Neferine suppresses autophagy-induced inflammation,oxidative stress and adipocyte differentiation in Graves' orbitopathy

Previous studies in Graves’ orbitopathy (GO) patient-derived fibroblasts showed that inhibition of autophagy suppresses adipogenic differentiation. Autophagy activation is associated with inflammation, production of reactive oxygen species and fibrosis. Neferine is an alkaloid extracted from Nelumbo nucifera, which induces Nrf2 expression and inhibits autophagy. Here, we have elucidated the role of neferine on interleukin (IL)-13-induced autophagy using patient-derived orbital fibroblasts as an in vitro model of GO. GO patient-derived orbital fibroblasts were isolated and cultured to generate an in vitro model of GO. Autophagy was determined by Western blot detection of the markers such as Beclin-1, Atg-5 and LC3 and by immunofluorescence detection of autophagosome formation. Analysis of differentiation towards an adipogenic lineage was performed by Oil red O staining. The expression of inflammatory factors was detected by ELISA and semiquantitative RT-PCR. Neferine inhibited autophagy in GO orbital fibroblasts, as indicated by the suppression of IL-13-induced autophagosome formation, overexpression of autophagy markers, increased LC3-II/LC3-I levels and finally down-regulation of p62. Neferine suppressed IL-13-induced inflammation, ROS generation, fibrosis and adipogenic differentiation in GO patient-derived orbital fibroblasts. The anti-inflammatory, antioxidant and antiadipogenic effects of neferine were accompanied by the up-regulation of Nrf2. These results indicated that orbital tissue remodelling and inflammation in GO may be mediated by autophagy, and neferine suppressed autophagy-related inflammation and adipogenesis through a mechanism involving Nrf2.     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.     

Myostatin regulates preadipocyte differentiation and lipid metabolism of adipocyte via ERK1/2

Myostatin is known as an inhibitor of muscle development, but its role in adipogenesis and lipid metabolism is still unclear, especially the underlying mechanisms. Here, we demonstrated that myostatin inhibited 3T3-L1 preadipocyte differentiation into adipocyte by suppressing C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome-proliferator-activated receptor γ), also activated ERK1/2 (extracellular-signal-regulated kinase 1/2). Furthermore, myostatin enhanced the phosphorylation of HSL (hormone-sensitive lipase) and ACC (acetyl-CoA carboxylase) in fully differentiated adipocytes, as well as ERK1/2. Besides, we noted that myostatin markedly raised the levels of leptin and adiponectin release and mRNA expression during preadipocyte differentiation, but the levels were inhibited by myostatin treatments in fully differentiated adipocytes. These results suggested that myostatin suppressed 3T3-L1 preadipocyte differentiation and regulated lipid metabolism of mature adipocyte, in part, via activation of ERK1/2 signalling pathway.     

Characterization of selenoprotein P as a selenium supply protein.

Selenium (Se) is well known to be essential for cell culture when using a serum-free medium, but not when a medium containing serum is used. This finding suggests that serum contains some usable form of Se. To identify the Se-supplier, T-lymphoma (Jurkat) cells were cultured for 3 days in the presence of human serum immunodepleted of Se-containing serum protein, selenoprotein P or extracellular glutathione peroxidase. The Se-dependent enzyme activities (glutathione peroxidases and thioredoxin reductase) and Se content within the cells markedly decreased only when cultured with selenoprotein P-depleted serum. Compared with other Se-containing proteins, the addition of purified selenoprotein P to the selenoprotein P-depleted serum or a serum-free medium was the most effective for the recovery of cellular glutathione peroxidase activity (index of Se status). These results suggest that selenoprotein P functions as a Se-supply protein, delivering Se to the cells.     

