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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 3  |  Page : 276-280

The effect of three acid-resistant isolated proteins from Lactobacillus casei on lipid and carbohydrate metabolism pathway-related genes: An In vitro study


1 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Cellular and Molecular Biology Research Center; Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Submission23-May-2021
Date of Acceptance12-Jul-2021
Date of Web Publication7-Sep-2021

Correspondence Address:
Dr. Mojgan Bandehpour
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran; Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_100_21

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  Abstract 


Background: The role of Lactobacillus casei on human health is well documented. However, little is known about their protein effects on food digestion. Therefore, in the present study, we aimed to investigate the efficacy of three proteins of L. casei on lipid and carbohydrate digestion that was identified at acidic pH in our previous study. Methods: Chaperonin (Ch), metal-dependent hydrolase (HYD), and lysozyme (LYS) proteins that were expressed by cultivated L. casei at pH 5 were extracted. HepG2 cell line was used to elucidate the effect of the considered three proteins on gene expression related to fat and glucose metabolism. The target genes were determined by Kyoto Encyclopedia of Genes and Genomes pathway analysis and extracted proteins were transfected into HepG2 cells. After 48 and 120 h, the mRNA expression of the following genes was analyzed using real-time polymerase chain reaction, insulin receptor substrate 2, (IRS-2), Protein kinase Bβ (AKT2), insulin-like growth factor 1, angiopoietin-like 4 (Angptl-4), and lipoprotein lipase (LPL). Results: The expression of all of the genes was significantly increased in comparison to control under the effect of Ch and metal-dependent HYDs after 48 h of culture. By increasing the duration of transfection from 48 h to 120 h, the expression of Angptl-4 from Ch and metal-dependent HYDs was reduced significantly, whereas the expression of LPL and Angptl-4 genes after 5 days was significantly increased in LYS compared to the last 3 days. Conclusions: L. casei secrets acidic proteins such as Ch, metal-dependent HYD, and LYS in bloodstream are involved in the digestion of carbohydrates and fats in the liver.

Keywords: Angiopoietin-like 4, chaperonin, insulin receptor substrate, insulin-like growth factor 1, Lactobacillus casei, lipoprotein lipase, lysozyme, metal-dependent hydroxylase, protein kinase Bβ, angiopoietin-like 4 protein (ptl-4)


How to cite this article:
Karimi G, Saadat M, Gheflat S, Kazemi B, Bandehpour M. The effect of three acid-resistant isolated proteins from Lactobacillus casei on lipid and carbohydrate metabolism pathway-related genes: An In vitro study. Biomed Biotechnol Res J 2021;5:276-80

How to cite this URL:
Karimi G, Saadat M, Gheflat S, Kazemi B, Bandehpour M. The effect of three acid-resistant isolated proteins from Lactobacillus casei on lipid and carbohydrate metabolism pathway-related genes: An In vitro study. Biomed Biotechnol Res J [serial online] 2021 [cited 2021 Dec 1];5:276-80. Available from: https://www.bmbtrj.org/text.asp?2021/5/3/276/325601




  Introduction Top


Probiotics are “live microorganisms” which, when administered in sufficient amounts, confer a health benefit on the host.[1] Lactobacillus casei is a Gram-positive facultative heterofermentative lactic acid bacterium which mainly uses in dairy products (fermented milk and cheese) as a nonstarter that improves flavor, ripening, and texture.[2],[3] We have reported the differences in protein expression of L. casei at pH 5 MRS (DE MAN, ROGOSA, SHARPE) media in comparison with pH 7. Among the seven identified proteins, three secreted ones (chaperonin [Ch], metal-dependent hydroxylase, and lysozyme [LYS]) with a role in metabolism were chosen.[4] Therefore, in the present study, we aimed to demonstrate the effects of secreted proteins of this strain at acidic pH (intestine pH) on the expression of genes related to carbohydrate and lipid metabolism.


