Lactobacillus plantarum LG42 isolated from gajami sik-hae decreases body and fat pad weights in diet-induced obese mice

Authors

  • J.-E. Park,

    1. Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, Korea
    2. Jeonju Makgeolli Research Center, Chonbuk National University, Jeonju, Korea
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  • S.-H. Oh,

    1. Department of Food and Biotechnology, Woosuk University, Jeonbuk, Korea
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  • Y.-S. Cha

    Corresponding author
    1. Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, Korea
    2. Jeonju Makgeolli Research Center, Chonbuk National University, Jeonju, Korea
    • Correspondence

      Youn-Soo Cha, Department of Food Science & Human Nutrition, Chonbuk National University, 664-14 Duckjin-dong, Jeonju, Jeonbuk 561-756, South Korea. E-mail: cha8@jbnu.ac.kr

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Abstract

Aims

This study investigated the antiobesity effect of lactic acid bacteria (Lactobacillus plantarum LG42) isolated from gajami sik-hae.

Methods and Results

Male C57BL/6J mice were divided into four groups (n = 10); NDC (normal diet & DW), HDC (high-fat diet & DW), LGLAB (high-fat diet & Lactobacillus plantarum LG42, 1 × 107 CFU per mouse), HGLAB (high-fat diet & L. plantarum LG42, 1 × 109 CFU per mouse). After 12 weeks, GLAB supplemented groups showed lower body weight, with a significant reduction in epididymal and back fat. Serum and hepatic triglyceride, serum insulin and leptin levels were significantly lowered in GLAB supplemented groups. The hepatic mRNA expression of PPARα and CPT-I were significantly increased in GLAB groups, whereas the level of ACC, SREBP-1 and LXRα were significantly decreased in GLAB groups compared with HDC group. Additionally, GLAB reduces the expression of PPARγ in the epididymal adipose tissue resulting in inhibition of genes regulated by PPARγ.

Conclusion

These results suggest that the Lactobacillus plantarum LG42 has antiobesity effects in high-fat-diet-induced obese mice.

Significance and Impact of the Study

These results may contribute to nutraceutical and food industries in developing functional food and probiotics based therapies for the treatment and prevention of obesity.

Introduction

Obesity threatens to become the twenty-first century's leading metabolic disease around the world (Formiguera and Cantón 2004). Obesity associated complications are major reasons behind morbidity and mortality. Obese people have a higher level of cholesterol and triglyceride putting them at risk of heart disease, hypertension, type 2 diabetes mellitus, cancer, respiratory complications and osteoarthritis (Kopelman 2000). The prevalence of obesity and its complications is a worldwide concern associated with high costs of treatment and lost productivity, and therefore, there are increasing interests in alternative nutritional interventions for preventing obesity (Finkelstein et al. 2008; Cawley and Meyerhoefer 2012).

Sik-hae is Korean's traditional fermented seafood that has long been used for seasoning and is the generic name for a class of Korean lactic acid–fermented fish products (Lee et al. 1993). Gajami sik-hae is one of the sik-hae produced by fermentation of the flat fish meat by lactic acid bacteria (LAB). The salted fishes are mixed with cooked millet, red pepper, garlic, ginger, malt meal and fermented at 5 or 20°C. The unique taste of sik-hae is due to the presence of the aforementioned ingredients and the fermenting action of various microorganism during the fermentation period (Lee 1997). LAB is the most predominant micro-organisms involved in sik-hae fermentation. The dominant microflora in gajami sik-hae is LAB; mainly composed of Lactobacillus spp. and Sterptococcaceae products (Lee et al. 1993). These viable bacteria beneficially influences the host by improving the intestinal microbial balance (Yanbo and Zirong 2006). Recently, LAB has been evaluated for their nutritional, physiological and pharmacological aspects (Turnbaugh et al. 2006; Liou et al. 2013), which have raised attention on the functional effect of Korean traditional foods including kimchi and joet-gal (Kim 2005; Hwang et al. 2007; Park et al. 2012).

Lactobacillus produces a greater health benefit for animals as well as for humans. Several studies have suggested that lactobacillus has potential health and nutritional benefits, including body weight controls and improvements from metabolic disorders such as obesity, diabetes, hypertension and hypercholesterolaemia (Lee et al. 2007; Bhathena et al. 2009; Lye et al. 2009). Additionally, the manipulation of gut microbial communities could be an alternative treatment for obesity, it has also been demonstrated that the human gut flora might be a contributing factor in body weight differences among individuals (Ley et al. 2005, 2006; Turnbaugh et al. 2006).

