AbstractDespite the widespread adoption of gluten-free diets for weight management, the relationship between gluten intake and obesity remains unclear because of the limited number of controlled studies available in the literature. Furthermore, there is ongoing debate regarding the impact of gluten-containing diets on the gut microbiota. This study aimed to investigate the effects of gluten consumption on the body weight and intestinal microbiota of mice fed a high-fat diet. Twenty-four Bagg albino laboratory-bred mice (BALB/c) were randomly divided into four groups for oral gavage feeding: standard diet control (SDC), standard diet + 5 mg/day gluten (SD + gluten), high-fat diet control (HFDC), and high-fat diet + 5 mg/day gluten (HFD + gluten). Each subject’s body weight was measured and recorded weekly. For microbiota analysis, fecal samples were collected weekly from the cages after overnight cage changes. The microbiota was analyzed using via the 16 S ribosomal ribonucleic acid (rRNA) method. Compared with the control diet, both gluten consumption and a high fat diet significantly increased weight gain (p < 0.05). No significant difference was observed in the total mesophilic aerobic bacterial count among the groups (p > 0.05). However, the addition of gluten to the diet positively affected Lactobacillus bulgaricus (p < 0.05). Conversely, gluten-containing diets negatively impacted the total coliform bacteria and Escherichia coli counts in the gut (p < 0.05). These findings suggest that gluten, when combined with either a normal diet or a high-fat diet, contributes to weight gain while exerting positive effects on the intestinal microbiota.
IntroductionChanges in the human microbiota that occur with industrialization are thought to be the underlying factor in the dramatic increase in diseases such as obesity and metabolic diseases. Microbiota describes the living microorganisms found in a defined environment, such as skin and gastrointestinal system, while the microbiome includes their genomes, metabolites and environmental factors1,2. Among these, the gut microbiota plays an important role in processes related to weight regulation3,4. Studies have shown that severe obesity is linked to reduced microbial diversity and abundance in the gut, with obese individuals having different microbiota compositions compared to lean individuals5,6. Obesity-associated microbiota may contribute to weight gain by increasing the production of short-chain fatty acids (SCFAs), such as butyrate, which provide extra energy as a result contributing to obesity7. The macro- and micronutrient contents of the diet are recognized as critical factors in shaping the gut microbiota8. However, the specific effects of protein intake from different dietary sources on obesity development and the composition of the gut microbiota are not fully understood9. Wheat gluten is considered a beneficial dietary protein, as it provides amino acids, such as glutamine and glutamic acid, which play a role in modulating gut immunity10. Previous studies have reported an increase in beneficial bacteria such as Lactobacillus while others have noted negative effects, including an increase in potentially pathogenic bacteria such as Clostridium XI alongside a decline in beneficial bacteria like Lactobacillus5,10,11,12. Research on celiac disease patients has shown that a gluten-free diet can lead to reduction in Lactobacillus while increase pathogens like Escherichia coli12,13. Gluten-free diets have been employed as potential strategies for antiobesity, anti-inflammatory, and antidiabetic effects10. Nevertheless, it remains unclear whether the effect of dietary gluten on body weight results from its direct impact on adipose tissue or from systemic inflammation triggered by its presence in the gastrointestinal system14. Animal studies have shown that gluten supplementation in high-fat diets may lead to obesity by reducing thermogenesis and browning of subcutaneous fat tissue and increasing adipocyte size11,15. In addition, studies in humans have shown that low-gluten diets are associated with increased thermogenesis due to increased levels of peptide YY and beta-aminoisobutyric acid (BAIBA) leading to weight loss12. Evidence also suggests that gluten’s influence on gut microbiota or its direct effects on intestinal barrier integrity may contribute to inflammation and subsequent weight gain. Gliadin, a gluten component, has been reported to trigger the release of zonulin, a protein that disrupts tight junctions like occludin and E-cadherin, leading to increased intestinal