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- _3 value "The presence of two bands (~45 and~50 kDa) in the sera may suggest processing or cleavage of vaspin by unknown serine proteases in the serum, which may be increased in states of insulin resistance or abdominal obesity. Alternatively, vaspin may have complexed to low Mr peptide in the blood to yield a high Mr ~50-kDa protein band. To ensure that vaspin circulating in the blood is produced exclusively by adipocytes, WATs of OLETF rats were separated into mature adipocytes and vascular stromal cells by collagenase digestion, and Western and Northern blot analyses were performed. Both mRNA and protein expression of vaspin were found in mature adipocytes (Fig. 3D). The expression of vaspin in the adipocytes was further confirmed by immunohistochemistry of MES WAT, where it was found to be localized in their cytoplasm and was absent in the stromal endothelial/vascular cells (Fig. 3 E and F). Vaspin Dose Not Inhibit Known Common Serine Proteases. Protein analysis using ExPASY Proteomics tools indicated that human vaspin consists of 395 (minus signal peptide) amino acids with a Mr of 45.2 kDa and theoretical pI 9.26. During the isolation of rhVaspin in the E. coli system, only ~10% vaspin was found in the soluble fraction and ~90% in the inclusion bodies. Both fractions yielded identical-size protein bands and thus were combined to investigate the biological activity of vaspin by using denatured and reduced hen-egg lysozyme as substrates. The reduced form is relatively sensitive to trypsin treatment (Fig. 4A), and it was used as a substrate for assay of rhVaspin inhibitory activity. Normally, proteolytic activity of trypsin toward reduced hen-egg lysozyme is inhibited by alpha1-antitrypsin (Fig. 4B). However, rhVaspin in the presence or absence of (His-6)-tag failed to inhibit protease activity of trypsin (Fig. 4B). Similarly, inhibitory activity of rhVaspin was not detected for other known common proteases, such as elastase, urokinase, factor Xa, collagenase, and dipeptidyl peptidase (data not shown). Vaspin Sensitizes Insulin Action in HFHS Chow-Induced Obese ICR Mice. Because vaspin expression is regulated in genetically obese OLETF rats (Figs. 1-4), the role of vaspin, causative or protective, was assessed in the development of type 2 diabetes in diet-induced obese ICR mice fed with HFHS chow. The ICR mice were used because they do not develop obesity when fed with normal chow. The ICR mice fed with HFHS chow developed hyperglycemia, hyperinsulinemia, and obesity at 18 wk of age (Fig. 5 A and B), which is reminiscent of metabolic syndrome in humans with no genetic influence. Administration of rhVaspin to 18-wk-old ICR mice with HFHS chow significantly reduced their glucose levels 120 min after tive" provenance.
- _3 wasQuotedFrom 16030142 provenance.