CD38 deficiency suppresses adipogenesis and lipogenesis in adipose tissues through activating Sirt1/PPARγ signaling pathway

It has been recently reported that CD38 was highly expressed in adipose tissues from obese people and CD38‐deficient mice were resistant to high‐fat diet (HFD)‐induced obesity. However, the role of CD38 in the regulation of adipogenesis and lipogenesis is unknown. In this study, to explore the roles of CD38 in adipogenesis and lipogenesis in vivo and in vitro, obesity models were generated with male CD38^?/? and WT mice fed with HFD. The adipocyte differentiations were induced with MEFs from WT and CD38^?/? mice, 3T3‐L1 and C3H10T1/2 cells in vitro. The lipid accumulations and the alternations of CD38 and the genes involved in adipogenesis and lipogenesis were determined with the adipose tissues from the HFD‐fed mice or the MEFs, 3T3‐L1 and C3H10T1/2 cells during induction of adipocyte differentiation. The results showed that CD38^?/? male mice were significantly resistant to HFD‐induced obesity. CD38 expressions in adipocytes were significantly increased in WT mice fed with HFD, and the similar results were obtained from WT MEFs, 3T3‐L1 and C3H10T1/2 during induction of adipocyte differentiation. The expressions of PPARγ, AP2 and C/EBPα were markedly attenuated in adipocytes from HFD‐fed CD38^?/? mice and CD38^?/? MEFs at late stage of adipocyte differentiation. Moreover, the expressions of SREBP1 and FASN were also significantly decreased in CD38^?/? MEFs. Finally, the CD38 deficiency‐mediated activations of Sirt1 signalling were up‐regulated or down‐regulated by resveratrol and nicotinamide, respectively. These results suggest that CD38 deficiency impairs adipogenesis and lipogenesis through activating Sirt1/PPARγ‐FASN signalling pathway during the development of obesity.     

Skp2 promotes adipocyte differentiation via a p27-independent mechanism in primary mouse embryonic fibroblasts

Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27^Kip1, a principal target of the SCF^Skp2 complex. Genetic ablation of p27^Kip1 in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27^Kip1 by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2^-/- MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), largely restored lipid accumulation and PPARγ gene expression in Skp2^−/− MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27^Kip1 expression.     

p27 is regulated independently of Skp2 in the absence of Cdk2

Cyclin-dependent kinase 2 (Cdk2) is dispensable for mitotic cell cycle progression and Cdk2 knockout mice are viable due to the compensatory functions of other Cdks. In order to assess the role of Cdk2 under limiting conditions, we used Skp2 knockout mice that exhibit increased levels of Cdk inhibitor, p27^Kip1, which is able to inhibit Cdk2 and Cdk1. Knockdown of Cdk2 abrogated proliferation of Skp2^−/− mouse embryonic fibroblasts, encouraging us to generate Cdk2^−/−Skp2^−/− double knockout mice. Cdk2^−/−Skp2^−/− double knockout mice are viable and display similar phenotypes as Cdk2^−/− and Skp2^−/− mice. Unexpectedly, fibroblasts generated from Cdk2^−/−Skp2^−/− double knockout mice proliferated at normal rates. The increased stability of p27 observed in Skp2^−/− MEFs was not observed in Cdk2^−/−Skp2^−/− double knockout fibroblasts indicating that in the absence of Cdk2, p27 is regulated by Skp2-independent mechanisms. Ablation of other ubiquitin ligases for p27 such as KPC1, DDB1, and Pirh2 did not restore stability of p27 in Cdk2^−/−Skp2^−/− MEFs. Our findings point towards novel and alternate pathways for p27 regulation.     

