Intestinal Microbiota and Development of NAFLD
Intestinal Microbiota and Development of NAFLD
Conventional mice of the strain C57BL/6J were freely fed a HFD for 16 weeks. HFD treatment led to varying body weights ranging from 33.6 to 47.0 g, with an average final body weight of 39.0±3.9 g (Table 1). Despite a majority of the mice developing metabolic disorders, several mice developed high levels of glycaemia, systemic inflammation and steatosis together and were considered as 'responders'. Conversely, several mice did not develop any metabolic disorders and were considered 'non-responders'. Therefore, we selected from this cohort one responder and one non-responder mouse to verify if we could transmit to GF mice the responder or non-responder phenotypes using gut microbiota transplants. To ensure reliable recovery of the initial gut microbiota in GF mice, transfer of microbiota from donor to receiver mice had to be performed using fresh caecal samples. As a consequence, selection of the donor mice were performed on the basis of parameters available the day of conventional mice euthanasia, including body weight, fat pad and liver weights, food intake, HOMA index and plasma concentrations of pro-inflammatory cytokines (Table 1). We intentionally selected two donor mice with similar body weights, fat pad masses and food intake to make sure that the differences in metabolic parameters were not the consequence of a different degree of obesity.
Two groups of GF male C57BL/6J mice were colonised with the inocula prepared from the two selected donor mice (conventionalisation). The two conventionalised groups were then named NRR and RR, and were subsequently maintained in isolators. After 16-week HFD feeding, both groups showed similar body weight gains (13.2±3.2 g and 14.6±2.6 g for NRR and RR, respectively, p=0.21) and final body weights (figure 1A). Daily food consumption was 2.52±0.92 g and 2.62±0.91 g for NRR and RR mice, respectively, indicating no differences in food intake. Accordingly, epididymal fat pad weights were not found to be different between these two groups (see supplementary table S1, available online only). Conversely, they displayed significantly different fasting glycaemia levels (104.4±26.6 mg/dl for NRR vs 134±34.1 mg/dl for RR) (figure 1B). Concurrently, fasting insulinaemia was lower in NRR mice than in RR mice (665.2±358.9 g/ml vs 1072.4±469.4 pg/ml) (figure 1C). The HOMA index (figure 1D) was consistently 2.4-fold greater in the RR group than in the NRR group, suggesting that the two groups developed different levels of insulin resistance. Leptinaemia (figure 1E) was 75% higher in RR than in NRR mice, whereas the level of resistin was not significantly different in the two groups (data not shown).
(Enlarge Image)
Figure 1.
Metabolic responses to high-fat diet of non-responder-receiver (NRR) and responder-receiver (RR) mice. (A) Body weight curves; (B) fasting glycaemia; (C) fasting insulinemia; (D) homeostasis model assessment—insulin resistance Index; (E) fasting leptinaemia. All mice were fasted for 6 h before blood sampling. Data are mean±SEM, n=18 for NRR and n=22 for RR mice. **p<0.01; ***p<0.001 (Student's t test).
The aspartate aminotransferase plasma concentration was three times higher in RR mice than in NRR mice (433.0±201.2 vs 135.9±73.7 UI/l, p=0.0058), whereas the difference did not achieve statistical significance (68.5±56.8 vs 38.1±34.4 UI/l, p=0.0786) for alanine aminotransferase concentrations. Fasting plasma concentrations of triglycerides, cholesterol and high-density lipoproteins were similar in the two groups (see supplementary table S2, available online only).
Concentrations and proportions of SCFA (acetate, butyrate, propionate, valerate, caproate) were found to be similar in the caecums of NRR and RR groups (see supplementary table S3, available online only). Consistently, the total SCFA concentration was not different in the two groups. Conversely, concentrations and proportions of isobutyrate and isovalerate were significantly higher in the caecum of RR mice (see supplementary table S4, available online only). These BCFA are compounds known to result from the bacterial fermentation of valine and leucine.
Liver histological analysis showed that the NRR group developed slight to mild steatosis (figure 2A), whereas the RR group developed marked mixed or macrovesicular steatosis (figure 2B). The steatosis score was found to be higher in RR than in NRR mice (3.00±0.87 vs 1.50±0.61) (figure 2C). Consistent with the morphological changes in the lipid deposition, the liver triacylglycerol concentration was 30% higher in RR mice (figure 2D). No inflammatory infiltrate was observed and no differences in liver weights were found between the two groups (see supplementary table S1, available online only).
(Enlarge Image)
Figure 2.
