IL-25 directly modulates adipocyte function and inflammation through the regulation of adiponectin.

OBJECTIVE: This study aimed to investigate the direct role of IL-25 in modulating adipocyte function during homeostasis and low-grade inflammation induced by lipopolysaccharide (LPS). METHODS: The 3T3-L1 preadipocyte cell lines and primary cultures of adipose-derived stromal vascular precursor cells of wild-type and IL-17RB-deficient mice were used to determine the direct function of IL-25. The expression of IL-17RB in differentiating adipocyte was determined using real-time PCR and flow cytometry analysis. The effect of IL-25 on lipid accumulation, triglyceride content, lipolysis, glucose uptake, and adipokine expression in the mature adipocytes was evaluated. IL-25 modulating the expression of inflammatory cytokines in adipocytes induced by low dose LPS was determined using real-time PCR and ELISA. RESULTS: The receptor for IL-25 was up-regulated during adipocyte differentiation and IL-25 directly modulated adipocyte function by reducing lipid accumulation and triglyceride concentration and enhancing lipolysis without affecting an insulin-stimulated glucose uptake. Interestingly, IL-25 induced adiponectin secretion through the PI3K/AKT signaling pathway. In 3T3-L1 adipocytes under low-grade inflammation, IL-25 attenuated the expression of IL-6 and CCL5 through the induction of adiponectin. CONCLUSION: Our studies suggest that IL-25 directly regulates adipocyte function by maintaining the adiponectin level during homeostasis and by alleviating inflammatory response through the regulation of adiponectin during low-grade inflammation in adipocytes.

Disulfide linkages in Plasmodium falciparum plasmepsin-i are essential elements for its processing activity and multi-milligram recombinant production yield.

Plasmodium falciparum plasmepsin-I (PM-I) has been considered a potential drug target for the parasite that causes fatal malaria in human. Determination of PM-I structures for rational design of its inhibitors is hindered by the difficulty in obtaining large quantity of soluble enzyme. Nearly all attempts for its heterologous expression in Escherichia coli result in the production of insoluble proteins in both semi-pro-PM-I and its truncated form, and thus require protein refolding. Moreover, the yields of purified, soluble PM-I from all reported studies are very limited. Exclusion of truncated semi-pro-PM-I expression in E. coli C41(DE3) is herein reported. We also show that the low preparation yield of purified semi-pro-PM-I with autoprocessing ability is mainly a result of structural instability of the refolded enzyme in acidic conditions due to incomplete formation of disulfide linkages. Upon formation of at least one of the two natural disulfide bonds, nearly all of the refolded semi-pro-PM-I could be activated to its mature form. A significantly improved yield of 10 mg of semi-pro-PM-I per liter of culture, which resulted in 6-8 mg of the mature PM-I, was routinely obtained using this strategy.

Effects of niacin and chromium on the expression of ATP-binding cassette transporter A1 and apolipoprotein A-1 genes in HepG2 cells.

The ATP-binding cassette transporter A1 (ABCA1) and apolipoprotein A1 (ApoA-1) are both involved in the regulation of cholesterol efflux from cells. The overexpression of ABCA1 and ApoA-1 genes are associated with increased high-density lipoprotein (HDL) levels. Previous studies have shown that niacin and chromium reduce plasma cholesterol while increasing HDL levels. The aim of the present study was to determine the effects of niacin and chromium on HDL formation by investigating the changes in ABCA1 and ApoA-1 transcription in the human hepatoblastoma cell line (HepG2 cells). Cells were treated with either niacin or chromium, or the combination of both. The expression of ABCA1 and ApoA-1 mRNA was measured by a relative quantitative real-time reverse transcriptase-polymerase chain reaction method. Results showed that niacin at concentrations of 1 and 5 mM significantly increased ABCA1 (1.3-1.7-fold), without affecting ApoA-1 (0.8-1.2-fold), whereas chromium at 3 mM significantly increased both ABCA1 (1.7+/-0.01-fold) and ApoA-1 (1.5+/-0.1-fold) transcription when compared to untreated cells. Niacin and chromium cotreatment significantly induced the expression of peroxisome proliferator-activated receptor-alpha (PPARalpha) mRNA by approximately 1.3-1.8-fold. It was likely that the increases observed for the ABCA1 transcript may be regulated by the increases in PPARalpha transcription. A combination of niacin and chromium chloride did not significantly increase (3+1 mM) but instead reduced (1+3 mM) ABCA1 gene expression. In the case of ApoA gene, the combination of niacin and chromium chloride at concentrations of 1+3 mM significantly elevated expression; however, this effect was not observed at concentrations of 3+1 mM. When cells were treated with the combination at both concentrations, only slight increases in PPARalpha mRNA was observed. Niacin, but not chromium, significantly reduced intracellular cholesterol. We hypothesize that the stimulation of ABCA1 gene expression causes an enhanced cholesterol efflux, perhaps mediated by PPARalpha pathway(s).

The trans-10, cis-12 isomer of conjugated linoleic acid decreases adiponectin assembly by PPARgamma-dependent and PPARgamma-independent mechanisms.

The adipocyte-derived secretory protein adiponectin functions as an insulin-sensitizing agent. In plasma, adiponectin exists as low, medium, and high molecular weight oligomers. Treatment with trans-10, cis-12 conjugated linoleic acid (t-10, c-12 CLA) reduces levels of adiponectin as well as triglyceride (TG) in mice and adipocyte cell culture models. The aim of this study was to determine whether the effects of t-10, c-12 CLA on adiponectin and TG are mediated through modulation of the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). 3T3-L1 cells were treated either during or after differentiation into adipocytes with 100 microM t-10, c-12 CLA with or without 10 microM troglitazone, a PPARgamma agonist, or 1 microM GW9662, a PPARgamma antagonist, and adiponectin and TG levels were analyzed. Treatment with t-10, c-12 CLA reduced TG as well as cellular and secreted adiponectin levels and impaired the assembly of adiponectin oligomers. These changes were accompanied by decreases in PPARgamma mass. Troglitazone was able to reverse the t-10, c-12 CLA-mediated decrease in TG levels and restore the assembly of adiponectin oligomers but was unable to restore adiponectin synthesis. Conversely, treatment with GW9662 decreased TG mass and impaired adiponectin oligomer assembly but did not decrease total adiponectin mass. In a reporter assay, t-10, c-12 CLA appeared to be a partial PPARgamma agonist and prevented the stimulation of reporter activity by troglitazone. Therefore, the t-10, c-12 CLA isomer appears to alter adipocyte adiponectin metabolism through PPARgamma-dependent and PPARgamma-independent mechanisms.

Active site contribution to specificity of the aspartic proteases plasmepsins I and II.

Plasmepsins I and II (PM I and II) are aspartic proteases involved in the initial steps of Plasmodium hemoglobin degradation. They are attractive targets for antimalarial drug development. The two enzymes are 73% identical, yet have different substrate and inhibitor specificities. The x-ray structures of proform and mature PM II have been determined, but models of PM I do not adequately explain the selectivity of the two proteases. To better understand the basis of these recognition differences, we have identified nine residues of PM II that are in proximity to the inhibitor pepstatin in the crystal structure and differ in PM I. We mutated these residues in PM II to the cognate amino acids of PM I. Kinetic parameters for substrate and inhibitors for the PM II-mutant were similar to those of PM II-wild type (WT). Cleavage specificity was assessed using hemoglobin or a random decamer peptide library as substrate. Again, PM II-mutant behaved like PM II-WT rather than PM I-WT. These results indicate that differences in plasmepsin specificity depend more on conformational differences from distant sites than on specific active site variation.