Skp2 controls adipocyte proliferation during the development of obesity

The increase in the mass of adipose tissue during the development of obesity can arise through an increase in cell size, an increase in cell number, or both. Here we show that long term maintenance of C57BL/6 mice on a high fat diet (for approximately 25 weeks) induces an initial increase in adipocyte size followed by an increase in adipocyte number in white adipose tissue. The latter effect was found to be accompanied by up-regulation of expression of the gene for the F-box protein Skp2 as well as by downregulation of the cyclin-dependent kinase inhibitor p27(Kip1), a principal target of the SCF(Skp2) ubiquitin ligase, in white adipose tissue. Ablation of Skp2 protected mice from the development of obesity induced either by a high fat diet or by the lethal yellow agouti (A(y)) mutation, and this protective action was due to inhibition of the increase in adipocyte number without an effect on adipocyte hypertrophy. The reduction in the number of adipocyte caused by Skp2 ablation also inhibited the development of obesity-related insulin resistance in the A(y) mutant mice, although the reduced number of beta cells and reduced level of insulin secretion in Skp2-deficient mice resulted in glucose intolerance. Our observations thus indicate that Skp2 controls adipocyte proliferation during the development of obesity.     

CD36 is important for adipocyte recruitment and affects lipolysis

Objective: The scavenger receptor CD36 facilitates the cellular uptake of long‐chain fatty acids. As CD36‐deficiency attenuates the development of high fat diet (HFD)‐induced obesity, the role of CD36‐deficiency in preadipocyte recruitment and adipocyte function was set out to characterize. Design and Methods: Fat cell size and number were determined in gonadal, visceral, and subcutaneous adipose tissue of CD36^?/? and WT mice after 6 weeks on HFD. Basal lipolysis and insulin‐inhibited lipolysis were investigated in gonadal adipose tissue. Results: CD36^?/? mice showed a reduction in adipocyte size in all fat pads. Gonadal adipose tissue also showed a lower total number of adipocytes because of a lower number of very small adipocytes (diameter <50 μm). This was accompanied by an increased pool of preadipocytes, which suggests that CD36‐deficiency reduces the capacity of preadipocytes to become adipocytes. Regarding lipolysis, in adipose tissue from CD36^?/? mice, cAMP levels were increased and both basal and 8‐bromo‐cAMP stimulated lipolysis were higher. However, insulin‐mediated inhibition of lipolysis was more potent in CD36^?/? mice. Conclusions: These results indicate that during fat depot expansion, CD36‐deficiency negatively affects preadipocyte recruitment and that in mature adipocytes, CD36‐deficiency is associated with increased basal lipolysis and insulin responsiveness.     

Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA^F/F) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA^F/F MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT mice contain significantly more adipocytes than those isolated from PCNA^F/F mice. This study identifies a critical role for PCNA in adipose tissue development, and for the first time identifies a role of the core DNA replication machinery at the interface between proliferation and differentiation.     

Sphk2−/− mice are protected from obesity and insulin resistance

Sphingosine kinases phosphorylate sphingosine to sphingosine 1?phosphate (S1P), which functions as a signaling molecule. We have previously shown that sphingosine kinase 2 (Sphk2) is important for insulin secretion. To obtain a better understanding of the role of Sphk2 in glucose and lipid metabolism, we have characterized 20- and 52-week old Sphk2^?/? mice using glucose and insulin tolerance tests and by analyzing metabolic gene expression in adipose tissue. A detailed metabolic characterization of these mice revealed that aging Sphk2^?/? mice are protected from metabolic decline and obesity compared to WT mice. Specifically, we found that 52-week old male Sphk2^?/? mice had decreased weight and fat mass, and increased glucose tolerance and insulin sensitivity compared to control mice. Indirect calorimetry studies demonstrated an increased energy expenditure and food intake in 52-week old male Sphk2^?/? versus control mice. Furthermore, expression of adiponectin gene in adipose tissue was increased and the plasma levels of adiponectin elevated in aged Sphk2^?/? mice compared to WT. Analysis of lipid metabolic gene expression in adipose tissue showed increased expression of the Atgl gene, which was associated with increased Atgl protein levels. Atgl encodes for the adipocyte triglyceride lipase, which catalyzes the rate-limiting step of lipolysis. In summary, these data suggest that mice lacking the Sphk2 gene are protected from obesity and insulin resistance during aging. The beneficial metabolic effects observed in aged Sphk2^?/? mice may be in part due to enhanced lipolysis by Atgl and increased levels of adiponectin, which has lipid- and glucose-lowering effects.     

