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author:("Chen, weilin")
1.  Altered Lipid Metabolism in Residual White Adipose Tissues of Bscl2 Deficient Mice 
PLoS ONE  2013;8(12):e82526.
Mutations in BSCL2 underlie human congenital generalized lipodystrophy type 2 disease. We previously reported that Bscl2−/− mice develop lipodystrophy of white adipose tissue (WAT) due to unbridled lipolysis. The residual epididymal WAT (EWAT) displays a browning phenotype with much smaller lipid droplets (LD) and higher expression of brown adipose tissue marker proteins. Here we used targeted lipidomics and gene expression profiling to analyze lipid profiles as well as genes involved in lipid metabolism in WAT of wild-type and Bscl2−/− mice. Analysis of total saponified fatty acids revealed that the residual EWAT of Bscl2−/− mice contained a much higher proportion of oleic18:1n9 acid concomitant with a lower proportion of palmitic16:0 acid, as well as increased n3- polyunsaturated fatty acids (PUFA) remodeling. The acyl chains in major species of triacylglyceride (TG) and diacylglyceride (DG) in the residual EWAT of Bscl2−/− mice were also enriched with dietary fatty acids. These changes could be reflected by upregulation of several fatty acid elongases and desaturases. Meanwhile, Bscl2−/− adipocytes from EWAT had increased gene expression in lipid uptake and TG synthesis but not de novo lipogenesis. Both mitochondria and peroxisomal β-oxidation genes were also markedly increased in Bscl2−/− adipocytes, highlighting that these machineries were accelerated to shunt the lipolysis liberated fatty acids through uncoupling to dissipate energy. The residual subcutaneous white adipose tissue (ScWAT) was not browning but displays similar changes in lipid metabolism. Overall, our data emphasize that, other than being essential for adipocyte differentiation, Bscl2 is also important in fatty acid remodeling and energy homeostasis.
PMCID: PMC3865019  PMID: 24358199
2.  Berardinelli-Seip Congenital Lipodystrophy 2/Seipin Is a Cell-Autonomous Regulator of Lipolysis Essential for Adipocyte Differentiation 
Molecular and Cellular Biology  2012;32(6):1099-1111.
Mutations in BSCL2 underlie human congenital generalized lipodystrophy. We inactivated Bscl2 in mice to examine the mechanisms whereby absence of Bscl2 leads to adipose tissue loss and metabolic disorders. Bscl2−/− mice develop severe lipodystrophy of white adipose tissue (WAT), dyslipidemia, insulin resistance, and hepatic steatosis. In vitro differentiation of both Bscl2−/− murine embryonic fibroblasts (MEFs) and stromal vascular cells (SVCs) reveals normal early-phase adipocyte differentiation but a striking failure in terminal differentiation due to unbridled cyclic AMP (cAMP)-dependent protein kinase A (PKA)-activated lipolysis, which leads to loss of lipid droplets and silencing of the expression of adipose tissue-specific transcription factors. Importantly, such defects in differentiation can be largely rescued by inhibitors of lipolysis but not by a gamma peroxisome proliferator-activated receptor (PPARγ) agonist. The residual epididymal WAT (EWAT) in Bscl2−/− mice displays enhanced lipolysis. It also assumes a “brown-like” phenotype with marked upregulation of UCP1 and other brown adipose tissue-specific markers. Together with decreased Pref1 but increased C/EBPβ levels, these changes highlight a possible increase in cAMP signaling that impairs terminal adipocyte differentiation in the EWAT of Bscl2−/− mice. Our study underscores the fundamental role of regulated cAMP/PKA-mediated lipolysis in adipose differentiation and identifies Bscl2 as a novel cell-autonomous determinant of activated lipolysis essential for terminal adipocyte differentiation.
