The authors have declared that no competing interests exist.
Conceived and designed the experiments: GJ MN. Performed the experiments: GJ. Analyzed the data: GJ JX. Contributed reagents/materials/analysis tools: GM SA MN. Wrote the paper: GJ JX GM SA MN.
Obesity is linked to type 2 diabetes and risk factors associated to the metabolic syndrome. Consumption of dietary fibres has been shown to have positive metabolic health effects, such as by increasing satiety, lowering blood glucose and cholesterol levels. These effects may be associated with short-chain fatty acids (SCFAs), particularly propionic and butyric acids, formed by microbial degradation of dietary fibres in colon, and by their capacity to reduce low-grade inflammation.
To investigate whether dietary fibres, giving rise to different SCFAs, would affect metabolic risk markers in low-fat and high-fat diets using a model with conventional rats for 2, 4 and 6 weeks.
Conventional rats were administered low-fat or high-fat diets, for 2, 4 or 6 weeks, supplemented with fermentable dietary fibres, giving rise to different SCFA patterns (pectin – acetic acid; guar gum – propionic acid; or a mixture – butyric acid). At the end of each experimental period, liver fat, cholesterol and triglycerides, serum and caecal SCFAs, plasma cholesterol, and inflammatory cytokines were analysed. The caecal microbiota was analysed after 6 weeks.
Fermentable dietary fibre decreased weight gain, liver fat, cholesterol and triglyceride content, and changed the formation of SCFAs. The high-fat diet primarily reduced formation of SCFAs but, after a longer experimental period, the formation of propionic and acetic acids recovered. The concentration of succinic acid in the rats increased in high-fat diets with time, indicating harmful effect of high-fat consumption. The dietary fibre partly counteracted these harmful effects and reduced inflammation. Furthermore, the number of
Obesity/overweight is a serious health risk and related to many metabolic diseases, such as type 2 diabetes, insulin resistance and coronary heart disease, stroke and cancer
It is generally accepted that soluble fibres improve glycaemia and insulin sensitivity in both healthy and diabetic subjects, and oat β-glucan may lower plasma cholesterol levels
Different fibres give rise to different amounts and patterns of the main SCFAs: acetic, propionic and butyric acids. Pectin forms high amounts of acetic acid, while guar gum yields propionic acid and β-glucan, fructo-oligosaccharides, some types of resistant starch and mixtures of dietary fibres form high amounts of butyric acid
Obesity and high-fat diet is thought to trigger low-grade inflammation and possibly the development of obesity-associated diseases
In the present study, two fermentable dietary fibres, pectin (acetic acid producer) and guar gum (propionic acid producer), and a mixture of the two (butyric acid producer), were chosen
Fermentable dietary fibres, pectin (esterification 70–75%) isolated from apples, and guar gum (viscosity 3.025 mPaS at 1% (w/v) and 25°C), isolated from guar bean (Sigma Aldrich, St. Louis, MO, USA), individually or as a mixture, were included in low-fat diet (LFD) or high-fat diet (HFD) (
Low-fat | High-fat | |||
Fibre diets | Fibre-free diets | Fibre diets | Fibre-free diets | |
Basal diet |
409.2 | 409.2 | 409.2 | 409.2 |
Lard | - | - | 230 | 230 |
Cholesterol | - | - | 20 | 20 |
Dietary fibre |
87.9–100 | - | 87.9–100 | - |
Wheat starch¥ | 490.8–502.9 | 590.8 | 240.8–252.9 | 340.8 |
Fat | 11.5–11.6 | 10.9 | 50.7–50.9 | 48.7 |
Protein Car | 21.1-21.4 | 20.0 | 21.1–21.4 | 16.0 |
Carbohydrate | 63.1–63.4 | 69.1 | 29.3 | 35.3 |
Dietary fibre | 3.9–4.0 | - | 3.1 | - |
Containing (g/kg, dwb): 200 casein (Sigma Aldrich, St. Louis, MO, USA), 50 rapeseed oil (Zeta, Stockholm, Sweden), 1.2 DL-methionine (Sigma Aldrich, St. Louis, MO, USA), 100 sucrose (Nordic sugar, Copenhagen, Denmark), 8 vitamin mixture§, 2 choline chloride (Sigma Aldrich, St. Louis, MO, USA), 48 mineral mixture‡.