Heat shock factor 1 deficiency via its downstream target gene αB‐crystallin (Hspb5) impairs p53 degradation

Heat shock factor Hsf1 regulates the stress‐inducibility of heat shock proteins (Hsps) or molecular chaperones. One of the functions attributed to Hsps is their participation in folding and degradation of proteins. We recently showed that hsf1^?/? cells accumulate ubiquitinated proteins. However, a direct role for Hsf1 in stability of specific proteins such as p53 has not been elucidated. We present evidence that cells deficient in hsf1 accumulate wild‐type p53 protein. We further show that hsf1^?/? cells express lower levels of αB‐crystallin and cells deficient in αB‐crystallin also accumulate p53 protein. Reports indicate that αB‐crystallin binds to Fbx4 ubiquitin ligase, and they target cyclin D1 for degradation through a pathway involving the SCF (Skp1‐Cul1‐F‐box) complex. Towards determining a mechanism for p53 degradation involving αB‐crystallin and Hsf1, we have found that ectopic expression of Fbx4 in wild‐type mouse embryo fibroblasts (MEFs) expressing mutant p53 (p53R175H) leads to increase in its degradation, while MEFs deficient in hsf1 or αBcry are defective in degradation of this p53 protein. In addition, immunoprecipitated p53R175H from wild‐type MEFs is able to pull‐down both αB‐crystallin and Fbx4. Finally, immunoprecipitated wild‐type p53 from doxorubicin treated U2OS cells can pull‐down endogenous αB‐crystallin and Fbx4. These results indicate that hsf1‐ and αBcry‐deficient cells accumulate p53 due to reduced levels of αB‐crystallin in these cells. Elevated levels of p53 in hsf1‐ and αBcry‐deficient cells lead to their increased sensitivity to DNA damaging agents. These data reveal a novel mechanism for protein degradation through Hsf1 and αB‐crystallin. J. Cell. Biochem. 107: 504–515, 2009. © 2009 Wiley‐Liss, Inc.     

Dietary alpha‐ketoglutarate promotes beige adipogenesis and prevents obesity in middle‐aged mice

Aging usually involves the progressive development of certain illnesses, including diabetes and obesity. Due to incapacity to form new white adipocytes, adipose expansion in aged mice primarily depends on adipocyte hypertrophy, which induces metabolic dysfunction. On the other hand, brown adipose tissue burns fatty acids, preventing ectopic lipid accumulation and metabolic diseases. However, the capacity of brown/beige adipogenesis declines inevitably during the aging process. Previously, we reported that DNA demethylation in the Prdm16 promoter is required for beige adipogenesis. DNA methylation is mediated by ten–eleven family proteins (TET) using alpha‐ketoglutarate (AKG) as a cofactor. Here, we demonstrated that the circulatory AKG concentration was reduced in middle‐aged mice (10‐month‐old) compared with young mice (2‐month‐old). Through AKG administration replenishing the AKG pool, aged mice were associated with the lower body weight gain and fat mass, and improved glucose tolerance after challenged with high‐fat diet (HFD). These metabolic changes are accompanied by increased expression of brown adipose genes and proteins in inguinal adipose tissue. Cold‐induced brown/beige adipogenesis was impeded in HFD mice, whereas AKG rescued the impairment of beige adipocyte functionality in middle‐aged mice. Besides, AKG administration up‐regulated Prdm16 expression, which was correlated with an increase of DNA demethylation in the Prdm16 promoter. In summary, AKG supplementation promotes beige adipogenesis and alleviates HFD‐induced obesity in middle‐aged mice, which is associated with enhanced DNA demethylation of the Prdm16 gene.     