  Methods Top


Selected bacterial proteins

According to our previous study[4], we selected three secreted proteins of L. casei in culture media with pH 5. They were Ch, metal-dependent hydroxylase, and LYS with 80, 60, and 70 kDa molecular weights, respectively. The identified protein spots in stained two-dimensional gel were excised and crushed using the Amicon Ultra-0.5 Centrifugal Filter Devices, and the Phosphate Buffer Saline (PBS) solution with 50 μg/μL protein was prepared for further analysis. Following the ethics guidelines of the principles of research , Shahid Beheshti University of Medical Sciences (IR.SBMU.RETECH.REC.1397.1345) approved this research.

Cell line, culture conditions, and treatment with proteins

Human hepatocellular carcinoma (HepG2) cell line as a liver source cell for metabolic study was purchased from the Pasteur Institute of Iran and cultured in roswell park memorial institute (RPMI) with 10% fetal bovine serum and 100 unit/mL penicillin–100 μg/mL streptomycin. Cells were incubated at 37°C under a 5% CO2/95% air atmosphere. A hemocytometer (Bright-Line Hemacytometer, Sigma-Aldrich, Mississauga, ON, Canada) was used to count the cells. After counting and viability assay by trypan blue solution, cultured cells were plated on a 6-well plate. The media of cells were changed every alternative day until transfection. Then, 50 μg of each protein solution extracted from the gel was transfected into 106 trypsinized cells under 400V voltage in 5 ms five times with a 10-s interval for each time.

Lipid and carbohydrate pathway analysis and gene selection

All genes selected in this study were collected from Comparative Toxicogenomics Database and GeneCards resources. These databases provide information about interactions between genes and proteins. Functional analysis was performed using the Enrichr database (http://amp. pharm.mssm.edu/Enrichr).

The reviewed criteria include Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), Cellular Component, and online mendelian inheritance in man (OMIM). All found genes from the different sources were fed into STRING database (https://string-db.org) to construct the networks.

RNA extraction, strand-specific real-time polymerase chain reaction assay, and quantitative real-time polymerase chain reaction

Total RNA was extracted from the HepG2 cells using an RNA extraction kit (GeneAll, Qiagen, Germany) after 48 and 120 h from the transfection process. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in Eppendorf Mastercycler Real Lex using Roche Applied Science, Mannheim, Germany kit. cDNA was synthesized as follows; 10 μL RNA,0.5 μL oligo-dT, 0.2 μL deoxyribonucleotide triphosphate (dNTP), 0.5 μL M-MuLV enzyme, 6 μL buffer RT, and up to 30 μL diethyl pyrocarbonate (DEPC) water. Samples were subjected to 95°C for 5 min, followed by 37°C for 30 min, 42°C for 1 h, and 70°C for 10 min. The list of primers used in this study is described in [Table 1]. The reactions were prepared in the mixture of 6 μL Ampliqon real-time master mix, 0.25 μL carboxyrhodamine (ROX), 2 μL cDNA, 2 μL primer mixed, and 3 μL deionized water. Thermal cycling conditions were 10 s at 95°C followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 54°C for 15 s, and extension at 72°C for 20 s. The average cycle threshold (Ct) was determined for each sample and normalized to β-actin housekeeping gene. The relative fold change was analyzed by REST 2009 Software.
Table 1: List of primers used for quantitative real-time polymerase chain reaction

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Statistical analysis

Data analysis was carried out with SPSS version 20 software (IBM, Chicago, IL, USA). Means were analyzed by one-way ANOVA, and the Tukey's test was used to calculate the mean of the data. REST 2009 Software (Qiagen, Hilden, Germany) was used to analyze qRT-PCR data. P < 0.05 indicated statistical significance.


  Results Top


Metabolic pathway of the lipid and carbohydrate

The Enrichr analysis in the pathway section demonstrated that the most common lipid and carbohydrate metabolism pathways are linked in OMIM analysis. The top five results of KEGG database analysis contained regulation of lipolysis in adipocytes, insulin signaling pathway, glycosphingolipid biosynthesis, carbohydrate digestion and absorption, glycolysis, and gluconeogenesis.