The γ-Aminobutyric acid (GABA) is a four-carbon nonprotein amino acid conserved from bacteria to plants and vertebrates. The consumption of GABA-enriched foods, such as milk, soybean and gabaron tea, has been reported to suppress the elevation of systolic blood pressures in spontaneously hypertensive rats (SHRs) (Omori et al. 1987; Aoki et al. 2003; Liu et al. 2011). Recently, we observed that Lactobacillus plantarum LG42 strain efficiently produces GABA in MRS broth containing 1% MSG. TLC (Thin-Layer Chromatography) analysis indicated that the cultured medium and cell-free culture supernatant of L. plantarum LG42 cells contained GABA but not the cytoplasmic fraction. Although the cytoplasmic fraction contains a negligible amount of GABA, it was highly effective in inhibiting the intracellular lipid accumulations in the differentiating adipocytes (Park et al. 2013).

In our previous study, the L. plantarum LG42 (GLAB) isolated from gajami sik-hae exerts antiadipogenic effects via the modulations of intracellular signalling of lipid metabolism in 3T3-L1 adipocytes. However, the antiobesity effects need to be illustrated under in vivo conditions. Therefore, in this study, we assessed the antiobesity effects of GLAB in mice fed with a high-fat diet.

Materials and methods

Preparation of bacterial cultures

Lactobacillus plantarum LG42, a lactic acid bacteria having GABA-producing capacity, isolated from gajami sik-hae (GLAB), was provided by Genetic Engineering Laboratory, Woosuk University, Korea. The GLAB was incubated at 37°C for 16–18 h in MRS medium (Difco, Detroit, MI, USA). The strains were harvested by centrifugation at 1800 × g for 20 min; washed twice with neutral saline. And resuspended at 5 × 107 or 5 × 109 CFU ml−1, and 200 μl bacterial solution was administered to each mouse by intragastric gavages. According to earlier study, 107–1011 CFU day−1 of Lactobacillus intake found to be clinically safe, and it is not associated with any intolerance problems (Connolly (2004); Hrnčár et al. 2013). In our study, the dosages of LG42 were used based on the earlier approaches and criteria as in most of the clinical trials and animal study using Lactobacillus.

Animal and diets

Male C57BL/6J mice, aged 4 weeks, were purchased from Charles River Laboratories (Tokyo, Japan). The animals were maintained on a pellet diet (Research Diets, New Brunswick, NJ, USA) for 1 week, then randomly divided into four groups: normal diet (NDC, distilled water per day), high-fat diet (HDC, distilled water per day), high-fat diet with low dose GLAB (LGLAB, 1 × 107 CFU per 200 μl day−1), high-fat diet with high dose GLAB (HGLAB, 1 × 109 CFU per 200 μl day−1) every morning (AM 10 : 00–11 : 00). The compositions of the experimental diets are shown in Table 1. The animals were randomized into groups (n = 10) such that the average weight in each groups were comparable. The animals were housed in a temperature-controlled environment with a 12-h light per dark cycle. The animals were given free access to food and water during the entire experimental period of 12 weeks. The food consumption and body weight were measured daily and weekly, respectively. The experimental protocol was approved by the Animal and Use Committee of Chonbuk National University.

Table 1. The composition of experimental diets
 Normal dietaHigh-fat dietb
g%Kcal%g%Kcal%
  1. a

    AIN-93 Modified diet with 4% fat (10% fat calories) content.

  2. b

    AIN-93 Modified high-fat diet with 35% fat (60% fat calories) content. DW; distilled water (1 ml kg−1 bw), NDC; Normal diet control, HDC; High-fat diet control, LGLAB; High-fat diet and 1 × 107 CFU of GLAB per mouse, HGLAB; High-fat diet and 1 × 109 CFU of GLAB per mouse.

Protein19·2202620
Carbohydrate67·3702620
Fat4·3103560
Total 100 100
Ingredients (g)
Casein, lactic200200200200
l-cystine3333
Corn starch315
Maltodextrin35125125125
Sucrose35068·868·868·8
Cellulose50505050
Soybean oil25252525
Lard20245245245
Mineral mix10101010
Dicalcium phosphate13131313
Calcium carbonate5·55·55·55·5
Potassium citrate16·516·516·516·5
Vitamin mix10101010
Choline bitarate2222
FD&C yellow dye no. 50·05
FD&C blue dye no. 10·050·050·05
Total1055·05773·85773·85773·85
Kcal4057405740574057
Kcal g−13·85·25·25·2
AdministrationDWDW1 × 107 CFU1 × 109 CFU

Collection of serum and tissue samples

Feed was removed 12 h before sacrificing. Blood samples were collected from each mouse by orbital vein puncture and placed in ice water for 1 h. Serum was separated from the blood by centrifugation (Micro 17R; Hanil Science In Co, Ltd, Gangneung, Korea) at 1100 × g for 15 min at 4°C and stored at −80°C until analysed. The liver, kidney, back fat and epididymal fat were removed, rinsed with a phosphate-buffered saline solution, wiped with a paper towel, weighed quickly, frozen in liquid nitrogen and stored at −80°C until assayed.

