research article effect of lactobacillus plantarum strain k21 on...

Research ArticleEffect of Lactobacillus plantarum Strain K21 on High-FatDiet-Fed Obese Mice

Chien-Chen Wu12 Wei-Lien Weng1 Wen-Lin Lai34 Hui-Ping Tsai1

Wei-Hsien Liu1 Meng-Hwan Lee5 and Ying-Chieh Tsai12

1 Institute of Biochemistry and Molecular Biology National Yang-Ming University Taipei 11221 Taiwan2Probiotics Research Center National Yang-Ming University Taipei 11221 Taiwan3School of Medical Laboratory and Biotechnology Chung Shan Medical University Taichung 40201 Taiwan4Clinical Laboratory Chung Shan Medical University Hospital Taichung 40201 Taiwan5Division of Animal Technology Animal Technology Laboratories Agricultural Technology Research Institute Zhunan TownshipMiaoli County 35053 Taiwan

Correspondence should be addressed to Ying-Chieh Tsai tsaiycymedutw

Received 23 September 2014 Revised 14 January 2015 Accepted 15 January 2015

Academic Editor Jian-Guo Chen

Copyright copy 2015 Chien-Chen Wu et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Recent studies have demonstrated beneficial effects of specific probiotics on alleviating obesity-related disorders Here we aimedto identify probiotics with potential antiobesity activity among 88 lactic acid bacterial strains via in vitro screening assays anda Lactobacillus plantarum strain K21 was found to harbor abilities required for hydrolyzing bile salt reducing cholesterol andinhibiting the accumulation of lipid in 3T3-L1 preadipocytes Furthermore effects of K21 on diet-induced obese (DIO) micewere examined Male C57Bl6J mice received a normal diet high-fat diet (HFD) or HFD with K21 administration (109 CFU in02mLPBSday) for eightweeks Supplementation ofK21 but not placebo appeared to alleviate bodyweight gain and epididymal fatmass accumulation reduce plasma leptin levels decrease cholesterol and triglyceride levels andmitigate liver damage inDIOmiceMoreover the hepatic expression of peroxisome proliferator-activated receptor-120574 (PPAR-120574) related to adipogenesis was significantlydownregulated in DIO mice by K21 intervention We also found that K21 supplementation strengthens intestinal permeability andmodulates the amount of Lactobacillus spp Bifidobacterium spp and Clostridium perfringens in the cecal contents of DIO miceIn conclusion our results suggest that dietary intake of K21 protects against the onset of HFD-induced obesity through multiplemechanisms of action

1 Introduction

Metabolic syndrome is a common metabolic disorder thatresults from the increasing prevalence of obesity [1] It com-bines disturbances in glucose and insulin metabolism excessweight mild dyslipidemia a proinflammatory state hyper-tension subsequent development of type 2 diabetes (T2D)nonalcoholic fatty liver disease (NAFLD) and cardiovasculardisease [1ndash3] Accumulating evidence indicates that the gutmicrobiota is associated with the development of obesity andrelated metabolic disorders [4ndash6] Moreover colonization ofgerm-free mice with gut microbes from obese mice resultsin a greater weight gain and body fat accumulation thancolonization with that from lean mice [7 8] indicating theimportance of gut microbiota on host metabolism

The modulation of gut microbiota affects host metab-olism and has an impact on energy homeostasis and ectopicfat deposition [9]The advent of probiotic treatments appearsto be a promising approach to reverse the dysbiosis-linkedhost metabolic alterations observed in obesity and relateddisorders [10] Probiotics are defined as living microor-ganisms that when administered in adequate amountsconfer health benefits to the host [11] Most microorganismsidentified to date as probiotics are Gram-positive bacteriaand belong to the genera Lactobacillus or Bifidobacteriumwhich have been used for centuries because of their benefitsto human health [12] Although the molecular mechanismof probiotic action is not fully elucidated many effects mayprove beneficial in obesity and in related disorders these

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 391767 9 pageshttpdxdoiorg1011552015391767

2 Evidence-Based Complementary and Alternative Medicine

effects include the modulation of the intestinal microbiotamodification of the local microenvironment inside the gutenhancement of the epithelial barrier function and theregulation of the host immune response [13 14]

In this study we aimed to identify probiotic strains withpotential antiobesity activities by in vitro assays and a spe-cific Lactobacillus plantarum strain K21 was thus identifiedMoreover K21 was found to inhibit lipid accumulation in3T3-L1 preadipocytes To further investigate if K21 possessesantiobesity effects in vivo a diet-induced obese (DIO) mousemodel was usedMale C57Bl6J mice were fed with aWesterndiet to induce obesity and obesity-related disorders themicewere simultaneously supplemented with K21 to examine itseffects on the progression of obesity hyperlipidemia liverdamage and hyperglycemia

2 Materials and Methods

21 Bacterial Strain Media and Growth Conditions L plan-tarumK21was isolated from locally fermented vegetables andpreserved in our lactic acid bacterial bank K21 was staticallygrown in Man Rogosa Sharpe (MRS BD Difco FranklinLakes NJ) broth at 37∘C for 18ndash20 h For in vivo assaysthe K21 culture was harvested using centrifugation (1500timesg10min) washed twice with sterile PBS and resuspended to afinal concentration of approximately 1010 CFUmL

22 Bile Salt Hydrolyzing Ability The bacterial ability tohydrolyze bile salt was assessed by performing a plate assay[15] Bile salt plates were prepared using 05 (wv) tau-rodeoxycholic acid sodium salt (TDCA Sigma St LouisMO)and 0037 (wv) CaCl

2in MRS plate [15] Briefly bacterial

cultures grown overnight were respectively inoculated inMRS and bile salt platesThe inoculated plates were incubatedanaerobically at 37∘C for 72 h and the colonial morphologywas observed A bacterial strain with ability to hydrolyzeTDCA resulted in white precipitates on the bile salt platebut not on the MRS plate while strains without the abilityproduced similar colony types on MRS and bile salt platesThe result was reproduced in triplicate in three independentexperiments

