1-Deoxynojirimycin isolated from Bacillus subtilis improves hepatic lipid metabolism and...

1-Deoxynojirimycin isolated from Bacillus subtilis improves hepaticlipid metabolism and mitochondrial function in high-fatndashfed miceHyun Ju Do a Ji Hyung Chung b Ji Won Hwang ac Oh Yoen Kim d Jae-Yeon Lee eMin-Jeong Shin acfa Department of Food and Nutrition Korea University Seoul 136-704 Republic of Koreab Department of Applied Bioscience CHA University Gyeonggi-do 463-400 Republic of Koreac Department of Public Health Sciences Graduate School Korea University Seoul 136-703 Republic of Koread Department of Food Science and Nutrition Dong-A University Busan Republic of Koreae RampD Center for Natural Science Biotopia Co Ltd Chuncheon 200-883 Republic of Koreaf Korea University Guro Hospital Korea University Seoul 152-703 Republic of Korea

A R T I C L E I N F O

Article historyReceived 28 April 2014Accepted 1 November 2014Available online 10 November 2014

KeywordsDNJObesityLipogenesisMitochondriaAMPK

A B S T R A C T

The aim of this study was to determine whether 1-deoxynojirimycin (DNJ) isolated from Bacillus subtilisMORI beneficially influences lipid metabolism and mitochondrial function in the liver of mice fed a high-fat diet in addition to the anti-obesity properties of DNJ Male C57BL6 mice (n = 29 5 weeks old) wererandomly assigned to three groups normal control diet (CTL n = 10) high-fat diet (HF n = 10) and high-fat diet supplemented with DNJ (DNJ n = 9) After 12 weeks the HF group exhibited higher overall weightgain of the liver and of various fat pads than the CTL and DNJ groups did The HF group also showedgreater expression of CEBPα and CD36 mRNA in the liver than that in the CTL andor DNJ groups Inaddition mRNA expressions of AAC and FAS were lower while mRNA expression of PGC-1β was higherin the liver of the DNJ group than that of the HF group The hepatic expression of p-AMPKAMPK washigher in the DNJ group than in the HF group This study provides novel insight into the protectiveeffect of DNJ supplementation against obesity-induced hepatic lipid abnormalities and mitochondrialdysfunction

copy 2014 Elsevier Ltd All rights reserved

1 Introduction

1-Deoxynojirimycin (DNJ) is known to improve diabetic condi-tions by inhibiting the activity of α-glucosidase and the absorptionof glucose in the intestinal brush border (Kwon et al 2011 Li et al2013) This leads to improved postprandial glucose response andinsulin sensitivity (Li et al 2013) DNJ has been isolated from theroot and leaf of the mulberry tree (Morus alba) (He and Lu 2013)and has been studied for its role in glucose and lipid metabolism

For example it reduced the levels of insulin and glucose therebyimproving carbohydrate metabolism (Tsuduki et al 2013) whichmay be due to the inhibitory effect of DNJ on the intestinal absorp-tion of glucose accelerating hepatic glucose metabolism (Li et al2013) Recently it was reported that a limited number of micro-organisms such as Bacilli and Streptomyces produce high levels ofDNJ (Lee et al 2013 Niwa et al 1984 Onose et al 2013) Partic-ularly a Bacillus sp Bacterium (specifically Bacillus subtilis) was foundto strongly inhibit α-glucosidase and produce high levels of DNJ fromlsquoChungkookjangrsquo (fast-fermented soybean paste) a Korean tradi-tional food (Kim et al 2011) According to these and other reportsthe structure of DNJ isolated from B subtilis and that from plantsources is the same (Nakagawa et al 2007 Onose et al 2013Shibano et al 2004)

Regarding the role of DNJ in weight control only a few studieshave reported the anti-obesity effect of DNJ using Otsuka Long-Evans Tokushima Fatty (OLETF) rats (Fukaya et al 2009 Mochizukiet al 2011) and a mouse model for diet-induced obesity (Kong et al2008) According to a recent report DNJ stimulates the produc-tion of adiponectin in cultured 3T3-L1 adipocytes (Lee et al 2013)which is supported by another study showing that animals fed DNJ-supplemented diets exhibited increased expression of adiponectin

Abbreviations DNJ 1-deoxynojirimycin IR insulin resistance T2DM type 2 di-abetes WAT white adipose tissue CEBPα ccaat-enhancer-binding protein α ACCacetyl-CoA carboxylase FAS fatty acid synthase SCD-1 stearoyl CoA desaturase 1CD36 cluster of differentiation 36 PGC-1α peroxisome proliferator-activated re-ceptor γ coactivator-1α PGC-1β peroxisome proliferator-activated receptor γcoactivator-1 β (phospho)-AMP-activated protein kinase (p-)AMPK GAPDH glyc-eraldehyde 3-phosphate dehydrogenase GOT glutamate oxalacetate transaminaseGPT glutamate pyruvate transaminase PPARγ peroxisome proliferator-activated re-ceptor γ FABP fatty acid-binding protein

Corresponding author Department of Food and Nutrition Korea University Seoul136-703 Republic of Korea Tel +82 2 940 2857 fax +82 2 940 2850

E-mail address mjshinkoreaackr (M-J Shin)

httpdxdoiorg101016jfct2014110010278-6915copy 2014 Elsevier Ltd All rights reserved

Food and Chemical Toxicology 75 (2015) 1ndash7

Contents lists available at ScienceDirect

Food and Chemical Toxicology

journal homepage wwwelseviercom locate foodchemtox

mRNA in adipose tissue (Tsuduki et al 2013) However the un-derlying mechanism by which DNJ exerts these systemic effectsremains largely unknown Several studies have demonstrated thatobesity impairs hepatic glucose and lipid homeostasis and that met-abolic dysregulation can ultimately cause hepatic steatosis(Benhamed et al 2012 Fabbrini et al 2010 Ferreacute and Foufelle2010) Notably hepatic steatosis insulin resistance (IR) and obesityoccur frequently and simultaneously and the presence of these con-ditions synergistically increases the risk of type 2 diabetes (T2DM)(Burra 2013) In addition it was reported that defects in mitochon-drial metabolism are associated with hepatic steatosis IR and T2DM(Garciacutea-Ruiz et al 2013 Sato et al 2013) Given the close inter-relationship between obesity-induced hepatic steatosis and IR (Posticand Girard 2008) non-pharmacological attempts to attenuatehepatic steatosis might elicit systemic metabolic effects and affectobesity and insulin sensitivity

