key: cord-0959259-az03khng
authors: Lee, Chul Sang; Park, Mi Hyun; Kim, Sae Hun
title: Selection and Characterization of Probiotic Bacteria Exhibiting Antiadipogenic Potential in 3T3-L1 Preadipocytes
date: 2021-05-07
journal: Probiotics Antimicrob Proteins
DOI: 10.1007/s12602-021-09793-5
sha: a3d3215f75a131a5cea28ff5c1cf0e8df1d785de
doc_id: 959259
cord_uid: az03khng

Abnormal adipocyte growth, distinguished by an increase in cell numbers and cellular differentiation, is regarded as a major pathological characteristic of obesity. Thus, inhibition of adipogenic differentiation in adipocytes could prevent obesity. Recently, certain probiotic stains have been reported to regulate lipid metabolism in vitro and/or in vivo. In this backdrop, this study aimed to investigate basic probiotic properties and potential antiobesity characteristics of mouse 3T3-L1 preadipocytes. Six lactic acid bacteria (LAB) strains were prescreened for their cholesterol-lowering activity, antioxidant activity, and survival at low pH and in a solution containing bile salts. These six strains were investigated for antiadipogenic activity by employing 3T3-L1 mouse preadipocytes. 3T3-L1 cells were treated with selected strains during the differentiation process. Lactobacillus johnsonii 3121 and Lactobacillus rhamnosus 86 were found to be more capable of reducing triglyceride and lipid accumulation, as compared to control group, which are fully differentiated 3T3-L1 adipocytes. These strains also inhibited adipocyte differentiation by downregulating the adipogenic transcription factor in 3T3-L1 adipocytes. Taken together, these results indicate that L. johnsonni 3121 and L. rhamnosus 86 could potentially act as probiotic bacteria and prevent fat accumulation by regulating adipogenesis-related markers.

Obesity, characterized by excessive fat storage in tissues, presents excessive risks to an individual's health, and is, therefore, considered a major risk factor for morbidity and mortality worldwide. Moreover, obesity is associated with diseases such as type 2 diabetes, hypercholesterolemia, hypertension, and hepatic steatosis, termed as the metabolic syndrome (MS), which is a crucial problem that needs to be resolved for improved quality of life [1] . Obesity occurs from a combination of less active lifestyle and a failure to reduce dietary energy intake in line with reduced total energy expenditure arising from reduced physical activity [2] . Recently, lockdown due to the coronavirus disease 2019 (COVID-19) pandemic have caused disruptions in daily lifestyles, thereby increasing the global prevalence of obesity [3] .

Lactic acid bacteria (LAB) are lactic acid-producing gram-positive bacteria that have been exploited in association with various health benefits and are widespread in nature. The most common health-promoting LABs are classified as probiotics. Probiotics are live microorganisms that provide beneficial health effects to the host when administered in adequate amounts [4] . Numerous beneficial effects of probiotics, such as anticonstipation, antiosteoporotic, and antiobesity effects, are frequently suggested by many research studies [5] [6] [7] . The health benefits of probiotic bacteria are usually explained by the protective effects of the interaction of consumed probiotics with commensal gut microbiota in the host [8] . It is, therefore, essential for LABs to withstand low pH conditions in the stomach and the presence of secreted bile salts in the small intestine [9] . Moreover, all probiotics meant for consumption must undergo thorough safety checks, including the absence of harmful metabolite (such as urease and gelatinase) secretion [10] .

Gut microbiota (GM) is defined as the community of bacteria inhabiting the gastrointestinal (GI) tract that has a complicated and mutually beneficial relationship with the host. Recently, the control on host gut microbiota exerted by probiotics has emerged as an important factor affecting health in the host, including obesity [11] . Dysbiosis, a form of microbial imbalance induced by a high-fat diet, is one of the most important factors in the development of obesity [12] . Colonization of intestinal microbes from obese mice to germ-free mice resulted in increased body weight gain and fat accumulation in the latter, thereby providing robust evidence of the association between obesity and GM [13] . Taken together, employing probiotic bacteria to target and alter high-fat diet-induced disturbance of gut microbiota might be an effective approach to treat obesity.