23 Cholesterol-Lowering Ability The ability of bacterialcultures to assimilate cholesterol was determined using amodified method described by Danielson et al [16] Eachfreshly prepared strain culture was inoculated (1) in 1mLofMRS broth supplemented with 02 sodium thioglycollate(Sigma) 03 oxgall (BD Difco Franklin Lakes NJ) anda water-soluble form of cholesterol (cholesterol-methyl-120573-cyclodextrin Sigma) The final concentration of cholesterolin the broth was 60mgdL The tubes were anaerobicallyincubated at 37∘C for 24 h After incubation cells werecentrifuged for 10min at 12000timesg at 4∘C The amounts ofcholesterol in the spent broth and uninoculated sterile brothas a negative control were determined using an enzymaticmethod according to the manufacturerrsquos instructions (Ran-dox Crumlin UK)

24 Preadipocytes Differentiation Assay 3T3-L1 cell culturedifferentiation andOil Red stainingwere performed as previ-ously described [17] Briefly 3T3-L1 cells were cultured in 12-well plates (6 times 104well) in high-glucose DMEM containing10 FBS 100UmL penicillin and 01mgmL streptomycinAfter confluence (day 0) the cells were stimulated in adifferentiation medium (DM) containing 10 FBS 05mMIBMX 05 120583M dexamethasone and 10 120583gmL insulin in thepresence or absence of heat-killed K21 (107 CFUmL) After3 days of stimulation (day 3) the media were replaced by10 FBSDMEM containing 10 120583gmL insulin On day 9 theintracellular lipids of the 3T3-L1 cells were stained with OilRed-O and observed by a previously described method [17]

25 Animals Diets and Experimental Design In all 24male C57Bl6J mice (8 weeks old) were purchased from theNational Laboratory Animal Center (NLAC Taipei Taiwan)and housed in a controlled environment (22∘C 50minus60humidity and 12 h lightdark cycle) with free access to foodand water The mice were randomly selected and assignedto three groups (eight mice per group) according to thetype of diet and test-material Group 1 (normal diet ND)was fed with a standard chow diet (Autoclavable RodentDiet 5010 low fat diet 12 energy from fat LabDiet) andplacebo (02mL PBS via an orogastric tube daily) group 2(high-fat-diet HFD) with HFD (Western diet 5TJN 40 ofenergy from fat with 015 cholesterol TestDiet) and placeboand group 3 (HFD + K21) with HFD and L plantarum K21(approximately 109 CFU02mL via an orogastric tube daily)for eight weeks All the animal experimental proceduresfollowed the guidelines of Care and Use of LaboratoryAnimals and the study was approved by the Animal Careand Ethics Committee of the National Yang-Ming UniversityMice were sacrificed and the livers were rapidly excisedand fixed in 10 neutral-buffered formalin for paraffin-embedded sections and hematoxylin and eosin staining

26 Blood Chemistry Blood plasma was analyzed using theFUJI DRI-CHEM SYSTEM 3500s (Fuji Photo Film CO LtdTokyo Japan) formeasurement of glucose cholesterol NEFA(nonesterified fatty acids) triglycerides aspartate amino-transferase (AST) and alanine aminotransferase (ALT)Plasma leptin and adiponectin were determined using ELISAkits (Boster Wuhan China) plasma insulin was determinedusing an ELISA kit (Mercodia Uppsala Sweden) accordingto the manufacturerrsquos instructions

27 Quantitative Real-Time PCR (qRT-PCR) Total RNAfrom each mice liver was prepared using the RNeasy minikit (Qiagen Hilden Germany) and the cDNA was thensynthesized using an oligo (dT) 15 primer and SuperScriptII reverse transcriptase reagents (Invitrogen Carlsbad CA)Subsequently qRT-PCR was performed in a LightCyclerinstrument (Roche Basel Switzerland) using the DyNAmoCapillary SYBR Green qPCR kit (Finnzymes Espoo Fin-land) as per the manufacturerrsquos recommendations Theforward primer 51015840-TGC TGT TAT GG GTG AAA CTC TG-31015840 and the reverse primer 51015840-GAA ATC AAC TGT GGTAAA GGG C-31015840 were used to detect PPAR-120574 (GenBank

Evidence-Based Complementary and Alternative Medicine 3

number NM 011146) whereas the forward primer 51015840-GTATGA CTC CAC TCA CGG CAA A-31015840 and the reverseprimer 51015840-GGT CTC GCT CCT GGA AGA TG-31015840 were usedto detect GAPDH (GenBank number NM 008084) Thethermal cycling conditions were 6min at 95∘C followed by50 cycles of denaturation at 95∘C for 10 s annealing at 57∘Cfor 10 s and extension at 72∘C for 8 s The expression levels ofthe mRNAs of each sample were normalized with GAPDHserving as an internal control The results were expressed asrelative expression ratios with respect to the control group

28 Intestinal Permeability In Vivo This measure is basedon the intestinal permeability towards the 4 kDa fluorescentdextranndashFITC (DX-4000ndashFITC) (Sigma) as described [18]Briefly mice that had fasted for 12 h were given DX-4000ndashFITC by gavage (500mgkg body weight 50mgmL) After1 h 120120583L of blood was collected from the tip of the tail veinThe blood was centrifuged at 12 000timesg for 3min at 4∘C Theplasma was diluted in an equal volume of PBS and analyzedfor the DX-4000ndashFITC concentration with a fluorescencespectrophotometer (Tecan Mannedorf Switzerland) usingan excitation wavelength of 485 nm and emission wave-length of 535 nm Standard curves were obtained by dilutingthe FITCndashdextran in nontreated plasma diluted with PBS(1 3 vv)

29 Analysis of Cecal Microflora After the mice were sacri-ficed the cecum of each animal was removed and 01 g ofthe cecal content was weighed transferred into a tube with09mL of anaerobic diluent and homogenized by vortexingThe homogenates (diluted with appropriate dilution factors)were plated in selective culture medium in triplicate MRSagarwas used for Lactobacillus spp BIM-25 agarwas used forBifidobacterium spp and egg yolk-free TSC (tryptose-sulfite-cycloserine) agar was used for Clostridium perfringens [19]Subsequently the plates were anaerobically incubated at 37∘Cfor 48 h for CFU counting

210 Statistical Analysis The statistical significance wasdetermined using Prism5 (GraphPad San Diego CA) usingone-way ANOVA followed by the Bonferroni post hoccorrection A 119875 value of lt005 was considered significantin all cases All the assays were reproduced with at leastthree independent experiments Each sample was assayed intriplicate and the mean activity and the standard deviation(SD) were determined