Microarray analysis of gene expression during early adipocyte differentiation

The molecular mechanisms that regulate cellular differentiation during development and throughout life are complex. It is now recognized that precise patterns of differentially expressed genes ultimately direct a particular cell toward a given lineage and many of these are regulated during the earliest stages of differentiation. Using a microarray-based expression analysis, we have examined gene expression profiles during the first 24 h of 3T3-L1 adipocyte differentiation. RNA was isolated at times 0, 2, 8, 16, and 24 h following stimulation of differentiation and hybridized in duplicate to high density Affymetrix microarray gene chips containing a series of 13,179 cDNA/expressed sequence tag (EST) probe sets. Two hundred and eighty-five cDNA/ESTs were shown to have at least a fivefold change in expression levels during this time course and both hierarchical and self-organizing map clustering analysis was performed to categorize them by expression profiles. Several genes known to be regulated during this time period were confirmed and Western blot analysis of the proteins encoded by some of the identified genes revealed expression profiles similar to their mRNA counterparts. As expected, many of the genes identified have not been examined in such a critical time period during adipogenesis and may well represent novel adipogenic mediators.     

Autotaxin is released from adipocytes,catalyzes lysophosphatidic acid synthesis,and activates preadipocyte proliferation. Up-regulated expression with adipocyte differentiation and obesity

Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904-910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipase d-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obese-diabetic db/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies.     

Attenuation of hepatic expression and secretion of selenoprotein P by metformin

High serum selenium levels have been associated epidemiologically with increased incidence of type 2 diabetes. The major fraction of total selenium in serum is represented by liver-derived selenoprotein P (SeP). This study was undertaken to test for a hypothesized effect of hyperglycemia and the antihyperglycemic drug metformin on hepatic selenoprotein P biosynthesis. Cultivation of rat hepatocytes in the presence of high glucose concentrations (25 mmol/l) resulted in increased selenoprotein P mRNA expression and secretion. Treatment with metformin dose-dependently downregulated SeP mRNA expression and secretion, and suppressed glucocorticoid-stimulated production of SeP. Moreover, metformin strongly decreased mRNA levels of selenophosphate synthetase 2 (SPS-2), an enzyme essential for selenoprotein biosynthesis. Taken together, these results indicate an influence of metformin on selenium metabolism in hepatocytes. As selenoprotein P is the major transport form of selenium, metformin treatment may thereby diminish selenium supply to extrahepatic tissues.     

Spatiotemporal expression of the selenoprotein P gene in postimplantational mouse embryos

Selenoprotein P (Sepp) is an extracellular glycoprotein which functions principally as a selenium (Se) transporter and antioxidant. In order to assess the spatiotemporal expression of the Sepp gene during mouse embryogenesis, quantitative RT-PCR and in situ hybridization analyses were conducted in embryos and extraembryonic tissues, including placenta. Sepp mRNA expression was detected in all embryos and extraembryonic tissues on embryonic days (E) 7.5 to 18.5. Sepp mRNA levels were high in extraembryonic tissues, as compared to embryos, on E 7.5-13.5. However, the levels were higher in embryos than in extraembryonic tissues on E 14.5-15.5, but were similar in both tissues during the subsequent periods prior to birth. According to the results of in situ hybridization, Sepp mRNA was expressed principally in the ectoplacental cone and neural ectoderm, including the neural tubes and neural folds. In whole embryos, Sepp mRNA was expressed abundantly in nervous tissues on E 9.5-12.5. Sepp mRNA was also expressed in forelimb and hindlimb buds on E 10.5-12.5. In the sectioned embryos, on E 13.5-18.5, Sepp mRNA was expressed persistently in the developing limbs, gastrointestinal tract, nervous tissue, lung, kidney and liver. On E 16.5-18.5, Sepp mRNA expression in the submandibular gland, whisker follicles, pancreas, urinary bladder and skin was apparent. In particular, Sepp mRNA was detected abundantly in blood cells during all the observed developmental periods. These results show that Sepp may function as a transporter of selenium, as well as an antioxidant, during embryogenesis.     

