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Ardiansyah et al. BMC Complementary and Alternative MedicineAcclimatizedfor 7 days(2018) 18:3047Page 3 of 81421 22OGTTAnimal groupsC, distilled waterAll micereceived ahigh fat diet1. Sacrificed2. Blood collection forfurther analysesADN, 10 mg/L25WeekITT1. Sacrificed2. Blood and organcollecting forfurther analysesAMP, 13 mg/LFood and water intake was recordedevery 2 days. Body weight was measuredevery week during the experimentalperiod.Fig. 1 Workflow diagram of the experiments. C, control group; ADN, adenosine group; AMP, adenosine-5′-monophosphate group; OGTT, oralglucose tolerance test; ITT, insulin tolerance testsRNA preparation and quantitative RT-PCRTotal RNA was isolated from muscle tissue with the guanidine isothiocyanate-based reagent Isogen (Nippon Gene,Tokyo, Japan), according to the manufacturer’s instructions. The isolated RNA was treated with RNase-freeDNase (Qiagen, Hilden, Germany) for 10 min at roomtemperature before being purified using an RNeasy Minikit (Qiagen). The ratio of absorbance at wavelengths of260 and 280 nm was measured, and agarose gel electrophoresis was performed for quantitative and qualitativeanalysis of the isolated RNA. Four micrograms of totalRNA was used as a template to synthesize cDNA. TheRNA was denatured in the presence of oligo (dT), randomprimers, and 10 mmol/L dNTP (Amersham Biosciences,Tokyo, Japan) at 65 C for 5 min. It was then incubated ina 20-μL volume with 50 mmol/L Tris-HCl (pH 8.3)containing 0.1 mol/L DTT, 50 U SuperScript III reversetranscriptase (Invitrogen, Carlsbad, CA, USA), and 20 URNaseOUT RNase inhibitor (Invitrogen) at 25 C for5 min, 50 C for 60 min, and 70 C for 15 min. Aliquots ofthe resulting cDNA were used as templates in subsequentquantitative PCR using an Applied Biosystems (FosterCity, CA, USA) 7300 Real-Time PCR System and SYBRPremix Ex Taq (Takara Bio Inc., Kusatsu, Japan) accordingto the manufacturers’ instructions. Relative target gene expression levels were normalized to those of eukaryoticelongation factor-1α1 mRNA [23]. The target sequenceswere amplified using primers specific to the corresponding cDNA (Additional file 1: Table S1).Applied Science, Mannheim, Germany) and phosphatase(PhosSTOP Phosphatase Inhibitor Cocktail, RocheApplied Science). The lysate was centrifuged at 15,000 gfor 30 min for collection of the supernatant, the concentration of protein in which was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). Twentymicrograms of protein was mixed with SDS gel-loadingbuffer and resolved by SDS-polyacrylamide gel electrophoresis on 10–20% gels (Wako Pure Chemical Industries). The proteins were subsequently transferred onto apolyvinylidene fluoride membrane (Millipore, Billerica,MA, USA), which was then blocked for 1 h withTris-buffered saline-Tween 20 (10 mM Tris-HCl atpH 7.4, 150 mM NaCl, and 0.1% Tween 20) containing5% skim milk or 5% bovine serum albumin (Sigma, St.Louis, MO, USA) and incubated with antibodies againstAMPKα2 (Millipore) or phosphorylated AMPKα(Thr172) (Millipore), respectively. The membranes werealso probed with an antibody against α-tubulin (Sigma).Protein bands were visualized with Immobilon WesternDetection Reagent (Millipore) and an LAS-4000 mini luminescent image analyzer (Fujifilm, Tokyo, Japan). Therelative level of each protein was normalized to that ofα-tubulin.Statistical analysisData are presented as means SEM. Statistical analysiscomprised repeated-measures one-way ANOVA followedby the Tukey–Kramer test. A p-value 0.05 was considered to indicate a significant difference among means.Western blot analysisSkeletal muscle lysate was prepared after removal of adipose tissue by homogenizing the muscle in ice-coldphosphate-buffered saline containing inhibitors of proteinase (Complete Proteinase Inhibitor Cocktail, RocheResultsBody weight and daily ADN and AMP intakeBody weight, body weight gain, and the food efficiencyratio (FER) did not differ among the groups after