3 Results

31 In Vitro Screening for Lactic Acid Bacteria with PotentialAntiobesity Activity To identify potential probiotic strainswith antiobesity activity we performed qualitative assaysto screen for isolates with abilities to hydrolyze bile andcholesterol both of which have been correlated with antiobe-sity effects [20] A total number of 88 lactic acid bacterialstrains collected in our laboratory were screened Amongthese strains 50 and 27 strains were respectively foundto harbor abilities required for hydrolyzing the conjugated

bile acid TDCA on the MRS agar plate and reducingcholesterol (gt50 reduction) in the MRS broth A specificL plantarum strain K21 with abilities to hydrolyze TDCA(Figure 1(a)) and cause sim65 reduction of cholesterol inthe MRS broth was thus selected for further analyses Wealso investigated the effects of K21 on 3T3-L1 preadipocytedifferentiation The result showed that 3T3-L1 preadipocytesstimulated with differentiation medium (DM) resulted in anincreased intracellular lipid accumulation as assessed by OilRed-O staining which could be apparently inhibited by thetreatment of K21 (Figure 1(b))

32 L plantarum K21 Inhibits Body Weight Gain and FatWeight Accumulation in DIO Mice To investigate if K21possesses antiobesity activities in vivo mice were fed withnormal diet (ND group) high-fat diet (HFD group) or HFDsupplemented with K21 (HFD + K21) (109 CFU in 02mLPBSmouseday) for eight weeks the ND and HFD groupswere fed with placebo (02mL PBSmouseday) and theirbody weight was monitored After 8-week feeding the HFDgroup showed significantly increased body weight (119875 lt 001)compared with that of the ND group (Table 1) K21 supple-mentation ameliorated the increased bodyweight ofHFD-fedmice although the difference was not statistically significant(Table 1) As shown in Figure 2(a) the body weight gain ofthe HFD group was also increased (119875 lt 0001) compared tothe ND group and the K21 supplementation attenuated thisincrease (119875 lt 001) however the body weight gain of theHFD + K21 group is still higher (119875 lt 001) than that of theND group These results revealed that the supplementationof K21 moderately attenuated the increased body weightof HFD-fed mice On the other hand the epididymal fatmass was significantly higher in the HFD and HFD + K21groups than in the ND group (119875 lt 0001) (Figure 2(b)) Ascompared to HFD-fed mice the K21-treated mice showed asignificant reduction in the epididymal fat mass (119875 lt 005)In addition the food efficiency ratio (FER) total grams ofbody weight gained on a test food divided by the total gramsof food consumed during an animal feeding study of theHFD-fed mice was significantly increased compared withthat of the ND-fed mice (119875 lt 0001) suggesting greaterefficiency of HFD on weight gain (Table 1) On the contrarythe supplementation of K21 reduced the FER of HFD-fedmice (119875 lt 001) reflecting lower weight gain per grams offood consumed Although the amount of food intake wassignificantly reduced in the HFD and HFD + K21 groupscompared to the ND group the supplementation of K21 didnot influence the amount of food intake in HFD-fed mice(Table 1)

33 L plantarum K21 Attenuates Dyslipidemia in DIO MiceAs shown in Table 1 the plasma cholesterol and triglyceridelevels in the HFD group showed a significant increase (119875 lt0001) suggesting the onset of dyslipidemia by HFD feedingK21 supplementation was found to significantly mitigate thecholesterol and triglyceride levels (119875 lt 005 and 119875 lt 0001resp)The serumNEFA levels were not significantly differentamong the three groups In addition HFD significantly


NC +05 TDCA

(a)

Non DM DM + K21

(b)

Figure 1 Qualitative assessment of L plantarum K21 for hydrolyzing bile salt and inhibiting lipid accumulation in 3T3-L1 preadipocytes(a) Demonstration of the ability of K21 to hydrolyze bile salt The K21 strain was inoculated on an MRS agar plate (NC negative control) oran MRS agar plate containing 05 TDCA as indicated The plates were incubated at 37∘C under anaerobic conditions for three days andthe colonial morphology was observed In the assays shown precipitation in the agar is indicative of bacterial ability for hydrolyzing TDCA(b)The inhibitory effects of K21 on lipid accumulation in 3T3-L1 preadipocytes 3T3-L1 cells were differentiated with differentiation medium(DM) in the absence or presence of heat-killed K21 (sim107 CFUmL) as described in Materials and Methods Intracellular lipids were stainedwith Oil Red-O Non undifferentiated cells The results were confirmed with three independent experiments

Table 1 Effects of experimental diets and K21 administration on mice

Parameters assessed ND HFD HFD + K21Initial body weight (g) 2256 plusmn 146 2251 plusmn 130 2261 plusmn 179Final body weight (g) 2400 plusmn 12 2666 plusmn 174lowastlowast 2480 plusmn 18Food intake (gmouseday) 272 plusmn 004 218 plusmn 012lowastlowastlowast 197 plusmn 025lowastlowastlowast

Food efficiency ratio () 104 plusmn 026 413 plusmn 055lowastlowastlowast 281 plusmn 063lowastlowastlowast

Cholesterol (mgdL) 787 plusmn 97 1702 plusmn 165lowastlowastlowast 1487 plusmn 154lowastlowastlowast

Triglyceride (mgdL) 738 plusmn 83 1045 plusmn 53lowastlowastlowast 706 plusmn 88

NEFA (mmolL) 151 plusmn 025 139 plusmn 027 120 plusmn 042Leptin (ngmL) 6388 plusmn 1798 36708 plusmn 12727lowastlowastlowast 16164 plusmn 3937lowastlowastlowast

Adiponectin (ngmL) 265 plusmn 24 226 plusmn 24lowastlowast 243 plusmn 27Glucose (mgmL) 1048 plusmn 199 1892 plusmn 169lowastlowastlowast 1750 plusmn 331lowastlowastlowast

Insulin (mgmL) 1407 plusmn 937 14944 plusmn 2835lowastlowastlowast 9887 plusmn 4799lowast

Liver weight (g) 100 plusmn 005 095 plusmn 006 099 plusmn 003AST (UL) 666 plusmn 189 2855 plusmn 321lowastlowastlowast 744 plusmn 186

ALT (UL) 325 plusmn 50 618 plusmn 227lowastlowast 392 plusmn 146

Hepatic cholesterol (mgdL) 442 plusmn 43 1082 plusmn 262lowastlowastlowast 813 plusmn 205lowastlowastlowast

