OBJECTIVE: Here we use a novel ELISA that is specific for full-length visfatin (PBEF/NAMPT), compare it with the existing C-terminal based assay and use it to investigate associations of visfatin with metabolic parameters. DESIGN, PATIENTS AND MEASUREMENTS: We established the specificity and effectiveness of the new ELISA and evaluated the associations of full-length visfatin with clinical, anthropometric and metabolic parameters in a cross-sectional study of 129 Thai subjects, consisting of 50 outpatients with type 2 diabetes and 79 healthy volunteers. RESULTS: The new ELISA accurately recovered full-length recombinant visfatin and detected visfatin secreted by primary human and rat adipocytes. We found serum full-length visfatin was significantly higher in subjects with diabetes compared to their nondiabetic peers (median 2.75 vs. 2.22 ng/ml, P = 0.0142). After adjustment for age, gender and traditional metabolic risk factors, adjusted mean visfatin remained significantly higher in the diabetes group (3.80 vs. 2.10 ng/ml, P = 0.0021). On Spearman univariate correlation analysis, visfatin was significantly associated with resistin (r = 0.30, P = 0.0011), but not with any other anthropometric or metabolic variables, including adiponectin multimers. On multiple linear regression analysis, the only covariates independently associated with visfatin were diabetes (t = 3.11, P = 0.0024) and log resistin (t = 2.68, P = 0.0086). CONCLUSIONS: Circulating visfatin is independently associated with diabetes and resistin concentration, but is not related to adiponectin multimers or other metabolic covariates. These data are suggestive of a potential role of visfatin in subclinical inflammatory states.

We developed a coculture system comprising primary rat adipocytes and L6 rat skeletal muscle cells to allow investigation of the effects of physiologically relevant mixtures of adipokines. We observed that coculture, or adipocyte-conditioned media, increased glucose uptake in muscle cells. An adipokine that could potentially mediate this effect is adiponectin, and we demonstrated that small interfering RNA-mediated knockdown of adiponectin receptor-2 in muscle cells reduced the uptake of glucose upon coculture with primary rat adipocytes. Analysis of coculture media by ELISA indicated total adiponectin concentration of up to 1 microg/ml, and Western blotting and gel filtration analysis demonstrated that the adipokine profile was hexamer greater than high molecular weight much greater than trimer. We used the streptozotocin-induced rat model of diabetes and found that high-molecular-weight adiponectin levels decreased in comparison with control animals and this correlated with the fact that diabetic rat-derived primary adipocytes in coculture did not stimulate glucose uptake to the same extent as control adipocytes. Coculture induced phosphorylation of AMP-activated protein kinase (T172) and interestingly also insulin receptor substrate-1 (Y612) and Akt (T308 & S473), which could be attenuated after adiponectin receptor-2-small interfering RNA treatment. In summary, we believe that this coculture system represents an excellent model to study the effects of primary adipocyte-derived adipokine mixtures on skeletal muscle metabolism, and here we have established that in the context of physiologically relevant mixtures of adipokines, adiponectin may be an important determinant of positive cross talk between adipocytes and skeletal muscle.

AIM: To investigate the anti-cancer mechanisms of Korean mistletoe lectin (Viscum album coloratum agglutinin, VCA) using a human colon cancer cell line (COLO). METHODS: Cytotoxic effects of VCA on COLO cells were determined by 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay in vitro and tumor-killing effects in vivo. To study the mechanisms involved, the expression of various pro-caspases, anti-apoptotic proteins, and death receptors was determined by western blot. To determine which death receptor is involved in VCA-induced apoptosis of COLO cells, cytotoxicity was examined by MTT assay after treatment with agonists or antagonists of death receptors. RESULTS: VCA killed COLO cells in a time- and dose-dependent manner and induced complete regression of tumors in nude mice transplanted with COLO cells. Treatment of COLO cells with VCA activated caspase-2, -3, -8, and -9 and decreased expression of anti-apoptotic molecules including receptor interacting protein, nuclear factor-kappaB, X-linked inhibitor of apoptosis protein, and Akt/protein kinase B. We then examined the involvement of death receptors in VCA-induced apoptosis. Only tumor necrosis factor receptor 1, among the death receptors examined, was involved in apoptosis of COLO cells, evidenced by inhibition of VCA-induced apoptosis and decreased activation of caspases, particularly caspase-8, by tumor necrosis factor receptor 1 antagonizing antibody. CONCLUSION: VCA-induced apoptotic COLO cell death is due to the activation of caspases and inhibition of anti-apoptotic proteins, in part through the tumor necrosis factor receptor 1 signaling pathway.