New assay for the measurement of selenoprotein P as a sepsis biomarker from serum

Selenium (Se) is incorporated into selenoproteins as the 21st proteinogenic amino acid selenocysteine. Serum Se concentrations decline during critical illness and are indicative of poor prognosis. Serum Se is mainly contained in the hepatically derived selenoprotein P (SePP) which controls the expression of antioxidative selenoproteins. Here, we describe the development of an immunoluminometric sandwich assay that uses two polyclonal sheep antihuman SePP antibodies. After assessing the stability of the analyte, we determined SePP concentrations in samples from healthy individuals and patients with sepsis. The analytical detection limit was 0.016 mg SePP/L serum. The assay was linear on dilution. SePP was stable in serum at room temperature for at least 24 h and resistant to six freeze-thaw cycles. Median SePP concentration in healthy individuals was 3.04 mg SePP/L serum (25th–75th percentiles, 2.6–3.4 mg/L) which corresponded to 98.4 μg Se/L serum. The interlaboratory CV was <20% for SePP values >0.06 mg/L. There was no association with gender, but concentrations differed between young and older individuals. Median SePP concentrations were significantly (P<0.0001) decreased in patients with sepsis (n=60) compared to healthy controls (n=318). Since SePP contains the major fraction of serum Se, we conclude that downregulation of SePP biosynthesis or removal of circulating SePP from blood underlies the negative acute phase response of serum Se in critical illness.     

Reduced expression of the NADPH oxidase NOX4 is a hallmark of adipocyte differentiation

Adipocyte differentiation is a complex process regulated among other factors by insulin and the production of reactive oxygen species (ROS). NOX4 is a ROS generating NADPH oxidase enzyme mediating insulin's action in 3T3L1 adipocytes. In the present paper we show that NOX4 is expressed at high levels both in white and brown preadipocytes and that differentiation into adipocytes results in a decrease in their NOX4 mRNA content. These in vitro results were confirmed in vivo by demonstrating that in intact adipose tissue the majority of NOX4 expressing cells are localized within the preadipocyte containing stromal/vascular fraction, rather than in the portion consisting of mature adipocytes. In line with these observations, quantification of NOX4 mRNA in fat derived from different rodent models of insulin resistance indicated that alteration in NOX4 expression reflects changes in the ratio of adipocyte/interstitial fractions. In conclusion, we reveal that decreased NOX4 mRNA content is a hallmark of adipocyte differentiation and that NOX4 expression measured in whole adipose tissue is not an unequivocal indicator of intact or impaired insulin action.     

Analysis of gene expression profile during 3T3-L1 preadipocyte differentiation

Cellular differentiation is a process in which a group of differentiation specific genes is programmatically induced. This gene expression program leads to changes in both cellular morphological and physiological phenotypes. Using an 18,376-member cDNA/EST microarray, we analyzed the difference in gene expression profiles between differentiated 3T3-L1 adipocyte and non-differentiated 3T3-L1 preadipocyte. From our study a large number of genes and ESTs were identified as differentially induced or suppressed. In this paper we describe the changes of gene expression profile during 3T3-L1 cell differentiation.     

家兔前体脂肪细胞分化不同时期基因表达谱分析

脂肪的过度积累严重危害人类健康。前体脂肪细胞分化是脂肪发育的关键过程,研究前体脂肪细胞分化相关基因的表达有助于认识脂肪沉积的机理。尽管家兔是一种理想的研究脂肪发育的动物模型,但是针对其前体脂肪细胞分化不同时期基因表达谱的研究鲜见报道。本研究通过诱导家兔前体脂肪细胞分化,在分化第0 d、3 d和9 d收集脂肪细胞,利用转录组测序(RNA-seq),在分化第3 d样本与第0 d样本的比较中筛选出1352个差异表达基因(differentially expressed genes, DEGs),在分化第9 d样本与第3 d样本的比较中筛选出888个DEGs。GO (gene ontology)功能富集和KEGG (kyoto encyclopedia of genes and genomes)通路分析发现,0~3 d分化期上调的DEGs显著富集在PPAR信号通路和PI3K-Akt信号通路上,3~9d分化期上调的DEGs显著富集到与细胞周期调控有关的GO条目和KEGG信号通路,0~3d和3~9d阶段特异上调的DEGs可能分别作用于细胞质和细胞核。通过DEGs的蛋白-蛋白互作(protein-protein interaction, PPI)网络分析发现,筛选出的核心节点(hub node)基因可能通过调控细胞周期而影响家兔前体脂肪细胞分化。