Transcriptional Regulation Of Autophagy By C/EBPβ During Adipogenesis And The Role Of SCO1 In Obesity-Related Metabolic Disorders

obesity and associated metabolic diseases such as type 2 diabetes, cardiovascular disease and certain cancers represent a major threaten to public health worldwide. Environmental factors undoubtedly drive the rise in the prevalence of obesity; meanwhile, we should concern about individual difference in body weight within a population that share the same environment. It is therefore important to approach the genetic methods to dissect the mechanism involved in weight regulation and identify the obesity-related genes.A cDNA micmarray was approached in one research which published in 2004; these microarrays were used to study the differential gene expression profiles of visceral adipose tissues between obese patients and normal subjects. Another similar research was reported in 2007, in this study, the author using a DNA chip to analyzed gene expression in the livers of 21 patients with type 2 diabetes (10 obese and 11 nonobese patients). Very interesting, one gene named SCO1, which was found up-regulated in obese patients than non-obese patients in those papers. These results suggest that SCO1 may high related with the obesity and diabetes.SCO1 is a mitochondrial copper-binding protein involved in the biogenesis of the CuA site in the cytochrome c oxidase (CcO) subunit Cox2 and in the maintenance of cellular copper homeostasis, but the specific mechanism of this is still unclear. We use RT-PCR and western blotting to compare the expression of SCO1 in the B6-ob/ob and B6-db/db and HFD mice in inguinal adipose tissue, epididymal adipose tissue, and liver. Our results show that SCO1 was higher expressed both in mRNA and protein levels in inguinal adipose tissue, epididymal adipose tissue and liver of obesity mice than in the same tissues of the normal B6 mice. Surprisingly, SCO1 was also up-regulated in brown adipose tissue and heart of obesity mice than in the same tissues of the normal B6 mice. Those results conformed the results of the two above papers, suggest that SCO1 is high associated with obesity disease.The functions of SCO1 are inseparable from copper. As a trace element; copper is an indispensable catalytic and structural cofactor that drives a wide array of important biochemical processes that are essential for life. We used ICP-mass spectroscopy to compare the copper levels in tissues, serum and urine between control and ob/ob mice. We found that the copper levels in the blood and urine of obesity mice were higher than in the blood and urine of lean mice. These results are consistent with the clinic survey data about type 2 diabetes patients. The copper levels in inguinal adipose tissue, epididymal adipose tissue, liver and heart of obesity mice were lower than that in lean mice. These results indicate that these tissues were lack of copper. To examine the functions of SCO1 played in obesity disease, lentiviral-expressing SCO1 shRNA was injected subcutaneously adjacent to one inguinal fat pad site of ob/ob mice. We found that SCO1 was knockdown in inguinal adipose tissue, the results show that the copper levels in inguinal adipose tissue increase 28% in injected shSCOl ob/ob mice compare to injected shLacZ ob/ob mice, the copper levels in other tissues like epididymal adipose tissue, liver and heart of obesity mice were higher than that in injected shLacZ ob/ob mice (increase average 1-1.5 times).Consistently, the copper levels in serum and urine were decrease dramatically in injected shSCOl ob/ob mice than that in injected shLacZ ob/ob mice(decrease 3 times in serum and 2.5 times in urine). These results suggest that knockdown SCO1 can make the copper redistribution in body. The copper enter tissues which real need them, the copper levels in circulation and excretory system is significantly reduced. We tried to mimic the high expression of SCO1 in cells, Over-expressed pcDNA3.1 vector expressed SCO1 in 293T or 3T3-L1 cells, then detect the quantity and distribution of copper.We found that over-expressed SCO1 in 293T or 3T3-L1 cells, the copper levels in cells were dramatically decreased. The high expression of SCO1 may caused copper cannot stay within the cells, this conform the tissues level results. Simultaneously, the cholesterol levels were reduced in the serum when SCO1 was knockdown, consistent with the copper deficiency disease that cholesterol levels in the serum and liver are