The essential function of CARD9 in diet‐induced inflammation and metabolic disorders in mice

Inflammation and metabolic disorder are common pathophysiological conditions, which play a vital role in the development of obesity and type 2 diabetes. The purpose of this study was to explore the effects of caspase recruitment domain (CARD) 9 in the high fat diet (HFD)‐treated mice and attempt to find a molecular therapeutic target for obesity development and treatment. Sixteen male CARD9^?/? and corresponding male WT mice were fed with normal diet or high fat diet, respectively, for 12 weeks. Glucose tolerance, insulin resistance, oxygen consumption and heat production of the mice were detected. The CARD9/MAPK pathway‐related gene and protein were determined in insulin‐responsive organs using Western blotting and quantitative PCR. The results showed that HFD‐induced insulin resistance and impairment of glucose tolerance were more severe in WT mice than that in the CARD9^?/? mice. CARD9 absence significantly modified O₂ consumption, CO₂ production and heat production. CARD9^?/? mice displayed the lower expression of p38 MAPK, JNK and ERK when compared to the WT mice in both HFD‐ and ND‐treated groups. HFD induced the increase of p38 MAPK, JNK and ERK in WT mice but not in the CARD9^?/? mice. The results indicated that CARD9 absence could be a vital protective factor in diet‐induced obesity via the CARD9/MAPK pathway, which may provide new insights into the development of gene knockout to improving diet‐induced obesity and metabolism disorder.     

Effect of apoptosis‐associated speck‐like protein containing a caspase recruitment domain on vaccine efficacy: Overcoming the effects of its deficiency with aluminum hydroxide adjuvant

Host factors such as nutritional status and immune cell state are important for vaccine efficacy. Inflammasome activation may be important for triggering vaccine‐induced humoral and cell‐mediated immune responses. Formulations with alum as a typical adjuvant to overcome the effects of host factors have recently been shown to induce inflammasome activation, which augments vaccine efficacy. Apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) is one of the main components of inflammasomes, but it is not clear whether ASC affects the vaccine‐induced immune response. Herein, we used two types of vaccines: inactivated influenza vaccine not formulated with alum, and HPV vaccine formulated with alum. We gave the vaccines to ASC knockout (ASC^?/?) mice to investigate the role of ASC in vaccine efficacy. Influenza vaccine‐immunized ASC^?/? mice did not show antibody titers in week 2 after the first vaccination. After boosting, the antibody titer in ASC^?/? mice was about half that in wild type (WT) mice. Furthermore, a cytotoxic T‐lymphocyte response against influenza vaccine was not induced in ASC^?/? mice. Therefore, vaccinated ASC^?/? mice did not show effective protection against viral challenge. ASC^?/? mice immunized with alum‐formulated HPV vaccine showed similar antibody titers and T‐cell proliferation compared with immunized WT mice. However, the HPV vaccine without alum induced up to threefold lower titers of HPV‐specific antibody titers in ASC^?/? mice compared with those in WT mice. These findings suggest that alum in vaccine can overcome the ASC‐deficient condition.

     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Evidence for cytosolic p27(Kip1) ubiquitylation and degradation during adipocyte hyperplasia

Objective: Subcellular localization has been shown to play an important role in determining activity and accumulation of p27 protein during cell cycle progression. The purpose of this study was to examine p27 localization and ubiquitylation in relation to E3 ligase expression during adipocyte hyperplasia. Research Methods and Procedures: This study used the murine 3T3‐L1 preadipocyte model to examine p27 regulation during synchronous cell cycle progression. Cell lysates were isolated over time after hormonal stimulation, fractionated to cytosolic and nuclear compartments, and immunoblotted for relative protein determinations. Results: Data presented in this study show that p27 was present in the cytosol and nucleus in density‐arrested preadipocytes and that abundance in both compartments decreased in a phase‐specific manner as preadipocytes synchronously re‐entered the cell cycle during early phases of adipocyte differentiation. Blocking CRM1‐mediated nuclear export did not prevent degradation, nor did it cause nuclear accumulation of p27, suggesting that distinct mechanisms mediating cytosolic and nuclear p27 degradation were involved. Treating preadipocytes with a potent and specific proteasome inhibitor during hormonal stimulation prevented Skp2 accumulation and p27¹⁸⁷ phosphorylation, which are essential events for SCF^Skp2 E3 ligase activity and nuclear p27 ubiquitylation during S/G₂ phase progression. Proteasome blockade also resulted in the first evidence of cytosolic p27 ubiquitylation during late G₁ phase as preadipocytes undergo the transition from quiescence to proliferation. Discussion: These data are consistent with the postulate that p27 is ubiquitylated and targeted for degradation by the 26S proteasome in a phase‐specific manner by distinct ubiquitin E3 ligases localized to the cytosol and nucleus during adipocyte hyperplasia.     