Therefore we investigated whether diet supplementationwith DNJ from B subtilis MORI could beneficially influence lipidmetabolism and mitochondrial function in the liver of mice fed ahigh-fat diet in addition to the anti-obesity properties of DNJ

2 Materials and methods

21 Animals and experimental protocol

Male C57BL6 mice (n = 29 5 weeks old initial weight 173 g) were randomlyassigned to three groups after a 1-week adaptation period normal control diet (CTLn = 10) high-fat diet (HF n = 10) and high-fat diet supplemented with DNJ (DNJn = 9) (Lee et al 2013) DNJ was prepared in distilled water and administered orallybased on body weight (10 mgkg once daily) as determined previously (Li et al 20112013) The mice in the CTL and HF groups received equivalent volumes of vehicle(distilled water) The CTL was based on the AIN-76 rodent diet composition The HFwas identical to the CTL except for the addition of 200 g fatkg feed (170 g lard plus30 g corn oil) and 1 cholesterol The mice were housed under standard (18ndash24 degC50ndash60 humidity) laboratory conditions maintained on a 1212-h lightdark sched-ule (lights on at 800 AM) with access to food and water ad libitum for 12 weeksDaily feed intake and weekly body weight gain were routinely recorded through-out the experimental period using a computing scale (Acom Co Inc Korea) Allexperimental procedures were approved by the Committee on Animal Experimen-tation and Ethics of Korea University

22 Sample collection and storage

At the end of the experimental period mice were fasted overnight (12 h) Micewere anesthetized with a ZoletilregRompunreg mixture (40 and 5 mgkg body weightrespectively intraperitoneal (ip) injection) Blood samples were obtained from theabdominal inferior vena cava for biochemical parameter analysis The liver and whiteadipose tissue (WAT) were extracted weighed and rapidly frozen with liquid ni-trogen and stored at minus80 degC Plasma was obtained by centrifugation of the collectedblood at 1400 times g for 10 min at 4 degC aliquoted and stored at minus80 degC until use

23 Quantification of the metabolic parameters in plasma

Fasting concentrations of plasma glucose were determined using the hexoki-nase method and an Olympus AU400 Chemistry Analyzer (Japan) The plasmaconcentrations of triglycerides glutamate oxalacetate transaminase (GOT) and glu-tamate pyruvate transaminase (GPT) were determined using an Olympus AU400Chemistry Analyzer (Japan)

24 RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from the liver tissue using an RNeasy Lipid Tissue MiniKit (Qiagen Germany) according to the manufacturerrsquos protocol cDNA was synthe-sized from 1 μg of RNA using an oligo-dT and Superscript II reverse transcriptase(Invitrogen USA) The sequences of the designed primers are shown in Table 1 Thereal-time PCR (Step One Plus Applied Biosystems USA) conditions were as follows15 min at 95 degC followed by 40 cycles at 94 degC for 30 s 52ndash60 degC for 20 s and 72 degCfor 30 s The cycle at which the ΔRn value crossed the threshold was defined as theCT (ΔΔCT) The relative quantity of target mRNA in each sample by comparing thenormalized quantity of target mRNA in each sample with the normalized quantityof target mRNA in the control sample was determined by the software The real-time PCR data were compiled from and are representative of at least three independentexperiments GAPDH was used as the control in the comparative cycle threshold (Ct)method

25 Immunoblot analysis

Liver tissues were lysed with RIPA lysis buffer (Amresco Solon OH USA) con-taining a protease and phosphatase inhibitor cocktail (Sigma-Aldrich St Louis MOUSA) Tissue lysates were centrifuged at 14000 times g for 15 min at 4 degC Proteins werequantified using the BCA protein assay (Sigma-Aldrich) Protein lysates were re-solved on 10 sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and thentransferred to a polyvinylidene difluoride (PVDF) membrane Anti-phospho-AMP-activated protein kinase (AMPK) (Cell Signaling Danvers MA USA) anti-AMPK (CellSignaling) and anti-β-actin (Santa Cruz Biotechnology Santa Cruz CA USA) anti-bodies were used to detect p-AMPK AMPK and β-actin respectively β-actin wasused as protein loading control Blots were analyzed using a western blot detec-tion kit (AbFrontier Seoul Korea) The results are representative of at least threeindependent experiments Protein bands were quantified using the Alphaviewreg soft-ware (Alpha Innotech San Leandro CA USA)

26 Statistical analysis

Statistical analysis was performed using the SPSS software (version 1201SPSS Inc Chicago IL USA) The results are presented as means plusmn SE The differ-ences between the experimental groups were analyzed using one-way ANOVAfollowed by Bonferronirsquos post hoc test P values of lt 005 were considered statisti-cally significant

3 Results

31 The effect of DNJ diet supplementation on body weightfood intake liver weight and body fat

Significant differences in cumulative body weight gains were foundbetween the mouse groups over the 12-week study period (Table 2)The HF group gained more weight than the CTL and DNJ groups didThe HF and DNJ groups consumed remarkably less feed than the CTLgroup did (Table 2) Food efficiency ratios were significantly higher in

Table 1Mouse-specific primer sequences

Gene Primer sequence

CEBPα Forward 5prime-GTTGACCAGTGACAATGACC-3primeReverse 5prime-AATCTCCTAGTCCTGGCTTG-3prime

ACC Forward 5prime-GCCTCTTCCTGACAAACGAG-3primeReverse 5prime-TAAGGACTGTGCCTGGAACC-3prime

