The Metalloproteinase ADAM28 Promotes Metabolic Dysfunction in Mice

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].

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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 synthetic a 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).

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FigureFigure 2. Increased 2. Increased A Disintegrin A Disintegrin And And Metalloproteinase Metalloproteinase 2828 (ADAM28)(ADAM28) mRNA mRNA expression expression in inlivers livers of miceFigureof fed mice a high 2. fed Increased fata high diet fatA (HFD). Disintegrindiet (HFD). Expression And Expression Metalloproteinase of ADAM28 of ADAM28 mRNA 28 mRNA(ADAM28) in livers in livers mRNA from from mice expression mice fed fed either ineither livers a normal a of mice fed a high fat diet (HFD). Expression of ADAM28 mRNA in livers from mice fed either a chownormal or high chow fat diet.or high * p fat< 0.05;diet. *n p= < 12–14.0.05; n = 12–14. normal chow or high fat diet. * p < 0.05; n = 12–14. Figure 2. Increased A Disintegrin And Metalloproteinase 28 (ADAM28) mRNA expression in livers of mice fed a high fat diet (HFD). Expression of ADAM28 mRNA in livers from mice fed either a normal chow or high fat diet. * p < 0.05; n = 12–14.

Figure 3. Elevated ADAM28 expression levels in high fat fed mice. Active ADAM28 (42 kDa) protein Figurelevels were 3. Elevated measured ADA byM28 immunoblotting expression levels ( ) inand high quantified fat fed mice. based Active on densitometry ADAM28 (42 ( kDa)) in livers protein of Figure 3. Elevated ADAM28 expression levelsA in high fat fed mice. Active ADAM28B (42 kDa) protein levelsmice on were a high measured fat diet byfor immunoblotting 12 weeks * p < 0.05; (A) n and = 3 –quantified4 mice/group. based on densitometry (B) in livers of levels were measured by immunoblotting (A) and quantified based on densitometry (B ) in livers of mice on a high fat diet for 12 weeks * p < 0.05; n = 3–4 mice/group. mice2.2.Figure ADAM28 on a3. high Elevated Exp fatression diet ADA forM28 Is 12 Elevated expression weeks in * p Human levels< 0.05; in Monocytesn high= 3–4 fat mice/group.fed Treated mice. Active with Noradrenaline ADAM28 (42 kDa)(NA) protein 2.2.levels ADAM28 were measured Expression by Is immunoblotting Elevated in Human (A) and Monocytes quantified Treated based with on densitometry Noradrenaline (B (NA)) in livers of Activation of the sympathetic nervous system (SNS) is a cardinal feature of obesity, metabolic 2.2. ADAM28mice on a Expression high fat diet Is for Elevated 12 weeks in * p Human < 0.05; n Monocytes = 3–4 mice/group. Treated with Noradrenaline (NA) syndrome,Activation and oftype the 2 sympatheticdiabetes (T2DM) nervous and system is associated (SNS) iswith a cardinal disease featureprogression of obesity, [12]. In metabolic order to 2.2.Activationsyndrome,test ADAM28 our hypothesis andExp of ressionthetype sympathetic that2 Isdiabetes Elevated sympathetic (T2DM) in nervous Human nervous and Monocytes system is associatedsystem (SNS) Treated activation with is with a disease cardinal Noradrenalinemay progressionresult feature in (NA) elevated of [ 12 obesity,]. In ADAM28 order metabolic to testexpression, our hypothesis we treated that sympathetic human THP nervous-1 monocytes system activation with noradrenalinemay result in elevated(NA), theADAM28 main syndrome,Activation and type of the 2 sympathetic diabetes (T2DM) nervous and system is associated (SNS) is a cardinal with disease feature progressionof obesity, metabolic [12]. In order expression,neurotransmitter we oftreated the SNS. human Excitingly, THP -we1 havemonocytes now shown with fornoradrenaline the first time that(NA), NA the treatment main to testsyndrome, our hypothesis and type 2 that diabetes sympathetic (T2DM) and nervous is associated system with activation disease mayprogression result in[12 elevated]. In order ADAM28 to neurotransmittermay result in elevated of the ADAM28 SNS. Excitingly, expression we inhave a dose now-dependent shown for manner the first (Figure time that 4). TheNA differencetreatment expression,test our hypothesis we treated that human sympathetic THP-1 monocytesnervous system with activation noradrenaline may result (NA), in the elevated main neurotransmitterADAM28 maybetween result 0 andin elevated 1.0 µM NAADAM28 treatment expression groups inwas a doseclose- dependentto reaching manner significance (Figure (p = 4). 0.0698). The difference expression, we treated human THP-1 monocytes with noradrenaline (NA), the main of thebetween SNS. Excitingly, 0 and 1.0 µ weM NA have treatment now shown groups for was the close first to time reaching that NA significance treatment (p = may 0.0698). result in elevated ADAM28neurotransmitter expression of inthe a SNS. dose-dependent Excitingly, we manner have now (Figure shown4). Thefor the difference first time between that NA 0 treatment and 1.0 µ M NA treatmentmay result groups in elevated was close ADAM28 to reaching expression significance in a dose- (dependentp = 0.0698). manner (Figure 4). The difference between 0 and 1.0 µM NA treatment groups was close to reaching significance (p = 0.0698).

Figure 4. Noradrenaline treatment of human THP-1 monocytes promotes elevated ADAM28 mRNA Figureexpression. 4. Noradrenaline Cells were treated treatment for 48 of h. human THP-1 monocytes promotes elevated ADAM28 mRNA expression. Cells were treated for 48 h.

