Dietary supplementation with fermentable fiber suppresses adiposity and the associated parameters of metabolic syndrome. Microbiota-generated fiber-derived short-chain fatty acids (SCFAs) and free fatty acid receptors including GPR43 are thought to mediate these effects. We find that while fermentable (inulin), but not insoluble (cellulose), fiber markedly protected mice against high-fat diet (HFD)-induced metabolic syndrome, the effect was not significantly impaired by either inhibiting SCFA production or genetic ablation of GPR43. Rather, HFD decimates gut microbiota, resulting in loss of enterocyte proliferation, leading to microbiota encroachment, low-grade inflammation (LGI), and metabolic syndrome. Enriching HFD with inulin restored microbiota loads, interleukin-22 (IL-22) production, enterocyte proliferation, and antimicrobial gene expression in a microbiota-dependent manner, as assessed by antibiotic and germ-free approaches. Inulin-induced IL-22 expression, which required innate lymphoid cells, prevented microbiota encroachment and protected against LGI and metabolic syndrome. Thus, fermentable fiber protects against metabolic syndrome by nourishing microbiota to restore IL-22-mediated enterocyte function.

Obesity and its associated metabolic disorders, collectively referred to as metabolic syndrome, are among humanity’s most pressing public health problems (). Metabolic syndrome is increasingly viewed as a chronic inflammatory disease in which altered host-microbiota interactions in the gut contribute to disease development (). Accordingly, in humans, metabolic syndrome is associated with alterations in gut microbiota composition and gut bacteria infiltrating the inner mucus layer, thus encroaching upon gut epithelial cells in a manner that may promote inflammation (). Societal changes in dietary habits, especially increased consumption of processed foods lacking fiber, are thought to have impacted the microbiota and contributed to increased incidence of chronic inflammatory disease, including metabolic syndrome (). Accordingly, administering mice or humans supplements of the fermentable fiber inulin suppresses adiposity and associated parameters of metabolic syndrome (). However, the doses of inulin needed to provide a marked health benefit are difficult to achieve both because of logistical considerations and adverse effects, especially bloating and flatulence. Hence, it remains important to use tractable models to define mechanisms by which fermentable fiber protects against metabolic syndrome, thus providing the rationale for the central question addressed herein. Previous studies have reported a role for microbiota-generated inulin-derived short-chain fatty acids (SCFAs) in promoting beneficial aspects of host metabolism and reducing inflammation, acting in large part through free fatty acid receptor GPR43 (also known as Ffar2) (). In contrast, our results indicate that much of the ability of inulin to suppress diet-induced obesity (DIO) may not require SCFAs but is driven by inulin-elicited bacteria inducing interleukin-22 (IL-22), which fortifies the intestine, thus reducing microbiota encroachment and ameliorating metabolic syndrome.

In considering how IL-22 might protect against metabolic syndrome, we hypothesized that IL-22-induced fortification of the gut might protect against bacterial translocation that promotes LGI, which has classically been defined as mild elevations in pro-inflammatory gene expression in adipose tissue (). In accord with this possibility, we observed that, in the absence of IL-22, inulin no longer significantly induced Reg3γ ( Figure 7 A) and no longer prevented HFD-induced microbiota encroachment ( Figure 7 C). Rather, inulin-treated IL-22KO mice exhibited markedly enhanced microbial staining in the mucus layer, which appeared to have an increased tendency to breach intestinal defenses ( Figures 7 C and 7D). Specifically, supplementation of HFD with inulin resulted in a marked increase in levels of bacterial 16S RNA in the liver of IL-22KO, but not wild-type (WT), mice ( Figure 7 B), suggesting increased bacterial translocation. Furthermore, inulin reversed some indices of HFD-induced LGI, specifically, adipose tissue expression of CXCL1, tumor necrosis factor alpha, and IL-6, in WT, but not IL-22KO, mice ( Figures 7 E and 7F). Together, these results indicate that inulin impacts microbiota to induce expression of IL-22, which fortifies the intestine, resulting in reduced microbiota encroachment and pro-inflammatory gene expression, which, together, may underlie inulin’s protection against HFD-induced metabolic syndrome.