PMCID: PMC3295006  PMID: 22269949
3.  Isolation and Identification of Endophytic Fungi from Actinidia macrosperma and Investigation of Their Bioactivities 
Endophytic fungi from the Chinese medicinal plant Actinidia macrosperma were isolated and identified for the first time. This was the first study to evaluate their cytotoxic and antitumour activities against brine shrimp and five types of tumour cells, respectively. In total, 17 fungal isolates were obtained. Five different taxa were represented by 11 isolates, and six isolates were grouped into the species of Ascomycete Incertae sedis with limited morphological and molecular data. Cytotoxic activity has been found in most isolates except AM05, AM06, and AM10. The isolates AM07 (4.86 μg/mL), AM11 (7.71 μg/mL), and AM17 (14.88 μg/mL) exhibited significant toxicity against brine shrimp. The results of the MTT assay to assess antitumour activity revealed that 82.4% of isolate fermentation broths displayed growth inhibition (50% inhibitory concentration IC50< 100 μg/mL). Moreover, AM07, AM11, and AM17 showed strong antitumour activity in all the cell lines examined. These results suggest that endophytic fungi in A. macrosperma are valuable for the isolation and identification of novel cytotoxic and antitumour bioactive agents.
PMCID: PMC3235672  PMID: 22203869
4.  Pnpla3/adiponutrin deficiency in mice is not associated with fatty liver disease 
Hepatology (Baltimore, Md.)  2010;52(3):1134-1142.
PNPLA3 (Adiponutrin), a novel patatin-like phospholipase domain-containing enzyme, is expressed at high level in fat, but also in other tissues including liver. Polymorphisms in PNPLA3 have been linked to obesity and insulin sensitivity. Notably, a nonsynonymous variant rs738409(G) allele of the PNPLA3 gene was found to be strongly associated with both non-alcoholic and alcoholic fatty liver disease. We have generated Pnpla3−/− mice by gene targeting. Loss of Pnpla3 has no effect on body weight or composition, adipose mass or development, whether the mice were fed regular chow or high-fat diet or bred into Lepob/ob background. Plasma and liver triglyceride content and plasma aspartate aminotransferase and alanine aminotransferase levels were not different between Pnpla3+/+ and Pnpla3−/− mice while they were on regular chow, fed three different fatty liver-inducing diets, or after they were bred into Lepob/ob background. Hepatic Pnpla5 mRNA levels were similar in wild-type and Pnpla3−/− mice, though adipose Pnpla5 mRNA level was increased in Pnpla3−/− mice. A high sucrose lipogenic diet stimulated hepatic Pnpla3 and Pnpla5 mRNA levels to a similar degree, but it did not affect adipose or liver triglyceride lipase (ATGL, aka Pnpla2) mRNA in Pnpla3+/+ and Pnpla3−/− mice. Finally, Pnpla3+/+ and Pnpla3−/− mice displayed similar glucose tolerance and insulin tolerance tests while on regular chow or three different fatty liver-inducing diets. Conclusion: Loss of Pnpla3 does not cause fatty liver, liver enzyme elevation, or insulin resistance in mice.
PMCID: PMC2932863  PMID: 20648554
Pnpla3 deficiency; fatty liver disease; insulin resistance
5.  Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP) 
Carbohydrate Response Element Binding Protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.
PMCID: PMC2874883  PMID: 20382127
carbohydrate response element binding protein (ChREBP); Glucose-6-phosphate (G-6-P); Transcriptional activation
6.  Remodeling of Retinal Fatty Acids in an Animal Model of Diabetes 
Diabetes  2009;59(1):219-227.
The results of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications cohort study revealed a strong association between dyslipidemia and the development of diabetic retinopathy. However, there are no experimental data on retinal fatty acid metabolism in diabetes. This study determined retinal-specific fatty acid metabolism in control and diabetic animals.
Tissue gene and protein expression profiles were determined by quantitative RT-PCR and Western blot in control and streptozotocin-induced diabetic rats at 3–6 weeks of diabetes. Fatty acid profiles were assessed by reverse-phase high-performance liquid chromatography, and phospholipid analysis was performed by nano-electrospray ionization tandem mass spectrometry.