Corresponding to the content of dietary fibre in pectin and guar gum which was 800 g/kg (dwb) and 910 g/kg (dwb), respectively.
Containing (g/kg): 0.62 menadione, 2.5 thiamin hydrochloride, 2.5 riboflavin, 1.25 pyridoxine hydrochloride, 6.25 calcium pantothenate, 6.25 nicotinic acid, 0.25 folic acid, 12.5 inositol, 1.25 p-aminobenzoic acid, 0.05 biotin, 0.00375 cyanocobalamin, 0.187 retinol palmitate, 0.00613 calciferol, 25 d-α-tocopheryl acetate, 941.25 maize starch (Lantmännen, Stockholm, Sweden).
Containing (g/kg): 0.37 CuSO4·5H2O, 1.4 ZnSO4·7H2O, 332.1 KH2PO4, 171.8 NaH2PO4·2H2O, 324.4 CaCO3, 0.068 KI, 57.2 MgSO4, 7.7 FeSO4·7H2O, 3.4 MnSO4·H2O, 0.02 CoCl·6H2O, 101.7 NaCl, 0.019 chromium(III)chloride and 0.011 sodium selenate.
¥ Norfoods Sweden AB, Malmö, Sweden, varied according to the dietary fibre content of the test materials.
Male Wistar rats (Scanbur AB, Sollentuna, Sweden), with an initial weight of 129 g (SE 0.9), were randomly divided into sixteen groups of seven (one group per cage). The rats were housed in a room maintained at 22°C, with a 12 h light-dark cycle. Three test diets containing pectin, guar gum or a mixture of pectin and guar gum (mixture), and a fibre-free control diet, were prepared and given to the rats. The LFD contained 50 g fat per kg dry weight basis (dwb), while the HFD contained 280 g fat per kg (dwb) (
Schematic illustration of the study design.
After 5 d of acclimatizing, an experimental period of 2, 4 and 6 weeks followed. The experiment on rats fed LFD lasted for 2 weeks, while the experiment on rats fed HFD persisted for 2, 4 and 6 weeks. The weight of the animals was registered every week. At the end of the experimental period, the animals were anaesthetized by subcutaneous injection of a mixture (1∶1∶2) of Hypnorm (fentanyl citrate 0.315 mg/ml and fluanisone 10 mg/l) (Division of Janssen-Cilag Ltd, Janssen Phamaceutica, Beerse, Belgium), Dormicum (midazolam 5 mg/ml) (F. Hoffman-La Roche AG, Basel, Switzerland) and water at a dose of 0.15 ml/100 g body weight. Blood samples (serum and plasma) were collected from the portal vein and placed in plasma tubes containing EDTA (K2E 3.6 mg, Plus Blood Collection Tubes, BD, Plymouth, UK) and serum tubes (SST™ Advance, Plus Blood Collection Tubes, BD, Plymouth, UK). After blood has been collected from the portal vein, the rats were euthanized through an incision in the heart. The samples were centrifuged and stored at −40°C until the analysis of SCFAs, succinic acid, cytokines and cholesterol. The caecum was removed, weighed with and without its content, and the pH of the content was measured, before being stored at −40°C for analysis of SCFAs, lactic and succinic acids. A small part of the caecum content was weighed and collected in sterile tubes containing freezing medium (water, glycerol [98%], MgSO4-7H2O, Na-Citrate, KH2PO4, K2HPO4) and immediately frozen in liquid nitrogen for analysis of the microbiota composition. The spleen and the liver were weighed and the liver was frozen at −20°C for analysis of cholesterol, triglycerides and fat content.
Before analysis of fat content, the livers were lyophilized and mortared. The fat content was analysed using the SBR (Schmidt-Bondzynski-Ratzlaff) method. The liver samples were digested in 7.7 M HCl (Merck, Darmstadt, Germany) for 60 min at 75°C before being washed and extracted with ethanol (Kemetyl, Haninge, Sweden), diethylether (Merck, Darmstadt, Germany) and petroleumbensin (Merck, Darmstadt, Germany) for 30 min. The extracts were transferred to a clean beaker, washed twice with 1∶1 diethylether∶petroleumbensin, and allowed to settle for 30 min before the extracts were transferred to a beaker, air-dried and weighed.