Hepatic triglyceride (mgdL) 1612 plusmn 306 2441 plusmn 478lowastlowastlowast 1340 plusmn 268

C57Bl6J mice were fed a normal diet (ND) a high-fat diet (HFD) or a HFD + L plantarum K21 (109 CFU per day) for eight weeks Data represent the meansplusmn SD of eight mice per group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus the ND group 119875 lt 005 119875 lt 001 and

119875 lt 0001 versus the HFD group


ND HFD

Body

wei

ght g

ain

(g)

0

1

2

3

4

5

6

HFD + K21

lowastlowastlowast

lowastlowast

(a)

ND HFD

Epid

idym

al fa

t pad

(g)

00

02

04

06

08

10

12

HFD + K21

lowastlowastlowast

lowastlowastlowast

(b)

Figure 2 L plantarum K21 supplementation decreases HFD-induced body weight gain and fat mass accumulation (a) Change in bodyweight gain (b) weight of epididymal fat pad Values are means with SD lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the ND group 119875 lt 001 versusthe HFD group

increased the leptin level (119875 lt 0001) which was amelioratedby the K21 supplementation (119875 lt 0001) although the leptinlevel of the HFD + K21 group was still higher than that ofthe ND group (119875 lt 0001) Compared with the ND groupthe HFD group showed a decreased level of adiponectin (119875 lt001) while no statistically significant difference was foundbetween the HFD + K21 group and the other two groupsOn the other hand the fasting plasma glucose and insulinlevels were elevated more in the HFD group than in the NDgroup (119875 lt 0001) however the K21 supplementation did notsuppress these effects (Table 1)

34 L plantarum K21 Attenuates Liver Damage and RegulatesHepatic PPAR-120574 Expression in DIO Mice As shown in Fig-ure 3(a) the liver histology of the HFD group (unlike the NDgroup) showed clear evidence of micro- and macrovesicularsteatosis resulting from the accumulation of fat dropletswhich could be ameliorated by the K21 supplementation Tofurther characterize steatosis in mice the plasma levels ofaspartate AST and ALT (markers of hepatic dysfunction)were measured As shown in Table 1 the AST and ALTlevels were significantly higher (119875 lt 0001 and 119875 lt 001resp) in the HFD group than in the ND group and K21supplementation could mitigate the elevated levels of ASTand ALT (119875 lt 0001 and 119875 lt 005 resp) induced byHFD feeding (Table 1) In addition hepatic cholesterol andtriglyceride were obviously increased (119875 lt 0001) in the HFDgroup which could be ameliorated by the supplementationof K21 (Table 1) We also measured the hepatic mRNAlevels of PPAR-120574 which is an important regulator of lipidhomeostasisThe expression of PPAR-120574 is generally increasedin steatotic livers [21] As shown in Figure 3(b) the mRNAlevels of PPAR-120574 were found to significantly increase in theHFD group (119875 lt 0001) than in the ND group and theadministration of K21 significantly normalized this effect(119875 lt 0001)

35 L plantarum K21 Attenuates Increased Gut Permeabilityin DIO Mice Diet-induced obesity has been correlated toincreased intestinal permeability [22 23] and specific pro-biotics with beneficial effects on the gut barrier functionshave been reported [24] In order to study the effect ofK21 supplementation on the gut barrier functions in DIOmice the intestinal permeability was measured using FITC-dextran The serum levels of FITC-dextran were higher inthe HFD group compared to the ND group (119875 lt 0001)(Figure 4) thereby indicating an increased intestinal perme-ability in DIO mice K21 supplementation clearly attenuatedthis increase (119875 lt 0001)

36 L plantarum K21 Modulates Members of the CecalMicrobiota Specific probiotics are known to modulate thegut microbiota composition by increasing beneficial anddecreasing harmful bacteria to promote human health Todetermine whether HFD-feeding and K21 supplementationmodulate members of the gut microbiota in mice Lacto-bacillus spp and Bifidobacterium spp which are generallyregarded as beneficial bacteria and a potential gut pathogenC perfringens were measured from cecal contents by platecounting method As shown in Figure 5 Lactobacillus sppwas decreased in the HFD group than in the ND group(119875 lt 001) and K21 supplementation attenuated this effect(119875 lt 005) Unlike the HFD- and ND-fed mice the K21-supplemented mice showed a significant increase in the cecalamount of Bifidobacterium spp (119875 lt 001) The levels of Cperfringens were increased with HFD-feeding (119875 lt 001)but this effect could be attenuated by K21 supplementation(119875 lt 005)

4 Discussion

Clinical and experimental studies suggest that probioticsdiffer greatly in their efficacy and mechanisms of action [10]In an effort to treat obesity and obesity-related disorders


ND HFD HFD + K21

(a)

ND HFD

Relat

ive a

mou

nt o

f mRN

A

0

1

2

3

4

5

6

7

HFD + K21

lowastlowastlowast

(b)

Figure 3 L plantarum K21 attenuates liver damages and hepatic mRNA expression of PPAR-120574 in DIO mice (a) Effect of K21 on hepatichistology of mice fed with HFD for eight weeks The microphotographs of liver tissue sections were analyzed with HampE staining at 200x (b)qRT-PCR analysis of the hepatic mRNA expression of PPAR-120574 The mRNA expression of GAPDH was used as an internal control for datanormalization Values are means with SD lowastlowastlowast119875 lt 0001 versus the ND group 119875 lt 0001 versus the HFD group

ND HFD000

005

010

015

020

025

Seru

m F

ITC-

dext

ran

(120583g

mL)

lowastlowastlowast

HFD + K21

Figure 4 L plantarum K21 attenuates the increased intestinalpermeability in DIO mice Serum levels of 4-kDa FITC-dextranwere determined one hour after oral gavage in mice at eight weeksas described in Materials and Methods Values are means with SDlowastlowastlowast119875 lt 0001 versus the ND group 119875 lt 0001 versus the HFD

group

numerous probiotic strains have been evaluated for theirability to modulate gut microbiota [25] lower serum choles-terol and triglycerides [26] decrease fat storage [27] reduceadipocyte size [28 29] improve insulin resistance decreaseglucose intolerance [30] and ameliorate liver dysfunctionin chronic liver disease [24 31] It is well-known thatmost effects of probiotics are strain-specific and cannot beextended to other probiotics of the same genus or speciesHowever a meta-analysis study has indicated that the effectof Lactobacillus spp on weight is species dependent [32]The administration of Lactobacillus acidophilus Lactobacillusfermentum and Lactobacillus ingluviei is associated withweight gain whereas the administration of L plantarum andLactobacillus gasseri is associated with weight loss in obesehumans and animals [32] In our study supplementationwithK21 belonging to L plantarum inhibited weight gain in DIOmice which also supported the finding of the meta-analysismentioned above Moreover compared to other studies onsimilar probiotic strains K21 appeared to confer beneficialeffects on DIO mice through multiple mechanisms of action


