jurnal dk 2

629

Braz J Med Biol Res 39(5) 2006

Effect of Lactobacillus delbrueckii on atherosclerosisBrazilian Journal of Medical and Biological Research (2006) 39: 629-635ISSN 0100-879X

Effect of Lactobacillus delbrueckii oncholesterol metabolism in germ-freemice and on atherogenesis inapolipoprotein E knock-out mice

1Departamento de Bioqumica e Imunologia,2Departamento de Patologia Geral, 3Departamento de Microbiogia,Instituto de Cincias Biolgicas, Universidade Federal de Minas Gerais,Belo Horizonte, MG, Brasil

L.R. Portugal1,J.L. Gonalves1,

L.R. Fernandes1,H.P.S. Silva1,

R.M.E. Arantes2,J.R. Nicoli3, L.Q. Vieira1

and J.I. Alvarez-Leite1

Abstract

Elevated blood cholesterol is an important risk factor associated withatherosclerosis and coronary heart disease. Several studies have re-ported a decrease in serum cholesterol during the consumption oflarge doses of fermented dairy products or lactobacillus strains. Theproposed mechanism for this effect is the removal or assimilation ofintestinal cholesterol by the bacteria, reducing cholesterol absorption.Although this effect was demonstrated in vitro, its relevance in vivo isstill controversial. Furthermore, few studies have investigated the roleof lactobacilli in atherogenesis. The aim of the present study was todetermine the effect of Lactobacillus delbrueckii on cholesterol me-tabolism in germ-free mice and the possible hypocholesterolemic andantiatherogenic action of these bacteria using atherosclerosis-proneapolipoprotein E (apo E) knock-out (KO) mice. For this purpose,Swiss/NIH germ-free mice were monoassociated with L. delbrueckiiand fed a hypercholesterolemic diet for four weeks. In addition, apo EKO mice were fed a normal chow diet and treated with L. delbrueckiifor 6 weeks. There was a reduction in cholesterol excretion in germ-free mice, which was not associated with changes in blood or livercholesterol concentration. In apo E KO mice, no effect of L. delbrueckiiwas detected in blood, liver or fecal cholesterol. The atheroscleroticlesion in the aorta was also similar in mice receiving or not thesebacteria. In conclusion, these results suggest that, although L.delbrueckii treatment was able to reduce cholesterol excretion ingerm-free mice, no hypocholesterolemic or antiatherogenic effect wasobserved in apo E KO mice.

CorrespondenceJ.I. Alvarez-Leite

Laboratrio de Bioqumica

Nutricional

Departamento de Bioqumica e

Imunologia, ICB, UFMG

Caixa Postal 486

30161-970 Belo Horizonte, MG

Brasil

Fax: +55-31-3499-2614

E-mail: [email protected]

Research supported by FAPEMIG

(Orc CBB2818/97). L.Q. Vieira,

J.R. Nicoli, J.L. Gonalves, L.R.Fernandes, and J.I. Alvarez-Leite

are recipients of CNPq fellowships.

H.P.S. Silva is the recipient of aPROBIC/FAPEMIG fellowship. L.R.Portugal is the recipient of a CAPES

fellowship.

Received May 19, 2005

Accepted November 18, 2005

Key words Lactobacillus Atherosclerosis Apolipoprotein E Cholesterol Germ-free mice

630


L.R. Portugal et al.

Introduction

Elevated serum cholesterol is an impor-tant risk factor associated with atherosclero-sis and coronary heart disease. Ingestion ofprobiotics such as lactic acid bacteria is apotential natural method to treat and preventhypercholesterolemia.

Several studies (1-8) have reported a de-crease in serum cholesterol during the con-sumption of lactic bacteria such as Lactoba-cillus acidophilus and L. reuteri and theirfermented dairy products. However, theseresults cannot be extrapolated to the condi-tions of human consumption.