We next began to consider other potential mechanisms whereby inulin might impact the microbiota to promote epithelial cell proliferation and production of antimicrobial gene expression that, together, might reduce HFD-induced microbiota encroachment and associated parameters of metabolic syndrome. IL-22, induced by gut bacteria, is known to both promote epithelial cell proliferation and induce antimicrobials such as Reg3γ, and thereby promote intestinal restitution following acute inflammation (). We view HFD-induced metabolic syndrome as a state of LGI and thus envisaged that inulin’s impact on the microbiota might result in IL-22 production that could prevent or resolve such LGI. In accord with this hypothesis, genetic ablation of IL-22 receptor potentiates HFD-induced metabolic syndrome, while administration of recombinant IL-22 ameliorates it (). Hence, we next considered the possibility that inulin’s protective effect against HFD might be mediated by IL-22. In accord with this notion, HFD supplementation with inulin increased IL-22 production ( Figure 6 A). Such inulin-induced IL-22 production, which was detectable in the colon, but not serum, was dependent upon the presence of a microbiota that was absent in GF and antibiotic-treated mice ( Figures 6 A and S6 A), wherein it correlated with lack of induction of Reg3γ expression in GF mice ( Figure S6 B). IL-22 has been reported to be produced by neutrophils (), a subset of T helper lymphocytes (Th22 cells) (), and type 3 innate lymphoid cells (ILC3) (). We observed that inulin-induced IL-22 was not impacted by antibody-mediated neutrophil depletion (data not shown); was maintained in Rag1KO mice, which lack most populations of T and B lymphocytes; but was absent in Rag/IL2Rγ-DKO mice, which lack ILC3, thus indicating a key role for ILC3 in this response ( Figure S7 A). Next, we examined whether deficiency of IL-22 impacted inulin’s ability to restore gut mass and protect against HFD-induced metabolic syndrome. We observed that inability to produce IL-22 in response to consumption of an inulin-enriched diet, due to loss of the IL-22 gene or ILC3, markedly impaired inulin’s ability to restore colon health ( Figures 6 B, 6C, and S7 B–S7G) and to suppress indices of metabolic syndrome ( Figures 6 D–6I and S6 C).

(H and I) Mice (n = 5) were fasted 5 hr, the glucose was measured at 0 (H), 30, 60, and 90 min after being intraperitoneally injected with insulin (I). Data are expressed as mean ± SEM. Statistical significance was assessed by unpaired Student’s t test.p < 0.01; n.s., not significant. See also Figures S6 and S7

Next, we examined the extent to which direct administration of SCFAs might impact the intestinal atrophy and metabolic syndrome induced by HFD. A mixture of SCFAs was provided in drinking water to HFD-fed mice using two different doses that were shown to be anti-inflammatory or health-promoting in other studies (). In contrast to inulin enrichment in diet, direct administration of SCFAs did not significantly restore colonic mass or ameliorate metabolic syndrome induced by HFD ( Figures 5 G–5N). Finally, we sought a means to impede the actions of SCFAs, specifically by use of mice deficient in the free fatty acid receptor GPR43 (also referred to FFar2), which is reported to mediate many of the beneficial effects of SCFAs (). Our experimental design was not optimized to discern the role of GPR43 in mediating the response to HFD per se, on which there is conflicting literature (), but rather to define if this receptor was required for inulin’s promotion of colon mass and/or its ability to improve indices of metabolic syndrome. We observed that, irrespective of whether mice expressed GPR43 or not, inulin promoted intestinal mass and suppressed HFD-induced adiposity and dysglycemia ( Figures S5 G–S5K). SCFAs are reported to promote expansion of regulatory T cells, which dampen chronic inflammatory diseases (). Hence, in light of the notion that HFD-induced metabolic syndrome can be viewed as chronic inflammatory disease, we considered role for T cells in mediating inulin’s protective effect in metabolic syndrome. Inulin promoted colonic mass, suppressed adiposity, and improved glycemic control in HFD-fed Rag1 knockout (Rag1KO) mice, which lack T and B lymphocytes, arguing against this possibility ( Figures S5 L–S5P). Together, these results indicate that inulin’s promotion of colon mass and improvement of metabolic syndrome are not largely mediated by SCFA production.