FAS Forward 5prime-CTGAAGAGCCTGGAAGATCG-3primeReverse 5prime-GTCACACACCTGGGAGAGGT-3prime

SCD-1 Forward 5prime- CTTCAAGGGCAGTTCTGAG-3primeReverse 5prime-CAATGGTTTTCATGGCAGTG-3prime

CD36 Forward 5prime-TTTCTGTAGACCCAATTATCCTG-3primeReverse 5prime-TTATACTTATGCCATGTGCTGAC-3prime

PGC-1α Forward 5prime-CGGAAATCATATCCAACCAG-3primeReverse 5prime-TGAGGACCGCTAGCAAGTTTG-3prime

PGC-1β Forward 5prime-AACCCAACCAGTCTCACAGG-3primeReverse 5prime-ATGCTGTCCTTGTGGGTAGG-3prime

GAPDH Forward 5prime-AACTTTGGCATTGTGGAAGG-3primeReverse 5prime-ACACATTGGGGGTAGGAACA-3prime

CEBPα ccaat-enhancer-binding protein ACC acetyl-CoA carboxylase FAS fatty acidsynthase SCD-1 stearoyl CoA desaturase 1 CD36 cluster of differentiation 36 PGC-1α peroxisome proliferator-activated receptor γ coactivator-1α PGC-1β peroxisomeproliferator-activated receptor γ coactivator-1 β GAPDH glyceraldehyde 3-phosphatedehydrogenase

Table 2Cumulative body weight gain food intake and food efficiency ratio among theexperimental groups

Groups CTL HF DNJ p

Cumulative BWgain (g)

1315 plusmn 109a 2021 plusmn 116b 1669 plusmn 131c lt005

Food intake (gday) 293 plusmn 004a 274 plusmn 005b 263 plusmn 004b lt005Food efficiency

ratio (FER )005 plusmn 0004a 009 plusmn 0005b 008 plusmn 0006b lt005

Means plusmn SE Tested by analysis of variance (ANOVA) with Bonferronirsquos post hoc testValues sharing the same letters indicate no significant difference between groupsCTL control diet HF high fat diet DNJ high fat diet with 1-deoxinojirimycin

2 HJ Do et alFood and Chemical Toxicology 75 (2015) 1ndash7

the HF and DNJ groups than in the CTL group (Table 2) Final bodyweights were significantly higher in the HF group (3808 plusmn 118 g) thanin the CTL (305 plusmn 123 g) or DNJ (3401 plusmn 141 g) group (Fig 1A) Liverweight and the liver weightbody weight ratio were significantly higherin the HF group than in the CTL and DNJ groups (Fig 1B and C) Theepididymal perirenal retroperitoneal and mesenteric fat weight weresignificantly different between the groups (Fig 1DndashG) Specifically theywere significantly higher in the HF group than in the CTL and DNJ groups(Fig 1H) as was the total fat weight in the HF group (307 plusmn 024 g com-pared with 153 plusmn 010 and 233 plusmn 023 g for that in the CTL and DNJ grouprespectively)

32 The effect of DNJ diet supplementation on plasmametabolic parameters

The plasma concentrations of the liver damage markers GOT andGPT were significantly higher in the HF group than in the CTL grouphowever DNJ food supplementation restored them to the controllevels (Fig 2A and B) The plasma triglyceride concentrations in theDNJ group were significantly lower than in the CTL or HF group(Fig 2C) The plasma glucose concentrations in the DNJ group weresignificantly lower than in the HF group but were similar to thoseof the CTL group (Fig 2D)

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aa

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Fig 1 The effect of DNJ (10 mgkg body weight po once daily) on the final body weight (A) liver weight (B) and liver weightbody weight ratio (C) in mice Epididymalfat (D) perirenal fat (E) retroperitoneal fat (F) mesenteric fat (G) and total WAT weights (H) were significantly lower in the DNJ group compared with those in the HFgroup CTL (n = 10) normal control diet HF (n = 10) high-fat diet DNJ (n = 9) high-fat diet supplemented with DJN The results are expressed as means plusmn SE and wereanalyzed by one-way ANOVA followed by Bonferronirsquos post hoc test Values with the same superscript letter are not significantly different (andashc)

3HJ Do et alFood and Chemical Toxicology 75 (2015) 1ndash7

33 The effect of DNJ diet supplementation on the expression ofgenes related to liver lipogenesis and mitochondrial function

The expression of ccaat-enhancer-binding protein alpha (CEBPα) mRNA in the liver was significantly lower in the DNJ groupthan in the HF group (Fig 3A) The hepatic mRNA expression ofacetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) of the DNJgroup was significantly lower than that of the HF group and wasnot different from that of the CTL group (Fig 3B and C) Howeverstearoyl CoA desaturase 1 (SCD-1) mRNA expression was not signifi-cantly different between the three groups (Fig 3D) Cluster ofdifferentiation 36 (CD36) mRNA expression in the liver of the micein the HF group was significantly higher than that in the liver ofthe mice in the CTL or DNJ group (Fig 3E) In contrast liver peroxi-some proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNAexpression of the HF group was significantly lower than that of theCTL group but was not significantly different from that of the DNJgroup (Fig 3F) The expression of hepatic PGC-1β mRNA was sig-nificantly higher in the DNJ group than in the HF group (Fig 3G)

34 The effect of DNJ diet supplementation on hepaticp-AMPKαAMPKα expression

We next quantified the expression of phosphorylated AMPKα(p-AMPKα) at the Thr172 residue in the liver and found that phos-phorylation of AMPKα was significantly higher in the DNJ group thanin the HF and the CTL groups (Fig 4)