Lb Bf Cp0

2

4

6

8

10

NDHFD

CFU

mL

(log 10

)

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HFD + K21

Figure 5HFD and probioticmodulatemembers of the cecalmicro-biota inmiceThe amount ofLactobacillus spp (Lb)Bifidobacteriumspp (Bf) and C perfringens (Cp) in the ceca of mice at eight weekswere determined as described in Materials and Methods Values aremeans with SD lowast119875 lt 005 and lowastlowast119875 lt 001 between the indicatedgroups

BSH activity has been mostly observed in Gram-positivecommensals but generally not in the Gram-negative com-mensals of the gastrointestinal tract [20] The presence ofBSH in probiotics helps reduce the blood cholesterol level inthe host which also renders them more tolerant to bile salts[20] As shown in Figure 1(a) K21 appeared to harbor an invitro BSH activity and inhibited the blood cholesterol levelin DIO mice (Table 1) We also found that K21 possessed ahigher tolerance against 05 oxgall than did the two well-studied probiotic strains namely L casei Shirota and Lrhamnosus GG (data not shown) BSHs are very specific tocertain bile types and their duration of contact with bileensures bacterial survival in varying bile environments [20]In the genome of L plantarum WCFS1 four bsh genes wereidentified and these may confer additional advantages to thebacterial persistence in the gut [33] Whether K21 harborsmultiple genes encoding BSHs remains to be studied

Our results indicate that K21 supplementation amelio-rates metabolic alterations in DIO mice including suchparameters as obesity liver damages and glucose intoleranceWe also found that an increased expression of hepatic PPAR-120574 mRNA was reversed in response to K21 intervention inDIO mice (Figure 3(b)) PPAR-120574 is an important regulatorof lipid homeostasis [34] and it expresses most abundantlyin the adipose tissue where it plays a role in increasinginsulin sensitivity [35] Increased PPAR-120574 mRNA levels areusually observed in steatotic livers [21]Thus K21may inducemetabolic alterations in the livers of DIO mice by regulatingthe PPAR-120574 expression Previous reports have also describedan elevated expression of PPAR-120574 in the livers of DIO mice[25 36] Probiotic treatment attenuates liver steatosis inDIO mice but causes a superinduction of hepatic PPAR-120574

activities [25 36] Elevated PPAR120574 levels may decrease theproduction of proinflammatory cytokines in order to reducehepatic inflammation [37] These findings suggested thatprobiotics exert differential mechanisms of action althoughthey produce similar effects

Probiotics may provide a way to alter the gut microbiotanaturally which may partly explain the modulation of hostmetabolism in response to probiotic interventions [9] Asshown in Figure 5 the supplementation of K21 increasedthe amount of cecal Lactobacillus spp and Bifidobacteriumspp but decreased the amount of C perfringens suggestingthat K21 modulates the gut microbiota in DIO mice whichmay result into antiobesity effects Consistent with ourfinding supplementation of Lactobacillus curvatus HY7601and L plantarum KY1032 increased the endogenous Bifi-dobacterium pseudolongum in DIOmice which appears to becorrelated with the suppression of body weight gain or bodyfat reduction [25] However it has also been described that LacidophilusNCDC 13 supplementation significantly increasesthe amount of Bifidobacterium spp in both fecal and cecalcontents with no detectable effect on obesity parameters inDIOmice [38]Thus the correlation between themodulationof gut microbiota by probiotic interventions and its influenceon host metabolism warrants further investigations

In conclusion the probiotic L plantarum K21 whichharbors bile salt hydrolyzing and cholesterol-lowing abilitiesinhibits the differentiation of preadipocytes In DIO micesupplementing K21 inhibits body weight gain and fat massaccumulation decreases the level of leptin reduces liverdamage and inhibits the hepatic expression of PPAR-120574 Fur-thermore K21 also strengthens intestinal barrier functionsand modulates the gut microbiota These results providean opportunity to develop foods and identify other LABstrains with potential antiobesity activity through in vitroscreening assays Furthermore our findings show that Lplantarum strain K21 helps alleviate obesity and obesity-related metabolic syndrome in a mouse model and clinicaltrials to confirm these effects in humans should hence beconducted in the near future

Conflict of Interests

Theauthors report no conflict of interests related to this study

Acknowledgments

This work was supported by the Asian Probiotics and Pre-biotics Ltd and Be Frankly Be Well (FBW) Bio-MedicineServices Ltd The funders had no role in study design datacollection and analysis decision to publish or preparation ofthe paper

References

[1] R H Eckel S M Grundy and P Z Zimmet ldquoThe metabolicsyndromerdquoThe Lancet vol 365 no 9468 pp 1415ndash1428 2005

[2] G Marchesini M Brizi A M Morselli-Labate et al ldquoAssocia-tion of nonalcoholic fatty liver disease with insulin resistancerdquo


The American Journal of Medicine vol 107 no 5 pp 450ndash4551999

[3] C E Ndumele K Nasir R D Conceicao J A M Carvalho RS Blumenthal and R D Santos ldquoHepatic steatosis obesity andthe metabolic syndrome are independently and additively asso-ciated with increased systemic inflammationrdquo ArteriosclerosisThrombosis and Vascular Biology vol 31 no 8 pp 1927ndash19322011

[4] A M Caricilli and M J A Saad ldquoGut microbiota compositionand its effects on obesity and insulin resistancerdquo CurrentOpinion in Clinical Nutrition and Metabolic Care vol 17 no 4pp 312ndash318 2014

[5] V Tremaroli and F Backhed ldquoFunctional interactions betweenthe gut microbiota and host metabolismrdquo Nature vol 489 no7415 pp 242ndash249 2012

[6] R E Ley ldquoObesity and the human microbiomerdquo CurrentOpinion in Gastroenterology vol 26 no 1 pp 5ndash11 2010