Lyophilized and mortared liver tissues were analysed for cholesterol and triglycerides. The lipids were extracted and washed with a 3∶2 mixture of hexane (Sigma Aldrich, St. Louis, USA) and isopropanol (Merck, Darmstadt, Germany) containing 0.005% (v/w) BHT (2,6-Di-Tert-Butyl-4-Metylphenol) (Merck, Munich, Germany) on an orbital shaker followed by centrifugation, and the extracts were then transferred into a clean tube. This procedure was repeated four times. The lipid extracts were dried under N2 flow at room temperature and re-dissolved in isopropanol+1% (v/v) Trition X100 (Sigma Aldrich, St. Louis, MO, USA). Total liver cholesterol and triglycerides and plasma cholesterol were determined spectrophotometrically using Infinity™ Cholesterol and Infinity™ Triglycerides reagent and Cholesterol and Triglycerides Standard (Thermo Scientific, Middletown, VA, USA).
After centrifugation of the blood, the serum was transferred to a clean tube and analysed with regard to SCFAs (acetic, propionic, iso-butyric, butyric, iso-valeric and valeric acids) using GLC
Succinic acid was analysed by mixing 400 µl of serum with 100 µl of 10% (v/v) sulphosalicylic acid to precipitate high-molecular-weight proteins. The samples were vortexed for 30 sec and then centrifuged for 30 min, and the supernatant was filtered through PolyTetraFluoroEthylene (PTFE) Syringe Filters (Pore Size: 0.45 µm, Diameter: 13 mm) (Skandinaviska Genetec AB, Västra Frölunda, Sweden) before quantification with ion-exclusion chromatography (MIC-2 Advanced modular IC) (Metrohm AG, Herisau, Switzerland). The system comprised a serial double-piston high-pressure pumping unit (818 IC), a two-channel peristaltic pump with the Metrohm Suppressor Module, a separation centre (820 IC), a conductivity detector (819 IC), and an interface (830 IC) to connect with a computer. The Metrohm IC Net 2.3 software was used to analyse the chromatograms. Ion-exclusion chromatography with inverse suppression and conductivity detection was used to detect the succinic acid peak. Samples were injected via a 20 µl loop and eluted at a flow rate of 0.6 ml per min and at a pressure of 3.0 Mpa. The column used was Metrosep organic acids analytical column (6.1005.210, 250 mm×7.8 mm, particle size of 10 µm, with polystyrene-divinylbenzene copolymer packing material functionalized with sulphonic acid groups). The eluent solution was 0.5 mM sulphuric acid, which was degassed by nitrogen before use. The solution of 50 mM LiCl and water was pumped at the same speed, which regenerated the suppressor system. Running temperature was 70°C and the running time for each analysis was 25 min. The conductivity detector was operated in the positive mode at a full scale of 10.0 µS/cm. Standard solutions were made with serial dilutions, resulting in five different concentrations, and analysed using the same method as the serum samples (see above). A linear equation was produced, using the peak area and the different concentrations. Then the equation was used to calculate the concentration of succinic acid in the samples.
The SCFAs (acetic, propionic, iso-butyric, butyric, iso-valeric and valeric acids) in the caecal content were analysed using a GLC method
A Milliplex micro-beads array system was used to simultaneously measure serum levels of the following eight cytokines: interleukin (IL)-1α, IL-1β, IL-6, IL-10, IL-18, MCP-1, IFNγ, and TNFα. The assay was conducted according to the manufacturer's instructions, using Milliplex™ MAP rat cytokine kit assay technology (Millipore Corp., Billerica, MA, USA). The antibody specific to each cytokine was coupled to microspheres that were uniquely labelled with a fluorescent dye. The microspheres were incubated with standards, controls and samples in a 96-well filter plate overnight at 4°C. After incubation, the plate was washed to remove excess reagent, and detection antibodies, one for each of the eight cytokines, were added to the vials. After 2 h incubation at room temperature, streptavidin-phycoerythrin was added for a further 30 min. A final wash step was included before the beads were resuspended in buffer and read on the Luminex 200 instrument (Luminex Corporation, USA) to determine the level of the cytokine of interest. All specimens received were tested in replicate wells. Milliplex™ Analyst v. 3.4 (Millipore) was used for the evaluation of the results.
The T-RFLP analysis was performed on three groups fed HFD (fibre-free, pectin and guar gum diets) for 6 weeks. The reason for this was that the caecal microbiota is probably most affected and adapted to each diet after a longer experimental period.