Functional analysis by GeneMANIA

GeneMANIA database was also applied for functional analysis. [Figure 1] shows the network feature with the most critical nodes based on functional analysis of hub genes. They are determined in this network. Some of the nodes are colored differently to represent different biological functions. The hub genes with the highest number of colors represented important biological functions in the lipid and carbohydrate metabolism included lipoprotein lipase (LPL), insulin-like growth factor (IGF), AKT1, angiopoietin-like 4 (Angptl-4), and insulin receptor substrate (IRS).
Figure 1: The network feature with the most critical nodes based on functional analysis of hub genes by GeneMANIA

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The level of the metabolism pathway gene expression

The expression of LPL, Angptl-4, IGF, AKT, and IRS was significantly increased in comparison to control under the effect of Ch and metal-dependent hydrolase (HYD) proteins after 48 h of culture, whereas any changes were not seen in the mRNA expression of metabolic genes under the effect of LYS protein [Figure 2].
Figure 2: Comparative expression of genes of HepG2 cell line under the effect of three secreted proteins (chaperonin [Ch], metal-dependent hydrolases [HYDs], and lysozyme [LYS]) of Lactobacillus casei at pH 5 after 48 h of transfection. LPL: Lipoprotein lipase, Angptl-4: Angiopoietin-like protein-4, IGF: Insulin-like growth factor, AKT: Protein kinase Bβ, IRS: Insulin receptor substrate

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By increasing the duration of transfection from 48 h to 120 h, the expression of Angptl-4 from Ch and metal-dependent HYDs was reduced significantly, whereas the expression of LPL and Angptl-4 genes after 5 days were significantly increased in LYS compared to the last 3 days [Figure 3]. Ch and metal-dependent hydrolysis indicate a downregulation of IRS-2, AKT, and IGF-1 genes after a longer transfection (120 h), whereas the expression of these three genes was not shown changes in treatment with LYS protein.
Figure 3: Comparative expression of genes of HepG2 cell line under the effect of three secreted proteins (chaperonin [Ch], metal-dependent hydrolases [HYDs], and lysozyme [LYS]) of Lactobacillus casei at pH 5 after 120 h of transfection. LPL: Lipoprotein lipase, ANGPTL-4: Angiopoietin-like protein-4, IGF: Insulin-like growth factor, AKT: Protein kinase Bβ, IRS: Insulin receptor substrate

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  Discussion Top


The acid tolerance feature of L. casei makes it a good source for probiotic supplementation.[5] It has been clearly shown that L. casei leads to the improvement in immunity, allergies,[6],[7] hyperlipidemia,[8],[9] digestion,[10],[11] modulation of food intake, body weight, and body metabolic functions through the gastrointestinal pathway[12],[13]

The results of the present study showed a significant upregulation of Angptl-4, LPL, IGF-1, IRS, and AKT in Ch and metal-dependent hydrolysis after 48 h of the HepG2 cell line transfection. The expression of all the aforementioned genes except significantly reduced compared to the control group when the duration of the transfection increased from 48 to 120 h. In LYS, the expression of all genes was significantly lower than control after 48 h of transfection. A similar trend was observed for IRS, IGF-1, and AKT after 120 h, whereas LPL and Angptl-4 were both upregulated, and this upregulation remained lower than the control group.

Most recent studies have indicated the safety and potential ability of this bacterium in regulating the digestive system. It has been reported that L. casei supplementation improves insulin signaling and glucose transport through the regulation of IRS-1/AKT pathway.[14] Angptl-4 is a circulating LPL inhibitor that controls triglyceride deposition into adipocytes and has been reported to be regulated by gut microbes. Some earlier studies show that L. casei supplementation decreases fat storage by increasing Angptl-2.[15]

Although the efficacy of probiotics and different strains of bacteria on body health and functions has been investigated and well documented, our knowledge regarding the efficacy of proteins secreted from L. casei in response to different pH on food metabolisms is so scarce.