[7] P J Turnbaugh R E Ley M A Mahowald V MagriniE R Mardis and J I Gordon ldquoAn obesity-associated gutmicrobiomewith increased capacity for energy harvestrdquoNaturevol 444 no 7122 pp 1027ndash1031 2006

[8] P J Turnbaugh F Backhed L Fulton and J I Gordon ldquoDiet-induced obesity is linked to marked but reversible alterations inthe mouse distal gut microbiomerdquo Cell Host amp Microbe vol 3no 4 pp 213ndash223 2008

[9] P D Cani and N M Delzenne ldquoGut microflora as a target forenergy andmetabolic homeostasisrdquoCurrent Opinion in ClinicalNutrition and Metabolic Care vol 10 no 6 pp 729ndash734 2007

[10] T Arora S Singh and R K Sharma ldquoProbiotics interactionwith gut microbiome and antiobesity potentialrdquo Nutrition vol29 no 4 pp 591ndash596 2013

[11] FAOWHO ldquoHealth and nutritional properties of probioticsin food including powder milk with live lactic report of ajoint FAOWHO expert consultation on acid bacteria expertconsultation on evaluation of health and nutritional propertiesof probitics in food including powder milk with live lactic acidbacteriardquo Tech Rep 2001

[12] J Behnsen E Deriu M Sassone-Corsi and M RaffatelluldquoProbiotics properties examples and specific applicationsrdquoCold Spring Harbor Perspectives inMedicine vol 3 no 3 ArticleID a010074 2013

[13] A Iacono G M Raso R B Canani A Calignano and R MelildquoProbiotics as an emerging therapeutic strategy to treatNAFLDfocus on molecular and biochemical mechanismsrdquo Journal ofNutritional Biochemistry vol 22 no 8 pp 699ndash711 2011

[14] A M OrsquoHara and F Shanahan ldquoMechanisms of action ofprobiotics in intestinal diseasesrdquo The Scientific World Journalvol 7 pp 31ndash46 2007

[15] M P Dashkevicz and S D Feighner ldquoDevelopment of adifferential medium for bile salt hydrolase-active LactobacillusspprdquoApplied and Environmental Microbiology vol 55 no 1 pp11ndash16 1989

[16] A D Danielson E R Peo Jr K M Shahani A J Lewis PJ Whalen and M A Amer ldquoAnticholesteremic property ofLactobacillus acidophilus yogurt fed to mature boarsrdquo Journalof Animal Science vol 67 no 4 pp 966ndash974 1989

[17] Y Y Chen M H Lee C C Hsu C L Wei and Y C TsaildquoMethyl cinnamate inhibits adipocyte differentiation via activa-tion of the CaMKK2-AMPK pathway in 3T3-L1 preadipocytesrdquoJournal of Agricultural and Food Chemistry vol 60 no 4 pp955ndash963 2012

[18] P D Cani S Possemiers T van de Wiele et al ldquoChanges ingut microbiota control inflammation in obese mice througha mechanism involving GLP-2-driven improvement of gutpermeabilityrdquo Gut vol 58 no 8 pp 1091ndash1103 2009

[19] C-H Chiu T-Y Lu Y-Y Tseng and T-M Pan ldquoThe effects ofLactobacillus-fermented milk on lipid metabolism in hamstersfed on high-cholesterol dietrdquoApplied Microbiology and Biotech-nology vol 71 no 2 pp 238ndash245 2006

[20] A K Patel R R Singhania A Pandey and S B ChincholkarldquoProbiotic bile salt hydrolase current developments and per-spectivesrdquo Applied Biochemistry and Biotechnology vol 162 no1 pp 166ndash180 2010

[21] O Gavrilova M Haluzik K Matsusue et al ldquoLiver peroxisomeproliferator-activated receptor gamma contributes to hepaticsteatosis triglyceride clearance and regulation of body fatmassrdquo The Journal of Biological Chemistry vol 278 no 36 pp34268ndash34276 2003

[22] P D Cani R Bibiloni C Knauf et al ldquoChanges in gut micro-biota control metabolic endotoxemia-induced inflammation inhigh-fat diet-induced obesity and diabetes in micerdquo Diabetesvol 57 no 6 pp 1470ndash1481 2008

[23] P Brun I Castagliuolo V di Leo et al ldquoIncreased intestinalpermeability in obese mice new evidence in the pathogene-sis of nonalcoholic steatohepatitisrdquo The American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 292 no2 pp G518ndashG525 2007

[24] Y Ritze G Bardos A Claus et al ldquoLactobacillus rhamnosusGGProtects against non-alcoholic fatty liver disease in micerdquo PLoSONE vol 9 no 1 Article ID e80169 2014

[25] D Y Park Y T Ahn S H Park et al ldquoSupplementationof Lactobacillus curvatus HY7601 and Lactobacillus plantarumKY1032 in diet-induced obese mice is associated with gutmicrobial changes and reduction in obesityrdquo PLoS ONE vol 8no 3 Article ID e59470 2013

[26] YHuang andY Zheng ldquoThe probiotic Lactobacillus acidophilusreduces cholesterol absorption through the down-regulationof Niemann-Pick C1-like 1 in Caco-2 cellsrdquo British Journal ofNutrition vol 103 no 4 pp 473ndash478 2010

[27] L Aronsson Y Huang P Parini et al ldquoDecreased fat storageby Lactobacillus paracasei is associated with increased levels ofangiopoietin-like 4 protein (ANGPTL4)rdquo PLoS ONE vol 5 no9 Article ID e13087 2010

[28] N Takemura T Okubo and K Sonoyama ldquoLactobacillusplantarum strainNo 14 reduces adipocyte size inmice fed high-fat dietrdquo Experimental Biology and Medicine (Maywood) vol235 no 7 pp 849ndash856 2010

[29] J-H Kang S-I Yun M-H Park J-H Park S-Y Jeong andH-O Park ldquoAnti-obesity effect of Lactobacillus gasseri BNR17in high-sucrose diet-induced obesemicerdquo PLoS ONE vol 8 no1 Article ID e54617 2013

[30] H Y Huang M Korivi C-H Tsai J-H Yang and Y-C TsaildquoSupplementation of Lactobacillus plantarum K68 and fruit-vegetable ferment along with high fat-fructose diet attenuatesmetabolic syndrome in rats with insulin resistancerdquo Evidence-Based Complementary and Alternative Medicine vol 2013Article ID 943020 2013