permeability10.This study aimed to investigate the effects of gluten consumption and high-fat diets on body weight and gut microbiota composition using the BALB-C mouse model by 16 S ribosomal RNA-based analysis. While many previous studies focused on disease-specific conditions (such as celiac disease, prediabetic mice) and used gliadin alone, which is considered allergenic in gluten5,16, this study aimed to better isolate the effects of dietary interventions without any disease physiology.Materials and methodsResearch site and sampling selectionThis study was conducted at the Istanbul Medipol University Medical Research Center (MEDITAM) from November 2019 to October 2020. All authors comply with the ARRIVE guidelines, and all methods were performed in accordance with the relevant guidelines and regulations. Male BALB/c mice were obtained from the MEDİTAM. The research involved twenty-four male BALB/c mice, aged four weeks, with an average weight of 20–30 g, distributed homogeneously. Ethical approval for the study was obtained from the Istanbul Medipol University Animal Experiments Local Ethics Committee (Approval date: 30/09/2019, Approval number: 62).General plan of the studyAfter one week of acclimatization, mice were randomized into four groups (n = 6) based on the initial body weights and placed into four cages, with six mice in each cage (n = 6): standard diet control (SDC), standard diet + 5 mg/day gluten (SD + gluten), high-fat diet control (HFDC), and high-fat diet + 5 mg/day gluten (HFD + gluten). During the study period, the mice were fed ad libitum at a constant room temperature of 21 ± 2 °C, and their natural light-dark cycles were maintained17. The body weights of the mice were measured and recorded prior to the initiation of the gluten and high-fat diet interventions. Sterile gluten was dissolved in acetic acid at a concentration of 5 mg/day and administered by oral gavage three times a week. Administering gavage three times a week was chosen to enhance the feasibility of dose application and minimize the stress levels induced by the gavage method in mice. The dose for our study was determined based on the literature, the estimated daily gluten intake from gluten-enriched diets used in previous studies, and human gluten consumption using the simple weight scaling method 12,18,19,20,21. To ensure consistency, food access was restricted one hour before gavage. In the nongluten groups, acetic acid was gavaged three times a week to simulate the same stress conditions. The body weight of each subject was monitored and recorded on a weekly basis. The cages were changed overnight, and fecal samples were collected from the cages once a week. These samples were stored in a deep freezer at −80°C until DNA extraction9,12.Anthropometric measurementsEach mouse was tagged with an identification number for body weight measurement. Body weights were measured in grams using via the Weightlab WH-2002 instrument.Dietary interventionAfter the acclimatization period, the mice were assigned to different diet groups. The standard diet consisted of Altromin 1324 (Altromin GmbH, Lage, Germany) mouse feed. The high-fat diet was prepared by adding 19.5 g of butter to 100 g of standard laboratory feed. The butter was melted and evenly mixed into the feed, which was then roasted. The macronutrient composition of a high-fat diet, on the basis of energy content, is 65% fat, 24% protein, and 11% carbohydrates22,23.Isolation and identification procedureAfter the mice were fed according to the specified procedure, changes in the diversity of Lactobacillus, coliform group, and Escherichia coli bacteria in the intestines were assessed weekly.Lactobacillus sppIsolation was performed via appropriate dilutions on Man, Rogosa, and Sharpe (MRS) agar (Merck, Germany) for lactobacilli and Nutrient Agar (NA) (Merck, Germany) for enterococci, via the spread plate method. Following 48 h of incubation at 30 ˚C for MRS agar (pH 5.7 ± 0.2) and at 37 ˚C for NA (pH 7.0 ± 0.2), colonies exhibiting different morphological characteristics were selected. For enterococci, small, white or pale-colored, smooth-edged colonies were prioritized, whereas for lactobacilli, cream-colored, matte, smooth-edged colonies were preferred. The microscopic appearance, Gram reaction, and catalase activities of the isolates were examined for purity control. Cocci-shaped, gram-positive, catalase-negative isolates of various sizes were reserved for enterococci, whereas rod-shaped, gram-positive, catalase-negative isolates were reserved for lactobacilli-specific identification