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Monobutyrin and monovalerin improve gut–blood–brain biomarkers and alter gut microbiota composition in high-fat fed apolipoprotein-E-knockout rats

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Caecal bile acids

There were no major differences in caecal BAs between the two rat models used (groups N and HF) when fed the same type of high-fat diet, an exception was the proportion of β-MCA that tended to be lower in group HF than in group N (pa = 0.0666). Decreasing the amount of fat in diets fed to ApoE-/- rats (LF compared with HF) gave lower amounts of total BAs (Table 1, pa = 0.0797), α-MCA (pa = 0.0742) and the secondary BAs including their sum (DCA, pa = 0.0003; LCA, pa = 0.0043; UDCA, pa = 0.0214; total secondary BAs, pa = 0.0006), while the amount of the primary BA CDCA (pa = 0.012) was higher. Similar results were seen for the proportions of CDCA (pa < 0.0001), DCA (pa = 0.0004), LCA (pa = 0.0027) and UDCA (pa = 0.0856).

Table 1 Caecal amounts (mg/caecum) of bile acids (BAs).

Supplementing MB to the groups fed high-fat diets did not change the total caecal amount of BAs (pool) but altered the composition of BAs by significantly increasing the amount (pa = 0.0297) and proportion (pa = 0.0343) of CA. MB and MV tended to decrease both the total amount (p = 0.0642 for MB, p = 0.0927 for MV) and proportion (p = 0.0433, p = 0.0355) of UDCA.

Caecal microbiota composition

The Shannon index tended to be higher in the MB group compared with the N group (p = 0.0727).

Phylum level

The nine identified phyla were found in all five groups, where Firmicutes (45.3 ± 1.5%), Bacteroidetes (33.2 ± 1.5%) and Verrucomicrobia (20.5 ± 1.6%) were the most abundant, and there were no significant differences between the groups.

Conventional rats fed a high-fat diet (N) had more Deferribacteres (pa = 0.0162) in caecum than when the corresponding diet fed to ApoE-/- rats (HF) (Fig. 1a). Of the ApoE-/- rats the HF group had less Verrucomicrobia (p = 0.0482) than the LF group. Supplementing the high-fat diet with MV decreased the amount of Proteobacteria (p = 0.0355) but tended to possess higher TM7 (p = 0.0872) than the HF group. Tenericutes increased in groups fed both MB (p = 0.0066) and MV (p = 0.0438) compared with HF.

Figure 1
figure 1

Caecal microbiota at phylum level. (a) Deferribacteres, (b) Verrucomicrobia, (c) Proteobacteria and (d) Tenericutes in conventional rats (N) fed a high-fat diet and ApoE-/- rats fed either a low-fat diet (LF), a high-fat diet as it is (HF) or supplemented with 1% monobutyrin (MB) or monovalerin (MV). Box and whisker plots show minimum to maximum values, central lines presenting medians and means shown as “ + ”. All data are compared with the HF group. Significant differences compared with this group: *, p < 0.05; **, p < 0.01.

Genus level

Of 75 identified genera, 30 were important for the group separation (Supplementary Figure S1a). The five most important bacteria according to the VIP test (in descending order) were Turicibacter, Holdemania, an unidentified genus in the Christensenellaceae family, Mucispirillum and Eubacterium.

The HF group (Supplementary Figure S1b) had higher abundance of an unidentified genus in the S24-7 family (pa = 0.0091) compared with the N group, but lower Parabacteroides (p = 0.0095), Clostridium in the Peptostreptococcaceae family (p = 0.0474), Streptococcus (p = 0.0266), Mogibacteriaceae family (pa = 0.0333), Holdemania (pa = 0.0037), Mucispirillum (pa = 0.0162), Dorea (pa = 0.0218), Eubacterium (pa = 0.0657), Enterobacteriaceae family (pa = 0.0451), Ruminococcaceae family (pa = 0.0054), Anaerotruncus (p = 0.0759) and Blautia (p = 0.0524).