higher.Copper is incorporated into a variety of proteins and metalloenzymes which perform essential metabolic functions. For instance, Cu-ATPase, Ceruloplasmin, Cu/ Zn SOD (SOD1).all these enzymes need copper for catalytic function. SOD1 is an important antioxidant enzyme, lacking of copper can lead to reduce its antioxidant activity; the levels of oxidative stress were increased. To identify whether higher ROS levels in the tissues of obesity mice was result from the high expression of SCO1, we over-expressed SCO1 in 293T and 3T3-L1 cell line, ROS level in both cell lines were dramatically increased. We performed lentiviral-expressing SCO1 shRNA was injected to one inguinal fat pad site of ob/ob mice to knockdown SCO1, we found that the ROS level in this inguinal adipose tissue was deduced about 30%. This result was consistent with the copper level which was decrease 28%, and the ROS levels were dramatically ameliorated in other tissues (epididymal adipose tissue, liver and heart). Meanwhile, the expression of inflammatory cytokines (MCP1, IL6, TNFa) were decreased and the anti-inflammatory cytokines were increased in those tissues.To clerify whether copper deficiency which imparied the SOD1 acvivity which can increasing the ROS level. We used a lentiviral-expressing of shRNA by intraperitoneal injection to knockdown the SCO1 in the tissues of ob/ob and db/db mice.we found that the SOD1 activities were alleviated in which copper levels were increased tissues. These results suggest that knockdown SCO1 could reduce ROS level, because the activity of SOD1 has improved.In order to eliminate the limitation of local injection, we used a lentiviral-expressing of shRNA by intraperitoneal injection to knockdown the SCO1 in the tissues of ob/ob and db/db mice, this handle can lead the copper ions redistribution and enter into each tissues (reduce the copper ion content in the blood and urine, to alleviate the lack of tissue of copper, improve tissue antioxidant capacity, reduce cholesterol content in the liver and blood. These results are consistent with the results of inject shRNA to inguinal adipose tissue.Take together, we demonstrated that SCO1 was up-regulated in the adipose tissue,liver and heart tissue of obese mice, which caused the failure of copper to enter these tissues, the efflux of copper into the blood and urine, leading to the lack of copper in adipose tissue, liver and heart. Copper deficiency inhibited the activity of SOD1 which impaired the clearance of the ROS in these tissues; and the high ROS levels lead to inflammation. Lack of copper ion lead blood cholesterol increased.Obesity results from an imbalance between food intake and energy expenditure, which leads to an excess of white adipose tissue. The most direct reason for excess adipose tissue is adipocyte hypertrophy and cell hyperplasia.Adipocyte hyperplasia is caused by the abnormal regulation of adipogenesis. Thus, to clarify the molecular mechanism of adipocyte differentiation has very important guiding significance for clinical prevention and treatment of obesity related metabolic diseases.Adipocyte differentiation is a complex process regulated by a number of transcriptional factors acting coordinately. C/EBPβ is the key transcriptional factor which plays an important role in adipocyte differentiation. In order to further understand the mechanism of C/EBPβ played during adipocyte differentiation, we use ChIP-on-chip technique to screen the targets gene of C/EBPβ during adipocyte differentiation process in 3T3-L1 cell.We found a serial of autophagy-related gene includes:atg2a, atg4b, atg7, atg9a and atg10, which are very important in autophagy. Among these genes only the expression of Atg4b was significantly inhibited by C/EBPβ siRNA, suggesting that Atg4b might be subjected to transcriptional regulated by C/EBPβ. Furthermore, ChlP-qPCR and deletion or mutation experiment confirmed the significant binding site (bp-6143 to -6130) of C/EBPβ to the promoter of Atg4b in differentiating cells after induction (day 2). These results demonstrate that Atg4b is abona fide target of C/EBPβ during adipogenesis.Atg4b exposes glycine from the LC3 precursor at its C terminus to form LC3I. This initial proteolytic processing is required for the subsequent lipidation of LC3I to form LC3Ⅱ, which is essential for autophagosome formation.To test the role of Atg4b in adipocyte differentiation, we use siRNA to knockdown the expression of Atg4b in 3T3-L1 cell, and we found that