Noradrenaline treatment of human THP-1 monocytes promotes elevated ADAM28 mRNA FigureFigure 4. 4.Noradrenaline treatment of human THP-1 monocytes promotes elevated ADAM28 mRNA expression. Cells were treated for 48 h. expression. Cells were treated for 48 h.

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2.3. In Vivo Knock Knock-Down-Down of ADAM28 Ameliorated ParametersParameters of the Metabolic Syndrome A vast array of studies have highlighted the ability of siRNA therapy to improve numerousnumerous disease states [13–16].]. We havehave previouslypreviously shownshown ourour capacitycapacity toto successfullysuccessfully knock-downknock-down ADAM protein expression utilising siRNA technology [17].]. Our Our next next aim aim was was therefore to utilise siRNA targeting mouse ADAM28 to reducereduce ADAM28 expression inin vivo in our high high fat fat diet diet-induced-induced obesity obesity mouse model.model. OurOur resultsresults demonstrate demonstrate successful successful reduction reduction of ADAM28of ADAM28 protein protein expression expression in the in liver the liverof mice of mice treated treated with with siRNA siRNA targeting targeting mouse mouse ADAM28 ADAM28 (mADAM28 (mADAM28 siSTABLE siSTABLEsiRNA) siRNA) (Figure(Figure 55A).A). Interestingly, only the active form of ADAM28 was detected in the liver, suggesting all pools of ADAM28 areare activated activated in in the the liver. liver. The The pro-form pro-form and activeand active form ofform ADAM28 of ADAM28 are observed are observed in gonadal in whitegonadal adipose white tissueadipose (Figure tissue5B). (Figure Inﬁltrating 5B). Infiltrating inﬂammatory inflammatory cells may be cells contributing may be contributing to this expression to this expressionin gonadal in white gonadal adipose white tissue. adipose ADAM28 tissue. ADAM28 protein levels protein were levels mildly were decreased mildly decreased in white in adipose white adiposetissue of tissue mice treatedof mice with treated ADAM28 with ADAM28 siRNA. siRNA.

Figure 5. In vivo knock-downknock-down of ADAM28 in liver (A) and white adipose tis tissuesue ( B) of high fat diet fed mice. The siRNA was administered for the ﬁnalfinal 2 weeksweeks ofof thethe dietdiet ((n = 3 mice/group).

Mice treatedtreated with with mADAM28 mADAM28 siSTABLE siSTABLE siRNA siRNA also showed also showed improvements improvements in several parametersin several parametersof the metabolic of the syndrome metabolic including syndrome the including failure to the exhibit failure further to exhibit increases further in highincreases fat diet-induced in high fat weightdiet-induced gain compared weight gain to mice compared treated to with mice control treated siRNA with (non-targeted control siRNA siSTABLE (non-targeted siRNA) siSTABLE (Figure6). siRNA)In addition, (Figure mADAM28 6). In addition, siSTABLE mADAM28 siRNA treatedsiSTABLE mice siRNA displayed treated increased mice displayed glucose increased tolerance (Figureglucose7 A)tolerance and insulin (Figure sensitivity 7A) and insul (Figurein 7sensitivityB) compared (Figure to mice7B) compared treated with to mice control treated siRNA. with controlIn addition, siRNA. blood In urea,addition, which blood is indicative urea, which of kidney is indicative function of was kidney reduced function in the was serum reduced of ADAM28 in the serumsiRNA of treated ADAM28 mice (FiguresiRNA 8treatedA), suggesting mice (Figure that kidney 8A), suggesting function may that be kidney better function preserved may in ADAM28 be better preservsiRNA treateded in ADAM28 mice. Liver siRNA enzymes, treated which mice. are Liver indicative enzymes, of liverwhich injury, are indicative such as alkaline of liver phosphataseinjury, such (Figureas alkaline8B) andphosphatase aspartate (Figure aminotransferase 8B) and aspartate (AST) (Figureaminotransferase8C) were markedly (AST) (Figure reduced 8C) in were the serummarkedly of ADAM28reduced in siRNA the serum treated of ADAM28 mice. As ADAM28siRNA treated is a sheddase mice. Asof ADAM28 TNF-α protein, is a sheddase we measured of TNF- theα protein, TNF-α proteinwe measured levels the by TNF ELISA-α protein in the livers levels ofby mice ELISA treated in the withlivers either of mice non-targeted treated with siRNA either non or ADAM28-targeted siRNAsiRNA. or We ADAM28 found that siRNA. reducing We ADAM28 found that activity reducing resulted ADAM28 in reduced activity TNF- resultedα protein inlevels reduced in the TNF liver-α (Figureprotein 9levels). The in TNF- the liverα protein (Figure may 9). beThe cleaved TNF-α orprotein cytoplasmic may be derivedcleaved TNF-or cytoplasmicα. derived TNF-α.

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FigureFigure 6. 6.In In vivo vivoknock-down knock-down ofof mousemouse ADAM28ADAM28 reduces reduces high high fat fat diet diet-induced-induced mean mean weight weight gain. gain. Figure 6. In vivo knock-down of mouse ADAM28 reduces high fat diet-induced mean weight gain. (n (=n 3= mice/group).3 mice/group). (n Figure= 3 mice/group). 6. In vivo knock-down of mouse ADAM28 reduces high fat diet-induced mean weight gain. (n = 3 mice/group).