Analysis of lipid profiles

Total cholesterol in the serum was measured by an enzymatic method using a commercial kit (Asan Pharmaceutical Co., Seoul, Korea); the HDL-cholesterol fraction was determined by the dextran sulfate-Mg++method. Liver lipids were extracted from liver tissues according to the method of Folch (Folch et al. 1957). The serum and hepatic triglyceride were measured enzymatically using a commercial kit (Asan Phamaceutical Co.).

Analysis of insulin and leptin

Serum insulin and leptin were measured using commercially available kits, Mouse Insulin Elisa kit (Shibayagi, Shibukawa, Japan) and ELISA-based Quantikine® Immunoassay kit (R&D Systems, Minneapolis, MN, USA), respectively.

Analysis of carnitine

Serum and liver tissues were prepared for carnitine analysis using standard method. Briefly, liver tissues were homogenized by adding 50 mg in 99 volumes (1% homogenate) of cold distilled water, centrifuged at 1500 × g for 10 min. Nonesterified carnitine (NEC), acid-soluble acylcarnitine (ASAC), and acid-insoluble acylcarnitine (AIAC) in serum were determined by the radio immunoenzymatic procedure of Cederblad and Lindsted (Cederblad and Lindstedt 1972), as modified by Sachan et al. (1984). In this method, AIAC were precipitated with perchloric acid and centrifugation, leaving the ASAC and NEC in the supernatant. An aliquot of the supernatant was assayed to determine NEC, and another aliquot was hydrolysed with 0·5 mol l−1 KOH to assay all acid-soluble carnitine (ASAC + NEC). The ASAC value was calculated as the as the difference between the NEC and total acid-soluble carnitines. The pellets containing the AIAC were drained, washed and hydrolysed in 0·5 mol l−1 of KOH for 60 min in a hot water bath 60°C. In each case, carnitine was assayed using carnitine acetyltransferase (Sigma Chemical Co., St, Louis, MO, USA) to esterifies the carnitine to a [14C] acetate from [1-14C] acetyl-CoA (Amersham, Arlington Heights, IL, USA). Radioactivity of the sample was determined in a Beckman model LS3801 liquid scintillation counter (Beckman Instruments, Palo Alto, CA, USA).

Quantitative real-time PCR analysis

Total RNA was extracted by Trizol reagent (Invitrogen Life Technologies; Carlsbad, CA, USA). The concentration and purity of RNA was measured spectrophotometrically (Simandzu, Kyoto, Japan) by determining absorbance at 260 nm followed calculating the ratio of absorbance at 260 and 280 nm.

For the real-time PCR, 1 μg of extracted RNA was reverse transcribed into first-stand cDNA using high-capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster city, CA, USA). Then, the RNA expression level was quantified by quantitative real-time using SYBER Green PCR Master Mix (Applied Biosystems, Woolston, Warrington, UK) and the 7500 real-time PCR system (Applied Biosystems) according to the manufacture's protocol. Sequences of primers used for quantitative real-time PCR were as follows: acetyl CoA carboxylase (ACC) F: 5′-CTCCTGCTCATCACAGTATG-3′ and R: 5′-GCAAGGCTACTAAGGCAGG-3′; carnitine palmetonyltransferase- I (CPT-I) F: 5′-AAAGATCAATVGGACCCTAGACA-3′ and R: 5′-CAGCGAGTAGCGCATAGTCA-3′; peroxisome proliferator-activated receptor α (PPARα) F: 5′-GGATGTCACACAATGCAATTCGCT-3′ and R: 5′-TCACAGAACGGCTTCCTCAGGTT-3′; sterol regulatory element-binding protein-1 (SREBP-1) F: 5′-GCGGAGCCATGGATTGCAC-3′ and R: 5′-CTCTTCCTTGATACCAGGCCC-3′; liver X receptor α (LXRα) F: 5′-GCGTCCATTCAGAGCAAGTGT-3′ and R: 5′-TCACTCGTGGACATCCCAGAT-3′; peroxisome proliferator-activated receptor γ (PPAR γ) F: 5′-CAAGAATACCAAAGTGCGATCAA-3′ and R: 5′-GAGCTGGGTCTTTTCAGAATAATAAG-3′; fatty acid–binding protein (aP2) F: 5′-AGTGAAAACTTCGATGATTACATGAA-3′ and R: 5′-GCCTGCCACTTTCCTTGTG-3′; CCAAT/enhancer-binding protein α (C/EBPα) F: 5′-AGCAACGAGTACCGGGTACG-3′ and R: 5′-TGTTTGGCTTTATCTCGGCTC-3′; lipoprotein lipase (LPL) F: 5′-TAATTGGCTGCAGAAAACAC-3′, R: 5′-CG TCAGCAAACTCAAAGGT-3′; β-actin F: 5′-ATGGATG ACGATATCGCT-3′ and R: 5′-ATGAGGTAGTCTGT CAGGT-3′. Relative quantification of gene expression with real-time PCR data was calculated relative to β-actin. Relative quantitative measures the relative change in mRNA expression levels. It determines the changes in steady state mRNA levels of a gene across multiple samples and expresses it relative to the other levels of RNA (Wong and Medrano 2005).

