Microbiota of the small intestine

The microbiota of the small intestine was analyzed by qPCR and T-RFLP at 22 weeks. Two samples were also subjected to cloning and sequencing of the 16S rRNA genes. The PCR-quantified load of Lactobacillus differed between the groups after 22 weeks (p = 0.002). Pair wise comparison showed that the Lp + GT group had significantly more Lactobacillus than the control group (p = 0.002) and the GT group (p = 0.04) (Figure 1). No differences in the amount of the Gram-negative and mucin degrading genus Akkermansia (belonging to the phylum Verrucomicrobia) or total amount of bacteria were observed between the groups. The mean values of log copies/g for Akkermansia were 6.75 ± 0.27 (control), 6.85 ± 0.75 (Lp), 6.97 ± 0.52 (GT) and 7.09 ± 0.43 (Lp + GT). For the total bacteria the mean values of bacterial copies were 8.66 ± 0.38 (control), 8.81 ± 0.53 (Lp), 9.02 ± 0.40 (GT), and 9.09 ± 0.38 (Lp + GT) log copies/g. No Enterobacteriaceae could be detected, except for in one individual (data not shown).

Figure 1 Quantification (PCR-copies) of Lactobacillus in small intestinal tissue after 22 weeks. Significant differences were reached for Lactobacillus between the control group and the Lp + GT group (p = 0.002), and between the groups receiving GT and Lp + GT (p = 0.04). Ctrl = high fat control diet (HFD), Lp = HFD + L. plantarum in the drinking water, GT = HFD supplemented with 4% green tea powder, Lp + GT = HFD supplemented with 4% green tea powder and L. plantarum in the drinking water. Full size image

T-RFLP was used for analyzing the diversity of the gut microbiota. When using restriction endonuclease Msp I the number of T-RFs varied between the animals and there was a significant difference in diversity based on the number of T-RFs among the four groups (p < 0.001). The median values of the peak numbers of the groups were 3.5, 6.0, 6.5 and 8.0 for control, Lp, GT and Lp + GT respectively. Pair wise comparison of the groups revealed significant differences between control and GT (p < 0.05), and between control and Lp + GT (p < 0.001). Shannon (H') as well as Simpsons’s diversity index (D) showed a significant difference among the four groups (p = 0.007 and p = 0.006 respectively). Pair-wise comparison showed that the Lp + GT group had a significantly more diverse microbiota than the control group (p = 0.008) and the GT group according to the Shannon diversity index (p = 0.04) (Figure 2). According to the Simpson’s diversity index the Lp group had a significantly higher diversity than the GT group (p = 0.04) (data not shown). A T-RF corresponding to L. plantarum DSM 15313 was found in 5 individuals in the Lp group and in 4 individuals in the Lp + GT group. In order to identify some of the T-RFs in the T-RFLP profiles, the two mice with the highest number of T-RFs in the control and Lp + GT group were selected for bacterial cloning. The obtained sequences were edited and 55 sequences were analyzed and compared to the closest matches in the RDP database [36] (Table 1). Fiftyone out of fiftyfive clones belonged to the phylum Firmicutes. Within Firmicutes, sequences similar to the family Erysipelotrichaceae were dominating. These sequences were most similar to the genus Allobaculum, but the similarity was only 89%. In the mouse from the Lp + GT group, 25.9% of the clones were identified as Lactobacillus compared to only 3.6% from the control mouse. Clones identified as Lactobacillus were most similar to sequences belonging to Lactobacillus reuteri and Lactobacillus intestinalis. Due to differences between the theoretical and the actual sizes of T-RFs of the clones no attempt was made to identify the T-RFs except for Akkermansia and the given L. plantarum strain which showed fragments length of 265 and 568 bp, respectively. A comparison was made between T-RFLP and the numbers of copies in the qPCR. The mean copy value for Akkermansia obtained in the qPCR for the T-RFs detected in the T-RFLP was log 7.28 ± 0.52 compared to a mean of log 6.75 ± 0.27 for copy number that was not detected by T-RFLP. No microbial analyses of the small intestine were performed at 11 weeks.