Figure 7. Metabolic testing in mice treated with ADAM28 siRNA. In vivo knock-down of ADAM28 Figure 7. Metabolic testing in mice treated with ADAM28 siRNA. In vivo knock-down of ADAM28 Figurereduces 7. Metabolic high fat diet testing-induced in miceglucose treated intolerance with ADAM28(A), as demonstrated siRNA. In with vivo glucoseknock-down tolerance of ADAM28testing, reducesFigure high 7. Metabolic fat diet-induced testing glucosein mice intolerancetreated with (A ADAM28), as demonstrated siRNA. In with vivo glucose knock- tolerancedown of ADAM28testing, reducesand insulin high resistance fat diet-induced (B), as demonstrated glucose intolerance with insulin (A), tolerance as demonstrated testing in mice. with * glucose p < 0.05; tolerance (n = 3 andreduces insulin high resistance fat diet -(inducedB), as demonstrated glucose intolerance with insulin (A), as tolerancedemonstrated testing with in glucosemice. * tolerancep < 0.05; ( testing,n = 3 testing,mice/group). and insulin resistance (B), as demonstrated with insulin tolerance testing in mice. * p < 0.05; mice/group).and insulin resistance (B), as demonstrated with insulin tolerance testing in mice. * p < 0.05; (n = 3 (n = 3 mice/group). mice/group).

Figure 8. Metabolic benefits in mice treated with ADAM28 siRNA. Blood urea nitrogen (A); Alkaline

FigurePhosphatase 8. Metabolic (B); and benefits aspartate in mice aminotransferase treated with ADAM28(AST) activity siRNA. (C). Blood * p < 0.05; urea nnitrogen = 3 mice/group. (A); Alkaline PhosphataseFigure 8. Metabolic (B); and aspartatebenefits in aminotransferase mice treated with (AST) ADAM28 activity siRNA. (C). * Bloodp < 0.05; urea n = nitrogen 3 mice/group. (A); Alkaline Figure 8. Metabolic beneﬁts in mice treated with ADAM28 siRNA. Blood urea nitrogen (A); Alkaline Phosphatase (B); and aspartate aminotransferase (AST) activity (C). * p < 0.05; n = 3 mice/group. Phosphatase (B); and aspartate aminotransferase (AST) activity (C). * p < 0.05; n = 3 mice/group.

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FigureFigure 9. Decreasing 9. Decreasing ADAM28 ADAM28 expression expression reducesreduces liver TNF TNF--αα levelslevels in inhigh high fat diet fat diet(HFD) (HFD) fed mice. fed mice. Mean + SEM; * p < 0.05; n = 3 mice/group. Mean + SEM; * p < 0.05; n = 3 mice/group. Figure 9. Decreasing ADAM28 expression reduces liver TNF-α levels in high fat diet (HFD) fed mice. 2.4. Metabolic Benefits in ADAM28 Knock-Out (KO) Mice 2.4. MetabolicMean Benefits+ SEM; * inp < ADAM280.05; n = 3 mice/group. Knock-Out (KO) Mice We used ADAM28 knock-out (KO) mice to determine if the absence of ADAM28 promoted We2.4.metabolic Metabolic used ADAM28benefits. Benefits In in knock-outparticular, ADAM28 KnockADAM28 (KO)-Out mice (KO)KO to m Mice determineice are viable if as the adults absence with of a ADAM28normal life promotedspan. metabolicExcitingly,We benefits. used in agreementADAM28 In particular, knockwith our-out ADAM28 hypothesis, (KO) mice KO ADAM28to micedetermine are KO viable ifmice the on asabsence a adults normal of with ADAM28chow a diet normal promoted possess life a span. p Excitingly,metabolicreduced in body agreementbenefits. mass In at particular, with10 months our hypothesis,ADAM28of age (Figure KO ADAM28m 10A;ice are= viable0.05335). KOmice as adultsSerum on a withhigh normal -adensity normal chow lipoprotein life diet span. possess (HDL) cholesterol levels were significantly elevated in ADAM28 KO mice at 49 days (Figure 10B) and a reducedExcitingly, body in mass agreement at 10 monthswith our ofhypothesis, age (Figure ADAM28 10A; p KO= 0.05335). mice on a Serum normal high-density chow diet possess lipoprotein a 6 months of age (Figure 10C). Blood urea, which is indicative of kidney function, was reduced in the (HDL)reduced cholesterol body mass levels at were 10 months significantly of age (Figure elevated 10A; in ADAM28p = 0.05335). KO Serum mice high at 49-density days (Figure lipoprotein 10B) and serum of ADAM28 KO mice (Figure 10D), suggesting that kidney function may be better preserved 6 months(HDL) of cholesterol age (Figure levels 10 C).were Blood significantly urea, which elevated is indicativein ADAM28 of KO kidney mice at function, 49 days (Figure was reduced 10B) and in the 6in months ADAM28 of age KO (Figure mice. 10C). Liver Blood enzymes, urea, whichwhich is areindicative indicative of kidney of liver function, injury, was such reduced as alkaline in the serumphosphatase of ADAM28 (Figure KO mice10E) and (Figure aspartate 10D), aminotransferase suggesting that (AST) kidney (Figure function 10F), were may markedly be better reduced preserved in ADAM28serum KO of ADAM28 mice. Liver KO enzymes,mice (Figure which 10D), are suggesting indicative that of kidney liver injury,function such may as be alkaline better preserved phosphatase inin theADAM28 serum ofKO ADAM28 mice. Liver KO mice enzymes, at 49 days which of age are. indicative of liver injury, such as alkaline (Figure 10E) and aspartate aminotransferase (AST) (Figure 10F), were markedly reduced in the serum phosphatase (Figure 10E) and aspartate aminotransferase (AST) (Figure 10F), were markedly reduced of ADAM28 KO mice at 49 days of age. in the serum of ADAM28 KO mice at 49 days of age.

