Inulin prevents diet induced NAFLD
FPC diet intake for 7 months caused steatosis (Figure 1A). FPC diet-fed mice had macrovesicular fat with grade 3 steatosis, and inulin-fed mice had improved fat scores (Figure 1B). In addition, inulin-supplemented mice had normal liver weights and liver-to-body weight ratios (Figure 1B). Moreover, FPC diet-fed mice had elevated ALT and ALP, and inulin supplementation reversed these changes (Figure 1B).
Inulin improves hepatic metabolism in FPC diet-fed mice
FPC diet intake increased the expression of genes involved in fatty acid synthesis, uptake, and mobilization as evidenced by increased mRNA levels of Acetyl-CoA carboxylase 1 (Acc1), Stearoyl-CoA desaturase-1 (Scd1), Fatty acid synthase (Fasn), Cluster of differentiation 36 (Cd36), Fatty Acid Binding Protein 4 (Fabp4), and Sterol regulatory element-binding protein 1 (Srebp1c) as well as genes implicated in fatty acid oxidation such as Peroxisome proliferator-activated receptor α (Pparα), PPARα-regulated Cyp4a14 and Cyp4a10, and Cyp2e1. In contrast, inulin supplementation prevented these changes in FPC diet-fed mice (Figure 2A). Similarly, FPC diet increased protein levels of FASN, CD36, and SCD1, which were all reduced by inulin. Representative Western blots are shown in Figure 2B.
Inulin prevents FPC diet-induced inflammatory signaling
IL-17A is implicated in the development of chronic inflammatory diseases in the brain, skin, and liver as well as liver cancer [31-34]. FPC diet-fed mice had increased mRNA level of Rorγt, a transcriptional factor for Il-17 expression, accompanied by elevated hepatic protein levels of IL-17A and TNFα as well as mRNA levels of Il6, Il1β, and Tgfβ, which generate Th17 cells; inulin supplementation prevented these changes (Figure 3A, B). However, IL10 cytokine family member Il22 displayed opposite trends in response to FPC diet intake and inulin supplementation (Figure 3A). Other inflammatory and fibrosis-related genes such as Tnfα, Mcp-1, Col1a1, Timp1, Mmp2, and Mmp9 as well as macrophage or neutrophil genes such as F4/80, Cd11b, and Cd68 were induced by FPC diet intake and reduced by inulin (Figure 3A).
Inulin mitigates FPC-reduced hippocampal LTP and neuroplasticity
FPC diet-fed mice had reduced LTP at Schaffer collateral-CA1 synapses compared with that of CD-fed mice, and inulin-supplemented mice recovered such a reduction (Figure 4A, B). In addition, open field behavior study revealed that FPC diet intake increased travel distance and central time spending, and inulin supplementation prevented such increases accompanied by increased rearing (Figure 4C). Furthermore, FPC diet-fed mice had reduced postsynaptic density-95 (PSD-95), a potent regulator of synaptic strength, as well as brain derived neurotrophic factor (BDNF); however, inulin-supplementation prevented these negative effects caused by FPC intake (Figure 4D) and improved synaptic plasticity.
Inulin reverses FPC diet-induced microglia activation
Microglia have pivotal roles in the inflammatory process in various neurodegenerative conditions; however, little is known about the effect of inulin on microglia activation. In this study, FPC diet-fed mice had elevated mRNA levels of Rorγt, Il-17a, as well as Il22 and increased protein levels of inflammatory marker IL-17A, IL6, and TNFα. (Figure 5A, B). We showed that FPC diet intake increased the expressions of proinflammatory modulators Il1β, Il6, Tnfα, and Nos2 in the brain, and such inductions were reversed by inulin supplementation (Figure 5A). Similar expression trends were noted for genes implicated in inflammation or markers for macrophage such as Mcp-1, Ccl5, Ccl17, Ccl20, and Nos2 as well as F4/80 and Cx3cr1 (Figure 5A). The mRNA levels of microglia calcium-activated potassium and voltage-dependent potassium channels Kca3.1 and Kv1.2, which are involved in microglial activation and inflammation, were also elevated in FPC diet-fed mice and reversed by inulin supplementation (Figure 5A). Together, inulin prevented FPC diet-induced neuroinflammation and microglia activation.