The caecal contents were thawed on ice and centrifuged at 10 000 rpm for 5 min. The supernatant was discarded and the pellet was resuspended in 1–4 ml of 1XPBS (Oxoid, Basingstoke, UK) depending on the weight of the caecal content. Total DNA was extracted using EZ1 DNA tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The 16S rRNA genes were amplified using a fluorescently labelled forward primer ENV1 (5′-FAM-AGAGTTTGATIITGGCTCAG-3′) and an unlabelled reverse primer ENV2 (5′-CGGITACCTTGTTACGACTT-3′). The PCR reaction was prepared in a total volume of 25 µl containing 0.4 µM of FAM-ENV1 primer and 0.2 µM of primer ENV2, 2.5 µl of 10 x PCR reaction buffer (500 mM Tris-HCl, 100 mM KCl, 50 mM (NH4)2SO4, 20 mM MgCl2, pH 8.3), 0.2 mM of each deoxyribonucleotide triphosphate, 2.5 U of FastStart Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany), and 2 µl of template DNA. The PCR was performed in an Eppendorf MasterCycler (Eppendorf, Hamburg, Germany) using the following programme: 95°C for 3 min, 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 50°C for 45 sec, and 72°C for 2 min. Finally, an additional extension at 72°C for 7 min was done. Triplicate reactions were carried out for each sample and a negative control was included in all PCR runs. After the amplification, the 16S rDNA amplicons were verified by Agarose Gel Electrophoresis. The amplicons of each sample were then pooled and purified by MinElute PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The purified DNA concentration was measured by Nanodrop ND-1000 (Saveen Werner, Limhamn, Sweden). Then 200 ng of the purified DNA were digested with 10 U of the restriction endonucleases
The experimental design was randomized. The eight test diets contained three fibre groups, pectin, guar gum, or a mixture, and one fibre-free group. These four test diets were administered either as LFD or HFD, resulting in a total of eight diets. The experiments with LFD lasted for 2 weeks, while the experiment with HFD continued for 2, 4 or 6 weeks.
Two-way ANOVA was used to determine the effects of dietary fibre (Fibre), fat content (Fat) and their interaction (Fibre×Fat) for the groups fed LFD and HFD for 2 weeks (
Serum concentration (µmol/L) of butyric acid in rats fed the three dietary fibre diets for 2, 4 and 6 weeks (means±SEM, n = 7, with exception of groups pectin and fibre-free diets for 4 and 6 weeks, respectively, n = 6).
A: Weight gain (g), B: weight of caecal content (g), C: caecal tissue weight (g) and D: pH in rats fed the four HFD for 2, 4 and 6 weeks (means ± SEM, n = 7). Values with different letters are significantly different, p<0.05.
A: liver weight (g), B: spleen weight (g) C: liver fat content (g), D: liver cholesterol (g) and E: liver triglyceride (g) in rats fed the four HFD for 2, 4 and 6 weeks (means ± SEM, n = 7). Values with different letters are significantly different, p<0.05.
Serum concentration (µmol/L) of A: acetic acid, B: propionic acid and C: butyric acid in rats fed the four HFD for 2, 4 and 6 weeks (means ± SEM, n = 7, with exception of groups pectin and fibre-free diets for 4 and 6 weeks, respectively, n = 6). Values with different letters are significantly different, p<0.05.
Caecal pools (µmol) of A: acetic acid, B: propionic acid, C: butyric acid, D: lactic acid and E: succinic acid in rats fed the four HFD for 2, 4 and 6 weeks (means ± SEM, n = 7, with exception of group guar gum diet for 6 weeks, respectively, n = 6). Values with different letters are significantly different, p<0.05.
Concentration of succinic acid (µmol/L) in serum from rats fed the fibre-free LFD for 2 w and HFD for 2, 4 and 6 weeks (means ± SEM, n = 7, 6, 4 and 6 for LFD 2 w, HFD 2 w, 4 w and 6 w, respectively).
Concentration (ng/L) of MCP-1 in portal serum in rats fed the four LFD for 2 weeks and HFD for 2, 4 and 6 weeks (means ± SEM, n = 7, with exceptions of groups fed pectin, guar gum and mixture with low-fat content for 2 weeks, fibre-free with high-fat content for 4 weeks and fibre-free and pectin with high-fat content for 6 weeks, n = 6). Values with different letters are significantly different, p<0.05.