Figure 10. Metabolic benefits in female ADAM28 knock-out (KO) mice. Body weight at 10 months of age (A); High-density lipoprotein (HDL) cholesterol at 49 days of age (B); HDL cholesterol at 6 months of age (C); Blood urea nitrogen at 49 days of age (D); Alkaline Phosphatase at 49 days of age (E) and Figure 10. Metabolic benefits in female ADAM28 knock-out (KO) mice. Body weight at 10 months of FigureAST 10. activityMetabolic at 49 beneﬁtsdays of age in female(F). * p < ADAM280.05; n = 3– knock-out4 mice/group. (KO) mice. Body weight at 10 months of age (A); High-density lipoprotein (HDL) cholesterol at 49 days of age (B); HDL cholesterol at 6 months age (A); High-density lipoprotein (HDL) cholesterol at 49 days of age (B); HDL cholesterol at 6 months of age (C); Blood urea nitrogen at 49 days of age (D); Alkaline Phosphatase at 49 days of age (E) and of age (C); Blood urea nitrogen at 49 days of age (D); Alkaline Phosphatase at 49 days of age (E) and AST activity at 49 days of age (F). * p < 0.05; n = 3–4 mice/group. AST activity at 49 days of age (F). * p < 0.05; n = 3–4 mice/group.

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3. Discussion For the first time, we have examined the role of ADAM28 in the metabolic syndrome in an in vivo mouse model. We found ADAM28 mRNA and protein levels to be higher in steatotic livers of obese mice. In addition, noradrenaline, the major neurotransmitter of the sympathetic nervous system, results in elevated Adam28 mRNA expression in human monocytes. Using siRNA technology, we also demonstrated that downregulation of ADAM28 resulted in a lack of weight gain, promotion of insulin sensitivity/glucose tolerance and decreased liver TNF-α levels in our diet-induced obesity mouse model as well as improved kidney function, and reduced liver injury . Our study also highlighted the metabolic benefits in ADAM28 knock-out mice. An increasing number of studies suggest that ADAM28 plays a crucial role in the pathogenesis of several diseases [18–25], particularly in cancers such as breast cancer [19], prostate cancer [21], B-cell acute lymphoblastic leukaemia [25], chronic lymphatic leukaemia [23], head and neck squamous cell carcinoma [24] and other conditions such as lethal acute respiratory infections [18]. However, the role of ADAM28 in colorectal cancer remains controversial [20,22]. Here, we provide evidence to show in our current functional study that ADAM28 appears to also play a pathogenic role in the metabolic syndrome. It is known that ADAM28 is expressed in immune cells in mice and humans, primarily in the B-lymphocyte lineage [26,27]. It would be interesting to conduct future bone marrow transplantation studies using ADAM28 KO bone marrow to ascertain the role that ADAM28 expression in cells of the hematopoietic lineage has in the metabolic syndrome and the aforementioned diseases. Indeed, there is increasing evidence that obesity and T2D are associated with a chronic inflammatory state and the important role of metalloproteinases in this inflammatory paradigm is being increasingly recognized [9,28,29]. We have previously documented several mechanisms by which the metalloproteinase and disintegrin domains of ADAM28 may promote inflammation and ultimately metabolic dysfunction [9]. Our group has highlighted that major substrates of the metalloproteinase domain of ADAM28 include IGFBP-3 and TNF-α which may confer adipogenesis and inflammation, respectively [9,30]. Hence, based on our current study, it is plausible that therapeutic ADAM28 inhibition may reduce adipogenesis and inflammation due to diminished IGFBP-3 cleavage and TNF-α shedding. We did indeed demonstrate that in our in vivo studies in HFD-fed mice that silencing ADAM28 expression resulted in a marked reduction in TNF-α protein in the liver. This TNF-α protein may be cleaved or cytoplasmic derived. Our previous work [9] and that from other groups [20] have reported that ADAM28 is elevated in overweight and obese humans and correlates with several parameters of the metabolic syndrome. The results in our present in vivo mouse study indicated that siRNA mediated downregulation of ADAM28 promoted decreased high fat diet-induced weight gain, increased glucose tolerance/insulin sensitivity, decreased liver TNF-α levels, improved kidney function, and reduced liver injury. We also illustrate in ADAM28 knock-out mice that body weight is decreased, levels of protective high density lipoprotein cholesterol are significantly elevated, whilst kidney function is better preserved and liver injury is reduced. Therefore, our current data further supports ADAM28’s pathogenic role in the metabolic syndrome. Future studies should address the effect of ADAM28 expression on leptin levels, as leptin plays numerous beneficial metabolic roles such as appetite control [31,32]. There are currently numerous physical, clinical, and therapeutic strategies to manage obesity and obesity related disorders such as type 2 diabetes. These strategies include physical activity [6], bariatric surgery [33,34], metformin therapy [35], consumption of a Mediterranean diet [36,37], natural products [38] and herbal medications [39]. Reducing ADAM28 levels with siRNA technology resulted in no gains in body weight, increased glucose tolerance/insulin sensitivity, decreased liver TNF-α levels, improved kidney function, and reduced liver injury. The siRNA approach would reduce all domains of the ADAM28 protein, including the disintegrin domain. The disintegrin domain may be critically involved in the pathogenic Int. J. Mol. Sci. 2017, 18, 884 8 of 12 role of ADAM28. We have previously discussed how binding of the disintegrin domain of ADAM28 to integrin α4β1 and/or P-selectin glycoprotein ligand-1 on leukocytes may promote inflammation [9]. Our novel findings show that increased ADAM28 mRNA and protein expression in high fat diet-induced obesity is associated with promoting features of the metabolic syndrome in mice. Additionally, ablation of ADAM28 in mice on normal chow confers metabolic benefits. These results provide evidence that downregulation of ADAM28 could be a potential therapeutic target for anti-obesity agents.