tests24.Total coliform group bacteriaThe samples were collected and transported to the laboratory in sterile containers under aseptic conditions. The samples were subjected to dilution and homogenization procedures, followed by the standard spread plate method. This method was applied via use of Violet Red Bile (VRB) agar (Merck, Germany), which was previously prepared and poured into Petri dishes. After cultivation, a second layer of VRB agar was added to the Petri dishes, which were subsequently incubated at 37 °C for 24 h. The typical colonies that formed at the end of the incubation period were subsequently counted25.Escherichia coliDilution and homogenization procedures were applied to the samples. The standard spread plate method was subsequently used with TBX (Tryptone Bile X-glucuronide) agar, which was subsequently poured into preprepared Petri dishes. The Petri dishes were incubated at 44 °C for 24 h, and the typical colonies that formed at the end of the incubation period were counted26.DNA extractionThe DNA of all the isolates was extracted via a commercial DNA extraction kit following the manufacturer’s protocol. The extracts were stored at −20°C for use as target DNA in PCR procedures17.PCRThe primers designed specifically for the microbiological parameters to be analyzed in this study for use in PCR procedures are presented in Table 117.Lactobacillus spp. parameter, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus brevis, which are considered to have the most significant effects on obesity in this study, were included in the PCR procedure.Table 1 Primer sets planned to be used in our study and their characteristics.Full size tableThe PCR mixture was prepared as follows: 2 µl of DNA sample, 2.5 mM MgCl₂, 10 mM Tris-HCl (pH 8.0), 5 mM KCl (0.2 mM of each nucleotide), 0.8 µmol/ml of each primer, and 1 U of Taq DNA polymerase (final volume of 25 µl). Amplification procedures were carried out according to the protocol. The initial denaturation was set at 94 °C for 5 min, followed by 35 cycles of the following steps: 1 s at 94 °C for denaturation, 1 s at 55 °C for primer annealing, and 21 s at 72 °C for elongation. A final extension step was performed at 72 °C for 7 min17.ElectrophoresisThe PCR products were electrophoresed in 2% (wt/vol) agarose containing ethidium bromide, and the presence of specific bands was detected via a UV transilluminator17.Statistical analysisFor normality analysis, the Kolmogorov-Smirnov test and skewness/kurtosis measures were examined. For group comparisons, ANOVA and Welch tests were utilized in cases where parametric assumptions were met. In non-parametric situations, the Kruskal-Wallis test was employed. To explore differences between groups, LSD was applied for parametric tests, while the Mann-Whitney U test was used for non-parametric analyses. A significance level of 0.05 was adopted for all statistical tests. Blinding was ensured during the study by coding group assignments, with researchers (except the primary investigator) and the statistician unaware of group details until the analysis was completed. Blinding was ensured during the study by coding group assignments, with researchers (except the primary investigator) and the statistician unaware of group details until the analysis was completed.To determine the appropriate sample size for this study, a power analysis was conducted. The calculations indicated that a sample size of 21 mice would be sufficient to obtain statistically significant results.ResultsChanges in body weightAccording to the results obtained, the gluten and high-fat diet interventions caused statistically significant weight gain compared with the control group (p < 0.05). Both high-fat and gluten intake induced weight gain in the subjects (Table S2).Total mesophilic aerobic BacteriaThere was no statistically significant difference between the groups in terms of total mesophilic aerobic bacteria count (p > 0.05). The different dietary protocols applied had no effect on this microbiological parameter (Table S3).L. BulgaricusThe addition of gluten to the diets significantly stimulated the growth of L. bulgaricus (p < 0.05) (Table S4).Total coliform group BacteriaIt was found that both the high-fat diet and the gluten-containing diet groups inhibited the growth of total coliform group bacteria (p < 0.05). No significant difference was detected between the high-fat and high-fat + gluten groups in terms of total coliform bacteria count (p > 0.05) (Table S5).E.ColiIt was found that the high-fat diet inhibited the growth of E. coli in the intestine (p < 0.05). A negative effect of the gluten-containing diet groups on total E. coli growth in the intestine was also observed (p < 0.05). No significant difference was found between the high-fat and high-fat + gluten groups in terms of E. coli growth (p > 0.05) (Table S6).PCR analysisThe agarose gel images obtained from the PCR analysis of the microorganisms studied are presented in Figs. 1 and 2.Fig. 1Agarose gel electrophesis image of amplified PCR products of E. coli strains (1-SM, 3 / 6- E. coli 23 S / 450, 2-negative control; SM: standard marker).Full size imageFig. 