The present work was conducted in order to screen novel potential probiotics by testing basic probiotic properties such as resistance to low pH and bile salts. We have previously prescreened 61 strains out of 151 probiotic candidates based on their survival rate in a low pH environment. Therefore, the objective of this study was to confirm the survivability of these prescreened strains and explore their adaptation to the bile salt environment. Moreover, their cholesterollowering and antioxidant activities were probed to identify strains capable of exerting antiadipogenic effects in 3T3-L1 adipocyte cells in vitro.

The bacterial strains used in this study were obtained from the Probiotics Research Laboratory, CKDBio Research Institute (Ansan, South Korea) and the Department of Food Bioscience and Technology, Korea University (Seoul, Korea). Strains were individually grown in Man, Rogosa and Sharpe (MRS) broth (BD Co., Franklin Lakes, NJ, USA) for 18 h at 37 °C. The strains were sub-cultured thrice prior to each experiment.

Tolerance to acid and bile conditions was assessed as reported previously [14] , with minor modifications. Activated bacterial strains were harvested by centrifugation (10,000×g, 5 min, 4 °C) and washed twice in phosphatebuffered saline (PBS) prior to inoculation in acidified and bile-supplemented MRS broth. To evaluate the ability of the strains to grow under acidic conditions, MRS broth was acidified to pH 2.5 and supplemented with 1000 U mL −1 of pepsin (Sigma-Aldrich, St. Louis, MO, USA). The washed bacterial cells were inoculated into the acidified broth and incubated for 3 h at 37 °C. Acid tolerance was evaluated by comparing the number of colonies at 0 h with that at 3 h after incubation. The ability of strains to grow in the presence of bile was evaluated using MRS broth containing 0.3% oxgall (Sigma-Aldrich, St. Louis, MO, USA). The broth was inoculated with the washed bacterial cells and incubated for 24 h at 37 °C. The viability of strains in the bile condition was assessed by comparing the number of colonies at 0 h with that at 24 h after incubation. A 100-μL aliquot of each sample was spread in triplicate onto MRS agar plates in order to count the number of viable bacterial colonies on the surface. Acid and bile tolerance rates were evaluated by calculating the survival rate after 3 and 24 h of incubation at 37 °C, respectively.

To evaluate bacterial safety, urease and gelatin liquefaction activities of the strains were assessed. A gelatin medium containing 12% gelatin (Sigma-Aldrich, St. Louis, MO, USA) was inoculated with activated strains at a concentration of 1% and incubated for 48 h at 37 °C. Gelatin liquefaction of strains was assessed by storing medium in a refrigerator for 24 h and checking whether gelatin was hydrolyzed or not. Urease activity of the strains was assessed in urea agar media containing 2% urea (Sigma-Aldrich, St. Louis, MO, USA) and 0.0012% phenol red (Sigma-Aldrich, St. Louis, MO, USA). Each strain was streaked in urea agar media and incubated for 48 h at 37 °C. Media was kept under observation to check whether the color changed from yellow to pink. Change in media color to pink indicates that the strain has the ability to hydrolyze urea.

A colorimetric assay to assess the cholesterol-lowering activity of strains was conducted [15] , with minor modifications. MRS-THIO broth was prepared by adding 0.2% sodium thioglycolate (Sigma Aldrich, St. Louis, MO, USA) and 0.3% oxgall (Sigma-Aldrich, St. Louis, MO, USA) to MRS broth. The broth was further supplemented with filtersterilized cholesterol micelles (1 mg mL −1 in 0.4 M sucrose solution) at a final concentration of 100 µg mL −1 . The broth was inoculated with 1% of the selected strains and incubated for 20 h at 37 °C. Uninoculated broth was used as a negative control. After incubation, cells were removed by centrifugation (10,000×g, 10 min, 4 °C) and total cholesterol content of the supernatant was measured using a colorimetric method [16] , with minor modifications. The supernatant (2 mL) was mixed with 3 mL of 97% ethanol (Sigma-Aldrich, St. Louis, MO, USA) and 2 mL of 50% potassium hydroxide (w/v), and then heated at 60 °C for 10 min. After cooling to room temperature, 5 mL of hexane was added and the resultant mixture was vortexed. Next, 4 mL of the hexane layer was transferred into a tube and dried using nitrogen gas. o-Phthalaldehyde (Sigma-Aldrich, St. Louis, MO, USA) (0.55 mg dissolved in 1 mL of acetic acid) was added to the tube and incubated for 10 min. Further, 2 mL of sulfuric acid was added and mixed for 1 min. Absorbance was measured spectrophotometrically at 550 nm using a VersaMax™ Tunable Microplate Reader (Molecular Devices, San Jose, CA, USA). To determine the concentration of cholesterol, the recorded A 550 values were compared with a standard curve. Results were expressed as micrograms of cholesterol per milliliter. The cholesterol-lowering activity was calculated using the following equation:

To investigate the antioxidant activity of selected strains, live cells of activated strains were assessed by the DPPH method [17] , with minor modifications. An aliquot of bacterial live cell (800 μL) was added to 1 mL of a 0.2 mM methanolic solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich, St. Louis, MO, USA) reagent. The suspension was mixed thoroughly and allowed to stand for 30 min at 37 °C in dark. After incubation, the supernatant from each sample was collected by centrifugation (10,000×g, 1 min, 4 °C). The absorbance of the supernatant was measured spectrophotometrically at 517 nm using a VersaMax™ tunable microplate reader (Molecular Device, San Jose, CA, USA). For comparison, PBS and 0.2 mM ascorbic acid (Sigma-Aldrich, USA) were used as negative and positive controls, respectively. The antioxidant activity was expressed as the percentage of DPPH radical scavenging activity using the following equation:

For the cell experiments, Leuconoc mesenteroides 7 (99% identity: accession no. CP003101), Lactobacillus rhamnosus 86 (99% identity: accession no. NR_113332.1), Lactobacillus sakei 105 (99% identity: accession no. CR936503), Lactobacillus casei 911 (99% identity: accession no. KP326371.1), Lactobacillus johnsonii 3121 (99% identity: accession no. NR_117574.1), and Lactobacillus plantarum MA2 (99% identity: accession no. NR_113338.1) were selected based on their probiotic potentials. The mouse preadipocyte cell line 3T3-L1 was obtained from the American

DPPH radical scavenging activity (%) = OD 517(control) − OD 517(sample)

Type Culture Collection (ATCC, Rockville, MD, USA). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) (GE Healthcare, Chicago, IL, USA) containing 10% (v/v) fetal bovine serum (FBS) (Gibco, Dublin, Ireland) and 1% (v/v) antibiotic solution (penicillin and streptomycin solution, 100 U mL −1 ) (GE Healthcare, Chicago, IL, USA). Cells were incubated at 37 °C and 5% CO 2 in a humidified atmosphere, and the culture medium was replaced with fresh DMEM every 2 days. Cells were transferred at 60% confluence using 1 mL of trypsin-EDTA (GE Healthcare, Chicago, IL, USA). Cell differentiation was performed in 6-well cell culture plates (Sigma-Aldrich, St. Louis, MO, USA). Cells were seeded at a density of 8 × 10 4 cells/well in maintenance medium (DMEM containing 10% FBS and 1% antibiotic solution) and grown to 100% confluence. At 100% confluence, the culture medium was replaced with fresh maintenance medium and incubated for 48 h to induce cell arrest.

After 48 h, the maintenance medium was replaced with induction medium ( 

The cytotoxicity of the selected strains was assessed by MTT assay. The mouse pre-adipocyte 3T3-L1 cells were seeded into 96-well cell culture plates (Sigma-Aldrich, St. Louis, MO, USA) at a density of 1 × 10 4 cells/well and incubated at 37 °C and 5% CO 2 condition. After 24 h, the medium was replaced with maintenance medium containing each of the heat-killed strains at a concentration of 10 8 CFU mL −1 and incubated for 24 or 48 h. The medium was subsequently removed, and the remaining cells were used for the MTT assay. A 10-μL aliquot of a 12 mM stock solution of 

IBM SPSS Statistics software version 25.0 (IBM Corp, Armonk, NY, USA) was used to analyze the data. One-way analysis of variance was used to compare sample means. Multiple comparisons of means were performed using Tukey's post hoc test. P < 0.05 was considered statistically significant.