[31] X Ma J Hua and Z Li ldquoProbiotics improve high fat diet-induced hepatic steatosis and insulin resistance by increasinghepatic NKT cellsrdquo Journal of Hepatology vol 49 no 5 pp 821ndash830 2008

[32] M Million E Angelakis M Paul F Armougom L Leiboviciand D Raoult ldquoComparative meta-analysis of the effect of


Lactobacillus species on weight gain in humans and animalsrdquoMicrobial Pathogenesis vol 53 no 2 pp 100ndash108 2012

[33] P A Bron D Molenaar W M de Vos and M KleerebezemldquoDNA micro-array-based identification of bile-responsivegenes in Lactobacillus plantarumrdquo Journal of Applied Microbi-ology vol 100 no 4 pp 728ndash738 2006

[34] E Moran-Salvador M Lopez-Parra V Garcıa-Alonso et alldquoRole for PPARgamma in obesity-induced hepatic steatosis asdetermined by hepatocyte- and macrophage-specific condi-tional knockoutsrdquo The FASEB Journal vol 25 no 8 pp 2538ndash2550 2011

[35] C Dreyer G Krey H Keller F Givel G Helftenbein and WWahli ldquoControl of the peroxisomal beta-oxidation pathway bya novel family of nuclear hormone receptorsrdquo Cell vol 68 no5 pp 879ndash887 1992

[36] SWagnerberger A Spruss G Kanuri et al ldquoLactobacillus caseiShirota protects from fructose-induced liver steatosis a mousemodelrdquo Journal of Nutritional Biochemistry vol 24 no 3 pp531ndash538 2013

[37] C Hofmann K Lorenz S S Braithwaite et al ldquoAlteredgene expression for tumor necrosis factor-120572 and its receptorsduring drug and dietary modulation of insulin resistancerdquoEndocrinology vol 134 no 1 pp 264ndash270 1994

[38] T Arora J Anastasovska G Gibson et al ldquoEffect of Lactobacil-lus acidophilusNCDC 13 supplementation on the progression ofobesity in diet-induced obesemicerdquo British Journal of Nutritionvol 108 no 8 pp 1382ndash1389 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


effects include the modulation of the intestinal microbiotamodification of the local microenvironment inside the gutenhancement of the epithelial barrier function and theregulation of the host immune response [13 14]

In this study we aimed to identify probiotic strains withpotential antiobesity activities by in vitro assays and a spe-cific Lactobacillus plantarum strain K21 was thus identifiedMoreover K21 was found to inhibit lipid accumulation in3T3-L1 preadipocytes To further investigate if K21 possessesantiobesity effects in vivo a diet-induced obese (DIO) mousemodel was usedMale C57Bl6J mice were fed with aWesterndiet to induce obesity and obesity-related disorders themicewere simultaneously supplemented with K21 to examine itseffects on the progression of obesity hyperlipidemia liverdamage and hyperglycemia

2 Materials and Methods

21 Bacterial Strain Media and Growth Conditions L plan-tarumK21was isolated from locally fermented vegetables andpreserved in our lactic acid bacterial bank K21 was staticallygrown in Man Rogosa Sharpe (MRS BD Difco FranklinLakes NJ) broth at 37∘C for 18ndash20 h For in vivo assaysthe K21 culture was harvested using centrifugation (1500timesg10min) washed twice with sterile PBS and resuspended to afinal concentration of approximately 1010 CFUmL

22 Bile Salt Hydrolyzing Ability The bacterial ability tohydrolyze bile salt was assessed by performing a plate assay[15] Bile salt plates were prepared using 05 (wv) tau-rodeoxycholic acid sodium salt (TDCA Sigma St LouisMO)and 0037 (wv) CaCl

2in MRS plate [15] Briefly bacterial

cultures grown overnight were respectively inoculated inMRS and bile salt platesThe inoculated plates were incubatedanaerobically at 37∘C for 72 h and the colonial morphologywas observed A bacterial strain with ability to hydrolyzeTDCA resulted in white precipitates on the bile salt platebut not on the MRS plate while strains without the abilityproduced similar colony types on MRS and bile salt platesThe result was reproduced in triplicate in three independentexperiments

23 Cholesterol-Lowering Ability The ability of bacterialcultures to assimilate cholesterol was determined using amodified method described by Danielson et al [16] Eachfreshly prepared strain culture was inoculated (1) in 1mLofMRS broth supplemented with 02 sodium thioglycollate(Sigma) 03 oxgall (BD Difco Franklin Lakes NJ) anda water-soluble form of cholesterol (cholesterol-methyl-120573-cyclodextrin Sigma) The final concentration of cholesterolin the broth was 60mgdL The tubes were anaerobicallyincubated at 37∘C for 24 h After incubation cells werecentrifuged for 10min at 12000timesg at 4∘C The amounts ofcholesterol in the spent broth and uninoculated sterile brothas a negative control were determined using an enzymaticmethod according to the manufacturerrsquos instructions (Ran-dox Crumlin UK)

24 Preadipocytes Differentiation Assay 3T3-L1 cell culturedifferentiation andOil Red stainingwere performed as previ-ously described [17] Briefly 3T3-L1 cells were cultured in 12-well plates (6 times 104well) in high-glucose DMEM containing10 FBS 100UmL penicillin and 01mgmL streptomycinAfter confluence (day 0) the cells were stimulated in adifferentiation medium (DM) containing 10 FBS 05mMIBMX 05 120583M dexamethasone and 10 120583gmL insulin in thepresence or absence of heat-killed K21 (107 CFUmL) After3 days of stimulation (day 3) the media were replaced by10 FBSDMEM containing 10 120583gmL insulin On day 9 theintracellular lipids of the 3T3-L1 cells were stained with OilRed-O and observed by a previously described method [17]