4. Materials and Methods

4.1. Cell Culture Experiments THP-1 human monocyte cells (<6 passages) were purchased from the American Type Culture ◦ Collection (Manassas, VA, USA). Cells were cultured at 37 C, 5% CO2 in a humidiﬁed chamber. THP-1 cells were cultured in Dulbecco’s Modiﬁed Eagle Medium (DMEM) [high glucose; Gibco, Gaithersburg, MD, USA] + 5% foetal calf serum (FCS) and 1% penicillin/streptomycin. For noradrenaline treatments, cells were washed and changed to starvation media (High glucose DMEM + 1.0% FCS). Cells were treated with 0, 0.1, or 1.0 µM noradrenaline (Sigma, St. Louis, MO, USA) for 48 h.

4.2. Animals All experimental and animal handing activities were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Royal Perth Hospital, Western Australia. Animal ethics approval (#R522/13–16) for our experiments was received from the Royal Perth Hospital Animal Ethics Committee. Eight-week-old male specific pathogen free C57BL6/J mice were obtained from the Animal Resources Centre (ARC, Perth, WA, Australia). Mice were acclimatized for 7 days, housed under a 12-h light/dark cycle, and given a standard diet with free access to food and water. In our first experiments, mice were administered a normal chow diet (14.3 MJ/kg, 76% of energy from carbohydrate, 5% from fat, 19% from protein; Specialty Feeds, Glen Forrest, WA, Australia) or high fat diet, HFD (19 MJ/kg, 35% of energy from carbohydrate, 42% from fat, 23% from protein; Speciality Feeds, Glen Forrest, WA, Australia) for 12 weeks, and body weights were recorded weekly. At the end of the experiment, mice were sacrificed and livers were collected for paraffin embedding and snap frozen in liquid nitrogen for mRNA studies.

4.3. ADAM28 siSTABLE siRNA Treatment Eight-week-old male specific pathogen free C57BL6/J mice were placed on different diet/antibody treatment regiments: (1) Standard chow: administered non-targeted siSTABLE siRNA (n = 3); (2) Standard chow: administered siSTABLE mouse ADAM28 siRNA (n = 3); (3) High fat diet: administered non-targeted siSTABLE siRNA (n = 3); and (4) High fat diet: administered siSTABLE mouse ADAM28 siRNA (n = 3). The siRNA administration occurred at the end of week ten of the dietary regiment, as ADAM28 mRNA is increased at this time-point in tissues such as the liver. Mice received siRNA injections every five days via the tail vein for the final two weeks of feeding. For each time-point, 20 µg of siSTABLE siRNA (Dharmacon, Lafayette, CO, USA) was mixed with 200 µL DOTAP liposomal transfection reagent (Roche, Indianapolis, IN, USA). Body weight measurements were collected weekly. Glucose tolerance tests were performed at the start of week 12 and insulin tolerance tests were performed at the end of week 12, as indicated previously [10]. Liver and adipose tissue were collected and snap frozen. Blood urea nitrogen, alkaline phosphatase, and aspartate aminotransferase were measured in serum by PathWest LMWA (Murdoch, WA, Australia).

4.4. Western Blotting Liver and gonadal white adipose tissue were homogenised in cytosolic extraction buffer containing phosphatase and protease inhibitors. Protein levels were determined using a Bradford Int. J. Mol. Sci. 2017, 18, 884 9 of 12 protein assay. Cell lysates were cleared and protein concentration calculated using protein assay solution (Bio-Rad, Hercules, CA, USA). Protein lysates were solubilized in Laemmli sample buffer and boiled for 10 min, resolved by SDS–polyacrylamide gel electrophoresis on 10% polyacrylamide gels, transferred by semi-dry transfer to polyvinylidene diﬂuoride membrane, and then blocked with 5% milk powder. Membranes were then incubated overnight at 4 ◦C in anti-mouse ADAM28 monoclonal antibody (sc-514228 [H4], Santa Cruz Biotechnology, Paso Robles, CA, USA); α-tubulin (Santa Cruz Biotechnology; sc-5546) or β-actin (Abcam, Cambridge, UK; ab6276) using recommended dilutions. Membranes were washed three times in washing buffer and incubated for 60 min at room temperature with either anti-rabbit or anti-mouse horse-radish peroxidase (HRP; GE, Issaquah, WA, USA), respectively. Membranes were then washed and brieﬂy incubated in Amersham ECL Prime Western Blotting Detection Reagent (GE, Issaquah, WA, USA). The protein bands were detected using the Alpha Innotech MultiImage II Fluor Chem FC2 (Miami, FL, USA).