We found a dramatic difference between retinal and liver elongase and desaturase profiles with high elongase and low desaturase gene expression in the retina compared with liver. Elovl4, an elongase expressed in the retina but not in the liver, showed the greatest expression level among retinal elongases, followed by Elovl2, Elovl1, and Elovl6. Importantly, early-stage diabetes induced a marked decrease in retinal expression levels of Elovl4, Elovl2, and Elovl6. Diabetes-induced downregulation of retinal elongases translated into a significant decrease in total retinal docosahexaenoic acid, as well as decreased incorporation of very-long-chain polyunsaturated fatty acids (PUFAs), particularly 32:6n3, into retinal phosphatidylcholine. This decrease in n3 PUFAs was coupled with inflammatory status in diabetic retina, reflected by an increase in gene expression of proinflammatory markers interleukin-6, vascular endothelial growth factor, and intercellular adhesion molecule-1.
This is the first comprehensive study demonstrating diabetes-induced changes in retinal fatty acid metabolism. Normalization of retinal fatty acid levels by dietary means or/and modulating expression of elongases could represent a potential therapeutic target for diabetes-induced retinal inflammation.
PMCID: PMC2797925  PMID: 19875612
7.  Evidence for U-tail stabilization of gRNA/mRNA interactions in kinetoplastid RNA Editing 
RNA biology  2004;1(1):28-34.
The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent KD in the 5–10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.
PMCID: PMC2762388  PMID: 17194935
affinity constant; guide RNA; RNA editing; RNA-RNA interaction; Trypanosomes
9.  Anti-inflammatory Effect of Docosahexaenoic Acid on Cytokine-Induced Adhesion Molecule Expression in Human Retinal Vascular Endothelial Cells 
Docosahexaenoic acid (DHA22:6n3), the principal n3-polyunsaturated fatty acid (PUFA) in the retina, has been shown to have a pronounced anti-inflammatory effect in numerous in vivo and in vitro studies. Despite the importance of vascular inflammation in diabetic retinopathy, the anti-inflammatory role of DHA22:6n3 in cytokine-stimulated human retinal vascular endothelial cells (hRVECs) has not been addressed.
Cytokine-induced expression of cell adhesion molecules (CAMs) was assessed by Western blot. The effect of DHA22:6n3 on cytokine-induced nuclear factor (NF)-κB signaling was analyzed by Western blot analysis and electrophoretic mobility shift assay (EMSA).
Stimulation of hRVECs with VEGF165, TNFα, or IL-1β for 6 to 24 hours caused significant induction of intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression. Pretreatment of the cells with 100 μM of BSA-bound DHA22:6n3 for 24 hours remarkably inhibited cytokine-induced CAM expression. IL-1β, TNFα, and VEGF165 induced nuclear translocation and binding of p65 and p50 NF-κB isoforms to the VCAM-1 promoter. DHA22:6n3 pretreatment inhibited cytokine-induced NF-κB binding by 25% to 40%. Moreover, DHA22:6n3 diminished IL-1β induced phosphorylation of the inhibitor of nuclear factor (NF)-κB (I-κBα), thus preventing its degradation.
IL-1β, TNFα, and VEGF165 induced CAM expression in hRVECs through activation of the NF-κB pathway. DHA22:6n3 inhibited cytokine induced CAM expression through suppression of NF-κB nuclear translocation and upstream I-κBα phosphorylation and degradation. DHA22:6n3 could be an important anti-inflammatory agent in the face of increased cytokine production and CAM expression in the diabetic retina.
PMCID: PMC1378111  PMID: 16249517
10.  Aquaporin 7 Is a β-Cell Protein and Regulator of Intraislet Glycerol Content and Glycerol Kinase Activity, β-Cell Mass, and Insulin Production and Secretion▿ † 
Molecular and Cellular Biology  2007;27(17):6026-6037.