Chs are proteins that prevent aggregation by providing suitable conditions for other proteins to be folded correctly. One of the potent regulators of triglyceride metabolism in the plasma is Angptl-4, a 50-kD glycosylated secretory protein.[16],[17] Results of the in vitro and in vivo experiments suggest a downward trend of LPL activity as a result of Angptl-4. LPL, which is mainly synthesized in adipocytes and myocytes, is a key enzyme in the metabolism of triglyceride in plasma.[18],[19] In fact, the mechanism effect of Angptl-4 on LPL is as follows: Angptl-4 acts as an extracellular unfolding molecular chaperone and deactivates the LPL through dissociating the catalytically active LPL dimer into inactive LPL monomer.[20],[21] Other roles of Angptl-4 which can be taken into account despite its effect on triglyceride (TG) metabolism are glucose metabolism,[22] angiogenesis,[23],[24] cancer development,[25] and wound healing.[26] In the current study, after 48 h of transfection, the secretion of Angptl-4, an unfolding molecular chaperon of LPL, increased LPL expression also upregulated. LPL upregulation leading to a decrease in TG and increasing high-density lipoprotein (HDL). However, after 120 h of transfection, Angptl-4 and LPL expression decreased. Hence, the TG level in the plasma will be increased. This inhibitory effect of Angptl-4 on LPL is a way to produce energy during the fasting or starvation. This result is in line with the results of the earlier studies about the effects of L. casei on lipid profile during fasting and satiety through decreasing LPL.[17],[27] IRS-1 appears to have a key role in the insulin-stimulated signal transduction pathway and mutation in this gene may result in diabetes mellitus.[28] In addition, serine/threonine kinase protein kinase B (PKB/AKT) has been shown to play a critical role in divers and crucial cellular functions, including glycogen metabolism. Furthermore, recent studies showed that AKT impairment is involved in insulin resistance.[29] In the body, after ingestion of carbohydrates and glucose, insulin is secret from the pancreas and therefore glucose uptakes by the targeted tissues through binding to insulin receptors[30] and activating receptor autophosphorylation. As a result of this autophosphorylation, a downstream signaling cascade happens through the phosphorylation of the IRSs, IRS (IRS-1 or IRS-2), followed by AKT (AKT1 and AKT2),[31] consequently glucose transporter 4 translocate, and eases the glucose uptake[32] [Figure 2]. Based on the current study, after 48 h of transfection, the expression of IRS and AKT in Ch and metal-dependent hydrolysis significantly increased, which in turn the blood glucose would be decreased after consuming a high carbohydrate diet. Despite lower blood glucose in response to higher IRS and AKT, the glycogen content of muscle increase through higher glycogenesis.

IGF-1 is a polypeptide trophic factor[33] that, via binding to its receptor, causes receptor autophosphorylation and the activation of intrinsic tyrosine kinase. Activated receptor kinase phosphorylates proteins like IRS-2,[34] leading to the activation of multiple signaling pathways, including the PI3K/AKT pathways.[34],[35] The results of the current study showed a significant upregulation of genes related to the insulin signaling pathway. Therefore, it seems that both Ch and metal-dependent HYD can increase glycogen and protein synthesis while decreasing lipolysis and gluconeogenesis, which all these functions together reduce the risk of obesity, diabetes, and metabolic syndrome. The metal-dependent HYD is a kind of enzymes that catalyze carbohydrates, peptides/proteins, nucleotides, xenobiotic, and phosphodiesters. The function of this enzyme is through using a combination of the amino acid side chain and one or more metal ion cofactors.[36] The results for LYS are quite different. It seems that LYS which is an antimicrobial enzyme suppressed the insulin signaling pathway and lipolysis. Therefore, glycogen and protein synthesis stimulated whereas lipolysis and gluconeogenesis suppressed.


  Conclusions Top


A significant upregulation of Angptl-4, LPL, IGF-1, IRS, and AKT in Ch and metal-dependent hydrolysis after 48 h of the transfection was observed. The results of the current study showed that upregulation of genes from Ch and methyl-dependent hydrolysis after 48 h of transfection might increase the fat and carbohydrate metabolism, Which it leads to lower triglycerides and sugar and also increases HDL. These effects together will decrease the risk of obesity, diabetes mellitus, and metabolic syndrome.

Acknowledgment

The authors would like to thank the staff of Cellular and Molecular Research Center at Shahid Beheshti University of Medical Sciences.

Financial support and sponsorship

This study was supported by the National Elites Foundation (Grant number 34787) and Shahid Beheshti University of Medical Sciences (Grant number 13099).

Conflicts of interest

There are no conflicts of interest.



 
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