4 Discussion

The present study demonstrates that diet supplementation withDNJ from B subtilis MORI ameliorates hepatic metabolic abnor-malities in mice with diet-induced obesity and systemically affectsbody weight and adipose tissue To the best of our knowledge oursis the first study to provide novel information on the protective effectof DNJ diet supplementation against obesity-induced hepatic lipidabnormalities and mitochondrial dysfunction Mice on a HF dietsupplemented with DNJ exhibited lower overall weight gain of the

liver and of various fat pads than the animals in the HF group didbut showed a weight pattern similar to that of the mice in the CTLgroup The DNJ group also had a lower liver CEBPα and CD36 mRNAexpression than that of the HF group Although the hepatic mRNAexpression of ACC and FAS was lower in the DNJ group that ofPGC-1β was higher in this group than in the HF group

It has been reported that CEBPα and peroxisome proliferator-activated receptor γ (PPARγ) cooperatively and directly affect thedifferentiation of adipocytes at the final phase by activating the ex-pression of adipocyte-specific genes (ie FAS fatty acid-binding protein(FABP) leptin and adiponectin) (Farmer 2006) In fact CEBPβ andCEBPδ are expressed in early phases of cell differentiation and sub-sequently induce the transcription of CEBPα and PPARγ two masterregulators of adipocyte differentiation (Cao et al 1991) Previousreports have shown that CEBP family members (ie CEBPα β andδ) play important roles in the processes of the clonal expansion ofhepatocytes after partial hepatectomy (Buck et al 1999) whichshares many features with the mitotic clonal expansion of adipocytedifferentiation (Tang et al 2003 Yeh et al 1999) Furthermore de-letion of CEBPβ may reduce adiposity hepatic steatosis and the riskof diabetes in the Lepr (dbdb) mice (Schroeder-Gloeckler et al2007) According to Li et al (2011) DNJ was detected in plasmaliver and adipose tissues from mice treated with DNJ and waspresent in higher concentrations in the liver than in the plasma andadipose tissue Reports on the bioavailability of DNJ (Kim et al 2010Kimura et al 2007) based on the analysis of the concentration ofDNJ in plasma feces and urine of rats that had received isolatedDNJ as well as mulberry water extract showed that DNJ was dose-dependently absorbed from the digestive tract into rat plasma Inaddition metabolism of DNJ in liver can be partially explained bythe report of Faber et al (1994) They studied the hepatic disposi-tion of two glycosidase inhibitors in the isolated perfused rat liverand after subcellular fractionation lsquo1-deoxymannojirimycin (DMJ)rsquoand lsquoN-methyl-1-deoxynojirimycin (MedN-DNJ)rsquo one of DNJ de-rivatives exhibited minimal binding to albumin and reached liverconcentrations that approximately equaled their medium concen-trations after 30 min (MedN-DNJ) or 90 min (DMJ) Within 2 hourssmall percentage of the dose of MedN-DNJ (05) and DMJ (29)

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gdL

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(m

gdL

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A B

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aa

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aab

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Fig 2 The effect of DNJ (10 mgkg body weight po once daily) on plasma GOT (A) GPT (B) triglycerides (C) and glucose (D) in mice CTL (n = 10) normal control diet HF(n = 10) high-fat diet DNJ (n = 9) high-fat diet supplemented with DNJ The results are expressed as means plusmn SE and were analyzed by one-way ANOVA followed by Bonferronirsquospost hoc test Values with the same superscript letter are not significantly different (a b)


were excreted in bile No metabolites were found for MedN-DNJbut minor degradation was inferred for DMJ After subcellular frac-tionation DMJ and MedN-DNJ were found predominantly in thecytosolic fraction Compared to the other particulate fractions MedN-DNJ was elevated in the microsomal fraction and both compoundswere slightly enriched in the lysosomal fraction Based on theseresults they suggested that MedN-DNJ and DMJ will likely inhibitliver enzymes when sufficiently high plasma levels are reached Itwas also reported that administration in vivo of the alpha-glucosidaseinhibitors resulted in a dose- and time-dependent decrease in therate of hepatic glycogenolysis induced by glucagon (Bollen et al1988) This represents a direct effect on the liver since it could be

reproduced on isolated hepatocytes Taken together presumablyDNJ is involved in the modulation of hepatic metabolism partiallythrough its metabolism in the liver However it was not experi-mentally proven in the present study Further study is needed totest the metabolism of DNJ in the liver explaining the functionalproperties We assume that the higher mRNA expression of CEBPαin the liver of HF-fed mice compared with that in the liver of themice of the CTL and the DNJ groups shows that CEBPα partici-pates in hepatic adipogenesis Moreover the reduced hepatic mRNAexpression of CEBPα observed in the DNJ group may indicate thatDNJ controls the expression of CEBPα and potentially that of CEBPβthereby beneficially regulating hepatic adipogenesis In addition

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Fig 3 The effect of DNJ (10 mgkg body weight po once daily) on hepatic CEBPα (A) ACC (B) FAS (C) SCD-1 (D) CD36 (E) PGC-1α (F) and PGC-1β (G) mRNA expres-sion CTL (n = 10) normal control diet HF (n = 10) high-fat diet DNJ (n = 9) high-fat diet supplemented with DNJ The results are expressed as means plusmn SE and were analyzedby one-way ANOVA followed by Bonferronirsquos post hoc test Values with the same superscript letter are not significantly different (andashc)


hepatic mRNA expression of ACC and FAS both of which areadipogenic genes that are activated by PPARγ and CEBPα (Huanget al 2011 Rosen and MacDougald 2006 Yim et al 2011) waslower in the DNJ-treated mice compared with that in the HF dietmice Our results may indicate that a diet supplementation with DNJleads to the down-regulation of CEBPα in hepatocytes by modu-lating the expression of the ACC and FAS genes thereby potentiallyinhibiting lipid accumulation in the liver and the development ofa fatty liver Furthermore DNJ diet supplementation suppressed theelevated hepatic mRNA level of CD36 in HF-fed mice CD36 is a scav-enger receptor with multiple ligands and cellular functions CD36also facilitates the cellular uptake of free fatty acids which ulti-mately contributes to the development of hepatic steatosis (Su andAbumrad 2009) Along with the modulation of genes responsiblefor hepatic lipogenesis the reduction of hepatic CD36 expressionby DNJ may further support the beneficial effects of DNJ on hepaticmetabolic abnormalities On the other hand we could not find sig-nificant differences in the hepatic mRNA expression of SCD-1 a keycomponent in the formation of triglycerides between the groupsafter the 12-week study period However its expression seemed tobe slightly lower in the DJN than in the HF group and was similarbetween the DJN and the CTL group