Within ApoE-/- groups, the LF group (Supplementary Figure S1c) had lower Allobaculum (pa = 0.0182), Prevotella (p = 0.0332), an unidentified genus in the S24-7 family (p = 0.0224), an unidentified genus of the RF32 order (p = 0.043), an unidentified genus in the Christensenellaceae family (pa = 0.0058), and Sutterella (p = 0.0748) than the HF group, whereas there was higher abundance of Coprococcus (pa = 0.0325), Lactococcus (pa = 0.014), an unidentified genus (pa = 0.0298) and Ruminococcus (pa = 0.054) in the Ruminococcaceae family, Ruminococcus in the Lachnospiraceae family (pa = 0.074), Streptococcus (pa = 0.047), an unidentified genus in the Erysipelotrichaceae family (p = 0.0385), Holdemania (p = 0.0608), Enterobacteriaceae family (pa = 0.0441), Anaerotruncus (p = 0.0325), and Akkermansia (p = 0.0482).

Adding MB (Supplementary Figure S1d) to a high-fat diet increased Adlercreutzia (pa = 0.0689), Turicibacter (p = 0.0323), Clostridia class (pa = 0.0956), Coprococcus (pa = 0.0518), Tissierella (p = 0.0956), an unidentified genus in the Erysipelotrichaceae family (pa = 0.0372), an unidentified genus of the RF39 order (pa = 0.0235), and Ruminococcaceae family (pa = 0.0174) in this group compared with the HF group but decreased Clostridium in the Clostridiaceae family (p = 0.0781).

Supplementing high-fat diets with MV (Supplementary Figure S1e) exhibited higher Parabacteroides (p = 0.0108), Turicibacter (pa = 0.0186), Coprobacillus (p = 0.0991), Clostridium in the Ruminococcaceae family (pa = 0.0586), Anaerotruncus (p = 0.0542), Mogibacteriaceae family (p = 0.0499), an unidentified genus in the Erysipelotrichaceae family (p = 0.0313), an unidentified genus in the F16 family (p = 0.0872), and an unidentified genus of the RF39 order (p = 0.0438) in the MV group than the HF group, but gave lower abundance of Allobaculum (p = 0.0409), Prevotella (p = 0.068), Sutterella (p = 0.0657), and Clostridium in the Clostridiaceae family (pa = 0.0654).

Serum corticosterone

Serum corticosterone (Fig. 2a) was significantly lower in the MB group than in the HF (p = 0.0123), N (p = 0.0033) and LF (p = 0.0675) groups. For MV, this level was also lower when compared with the N group (p = 0.021) but not with the HF or LF groups.

Figure 2
figure 2

Blood and brain markers. (a) Serum corticosterone (nM) and (b) brain gamma-aminobutyric acid (GABA, µmol/g) in conventional rats (N) fed a high-fat diet or in ApoE-/- rats fed either a low-fat diet (LF), or a high-fat diet as it is (HF) or supplemented with 1% monobutyrin (MB) or monovalerin (MV). Data are shown as means and their standard errors. Significant differences compared with the HF group: *, p < 0.05; ** p < 0.01.

GABA in the brain

Brain concentrations of GABA (Fig. 2b) were significantly higher in the LF (0.84 µmol/g, pa = 0.0418), MB (0.82 µmol/g, p = 0.0279) and MV (0.81 µmol/g, p = 0.0183) groups in comparison with the HF group (0.70 µmol/g). There was no difference in GABA concentration between the N (0.76 µmol/g) and HF group.

Correlation of caecal microbiota with metabolites

Bile acids

The amounts of caecal CA and UDCA were associated with the abundance of 17 and 12 bacteria in the MB and MV group, respectively. UDCA was linked to a higher number of bacteria than CA, that can be seen in Fig. 3a,b and more details in the Supplementary materials.