The cholesterol-lowering potential ofL. acidophilus has been extensively studiedin humans. Massey (9) showed that, ini-tially, yogurt consumption significantly re-duced cholesterol by 10 to 12% in humanadult males, but 2 weeks later concentra-tions returned to the control values evenwith continued yogurt consumption. Linet al. (10) conducted a large double-blind,placebo-controlled trial with a crossover de-sign on 334 volunteers. The results did notshow a significant effect of lactobacilli onserum cholesterol. de Roos et al. (11) foundno effect of the consumption of yogurt en-riched with L. acidophilus L-1 at a doseof 1010 colony-forming units/day (CFU/day)in a placebo-controlled study of 78 healthymen and women. Thus, the effects of lacto-bacilli on blood cholesterol are not consist-ent.

The present study investigated theeffect of L. delbrueckii on total blood cho-lesterol and total lipids and on cholesterol inliver and feces in germ-free mice fed ahypercholesterolemic diet. In addition, weinvestigated the effect of L. delbrueckiion cholesterol metabolism, on the lipopro-tein fractions and atherosclerotic lesionsof the aorta in apolipoprotein E (apo E)knock-out (KO) mice that spontaneouslydevelop hypercholesterolemia and athero-sclerosis.

Material and Methods

Animals and diet

Thirty-one 8-week-old germ-free micefed a hypercholesterolemic diet (1% choles-terol and 0.5% cholic acid) were divided intothree groups: control group (N = 10) kept inthe axenic condition, EL group (N = 13)simultaneously receiving Escherichia coliEMO and L. delbrueckii in a mixture of DeMan, Rogosa and Sharpe (MRS) and brainheart infusion (BHI) culture broth contain-ing 1012 CFU/mL of both bacteria, and Ecgroup (N = 8) receiving 1012 CFU/mL E. coliEMO (as a control of colonization with abacterial strain with no hypocholesterolemiceffect). The animals were kept in Trexlertype isolators (Standard Safety Company,McHenry, IL, USA) in rooms on a 12-hlight-dark cycle and constant temperature(22C). The animals had free access to ster-ile diet and water. After confirmation of cellviability, culture broth was introduced asep-tically in the isolators and given orally to themice separately or mixed with water andfood. The experimental diet was introducedonly after confirmation of intestinal coloni-zation by feces culture. All animals weresacrificed under anesthesia after 4 weeks ofthe experiment.

To investigate atherosclerosis develop-ment, 4-week-old apo E KO mice with simi-lar body weight (about 17 g) and serumcholesterol concentrations (about 300 mg/dL) were used. Since these animals sponta-neously develop hypercholesterolemia andatherosclerosis, they received commercialdiet (Nuvital, Curitiba, PR, Brazil) ad libi-tum during the 6 weeks of the experiment.Body weight and food intake were measuredweekly. The animals were divided into acontrol group (N = 7) receiving water and aLac group (N = 7) receiving saline solutioncontaining L. delbrueckii (1012 CFU/mL).

All animals received humane care as out-lined in the Guide for the Care and Use of

631


Effect of Lactobacillus delbrueckii on atherosclerosis

Laboratory Animals of the National ResearchCouncil (12).

Bacteria

L. delbrueckii UFV H2B20 and E. coliEMO were isolated and identified at theFederal University of Viosa (MG, Brazil)and in the Laboratory of Microbial Ecology(INRA, Jouy-en-Josas, France), respective-ly. The strains were inoculated respectivelyinto MRS and BHI culture media and incu-bated at 37C for 18 h before inoculation.

Blood, liver and feces samples. Serumlipoproteins were separated by fast proteinliquid chromatography (FPLC) as previouslydescribed (13). The liver was removed,washed in saline solution, dried on filterpaper, weighed, and frozen at -20C. Fecesfrom the last experimental day were col-lected, homogenized and frozen at -20C.The hepatic and fecal lipids were extractedas described (14). Total blood cholesteroland cholesterol in FPLC fractions and inhepatic and fecal extracts were determinedusing commercial kits (KATAL, Belo Hori-zonte, MG, Brazil)