It has been suggested that perturbation in mitochondrial energymetabolism can cause hepatic steatosis IR and T2DM (Lehman et al2000 Vega et al 2000 Wu et al 1999) This study showed thatDNJ significantly upregulated the mRNA expression of PGC-1β inthe liver of HF-fed mice but not that of PGC-1α PGC-1 is involvedin mitochondrial biogenesis adaptive thermogenesis fatty acidβ-oxidation and hepatic gluconeogenesis (Wu et al 1999) More-over PGC-1 is a key transcriptional cofactor that regulates theexpression of PPARα (Lehman et al 2000 Vega et al 2000) Pre-vious studies have suggested that PGC-1β exhibits a higher abilityto regulate mitochondrial function than PGC-1α does (Vianna et al2006) Based on these previous reports our results may indicate thatDNJ improved mitochondrial function predominantly through theupregulation of PGC-1β thereby ameliorating abnormal hepatic

metabolism in a diet-induced obesity mouse model (ie im-proved lipid accumulation insulin sensitivity and glucose transport)We also found enhanced hepatic expression of p-AMPKαAMPKαafter DNJ diet supplementation AMPK acts as an intracellular energysensor and plays a pivotal role in maintaining the intracellular energybalance (Carling 2004 Hardie 2007) It also plays a critical role inmitochondrial biogenesis in response to energy deprivation (Suwaet al 2003) and modulates the activity of downstream target genesfor example the transcription factors PGC-1 Fork head box O1 andO3 (Cantoacute et al 2009) Emerging evidence indicates that p-AMPKis critical for maintaining cellular energy reserves by turning on cata-bolic pathways that generate adenosine triphosphate (ATP) andstimulate the biogenesis of mitochondria and switching-off ana-bolic pathways that utilize ATP (Cantoacute and Auwerx 2009 Zong et al2002) Therefore the increased hepatic expression of PGC-1 mRNAand p-AMPKαAMPKα protein after DNJ diet supplementation in-dicates that DNJ might improve mitochondrial function (Spiegelman2007)

In summary the findings of this study may be the first to providenovel information on how DNJ diet supplementation might exertprotective effects against obesity-induced hepatic lipid abnormali-ties and mitochondrial dysfunction Moreover these data suggesta potential therapeutic role for DNJ in obesity and obesity-relatedcomplications Taken together our data suggest that DNJ attenu-ates HF-induced hepatic steatosis by modulating the expression ofhepatic genes involved in lipogenesis and mitochondrial function

Conflict of interest

The authors declare that there are no conflicts of interest

Transparency document

The Transparency document associated with this article can befound in the online version

Acknowledgements

This research was supported by the Basic Science ResearchProgram through the National Research Foundation of Korea (NRF)funded by the Ministry of Education Science and Technology (NRF-2013R1A1A2A10006101)

References

Benhamed F Denechaud PD Lemoine M Robichon C Moldes MBertrand-Michel J et al 2012 The lipogenic transcription factor ChREBPdissociates hepatic steatosis from insulin resistance in mice and humans J ClinInvest 122 2176ndash2194

Bollen M Vandebroeck A Stalmans W 1988 1-Deoxynojirimycin and relatedcompounds inhibit glycogenolysis in the liver without affecting the concentrationof phosphorylase a Biochem Pharmacol 37 905ndash909

Buck M Poli V Geer PVD Chojkier M Hunter T 1999 Phosphorylation of ratserine 105 or mouse threonine 217 in CEBP beta is required for hepatocyteproliferation induced by TGF alpha Mol Cell 4 1087ndash1092

Burra P 2013 Liver abnormalities and endocrine diseases Best Pract Res ClinGastroenterol 27 553ndash563

Cantoacute C Auwerx J 2009 Caloric restriction SIRT1 and longevity Trends EndocrinolMetab 20 325ndash331

Cantoacute C Gerhart-Hines Z Feige JN Lagouge M Noriega L Milne JC et al 2009AMPK regulates energy expenditure by modulating NAD(+) metabolism and SIRT1activity Nature 458 1056ndash1060

Cao Z Umek RM McKnight SL 1991 Regulated expression of three CEBP isoformsduring adipose conversion of 3T3-L1 cells Genes Dev 5 1538ndash1552

Carling D 2004 The AMP-activated protein kinase cascade ndash a unifying system forenergy control Trends Biochem Sci 29 18ndash24

Fabbrini E Sullivan S Klein S 2010 Obesity and nonalcoholic fatty liver diseasebiochemical metabolic and clinical implications Hepatology 51 679ndash689

Faber ED Proost JH Oosting R Meijer DK 1994 Disposition of glycosidaseinhibitors in the isolated perfused rat liver hepatobiliary and subcellularconcentration gradients of 1-deoxymannojirimycin and N-methyl-1-deoxynojirimycin Pharm Res 1 144ndash150

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b-actin

CTL HF DNJ

Fig 4 The effect of DNJ (10 mgkg body weight po once daily) on hepatic p-AMPKαAMPKα protein expression CTL (n = 10) normal control diet HF (n = 10) high-fatdiet DNJ (n = 9) high-fat diet supplemented with DNJ The results are expressedas means plusmn SE and were analyzed by one-way ANOVA followed by Bonferronirsquos posthoc test Values with the same superscript letter are not significantly different(a b)


Farmer SR 2006 Transcriptional control of adipocyte formation Cell Metab 4263ndash273

Ferreacute P Foufelle F 2010 Hepatic steatosis a role for de novo lipogenesis and thetranscription factor SREBP-1c Diabetes Obes Metab 12 83ndash92