Loading Bi plot of the grouping of the caecal microbiota and analytical markers in rats fed fibre-free (dots), pectin (triangles) and guar gum (squares) diets with high-fat content for 6 weeks (n = 7).
Peak area of T-RFLP peaks for
Fat | Diet | Weight gain (g) | Caecal content (g) | Caecal tissue (g) | Caecal pH | Liver weight (g) | Spleen weight (g) |
Fibre-free | 59±2 | 1.4±0.1a | 0.6±0.02a | 6.9±0.11 | 8.3±0.3 | 0.58±0.04 | |
Pectin | 58±3 | 3.2±0.3b | 0.8±0.04b | 7.0±0.05 | 7.1±0.5 | 0.59±0.2 | |
Guar gum | 61±3 | 3.8±0.3bc | 1.0±0.04c | 7.0±0.08 | 7.6±0.3 | 0.58±0.03 | |
Mixture | 60±3 | 2.8±0.3b | 0.9±0.04bc | 7.1±0.04 | 7.1±0.1 | 0.59±0.02 | |
Fibre-free | 78±4a ** | 1.7±0.1a | 0.7±0.03a | 7.1±0.06 | 10.6±0.4* | 0.64±0.03 | |
Pectin | 62±4b | 2.9±0.4ab | 1.1±0.08b * | 7.0±0.1 | 8.6±0.3 | 0.58±0.03 | |
Guar gum | 73±2 | 4.3±0.6b | 1.1±0.06b | 7.2±0.07 | 9.5±0.3* | 0.58±0.02 | |
Mixture | 68±4 | 4.5±0.5b * | 1.2±0.09b * | 6.9±0.1 | 9.6±0.6*** | 0.61±0.02 |
Mean values within a column, for different fat levels, with unlike superscripts are significantly different (P<0.05).
Mean values were significantly different from those of rats fed the corresponding low-fat diet: * P<0.05, ** P<0.01, ***P<0.001.
1 n = 5.
Fat content | Cholesterol | Triglyceride | Cholesterol | ||
Fat | Diet | Liver (g) | Liver (mg) | Liver (mg) | Plasma (mmol/L) |
Fibre-free | 0.31±0.03a | 79±3a | 168±19a | 2.9±0.1 | |
Pectin | 0.23±0.02b | 65±6ab | 128±10ab | 3.0±0.1 | |
Guar gum | 0.24±0.01ab | 65±3ab | 112±5b | 2.8±0.07 | |
Mixture | 0.23±0.01b | 63±1b | 106±2b | 2.7±0.08 | |
Fibre-free | 1.59±0.1a*** | 422±26a*** | 503±52a*** | 4.0±0.2a*** | |
Pectin | 0.86±0.05b*** | 252±13b*** | 204±12b** | 3.3±0.1b | |
Guar gum | 1.10±0.1b*** | 327±26b*** | 296±25b*** | 3.4±0.1b** | |
Mixture | 0.90±0.02b*** | 293±17b*** | 282±14b*** | 3.8±0.1ab*** |
Mean values within a column, for different fat levels, with unlike superscripts are significantly different (P<0.05).
Mean values were significantly different from those of rats fed the corresponding low-fat diet: * P<0.05, ** P<0.01, ***P<0.001.
Fat | Diet | Acetic acid | Propionic acid | Butyric acid | Minor acids | Total SCFA |
Fibre-free | 723±46a | 49±7a | 26±4 | 78±2a | 876±59a | |
Pectin | 1111±81b | 86±9ab | 45±5 | 94±7ab | 1336±97b | |
Guar gum | 723±39a | 129±20b | 48±7 | 126±16b | 1026±73a | |
Mixture | 766±24a | 71±11a | 36±8 | 93±8ab | 982±53a | |
Fibre-free | 648±27a | 53±4a | 27±2 | 94±5 | 823±32a | |
Pectin | 1033±61b | 82±13ab | 28±3 | 83±11 | 1226±79b | |
Guar gum | 655±22a | 84±9ab | 32±5 | 87±6 | 859±32a | |
Mixture | 891±54b | 115±13b | 39±5 | 72±4 | 1116±71b |
a,b Mean values within a column, for different fat levels, with unlike superscripts are significantly different (P<0.05).