Representative H&E stains of paraformaldehyde-fixed liver sections prepared from non-responder-receiver (NRR) (A) and responder-receiver (RR) mice (B). The steatosis score corresponds to the percentage of hepatocytes presenting with steatosis multiplied by the intensity of the steatosis of the concerned hepatocytes (C). (D) Hepatic triglyceride concentrations (mmol of triglycerides per milligram of liver). Data are shown as mean±SEM, n=18 for NRR and n=22 for RR mice. *p<0.05; **p<0.01 (Student's t test).
We analysed the hepatic expression of genes involved in lipid uptake, lipogenesis, fatty acid catabolism and very low-density lipoprotein export. The relative expressions of the transcription factors sterol regulatory binding protein (SREBP) 1c, liver X receptor and carbohydrate response element binding protein (ChREBP) are shown figure 3A. These nuclear factors are known to promote de-novo lipogenesis. The expression of SREBP1c and ChREBP was 1.97 and 2.02 greater in the RR group than in the NRR group, whereas no difference was found in the expression of liver X receptor between the two groups. The relative expressions of three lipogenic enzymes (acetyl-CoA carboxylase 1, stearoyl-CoA desaturase 1 and fatty acid synthase) are shown figure 3B. Stearoyl-CoA desaturase 1 and fatty acid synthase were not differently expressed in the two groups of mice. Conversely, acetyl-CoA carboxylase 1 appeared to be upregulated in RR mice (fold change 1.97). CD36, which imports a large variety of lipids and lipoproteins, was more highly expressed in RR than in NRR mice (fold change 2.32). On the contrary, fatty acid transport protein 5, which essentially transports long-chain fatty acids, was slightly but significantly downregulated (fold change 0.80). No differences were found between the two groups of mice in the messenger RNA levels of carnitine palmytotransferase 1a, a transport protein that regulates mitochondrial β-oxidation, and membrane transport protein, a protein exerting a central regulatory role in lipoprotein assembly and secretion (figure 3C).
(Enlarge Image)
Figure 3.
Relative hepatic gene expression analyses of transcription factors (A), lipogenic enzymes (B) and lipids transporters (C). Results were normalised on the non-responder-receiver (NRR) group. Data are shown as mean±SEM, n=14 for NRR and n=16 for responder-receiver (RR) mice. *p<0.05; ***p<0.001. ACC, acetyl-CoA carboxylase; ChREBP, carbohydrate response element binding protein; CPT1a, carnitine palmytotransferase 1a; FAS, fatty acid synthase; FATP5, fatty acid transport protein 5; LXR, liver X receptor; MTP, membrane transport protein; SCD1, stearoyl-CoA desaturase 1; SREBP, sterol regulatory binding protein.
Systemic inflammation was evaluated by assaying the plasma concentration of pro-inflammatory cytokines. No significant differences in the plasma concentrations of these cytokines were found between the two groups of receiver mice (see supplementary table S2, available online only). We then focused specifically on liver inflammation by assaying the hepatic expression of cytokines, markers of liver macrophages and Toll-like receptors (TLR). The relative expression of TNFα, IL-1β, IL-6, IL-10 and transforming growth factor β were found to be similar in the two groups of mice (see supplementary table S5, available online only). Likewise, expressions of CD68 (a marker of macrophages), and TLR (TLR-2, TLR-4, TLR-5 and TLR-9), which play a central role in the innate immune system by recognising bacterial components leading to activation of immune response, appeared to be equivalent in NRR and RR mice (figure 4A,B). Altogether these results indicate that systemic and hepatic inflammation was similar in RR and NRR mice.
(Enlarge Image)
Figure 4.
Relative hepatic gene expression analyses of CD68 (A), and Toll-like receptors (TLR) 2, 4, 5 and 9 (B). Results were normalised on the non-responder-receiver (NRR) group. Data are shown as mean±SEM, n=14 for NRR and n=16 for responder-receiver (RR) mice.