Figure 2 Bacterial diversity in the small intestine after 22 weeks. The data is based on T-RFLP-profiles and Msp 1 digestion. The area of each peak, expressed as the proportion of the total area, was used for calculation of the Shannon diversity index. Control vs. Lp + GT (p = 0.008), GT vs. Lp + GT (p = 0.04). Full size image

Table 1 Direct identification of 16S rRNA genes from small intestinal tissue by PCR-amplification, cloning and sequencing Full size table

Caecum weight and microbiota

After 11 weeks, the caecum weights were significantly higher in mice from the GT groups compared to mice in the control and Lp groups (control: 0.126 ± 0.006, Lp: 0.133 ± 0.007 GT: 0.225 ± 0.015, Lp + GT: 0.229 ± 0.015 g). After 22 weeks, the difference in caecum weights was even more pronounced (control: 0.125 ± 0.013, Lp: 0.148 ± 0.008, GT: 0.291 ± 0.027, Lp + GT: 0.332 ± 0.022 g). The caecum microbiota was analyzed by cultivation and by T-RFLP after 11 and 22 weeks. The viable count of lactobacilli in the caecum content was significantly higher in the Lp group (p <0.01) than in the control group at 11 weeks while the Lp + GT group had significant more lactobacilli than control (p < 0.01) and the GT-group (p < 0.05) after 22 weeks (see Additional file 6). L. plantarum DSM 15313 was found at both 11 and 22 weeks in the two groups fed L. plantarum. L. plantarum could be reisolated from 77% and 100% of the mice in the Lp + GT and Lp groups, respectively (data not shown). No significant difference in the viable count of Enterobacteriaceae was seen after 11 weeks in the three treatment groups compared to the control. After 22 weeks, the viable count of Enterobacteriaceae had decreased below the detection limit in more than 40% of the animals and no statistical analysis was made (data not shown).

T-RFLP analysis of the caecum content generated in total 72 different T-RFs after 11 weeks and 82 T-RFs after 22 weeks in the T-RFLP-profile using Msp I. At 11 weeks the GT group had significantly more T-RFs than the control when using Msp I (p < 0.01). For Alu I digestions, both the GT group and the Lp + GT group had significantly more T-RFs than the control group (p < 0.05). After 22 weeks there were no differences compared to the control with either Msp I or Alu I (data not shown). Shannon and Simpson diversity indices were calculated for each sample using the relative T-RF area. After 11 weeks both the Shannon and the Simpson indices showed a significantly higher diversity of the microbiota in the GT group compared to the control when using Alu 1 digestion for the former and either Msp I or Alu 1 digestion for the later (see Additional file 7). After 22 weeks, no significant differences in the bacterial diversity of the caecum content could be seen between the treatment groups and the control (see Additional file 7). In order to perform a putative identification of T-RFs from the caecum content, a pure culture of L. plantarum DSM 15313 was analyzed by T-RFLP, showing a single T-RF of 568 base pairs. This T-RF was not found in the control group or in the GT group but in all animals of the Lp group and in 75% of the mice in the Lp + GT group. Also, a T-RF (265 bp) corresponding to Akkermansia was found in 27 mice of 29 after 11 weeks and in 40 mice of 43 after 22 weeks.

Adiposity

Body weight gain and relative body fat content was significantly reduced in mice of the GT groups compared to control mice (Figure 3A, C). A supplement of Lp had no significant effect on body weight or body fat content. The mean energy intake was higher in mice fed GT compared to mice fed a diet without GT (Figure 3B). In addition, periovarian white adipose tissue depots (Figure 3E) and circulating leptin (Figure 3F) were strongly reduced in mice receiving GT. No significant difference in lean body mass was observed between groups (Figure 3D). The amount of Akkermansia in the small intestine correlated negatively with body fat content (rho = −0.43; p = 0.04), periovarian white adipose tissue (rho = −0.43; p = 0.03) as well as plasma leptin (rho = −0.45; p = 0.03). The total amount of bacteria in the small intestine correlated negatively with periovarian white adipose tissue (rho = −0.41; p = 0.04) and showed a tendency towards a negative correlation with total body fat (rho = −0.41; p = 0.07) and plasma leptin (rho = −0.35; p = 0.09). The mean faecal TAG excretion was significantly elevated in the GT groups compared to control (control: 0.08 ± 0.01, Lp: 0.18 ± 0.05, GT: 0.27 ± 0.04, Lp + GT: 0.31 ± 0.04 mg/mouse/24 h). The total amount of faeces was not significantly different between the groups although a tendency towards increased excretion was observed in the GT groups compared to the control group (ctrl: 0.27 ± 0.02, Lp: 0.30 ± 0.04, GT: 0.39 ± 0.06, Lp + GT: 0.40 ± 0.06 mg/mouse/24 h).