Ardiansyah et al. BMC Complementary and Alternative Medicine(2018) 18:304administration of ADN or AMP for 14 or 25 weeks(Additional file 2: Table S2) when compared with thecontrol group. However, the FER in the ADN group wassignificantly higher compared with that in the AMPgroup at 14 weeks. In addition, no differences betweenthe groups were noted in food and water intake andrelative organ weights (data not shown). ADN and AMPintake, calculated according to the volume of water consumed, was 25 0.1 and 34 0.3 μg·day 1, respectively.Serum parametersAfter 6 h of fasting, serum glucose levels at the 14thweek of treatment were significantly lower in the ADNgroup than in the AMP group but did not significantlydiffer from those in the control group (Fig. 2a). Inaddition, no differences were noted among the control,ADN, and AMP groups in terms of serum levels of insulin, triacylglycerol, non-esterified fatty acids, and adiponectin (Fig. 2b-e).OGTTWe performed OGTT at the 14th week of administration. Plasma glucose levels in both the ADN and AMPgroups were significantly lower than those in the controlgroup 30 and 60 min after glucose administration (Fig. 3a).Page 4 of 8Furthermore, the incremental area under the curve calculated for the ADN and AMP treatments was significantlysmaller than that for the control (Fig. 3b). ITT werecarried out at the 15th week of treatment. Plasmaglucose levels were significantly lower in the ADNgroup than in the control group 60 min after administration of insulin (Fig. 4).AMPK phosphorylation and gene expressionWe measured levels of phosphorylated and total AMPKα-subunit (AMPKα) protein in skeletal muscle by western blotting to evaluate AMPK activation in this tissue(Fig. 5a, b). The relative quantity of total AMPKα(t-AMPKα) at the 14th week of administration was significantly higher in the AMP group than in the controlgroup (Fig. 5c), and phosphorylated AMPKα (p-AMPKα)levels in the ADN and AMP groups were significantlyhigher than those in the control group at both week 14and 25 (Fig. 5d). Moreover, the ratio of p-AMPKα tot-AMPKα was significantly higher in the ADN and AMPgroups than in the control group after 14 and 25 weeks oftreatment (Fig. 5e). These results suggest that administration of ADN or AMP enhanced AMPK activity in skeletalmuscle. mRNA levels of peroxisome proliferator-activatedreceptor α (Pparα) were significantly increased afterFig. 2 Serum biochemical parameters of mice with high-fat diet-induced obesity administered ADN or AMP after fasting 6 h. a Glucose; (b)insulin; (c) triacylglycerol; (d) non-essential fatty acids; (e) adiponectin. Values are means SEM, n 5 or 6. *p 0.05 versus the control group.Different letters in the same panel represent a significant difference (p 0.05). C, control group; ADN, adenosine group; AMP, adenosine-5′-monophosphategroup

Ardiansyah et al. BMC Complementary and Alternative Medicine(2018) 18:304aPage 5 of 8bFig. 3 Oral glucose tolerance tests of mice with high-fat diet-induced obesity administered ADN or AMP. a Blood glucose changes; (b)incremental areas under the curve (iAUC). Values are means SEM, n 5 or 6. *p 0.05, **p 0.01, ***p 0.001 versus the control group. C,control group; ADN, adenosine group; AMP, adenosine-5′-monophosphate group14 weeks of ADN or AMP treatment. In addition, those ofacyl-CoA synthase (Acs), very long chain acyl-CoA dehydrogenase (Vlcad), and PPAR gamma coactivator 1α(Pgc1α) were significantly higher in the AMP group than inthe control group at week 14 (Additional file 3: Table S3).However, no significant differences in the levels ofthese transcripts were observed between the groups atthe 25th week of administration.DiscussionIn our previous work, we investigated the capacity ofADN and AMP to ameliorate metabolic syndrome-relateddiseases using a stroke-prone spontaneously hypertensiverat model of non-obesity-associated insulin resistance andhypertension-related disorders similar to human essentialhypertension [20, 21]. ADN administration reduced bloodglucose and insulin levels measured under fastingADNGlucose (% of baseline)CAMP**0153060Time after administration (min)Fig. 4 Changes in blood glucose during insulin tolerance tests ofmice with high-fat diet-induced obesity administered ADN or AMP.Values are means SEM, n 5 or 6. **p 0.01 versus the controlgroup. C, control group; ADN, adenosine group; AMP,adenosine-5′-monophosphate groupconditions, as well as glucose and insulin tolerance. Inaddition, we showed for the first time that ingestion ofAMP increases plasma adiponectin concentration, upregulates hepatic Prkaa1 mRNA expression, and