Caecal samples used for inoculation as well as faecal samples from receiver mice after 3 (T3) and 16 (T16) weeks of HFD were analysed by pyrosequencing. A total of 188 058 sequences was obtained and after trimming, 120 241 sequences were further analysed (approximately 3000 sequences/sample). To evaluate similarity among the samples, interclass PCA was performed based on their microbial composition. The main genera between caecal samples from donor mice and faecal samples from corresponding receiver mice were conserved but showed different proportions (see supplementary figure S1, available online only). These differences may relate to sampling location or specific selection within receivers. At a genus level, the microbiota from NRR and RR mice significantly clustered separately at T3 (Monte Carlo test; p=0.001) but adopted a closer configuration at T16 (figure 5A). Barnesiella and Roseburia genera were represented more in RR mice at T3 and T16, whereas Allobaculum was higher in NRR mice (figure 5B). Moreover, HFD treatment increased Barnesiella and Allobaculum and decreased Lactobacilli in the two groups (figure 5B). The microbial phylotype richness assessed by the Chao1 estimator was very similar between the two groups whatever the sampling time (figure 5C). At the phylum level, RR mice harboured significantly increased numbers of sequences belonging to Firmicutes at T3 (p=0.0041; figure 5D). Finally, two main bacterial species (Lachnospiraceae bacterium 609 and a relative of Barnesiella intestinihominis), corresponding to more than 10% of sequences, were found to be significantly overrepresented in RR mice at T3 (p=2.16e-5 and p=0.00025, respectively) and to a lesser extent at T16. Conversely, a significantly increased number of sequences related to Bacteroides vulgatus was found in NRR mice (p=0.00041; figure 5E).
(Enlarge Image)
Figure 5.
Microbiota composition and diversity in non-responder-receiver (NRR) and responder-receiver (RR) mice (A) Interclass principal component analyses with phenotype as environmental factor was performed based on the genera abundance. Mice (light green circles, NRR_T3; green circles, NRR_T16, light red, RR_T3 and red, RR_T16) were clustered (ellipses) and the centre of gravity computed for each class. The p value of the link between phenotype and genera abundance was assessed using a Monte Carlo test (999 replicates) p=0.001. (B) Main genus composition as a percentage of total assigned sequences (sequences unassigned to any genus represent 46% of total sequences in average, SD 13.94%). (C) Estimates of bacterial richness as assessed by the Chao1 estimate (median number of OTU NRR_T3: 1052; NRR_T16: 1230; RR_T3: 1080; RR_T16: 1146). (D) Bacterial phyla distribution as a percentage of total sequences. (E) Differentially represented bacterial species (or relatives of bacterial isolates). All presented results are statistically significant (p<0.05) as assessed by the Wilcoxon test after applying Bonferroni correction.
Results
Selection of the Responder and Non-responder Donor Mice
Conventional mice of the strain C57BL/6J were freely fed a HFD for 16 weeks. HFD treatment led to varying body weights ranging from 33.6 to 47.0 g, with an average final body weight of 39.0±3.9 g (Table 1). Despite a majority of the mice developing metabolic disorders, several mice developed high levels of glycaemia, systemic inflammation and steatosis together and were considered as 'responders'. Conversely, several mice did not develop any metabolic disorders and were considered 'non-responders'. Therefore, we selected from this cohort one responder and one non-responder mouse to verify if we could transmit to GF mice the responder or non-responder phenotypes using gut microbiota transplants. To ensure reliable recovery of the initial gut microbiota in GF mice, transfer of microbiota from donor to receiver mice had to be performed using fresh caecal samples. As a consequence, selection of the donor mice were performed on the basis of parameters available the day of conventional mice euthanasia, including body weight, fat pad and liver weights, food intake, HOMA index and plasma concentrations of pro-inflammatory cytokines (Table 1). We intentionally selected two donor mice with similar body weights, fat pad masses and food intake to make sure that the differences in metabolic parameters were not the consequence of a different degree of obesity.
The Two Groups of Receiver Mice Developed Comparable Obesity but Different Metabolic Statuses
Two groups of GF male C57BL/6J mice were colonised with the inocula prepared from the two selected donor mice (conventionalisation). The two conventionalised groups were then named NRR and RR, and were subsequently maintained in isolators. After 16-week HFD feeding, both groups showed similar body weight gains (13.2±3.2 g and 14.6±2.6 g for NRR and RR, respectively, p=0.21) and final body weights (figure 1A). Daily food consumption was 2.52±0.92 g and 2.62±0.91 g for NRR and RR mice, respectively, indicating no differences in food intake. Accordingly, epididymal fat pad weights were not found to be different between these two groups (see supplementary table S1, available online only). Conversely, they displayed significantly different fasting glycaemia levels (104.4±26.6 mg/dl for NRR vs 134±34.1 mg/dl for RR) (figure 1B). Concurrently, fasting insulinaemia was lower in NRR mice than in RR mice (665.2±358.9 g/ml vs 1072.4±469.4 pg/ml) (figure 1C). The HOMA index (figure 1D) was consistently 2.4-fold greater in the RR group than in the NRR group, suggesting that the two groups developed different levels of insulin resistance. Leptinaemia (figure 1E) was 75% higher in RR than in NRR mice, whereas the level of resistin was not significantly different in the two groups (data not shown).