Fat | Diet | Acetic acid | Propionic acid | Butyric acid | Minor acids | Total SCFA | Lactic acid | Succinic acid |
Fibre-free | 77±6a | 12±2a | 10±0.7a | 5±0.8a | 90±9a | 0.7±0.1 | 0.4±0.05a | |
Pectin | 198±24c | 30±4bc | 22±2b | 10±0.8b | 260±30b | 1.8±0.4 | 0.9±0.2b | |
Guar gum | 135±19bc | 42±8c | 23±3b | 11±1b | 210±31b | 2±0.4 | 1±0.1b | |
Mixture | 123±13ab | 22±3ab | 22±5b | 8±1ab | 176±20ab | 1±0.3 | 0.9±0.1b | |
Fibre-free | 64±6a | 17±1a | 13±1a | 7±0.7ab | 114±9a | 0.9±0.1a | 0.6±0.1a | |
Pectin | 146±31ab | 25±5b | 14±3a | 6±0.9a | 191±39ab | 2±0.4ab | 4±2ab | |
Guar gum | 131±15ab | 38±7bc | 22±3ab | 10±1b | 201±24ab | 2±0.8ab | 3±1ab | |
Mixture | 192±27b* | 46±8c* | 25±3b | 9±1ab | 272±36b | 3±0.8b | 17±5b* |
Mean values within a column, for different fat levels, with unlike superscripts are significantly different (P<0.05).
Mean values were significantly different from those of rats fed the corresponding low-fat diet: * P<0.05.
The concentration of each SCFA (µmol/g) and the amount of liver cholesterol and triglyceride (mg/g and g/g) was multiplied by either the caecal content weight and liver weight, to obtain the caecal pool of SCFA (µmol) and the total amount of liver cholesterol and triglyceride (mg and g). P-values close to significance (p≤0.1) were defined as tendency.
Body weight gain was similar for all groups, and thus independent of whether dietary fibre was added to the diets or not (58–61 g) (
Rats fed pectin and the mixture had lower content of fat in the liver compared with the fibre-free group (0.23±0.01 g vs. 0.31 g, p<0.05) (
The concentration of acetic acid in portal serum of the rats was between 723 and 1111 µmol/L, of propionic acid between 49 and 129 µmol/L, and of butyric acid between 26 and 48 µmol/L (
The main SCFA in the caecal pool was acetic acid (77–198 µmol), followed by propionic acid (12–42 µmol) and butyric acid (10–23 µmol) (
The average body weight was higher in rats fed the HFD than rats fed the LFD (mean 70±2 g vs. mean 60±1 g, p<0.001) (
The average weight of the caecal content and tissue was higher in groups fed HFD than LFD (mean 3.3±0.3 g vs. mean 2.8±0.2 g for caecal content and mean 1.0±0.05 g vs. mean 0.8±0.05 g for caecal tissue, p<0.001). The weights were also higher with dietary fibre in the diets than without any fibre (p<0.001). When comparing the individual groups, only the mixture gave a higher caecal content weight with HFD than with corresponding LFD (4.5 g vs. 2.8 g, p = 0.039). Caecal tissue weight was higher in rats fed pectin and the mixture compared with corresponding LFD (1.1 g vs. 0.8 g, p = 0.016 for pectin and 1.2 vs. 0.9 g, p = 0.032 for the mixture). No differences in caecal pH were observed between LFD and HFD.
The average liver weight was higher in groups fed the HFD compared with LFD (mean 9.6±0.2 g vs. mean 7.5±0.2 g, p<0.001). Pectin was most effective in lowering the liver weight in the groups fed HFD (from 10.6 to 8.6 g, p = 0.011). When comparing rats fed corresponding LFD and HFD, all groups with the exception of pectin had significantly heavier liver weight with HFD (p = 0.0017 [fibre-free], p = 0.0106 [guar gum] and p = 0.0007 [mixture]) compared with LFD. The appearance of the liver tissue was also affected by the fat content, and the colour was light and yellow with HFD, while rats fed LFD had dark red livers. No difference could be seen for the spleen weights with any of the HFD, and not compared with LFD either.
The fat and cholesterol content of the liver was four to five times higher with HFD than with LFD for all groups (p<0.001), even though the values were significantly lower with dietary fibre in the diet (
Similar results could be seen in liver triglycerides. The total amount of triglycerides in the liver was higher in the rats fed HFD compared with the LFD (p<0.01). This was valid for all groups, but the difference was highest between the fibre-free groups, i.e. the addition of dietary fibre reduced the effect of fat in the diet (p<0.001).