25 Animals Diets and Experimental Design In all 24male C57Bl6J mice (8 weeks old) were purchased from theNational Laboratory Animal Center (NLAC Taipei Taiwan)and housed in a controlled environment (22∘C 50minus60humidity and 12 h lightdark cycle) with free access to foodand water The mice were randomly selected and assignedto three groups (eight mice per group) according to thetype of diet and test-material Group 1 (normal diet ND)was fed with a standard chow diet (Autoclavable RodentDiet 5010 low fat diet 12 energy from fat LabDiet) andplacebo (02mL PBS via an orogastric tube daily) group 2(high-fat-diet HFD) with HFD (Western diet 5TJN 40 ofenergy from fat with 015 cholesterol TestDiet) and placeboand group 3 (HFD + K21) with HFD and L plantarum K21(approximately 109 CFU02mL via an orogastric tube daily)for eight weeks All the animal experimental proceduresfollowed the guidelines of Care and Use of LaboratoryAnimals and the study was approved by the Animal Careand Ethics Committee of the National Yang-Ming UniversityMice were sacrificed and the livers were rapidly excisedand fixed in 10 neutral-buffered formalin for paraffin-embedded sections and hematoxylin and eosin staining

26 Blood Chemistry Blood plasma was analyzed using theFUJI DRI-CHEM SYSTEM 3500s (Fuji Photo Film CO LtdTokyo Japan) formeasurement of glucose cholesterol NEFA(nonesterified fatty acids) triglycerides aspartate amino-transferase (AST) and alanine aminotransferase (ALT)Plasma leptin and adiponectin were determined using ELISAkits (Boster Wuhan China) plasma insulin was determinedusing an ELISA kit (Mercodia Uppsala Sweden) accordingto the manufacturerrsquos instructions

27 Quantitative Real-Time PCR (qRT-PCR) Total RNAfrom each mice liver was prepared using the RNeasy minikit (Qiagen Hilden Germany) and the cDNA was thensynthesized using an oligo (dT) 15 primer and SuperScriptII reverse transcriptase reagents (Invitrogen Carlsbad CA)Subsequently qRT-PCR was performed in a LightCyclerinstrument (Roche Basel Switzerland) using the DyNAmoCapillary SYBR Green qPCR kit (Finnzymes Espoo Fin-land) as per the manufacturerrsquos recommendations Theforward primer 51015840-TGC TGT TAT GG GTG AAA CTC TG-31015840 and the reverse primer 51015840-GAA ATC AAC TGT GGTAAA GGG C-31015840 were used to detect PPAR-120574 (GenBank






3 Results






NC +05 TDCA

(a)

Non DM DM + K21

(b)

















ND HFD

Body

wei

ght g

ain

(g)

0

1

2

3

4

5

6

HFD + K21

lowastlowastlowast

lowastlowast

(a)

ND HFD

Epid

idym

al fa

t pad

(g)

00

02

04

06

08

10

12

HFD + K21

lowastlowastlowast

lowastlowastlowast

(b)






4 Discussion



ND HFD HFD + K21

(a)

ND HFD

Relat

ive a

mou

nt o

f mRN

A

0

1

2

3

4

5

6

7

HFD + K21

lowastlowastlowast

(b)


ND HFD000

005

010

015

020

025

Seru

m F

ITC-

dext

ran

(120583g

mL)

lowastlowastlowast

HFD + K21


group



Lb Bf Cp0

2

4

6

8

10

NDHFD

CFU

mL

(log 10

)

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HFD + K21









Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results






NC +05 TDCA

(a)

Non DM DM + K21

(b)

















ND HFD

Body

wei

ght g

ain

(g)

0

1

2

3

4

5

6

HFD + K21

lowastlowastlowast

lowastlowast

(a)

ND HFD

Epid

idym

al fa

t pad

(g)

00

02

04

06

08

10

12

HFD + K21

lowastlowastlowast

lowastlowastlowast

(b)






4 Discussion



ND HFD HFD + K21

(a)

ND HFD

Relat

ive a

mou

nt o

f mRN

A

0

1

2

3

4

5

6

7

HFD + K21

lowastlowastlowast

(b)


ND HFD000

005

010

015

020

025

Seru

m F

ITC-

dext

ran

(120583g

mL)

lowastlowastlowast

HFD + K21


group



Lb Bf Cp0

2

4

6

8

10

NDHFD

CFU

mL

(log 10

)

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HFD + K21









Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC +05 TDCA

(a)

Non DM DM + K21

(b)

















ND HFD

Body

wei

ght g

ain

(g)

0

1

2

3

4

5

6

HFD + K21

lowastlowastlowast

lowastlowast

(a)

ND HFD

Epid

idym

al fa

t pad

(g)

00

02

04

06

08

10

12

HFD + K21

lowastlowastlowast

lowastlowastlowast

(b)






4 Discussion



ND HFD HFD + K21

(a)

ND HFD

Relat

ive a

mou

nt o

f mRN

A

0

1

2

3

4

5

6

7

HFD + K21

lowastlowastlowast

(b)


ND HFD000

005

010

015

020

025

Seru

m F

ITC-

dext

ran

(120583g

mL)

lowastlowastlowast

HFD + K21


group



Lb Bf Cp0

2

4

6

8

10

NDHFD

CFU

mL

(log 10

)

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HFD + K21









Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ND HFD

Body

wei

ght g

ain

(g)

0

1

2

3

4

5

6

HFD + K21

lowastlowastlowast

lowastlowast

(a)

ND HFD

Epid

idym

al fa

t pad

(g)

00

02

04

06

08

10

12

HFD + K21

lowastlowastlowast

lowastlowastlowast

(b)






4 Discussion



ND HFD HFD + K21

(a)

ND HFD

Relat

ive a

mou

nt o

f mRN

A

0

1

2

3

4

5

6

7

HFD + K21

lowastlowastlowast

(b)


ND HFD000

005

010

015

020

025

Seru

m F

ITC-

dext

ran

(120583g

mL)

lowastlowastlowast

HFD + K21


group



Lb Bf Cp0

2

4

6

8

10

NDHFD

CFU

mL

(log 10

)

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HFD + K21









Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ND HFD HFD + K21

(a)

ND HFD

Relat

ive a

mou

nt o

f mRN

A

0

1

2

3

4

5

6

7

HFD + K21

lowastlowastlowast

(b)


ND HFD000

005

010

015

020

025

Seru

m F

ITC-

dext

ran

(120583g

mL)

lowastlowastlowast

HFD + K21


group



Lb Bf Cp0

2

4

6

8

10

NDHFD

CFU

mL

(log 10

)

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HFD + K21









Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Lb Bf Cp0

2

4

6

8

10

NDHFD

CFU

mL

(log 10

)

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HFD + K21









Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article effect of lactobacillus plantarum strain k21 on...

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