Cell. Mol. Life Sci. DOI 10.1007/s00018-017-2658-y Cellular and Molecular LifeSciences ORIGINAL ARTICLE Ablation of kallikrein 7 (KLK7) in adipose tissue ameliorates metabolic consequences of high fat diet‑induced obesity by counteracting adipose tissue infammation in vivo Konstanze Zieger1 · Juliane Weiner1,2 · Anne Kunath2,3 · Martin Gericke4 · Kerstin Krause2 · Matthias Kern3 · Michael Stumvoll2 · Nora Klöting3,5 · Matthias Blüher2,5 · John T. Heiker1,2,5 Received: 21 April 2017 / Revised: 4 September 2017 / Accepted: 13 September 2017 © The Author(s) 2017. This article is an open access publication Abstract Vaspin is an adipokine which improves glu- cytokine expression was signifcantly reduced in combina- cose metabolism and insulin sensitivity in obesity. Kal- tion with an increased percentage of alternatively activated likrein 7 (KLK7) is the frst known protease target inhib- (anti-infammatory) M2 macrophages in epigonadal adipose / ited by vaspin and a potential target for the treatment of tissue of ATKlk7− −. Taken together, by attenuating adipose metabolic disorders. Here, we tested the hypothesis that tissue infammation, altering adipokine secretion and epigo- inhibition of KLK7 in adipose tissue may benefcially afect nadal adipose tissue expansion, Klk7 defciency in adipose glucose metabolism and adipose tissue function. Therefore, tissue partially ameliorates the adverse efects of HFD- we have inactivated the Klk7 gene in adipose tissue using induced obesity. In summary, we provide frst evidence for conditional gene-targeting strategies in mice. Klk7-defcient a previously unrecognized role of KLK7 in adipose tissue / mice (ATKlk7− −) exhibited less weight gain, predominant with efects on whole body energy expenditure and insulin expansion of subcutaneous adipose tissue and improved sensitivity. whole body insulin sensitivity under a high fat diet (HFD). / ATKlk7− − mice displayed higher energy expenditure and Keywords Adiposity · Insulin resistance · Metabolic food intake, most likely due to altered adipokine secre- syndrome · Serine proteases · Serpin tion including lower circulating leptin. Pro-infammatory Abbreviations KLK7 Kallikrein 7 Electronic supplementary material The online version of this AT Adipose tissue article (doi:10.1007/s00018-017-2658-y) contains supplementary material, which is available to authorized users. HFD high fat diet ATM Adipose tissue macrophage * Matthias Blüher [email protected]‑leipzig.de * John T. Heiker Introduction jheiker@uni‑leipzig.de A primary risk factor associated with obesity is adipose tis- 1 Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Brüderstr. 34, sue (AT) dysfunction which is characterized by extensive 04103 Leipzig, Germany visceral and ectopic fat accumulation, adipocyte hypertro- 2 Department of Medicine, University of Leipzig, Liebigstr. 20, phy, a chronic low-grade AT infammation with increased 04103 Leipzig, Germany numbers of infltrating AT macrophages (ATM) and initially 3 German Center for Diabetes Research (DZD), Munich, local but ultimately systemic insulin resistance [1]. In con- Germany sequence, there is a causal relationship between obesity and 4 Institute of Anatomy, University of Leipzig, Leipzig, several major adverse health outcomes, such as type 2 dia- Germany betes, dyslipidemia, hypertension, cardiovascular and fatty 5 IFB Adiposity Diseases, University of Leipzig, Leipzig, liver disease as well as kidney diseases [2, 3]. Adipose tissue Germany expresses and secretes a variety of adipokines, i.e., enzymes, Vol.:(0123456789)1 3 K. Zieger et al. cytokines, hormones, peptides and other biologically active may benefcially afect glucose metabolism and adipose tis- molecules, which actively regulate whole body metabo- sue function. lism, energy homeostasis and infammatory processes [4]. In consequence, AT dysfunction is refected by an adipokine secretion pattern, promoting insulin resistance and a pro- Research design and methods infammatory state. The adipokine visceral AT serine protease inhibitor Animal studies (vaspin) has been shown to improve glucose metabolism and insulin sensitivity and reduces food intake upon admin- All experiments conformed to the animal ethical law of the istration in pharmacological doses in diferent mouse models state Saxony, Germany, and were approved by the local ani- [5–9]. Genetically or high fat diet-induced insulin resistance mal ethics review board (Landesdirektion Sachsen, Leipzig, is accompanied by increased expression of vaspin [7, 10]. TVV23/12, T03/12, T08/15, Germany). Male mice were Transgenic overexpression of vaspin in AT in mice markedly housed in a pathogen-free facility with a daylight cycle from ameliorated glucose and insulin tolerance as well as