Plasma cholesterol concentrations were higher with HFD than with LFD (p = 0.005), in all groups except those fed pectin. Comparing the different HFD, groups fed either pectin or guar gum had lower plasma concentrations of cholesterol than the group fed a fibre-free diet (p<0.03).
Similar concentrations of acetic acid could be seen with HFD as for LFD (
The mixture generated the highest pool of acetic, propionic and butyric acids in rats fed HFD (p<0.05) (
Rats given dietary fibre had a lower weight gain than those given a fibre-free diet throughout the experimental period, and pectin had most pronounced effects (62 g vs. 78 g for the fibre-free diet, p = 0.0218 after 2 weeks, 172 g vs. 196 g for the fibre-free diet after 4 weeks, p = 0.0314 and 239 g vs. 282 g for the fibre-free diet after 6 weeks, p = 0.0036) (
Both the caecal content and tissue weight were quite similar for the groups fed the fibre-free diet throughout the experiment, but lower compared with the other groups. The caecal content weight was 1.7 g vs. mean 3.9±0.3 g for the fibre groups after 2 weeks; 2.6 g vs. mean 10.0±0.4 g for the fibre groups after 4 weeks; and 2.2 g vs. mean 8.4±0.4 g for the fibre groups after 6 weeks (p<0.01). Similar results could be seen for the tissue weight (p<0.001) when compared with the dietary fibre diets. For groups fed dietary fibre, the caecal content and tissue weights were considerably higher after 4 weeks than after 2 weeks, while the weights remained unchanged or even decreased after 6 weeks. Groups fed pectin generally had a lower caecal content weight than the other dietary fibre groups but, at 6 weeks, caecal content in rats fed the mixture also weighed less than rats fed guar gum (8.3 g vs. 10.7 and 11.1 g for guar gum [p = 0.0052], and the mixture [p = 0.0009], respectively at 4 weeks and 7.9 vs. 10.0 for guar gum at 6 weeks, [p = 0.0366]).
No difference in pH between groups was seen after 2 weeks, but at 4 and 6 weeks the fibre-free group had a higher pH than the other groups (6.9 vs. mean 6.1±0.06 at 4 weeks, p<0.001 and 6.9 vs. mean 6.5±0.06 at 6 weeks, p<0.001). Groups fed guar gum gave the lowest pH, which was significantly lower compared with pectin (5.8 vs. 6.3, p = 0.0046 after 4 weeks).
The liver weights increased with the length of the experiment (
No difference was seen in the spleen weight after 2 or 4 weeks, but after 6 weeks the fibre-free group had a higher spleen weight than the guar gum group (1.09 g vs. 0.87 g, p = 0.043).
The length of the experiment had an influence on liver cholesterol, triglycerides and fat content, and the amount increased with time (correlation factor 0.86, 0.82 and 0.84, respectively, p<0.001). The addition of dietary fibre counteracted this to some extent (
The fibre-free group had more fat in the liver than the groups fed the various fibre diets (p<0.005, p<0.001 and p<0.001, respectively after 2, 4 and 6 weeks). Similar results were seen with cholesterol and triglycerides. The fibre-free groups had the highest amount of liver cholesterol at the end of each experimental period (0.4 g vs. mean 0.3 ± 0.01 g [p<0.05], 0.9 g vs. mean 0.6 ± 0.02 g [p<0.001] and 1.9 g vs. mean 1.2 ± 0.05 g [p<0.001], after 2, 4 and 6 weeks, respectively). No difference was seen between the different fibre groups. The amount of triglyceride was highest in the groups fed the fibre-free diet, 0.5 g vs. mean 0.3 ± 0.02 g (p<0.001) after 2 weeks, 1.5 g vs. mean 0.9 ± 0.07 g (p<0.001) after 4 weeks and 2.0 g vs. mean 1.3 ± 0.04 g for after 6 weeks (p<0.05).
No correlation could be found between plasma cholesterol concentrations and a longer experimental period (data not shown). However, after 2 weeks, pectin and guar gum had lower concentrations than the group fed the fibre-free diet (3.3 and 3.4 vs. 4.0 mmol/L, p<0.05) and, after 4 weeks, rats fed the mixture had the lowest concentrations (2.9 vs. 3.8 mmol/L for the fibre-free group, p = 0.0026). Remarkably, the group fed guar gum had the highest plasma cholesterol concentrations after 6 weeks (4.2 vs. 3.2 ± 0.09 mmol/L for the other groups, p<0.05).
