Vaspin inhibits kallikrein 7 by serpin mechanism

Authors: John T Heiker Nora Klöting Peter Kovacs E Bartholomeus Kuettner Norbert Sträter Stephan Schultz Matthias Kern Michael Stumvoll Matthias Blüher Annette G BeckSickinger

Publish Date: 2013/01/31

Volume: 70, Issue: 14, Pages: 2569-2583

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Abstract

The molecular target of the adipokine vaspin visceral adipose tissuederived serpin serpinA12 and its mode of action are unknown Here we provide the vaspin crystal structure and identify human kallikrein 7 hK7 as a first protease target of vaspin inhibited by classical serpin mechanism with high specificity in vitro We detect vaspin–hK7 complexes in human plasma and find coexpression of both proteins in murine pancreatic βcells We further demonstrate that hK7 cleaves human insulin in the A and Bchain Vaspin treatment of isolated pancreatic islets leads to increased insulin concentration in the media upon glucose stimulation without influencing insulin secretion By application of vaspin and generated inactive mutants we find the significantly improved glucose tolerance in C57BL/6NTac and db/db mice treated with recombinant vaspin fully dependent on the vaspin serpin activity and not related to vaspinmediated changes in insulin sensitivity as determined by euglycemichyperinsulinemic clamp studies Improved glucose metabolism could be mediated by increased insulin plasma concentrations 150 min after a glucose challenge in db/db mice supporting the hypothesis that vaspin may inhibit insulin degradation by hK7 in the circulation In conclusion we demonstrate the inhibitory serpin nature and the first protease target of the adipose tissuederived serpin vaspin and our findings suggest hK7 inhibition by vaspin as an underlying physiological mechanism for its compensatory actions on obesityinduced insulin resistanceObesity represents a fastgrowing health problem that is reaching epidemic proportions worldwide 1 and is associated with an increased risk of premature death 2 Obesity significantly increases the risk of developing type 2 diabetes mellitus hypertension coronary heart disease stroke and several types of cancer 3 Understanding the molecular mechanisms linking adipose tissue to these obesity related pathologies is of enormous scientific and medical importance Cells of adipose tissue produce and secrete a variety of cytokines and cytokinelike molecules termed adipokines which have effects on insulin sensitivity glucose and lipid levels satiety and appetite 4Previously visceral adipose tissuederived serpin referred to as vaspin in this paper serpinA12 according to the serpin nomenclature 5 was identified as a putative member of the serine protease inhibitor family which was expressed in visceral adipose tissue of Otsuka LongEvans Tokushima Fatty OLETF rats at the age when obesity and insulin plasma concentrations reach a peak 6 Vaspin expression was shown to decrease with worsening of diabetes and body weight loss whereas vaspin serum levels could be normalized by insulin or pioglitazone treatment Consistent with that we found increased vaspin mRNA expression in human adipose tissue associated with obesity insulin resistance and type 2 diabetes 7Administration of vaspin to obese mice improves glucose tolerance insulin sensitivity and altered gene expression of candidate genes for insulin resistance 6 In addition we could demonstrate that central vaspin administration leads to reduced food intake and has sustained blood glucoselowering effects in mice 8 and this has since been confirmed in rats 9 We and others have recently reported that elevated vaspin serum concentrations are associated with obesity and impaired insulin sensitivity in humans 10 11It has therefore been postulated that increased vaspin expression and secretion could represent a compensatory mechanism associated with obesity severe insulin resistance and type 2 diabetes 6 Althoughprotease inhibitor properties have been suggested as mechanism of vaspin action until now a protease target of vaspin has not yet been identified Here we demonstrate the inhibitory serpin nature of vaspin by identifying human kallikrein 7 hK7 as the first protease target of vaspin and confirm the vaspin–hK7 interaction in human plasma We find human insulin as a substrate of hK7 and coexpression of vaspin and hK7 in pancreatic islets We provide evidence that the serpin function of vaspin is essential for its physiologic effects and demonstrate that the antidiabetic vaspin effects in vivo do not result from increased insulin sensitivity but are rather based on an insulinstabilizing effect most likely by inhibiting hK7mediated insulin degradation Thus our results give insight into the molecular mechanism of reported beneficial vaspin effects in obesityrelated metabolic dysfunctionExpression of native human vaspin cloned into the pET16b bacterial expression vector 10×histidin tag Qiagen Hilden Germany and variants vaspinT365R and vaspinA369P in E coli BL21DE3pLysRARE was induced by the addition of isopropyl βthiogalactopyranoside 1 mM to the growth medium Bacterial extracts were prepared using standard methods and the fusion protein was purified by immobilized metal ion affinity chromatography NiNTA FastStart kit Qiagen Supplementary Fig S1A–D Protein identity and purity were affirmed by SDS–PAGE HPLC Western blot and MALDITOF mass spectrometry ActiClean Etox affinity columns Sterogene Bioseparations Carlsbad CA USA were used to remove potential endotoxin contaminations

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