knockdown Atg4b blocked the autophagy and the terminal differentiation of 3T3-L1 cells. Forced expression of human Atg4b rescued autophagy and adipogenesis, the rescue effect was dependent on the ectopic expression level of Atg4b. Those results reinforce the idea that transactivation of Atg4b by C/EBPP is required for autophagy and adipogenesis.A substantial increase of autophagic activity was detected from day2 to day4 of 3T3-L1 adipocyte differentiation; this time course suggests an important role of autophagy during this period. Because autophagy is one of the main pathway sutilized by cells to degrade intracellular proteins, it is hypothesized that autophagy promoted adipocyte differentiation via controlling the degradation of negative regulators. To address this possibility, the effect of autophagy inhibition on the degradation of a series of well-characterized negative regulators was examined.The 3-MA treatment impeded the turnover of Klf2 and Klf3 but had little effect on the degradation of the other factors. In contrast, MG132, an inhibitor of proteasomes, had a minor role in the degradation of Klf2 and Klf3.We further use siRNAs to knockdown Klf2 and Klf3 and found that significant reversal of the inhibition of adipogenesis in the presence of the autophagy inhibitor 3-MA or siAtg4b. Collectively, these results indicate that the turnover of Klf2 and KID by autophagy plays an important role in adipocyte differentiation.A recent study indicated that p62, an autophagy-specific substrate, bound to and recruited the polyubiquitinated oncoprotein PML-retinoic acid receptor (RARa) to autophagosomes for degradation, resulting in myeloid cell differentiation. Therefore, the potential role of p62 in regulating the turnover of Klf2 and Klf3 and in adipocyte differentiation was explored.A significant interaction between p62 and Klf2/3 was detected on day3 of differentiation.Colocalization between GFP-tagged Klf2/3and LC3 puncta was detected by confocal microscopic analysis, which was significantly impaired by the treatment siP62-post.Ectopic expression of wild-type human p62, which is resistant to p62siRNA of mouse origin, rescued the inhibition of adipogenesis and impairment of Klf2/3 decline mediated by siP62-post.In contrast, ectopic expression of mutant human p62,which lacks the LC3-interactingregion, failed to rescueit, supporting the critical role of the p62-LC3 interaction in mediating the removal of Klf2/3. Collectively, these studies implicate an important role of p62 in promoting the degradation of Klf2and Klf3 by autophagy during adipocyte differentiation.To verify the above-described findings in vivo, experiments were further performed in mice. C/EBPβ binding to the distal promoter of Atg4b was confirmed with ChIP analysis on mouse white adipose tissue.Recombinant adenovirus expressing Atg4bshRNA was injected to one inguinal fat pad site whish resulted in autophagy inhibition and an increase in Klf2/3 and a decrease in PPARy and C/EBPa. Moreover, the amount of WAT was significantly reduced by Atg4b knockdown. Histologic alanalyses showed the diminished size of adipocytes in fat pads injected with Atg4bshRNA, indicating decreased lipid content in adipocyte. In addition, the percentage of adipocytes in WAT was reduced in Atg4bshRNA-treated fat pads suggesting impaired adipocyte differentiation. Compared with the WAT of regular-diet (RD)-fed mice, the elevated level of C/EBPβ, PPARy, C/EBPa, Atg4b and autophagy and decreased level of Klf2/3 were detected in WAT from HFD-fed mice. These data correlate well with the results described above and further confirm the important function of autophagy during adipocyte differentiation, which is regulated by C/EBPβ.Take together; we reported that C/EBPβ transactivates the expression of Atg4b to promote autophagy, which mediates the degradation of Klf2/3 in a p62-and ubiquitination -dependent manner, thereby relieving the repression of PPARγ and C/EBPα transcription.Meanwhile,C/EBPβ is involved in the transactivation of PPARγ and C/EBPα.These two effects coordinately promote the expression of PPARr and C/EBPα and facilitate adipogenesis.The findings presented in this study provide new insights into the role of C/EBPβ and shed light on the mechanism of autophagy during adipocyte differentiation. Modulation of this regulatory pathway maybe of the rapeutic value for intervening in the overexpansion of adipose mass, thereby preventing obesity and its related metaboli ccomplications.