By use of its affinity to gelatin-Cellulofine, a novel protein, GBP28 (gelatin-binding protein of 28 kDa), was obtained from human plasma. GBP28 bound to gelatin-Cellulofine could be eluted with 1 M NaCl. By analysis of its amino-terminal amino acid sequences and the peptides obtained by protease digestion, GBP28 was identified as a novel protein. After repeated gel chromatography of the 1 M NaCl eluate from gelatin-Cellulofine, about 50 micrograms of GBP28 was purified from 500 ml of human plasma. On gel chromatography, the protein migrated as a molecule of about 420 kDa. On SDS-PAGE, its molecular mass was 28 kDa under reducing conditions and 68 kDa under nonreducing conditions. Recently, human mRNA specific to adipose tissue, cDNA clone apM1, has been registered [Maeda, K., Okubo, K., Shimomura, I., Funahashi, T., Matsuzawa, Y., and Matsubara, K. (1996) Biochem. Biophys. Res. Commun. 221, 286-289]. The assumed amino acid sequence of cDNA clone apM1 contained all the sequences of GBP28 and its peptides. Therefore, it is evident that the cDNA clone apM1 encodes GBP28 and the protein is specific to adipose tissue. The clone encodes a polypeptide of 244 amino acids with a secretory signal sequence at the amino terminus, a small non-helical region, a stretch of 22 collagen repeats and a globular domain. Thus, GBP28 appears to belong to a family of proteins possessing a collagen-like domain through which they form homo-trimers, which further combine to make oligomeric complexes. Although its biological function is presently unclear, its adipocyte-specific expression suggests that GBP28 may function as an endogenous factor involved in lipid catabolism and storage or whole body metabolism.

Adiponectin (Acrp30), an adipocyte-derived protein, exists in serum as a trimer, a hexamer, and a high-molecular weight (HMW) form, including 12-18 subunits. Because HMW adiponectin may be biologically active, we measured it in serum using a novel enzyme-linked immunosorbent assay (ELISA) confirmed by gel filtration chromatography that the ELISA detected mainly adiponectin with 12-18 subunits, and we compared HMW with total adiponectin concentration in patients with type 2 diabetes. We next investigated the relationship between serum HMW and coronary artery disease (CAD) in 280 consecutive type 2 diabetic patients, including 59 patients with angiographically confirmed CAD. Total adiponectin was measured in serum by a commercially available ELISA. Like serum total adiponectin, HMW adiponectin correlated positively with HDL cholesterol and negatively with triglyceride, insulin sensitivity, creatinine clearance, and circulating inflammatory markers. Total and HMW adiponectin were significantly higher in women than in men, as was the HMW-tototal adiponectin ratio. Serum HMW and the HMW-to-total adiponectin ratio were significantly lower in men with than without CAD (P < 0.05, respectively). In women, the ratio, but neither total nor HMW adiponectin, tended to be lower when CAD was present. In conclusion, determination of HMW adiponectin, especially relative to total serum adiponectin, is useful for evaluating CAD in type 2 diabetic patients. Diabetes

Background-Overeating and obesity are major health problems in developed countries. Caloric restriction (CR) can counteract the deleterious aspects of obesity-related diseases and prolong lifespan. We have demonstrated that short-term CR improves myocardial ischemic tolerance and increases adiponectin levels. Here, we investigated the specific role of adiponectin in CR-induced cardioprotection. Methods and Results-Adiponectin antisense transgenic (Ad-AS) mice and wild-type (WT) mice were randomly assigned to a group fed ad libitum and a CR group (90% of caloric intake of ad libitum for 3 weeks, then 65% for 2 weeks). Isolated perfused mouse hearts were subjected to 25 minutes of ischemia, followed by 60 minutes of reperfusion. CR increased serum adiponectin levels by 84% in WT mice. Gel filtration analysis of the oligomeric complex distribution showed that CR produced a marked increase in the high-molecular-weight complex of adiponectin in WT mice; in contrast, CR did not change serum adiponectin levels or their oligomeric pattern in Ad-AS mice. CR improved the recovery of left ventricular function after ischemia/reperfusion and limited infarct size in WT mice; these effects were completely abrogated in Ad-AS mice. CR also increased the phosphorylated form of AMP-activated protein kinase and acetyl-CoA carboxylase in WT but not in Ad-AS mice. Recombinant adiponectin restored CR-induced cardioprotection in Ad-AS mice, and inhibition of AMP-activated protein kinase phosphorylation completely abrogated CR-induced cardioprotection in WT mice. Conclusion-The cardioprotective effects of short-term CR are mediated by increased production of adiponectin and the associated activation of AMP-activated protein kinase.