Probiotic functionality depends on the ability of the strain to confer health advantages on a host upon oral consumption of viable cells. Therefore, we studied the tolerance of bacterial strains to acidic conditions (artificial gastric juice) and the presence of bile salt (artificial intestinal stress). We set a cutoff value of 0.5 for the log CFU value at 3 h subtracted from that at 0 h, to indicate intolerance to low pH conditions. The same cutoff value was also considered for the bile salt condition. Previously, we have prescreened 61 strains out of 151 candidates that qualified for further investigation based on this cutoff value for resistance to acid and bile salts (data not shown). In order to increase the accuracy of identifying potential probiotics from the prescreened strains, we double-checked the acid and bile salt tolerance of the 61 prescreened strains (Figs. 1a-c  and 2a-c) . In the main screening process, the tolerance rates to acid and bile conditions were evaluated by calculating the survival rate after 3 h in acidic broth and 24 h in bile-supplemented broth. Twenty out of the 61 strains exhibited a high survival rate (more than 95%) after incubation in an acidic environment for 3 h (Fig. 1a) . Moreover, these 20 strains also showed adaptability to high bile conditions, as evident from the more than 120% survival rate (Fig. 2a) . Thereafter, the safety of the prescreened strains was verified using urease and gelatinase agar, and all 61 strains were evaluated to be safe ( Table 2 ).

In order to confirm the functionality of the 61 strains, we assessed their cholesterol lowering activity in the presence of 0.3% oxgall (Fig. 3a-c) . All strains were capable of removing cholesterol from the MRS-THIO broth after 18 h of incubation. However, the extent of cholesterol reduction was strain-specific. The top 20 of the 61 strains exhibited over 27% reduction in cholesterol concentration (Fig. 3a) . Moreover, Lactobacillus plantarum MA2 and L. johnsonni 3121 were identified to exhibit the highest cholesterol-lowering activities (over 50%) in MRS-THIO broth.

We further examined the functional properties of the 61 prescreened strains by measuring the radical scavenging activity of DPPH (Fig. 4a-c) . DPPH is a widely used chemical compound for evaluating the antioxidant activity of cell-free supernatant of bacteria. Leuconostoc mesenteroides 7, Lactobacillus casei 911, and Lactobacillus sakei 105 exhibited over 55% DPPH scavenging activities. Moreover, the top 20 strains were also characterized by over 30% DPPH scavenging capacity, which was taken as the minimum cutoff value for in vitro experiments (Fig. 4a) .

Further investigations were continued by narrowing down the 61 strains to 6 probiotic candidates that qualified in the top 20 with respect to the parameters of functionality (acid tolerance, bile tolerance, cholesterol-lowering activity, and DPPH scavenging activity) explored in this study. Therefore, L. johnsonii 3121, L. rhamnosus 86, L. plantarum MA2, L. sakei 105, L. casei 911, and Leuc. mesenteroides 7 were selected for in vitro experiments using 3T3-L1 cells. The MTT assay was used to determine cytotoxic effect of the six heat-killed probiotic candidates on 3T3-L1 cell proliferation (Fig. 5) . Our results showed that none of the six heat-killed strains affected the viability of 3T3-L1 cells. Culturing cells with these six heat-killed strains did not result in significant differences, compared to the control group, at both 24 and 48 h of incubation (P > 0.05). These data indicate that the six probiotic candidates displayed negligible cytotoxicity and caused no damage to the cells.

