The Mechanism Studies Of Browning Of White Adipose Tissue In Adipose Specific Dicer Knock-Out Mice

Objective To observe the effects of adipose tissue specific knock-out Dicer on body weight and glucose tolerance of mice, and further test the browning of primary white adipose cells to investigate the relationship between Dicer gene and energy metabolism in vitro.Methods 1. We generated adipose tissue specificity Dicer knock-out mice, the Adiponectin-Cre- Dicer flox/flox and the control mouse Dicer flox/flox. 2. The body weight has been recorded and IGTT has been investigated in 6 week old adipose specific Dicer knockout mice and control mice. The visceral adipose tissue was fixed with 4% PFA for HE staining, and RNA of visceral adipose tissues was extracted by Trizol. 3. In order To investigate the effects of Dicer gene on browning of primary white adipose cells in vitro, we separated the precursor cells of white adipose tissue from conditional knockout mice(Dicer flox/flox) and infected the LV-Cre-GFP lentivirus and control LV-GFP Lentivirus. The differentiation of brown fat cells was observed by fat cells morphology, oil red O staining and real-time quantitative PCR(Real-time q PCR) of specific gene expression of brown fat tissue. 4. Real-time q PCR gene expression of heat production and mitochondrial genes was observed to detect the formation of brown fat. Western Blot was used to detect the protein expression of the uncoupling protein UCP1, the brown fat specific protein,Result: 1. The body weight and the visceral fat mass was less in adipose tissue specific Dicer knock-out mice than in the wild type mouse(p<0.01). The Dicer mutant mice had glucose higher tolerance(p<0.01) and different HE staining of adipose morphology. 2. The size of fat cells was smaller in in adipose tissue specific Dicer knockout mice than in the wild type mouse(p<0.01), and the UCP1 gene expression of Dicer mutant mice increased significantly(p <0.005) by two separate experiments. 3. There was no significant morphology difference between LV-Cre-GFP and control LV-GFP infected cells. The two group cells all had many lipid droplets by oil red O staining. But further experiments found that the LV-Cre-GFP infected cells had higher expression of Prdm16 and pgc1, the transcription factor, ucp1, the thermogenic genes, and cox8b, cox4 and mitochondrial marker gene which increased more than 30 times(p <0.005).Conclusion: White adipose specific knock-out of Dicer contributes to more browning of visceral adipose tissue, which might promote energy metabolism and alleviate obesity.