4.5. Adam28 mRNA Expression Studies in Human THP-1 Monocytes and Livers of Mice Fed Normal Chow or High Fat Diet (HFD) RNA from human THP-1 monocytes and livers of mice (fed normal chow or HFD) was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA synthesis was performed using the High Capacity RNA-to-cDNA kit (Thermofisher Scientific, Waltham, MA, USA). Real-time PCR to determine the mRNA abundance of human or mouse Adam28 and 18S rRNA (house-keeper gene) was performed using a Rotor-gene real-time PCR machine (Qiagen, Germantown, MD, USA) using pre-developed TaqMan probe and primer sets for human Adam28 (Hs00248020_m1), human 18S (Hs03928990_g1), mouse Adam28 (Mm00456637_m1), and eukaryotic 18S rRNA (4310893E) (Thermoﬁsher Scientiﬁc, Waltham, MA, USA). Quantitation was conducted as previously described [40].

4.6. ADAM28 KO Mice An Academic Delta One licence agreement was obtained from Deltagen (San Mateo, CA, USA) to access phenotypic data for female ADAM28 knock-out (KO) mouse fed a normal chow diet (t137). Permission to publish the data was obtained from Robert Driscoll.

4.7. TNF-α ELISA on Murine Liver Protein Liver tissue was homogenised in cytosolic extraction buffer containing phosphatase and protease inhibitors. Protein levels were determined using a Bradford protein assay and TNF-α was measured in lysates using a mouse TNF-α ELISA (ELISAkit.com, Caribbean Park, Scoresby, VIC, Australia).

4.8. Statistics All in vitro and in vivo results are expressed as the mean + and/or − standard error of the mean (SEM). Data were analysed for differences by Students t-test for unpaired samples where appropriate. Data was considered to be statistically signiﬁcant when p < 0.05. T values were also calculated to further verify signiﬁcance (Table S1).

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/18/4/884/s1. Acknowledgments: The authors acknowledge the facilities and scientific and technical expertise of CELLCentral, School of Anatomy, Physiology and Human Biology, University of Western Australia. This study was supported by grants from the University of Western Australia (Safety Net Grant), Diabetes Australia Research Program (DARP), Rebecca L Cooper Medical Research Foundation, and the Royal Perth Hospital Medical Research Foundation. Author Contributions: Lakshini Herat and Caroline Rudnicka conducted experimental work and drafted the manuscript. Yasunori Okada and Satsuki Mochizuki drafted the manuscript. Markus Schlaich drafted the manuscript. Vance Matthews funded, conceived, supervised, and conducted experimental work and drafted the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Int. J. Mol. Sci. 2017, 18, 884 10 of 12

Abbreviations

ADAM28 A Disintegrin And Metalloproteinase 28 AST Aspartate aminotransferase DMEM Dulbecco’s Modiﬁed Eagle Medium FCS Foetal Calf Serum HDL High Density Lipoprotein HFD High fat diet KO Knock-out mRNA Messenger RNA NA Noradrenaline SAFHS San Antonio Family Heart Study SEM Standard error of the mean siRNA Small interfering RNA SNS Sympathetic nervous system T2D Type 2 Diabetes TNF-α tumour necrosis factor-α

References

1. Alwahsh, S.M.; Dwyer, B.J.; Forbes, S.; Thiel, D.H.; Lewis, P.J.; Ramadori, G. Insulin production and resistance in different models of diet-induced obesity and metabolic syndrome. Int. J. Mol. Sci. 2017, 18, 285. [CrossRef] [PubMed] 2. Guilherme, A.; Virbasius, J.V.; Puri, V.; Czech, M.P. Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes. Nat. Rev. Mol. Cell Biol. 2008, 9, 367–377. [CrossRef][PubMed] 3. Abdelmalek, M.F.; Lazo, M.; Horska, A.; Bonekamp, S.; Lipkin, E.W.; Balasubramanyam, A.; Bantle, J.P.; Johnson, R.J.; Diehl, A.M.; Clark, J.M.; et al. Higher dietary fructose is associated with impaired hepatic adenosine triphosphate homeostasis in obese individuals with type 2 diabetes. Hepatology 2012, 56, 952–960. [CrossRef][PubMed] 4. Alwahsh, S.M.; Xu, M.; Seyhan, H.A.; Ahmad, S.; Mihm, S.; Ramadori, G.; Schultze, F.C. Diet high in fructose leads to an overexpression of lipocalin-2 in rat fatty liver. World J. Gastroenterol. 2014, 20, 1807–1821. [CrossRef][PubMed] 5. Bray, G.A.; Popkin, B.M. Dietary sugar and body weight: Have we reached a crisis in the epidemic of obesity and diabetes?: Health be damned! Pour on the sugar. Diabetes Care 2014, 37, 950–956. [CrossRef][PubMed] 6. Fan, W.; Evans, R.M. Exercise mimetics: Impact on health and performance. Cell Metab. 2017, 25, 242–247. [CrossRef][PubMed] 7. Edwards, D.R.; Handsley, M.M.; Pennington, C.J. The ADAM metalloproteinases. Mol. Asp. Med. 2008, 29, 258–289. [CrossRef][PubMed] 8. Fourie, A.M.; Coles, F.; Moreno, V.; Karlsson, L. Catalytic activity of ADAM8, ADAM15, and MDC-l (ADAM28) on synthetic peptide substrates and in ectodomain cleavage of CD23. J. Biol. Chem. 2003, 278, 30469–30477. [CrossRef][PubMed] 9. Jowett, J.B.; Okada, Y.; Leedman, P.J.; Curran, J.E.; Johnson, M.P.; Moses, E.K.; Goring, H.H.; Mochizuki, S.; Blangero, J.; Stone, L.; et al. ADAM28 is elevated in humans with the metabolic syndrome and is a novel sheddase of human tumour necrosis factor-α. Immunol. Cell Biol. 2012, 90, 966–973. [CrossRef][PubMed] 10. Matthews, V.B.; Allen, T.L.; Risis, S.; Chan, M.H.; Henstridge, D.C.; Watson, N.; Zaffino, L.A.; Babb, J.R.; Boon, J.; Meikle, P.J.; et al. Interleukin-6-deficient mice develop hepatic inflammation and systemic insulin resistance. Diabetologia 2010, 53, 2431–2441. [CrossRef][PubMed] 11. Musso, G.; Gambino, R.; Cassader, M.; Pagano, G. Meta-analysis: Natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity. Ann. Med. 2011, 43, 617–649. [CrossRef][PubMed] 12. Schlaich, M.; Straznicky, N.; Lambert, E.; Lambert, G. Metabolic syndrome: A sympathetic disease? Lancet Diabetes Endocrinol. 2015, 3, 148–157. [CrossRef] 13. Zuckerman, J.E.; Davis, M.E. Clinical experiences with systemically administered siRNA-based therapeutics in cancer. Nat. Rev. Drug Discov. 2015, 14, 843–856. [CrossRef][PubMed] Int. J. Mol. Sci. 2017, 18, 884 11 of 12