The concentrations of acetic and propionic acids were considerably higher at week 4 than week 2, while the concentrations at week 6 were similar to the ones at week 4 (
The caecal pool of acetic, propionic, butyric and succinic acids increased with the length of the experimental period (correlation factors 0.50, 0.42, 0.35 and 0.51, respectively, and p<0.001) (
The lowest concentration of MCP-1 was seen in the group fed guar gum in LFD (23.5 ng/L) (
The T-RFLP profiles for the individual animals in the three high-fat groups, given pectin, guar gum and the fibre-free diet for 6 weeks, were compared by Principal Component (PC) Analysis. The three groups formed separate clusters (
Overweight, obesity and NAFLD are increasing rapidly, and consequently the rate of related public health diseases, like type 2 diabetes and cardiovascular disease is increasing as well
In order to see any effects on weight gain, the rats had to be fed HFD. The weight gain of the rats was on average higher with the HFD than with the LFD, but this effect was partially counteracted by dietary fibre (p = 0.057). When comparing the dietary fibres, pectin had the most noticeable effect, and the effect was greater with a prolonged experimental time. Similar results on rats have been reported elsewhere, with pectin resulting in lower weight gain
Dietary fibre increased both the caecal content and the caecal tissue weight in the rats already after 2 weeks on LFD, and many studies have reported similar results when supplementing the diet with dietary fibre
The abundance of fat reduced the amount of SCFAs formed in rats fed all the diets, with the exception of the mixture, indicating a lower bacterial activity in the colon. Similar results have been seen in pigs, with high-fat diets suppressing SCFA formation
Administration of dietary fibre in LFD for only 2 weeks tended to give lower liver weights (p = 0.078), which is quite surprising. Furthermore, contents of fat, cholesterol and triglycerides in the liver together with dietary fibre were lower than without these components, while there were no differences in plasma cholesterol concentrations between groups. Similar effects could be seen with dietary fibre in HFD, but the effects were much more pronounced. Lower plasma cholesterol concentrations with pectin and guar gum could also be seen in the rats compared with those fed a fibre-free control diet. The differences persisted over time. All this indicates that the consumption of dietary fibre, even at low fat content (5%), has beneficial effect on lipid metabolism. In an attempt to rank the dietary fibres in LFD, the mixture seemed to have most pronounced effects on liver fat, cholesterol and triglycerides (significant for all parameters), followed by guar gum (tendency for cholesterol and p<0.01 for triglycerides) and then pectin (tendency for cholesterol and p<0.05 for triglycerides). Butyric acid and, to some extent, also propionic acid have been suggested to have effects on lipid metabolism
After 6 weeks a difference in the spleen weight could be seen when comparing the groups fed guar gum and the fibre-free diets, where the fibre-free gave larger spleens (p = 0.043). A higher spleen weight has been proposed to be a marker for systemic inflammation
Interestingly, the microbiota composition in groups fed pectin, guar gum and the fibre-free diet was completely different, indicating that the different types of dietary fibre affect the composition of the microbiota. When the gut microbiota, represented by the TRFs, of the individual rats were linked to specific analytical parameters, guar gum gave abundance of
In conclusion, supplementation with fermentable dietary fibre affected weight gain, fat content of the liver and also the amount of cholesterol and triglycerides in the liver. The effects were more pronounced in HFD than in LFD and the differences increased with time. Pectin and the mixture seem to be especially prone to lower these parameters. Dietary fibre also seemed to decrease systemic inflammation, as judged by the lower MCP-1 values and lower spleen weights. High-fat content in the diet resulted initially in a reduced formation of SCFAs in caecum and in the circulation, but gradually recovered after a longer experimental period. There was an increase in concentration of succinic acid in rats fed a HFD and it increased markedly with a prolonged experimental time, while butyric acid concentration decreased, indicating a change in the caecal microbiota composition. However, guar gum and the mixture counteracted this effect and stimulated the butyric acid formation after 6 weeks. A correlation between the caecal microbiota and SCFA, liver cholesterol and triglycerides, inflammatory cytokine and pH was seen. It might therefore be speculated whether some microbial metabolites could serve as markers for e.g. cholesterol metabolism or SCFA formation.