Histological analysis. The heart andproximal section of the aorta of apo E KOmice were removed and cleaned of adventi-tial tissue. The top half of the hearts wasobtained under stereoscopic observation andfixed by immersion in 4% paraformalde-hyde in 0.1 M phosphate-buffered saline atroom temperature. The specimens were rou-tinely processed for paraffin embedding andanalyzed by the method of Paigen et al. (15)with modifications, as briefly described be-low. Ten-micrometer thick cross-sectionswere cut with a microtome (Spencer, #820,Buffalo, NY, USA) all along the aortic rootarea (300 m), stained with hematoxylin-eosin, coded and examined by a single pa-thologist, who was unaware of the experi-mental conditions for each group. The slideswere decoded only after the report had beenwritten. The aortic root area was recognized

by the proximal presence of aortic valveleaflets. One of every three sections (for atotal of ten sections per mouse) was kept formorphometric analysis and the images ob-tained from a microscope coupled to a digi-tal camera were processed with an imageanalyzer (Kontron Electronic 300 software,Fremont, CA, USA). The total lesion area ofeach animal was the sum of lesion areasobtained from the ten selected sections. Dataare reported as mean total lesion area pergroup of 7 control and 7 Lactobacillus-treatedanimals.

Statistical analysis

Student t-test was used to compare con-trol and experimental groups, with the levelof significance set at P < 0.05.

Results

Germ-free mice

To examine the effect of L. delbrueckiion cholesterol absorption and metabolism,germ-free mice or germ-free mice colonizedwith E. coli EMO (without a hypocholester-olemic effect) or E. coli plus L. delbrueckiiwere fed a hypercholesterolemic diet for 4weeks. Feces culture confirmed the axenicstatus of control animals, the monoassocia-tion in the Ec group and the diassociation inthe EL group until the end of experiment.

Body weight was similar in both groups,suggesting no differences in food intake ornutritional alterations caused by bacterialcolonization (data not shown).

At the end of the experiment, no differ-ences in blood cholesterol were observedbetween groups. The means SEM of con-trol (N = 10), Ec (N = 8) and EL (N = 13)groups were 188 13, 163 13 and 193 17mg/dL, respectively. In the liver, total lipidswere reduced in the EL group comparedto the Ec group and reduced in bothwhen compared to the control axenic group

632



(Table 1). However, although the liver cho-lesterol level was lower in both colonizedgroups than in the control group, there wasno difference between the Ec and EL groups,suggesting an effect of colonization but notspecifically of L. delbrueckii on liver cho-lesterol. These results suggest that, althoughit had no effect on blood cholesterol, L.delbrueckii may be able to reduce the ab-sorption or increase the release of lipidsfrom the systemic circulation.

A hypercholesterolemic diet can contami-nate the feces and consequently cause anoverestimation of the fecal lipid and choles-terol concentration. For this reason, we ana-lyzed lipids and cholesterol in cecum con-tents. No differences were observed in theconcentration of cecal lipids among groups(Table 1). However, the EL group showed areduction of cecal cholesterol concentrationcompared to the Ec group (P = 0.047).

Apolipoprotein E knock-out mice

As germ-free Swiss/NIH mice do not de-velop spontaneous atherosclerosis, we studiedthe effect of L. delbrueckii in a model ofatherosclerosis. Apo E KO animals fed on aregular chow diet develop spontaneous hyper-cholesterolemia and aortic atherosclerotic le-sions after the age of 8 weeks. In this experi-ment, the animals received L. delbrueckii dailyto maintain the intestinal colonization.

There was no difference between groupswhen food and water intake and body weightwere analyzed (data not shown).

Liver lipids and cholesterol were un-changed after lactobacillus supplementation(Table 2). Similarly, fecal lipids and 3--hydroxy sterols (as indicators of cholesterolexcretion) were also similar when the Lacgroup was compared to control animals, sug-gesting that L. delbrueckii has no effect oncholesterol absorption and metabolism un-der these experimental conditions.

As expected, blood cholesterol was higherthan in wild-type animals (about 80 mg/dL),confirming the spontaneous hypercholester-olemia occurring in these animals. However,no statistically significant differences wereseen between groups at the beginning (300mg/dL for both groups) or after 6 weeks of theexperiment (356 26 and 399 35 mg/dL forcontrol and L. delbrueckii-supplementedgroups, respectively). These results demon-strate, as observed in germ-free mice, no hy-pocholesterolemic effect of L. delbrueckii.Likewise, lipoprotein analysis showed a simi-lar profile of atherogenic (VLDL + IDL +LDL) fractions and also of the protective (HDL)fraction for the various groups (Figure 1).