adipose 6.00 to 18.00 hours and 22 ± 2 °C, in groups of three to fve tissue infammation under high fat diet-induced obesity [11]. animals. Animals were given a standard chow diet (chow; Importantly, non-inhibitory vaspin mutants failed to improve EV153, 3.3% from fat) or a Western diet (HFD) contain- glucose tolerance in insulin-resistant mice [9]. ing 55% calories from fat (E15772-34, Ssnif®, Germany). The serine protease kallikrein-related peptidase 7 (KLK7) Mice permanently had free access to water and food; food is the only known target protease of vaspin so far and may restriction was only performed if required for an experiment. therefore mediate vaspin´s efects in AT [9]. KLK7 (uniprot Female mice were investigated in parallel and showed a accession number P49862) is a member of the kallikrein similar phenotype for key parameters of this study (Sup- subfamily of 15 closely related (chymo)trypsin-like serine plementary Fig. 1). proteases located on chromosome locus 19q13.4 in humans [12]. In rodents, the kallikrein gene family consists of more Generation of adipocyte‑specifc Klk7 knockout (KO) than 20 genes, including pseudogenes, but they share many mice (ATKlk7−/−) similarities with respect to tissue-specifc expression, regula- tory mechanisms and function that demonstrate the evolu- The inactivation of the Klk7 gene in adipose tissue (KO, tionary conservation across these species (reviewed in [13]). ATKlk7−/−) was obtained via the Cre-lox system for con- KLKs are known to participate in pathways regulating skin ditional gene-targeting strategies using animals with con- / desquamation, kidney function, seminal liquefaction, syn- ditional (floxed) Klk7 (Klk7− − by Taconic, Cologne, aptic neural plasticity and brain function (reviewed in [14]). Germany) and adipose tissue-specifc expression of Cre- KLK activities are tightly regulated and multiple mecha- recombinase under the fatty acid-binding protein 4 (Fabp4) / nisms, such as pro-enzyme activation cascades, peptide as promoter (Fabp4-Cre+_Klk7− −). In AT, Cre-recombinase well as protein inhibitors (e.g., serpins and LEKTI), pH and mediates the deletion of all foxed alleles (Fig. 1a). ATKlk7 metal ions fne-tune tissue-specifc KLK activity (reviewed mice were maintained on a C57BL/6/NTac background. in [15]). As KLK proteases exert various important regu- Littermates without conditional Klk7 (WT or Fabp4-Cre+_ / / latory functions, they are promising targets in potential Klk7+ +) or without Cre-recombinase (Fabp4-Cre−_Klk7− −) therapies of several diseases including respiratory diseases, were used as controls (termed ATKlk7+/+ throughout the neurodegeneration, skin-barrier dysfunction, infammation manuscript). and cancer (reviewed in [16]). KLK7 was frst identifed as a protease involved in the Genotyping of ATKlk7−/− mice desquamation process in the outermost layer of the skin [17, 18] and transgenic mice overexpressing human KLK7 Genotyping was accomplished by isolating genomic DNA display hyperkeratosis, epidermal thickness and cumulative from tail tips, using the INVISORB spin tissue mini kit appearances of immune cells in the dermis [19, 20]. Thus, its (Stratec, Berlin, Germany) and quantified afterward by function in skin desquamation and role in the pathogenesis PCR. The following primers were used: Klk7 loxP sites, of infammatory skin diseases such as psoriasis [21] and 5′-GGG ATG TAG GAT TAT GAG TGAGC-3′ (forward) and acne rosacea [22] are fairly well understood. 5′-CAG TCC AGT GAA CTG CTC ACC-3′ (reverse), as well But, although it has been suggested that Klk7 may medi- as Cre-recombinase, 5′-GCG GTC TGG CAG TAA AAA ate benefcial efects of recombinant vaspin in mouse models CTATC-3′ (forward) and 5′-GTG AAA CAG CAT TGC TGT of obesity and diabetes, the role of KLK7 in adipose tissue CACTT-3′ (reverse). PCR was performed for 35 cycles, has not been systematically studied in vivo yet. Here, we 95 °C for denaturation (loxP sites and Cre), 60 °C (loxP tested the hypothesis that ablation of KLK7 in adipose tissue sites) or 56 °C (cre) for annealing and 72 °C (loxP sites and 1 3 Ablation of kallikrein 7 (KLK7) in adipose tissue ameliorates metabolic consequences of high… / Fig. 1 Targeting strategy for the generation of ATKlk7− − mice. a PCR product size of 276 bp), heterozygous foxed (Klk7+/−, predicted Schematic representation of the loxP-fanked Klk7 allele before and PCR products of 276 and 402 bp) or homozygous foxed Klk7 mice after crossing with transgenic animals expressing Cre-recombinase (Klk7−/−, predicted PCR product of 402 bp) was used as template for under the Fabp4 promoter. The targeting vector consisted of 2 kb of PCR of the foxed Klk7 allele and from Fabp4-Cre+ mice (Cre+, pre- the murine Klk7 gene, comprising exon