Statistical analysis

Data from individual experiments are expressed as the mean ± SD deviation. The data were analysed by one-way anova using spss 12.0.1 program (SPSS Inc., Chicago, IL, USA), and the differences between the means assessed using Duncan's multiple range test. Statistical significance was considered at P-value < 0·05.

Result

Weight gain, food intake and feed efficiency in mice

The weight gain, food intake and feed efficiency during the experimental period are shown in Table 2. The results showed that the food intake was significantly decreased in the LGLAB and HGLAB groups compared with HDC and NDC groups (< 0·05). Body weight was significantly increased in the HDC group compared with NDC group (< 0·05), while GLAB supplement in HD group resulted in significant decrease in the weight gain as compared to HDC group (< 0·05). The feed efficiency ratio was significantly higher in the HDC group when compared with the NDC group, and GLAB supplement groups were significantly lower as compared to HDC group (< 0·05).

Table 2. The effects of lactic acid bacteria isolated gajami sik-hae on weight gain, food intake and feed efficiency ratios in mice
GroupsNDCHDCLGLABHGLAB
  1. Results are Mean ± SD of 10 mice per group. (a–c); Values with different superscripts are significantly different by anova with Duncan's multiple range test at < 0·05.

  2. NDC, Normal diet control; HDC, High-fat diet control; LGLAB, High-fat diet and 1 × 107 CFU of GLAB per mouse; HGLAB, High-fat diet and 1 × 109 CFU of GLAB per mouse; Feed efficiency ratio, weight gain day−1/dietary intake day−1.

Initial weight (g)22·21 ± 1·0622·68 ± 0·9422·14 ± 0·9822·37 ± 1·18
Weight gain (g)4·40 ± 1·56c22·62 ± 1·24a16·62 ± 3·74b14·08 ± 6·11b
Food intake (g day−1)2·15 ± 0·21a2·02 ± 0·31a1·79 ± 0·40b1·73 ± 0·11b
Feed efficiency ratio0·02 ± 0·07c0·13 ± 0·01a0·11 ± 0·07b0·10 ± 0·04b

Adipose tissue weight in mice

As shown in Fig. 1, epididymal fat pad weight was significantly greater in the HDC group than the NDC group, and was significantly lower in the HGLAB group than in the HDC group (< 0·05). Back fat weight was significantly decreased by the GLAB supplemented groups as compared with the HDC group (< 0·05).

Figure 1.

Effect of lactic acid bacteria isolated gajami sik-hae on epididymal fat and back fat weight in mice. Results are mean ± SD of 10 mice per group. (a–c); Values with different superscripts are significantly different by anova with Duncan's multiple range test at < 0·05. NDC; Normal diet control, HDC; High-fat diet control, LGLAB; High-fat diet and 1 × 107 CFU of GLAB per mouse, HGLAB; High-fat diet and 1 × 109 CFU of GLAB per mouse.

Serum and liver lipid levels

The effects of GLAB supplementation on serum and liver lipid profiles are shown in Table 3. Serum triglyceride (TG) concentration was significantly higher in the HDC group as compared with the NDC group (< 0·05), and significantly lower in the GLAB supplemented groups than in the HDC group. The serum TG concentration was almost similar in LGLAB and HGLAB to that of NDC group. Serum total cholesterol (TC) concentrations were significantly higher in the LGLAB and HGLAB groups than in the NDC and the HDC groups (< 0·05). Serum HDL-cholesterol concentrations were higher in HGLAB group as compared to other groups, although this effect was not significant and HDL-c/TC was the higher in the NDC and the HDC groups (< 0·05). Hepatic TGs in of the animals in HGLAB group were significantly lower as compared with those in the HDC group and were normalized to be the same as the NDC group (< 0·05). There were no significant effects of GLAB supplementation on liver total cholesterol concentration (< 0·05).

Table 3. The effect of lactic acid bacteria isolated gajami sik-hae on lipid profiles in mice
GroupsNDCHDCLGLABHGLAB
  1. NDC, Normal diet control; HDC, High-fat diet control; LGLAB, High-fat diet and 1 × 107 CFU of GLAB per mouse; HGLAB, High-fat diet and 1 × 109 CFU of GLAB per mouse.

  2. Results are Mean ± SD of 10 mice per group. (a–c); Values with different superscripts are significantly different by anova with Duncan's multiple range test at < 0·05.