14. Fitzgerald, K.; White, S.; Borodovsky, A.; Bettencourt, B.R.; Strahs, A.; Clausen, V.; Wijngaard, P.; Horton, J.D.; Taubel, J.; Brooks, A.; et al. A highly durable RNAi therapeutic inhibitor of PCSK9. N. Engl. J. Med. 2017, 376, 41–51. [CrossRef][PubMed] 15. Nguyen, Q.D.; Schachar, R.A.; Nduaka, C.I.; Sperling, M.; Basile, A.S.; Klamerus, K.J.; Chi-Burris, K.; Yan, E.; Paggiarino, D.A.; Rosenblatt, I.; et al. Dose-ranging evaluation of intravitreal siRNA PF-04523655 for diabetic macular edema (the DEGAS study). Investig. Ophthalmol. Vis. Sci. 2012, 53, 7666–7674. [CrossRef][PubMed] 16. Wang, X.; Oka, T.; Chow, F.L.; Cooper, S.B.; Odenbach, J.; Lopaschuk, G.D.; Kassiri, Z.; Fernandez-Patron, C. Tumor necrosis factor-α-converting enzyme is a key regulator of agonist-induced cardiac hypertrophy and fibrosis. Hypertension 2009, 54, 575–582. [CrossRef][PubMed] 17. Weerasekera, L.; Rudnicka, C.; Sang, Q.-X.; Curran, J.E.; Johnson, M.P.; Moses, E.K.; Göring, H.H.H.; Blangero, J.; Hricova, J.; Schlaich, M.; et al. ADAM19: A novel target for metabolic syndrome in humans and mice. Mediat. Inflamm. 2017, 2017, 9. [CrossRef][PubMed] 18. Baurakiades, E.; Costa, V.H., Jr.; Raboni, S.M.; de Almeida, V.R.; Larsen, K.S.; Kohler, J.N.; Gozzo Pdo, C.; Klassen, G.; Manica, G.C.; de Noronha, L. The roles of ADAM33, ADAM28, IL-13 and IL-4 in the development of lung injuries in children with lethal non-pandemic acute infectious pneumonia. J. Clin. Virol. 2014, 61, 585–589. [CrossRef][PubMed] 19. Mitsui, Y.; Mochizuki, S.; Kodama, T.; Shimoda, M.; Ohtsuka, T.; Shiomi, T.; Chijiiwa, M.; Ikeda, T.; Kitajima, M.; Okada, Y. ADAM28 is overexpressed in human breast carcinomas: Implications for carcinoma cell proliferation through cleavage of insulin-like growth factor binding protein-3. Cancer Res. 2006, 66, 9913–9920. [CrossRef][PubMed] 20. Nowakowska-Zajdel, E.; Mazurek, U.; Wierzgon, J.; Kokot, T.; Fatyga, E.; Ziolko, E.; Klakla, K.; Blazelonis, A.; Waniczek, D.; Glogowski, L.; et al. Expression of ADAM28 and IGFBP-3 genes in patients with colorectal cancer—A preliminary report. Int. J. Immunopathol. Pharmacol. 2013, 26, 223–228. [CrossRef][PubMed] 21. Rudnicka, C.; Mochizuki, S.; Okada, Y.; McLaughlin, C.; Leedman, P.J.; Stuart, L.; Epis, M.; Hoyne, G.; Boulos, S.; Johnson, L.; et al. Overexpression and knock-down studies highlight that a disintegrin and metalloproteinase 28 controls proliferation and migration in human prostate cancer. Medicine 2016, 95, e5085. [CrossRef][PubMed] 22. Wang, J.; Li, H.; Wang, Y.; Wang, L.; Yan, X.; Zhang, D.; Ma, X.; Du, Y.; Liu, X.; Yang, Y. MicroRNA-552 enhances metastatic capacity of colorectal cancer cells by targeting a disintegrin and metalloprotease 28. Oncotarget 2016, 7, 70194–70210. [CrossRef][PubMed] 23. Wong, K.K.; Zhu, F.; Khatri, I.; Huo, Q.; Spaner, D.E.; Gorczynski, R.M. Characterization of CD200 ectodomain shedding. PLoS ONE 2016, 11, e0152073. [CrossRef][PubMed] 24. Wood, O.; Woo, J.; Seumois, G.; Savelyeva, N.; McCann, K.J.; Singh, D.; Jones, T.; Peel, L.; Breen, M.S.; Ward, M.; et al. Gene expression analysis of TIL rich HPV-driven head and neck tumors reveals a distinct B-cell signature when compared to HPV independent tumors. Oncotarget 2016, 7, 56781–56797. [CrossRef] [PubMed] 25. Zhang, X.H.; Wang, C.C.; Jiang, Q.; Yang, S.M.; Jiang, H.; Lu, J.; Wang, Q.M.; Feng, F.E.; Zhu, X.L.; Zhao, T.; et al. ADAM28 overexpression regulated via the PI3K/AKT pathway is associated with relapse in de novo adult B-cell acute lymphoblastic leukemia. Leuk. Res. 2015.[CrossRef][PubMed] 26. McGinn, O.J.; English, W.R.; Roberts, S.; Ager, A.; Newham, P.; Murphy, G. Modulation of integrin α4β1 by ADAM28 promotes lymphocyte adhesion and transendothelial migration. Cell Biol. Int. 2011, 35, 1043–1053. [CrossRef][PubMed] 27. Bret, C.; Hose, D.; Reme, T.; Kassambara, A.; Seckinger, A.; Meissner, T.; Schved, J.F.; Kanouni, T.; Goldschmidt, H.; Klein, B. Gene expression profile of adams and adamtss metalloproteinases in normal and malignant plasma cells and in the bone marrow environment. Exp. Hematol. 2011, 39, 546–557. [CrossRef] [PubMed] 28. Masaki, M.; Kurisaki, T.; Shirakawa, K.; Sehara-Fujisawa, A. Role of meltrin α(ADAM12) in obesity induced by high-fat diet. Endocrinology 2005, 146, 1752–1763. [CrossRef][PubMed] 29. Serino, M.; Menghini, R.; Fiorentino, L.; Amoruso, R.; Mauriello, A.; Lauro, D.; Sbraccia, P.; Hribal, M.L.; Lauro, R.; Federici, M. Mice heterozygous for tumor necrosis factor-α converting enzyme are protected from obesity-induced insulin resistance and diabetes. Diabetes 2007, 56, 2541–2546. [CrossRef][PubMed] Int. J. Mol. Sci. 2017, 18, 884 12 of 12