(Enlarge Image)
Figure 1.
Metabolic responses to high-fat diet of non-responder-receiver (NRR) and responder-receiver (RR) mice. (A) Body weight curves; (B) fasting glycaemia; (C) fasting insulinemia; (D) homeostasis model assessment—insulin resistance Index; (E) fasting leptinaemia. All mice were fasted for 6 h before blood sampling. Data are mean±SEM, n=18 for NRR and n=22 for RR mice. **p<0.01; ***p<0.001 (Student's t test).
The aspartate aminotransferase plasma concentration was three times higher in RR mice than in NRR mice (433.0±201.2 vs 135.9±73.7 UI/l, p=0.0058), whereas the difference did not achieve statistical significance (68.5±56.8 vs 38.1±34.4 UI/l, p=0.0786) for alanine aminotransferase concentrations. Fasting plasma concentrations of triglycerides, cholesterol and high-density lipoproteins were similar in the two groups (see supplementary table S2, available online only).
Concentrations and proportions of SCFA (acetate, butyrate, propionate, valerate, caproate) were found to be similar in the caecums of NRR and RR groups (see supplementary table S3, available online only). Consistently, the total SCFA concentration was not different in the two groups. Conversely, concentrations and proportions of isobutyrate and isovalerate were significantly higher in the caecum of RR mice (see supplementary table S4, available online only). These BCFA are compounds known to result from the bacterial fermentation of valine and leucine.
RR Mice Accumulated More Triglycerides in the Liver Than NRR Mice
Liver histological analysis showed that the NRR group developed slight to mild steatosis (figure 2A), whereas the RR group developed marked mixed or macrovesicular steatosis (figure 2B). The steatosis score was found to be higher in RR than in NRR mice (3.00±0.87 vs 1.50±0.61) (figure 2C). Consistent with the morphological changes in the lipid deposition, the liver triacylglycerol concentration was 30% higher in RR mice (figure 2D). No inflammatory infiltrate was observed and no differences in liver weights were found between the two groups (see supplementary table S1, available online only).
(Enlarge Image)
Figure 2.
Representative H&E stains of paraformaldehyde-fixed liver sections prepared from non-responder-receiver (NRR) (A) and responder-receiver (RR) mice (B). The steatosis score corresponds to the percentage of hepatocytes presenting with steatosis multiplied by the intensity of the steatosis of the concerned hepatocytes (C). (D) Hepatic triglyceride concentrations (mmol of triglycerides per milligram of liver). Data are shown as mean±SEM, n=18 for NRR and n=22 for RR mice. *p<0.05; **p<0.01 (Student's t test).
RR Mice Displayed a Steatosis-prone Hepatic Metabolism in Contrast to NRR Mice
We analysed the hepatic expression of genes involved in lipid uptake, lipogenesis, fatty acid catabolism and very low-density lipoprotein export. The relative expressions of the transcription factors sterol regulatory binding protein (SREBP) 1c, liver X receptor and carbohydrate response element binding protein (ChREBP) are shown figure 3A. These nuclear factors are known to promote de-novo lipogenesis. The expression of SREBP1c and ChREBP was 1.97 and 2.02 greater in the RR group than in the NRR group, whereas no difference was found in the expression of liver X receptor between the two groups. The relative expressions of three lipogenic enzymes (acetyl-CoA carboxylase 1, stearoyl-CoA desaturase 1 and fatty acid synthase) are shown figure 3B. Stearoyl-CoA desaturase 1 and fatty acid synthase were not differently expressed in the two groups of mice. Conversely, acetyl-CoA carboxylase 1 appeared to be upregulated in RR mice (fold change 1.97). CD36, which imports a large variety of lipids and lipoproteins, was more highly expressed in RR than in NRR mice (fold change 2.32). On the contrary, fatty acid transport protein 5, which essentially transports long-chain fatty acids, was slightly but significantly downregulated (fold change 0.80). No differences were found between the two groups of mice in the messenger RNA levels of carnitine palmytotransferase 1a, a transport protein that regulates mitochondrial β-oxidation, and membrane transport protein, a protein exerting a central regulatory role in lipoprotein assembly and secretion (figure 3C).
(Enlarge Image)
Figure 3.