Serum (mg dl−1)
TG148·3 ± 28·9b186·4 ± 27·9a151·0 ± 14·3b151·0 ± 14·3b
TC199·3 ± 31·1c319·1 ± 30·5b390·5 ± 25·1a395·9 ± 32·5a
HDL-c113·3 ± 24·8c168·7 ± 33·3ab135·0 ± 30·5bc187·9 ± 27·0a
HDL-c/TC56·8 ± 0·8a51·6 ± 1·3a34·7 ± 1·2b47·5 ± 0·8ab
Liver (mg g−1)
TG2·55 ± 0·44b5·02 ± 1·04a4·08 ± 0·76ab3·77 ± 0·44b
TC0·16 ± 0·020·16 ± 0·020·17 ± 0·020·17 ± 0·03

Serum insulin and leptin levels

GLAB supplements showed significant effects on serum insulin and leptin levels. As shown in Fig. 2, the insulin and leptin levels were significantly higher in the HDC group as compared with the NDC group. However, in contrast to the HDC group, the GLAB supplemented groups showed a significant decrease in both serum insulin and leptin levels.

Figure 2.

Effect of lactic acid bacteria isolated gajami sik-hae on serum insulin and leptin in mice. Results are mean ± SD of 10 mice per group. (a–c); Values with different superscripts are significantly different by anova with Duncan's multiple range test at < 0·05. NDC; Normal diet control, HDC; High-fat diet control, LGLAB; High-fat diet and 1 × 107 CFU of GLAB per mouse, HGLAB; High-fat diet and 1 × 109 CFU of GLAB per mouse.

Serum and liver carnitine levels

GLAB supplementation tended to increase serum carnitine concentrations (Table 4). Especially, the HGLAB group exhibited higher concentrations with all serum parameters (NEC, AIAC, ASAC, TCNE) as compared to the HDC group. Liver NEC, ASAC and TCNE levels significantly decreased in the HDC group as compared with the NDC group (< 0·05) (Table 4) and significantly increased in the GLAB supplemented groups when compared with in the HDC group. Hepatic AIAC level was not significantly different between groups (< 0·05).

Table 4. The effects of lactic acid bacteria isolated gajami sik-hae on carnitine concentrations in mice
GroupsNDCHDCLGLABHGLAB
  1. NDC, Normal diet control; LGLAB, High-fat diet and 1 × 107 CFU of GLAB per mouse; HGLAB, High-fat diet and 1 × 109 CFU of GLAB per mouse; NEC, non-esterified carnitine; ASAC, acid-soluble acyl carnitine; AIAC, acid-insoluble acyl carnitine; TCNE, total carnitine.

  2. Results are Mean ± SD of 10 mice per group. (a–c); Values with different superscripts are significantly different by anova with Duncan's multiple range test at < 0·05.

Serum (μmol dl−1)
NEC0·47 ± 0·08b0·45 ± 0·11b0·51 ± 0·10b0·67 ± 0·12a
ASAC1·25 ± 0·20a0·95 ± 0·21b1·12 ± 0·24a1·18 ± 0·12a
AIAC0·22 ± 0·04b0·23 ± 0·02b0·24 ± 0·02ab0·28 ± 0·06a
TCNE1·88 ± 0·24a1·54 ± 0·17b1·92 ± 0·24a2·04 ± 0·25a
Liver (μmol dl−1)
NEC11·26 ± 2·02a7·69 ± 1·22b11·60 ± 1·45a11·93 ± 3·05a
ASAC6·88 ± 1·39a4·20 ± 1·23b6·88 ± 1·02a6·88 ± 1·89a
AIAC0·05 ± 0·030·02 ± 0·020·02 ± 0·010·04 ± 0·01
TCNE18·87 ± 3·36a11·59 ± 2·36b18·49 ± 2·39a17·91 ± 4·96a

Hepatic mRNA levels

The hepatic mRNA levels of ACC, LXRα and SREBP-1 were reduced in both of the supplemented groups as compared with the HDC group (< 0·05) (Fig. 3). CPT-I and PPARα mRNA levels, which are associated with fatty acid β-oxidation, were higher in the supplemented groups than in HDC group (< 0·05) (Fig. 3).

Figure 3.

Effect of lactic acid bacteria isolated gajami sik-hae on mRNA levels of lipid metabolism-related genes in the liver of C57BL/6J mice. Results are Mean ± SD of 10 mice per group. (a–c); Values with different superscripts are significantly different by anova with Duncan's multiple range test at < 0·05. NDC; Normal diet control, HDC; High-fat diet control, LGLAB; High-fat diet and 1 × 107 CFU of GLAB per mouse, HGLAB; High-fat diet and 1 × 109 CFU of GLAB per mouse. ACC; acetyl CoA carboxylase, CPT-I; carnitine palmitoyltransferase-I, LXRα; liver X receptor α, PPARα; Peroxisome Proliferator-activated Receptor α, SREBP-1; sterol regulatory element-binding protein-1. (image) NDC; (image) HDC; (image)LGLAB; (image) HGLAB.