30. Mochizuki, S.; Shimoda, M.; Shiomi, T.; Fujii, Y.; Okada, Y. ADAM28 is activated by MMP-7 (matrilysin-1) and cleaves insulin-like growth factor binding protein-3. Biochem. Biophys. Res. Commun. 2004, 315, 79–84. [CrossRef][PubMed] 31. Maier, I.B.; Ozel, Y.; Engstler, A.J.; Puchinger, S.; Wagnerberger, S.; Hulpke-Wette, M.; Bischoff, S.C.; Bergheim, I. Differences in the prevalence of metabolic disorders between prepubertal boys and girls from 5 to 8 years of age. Acta Paediatr. 2014, 103, e154–e160. [CrossRef][PubMed] 32. Alwahsh, S.M.; Xu, M.; Schultze, F.C.; Wilting, J.; Mihm, S.; Raddatz, D.; Ramadori, G. Combination of alcohol and fructose exacerbates metabolic imbalance in terms of hepatic damage, dyslipidemia, and insulin resistance in rats. PLoS ONE 2014, 9, e104220. [CrossRef][PubMed] 33. Alwahsh, S.; Ramadori, G. How does bariatric surgery improve type II diabetes. The neglected importance of the liver in clearing glucose and insulin from the portal blood. J. Obes. Weight Loss Ther. 2015, 5, 5–8. [CrossRef] 34. Dib, N.; Kiciak, A.; Pietrzak, P.; Ferenc, K.; Jaworski, P.; Kapica, M.; Tarnowski, W.; Zabielski, R. Early-effect of bariatric surgery (scopinaro method) on intestinal hormones and adipokines in insulin resistant wistar rat. J. Physiol. Pharmacol. 2013, 64, 571–577. [PubMed] 35. Spruss, A.; Kanuri, G.; Stahl, C.; Bischoff, S.C.; Bergheim, I. Metformin protects against the development of fructose-induced steatosis in mice: Role of the intestinal barrier function. Lab. Investig. 2012, 92, 1020–1032. [CrossRef][PubMed] 36. Godos, J.; Federico, A.; Dallio, M.; Scazzina, F. Mediterranean diet and nonalcoholic fatty liver disease: Molecular mechanisms of protection. Int. J. Food Sci. Nutr. 2017, 68, 18–27. [CrossRef][PubMed] 37. Cakir, M.; Akbulut, U.E.; Okten, A. Association between adherence to the mediterranean diet and presence of nonalcoholic fatty liver disease in children. Child. Obes. 2016, 12, 279–285. [CrossRef][PubMed] 38. Chen, Q.; Wang, T.; Li, J.; Wang, S.; Qiu, F.; Yu, H.; Zhang, Y.; Wang, T. Effects of natural products on fructose-induced nonalcoholic fatty liver disease (NAFLD). Nutrients 2017, 9, 96. [CrossRef][PubMed] 39. Alwahsh, S.M.; Gebhardt, R. Dietary fructose as a risk factor for non-alcoholic fatty liver disease (NAFLD). Arch. Toxicol. 2016, 1–19. [CrossRef][PubMed] 40. Chan, M.; Carey, A.; Watt, M.; Febbraio, M. Cytokine gene expression in human skeletal muscle during concentric contraction: Evidence that IL-8, like IL-6, is inﬂuenced by glycogen availability. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2004, 287, 322–327. [CrossRef][PubMed]

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