We then analyzed atherosclerotic lesionsin the aorta of mice from both groups. Whenthe aortic valve was analyzed, no reductionin lesion area was observed in mice from theLac group (278 76 x 1000 m2) comparedto control (199 95 x 1000 m2). The ath-erosclerotic lesions in mice of both groupsshowed an initial lesion (fatty streak) com-

Table 1. Total lipids and cholesterol in liver and cecum of NIH mice colonized withEscherichia coli or E. coli and Lactobacillus delbrueckii for 4 weeks or maintainedaxenic (control).

Control E. coli E. coli + L. delbrueckii(N = 10) (N = 8) (N = 13)

Liver lipids 322.0 4.16a 194.2 21.81b 105.3 5.01c

Liver cholesterol 24.4 2.5a 9.6 0.8b 8.1 1.91b

Cecum lipids ND 20.0 4.08 16.6 0.55Cecum cholesterol ND 2.4 0.22a 1.9 0.14b

Data are reported as mean SEM in mg/g. ND = not determined. Different letters onthe same line indicate statistically significant differences (P < 0.05, Student t-test).

Table 2. Hepatic and fecal total lipid and cholesterol of apolipoprotein E knock-outmice receiving Lactobacillus delbrueckii for 6 weeks (N = 7 mice/group).

Parameter Liver Feces

Control L. delbrueckii Control L. delbrueckii

Total lipid 112 9.2 96 6.8 30 13.9 30 10.1Cholesterol 4.1 0.2 4.1 0.2 4.1 0.2 3.8 0.5

Data are reported as mean SEM in mg/g. The controls received vehicle. There wereno statistical differences between control and L. delbrueckii-treated mice (Student t-test).

633



posed of well-defined foam cells and sparseinflammatory cells (Figure 2). Since therewas no difference in blood cholesterol or inatherosclerotic lesions between groups, itwas concluded that L. delbrueckii does nothave a positive effect in preventing athero-sclerotic lesions under these experimentalconditions.

Discussion

We investigated the effect of L. delbru-eckii on cholesterol metabolism in two dif-ferent models. In germ-free mice fed a hy-percholesterolemic diet, we studied the spe-cific effects of this bacterium on cholesterolabsorption and liver and blood cholesterolconcentrations without the interference ofindigenous microbiota. However, wild-type(conventional or germ-free) mice are not thebest models to study atherogenesis, sincethey do not develop atherosclerosis becauseof their high levels of the protective lipopro-tein fraction (HDL). On the other hand, apoE KO mice are good models to study athero-sclerosis, since their profile of blood choles-terol and lipoprotein fractions as well asatherosclerotic lesions are closely similar tothe human (16). For this reason, we used apoE KO mice to study the influence of L.delbrueckii specifically on atherogenesis.Our results showed that colonization by L.delbrueckii did not reduce blood cholesterolin germ-free mice, although a reduction ofcholesterol excretion was seen in monoasso-ciated animals. The reduction of fecal excre-tion may indicate a higher intestinal absorp-tion or assimilation of cholesterol by bacte-ria. As the liver and blood cholesterol con-centration was not increased in our experi-ment, it is probable that L. delbrueckii as-similated cholesterol in the intestines.

It has been reported that cholesterol can beremoved from culture media by precipitationwith free bile acids because of the activity ofthe bacterial enzyme bile salt hydrolase (17).Enhanced bile salt hydrolase activity in vivo

may increase cholesterol excretion. However,this hypothesis was not confirmed by our invivo experiment since there was a reduction ofcecal cholesterol excretion. Several in vitrostudies have shown that some bacteria such aslactobacilli assimilate cholesterol from cul-ture media (18-20). This indicates that certainstrains could act directly on cholesterol in thegastrointestinal tract, assimilating it from the

0

10

20

30

40

1 4 7

VLDL

LDL + IDL

HDL

10 13 16 19 22 25 28

Cho

lest

erol

(m

g/fr

actio

n)

Fractions

Figure 1. Lipoprotein profile ofcontrol (pool of 7 mice, fine line)or Lactobacillus delbrueckii apo-lipoprotein E knock-out mice(pool of 7 mice, bold line) after 6weeks of the experiment (VLDL,fractions 6 to 9; LDL + IDL, frac-tions 10 to 19; HDL, fractions 20to 26).