elevateslevels of p-AMPKα, leading to enhanced expression ofgenes associated with β-oxidation in the liver.In the current study, we examined the effect of ADNand AMP administration on glucose metabolism usingmice fed a high-fat diet as an animal model of obesity.Human and animal studies have demonstrated that adiet high in fat induces increased lipid accumulation intissues including adipose tissue and skeletal muscle,followed by insulin resistance [24]. Increased levels ofnon-esterified fatty acids in the blood and diacylglyceroland palmitoyl-CoA in organs result in activation ofc-Jun N-terminal kinase (JNK) and inhibitor of kappalight polypeptide gene enhancer in B-cells, kinase β(IKKβ). Activation of JUN and IKKβ leads to serinephosphorylation of insulin receptor substrate, interferingwith insulin signaling via the insulin receptor [25, 26].We speculate that the effect of ADN and AMP is morepronounced in the early stage of diabetes. Future studiesare needed to clarify the detailed mechanism, but ADNand AMP are promising candidates for the prevention ofdiabetes in both mice and humans.Even though blood glucose levels in fasted state werenot different among the groups (Fig. 1a), tolerance testsrevealed that ADN and AMP improved the abnormalglucose tolerance (Fig. 3) and insulin sensitivity (Fig. 4)observed in the mouse model of obesity used in thisstudy. These results indicate that administration of ADNor AMP can mitigate insulin resistance in mice fed ahigh-fat diet. ADN and AMP treatment may have theability to reduce blood glucose in postprandial, but notin fasted state.In this study, we found that oral administration ofADN or AMP can activate AMPK in skeletal muscle(Fig. 5). This is the first in vivo demonstration of AMPKactivation by ADN or AMP in the high-fat diet-inducedobese mouse model of insulin resistance. AMPK acts as

Ardiansyah et al. BMC Complementary and Alternative Medicine(2018) 18:304Page 6 of 8Fig. 5 Representative immunoblot images and quantitative analyses of total AMPKα, phosphorylated AMPKα, and α-tubulin. a Western blotimages at 14 weeks; (b) western blot images at 25 weeks; (c) expression of total AMPKα (t-AMPKα); (d) expression of phosphorylated AMPKα(p-AMPKα); and (e) ratio of t-AMPKα to p-AMPKα. Values are means SEM, n 5 or 6. *p 0.05, **p 0.01, ***p 0.001 versus the control group.C, control group; ADN, adenosine group; AMP, adenosine-5′-monophosphate groupa sensor, and when activated, stimulates mitochondrialbiogenesis, energy production, lipid oxidation, and glucose influx, alleviating insulin resistance via activation ofsirtuin 1 and PGC1α [27]. AMPK activation in musclewas enhanced after both 14 and 25 weeks of ADN orAMP administration independently of adiponectin.Thus, ADN and AMP may indeed suppress the progression of high-fat diet-induced insulin resistance relativelyearly in the development of diabetes.mRNA expression of the gene encoding PPARα, anAMPK target in skeletal muscle, was increased in theAMP and ADN groups after 14 weeks of administration.Furthermore, levels of Acs and Vlcad mRNA were alsohigher in the AMP group at this time point (Additionalfile 3: Table S3). However, these differences were nolonger evident after 25 weeks of treatment, despitecontinued AMPK activation in the skeletal muscle ofADN- or AMP-treated mice. Thus, although at 14 weeks(Additional file 3: Table S3), activation of AMPK inskeletal muscle stimulated the expression of PPARαand its target genes; it suggests that an adaption hadoccurred by the 25th week of administration(Additional file 3: Table S3). Skeletal muscle canadapt its metabolic properties in response to a number of physiologic conditions by activation and repression of signaling events that affect the metabolicpathways of several genes related to the oxidationprocess. The first evidence linking AMPK to theregulation of glucose metabolism in skeletal musclewas provided by treatment with AICAR, an activator