2Agarose gel electrophoresis image of Lactobacillus spp. amplified PCR products (1-SM, 5 / 8- L. delbrueckii subs bulgaricus 16s rRNA, 3 / 4- L. acidophilus 16s rRNA, 2-negative control; SM: standard marker).Full size imageDiscussionThe prevalence of gluten-free diets is increasing, with nearly one in three people in the United States attempting to eliminate gluten from their diet in the past decade. This trend is reportedly driven by the belief that gluten-free diets are beneficial for overall health and promote faster weight loss27. Gluten-free diets are also widely used for weight control and the treatment of intestinal diseases. However, a small number of controlled studies have reported inconsistent findings regarding the relationships among gluten, obesity, and the gut microbiota14,28,29. Although some studies in mice have investigated the combined effects of high-fat diets and gluten intake, conflicting findings highlight the need for further research. Additionally, many previous studies have focused on disease-specific conditions (such as celiac disease, prediabetic mice) and disease physiology is known to influence the microbiota, and some studies have used gliadin alone, which is considered an allergen in gluten5,11,30.This study aimed to investigate the effects of gluten consumption and high-fat diets on body weight and gut microbiota composition using the BALB-C mouse model. The analysis was performed by 16 S ribosomal RNA-based sequencing to better isolate the effects of dietary interventions using a healthy animal model without any disease physiology. In our study, high-fat diet interventions caused statistically significant weight gain compared to the control group (p< 0.05). Our observations are consistent with earlier research findings. Chronic exposure to high-fat diets (HFDs) disrupts glucose homeostasis and energy metabolism, leading to increased body weight. For example, in a study in which mice were fed an HFD for 20 weeks, significant weight gain was observed31.Our study revealed that gluten contributes to weight gain both independently and when combined with a high-fat diet (p< 0.05). These observations support the conclusions of previous studies. For instance, mice fed a Western diet containing gluten were found to exhibit the highest body weight gain, adiposity, and larger adipocyte size11. In the referenced study, unlike ours, the mice were fed a high-fat and high-sucrose Western diet. Sucrose is known to trigger weight gain and fat accumulation32. Evidence from another experiment demonstrated that mice fed a high-fat diet with added gluten showed greater body weight and reduced energy expenditure33. Furthermore, a study conducted in Denmark reported that a low-gluten diet led to an average weight loss compared to a high-gluten diet, despite unchanged energy intake12. However, caution should be exercised when comparing these findings to human studies, given the fundamental interspecies differences.The impact of dietary gluten on body weight remains uncertain, whether due to its direct effects on adipose tissue or the systemic inflammation it triggers in the gastrointestinal system. In a mice study, reported that gluten intake leads to weight gain by decreasing browning and thermogenesis markers in subcutaneous fat32. Specifically, gluten consumption decreases the levels of UCP1 (uncoupling protein 1) in brown adipose tissue (BAT), a key regulator of thermogenesis, thereby lowering energy expenditure. Additionally, it suppresses the expression of BMP7, a protein that promotes the browning of white adipose tissue, further limiting energy dissipation12,14.Low-gluten intake has been associated with increased concentrations of peptide YY (PYY), a hormone that suppresses appetite postprandially and beta-aminoisobutyric acid (BAIBA) which is known to increase thermogenesis by promoting the conversion of white fat tissue into brown fat tissue. These factors may help increase thermogenesis and reduce weight gain. In contrast, gluten consumption