Figure 2. Lesion area of theaorta root of the apolipoproteinE knock-out mice receiving Lac-tobacillus delbrueckii (A) or ve-hicle (B) for 6 weeks, showinginitial lesion (fatty streak, ar-rows) composed of well-definedfoam cells and sparse inflam-matory cells. N = 7 animals inboth groups. There were no sta-tistical differences (Student t-test).

634



intestines and this could be associated with areduction in blood cholesterol levels. Thismechanism is consistent with our results show-ing a reduction of cholesterol excretion in ELanimals. However, in our experiment this ef-fect was not associated with lower blood cho-lesterol levels.

It is well known that hepatic cholesterolconcentration depends on liver uptake oflipoproteins (via LDL and LRP receptors),cholesterol synthesis (via HMG CoA reduc-tase activity), and bile acid synthesis (viacholesterol 7--hydroxylase activity). Webelieve that after 4 weeks the reduction ofexogenous cholesterol following L. delbru-eckii cholesterol assimilation was compen-sated mostly by a higher hepatic cholestero-genesis that kept liver cholesterol at levelssimilar to those of the control group, pre-venting a reduction of blood cholesterol.

Zacconi et al. (21) investigated the effectof L. acidophilus N5 in axenic mice and inconventionalized flora. Lactobacillus in-duced a discrete decrease of blood choles-terol in axenic mice, but had no effect inconventionalized mice. The discrepancy be-tween this result and ours may be due to thedifferent species of lactobacillus used in eachexperiment (L. acidophilus and L. delbru-eckii, respectively).

Despite the large number of studies asso-ciating lactobacilli and blood cholesterol,there are no studies addressing the effect ofthese bacteria on atherogenesis. For this rea-son, we investigated the effect of L. delbru-eckii in apo E KO mice that spontaneouslydevelop atherosclerosis. To the best of ourknowledge, this is the first study directlycorrelating Lactobacillus inoculation and thedevelopment of atherosclerosis.

Contrary to the results obtained for germ-free mice, we did not detect differences infecal cholesterol excretion in apo E KO mice.This may be due to the differences betweenmouse strains and diets: germ-free mice feda hypercholesterolemic diet have a higherconcentration of cholesterol in the intestines

compared to apo E KO mice fed a regularchow diet. Moreover, germ-free mice werediassociated (E. coli and L. delbrueckii) whileconventional apo E KO mice had an indig-enous microbiota. Consequently, in germ-free mice, the effect of cholesterol assimila-tion could be more evident due to the highercholesterol concentration in the diet and couldbe detected as a reduction of fecal excretion.In apo E mice, the lower intestinal concen-tration of cholesterol and the presence ofother cholesterol-metabolizing bacterialstrains possibly masked the effect of L.delbrueckii observed with germ-free mice.

Our main finding was that L. delbrueckiidid not affect the lipoprotein profile or thedevelopment of aortic lesion. We directlyshowed that the atherosclerotic lesions of L.delbrueckii-colonized animals were qualita-tively and quantitatively similar to those ofcontrol mice. It is possible that other lactoba-cillus strains have a more potent effect oncholesterol metabolism (or on other compo-nents of plaque development) and would beable to impair atherosclerosis formation. Forthis reason, more studies on bacterial inocula-tion and atherosclerotic lesions should be per-formed.

In conclusion, we believe that L. del-brueckii can assimilate intestinal cholesterol,but this effect did not impair atherogenesisin apo E KO mice. Since experimental mod-els of atherosclerosis are available, the studyof the hypocholesterolemic effect of probioticbacteria should be performed in these mod-els to determine both the prevention of riskfactors and the reduction of atherosclerosis.

Acknowledgments

We are indebted to Antonio Vaz Mesquitaand Maria Helena Alves for excellent care ofthe germ-free and apo E knock-out mousecolonies, respectively. We also thank KATALfor the donation of cholesterol and HDL kitsused in this study, and FAPEMIG, CAPESand CNPq for financial support.

635



References

1. Mann GV (1974). Studies of a surfactant and cholesteremia in theMaasai. American Journal of Clinical Nutrition, 27: 464-469.