In order to evaluate the antiadipogenic effects of the heatkilled bacterial strains, the 3T3-L1 pre-adipocytes were treated with or without the heat-killed probiotic candidates for 8 days (Fig. 6a-c) . The differentiated 3T3-L1 cells were subsequently stained with oil red O for visualization of lipid droplets and treatment with L. rhamnosus 86 and L. johnsonii 3121 were found to significantly inhibit lipid accumulation (Fig. 6c) . The microscopybased imaging and examination of oil red O-stained lipid droplets were performed by eluting in isopropanol. These data showed that L. rhamnosus 86 and L. johnsonii 3121 significantly downregulated the intracellular lipid content compared to control 3T3-L1 cells (P < 0.05) (Fig. 6b) . Moreover, the decreased lipid accumulation activity was also supported by trends in the TG content in 3T3-L1 cells (Fig. 6a) . Both L. rhamnosus 86 and L. johnsonii 3121 were capable of significantly reducing the TG content compared to the control (Fig. 6a) . Taken together, these results suggest that treating 3T3-L1 cells with heatkilled L. rhamnosus 86 and L. johnsonii 3121 during differentiation could lead to inhibition of adipogenesis by suppressing lipid accumulation. enhancer-binding protein α (C/EBPα), and adipocyte protein 2 (aP2), were evaluated in 3T3-L1 preadipocytes treated with L. rhamnosus 86 and L. johnsonii 3121 (Fig. 7) . All three genes were significantly downregulated in pre-adipocytes, which confirms that the differentiation process was successfully induced in mature adipocytes. Interestingly, 3T3-L1 cells treated with L. rhamnosus 86 and L. johnsonii 3121 showed significantly downregulated gene expression levels of PPARγ, C/EBPα, and aP2 (P < 0.05), which represent the key adipogenic transcription factors related to adipocyte differentiation. These results indicate that L. rhamnosus 86 and L. johnsonii 3121 decrease the amount of intracellular lipid accumulation in 3T3-L1 adipocytes by downregulating the expression levels of genes related to adipogenesis.

The present study was designed to identify novel probiotics with antiadipogenic capacity. Bacteria must be able to survive acidic conditions in the stomach and resist bile acids in order to exert their beneficial effect on the host and function as probiotics [9] . Therefore, in this study, artificial stress conditions of the human GI tract were mimicked. The first stress condition that probiotic bacteria face is the low pH environment in the stomach where gastric acid is secreted [22] . The average pH of secreted gastric acid is 2.5. This can damage the cell membrane of the bacteria and kill it before it reaches the gut and exerts its beneficial effects. Next, bacteria must resist the acidic bile conditions prevalent at the start of the small intestine; this is another crucial requirement for bacteria to act as probiotics. Bile is known to play an essential role in specific and nonspecific gut defense systems, and the intensity of its inhibitory effect is determined primarily by the bile salt concentration [23] . On the other hand, bile salts can disrupt bacterial cell membranes, which is toxic to living bacterial cells; therefore, bile tolerance is considered an important characteristic of probiotics, which enables bacterial survival, growth, and subsequent positive action in the GI tract [24] . We have previously identified 61 strains, out of 151 candidates, that exhibit high resistance to bile and low pH conditions. The present work was carried out with the objective of selecting the top 20 strains out of the 61 prescreened ones, which show the highest tolerance to both low pH and bile salt environments. Several studies have demonstrated that some LABs have high survivability in bile salt containing medium due to the presence of genes coding for bile salt hydrolases [25] . Moreover, safety evaluation is another important characteristic needed to be considered while screening for potential probiotics. The absence of urease and gelatinase activities represents one of the crucial factors that underscore the safety of probiotic consumption. Urease is an enzyme that catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thereby playing a key role in urea-related diseases such as urolithiasis [26] . Gelatinase is a protease that is capable of hydrolyzing gelatin, which may lead to tissue necrosis and damage to the body [27] . In our study, all 61 prescreened strains showed negligible action with respect to both of these factors. In order to confirm the functional characteristics of these 61 strains, their antioxidant and cholesterol-lowering activities were evaluated. We anticipated that the most important functional ability needed for LABs to exert antiobesity effects could be cholesterol reducing activity. Kumar and colleagues have earlier suggested that cholesterol-lowering probiotics could be potential biotherapeutics for treating metabolic diseases [28] . Several LAB strains have been reported to possess the ability to assimilate and uptake cholesterol by adsorbing cholesterol into the cell wall, thereby assimilating it into the cell membrane [29] [30] [31] . Our study demonstrated that the top 20 strains exhibited over 27% reduction in cholesterol. Moreover, L. plantarum MA2 and L. johnsonni 3121 were capable of lowering cholesterol levels by more than 50%. For the second functionality, we evaluated the radical scavenging activity of the 61 strains. Since chronic oxidative stress is a major cause of metabolic diseases such as obesity, the antioxidant activity of LAB strains could be an ideal therapeutic method for treating obesity. Previous studies have shown that oxidative stress in accumulated fat mediates the development of obesity-associated MS via the dysregulation of adipocytokine production and increased ROS production [32] [33] [34] . 20 . Since the objective of this study was to screen for probiotics with antiobesity effects, the cytotoxicity and lipid accumulation levels were examined using 3T3-L1 mouse pre-adipocyte cells. The proliferation of 3T3-L1 cells was not affected by the six strains, indicating that none of these strains were cytotoxic to adipocytes. Further examination of the six strains was performed by confirming the decreased TG content and downregulated lipid accumulation in 3T3-L1 cells. Among the six strains, only L. johnsonii 3121 and L. rhamnosus 86 were capable of significantly decreasing the TG content and lipid accumulation in fully differentiated adipocytes. To confirm this antiadipogenic effect, the expression of adipogenesis-related genes was examined by qRT-PCR. Several studies have demonstrated the inhibition of adipogenesis in adipocytes on treatment with functional bacterial strains, which regulate adipogenic transcription factors [35, 36] . Similar findings were observed in our study. The qRT-PCR analysis revealed that both L. johnsonii 3121 and L. rhamnosus 86 significantly decreased the gene expression levels of PPARγ, C/EBPα, and aP2, which are known to control adipocyte differentiation [37] . The early stage of adipocyte differentiation is known to be regulated by PPARγ and C/EBPα, which are critical transcription factors in adipogenesis [38] . Moreover, PPARγ regulates expression of genes involved in lipid metabolism (such as aP2), thereby triggering intracellular fat accumulation [39] . Further, aP2 is a carrier protein of fatty acids which is expressed in mature adipocytes. Interestingly, mRNA expression and protein levels of aP2 are known to markedly increased on increased PPARγ gene expression, which highlights the role of PPARγ as an aP2 regulator [40] . Based on these results, our study clearly indicates that the inhibited adipogenesis and lipid accumulation effects exerted by heat-killed L. johnsonii 3121 and L. rhamnosus 86 in mouse preadipocyte cells are mediated by the suppression of key adipogenic transcription factors related to adipocyte differentiation.