Figure 3 Decreased adiposity in mice fed a diet supplemented with green tea. (A) Weekly body weight registration during the 22 week study. (B) Average energy intake calculated as KJ/mouse/day. (C) Relative body fat content (%) recorded using DEXA scan technique at week 0, 5, 11 and 22. (D) Lean body mass measured with DEXA at 0, 5, 11 and 22 weeks. (E) Periovarian white adipose tissue weight and (F) plasma leptin concentration after 22 weeks on the different diets. Ctrl = high fat control diet (HFD), Lp = HFD + L. plantarum in the drinking water, GT = HFD supplemented with 4% green tea powder, Lp + GT = HFD supplemented with 4% green tea powder and L. plantarum in the drinking water. Data are means ± SEM n = 21 at 11 weeks and n = 9–11 at 22 weeks. *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

Blood glucose control

Oral glucose tolerance tests were performed at week 8 and week 21. At week 8, the mice in the two GT groups had a significantly increased insulin response compared to control mice (Figure 4B). Also, the Lp + GT group had a significantly increased glucose response compared to the control and the Lp group (Figure 4A). The same tendencies were seen at week 21, however, the differences did no longer reach statistical significance (Additional file 8). An insulin tolerance test was performed at week 15. No significant differences were observed between any of the groups (Figure 4C). After 22 weeks, fasting plasma glucose, insulin and fructosamine were lower in the mice from the GT groups, while insulin and fructosamine were significantly lower already after 11 weeks (Table 2). In addition, after 22 weeks, GT resulted in a lower HOMA index of insulin resistance (Figure 4D). The amount of Akkermansia in the small intestine correlated negatively to plasma insulin (rho = −0.47; p = 0.03) and there was also a tendency towards a negative correlation between the total amount of bacteria and plasma insulin (rho-0.35; p = 0.07). No differences in plasma glucose or insulin were observed between the groups at the initiation of the study (data not shown).

Figure 4 Altered glucose and insulin tolerance by supplementing the diet with green tea. (A) Plasma glucose and (B) insulin concentrations in an oral glucose tolerance test performed at week 8. Mean values and SEM for 11 mice in each group. (C) Plasma glucose expressed as % of basal after an intraperitoneal injection of insulin at week 15. Mean values and SEM for 9–11 mice in each group. No differences were detected between any of the four groups. The areas under the curves (AUC) are shown in the insets (D) HOMA-IR index at week 22. The index was calculated by multiplying fasting glucose (mM) and fasting insulin (μU/ml) divided by 22.5. Ctrl = high fat control diet (HFD), Lp = HFD + L. plantarum in the drinking water, GT = HFD supplemented with 4% green tea powder, Lp + GT = HFD supplemented with 4% green tea powder and L. plantarum in the drinking water. Data are means ± SEM for 9–11 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

Table 2 Plasma profile after indicated time of dietary treatment Full size table

Lipid metabolism

Both the GT and Lp + GT groups had significantly lower plasma TAG compared to control after 22 weeks (Table 2). No differences in plasma TAG were observed at the start of the study. Liver weights were significantly lower in mice from both GT groups compared to control mice after both 11 and 22 weeks (Figure 5A and B). The liver weight was also significantly lower in the Lp + GT group compared to the Lp group. Also the liver TAG content related to liver mass was decreased in the groups fed GT and showed the same picture regarding significance as the liver weight (Figure 5C and D). Between 11 and 22 weeks, there was a general increase in liver TAG accumulation in all groups but the GT group. Moreover, the liver enzyme ALT was decreased in plasma from mice in the GT groups compared to the mice in the control group (Figure 5E and F). However, plasma ALT was generally higher in all groups after 22 weeks compared to 11 weeks. After 22 weeks the mRNA expression of the lipogenic transcription factors SREBP1c and PPARγ was significantly down-regulated in both groups of mice fed GT (Figure 6A and B). The mRNA expression of the lipogenic enzyme ACC was decreased only in mice receiving GT compared to control, and the same trend was observed for the expression of FAS mRNA (Figure 6C and D). The hepatic mRNA expression of PPARα, PGC-1α, CD36, ACADL, LXR, PXR, chREBP, XBP1, PEPCK, CREB and GK is shown in the Additional file 9. A significant difference in the mRNA expression of PPARα, which plays a role in the control of fatty acid oxidation, was observed between mice in the GT group compared to control mice at 22 weeks. Also, a decreased mRNA expression of CD36 and LXR, both involved in the control of lipogenesis, was observed in mice from the Lp + GT group compared to control, after 11 and 22 weeks respectively (Additional file 9). The amount of Akkermansia correlated negatively with the TAG content in the liver (rho = −0.44; p = 0.03) and there was also a tendency towards a negative correlation between the total amount of bacteria and liver TAG (rho-0.36; p = 0.07).