2. McNamara DJ, Lowell AM & Sabb JE (1989). Effect of yogurt intakeon plasma lipid and lipoprotein levels in normolipidemic males.Atherosclerosis, 79: 167-171.

3. Rao DR, Chawan CB & Pulusani SR (1981). Influence of milk andthermophilus milk on plasma cholesterol levels and hepaticcholesterogenesis in rats. Journal of Food Science, 46: 1339-1341.

4. De Rodas BZ, Gilliland SE & Maxwell CV (1996). Hypocholesterol-emic action of Lactobacillus acidophilus ATCC 43121 and calciumin swine with hypercholesterolemia induced by diet. Journal of DairyScience, 79: 2121-2128.

5. Akalin AS, Gonc S & Duzel S (1997). Influence of yogurt andacidophilus yogurt on serum cholesterol levels in mice. Journal ofDairy Science, 80: 2721-2725.

6. Fukushima M & Nakano M (1996). Effects of a mixture of organisms,Lactobacillus acidophilus or Streptococcus faecalis on cholesterolmetabolism in rats fed on a fat- and cholesterol-enriched diet. BritishJournal of Nutrition, 76: 857-867.

7. Taranto MP, Medici M, Perdigon G et al. (2000). Effect of Lactobacil-lus reuteri on the prevention of hypercholesterolemia in mice. Jour-nal of Dairy Science, 83: 401-403.

8. Taranto MP, Medici M, Perdigon G et al. (1998). Evidence forhypocholesterolemic effect of Lactobacillus reuteri in hypercholes-terolemic mice. Journal of Dairy Science, 81: 2336-2340.

9. Massey L (1984). Effect of changing milk and yogurt consumptionon human nutrient intake and serum lipoprotein. Journal of DairyScience, 67: 255-262.

10. Lin SY, Ayres JW, Winkler Jr W et al. (1989). Lactobacillus effectson cholesterol: in vitro and in vivo results. Journal of Dairy Science,72: 2885-2899.

11. de Roos NM, Schouten G & Katan MB (1999). Yogurt enriched withLactobacillus acidophilus does not lower blood lipids in healthy menand women with normal to borderline high serum cholesterol levels.

European Journal of Clinical Nutrition, 53: 277-280.12. National Research Council (1996). Guide for the Care and Use of

Laboratory Animals. National Research Council, Washington, DC,USA.

13. Peluzio MC, Miguel Jr E, Drumond TC et al. (2003). Monocytechemoattractant protein-1 involvement in the alpha-tocopherol-in-duced reduction of atherosclerosis lesions in apo E knock out mice.British Journal of Nutrition, 90: 3-11.

14. Oliveira DR, Portugal LR, Cara DC et al. (2001). Gelatin intakeincreases the atheroma formation in apo E knock out mice. Athero-sclerosis, 154: 71-77.

15. Paigen B, Morrow A, Holmes PA et al. (1987). Quantitative assess-ment of atherosclerosis lesions in mice. Atherosclerosis, 68: 231-240.

16. Nakashima Y, Plump S, Raines W et al. (1994). Apo E-deficientmice develop lesions of all phases of atherosclerosis throughout thearterial tree. Arteriosclerosis and Thrombosis, 14: 133-140.

17. Klaver FA & van der Meer R (1993). The assumed assimilation ofcholesterol by Lactobacilli and Bifidobacterium bifidum is due totheir bile salt-deconjugating activity. Applied and EnvironmentalMicrobiology, 59: 1120-1124.

18. Gilliland SE (1990). Health and nutritional benefits from lactic acidbacteria. FEMS Microbiology Reviews, 7: 175-188.

19. Gilliland SE, Nelson CR & Maxwell C (1985). Assimilation of choles-terol by Lactobacillus acidophilus. Applied and Environmental Mi-crobiology, 49: 377-381.

20. Gilliland SE & Walker DK (1990). Factors to consider when selectinga culture of Lactobacillus acidophilus as a dietary adjunct to producea hypocholesterolemic effect in humans. Journal of Dairy Science,73: 905-911.

21. Zacconi C, Bottazzi V, Rebecchi A et al. (1992). Serum cholesterollevels in axenic mice colonized with Enterococcus faecium andLactobacillus acidophilus. Microbiologica, 15: 413-417.

jurnal dk 2

Documents