Akt2Regulates Porcine Preadipocyte Differentiation

Akt, called PKB or protein kinase B, is a serine/threonine kinase and as a keycomponent of insulin signaling. Akt family has three isoforms in mammals: Akt1,Akt2, Akt3and their expression profile and the biology functions (cell metabolism,growth, apoptosis) are distinction in different tissue. Akt1has a wide tissuedistribution, whereas Akt2is highly expressed in insulin targeted tissues (liver, muscle,adipose tissue).The distribution of Akt3is more restricted with expression mainlyfound in brain. Recently, many researches indicate Akt2playes important roles inresponse to insulin, moreover adipocyte differentiation is closely related to regulationof insulin. So we speculate that Akt2would also play a key role in adipocytedifferentiation. In this study, we constructed plentiH1-Akt2shRNA recombinantlentivirus vector of pig and mouse. Vrius particles were obtained by transfectingHEK293T cells and had used to infect the porcine preadipocyte and3T3-L1cell,respectively, for detecting interference efficiency. Furthermore, we selected porcinepreadipocytes as material to further study effects of Akt2on adipocyte differentiation.Results are as follows:(1) The levels of Akt2protein and mRNA were much higher in muscle and adiposetissue of either180-day-old pig or3-day-old piglets. Akt2expression graduallyincreased during the process of adipocyte differentiation.(2) We constructed successfully recombinant lentivirus vectors of pig Akt2:pLentiH1-Akt2shRNA1， shRNA2， shRNA3and mouse Akt2: pLentiH1-Akt2shRNA4，shRNA5，shRNA6and obtained the transfected lentivirus particles. Thentheir interference efficiency was measured. The interference efficiency ofpLentiH1-Akt2shRNA3and shRNA6was above70%and50%, respectively.(3) The porcine preadipocyte of Akt2knockdown is induced to differentiation withMDI for7days. The results of Oil red O and Bodipy stainning showed that the sizeand number of lipid droplets were reduced. The results of Western Blotting analysisindicated that protein levels of PPARγ and aP2were significantly decreased.