Fukaya N Mochizuki K Tanaka Y Kumazawa T Jiuxin Z Fuchigami M et al2009 The alpha-glucosidase inhibitor miglitol delays the development of diabetesand dysfunctional insulin secretion in pancreatic beta-cells in OLETF rats EurJ Pharmacol 624 51ndash57

Garciacutea-Ruiz C Baulies A Mari M Garciacutea-Roveacutes PM Fernandez-Checa JC 2013Mitochondrial dysfunction in non-alcoholic fatty liver disease and insulinresistance cause or consequence Free Radic Res 47 854ndash868

Hardie DG 2007 AMP-activatedSNF1 protein kinases conserved energy Nat RevMol Cell Biol 8 774ndash785

He H Lu YH 2013 Comparison of inhibitory activities and mechanisms of fivemulberry plant bioactive components against α-glucosidase J Agric Food Chem61 8110ndash8119

Huang B Yuan HD Kim do Y Quan HY Chung SH 2011 Cinnamaldehydeprevents adipocyte differentiation and adipogenesis via regulation of peroxisomeproliferator-activated receptor-γ (PPARγ) and AMP-activated protein kinase(AMPK) pathways J Agric Food Chem 59 3666ndash3673

Kim HS Lee JY Hwang KY Cho YS Park YS Kang KD et al 2011 Isolationand identification of a Bacillus sp producing α-glucosidase inhibitor1-deoxynojirimycin Korean J Microbiol Biotechnol 39 49ndash55

Kim JY Kwon HJ Jung JY Kim DR Kim YS Kwon O 2010 Comparison forbioavailability of 1-deoxynojirimycin with mulberry water extract in rat FASEBJ 24 540ndash549

Kimura T Nakagawa L Kubota H Kojima Y Goto Y Yamagishi K et al 2007Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humans J Agric Food Chem 555869ndash5874

Kong WH Oh SH Ahn YR Kim KW Kim JH Seo SW 2008 Antiobesity effectsand improvement of insulin sensitivity by 1-deoxynojirimycin in animal modelsJ Agric Food Chem 56 2613ndash2619

Kwon HJ Chung JY Kim JY Kwon O 2011 Comparison of 1-deoxynojirimycinand aqueous mulberry leaf extract with emphasis on postprandial hypogly-cemic effects in vivo and in vitro studies J Agric Food Chem 59 3014ndash3019

Lee SM Do HJ Shin MJ Seong SI Hwang KY Lee JY et al 20131-Deoxynojirimycin isolated from a Bacillus subtilis stimulates adiponectinand GLUT4 expressions in 3T3-L1 adipocytes J Microbiol Biotechnol 23637ndash643

Lehman JJ Barger PM Kovacs A Saffitz JE Medeiros DM Kelly DP 2000Peroxisome proliferator-activated receptor g coactivator-1 promotes cardiacmitochondrial biogenesis J Clin Invest 106 847ndash856

Li YG Ji DF Zhong S Lv ZQ Lin TB Chen S et al 2011 Hybrid of1-deoxynojirimycin and polysaccharide from mulberry leaves treat diabetesmellitus by activating PDX-1insulin-1 signaling pathway and regulating theexpression of glucokinase phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in alloxan-induced diabetic mice J Ethnopharmacol 134 961ndash970

Li YG Ji DF Zhong S Lv ZQ Lin TB 2013 Cooperative anti-diabetic effects ofdeoxynojirimycin-polysaccharide by inhibiting glucose absorption andmodulating glucose metabolism in streptozotocin-induced diabetic mice PLoSONE 8 e65892

Mochizuki K Fukaya N Tanaka Y Fuchigami M Goda T 2011 Treatment withthe α-glucosidase inhibitor miglitol from the preonset stage in Otsuka Long-EvansTokushima Fatty rats improves glycemic control and reduces the expression ofinflammatory cytokine genes in peripheral leukocytes Metabolism 60 1560ndash1565

Nakagawa K Kubota H Kimura T Yamashita S Tsuzuki T Oikawa S et al 2007Occurrence of orally administered mulberry 1-deoxynojirimycin in rat plasmaJ Agric Food Chem 55 8928ndash8933

Niwa T Tsuruoka T Goi H Kodama Y Itoh J Inouye S et al 1984 Novelglycosidase inhibitors nojirimycin B and D-mannonic-delta-lactam Isolationstructure determination and biological property J Antibiot (Tokyo) 37 1579ndash1586

Onose S Ikeda R Nakagawa K Kimura T Yamagishi K Higuchi O et al 2013Production of the α-glycosidase inhibitor 1-deoxynojirimycin from Bacillusspecies Food Chem 138 516ndash523

Postic C Girard J 2008 The role of the lipogenic pathway in the development ofhepatic steatosis Diabetes Metab 34 643ndash648

Rosen ED MacDougald OA 2006 Adipocyte differentiation from the inside outNat Rev Mol Cell Biol 7 885ndash896

Sato C Saito T Misawa K Katsumi T Tomita K Ishii R et al 2013 Impairedmitochondrial β-oxidation in patients with chronic hepatitis C relation with viralload and insulin resistance BMC Gastroenterol 13 112

Schroeder-Gloeckler JM Rahman SM Janssen RC Qiao L Shao J Roper Met al 2007 CCAATenhancer-binding protein beta deletion reduces adiposityhepatic steatosis and diabetes in Lepr(dbdb) mice J Biol Chem 282 15717ndash15729

Shibano M Fujimoto Y Kushino K Kusano G Baba K 2004 Biosynthesis of1-deoxynojirimycin in Commelina communis a difference between themicroorganisms and plants Phytochemistry 65 2661ndash2665

Spiegelman BM 2007 Translational control of mitochondrial energy metabolismthrough PGC1 coactivators Novartis Found Symp 287 60ndash63

Su X Abumrad NA 2009 Cellular fatty acid uptake a pathway under constructionTrends Endocrinol Metab 20 72ndash77

Suwa M Nakano H Higaki Y Nakamura T Katsuta S Kumagai S 2003 Increasedwheel-running activity in the genetically skeletal muscle fast-twitch fiberdominant rats J Appl Physiol 94 185ndash192

