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Adiponectin is an adipocyte-derived hormone and known to form several species of multimer, however, the precise components of each multimer have not been fully determined. We purified each multimer adiponectin selectively from human plasma and characterized them by affinity columns using anti-adiponectin, gelatin, or anti-albumin antibody and gel filtration. We found that adiponectin exists as four species of multimers in human plasma. According to their migrating mobility and N-terminal amino acid analysis, we defined them as a trimer, albumin-binding trimer, hexamer, and HMW. Low pH shifted HMW to hexamer, raising the possibility that HMW is a 12 mer or larger multimer. We also showed that HMW had the highest binding activity to the membrane fractions of C2C12 myocytes and activated AMPK most potently. Our results indicate that adiponectin forms diverse multimer species and at least some of the functional properties are dependent on a multimer status.

Adiponectin is an anti-diabetic and anti-atherogenic hormone that is exclusively secreted from fat cells. Serum adiponectin levels are reduced in obese patients and obese model mice, despite increased adipose tissue mass. Elucidation of the mechanism(s) by which plasma adiponectin levels are decreased in obese and diabetic patients would provide insight into the cause of obesity-induced diabetes and the development of therapeutic advances. In the present study, the regulation of adiponectin secretion was investigated using 3T3-L1 adipocytes and a diabetic-/obese-mouse model. A novel insulin sensitizer, IkappaB kinase beta (IKKbeta) inhibitor, ameliorated insulin resistance and up-regulated plasma levels of adiponectin without producing a significant change in body weight in KKAy mice that were fed a high-fat diet. The IKKbeta inhibitor cancelled the TNFalpha-mediated down-regulation of adiponectin secretion and simultaneously up-regulated the phosphorylation of Akt in 3T3-L1 adipocytes. Using dominant-negative mutants of Akt or PKClambda (downstream effectors of phosphoinositide 3-kinase), insulin-stimulated Akt activity was found to be important in the regulation of adiponectin secretion by insulin in 3T3-L1 adipocytes. These observations suggest that "insulin-stimulated Akt activity in adipocytes" may play an important role in the regulation of adiponectin secretion., (Copyright 2004 Elsevier Inc.)

The acute lymphoblastic leukemia cell line CCRF-CEM is sensitive to Ara-C and undergoes apoptosis. In contrast, the chronic myelogenous leukemia (CML) cell line K562 is highly resistant to Ara-C, which causes the cells to differentiate into erythrocytes before undergoing apoptosis. We used cDNA microarrays to monitor the alterations in gene expression in these two cell lines under conditions leading to apoptosis or differentiation. Ara-C-treated CCRF-CEM cells were characterized by a cluster of down-regulated chaperone genes, whereas Ara-C-treated K562 cells were characterized by a cluster of up-regulated hemoglobin genes. In K562 cells, Ara-C treatment induced significant down-regulation of the asparagine synthetase gene, which is involved in resistance to L-asparaginase. Sequential treatment with Ara-C and L-asparaginase had a synergistic effect on the inhibition of K562 cell growth, and combination therapy with these two anticancer agents may prove effective in the treatment of CML, which cannot be cured by either drug alone.

Sphingosine 1-phosphate (S1P) is known to regulate cell proliferation, apoptosis, and motility. Recently, we have reported that S1P and its analogue dihydro-S1P (DHS1P) promote proliferation of rat cultured mesangial cells. To investigate the signaling mechanisms underlying S1P- and DHS1P-induced mesangial cell proliferation, we performed cDNA microarray analysis of gene expression during mesangial cell proliferation. In terms of the overall pattern, gene expression waves induced by S1P and DHS1P were similar to those induced by a potent mesangial mitogen platelet-derived growth factor (PDGF), whereas we found several genes, such as two growth factors, connective tissue growth factor (CTGF) and heparin-binding EGF-like growth factor (HB-EGF), which were induced by the sphingolipids, but not by PDGF. Cluster analysis also identified calcium-dependent molecules highly expressed in DHS1P-stimulated cells compared to S1P-stimulated cells. Calcium mobilization analysis showed that DHS1P had higher magnitudes of intracellular calcium mobilization than S1P, suggesting that S1P and DHS1P differentially regulate intracellular calcium mobilization, possibly leading to different gene expression in mesangial cells. The large-scale monitoring of gene expression performed here allows us to identify S1P-induced transcriptional properties during mesangial cell proliferation.