Defective ATM‐Kap‐1‐mediated chromatin remodeling impairs DNA repair and accelerates senescence in progeria mouse model

ATM‐mediated phosphorylation of KAP‐1 triggers chromatin remodeling and facilitates the loading and retention of repair proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs) derived from Zmpste24^?/? mice undergo early senescence, attributable to delayed recruitment of DNA repair proteins. Here, we show that ATM‐Kap‐1 signaling is compromised in Zmpste24^?/? MEFs, leading to defective DNA damage‐induced chromatin remodeling. Knocking down Kap‐1 rescues impaired chromatin remodeling, defective DNA repair and early senescence in Zmpste24^?/? MEFs. Thus, ATM‐Kap‐1‐mediated chromatin remodeling plays a critical role in premature aging, carrying significant implications for progeria therapy.     

VDAC2 is required for truncated BID‐induced mitochondrial apoptosis by recruiting BAK to the mitochondria

Truncated BID (tBID), a proapoptotic BCL2 family protein, induces BAK/BAX‐dependent release of cytochrome c and other mitochondrial intermembrane proteins to the cytosol to induce apoptosis. The voltage‐dependent anion channels (VDACs) are the primary gates for solutes across the outer mitochondrial membrane (OMM); however, their role in apoptotic OMM permeabilization remains controversial. Here, we report that VDAC2^?/? (V2^?/?) mouse embryonic fibroblasts (MEFs) are virtually insensitive to tBID‐induced OMM permeabilization and apoptosis, whereas VDAC1^?/?, VDAC3^?/? and VDAC1^?/?/VDAC3^?/? MEFs respond normally to tBID. V2^?/? MEFs regain tBID sensitivity after VDAC2 expression. Furthermore, V2^?/? MEFs are deficient in mitochondrial BAK despite normal tBID–mitochondrial binding and BAX/BAK expression. tBID sensitivity of BAK^?/? MEFs is also reduced, although not to the same extent as V2^?/? MEFs, which might result from their strong overexpression of BAX. Indeed, addition of recombinant BAX also sensitized V2^?/? MEFs to tBID. Thus, VDAC2 acts as a crucial component in mitochondrial apoptosis by allowing the mitochondrial recruitment of BAK, thereby controlling tBID‐induced OMM permeabilization and cell death.     

Leukocyte Migration in Adipose Tissue of Mice Null for ICAM‐1 and Mac‐1 Adhesion Receptors

Objective: To determine whether the leukocyte adhesion receptors ICAM‐1 and Mac‐1, regulators of immune cell migration, have an intrinsic role within adipose tissue by 1) analyzing the expression of ICAM‐1 in adipose tissue, 2) identifying leukocyte populations within adipose tissue, and 3) determining whether ICAM‐1 and Mac‐1 mutant mice exhibit abnormal numbers of adipose tissue leukocytes. Research Methods and Procedures: Wild‐type, ICAM‐1^?/?, and Mac‐1^?/? mice were fed a long‐term high‐fat diet. ICAM‐1 expression was analyzed by Northern blot and immunohistochemistry. Leukocytes within adipose tissue were identified by immunohistochemistry and flow cytometry. Results: ICAM‐1 was expressed in adipose tissue and localized to the vascular endothelium. Macrophages and lymphocytes were prevalent within the stromal‐vascular cell fraction of adipose tissue, and gender‐specific differences were observed, with adipose tissue from female mice containing significantly more macrophages than tissue from male mice. Numbers of leukocytes in ICAM‐1^?/? and Mac‐1^?/? mice were not different from wild‐types, however, indicating that these adhesion receptors are not required for leukocyte migration into adipose tissue. Discussion: Our results documented leukocyte populations within adipose tissue, which may be involved in the development of heightened inflammation that is characteristic of obesity.     