FPC and inulin supplementation modulate intestinal microbiota
Firmicutes and Bacteroidetes are the most abundant gut bacterial phyla. FPC diet-fed mice had reduced Firmicutes while inulin-supplemented mice had increased Bacteroidetes (Figure 6A). FPC diet intake resulted in increased Proteobacteria, Actinobacteria, and Verrucomicrobia, and inulin intake only reduced the abundance of Actinobacteria (Figure 6A).
The reduction of Firmicutes in FPC diet-fed mice is a direct result of the reduction in the families Lachnospiraceae and Lactobacillaceae, which promote gut health and fight inflammation; however, inulin supplementation increased their abundances (Figure 6B) and reduced inflammation. In contrast, Erysipelotrichaceae, which is involved in cholesterol metabolism, as well as Peptostreptococcaceae, Clostridiaceae, and Eubacteriaceae genera under Firmicutes were enriched by FPC diet, however inulin reduced them (Figure 6B). Moreover, inulin-fed mice had an increased family under Bacteroidetes, i.e., Porphyromonadaceae, which protects the gut from infection in mouse models [35] (Figure 6B). The family Helicobacteraceae under Proteobacteria, which is implicated in gastrointestinal inflammation [35], increased in FPC diet-fed mice, and inulin supplementation reduced it (Figure 6B). Additionally, increased Proteobacteria in FPC diet-fed mice could be attributed to increased Sutterellaceae, which is implicated in gut inflammation as well as autism and Down syndrome [37, 38]. Under Actinobacteria phylum, Coriobacteriaceae, which has a role in cholesterol metabolism, increased with FPC diet intake and reduced with inulin supplementation (Figure 6B).
At the genus level, FPC diet-fed mice had increased levels of Allobaculum and Holdemanella (Erysipelotrichaceae family), Romboutsia (Peptostreptococcaceae family), Romboutsia (Peptostreptococcaceae family), Olsenella and Clostridium XVIII (Coriobacteriaceae family), Lactonifactor (Clostridiaceae family), Eubacterium (Eubacteriaceae family), Enterococcus (Enterococcaceae family), Parasutterella (Sutterellaceae family), and Dorea and Acetatifactor (Lachnospiraceae family); inulin supplementation resulted in reductions in these genus except for Acetatifactor (Figure 6C). In contrast, FPC diet reduced Lactobacillus (Lactobacillaceae family), Eisenbergiella (Lachnospiraceae family), Butyrivibrio (Lachnospiraceae family), Clostridium XIVa (Clostridiaceae family), and Bifidobacterium (Bifidobacteriaceae family), and inulin supplementation resulted in their increases except for Bifidobacterium. Under the family Porphyromonadaceae, inulin enriched Barnesiella and Coprobacter (Figure 6C).
Inulin ameliorates diet-induced dysregulated BA signaling
BA synthesis is jointly regulated by host and gut microbes. Dysregulated BA synthesis is implicated in hepatic inflammation and neurological diseases [24, 39]. We therefore studied the expression of genes that regulate BA homeostasis. FPC diet-fed mice had reduced mRNA and protein levels of FXR and its target gene small heterodimer partner (Shp) as well as its encoded protein, which were reversed by inulin supplementation (Figure 7A, B). In consistency, FXR-regulated genes that are responsible for BA synthesis such as hepatic Cyp7b1 and Cyp27a1 had the same expression patterns (Figure 7A). Additionally, the level of Cyp7a1, a rate-limiting enzyme for BA synthesis, was increased in the steatotic livers but reduced by inulin supplementation (Figure 7A). Moreover, the mRNA and protein levels of hepatic TGR5, another FXR target gene, were reduced in FPC diet-fed mouse livers but increased with inulin supplementation (Figure 7A, B). Further, BA uptake genes Slc10a1 and Slc01b2 as well as transporter genes Abbc1 and Abbc4 were induced in FPC diet-fed mice, and inulin supplementation reversed these inductions, suggesting normalization of BA homeostasis (Figure 7A). Lastly, hepatic HNF4α was reduced by FPC diet intake and increased by inulin supplementation, which likely contributes to normalizing the hepatic transcriptional machinery (Figure 7A, B). Together, diet-induced metabolic overburden inactivates FXR signaling, and inulin normalizes those changes.
The copy number of bacterial genes that produce BAs was also quantified using cecal DNA. FPC diet-fed mice had increased BA inducible operon J (baiJ), a gene involved in BA 7α-dehydroxylation, and inulin supplementation reduced it (Figure 7C). However, the abundance of bile salt hydrolase gene (bsh), which hydrolyzes conjugated BAs into free BAs, was unchanged by FPC diet intake but was increased by inulin (Figure 7C).
