International Journal of Molecular Sciences Article The Metalloproteinase ADAM28 Promotes Metabolic Dysfunction in Mice Lakshini Herat 1, Caroline Rudnicka 2, Yasunori Okada 3, Satsuki Mochizuki 4, Markus Schlaich 1,5 and Vance Matthews 1,* 1 Dobney Hypertension Centre, School of Medicine and Pharmacology, University of Western Australia, Crawley WA 6009, Australia; [email protected] (L.H.); [email protected] (M.S.) 2 Research Centre, Royal Perth Hospital, Perth WA 6000, Australia; [email protected] 3 Department of Pathophysiology for Locomotive and Neoplastic Diseases, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan; [email protected] 4 Department of Surgery, National Defense Medical College, Saitama 359-8513, Japan; [email protected] 5 Department of Cardiology and Department of Nephrology, Royal Perth Hospital, Perth WA 6000, Australia * Correspondence: [email protected]; Tel.: +61-8-9224-0239; Fax: +61-8-9224-0374 Academic Editor: Masatoshi Maki Received: 17 February 2017; Accepted: 18 April 2017; Published: 21 April 2017 Abstract: Obesity and diabetes are major causes of morbidity and mortality globally. The current study builds upon our previous association studies highlighting that A Disintegrin And Metalloproteinase 28 (ADAM28) appears to be implicated in the pathogenesis of obesity and type 2 diabetes in humans. Our novel study characterised the expression of ADAM28 in mice with the metabolic syndrome and used molecular inhibition approaches to investigate the functional role of ADAM28 in the pathogenesis of high fat diet-induced obesity. We identified that ADAM28 mRNA and protein expression was markedly increased in the livers of mice with the metabolic syndrome. In addition, noradrenaline, the major neurotransmitter of the sympathetic nervous system, results in elevated Adam28 mRNA expression in human monocytes. Downregulation of ADAM28 with siRNA technology resulted in a lack of weight gain, promotion of insulin sensitivity/glucose tolerance and decreased liver tumour necrosis factor-α (TNF-α) levels in our diet-induced obesity mouse model as well as reduced blood urea nitrogen, alkaline phosphatase and aspartate aminotransferase. In addition, we show that ADAM28 knock-out mice also displayed reduced body weight, elevated high density lipoprotein cholesterol levels, and reductions in blood urea nitrogen, alkaline phosphatase, and aspartate aminotransferase. The results of this study provide important insights into the pathogenic role of the metalloproteinase ADAM28 in the metabolic syndrome and suggests that downregulation of ADAM28 may be a potential therapeutic strategy in the metabolic syndrome. Keywords: ADAM28; type 2 diabetes (T2D); obesity; metabolic syndrome; metalloproteinases 1. Introduction Obesity is one of the most prevalent metabolic diseases globally and is an established risk factor for type 2 diabetes (T2D). Excess weight correlates directly with glucose intolerance and insulin resistance, which may ultimately lead to the development of T2D [1]. Recent studies have identified associations between obesity and T2D involving pro-inflammatory cytokines (tumour necrosis factor-α (TNF-α) and interleukin-6), insulin resistance, deranged fatty acid metabolism, and cellular processes such as mitochondrial dysfunction and endoplasmic reticulum stress [2]. Risk factors for obesity and fatty liver disease may include consumption of diets high in fat and fructose [3–5] and low physical activity [6]. Int. J. Mol. Sci. 2017, 18, 884; doi:10.3390/ijms18040884 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 884 2 of 12 Int. J. Mol. Sci. 2017, 18, 884 2 of 12 obesity and fatty liver disease may include consumption of diets high in fat and fructose [3–5] and low physical activity [6]. AA Disintegrin Disintegrin And And Metalloproteinases Metalloproteinases,, or or ADAMs, ADAMs, are are a a group group of of transmembrane transmembrane and and secreted secreted proteinsproteins which which play play an an important important role role in in regulating regulating cell cell phenotype phenotype via via their their effects effects on on cell cell adhesion, adhesion, migration,migration, proteolysis, proteolysis, and and signalling signalling [7 []7.]