Ardiansyah et al. BMC Complementary and Alternative Medicine(2018) 18:304of AMPK that enhances fatty acid oxidation [28]. Ourresults suggest that ADN and AMP contribute to improving the regulation of glucose metabolism throughexpression of PPARα and its target genes.Lipid accumulation in the skeletal muscle of obese animals induces abnormal glucose metabolism accompaniedby dysregulated insulin signaling and skeletal muscle function. Upregulation of lipid oxidation in skeletal muscle isconsidered a therapeutic strategy to combat insulin resistance. Unsaturated fatty acids, such as oleic acid, and agonists of PPARα enhance lipid oxidation and are expectedto have therapeutic effects in diabetes [29–31]. In ourstudy, administration of ADN or AMP increased the expression of transcripts associated with lipid oxidation inskeletal muscle. As muscle is the major site of ATPproduction and consumption, enhanced lipid oxidation inskeletal muscle may be involved in the mitigation of abnormal glucose metabolism by ADN and AMP in micefed a high-fat diet.Although this study was carefully and well prepared,we were still aware of its limitations. We need to measure locomotor activity of mice. Various studies haveshown that in rodent models of obesity locomotor activity is necessary to evaluate. Further studies are neededto evaluate the behavior of mice after administration ofADN and AMP. In addition, further studies are neededto elucidate the complete physiological effect of ADNand AMP to confirm its detailed mechanism of action atthe molecular level.ConclusionsHere, we showed that administration of ADN or AMPto obese mice ameliorates abnormal glucose metabolisminduced by a high-fat diet. ADN or AMP treatment alsoincreases the level of activated AMPK in skeletal muscle.Activation of AMPK may upregulate lipid oxidation andattenuates insulin resistance caused by obesity.Additional filesAdditional file 1: Table S1. Sequences of primers used for quantitativeRT-PCR. (DOCX 18 kb)Additional file 2: Table S2. Effect of ADN and AMP on body weight,weight gain, and food efficiency ratio. (DOCX 13 kb)Additional file 3: Table S3. Quantitative RT-PCR measurements of mRNAlevels (fold changes) after ADN and AMP administration. (DOCX 17 kb)AbbreviationsADN: Adenosine; AMP: Adenosine-5′-monophosphate; AMPK: AMP-activatedprotein kinase; ITT: Insulin tolerance tests; OGTT: Oral glucose tolerance testsAcknowledgmentsWe are thankful to Yamasa Co. (China, Japan), which kindly provided theAMP used in this study.Page 7 of 8FundingThis work was partially supported by the JSPS Core-to-Core Program A (AdvancedResearch Networks) entitled “Establishment of international agricultural immunology research-core for a quantum improvement in food safety.”Availability of data and materialsThe datasets used and/or analyzed during the current study are availablefrom the corresponding author on reasonable request.Authors’ contributionsA and YI performed the experiments and prepared the manuscript. TK, AZA,II, and TG contributed to the design of the experiments and participated inthe discussion of the results. MK and HS supervised the entire design of theexperiments and drafted the manuscript. All authors read and approved thefinal manuscript.Ethics approvalThe experimental design of the present study was approved by the AnimalResearch-Animal Care Committee of Tohoku University. All experiments wereconducted in accordance with the guidelines issued by this committee andJapanese legislation (2005).Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interest associated withthis publication.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Laboratory of Nutrition, Graduate School of Agricultural Science, TohokuUniversity, Sendai 980-0845, Japan. 2Department of Food Science andTechnology, Universitas Bakrie, Jakarta 12920, Indonesia. 3Faculty ofAgriculture, Yamagata University, Tsuruoka 997-8555, Japan. 4Laboratory ofFood and Biomolecular Science, Graduate School of Agricultural Science,Tohoku University, Sendai 980-0845, Japan. 5International Education andResearch Center for Food Agricultural Immunology, Graduate School ofAgricultural Science, Tohoku University, Sendai 980-0845, Japan.Received: 25 April 2018 Accepted: 30 October 2018References1. 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metabolism in mice with high-fat diet-induced obesity. Methods: Seven-week-old C57BL/6 J mice were administered distilled water (as a control), 10 mg/L ADN, or 13 mg/L AMP via their drinking water for 14 or 25 weeks, during which they were fed a high-fat diet. Oral glucose tolerance test (OGTT) was conducted on 21-week-old mice fasted for 16 h.