Figure 5 Improved liver phenotype in mice fed a diet supplemented with green tea. Liver weights after 11 (A) and 22 (B) weeks on the different diets. Triacylglycerol (TAG) content in liver after 11 (C) and 22 (D) weeks, respectively. The concentration of the liver enzyme ALT measured in plasma after 11 (E) and 22 (F) weeks. Ctrl = high fat control diet (HFD), Lp = HFD + L. plantarum in the drinking water, GT = HFD supplemented with 4% green tea powder, Lp + GT = HFD supplemented with 4% green tea powder and L. plantarum in the drinking water. Data are means ± SEM for n = 21 at 11 weeks and n = 9–11 at 22 weeks. *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

Figure 6 Decreased lipogenic gene expression in liver of mice fed a diet supplemented with green tea. Hepatic mRNA expression of sterol regulatory-binding protein 1c (SREBP1c, A), peroxisome proliferator-activated receptor γ (PPARγ, B), acetyl CoA carboxylase (ACC, C) and fatty acid synthase (FAS, D) was quantified with real-time PCR after 22 weeks of the different diets. Ctrl = high fat control diet (HFD), Lp = HFD + L. plantarum in the drinking water, GT = HFD supplemented with 4% green tea powder, Lp + GT = HFD supplemented with 4% green tea powder and L. plantarum in the drinking water. Data are means ± SEM. n = 9–11 at 22 weeks. *p < 0.05, **p < 0.01. Full size image

Cholesterol metabolism

Plasma cholesterol was significantly higher in the Lp + GT group after 11 weeks but after 22 weeks it was significantly lower in both GT groups compared to control (Table 2). No differences in plasma cholesterol were observed between the groups at the start of the study (data not shown). The liver cholesterol content was decreased in both groups receiving GT after 11 weeks, but after 22 weeks it was also decreased in the Lp group compared to control (Figure 7A and B). Liver cholesterol decreased over time in all groups but Lp + GT, which showed unchanged cholesterol content between 11 and 22 weeks. After 11 weeks, the key enzyme in cholesterol synthesis, HMGCoA reductase was upregulated in the Lp + GT group but no significant changes in the mRNA expression of the cholesterol-regulating transcription factor SREBP2 were observed between the groups (Figure 7C and E). However, after 22 weeks, HMGCoA reductase was significantly up-regulated in the Lp + GT group compared to the other groups (Figure 7D) and SREBP2 was upregulated compared to control and Lp (Figure 7F). The hepatic expression of SR-B1, LDLR and CYP7A1, genes involved in reverse cholesterol transport, is shown in Additional file 9. SR-B1, an HDL receptor, was significantly upregulated in mice fed GT compared to control mice. The mRNA expression of the LDL receptor was down-regulated in mice in the GT group compared to control mice but the significance was eliminated with addition of Lp. Total cholesterol excretion was increased in the GT groups compared to control (control: 0.64 ± 0.1, Lp: 0.65 ± 0.11, GT: 1.56 ± 0.11, Lp + GT: 1.55 ± 0.18 mg/mouse/24 h). The hepatic cholesterol content and plasma cholesterol correlated negatively with the total amount of bacteria (rho = −0.43; p = 0.04 and rho = −0.44; p = 0.04 respectively).