Relative hepatic gene expression analyses of transcription factors (A), lipogenic enzymes (B) and lipids transporters (C). Results were normalised on the non-responder-receiver (NRR) group. Data are shown as mean±SEM, n=14 for NRR and n=16 for responder-receiver (RR) mice. *p<0.05; ***p<0.001. ACC, acetyl-CoA carboxylase; ChREBP, carbohydrate response element binding protein; CPT1a, carnitine palmytotransferase 1a; FAS, fatty acid synthase; FATP5, fatty acid transport protein 5; LXR, liver X receptor; MTP, membrane transport protein; SCD1, stearoyl-CoA desaturase 1; SREBP, sterol regulatory binding protein.
No Major Differences in Systemic and Hepatic Inflammation Were Detected Between the Two Groups of Receiver Mice
Systemic inflammation was evaluated by assaying the plasma concentration of pro-inflammatory cytokines. No significant differences in the plasma concentrations of these cytokines were found between the two groups of receiver mice (see supplementary table S2, available online only). We then focused specifically on liver inflammation by assaying the hepatic expression of cytokines, markers of liver macrophages and Toll-like receptors (TLR). The relative expression of TNFα, IL-1β, IL-6, IL-10 and transforming growth factor β were found to be similar in the two groups of mice (see supplementary table S5, available online only). Likewise, expressions of CD68 (a marker of macrophages), and TLR (TLR-2, TLR-4, TLR-5 and TLR-9), which play a central role in the innate immune system by recognising bacterial components leading to activation of immune response, appeared to be equivalent in NRR and RR mice (figure 4A,B). Altogether these results indicate that systemic and hepatic inflammation was similar in RR and NRR mice.
(Enlarge Image)
Figure 4.
Relative hepatic gene expression analyses of CD68 (A), and Toll-like receptors (TLR) 2, 4, 5 and 9 (B). Results were normalised on the non-responder-receiver (NRR) group. Data are shown as mean±SEM, n=14 for NRR and n=16 for responder-receiver (RR) mice.
Gut Microbiota Differs Between RR and NRR Mice
Caecal samples used for inoculation as well as faecal samples from receiver mice after 3 (T3) and 16 (T16) weeks of HFD were analysed by pyrosequencing. A total of 188 058 sequences was obtained and after trimming, 120 241 sequences were further analysed (approximately 3000 sequences/sample). To evaluate similarity among the samples, interclass PCA was performed based on their microbial composition. The main genera between caecal samples from donor mice and faecal samples from corresponding receiver mice were conserved but showed different proportions (see supplementary figure S1, available online only). These differences may relate to sampling location or specific selection within receivers. At a genus level, the microbiota from NRR and RR mice significantly clustered separately at T3 (Monte Carlo test; p=0.001) but adopted a closer configuration at T16 (figure 5A). Barnesiella and Roseburia genera were represented more in RR mice at T3 and T16, whereas Allobaculum was higher in NRR mice (figure 5B). Moreover, HFD treatment increased Barnesiella and Allobaculum and decreased Lactobacilli in the two groups (figure 5B). The microbial phylotype richness assessed by the Chao1 estimator was very similar between the two groups whatever the sampling time (figure 5C). At the phylum level, RR mice harboured significantly increased numbers of sequences belonging to Firmicutes at T3 (p=0.0041; figure 5D). Finally, two main bacterial species (Lachnospiraceae bacterium 609 and a relative of Barnesiella intestinihominis), corresponding to more than 10% of sequences, were found to be significantly overrepresented in RR mice at T3 (p=2.16e-5 and p=0.00025, respectively) and to a lesser extent at T16. Conversely, a significantly increased number of sequences related to Bacteroides vulgatus was found in NRR mice (p=0.00041; figure 5E).
(Enlarge Image)
Figure 5.
Microbiota composition and diversity in non-responder-receiver (NRR) and responder-receiver (RR) mice (A) Interclass principal component analyses with phenotype as environmental factor was performed based on the genera abundance. Mice (light green circles, NRR_T3; green circles, NRR_T16, light red, RR_T3 and red, RR_T16) were clustered (ellipses) and the centre of gravity computed for each class. The p value of the link between phenotype and genera abundance was assessed using a Monte Carlo test (999 replicates) p=0.001. (B) Main genus composition as a percentage of total assigned sequences (sequences unassigned to any genus represent 46% of total sequences in average, SD 13.94%). (C) Estimates of bacterial richness as assessed by the Chao1 estimate (median number of OTU NRR_T3: 1052; NRR_T16: 1230; RR_T3: 1080; RR_T16: 1146). (D) Bacterial phyla distribution as a percentage of total sequences. (E) Differentially represented bacterial species (or relatives of bacterial isolates). All presented results are statistically significant (p<0.05) as assessed by the Wilcoxon test after applying Bonferroni correction.
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