This study aimed to screen functional probiotic strains with antiobesity effects and identified L. johnsonii 3121 and L. rhamnosus 86 as potentially effective antiobesity probiotic strains. We established five steps to screen potential probiotic strains with antiobesity functions (Fig. 8) .

Sixty-one novel strains were prescreened from 151 candidates for their ability to show resistance to acidic and bilecontaining environment. The functional aspects of these 61 strains were assessed in terms of their cholesterol-lowering and antioxidant activities. Based on the data regarding acid tolerance, bile tolerance, cholesterol reduction, and antioxidant activity, L. johnsonii 3121, L. rhamnosus 86, L. plantarum MA2, L. sakei 105, L. casei 911, and Leuc. mesenteroides 7 were selected for further studies, since these strains exhibited their probiotic potential by qualifying among the top 20 strains in all four parameters measured. Of the six strains, only L. johnsonii 3121 and L. rhamnosus 86 were found to be capable of lowering TG content and lipid accumulation in 3T3-L1 cells. Further, L. johnsonii 3121 and L. rhamnosus 86 could inhibit adipogenic differentiation in 3T3-L1 cells by downregulating the expression levels of adipogenic transcription factors PPARγ, C/EBPα, and aP2. Taken together, these results identify L. rhamnosus 86 and L. johnsonii 3121 as potential therapeutic agents possessing antiobesity effects. Moreover, in vivo trials are under progress in animal models of high-fat diet-induced obesity in order to further confirm their functionality as antiobesity probiotics.

Funding This work was financially supported by grants funded by the Chong Kun Dang Bio and Korea University Grant.

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics Approval This article does not contain any studies with human or animal subjects.

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The authors have no conflicts of interest to declare that are relevant to the content of this article.