytes.2. This study showed that selective beta 3-adrenergic receptor agonists BRL37344 could promote mRNA and protein expression of UCP1 and GLUT4 in human omental adipocytes, then increased the energy expenditure and glucose transportation, alleviated energy accumulation and abnormal glucose metabolization. This study lay the basis for further probing into obesity, type 2 diabetes and related diseases.