Figure 3
figure 3

Spearman correlation between caecal bacterial genera and biomarkers. ApoE-/- rats fed a high-fat diet as it is (c, HF) or supplemented with 1% monobutyrin (a, MB) or monovalerin (b, MV). Colour indicator shows positive/negative correlation in blue/orange. Only significant correlations are shown in these figures, which were part of a correlation matrix. Significant values: *, p < 0.05.

In the HF group (Fig. 3c), only the phylum TM7 and its family F16 (r = − 0.7622, p = 0.0136) were negatively correlated with CA, while UDCA was not connected with any bacteria.

Corticosterone

In the MB group (Fig. 3a), corticosterone was positively correlated to Actinobacteria phylum and its genus Adlercreutzia (r = 0.6512, p = 0.0476), Turicibacter (r = 0.7509, p = 0.0162), and Eubacterium (r = 0.653, p = 0.0481), and negatively related to Prevotella in the Paraprevotellaceae family (r = -0.7006, p = 0.0222).

In the MV group (Fig. 3b), corticosterone was negatively related to Proteobacteria phylum (r = -0.7091, p = 0.0268) and its RF32 order (r = − 0.676, p = 0.0373). No associations between corticosterone and bile acids were seen for the MB and MV groups.

In the HF group (Fig. 3c), corticosterone was positively correlated to Bilophila (r = 0.6918, p = 0.0341), and CA proportion (r = 0.7455, p = 0.0174), and negatively correlated to the amounts of CDCA (r = − 0.7697, p = 0.0126) and LCA (r = − 0.6848, p = 0.0347).

In the N group, corticosterone was found to positively associate with Blautia (r = 0.8182, p = 0.0058) and negatively correlated with an unidentified genus in the Christensenellaceae family (r = − 0.7173, p = 0.0237), TM7 phylum and its unidentified genus in the F16 family (r = − 0.7006, p = 0.0304), and CDCA proportion (r = − 0.6485, p = 0.049).

GABA

In the MB group (Fig. 3a), GABA positively correlated to Actinobacteria phylum and its genus Adlercreutzia (r = 0.8088, p = 0.004), and negatively to Parabacteroides (r = − 0.7333, p = 0.0311).

In the MV group (Fig. 3b), GABA positively correlated with rc4-4 (r = 0.7167, p = 0.0369), and Enterobacteriaceae family (r = 0.887, p = 0.0025).

In the HF group, GABA only associated with Holdemania (r = 0.7006, p = 0.0222).

In the LF group, GABA was positively correlated with α-MCA proportion (r = 0.6727, p = 0.039), and negatively with the amount of CDCA (r = − 0.6606, p = 0.0438) and Candidatus Arthromitus (r = -0.6786, p = 0.0289).

Summary of all data

Based on data from all groups, variables important for group separation are presented in Fig. 4. The top 12 variables (marked as red 4-point stars) were CDCA (relative and absolute amounts, % and mg), DCA (% and mg), UDCA (%), LCA (%), Holdemania, an unidentified genus in the Christensenellaceae family, Oscillospira, Coprococcus, an unidentified genus in the Erysipelotrichaceae family, and corticosterone. The N and HF groups were closely associated with corticosterone and UDCA. The LF group had more CDCA but less DCA, LCA and an unidentified genus in the Christensenellaceae family. MB and MV groups were diverged from the N and HF groups, with similar patterns like the LF group for some variables, including lower corticosterone and higher brain GABA, Coprococcus, and an unidentified genus in the Erysipelotrichaceae family (both belonging to the Firmicutes phylum).

Figure 4
figure 4

Important variables contributing to group separation. Conventional rats (N) fed a high-fat diet or ApoE-/- rats fed either a low-fat diet (LF), a high-fat diet as it is (HF) or supplemented with 1% monobutyrin (MB) or monovalerin (MV). Groups are shown as red circles, bacteria as 4-point stars. Large red stars are top important variables influencing group separation. Cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), gamma-aminobutyric acid (GABA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), α-muricholic acid (α-MCA) and β-muricholic acid (β-MCA).


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