TGR5 is abundantly expressed in the macrophage [40, 41]. We thus studied TGR5-regulated signaling in freshly isolated microglia. In consistency with the data generated from the digestive tract, FPC diet-fed mice had reduced Tgr5 as well as TGR5-regulated Nos1 and Dio2 (Figure S2). The expressions of FXR and its target gene Shp were also reduced in the microglia of FPC-fed mice, and inulin supplementation increased their expression levels (Figure S2). Thus, diet and inulin have an impact on BA-regulated signaling at the systemic level.
The effect of FPC diet and inulin on gut metabolites
Metabolomics profiling was performed using cecum content by GC-TOF-MS, which identified 273 known metabolites. As per sparse partial-least-squares discriminant analysis (sPLS-DA), metabolites had different clusters (Figure S3). Pathway analysis revealed that the most significant difference between CD- and FPC diet-fed mice occurred in lipid metabolism that includes steroid hormone, BA, steroid, and fatty acid biosynthesis (Supplementary table 2, Figure S3). However, inulin supplementation markedly changed sucrose metabolism in addition to amino acid and carbohydrate metabolism pathways, arginine, proline, butanoate, and starch. The top 15 most significantly changed pathways in response to FPC diet intake and inulin supplementation are listed in Supplementary Table 2 and Figure S3.
Fold changes in the cecal metabolites are shown in the volcano plots (Figure 8A). Zymosterol and 2, 8-dihydroxyquinoline (2, 8-DHQ) are commonly found in both FPC vs. CD and FPC + inulin vs. FPC plots. Zymosterol, the precursor of cholesterol, was increased in FPC diet-fed mice but was reduced by inulin intake. In contrast, 2, 8-DHQ was markedly reduced in FPC diet-fed mice, and inulin supplementation increased it (Figure 8A). Additionally, glucose 6-phosphate, fructose 6-phosphate, galactose 6-phosphate, xylose, and lyxose had reduced concentrations in FPC diet-fed mice (Figure 8A). 3-Hydroxyphenylacetic acid, a metabolite of the flavonoid rutin which protects against glucose intolerance and obesity, was decreased due to FPC diet intake. In contrast, tocopherols (β, γ, δ) and digalacturonic acid were increased by FPC diet intake (Figure 8A). Supplementation of inulin increased trans-4-hydroxyproline, β-sitosterol, isothreonic acid, and adenine (Figure 8A).
Chemical similarity enrichment analysis shows that FPC diet-fed mice had reduced hexose phosphates, purine nucleosides, pyrimidinones, and sugar alcohols, which were increased by inulin supplementation (Figure 8B). In contrast, cholestanes, a cholesterol metabolite, was increased in FPC diet-fed mice, and inulin supplementation reduced it (Figure 8B). In addition, butyrate, a major SCFA produced by fermentation of inulin, was decreased by FPC diet intake and increased by inulin supplementation (Figure 8B).
The relationships between metabolites and the gut microbiota
Among the 273 identified metabolites, 104 had significant changes in their scaled intensity due to FPC diet intake or inulin supplementation. These metabolites and 59 bacterial genera found in the mouse cecum were subjected to Spearman’s correlation analysis (Figure 9).
Genus Allobaculum, which was increased in FPC diet-fed mice, was positively associated with cholesterol and zymosterol, but negatively correlated with sugar alcohols (xylose, and lyxose); glycolysis pathway metabolites (glucose-6-phosphate and fructose-6-phosphate); and pinitol, which has a role in regulating insulin function (Figure 9). In addition, genus Holdemanella was positively correlated with the scaled intensity of cecum fatty acids stearic acid and palmitic acid as well as fatty acid byproduct monostearin (Figure 9). Genus Barnesiella, Coprobacter, Clostridium XIVa, and Butyrivibrio, which are negatively correlated with tocopherol, zymosterol, and monostearin, had increased abundances in inulin-fed mice (Figure 9). Moreover, Lactobacillus and Eisenbergiella genus, which also increased due to inulin supplementation, were negatively associated with cholesterol, zymosterol, and fatty acids D-erythrosphingosine, palmitic acid, stearic acid, as well as arachidonic acid (Figure 9). These fatty acids are clustered together and positively associated with the bacteria that were enriched in FPC diet-fed mice (Figure 9).