in mice has been shown to reduce oxygen consumption and energy expenditure during fasting, leading to decreased thermogenesis. Moreover, the expression of thermogenic genes was observed to decrease across all adipose tissues in gluten-fed mice, potentially explaining the observed weight gain12,14.The potential impact of gluten on weight gain through its effects on the gut has been explained in several studies, suggested that gluten administration may lead to gut barrier dysfunction5. Furthermore, a mouse study revealed that duodenal mucosal damage and reduced expression of tight junction proteins was linked to weight gain31.In our study, it was determined that gluten intervention stimulated the production of Lactobacillus bulgaricus, a genus thought to be beneficial for the intestinal microbiota (p < 0.05), and had a statistically significant inhibitory effect on the growth of Escherichia coli and total coliform bacteria, both of which are considered pathogens (p < 0.05). In addition, no statistically significant difference was detected between the groups in terms of the total mesophilic aerobic bacteria count parameter (p > 0.05).In a 23-week study examining the effects of gliadin in mice fed a high-fat diet found increased opportunistic pathogens such as Clostridium XI, Dorea, and Coriobacteriaceae, while Akkermansia muciniphila (considered beneficial) was increased, but Lactobacillus, a beneficial genus particularly important for gut health, was significantly decreased5. The differences in study results may be due to the longer experimental period with gliadin instead of gluten5. A study investigating wheat gluten intake with a high-fat diet showed a reduction in the relative abundances of Firmicutes and Lactobacillus, but an increase in the relative abundances of Bacteroidales S24-7 and Ruminococcaceae34. However, the findings of this study may differ from ours, as the comparator group was fed casein. Casein is unique in that it contains a high proportion of branched-chain amino acids (BCAAs), which can affect the microbiota35.Furthermore another study demonstrated that healthy bacteria, such as Bifidobacterium and Lactobacillus, were reduced, whereas unhealthy bacteria (especially E. coli) increased in the fecal microbiota of healthy adults following a gluten-free diet28. Our research revealed similar trends as those highlighted in this study. Still, we need to be careful when comparing these results to humans because of the natural differences between species.High-fat diets are known to affect the gut microbiota. A study investigating the effects of a high-fat diet (HFD) on the gut microbiota found that Escherichia-Shigella was increased in the high-fat diet group, while conversely, the abundance of Staphylococcus, Ralstonia, Vagococcus, and Streptococcuswas significantly reduced36.Our findings are different from previous studies, as it has been determined that the high-fat diet in our study significantly reduced the growth of Escherichia coli and total coliform bacteria in the gut (p< 0.05). This difference may be due to the shorter study duration (4 weeks) or the role of high-fat diets in increasing bile acid secretion. It has been observed that higher bile acid levels lead to the production of secondary bile acids, which are known to suppress some Gram-negative pathogens. However, while E. coli has developed mechanisms to resist bile-induced stress, its survival may decrease under nutrient competition and environmental stress37.In addition, it has been determined that there was no significant difference in the total counts of mesophilic aerobic bacteria between groups (p > 0.05), which may be due to the broad diversity of this bacterial group. Future studies focusing specifically on mesophilic aerobic bacteria could provide clearer information about the effects of high-fat diets.In a study investigating the effects of prebiotics alongside the effects of gluten described in the literature, prebiotics were found to reduce fat accumulation, restore fat-burning markers, and increase beneficial bacteria which breaks down gluten peptides and reduces inflammation. Additionally, prebiotics inhibited the growth of obesity-associated bacteria, highlighting their potential to counteract the negative effects of gluten and improve gut health. Furthermore, in human diets, gluten is primarily consumed through wheat-based products, which also supply carbohydrates and prebiotics. These additional components may influence weight-related outcomes and microbiota composition, potentially affecting the observed effects11.LimitationsThe