Epididymal adipose tissue mRNA levels

The mRNA levels of adipocyte-specific genes, PPARγ, aP2, C/EBPα, LPL and LXRα, associated with adipocyte differentiation, were significantly higher in the HDC group as compared with the NDC group. However, in the GLAB supplemented groups, the expressions of these genes were significantly decreased than in other groups (< 0·05) (Fig. 4).

Figure 4.

Effect of lactic acid bacteria isolated gajami sik-hae on mRNA levels of lipid metabolism-related genes in the adipose tissue of C57BL/6J mice. Results are Mean ± SD of 10 mice per group. (a–c); Values with different superscripts are significantly different by anova with Duncan's multiple range test at < 0·05. NDC; Normal diet control, HDC; High-fat diet control, LGLAB; High-fat diet and 1 × 107 CFU of GLAB per mouse, HGLAB; High-fat diet and 1 × 109 CFU of GLAB per mouse. PPARγ; peroxisome proliferator-activated receptor γ, aP2; Fatty acid–binding protein, C/EBPα; CCAAT/enhancer-binding protein, LPL; lipoprotein lipase, LXRα; liver X receptor α. (image) NDC; (image) HDC; (image) GLAB; (image) HGLAB.

Discussion

Lactic acid bacteria (LAB) used for fermenting foods has therapeutic effects on human health. Many studies reported that intakes of lactobacilli had no apparent clinical side effects (Taranto et al. 1998; Wullt et al. 2003; Connolly 2004; Hrnčár et al. 2013). The FAO/WHO definition of a probiotic is the live micro-organism that administrated in adequate amounts and conferred a health benefit to the host (Connolly 2004). Intake of LAB resulted in greater improvement for diverse kinds of gastrointestinal disorders; it also acts as a beneficial immunomodulator and plays a role in prevention of cancer (Rolfe 2000; Adolfsson et al. 2004; de Moreno de LeBlanc et al. 2007). Antiobesity effects of probiotic have been reported by several investigators (Arora et al. 2012; Moon et al. 2012; Zhao et al. 2012). Zhao et al. (2012) demonstrated that obese mice supplemented with high-fat diets containing probiotic significantly lowers (P < 0·05) body weights and liver lipid contents (triglyceride and cholesterol) than the controls (Zhao et al. 2012). High-fat diet–induced obesity are characterized by adipocyte hypertrophy and insulin resistance (Kubota et al. 1999). In our study, mice fed a high-fat diet gained significantly more weight than those fed a normal diet. However, the animals in GLAB supplemented groups showed lesser body weight gains and food intakes as compared to the HDC group (Table 2). The reduction in food intake by LGLAB and HGLAB might also influence the decrease in body weight gain compared with HDC group. Changes in body weight correlated with body fat depositions. The back fat resembled subcutaneous fat in humans and are strongly related with plasma leptin (Takahashi et al. 1996). In animals, the back fat thickness to liver weight ratio showed a significant linear relationship with plasma leptin (Blache et al. 2000). To measure the subcutaneous fat of mice, we measured the back fat weight of these animals. Therefore, we measured the weight of epididymal and back fat pad to ensure that GLAB supplementation prevented weight gain by reducing the hypertrophic and hyperplastic growth of adipose tissue (Fig. 1). The epididymal adipose tissue mass revealed that higher doses of GLAB significantly prevented epididymal adipose tissue mass gain in the HGLAB group as compared to the HDC group. In a study on diet-induced obese subjects, it was observed that supplementation of Lactobacillus aided body weights and abdominal fat reductions (Arora et al. 2012). These data suggested that the GLAB regulates body weight and body fat through effects on metabolism and appetite. Several animal studies reported that supplementation with prebiotics lowered energy intakes, reduced weight gains and influenced gut hormones which regulates appetite and satiety (Paineau et al. 2008; Ruxton 2012), thereby suggesting that the adaptation of the gut microbiota is required to have a physiological relevance in the control of food intakes. Thus, it could be possible that GLAB might influence the intestinal micribiota population. Therefore, leptin and insulin are involved in the regulation of energy balance and food intake (Weight 1995; Heini et al. 1998; Keim et al. 1998; Schwartz et al. 2000; Verdich et al. 2001) and suppressed food intakes partly through suppression of nueropeptite Y and agouui-related protein (Wahlestedt et al. 1993; Ebihara et al. 1999).

