Vaspin is involved in the pathophysiology of type 2 diabetes by regulating insulin sensitivity

Abstract

Background and aims: Visceral adipose tissue derived serine protease inhibitor (vaspin) is a novel adipokine that may link obesity, insulin resistance (IR) and type 2 diabetes (T2D), but so far its pathophysiological role remains largely unknown. The first aim was to study the effects of recombinant vaspin treatment on insulin sensitivity in db/db mice. In addition, we investigated the role of genetic variation in the human vaspin gene in the pathogenesis of T2D. Materials and methods: Animal studies: After recombinant vaspin administration (1mg/kg body weight i.p.; at 6 pm and at 6 am prior to the tests), we performed glucose tolerance tests (2g/kg body weight i.p.) and hyperinsulinemic- euglycemic clamps in db/db mice (N=5 for each test). Human genetic studies: Vaspin (exons, exon-intron boundaries, 5' and 3' UTRs) was sequenced in DNA samples from 48 unrelated Caucasian subjects (ABI PRISM 3100 Avant; Applied Biosystems Inc.). Six single nucleotide polymorphisms (SNPs) identified by sequencing and 22 haplotype tagging SNPs representative for their linkage disequilibrium groups (r2>0.8 and minor allele frequencies >0.05) were genotyped in 1046 clinically well-characterized Sorbs from Germany for subsequent association studies on metabolic traits including insulin resistance and secretion indices (e.g. fasting Belfiore, Stumvoll index, HOMA-IR) based on glucose tolerance test in non diabetic subjects. P values <0.05 were considered to be of nominal statistical significance. For in vitro analyses of the effects of the stop codon mutation (p.R211X), full-length (wild type) and short-length (carrying the mutation) vaspin was cloned into p3xFLAG-myc-CMV expression vector (Sigma-Aldrich) and transfected into HEK-cells (Fugene HD Transfection Kit; Roche). Proteins were detected by western blot. Results: Animal studies: Vaspin administration in db/db mice resulted in improved glucose tolerance (P<0.05). Consistently, glucose infusion rate (GIR) during the steady state of the clamp significantly increased after vaspin treatment (P<0.05). Human genetic studies: Sequencing of the vaspin gene revealed one SNP (rs61757459) in exon 3 resulting in a STOP-codon (p.R211X). Western blot experiments showed that full-length and short-length vaspin were expressed in eukaryotic cells. Short-length vaspin yielded in a prominent ~25-kDa band. Several SNPs were nominally associated with WHR (waist-to-hip-ratio), 30min glucose levels or 2-hr-insulin levels (adj. for age, sex and BMI). Furthermore one SNP (rs2236242) showed additional associations with AUCGlucose, insulin sensitivity and insulin resistance indices. Conclusion: In conclusion, our data demonstrate the substantial insulin sensitizing effect of vaspin and suggest a role of vaspin genetic variants in the pathophysiology of insulin resistance and T2D.