. These These proteins proteins have have a major a major impact impact on on the the pathogenesis pathogenesis of numerousof numerous diseases. diseases. The The metalloproteinase metalloproteinase ADAM28, ADAM28, also also known known as as lymphocyte lymphocyte metalloprotease metalloprotease MDCMDC-L,-L, was was first first identified identified on lymphoid cells and has two isoforms: (i) (i) a membrane membrane-type-type form (ADAM28m)(ADAM28m) and and (ii) (ii) a a secreted secreted soluble form form (ADAM28s). A A number number of of early early studies studies suggested suggested that that ADAM28 maymay be be important important in inflammationin inflammation and metabolismand metabolism [8,9]. One[8,9 known]. One substrateknown substrate for ADAM28 for ADAM28is the pro-inflammatory is the pro-inflammatory cytokine TNF- cytokineα. Interestingly, TNF-α. Interestingly, a number of synthetica number peptides of synthetic containing peptides the containingauthentic TNF- the authenticα shedding TNF site-α wereshedding shown site to were be cleaved shown to by be ADAM28 cleaved [by8]. ADAM28 Our recent [8] studies. Our recent have studiesconfirmed have that confirmed human ADAM28that human is ADAM28 a novel sheddase is a novel of sheddase human TNF- of humanα [9] andTNF reinforced-α [9] and reinforced the notion thethat notion ADAM28 that is ADAM28 a novel sheddase is a novel of onesheddase of the majorof one pro-inflammatory of the major pro cytokines-inflammatory involved cytokines in the involvedpathogenesis in the of pathogenesis the metabolic of syndrome. the metabolic syndrome. InIn our currentcurrent study,study, we we aimed aimed to to translate translate our our previous previous findings findings [9] into [9] anintoin an vivo inanimal vivo animal model modelto assess to theassess effects the effects of limiting of limiting ADAM28 ADAM28 activity activity on parameters on parameters of the metabolicof the metabolic syndrome. syndrome. In this Instudy, this westudy, establish we establish that ADAM28 that ADAM28 is pathogenic is pathogenic in the metabolic in the syndrome metabolic and syndrome provide evidenceand provide that evidencemetalloproteinase that metalloproteinase inhibition is a inhibitio potentialn therapeuticis a potential target therapeutic for anti-obesity target for agents. anti-obesity agents. 2.2. Results Results WeWe have previously reported that high expression of ADAM28 mRNA in peripheral blood mononuclearmononuclear cells cells from from the the San Antonio Family Heart Study (SAFHS) cohort ( n = 1240) correlated stronglystrongly withwith parametersparameters of of the the metabolic metabolic syndrome syndrome [9]. To[9] further. To further our previously our previously published published findings, findings,we conducted we conducted ADAM28 expressionADAM28 andexpression functional and studies functional in our studies murine in high ourfat murine diet-induced high fat obesity diet- inducedmodel. Mice obesity were m weighedodel. Mice weekly were weighed to confirm weekly obesity to (Figure confirm1). obesity High fat (Figure diet fed 1). mice High were fat diet glucose fed miceintolerant were andglucose insulin intolerant resistant and compared insulin to resistant their chow compared fed counterparts, to their chow as previously fed counterparts, reported [ 10as]. previouslyIn addition, reported livers of high[10]. fatIn addition, diet fed mice livers were of high markedly fat diet steatotic fed mice with were inflammatory markedly cellsteatotic infiltration, with inflammatoryas demonstrated cell in infiltration, our previous as demonstrated study [10]. in our previous study [10]. FigureFigure 1. HighHigh fat fat diet diet (HFD) (HFD) induced induced obesity obesity in in mice mice before before s smallmall interfering interfering RNA RNA (siRNA) (siRNA) administration.administration. nn == 9 9 mice/group, mice/group, * *p p< <0.05. 0.05. 2.1. ADAM28 mRNA Expression Is Significantly Elevated in the Liver of High Fat Diet Fed Mice 2.1. ADAM28 mRNA Expression Is Significantly Elevated in the Liver of High Fat Diet Fed Mice ADAM28 mRNA and protein expression in the liver was studied, as the liver is an organ well ADAM28 mRNA and protein expression in the liver was studied, as the liver is an organ well known for lipid and glucose metabolism [11]. We demonstrated that ADAM28 mRNA levels are known for lipid and glucose metabolism [11]. We demonstrated that ADAM28 mRNA levels are increased in the livers of mice fed a high fat diet for 12 weeks (Figure 2). In addition, active ADAM28 increased in the livers of mice fed a high fat diet for 12 weeks (Figure2). In addition, active ADAM28 (42 kDa) protein levels were also increased in the livers of mice fed a high fat diet, as evidenced by (42 kDa) protein levels were also increased in the livers of mice fed a high fat diet, as evidenced by western blotting (Figure 3). western blotting (Figure3). Int. J. Mol. Sci. 2017, 18, 884 3 of 12 Int. J. Mol. Sci. 2017, 18, 884 3 of 12 Int. J. Mol. Sci. 2017, 18, 884 3 of 12 Int. J. Mol. Sci. 2017, 18, 884 3 of 12 FigureFigure 2. Increased 2. Increased A Disintegrin A Disintegrin And And Metalloproteinase Metalloproteinase 2828 (ADAM28)(ADAM28) mRNA mRNA expression expression in inlivers livers