It was reported that circulating leptin levels increased for animals fed high-fat diets, leading to leptin resistances (Lin et al. 2000). In our study, serum leptin levels increased in the high-fat diet groups as compared to the normal diet group. However, the serum leptin levels decreased in the GLAB-supplemented groups. The circulating serum leptin levels are highly correlated with the proportion of adipose tissue masses and are secreted in higher amounts from the epididymal fat pad (Considine et al. 1996; Nazian 2001). Low serum leptin levels in the LGLAB and HGLAB groups were associated with reduced epididymal fat masses as compared to the HDC group, suggesting that lower epididymal fat masses in the GLAB groups could be the reason behind the decreased levels of circulating leptin.

In this study, mice fed the high-fat diets showed a remarkable increase in serum insulin levels, as compared to normal diet groups, and decrease in the GLAB supplement groups. Insulin signalling in adipose tissues plays important role in lipid storage, the regulation of glucose homoeostasis and adipocyte differentiation (Bluher et al. 2002). In obese mice, feeding of high-fat diets over long term was shown to modulate levels of leptin and insulin, consequently impairing the normal physiological status. This indicated that glucose and lipid metabolism can proceed to abnormality (Song et al. 2012). However, in our study, GLAB restored the abnormal or impaired levels of leptin and insulin and improved or delayed high-fat diets which induced metabolic abnormalities, thus indicated a beneficial effect of GLAB in averting insulin/leptin resistance.

Dietary fat intake have an impact on the amount of body fat, serum cholesterol levels and formation of fatty acids (Sanders et al. 1994). In this study, GLAB supplementation also reversed the HD-induced elevation of TG in serum and liver. Even though, the GLAB supplementation was significantly associated with an increase in serum TC, the HDL-c/TC ratio was not significant in the HGLAB group when compared with HDC group.

Carnitine (3-hydroxy-4-N-trimethyl-ammonium butyrate) transported fatty acids to the mitochondria, where fatty acids undergo β-oxidation, thus, played a vital role in activating oxidation of fatty acids (Vaz and Wanders 2002). Deficiency of carnitine slowed down oxidations of fatty acids and resulted in an increased levels of serum lipid. Carnitine was presented in biological materials for both the free or nonesterified form (NEC) and as the esterified carnitine (acylcarnitine) which is the metabolic products of reactions utilizing the acyl-CoA catalysed by carnitine acyltransferases (Sewell and Böhles 1995). In this study, all serum parameters were higher in the HGLAB group when compared to the HDC group (Table 4). Higher NEC level increased the availability of fatty acids as energy substrates and improved fatty oxidation efficiency. The serum AIAC and ASAC are considered very good markers of the activation of fatty acids (Aggett et al. 1991). These results suggested that GLAB also improves impaired metabolic functions such as fatty acid oxidation and ketogenesis. Carnitine was synthesized in the liver and kidney with 4-trimethylaminobutyraldehyde (TMABA) and butyrobetaine as intermediates in carnitine biosynthesis of the liver (Vaz and Wanders 2002). In this study, acyl/free carnitine, ASAC and TCNE concentrations were elevated in the liver after GLAB supplementations, which was due to the increased carnitine biosynthesis in the liver. Thus, we assumed that supplementations of GLAB increased β-oxidation of free fatty acids.

In an effort to assess the mRNA expression profiles of genes involved in lipid metabolism, we used RNA from both the liver and adipose tissues. ACC catalysed the ATP-dependent carboxylation of acetyl-CoA to malonyl CoA and acted as the rate limiting enzymes in fatty acid synthesis (Wakil et al. 1983), whereas the CPT-I and PPARα are involved in mitochondrial fatty acid oxidations. Fatty acid oxidation has been suggested as a target for the metabolic syndromes. The process of β-oxidation in mitochondria involved shuttling of long chain fatty acids that are released from adipose tissues across the mitochondrial membrane, a process that was regulated by the enzymes CPT-I and ACC. Both inhibitors and activators of CPT-I have been proposed to combat hyperglycaemia and the metabolic syndromes (Fåk and Bäckhed 2012). In this study, the hepatic mRNA expression level of ACC was significantly decreased in the GLAB supplement groups as compared to the HD group, while the CPT-I and PPAR-α genes responsible for β-oxidations were significantly increased in the GLAB-supplemented group, as compared to the HD group (Fig. 3). The CPT-I stimulations or interventions in the ACC system have had beneficial effects on insulin resistance and adiposity without apparent side effects (Fåk and Bäckhed 2012). Similarly, in the present study, the GLAB strain-induced expressions of CPT-I in the liver, increases fatty acid oxidations in the hepatic mitochondria by decreasing the synthesis of acetyl-CoA carboxylase, which might provide an alternate mechanism to combat obesity.