Tang QQ Otto TC Lane MD 2003 CCAATenhancer-binding protein beta isrequired for mitotic clonal expansion during adipogenesis Proc Natl Acad SciUSA 100 850ndash855

Tsuduki T Kikuchi I Kimura T Nakagawa K Miyazawa T 2013 Intake of mulberry1-deoxynojirimycin prevents diet-induced obesity through increases inadiponectin in mice Food Chem 139 16ndash23

Vega RB Huss JM Kelly DP 2000 The coactivator PGC-1 cooperates withperoxisome proliferator-activated receptor a in the transcriptional control ofnuclear genes encoding mitochondrial fatty acid oxidation enzymes Mol CellBiol 20 1868ndash1876

Vianna CR Huntgeburth M Coppari R Choi CS Lin J Krauss S et al 2006Hypomorphic mutation of PGC-1beta causes mitochondrial dysfunction and liverinsulin resistance Cell Metab 4 453ndash464

Wu Z Puigserver P Andersson U Zhang C Adelmant G Mootha V et al 1999Mechanisms controlling mitochondrial biogenesis and respiration through thethermogenic coactivator PGC-1 Cell 98 115ndash124

Yeh WC Cao Z Classon M McKnight SL 1999 Cascade regulation of terminaladipocyte differentiation by three members of the CEBP family of leucine zipperproteins Genes Dev 9 168ndash181

Yim MJ Hosokawa M Mizushina Y Yoshida H Saito Y Miyashita K 2011Suppressive effects of Amarouciaxanthin A on 3T3-L1 adipocyte differentiationthrough down-regulation of PPARγ and CEBPα mRNA expression J Agric FoodChem 59 1646ndash1652

Zong H Ren JM Young LH Pypaert M Mu J Birnbaum MJ et al 2002 AMPkinase is required for mitochondrial biogenesis in skeletal muscle in responseto chronic energy deprivation Proc Natl Acad Sci USA 99 15983ndash15987


1-Deoxynojirimycin isolated from Bacillus subtilis improves hepatic lipid metabolism and mitochondrial function in high-fatndashfed mice

Introduction

Materials and methods

Animals and experimental protocol

Sample collection and storage

Quantification of the metabolic parameters in plasma

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Immunoblot analysis

Statistical analysis

Results

The effect of DNJ diet supplementation on body weight food intake liver weight and body fat

The effect of DNJ diet supplementation on plasma metabolic parameters

The effect of DNJ diet supplementation on the expression of genes related to liver lipogenesis and mitochondrial function

The effect of DNJ diet supplementation on hepatic p-AMPKAMPK expression

Discussion



Acknowledgements

References

mRNA in adipose tissue (Tsuduki et al 2013) However the un-derlying mechanism by which DNJ exerts these systemic effectsremains largely unknown Several studies have demonstrated thatobesity impairs hepatic glucose and lipid homeostasis and that met-abolic dysregulation can ultimately cause hepatic steatosis(Benhamed et al 2012 Fabbrini et al 2010 Ferreacute and Foufelle2010) Notably hepatic steatosis insulin resistance (IR) and obesityoccur frequently and simultaneously and the presence of these con-ditions synergistically increases the risk of type 2 diabetes (T2DM)(Burra 2013) In addition it was reported that defects in mitochon-drial metabolism are associated with hepatic steatosis IR and T2DM(Garciacutea-Ruiz et al 2013 Sato et al 2013) Given the close inter-relationship between obesity-induced hepatic steatosis and IR (Posticand Girard 2008) non-pharmacological attempts to attenuatehepatic steatosis might elicit systemic metabolic effects and affectobesity and insulin sensitivity

Therefore we investigated whether diet supplementationwith DNJ from B subtilis MORI could beneficially influence lipidmetabolism and mitochondrial function in the liver of mice fed ahigh-fat diet in addition to the anti-obesity properties of DNJ

2 Materials and methods

21 Animals and experimental protocol

Male C57BL6 mice (n = 29 5 weeks old initial weight 173 g) were randomlyassigned to three groups after a 1-week adaptation period normal control diet (CTLn = 10) high-fat diet (HF n = 10) and high-fat diet supplemented with DNJ (DNJn = 9) (Lee et al 2013) DNJ was prepared in distilled water and administered orallybased on body weight (10 mgkg once daily) as determined previously (Li et al 20112013) The mice in the CTL and HF groups received equivalent volumes of vehicle(distilled water) The CTL was based on the AIN-76 rodent diet composition The HFwas identical to the CTL except for the addition of 200 g fatkg feed (170 g lard plus30 g corn oil) and 1 cholesterol The mice were housed under standard (18ndash24 degC50ndash60 humidity) laboratory conditions maintained on a 1212-h lightdark sched-ule (lights on at 800 AM) with access to food and water ad libitum for 12 weeksDaily feed intake and weekly body weight gain were routinely recorded through-out the experimental period using a computing scale (Acom Co Inc Korea) Allexperimental procedures were approved by the Committee on Animal Experimen-tation and Ethics of Korea University

22 Sample collection and storage

At the end of the experimental period mice were fasted overnight (12 h) Micewere anesthetized with a ZoletilregRompunreg mixture (40 and 5 mgkg body weightrespectively intraperitoneal (ip) injection) Blood samples were obtained from theabdominal inferior vena cava for biochemical parameter analysis The liver and whiteadipose tissue (WAT) were extracted weighed and rapidly frozen with liquid ni-trogen and stored at minus80 degC Plasma was obtained by centrifugation of the collectedblood at 1400 times g for 10 min at 4 degC aliquoted and stored at minus80 degC until use

23 Quantification of the metabolic parameters in plasma

Fasting concentrations of plasma glucose were determined using the hexoki-nase method and an Olympus AU400 Chemistry Analyzer (Japan) The plasmaconcentrations of triglycerides glutamate oxalacetate transaminase (GOT) and glu-tamate pyruvate transaminase (GPT) were determined using an Olympus AU400Chemistry Analyzer (Japan)