primary challenges in this study included the dissolution of gluten for administration to the mice and managing the subjects’ mobility of the subjects during gavage, which hindered the complete delivery of gluten to the stomach.ConclusionOur study demonstrated that gluten consumption contributes to weight gain under both normal and high-fat diet conditions. Unlike research that focuses on the effects of gluten in the context of disease states such as celiac disease, prediabetes, or colitis, we conducted our study using healthy mice. This provides a fresh perspective on how gluten affects weight and gut health in non-disease scenarios.Interestingly, our results revealed that gluten supplementation significantly inhibited the growth of harmful bacteria, such as Escherichia coli and total coliforms, while promoting the growth of Lactobacillus, a beneficial genus for gut health. Another surprising finding was that the high-fat diet itself suppressed the growth of these pathogenic bacteria. This novel observation underscores the complex ways in which diets influence the microbiota and calls for further research to understand the mechanisms involved.The link between gluten intake and weight gain may be driven by a combination of factors, such as impaired thermogenesis, altered gut permeability, and inflammatory pathways. These underlying mechanisms need further exploration in future studies. To counteract the potential weight-gain effects of gluten, prebiotic supplementation might be a viable dietary approach. Therefore, as our study was conducted in animal models, caution is necessary when extending these findings to humans. Future research, both experimental and clinical, should focus on these mechanisms and evaluate how prebiotic supplementation could mitigate gluten’s effects.
Data availability
The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request, provided that the manuscript is accepted and published.
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Download referencesAuthor informationAuthors and AffiliationsInstitute of Health Sciences, Department of Nutrition and Dietetic, Istanbul Medipol University, Göztepe Mahallesi, Atatürk Caddesi. No: 40/16, 34815, Beykoz, İstanbul, TürkiyeMerve Sayın DülgerCollege of Health Sciences, Department of Nutrition and Dietetic, Istanbul Medipol University, Cibali Mahallesi, Unkapanı, Atatürk Bulvarı, No: 27, 34083, Fatih, İstanbul, TürkiyeNihal Zekiye ErdemSchool of Veterinary Medicine, Department of Food Hygiene& Technology, Istanbul University Cerrahpaşa, Alkent 2000. Mahallesi, Yiğittürk Caddesi, Avcılar, İstanbul, TürkiyeEmek DümenAuthorsMerve Sayın DülgerView author publicationsYou can also search for this author in
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PubMed Google ScholarContributionsAuthors Merve Sayın Dülger and Nihal Zekiye Erdem have contributed equally to this work. All authors contributed to the study conception and design. Data collection and analysis were performed by Merve Sayın Dülger and Nihal Zekiye Erdem. The first draft of the manuscript was written by Merve Sayın Dülger and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Merve Sayın Dülger conducted the experiments, while Emek Dümen provided consultation and assistance with data analysis.Corresponding authorCorrespondence to
Merve Sayın Dülger.Ethics declarations
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The authors declare no competing interests.
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This research did not receive any specific grant from funding agencies in the public, commercial or not-forprofitsectors.
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All procedures involving animals were in compliance with the European Community Council Directive of 24 November 1986, and ethical approval was granted by the Istanbul Medipol University Animal Experiments Local Ethics Committee (Approval date: 30/09/2019, Approval number: 62).
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Reprints and permissionsAbout this articleCite this articleDülger, M.S., Erdem, N.Z. & Dümen, E. Effects of dietary gluten on body weight and gut microbiota in BALB-C mice using 16 S rRNA-Based analysis.
Sci Rep 15, 7959 (2025). https://doi.org/10.1038/s41598-025-92213-3Download citationReceived: 26 November 2024Accepted: 26 February 2025Published: 07 March 2025DOI: https://doi.org/10.1038/s41598-025-92213-3Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard
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KeywordsNutritionGlutenMicrobiotaBody weightHigh-fat diet