Preparation And Regulation. Adiponectin (gadiponectin), A Large Number Into The Function Of Osteoblast Differentiation

Adipose tissue and adipocytes were known as a principal storage depot for triglycerides(TG).Adipocytes,however,also secrete several proteins potentially important in homeostatic control of glucose and lipid metabolism.The Adiponectin, also known as AdipoQ,adipose most abundant gene transcript-1(apM1),gelatin binding protein of 28kDa(GBP28) and adipocyte complement-related protein of 30kDa(ACRP30),was one of the most important hormone secreted by adipocytes. The full length Adiponectin have two important domains,the one is the collagen-like domain containing 22 repeats that follow the Gly-X-Y or Gly-X-Pro consensus for proteins forming collagenous triple helices;the other one is globular domain of Adiponectin(gAdiponectin) at C-terminal.Adiponectin can be truncated to derive the gAdiponectin.The globular domain is important for preserving the vital biological functions of Adiponectin.Adiponectin stimulates free fatty acid oxidation in skeletal muscle,and cause profound and sustainable weight loss in fatty mice,without affecting food intake.The last report indicates Adiponectin acts in the brain to decrease body weight. Treatment of hyperlipidemic mice,caused either by high fat/glucose diet or i.v.of Intralip,with recombinant Adiponectin the elevated levels of plasma FFAs were significantly decrease.Overexpression of Adiponectin in transgenic mice ameliorates hyperglycemia and insulin resistance induced by high fat diet.Furthermore,in contrast,loss of the Adiponectin gene decreased insulin response in diet induced obese mice.Recombinant Adiponectin protects ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis.The inhibitory effects of gAdiponectin on accumulation of inflammatory macrophages and lipids in the arterial wall of ApoE-deficient mice have been suggested to result from its ability to supress the expression of class A scavenger receptor and TNF-α.Interestingly,Adiponectin and TNF-αhave unrelated amino-acid sequence and exert opposing effects on development of atheroscleosis,however,they display highly similar homo-trimeric three-dimensional structures.Recently,researchers had reported that Adiponectin mediates modulations of hypertrophic signal in the heart associated with diabetes and other obesity related disease.Recently,several groups had expressed recombinant gAdiponectin and Adiponectin in vitro.However,the expression level of recombinant gAdiponectin or Adiponectin was very low or the costs were significantly high.In order to product recombinant gAdiponectin by simple and economical way,we try to get high and soluble expression of gAdiponectin in Pichia.Pastoris through rebuilding the gAdiponectin cDNA by PCR based on Pichia.pastoris's optimal codons.The rebuilded gAdiponectin cDNA was inserted into the expression vector pPIC9K.The recombinant expression plasmids were transformed into Pichia pastoris strain(GS115) and gAdiponectin were highly expressed and secreted into culture medium as a soluble form.Recombinant gAdiponectin were purified and charactered. A rapid,effective and simple strategy for pilot production of gAdiponectin was established.To verify the activity of rh-gAdiponectin,we firstly established the STZ-induced diabetes mellitus mice model and Intralipid-induced hyperlipidemic model.And then we treated these mice with different doses of rh-gAdiponectin and the blood glucose and FFA level were significantly reduced.Some clinical observations that obesity protects from osteoporosis indicate that energy metabolism and bone mass could be regulated by the same hormones.To test this hypothesis,we'd like to investgate the effects of adipocyte-derived hormone Adiponectin on bone mass.To test the effects of gAdiponectin on the osteoblast differentiation,we established the MC3T3-E1 pre-osteoblasts differentiation cell model.After the induction of MC3T3-E1 pre-osteoblasts cell lines,the molecular levels of some osteoblast-specific genes were raise gradually,and the extracellular matrix were mineralized,too.However,the rh-gAdiponectin can significantly decrease the levels of the osteoblast-specific genes,such as ALP,Osteocalcin,and delay the mineralization of the extracellular matrix with a dose-dependent manner.In the process of osteoblasts differentiation,there are two osteobalst-specific transcriptional factors:Runx2 and Osterix.We found that the inhibitory role of gAdiponectin on osteoblast differentiation was mediated through Runx2 and Osterix, the gAdiponectin can down-regulate significantly the mRNA and protein levels of Runx2 and Osterix.