LXRs, LXRα and LXRβ, are nuclear receptors that regulate the metabolisms of several important lipids, including cholesterol and bile acids. Schultz et al. (2000) suggested that the increase in plasma lipids occurs via LXR-mediated inductions of the SREBP-1 lipogenic program (Schultz et al. 2000). Our data also showed parallel results of LXRα and SREBP-1 levels (Fig 3). In fatty acid biosynthesis, proteases releases nuclear SREBP-1, which activates transcription of the major genes of fatty acid synthesis including ACC, FAS, SCD-1, glycerol-3-phosphate acyltransferase, and others (Bennett et al. 1995; Lopez et al. 1996; Ericsson et al. 1997).

Furthermore, GLAB has been shown to modulate the expression of PPARγ, aP2, C/EBPα, LPL and LXRα mRNA levels of the epididymal adipose tissues in mice, which are associated with adipocyte differentiations (Fig. 4). PPARγ and C/EBPα played vital roles in the early stage of adipose differentiation (Rosen et al. 2000). PPARγ and C/EBPα regulated the expressions of adipogenic genes such as CD36, leptin, GPDH and aP2 triggering the accumulation of fat in adipose tissues (Kawada et al. 2001; Berger and Moller 2002). Our results indicated that PPARγ and C/EBPα were inhibited by GLAB resulting in reduced adipogenesis indirectly confirmed by the measurement of body fat mass. Therefore, it appeared that GLAB inhibits adipogenesis by reducing or suppressing the expressions of PPARγ and C/EBPα levels. It was also reasonable to articulate that GLAB acts directly on PPARγ and C/EBPα. Activation of PPARγ induced the expression of genes that controls adiposity fatty acid metabolisms, including LPL, aP2 and LXRα. In this study, we demonstrated that GLAB reduces the expression of PPARγ target genes during adipogenesis. Therefore, we evaluated this down-regulation to determine whether it was related to the decrease that was observed at the PPARγ mRNA level. We found that GLAB reduced the expression of PPARγ target genes through inhibitions. Our in vitro study (Park et al. 2013) as well as reports from other researchers (Kim et al. 2008) suggested that treatments of 3T3-L1 cell lines with lactic acid bacteria extract modulates the adipogenesis via decreasing the expression levels of adipogenic markers such as aP2, leptin, GPDH and CD36 significantly. Therefore, our data suggested that GLAB decreases body weight fat masses by down-regulating the expressions of adipogenic transcription factor, PPARγ and C/EBPα; and adipocyte-specific gene such as aP2, LPL and LXRα. It appeared to be mediated through hepatic steatosis by down-regulating the lipogenic process via reducing the expressions of LXRα, SREBP-1 and ACC while accelerating the β-oxidations by increasing the expression of CPT-I and PPARα.

Presently, it was not clear how the supplementations of GLAB affected the number of gut microflora in LGLAB and HGLAB groups. It has been shown that obesity is closely associated with gut microbiota (Ley et al. 2006; Turnbaugh et al. 2006). For example, the relative proportion of Bacteroidetes to Firmicutes in the human gut affected body weight losses suggesting that obesity is associated with gut microbes (Ley et al. 2006). There is no data on the population size of GLAB in colonized individuals and what factors affect their numbers. Johansson et al. (1993) reported that 19 lactobacilli in human were tested for colonization and only five strains were recovered from the faeces 1 day after administration, and of these five strains (including two Lactobacillus plantarum strains) were recovered from faeces 11 days after administration. Lactobacillus strains have a general ability to colonize human intestinal mucosa, independent of dietary and physiological differences among individuals. Future studies should be performed to investigate the compositions of the dominant gut microbiota and their metabolites (bioactivities compounds) in the LGLAB and HGLAB groups.

In conclusion, we have demonstrated that lactic acid bacteria isolated from gajami sik-hae has beneficial metabolic effects. It provided evidence that GLAB supplementation decreases the food intake, weight gain, serum and liver TG. The study also observed significant differences in carnitine concentrations among carnitine fractions. GLAB supplementations decreased hepatic LXRα, SREBP-1, and ACC mRNA levels, whereas it increased the hepatic PPARα and CPT-I mRNA levels. Our study suggested that GLAB may up-regulate genes encoding enzymes involved in hepatic fatty acid oxidations by activating the CPT-I and PPARα. Additionally, GLAB reduced the mRNA levels of lipid anabolism related genes in the adipose tissues. Finally, the study established the efficacy of L. plantaum LG42 isolated from gajami sik-hae for preventing obesity with no safety or intolerance problem. GLAB is a new strain for clinical study. At present, there are no data available that could suggest the colonization of GLAB in obese subjects. However, animal study showed that Lactobacillus colonizes the intestinal walls and proliferate in the intestine of diet-induced obese mice (Arora et al. 2012). Further clinical trials at a dose ranging 106–1011 CFU day−1 required to be conducted in this context.

Acknowledgements

This research was supported by Research and Development for Regional Industry (Grant no. 10027215) funded by Ministry of Knowledge Economy.

Conflict of Interest

We do not have any conflict of interest.

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