Distinct serum proteome profiles associated with collagen‐induced arthritis and complete Freund's adjuvant‐induced inflammation in CD38−/− mice: The discriminative power of protein species or proteoforms

Collagen‐type‐II‐induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38^?/? than in wild‐type (WT) mice. ProteoMiner‐equalized serum samples were subjected to 2D‐DiGE and MS‐MALDI‐TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38^?/? versus WT mice either with arthritis (CIA⁺), with no arthritis (CIA^?), or with inflammation (complete Freund's adjuvant (CFA)‐treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA⁺ from CIA^? mice, and WT from CD38^?/? mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA⁺ CD38^?/? mice from CIA⁺ WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38^?/? and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low‐abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 ( http://proteomecentral.proteomexchange.org/dataset/PXD001788 , http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071 ).     

Asiaticoside might attenuate bleomycin‐induced pulmonary fibrosis by activating cAMP and Rap1 signalling pathway assisted by A2AR

Asiaticoside (AS) has been reported to have protective effect on pulmonary fibrosis (PF). In this study, we aimed to explore the potential mechanism of the therapeutic role of AS and its relationship with A2AR in PF. Adenosine 2A receptor gene knockout (A2AR^?/?) mice and wild‐type (WT) mice were used to establish bleomycin (BLM)‐induced PF models and were then treated with AS (50 mg/kg/d). Pulmonary inflammation and fibrosis were observed in the PF model with much higher severity in A2AR^?/?mice than that in WT mice and AS significantly alleviated lung inflammation and fibrosis; however, it was less effective in A2AR^?/? mice than in WT mice via histopathological analysis. Using RNA sequencing analysis, we found up‐regulated differentially expressed genes (DEGs) in BLM group were enriched in immune and inflammation‐associated pathways compared with control group. There were 242 common DEGs between down‐regulated in BLM vs control group and up‐regulated in BLM + AS vs BLM group, which were enriched in cAMP and Rap1 signalling pathways. Furthermore, the expression of five key factors of these two pathways including adenylate cyclase (ADCY1, ADCY5, ADCY8, cAMP and Rap1) were confirmed up‐regulated by AS with the presence of A2AR. Therefore, AS might attenuate BLM‐induced PF by activating cAMP and Rap1 signalling pathways which is assisted by A2AR, making it a promising therapeutic optional for PF.     

Targeted deletion of autophagy-related 5 (atg5) impairs adipogenesis in a cellular model and in mice

Mammalian white adipocytes have a unique structure in which nearly the entire cell volume is occupied by a single large lipid droplet, while the surrounding cytoplasm occupies minimal space. The massive cytoplasmic remodeling processes involved in the formation of this unique cellular structure are poorly defined. Autophagy is a membrane trafficking process leading to lysosomal degradation of cytoplasmic components. Here, we investigated the functional role of atg5, a gene encoding an essential protein required for autophagy, in adipocyte differentiation in a cellular model and in mice. Massive autophagy was activated when wild type primary mouse fibroblasts (MEFs) were induced for adipocyte differentiation. Importantly, the autophagy deficient primary atg5-/- MEFs exhibited dramatically reduced efficiency in adipogenesis. Time-lapse microscopy revealed that atg5^-/- MEFs initially appeared to differentiate normally; however, a majority of the differentiating atg5^-/- cells ultimately failed to undergo further morphological transformation and eventually died, likely through apoptosis. Consistent with these in vitro results, histological analysis revealed that the atg5^-/- late-stage embryos and neonatal pups had much less subcutaneous perilipin A-positive adipocytes. Consistently, when treated with chloroquine, a functional inhibitor of autophagy, wild type MEFs exhibited drastically reduced efficiency of adipocyte differentiation. Taken together, these findings demonstrated that Atg5 is involved in normal adipocyte differentiation, suggesting an important role of autophagy in adipogenesis.