To investigate if intracellular glycerol content plays a role in the regulation of insulin secretion in pancreatic β cells, we studied the expression of the glycerol channels, or aquaglyceroporins, encoded by the aquaporin 3 (Aqp3), Aqp7, and Aqp9 genes in mouse islets. We found expression of Aqp7 only, not that of Aqp3 or Aqp9, in the endocrine pancreas at both the mRNA (by reverse transcription-PCR) and protein (by immunohistochemistry) levels. Immunohistochemistry revealed a complete overlap between insulin and Aqp7 immunostaining in the pancreatic islet. Inactivation of Aqp7 by gene targeting produced viable and healthy mice. Aqp7−/− mice harbored an increased intraislet glycerol concentration with a concomitant increase of the glycerol kinase transcript level and enzyme activity. The islet triglyceride content in the Aqp7−/− mice was also increased compared to that in the Aqp7+/+ mice. Interestingly, Aqp7−/− mice displayed reduced β-cell mass and insulin content but increased insulin-1 and insulin-2 mRNAs. The reduction of β-cell mass in Aqp7−/− mice can be explained at least in part by a reduction in cell proliferation through protein kinase C and the c-myc cascade, with a reduction in the transcript levels of these two genes. Concomitantly, there was a decreased rate of apoptosis, as reflected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase 3 and Bax expression in Aqp7−/− mice. Compared with Aqp7+/+ islets, islets isolated from Aqp7−/− mice secreted insulin at a higher rate under basal low-glucose conditions and on exposure to a high (450 mg/dl) glucose concentration. Aqp7−/− mice exhibited normal fasting blood glucose levels but elevated blood insulin levels. Their plasma glucose response to an intraperitoneal (i.p.) glucose tolerance test was normal, but their plasma insulin concentrations were higher than those of wild-type mice during the 2-h test. An i.p. insulin tolerance test showed similar plasma glucose lowering in Aqp7−/− and Aqp7+/+ mice, with no evidence of insulin resistance. In conclusion, we found that pancreatic β cells express AQP7, which appears to be a key regulator of intraislet glycerol content as well as insulin production and secretion.
PMCID: PMC1952143  PMID: 17576812
11.  Inhibition of Cytokine Signaling in Human Retinal Endothelial Cells through Modification of Caveolae/Lipid Rafts by Docosahexaenoic Acid 
Docosahexaenoic acid (DHA22:6,n3) is the principal n3 polyunsaturated fatty acid (PUFA) in the retina. The authors previously demonstrated that DHA22:6,n3 inhibited cytokine-induced adhesion molecule expression in primary human retinal vascular endothelial (hRVE) cells, the target tissue affected by diabetic retinopathy. Despite the importance of vascular inflammation in diabetic retinopathy, the mechanisms underlying anti-inflammatory effects of DHA22:6,n3 in vascular endothelial cells are not understood. In this study the authors address the hypothesis that DHA22:6,n3 acts through modifying lipid composition of caveolae/lipid rafts, thereby changing the outcome of important signaling events in these specialized plasma membrane microdomains.
hRVE cells were cultured in the presence or absence of DHA22:6,n3. Isolated caveolae/lipid raft–enriched detergent-resistant membrane domains were prepared using sucrose gradient ultracentrifugation. Fatty acid composition and cholesterol content of caveolae/lipid rafts before and after treatment were measured by HPLC. The expression of Src family kinases was assayed by Western blotting and immunohistochemistry.
Disruption of the caveolae/lipid raft structure with a cholesterol-depleting agent, methyl-cyclodextrin (MCD), diminished cytokine-induced signaling in hRVE cells. Growth of hRVE cells in media enriched in DHA22:6,n3 resulted in significant incorporation of DHA22:6,n3 into the major phospholipids of caveolae/lipid rafts, causing an increase in the unsaturation index in the membrane microdomain. DHA22:6,n3 enrichment in the caveolae/raft was accompanied by a 70% depletion of cholesterol from caveolae/lipid rafts and displacement of the SFK, Fyn, and c-Yes from caveolae/lipid rafts. Adding water-soluble cholesterol to DHA22:6,n3-treated cells replenished cholesterol in caveolae/lipid rafts and reversed the effect of DHA22:6,n3 on cytokine-induced signaling.
Incorporation of DHA22:6,n3 into fatty acyl chains of phospholipids in caveolae/lipid rafts, followed by cholesterol depletion and displacement of important signaling molecules, provides a potential mechanism for anti-inflammatory effect of DHA22:6,n3 in hRVE cells.
PMCID: PMC1975816  PMID: 17197511

Results 1-11 (11)