24 RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from the liver tissue using an RNeasy Lipid Tissue MiniKit (Qiagen Germany) according to the manufacturerrsquos protocol cDNA was synthe-sized from 1 μg of RNA using an oligo-dT and Superscript II reverse transcriptase(Invitrogen USA) The sequences of the designed primers are shown in Table 1 Thereal-time PCR (Step One Plus Applied Biosystems USA) conditions were as follows15 min at 95 degC followed by 40 cycles at 94 degC for 30 s 52ndash60 degC for 20 s and 72 degCfor 30 s The cycle at which the ΔRn value crossed the threshold was defined as theCT (ΔΔCT) The relative quantity of target mRNA in each sample by comparing thenormalized quantity of target mRNA in each sample with the normalized quantityof target mRNA in the control sample was determined by the software The real-time PCR data were compiled from and are representative of at least three independentexperiments GAPDH was used as the control in the comparative cycle threshold (Ct)method

25 Immunoblot analysis

Liver tissues were lysed with RIPA lysis buffer (Amresco Solon OH USA) con-taining a protease and phosphatase inhibitor cocktail (Sigma-Aldrich St Louis MOUSA) Tissue lysates were centrifuged at 14000 times g for 15 min at 4 degC Proteins werequantified using the BCA protein assay (Sigma-Aldrich) Protein lysates were re-solved on 10 sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and thentransferred to a polyvinylidene difluoride (PVDF) membrane Anti-phospho-AMP-activated protein kinase (AMPK) (Cell Signaling Danvers MA USA) anti-AMPK (CellSignaling) and anti-β-actin (Santa Cruz Biotechnology Santa Cruz CA USA) anti-bodies were used to detect p-AMPK AMPK and β-actin respectively β-actin wasused as protein loading control Blots were analyzed using a western blot detec-tion kit (AbFrontier Seoul Korea) The results are representative of at least threeindependent experiments Protein bands were quantified using the Alphaviewreg soft-ware (Alpha Innotech San Leandro CA USA)

26 Statistical analysis

Statistical analysis was performed using the SPSS software (version 1201SPSS Inc Chicago IL USA) The results are presented as means plusmn SE The differ-ences between the experimental groups were analyzed using one-way ANOVAfollowed by Bonferronirsquos post hoc test P values of lt 005 were considered statisti-cally significant

3 Results

31 The effect of DNJ diet supplementation on body weightfood intake liver weight and body fat

Significant differences in cumulative body weight gains were foundbetween the mouse groups over the 12-week study period (Table 2)The HF group gained more weight than the CTL and DNJ groups didThe HF and DNJ groups consumed remarkably less feed than the CTLgroup did (Table 2) Food efficiency ratios were significantly higher in

Table 1Mouse-specific primer sequences

Gene Primer sequence

CEBPα Forward 5prime-GTTGACCAGTGACAATGACC-3primeReverse 5prime-AATCTCCTAGTCCTGGCTTG-3prime

ACC Forward 5prime-GCCTCTTCCTGACAAACGAG-3primeReverse 5prime-TAAGGACTGTGCCTGGAACC-3prime

FAS Forward 5prime-CTGAAGAGCCTGGAAGATCG-3primeReverse 5prime-GTCACACACCTGGGAGAGGT-3prime

SCD-1 Forward 5prime- CTTCAAGGGCAGTTCTGAG-3primeReverse 5prime-CAATGGTTTTCATGGCAGTG-3prime

CD36 Forward 5prime-TTTCTGTAGACCCAATTATCCTG-3primeReverse 5prime-TTATACTTATGCCATGTGCTGAC-3prime

PGC-1α Forward 5prime-CGGAAATCATATCCAACCAG-3primeReverse 5prime-TGAGGACCGCTAGCAAGTTTG-3prime

PGC-1β Forward 5prime-AACCCAACCAGTCTCACAGG-3primeReverse 5prime-ATGCTGTCCTTGTGGGTAGG-3prime

GAPDH Forward 5prime-AACTTTGGCATTGTGGAAGG-3primeReverse 5prime-ACACATTGGGGGTAGGAACA-3prime

CEBPα ccaat-enhancer-binding protein ACC acetyl-CoA carboxylase FAS fatty acidsynthase SCD-1 stearoyl CoA desaturase 1 CD36 cluster of differentiation 36 PGC-1α peroxisome proliferator-activated receptor γ coactivator-1α PGC-1β peroxisomeproliferator-activated receptor γ coactivator-1 β GAPDH glyceraldehyde 3-phosphatedehydrogenase

Table 2Cumulative body weight gain food intake and food efficiency ratio among theexperimental groups

Groups CTL HF DNJ p

Cumulative BWgain (g)

1315 plusmn 109a 2021 plusmn 116b 1669 plusmn 131c lt005

Food intake (gday) 293 plusmn 004a 274 plusmn 005b 263 plusmn 004b lt005Food efficiency

ratio (FER )005 plusmn 0004a 009 plusmn 0005b 008 plusmn 0006b lt005

Means plusmn SE Tested by analysis of variance (ANOVA) with Bonferronirsquos post hoc testValues sharing the same letters indicate no significant difference between groupsCTL control diet HF high fat diet DNJ high fat diet with 1-deoxinojirimycin





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References





4 Discussion




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Acknowledgements

References



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Introduction






Immunoblot analysis


Results





Discussion



Acknowledgements

References









Acknowledgements


References











aa

b

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03

06

09

12

15

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CTL HF DNJ

Rel

ativ

e le

vels

of

p-A

MP

Ka

AM

PKa

prot

ein

expr

essi

on

p-AMPKa

AMPKa

b-actin

CTL HF DNJ









































Introduction






Immunoblot analysis


Results





Discussion



Acknowledgements

References







































Introduction






Immunoblot analysis


Results





Discussion



Acknowledgements

References

1-Deoxynojirimycin isolated from Bacillus subtilis improves hepatic lipid metabolism and...

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