Figure 7 Altered cholesterol homeostasis in mice fed a diet supplemented with green tea and Lactobacillus plantarum . Total cholesterol content in liver after 11 (A) and 22 (B) weeks on the different diets. Hepatic mRNA expression of hydroxy-methyl-glutaryl-CoA reductase (HMGCR, C and D) and sterol regulatory-binding protein 2 (SREBP2, E and F) after 11 and 22 weeks respectively. Ctrl = high fat control diet (HFD), Lp = HFD + L. plantarum in the drinking water, GT = HFD supplemented with 4% green tea powder, Lp + GT = HFD supplemented with 4% green tea powder and L. plantarum in the drinking water. Data are means ± SEM. n = 21 at 11 weeks and n = 9–11 at 22 weeks. *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

Markers of inflammation

Circulating PAI-1 was significantly decreased in mice in the Lp + GT group compared to mice in the Lp group (p < 0.05) after 22 weeks but not after 11 weeks (Figure 8A and B). Plasma PAI-1 was also negatively correlated with Akkermansia (rho = −0.49; p = 0.03) at 22 weeks. The cytokines IL-6 and MCP-1 were analyzed in the same multiplex assay as PAI-1, but the plasma concentrations were below the detection limit. The hepatic mRNA expression of MCP-1 and TNF-α showed no differences after 11 weeks but with a tendency of decreased MCP-1 in the Lp + GT group (Figure 8C and E). After 22 weeks MCP-1 decreased in the Lp + GT group compared to the Lp group and TNF-α decreased in the Lp + GT group compared to both the control and the GT group (p < 0.05) (Figure 8D and F). The hepatic mRNA expression of PAI-1, TLR4, MyD88 and F4-80 is shown in Additional file 9. A significant difference in the expression of TLR4 was observed between mice in GT and Lp + GT groups. Moreover, the spleen weights were decreased in the Lp + GT group compared to control group after 11 weeks (p < 0.05) (Figure 8G), and after 22 weeks, mice in the two GT groups had significantly smaller spleens compared to the control group (p < 0.001) (Figure 8H).

Figure 8 Decreased inflammatory markers in mice fed a combination of green tea and Lactobacillus plantarum . The inflammatory marker PAI-1 was analyzed in plasma after 11 (A) and 22 (B) weeks using Luminex Technology. Quantitative real-time PCR was used to analyze the liver mRNA expression of the inflammatory markers monocyte chemoattractant protein 1 (MCP-1, C and D) and tumor necrosing factor α (TNF-α, E and F) after 11 and 22 weeks of the study. Spleen masses were analyzed as a reflection of inflammatory activity (G and H). Ctrl = high fat control diet (HFD), Lp = HFD + L. plantarum in the drinking water, GT = HFD supplemented with 4% green tea powder, Lp + GT = HFD supplemented with 4% green tea powder and L. plantarum in the drinking water. Data are means ± SEM. n = 21 at 11 weeks and n = 9–11 at 22 weeks. *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

Multivariate data analysis

A PCA of the metabolic data was performed for the four groups of mice. The mice fed green tea clustered separately from the control and Lp groups (data not shown). An x/y plot showed that most of the variability in the model was explained by body fat (data not shown).

A PCA was applied on the T-RFLP data of the small intestine and the caecum, respectively, combined with the metabolic parameters, in order to reveal differences and similarities between and within the four groups. The combined data for the small intestine showed that mice in the GT and Lp + GT groups displayed more similarity to each other compared to the control and Lp groups (data not shown). The combined caecum data indicate that the mice in the Lp group clustered together while the mice in the three other groups were more different from each other (data not shown). A PLS analysis of the combined data was then performed to reveal any correlation between the T-RFs, i.e. microbiota, and the metabolic parameters. The obtained PLS loadings bi-plots showed that the control and the Lp groups were separated from the GT and Lp + GT groups (Figure 9A and B). Variables located closely together are more likely to have a positive correlation, while variables far away from each other, either along the first or the second principal component, are more likely to be negatively correlated. The T-RFs having the most influence on the model are indicated with their fragment size, and in the small intestine a high abundance of T-RFs 118 and 184 correlated positively to the increase of the metabolic markers while T-RF 271 was negatively correlated (Figure 9A). In the ceacum, T-RFs 270 and 307 correlated negatively with all the metabolic markers except SR-B1 expression and caecum weight while T-RF 568 and 497 correlated positively with the same data (Figure 9B). The T-RF 568 corresponds to the given strain L. plantarum DSM 15313 and the positive correlation is probably due to the high abundance of this strain in the Lp group.