Next, we examined the mechanism by which the microbiota mediate inulin’s restoration of colon mass and amelioration of metabolic syndrome. First, we considered the role of SCFAs, which have been reported to mediate many of the beneficial effects of fermentable fiber in general and inulin in particular (). In accord with this notion, the consumption of the HFD resulted in a marked loss of feces levels of acetate, propionate, and butyrate, which was fully restored by addition of inulin ( Figures S5 A–S5C). To investigate the role of inulin-induced SCFAs in promotion of colon mass and amelioration of metabolic syndrome, we sought a means to inhibit SCFAs without significantly altering bacterial loads per se. Hence, we utilized β acids, an ingredient derived from hops that have been shown to decrease bacterial-mediated SCFA production by gastrointestinal or fecal bacteria ex vivo (). We observed that administration of β acids to mice via drinking water potently blocked the inulin-induced increase in SCFAs in vivo ( Figures S5 D–S5F) but, importantly, did not significantly reduce inulin’s ability to restore microbiota growth (data not shown). Such β acid-mediated inhibition of SCFA production did not reduce inulin’s ability to promote intestinal mass or suppress adiposity and only mildly impaired inulin’s ability to improve glycemic control ( Figures 5 A–5F ), arguing against the notion that SCFA production is a limiting factor in inulin’s protective effects in this model.

HFD-induced metabolic syndrome is, itself, known to be largely dependent upon the presence of a microbiota (), particularly in GF mice, thus making it difficult to discern the extent to which amelioration of such a phenotype by a particular treatment is also dependent upon the microbiota. Such caveat notwithstanding, we sought to determine if, under conditions of microbiota ablation via antibiotics, inulin might retain any ability to improve parameters of metabolic syndrome in HFD-treated mice. We observed that, upon antibiotic cocktail-mediated suppression of the microbiota, inulin’s ability to reduce weight gain, adiposity, and improve glycemic control was not merely eliminated but, rather, inulin promoted adiposity and dysglycemia ( Figures 4 A, 4D–4I, and S4 H), perhaps reflecting that mice with minimal microbiota more readily digest inulin-enriched diets and harvest energy therefrom (). In any case, these results indicate that microbiota are necessary for both inulin’s promotion of colon mass and beneficial effects on parameters of metabolic syndrome. Lastly, in accord with the general notion that increased colonic mass is generally protective against metabolic syndrome, we note that, over a range of experimental conditions, colon mass negatively correlates with fasting blood glucose concentration ( Figure 4 J). To further investigate the role of inulin-induced changes in microbiota in impacting the gut and influencing metabolic parameters, fecal microbiota transplants were performed. Specifically, feces from mice fed chow or HFD supplemented with inulin or cellulose, were administered to 4-week-old GF mice, which were then maintained on autoclaved chow for an additional 4 weeks. We observed that mice transplanted with microbiota from inulin-fed mice exhibited a trend toward improved glycemic control, reduced adiposity, and increased colon mass ( Figures S4 I–S4O). That these effects were of modest magnitude and significance might reflect that the inulin-induced alterations in microbiota are not fully maintained when mice are fed a chow diet, but are nonetheless in accord with the notion that such alterations play a role in mediating inulin’s impact on the gut and metabolic phenotype.

We next sought to examine the extent to which inulin’s ability to restore HFD-induced loss of colon mass required the presence of a microbiota. First, we eliminated the contribution of the microbiota by using germ-free (GF) mice. These experiments utilized Swiss-Webster mice, which, unlike C57BL/6 mice, are readily maintained in a seemingly healthy state in GF conditions. While HFD is not autoclavable, irrespective of fiber content, we found that subjecting such diets to two rounds of γ-irradiation did not compromise the GF status of mice consuming these diets, as demonstrated by similar levels of fecal bacterial DNA before and after cessation of HFD ( Figure S4 A). The increase in levels of fecal bacterial DNA while consuming HFD reflects levels of bacterial products in these diets, which is not removed by irradiation, with the observation of bacterial DNA mainly being from Lactococcus ( Figure S4 A). The source in the purified HFDs was likely the casein, which is precipitated from skim milk by lactic cultures. Comparison of chow-fed conventional and GF Swiss-Webster mice revealed that the absence of a microbiota by itself on a chow diet resulted in colon atrophy, specifically reduced mass and crypt length, reminiscent of that exhibited by conventional mice fed a compositionally defined diet with cellulose as a sole source of fiber ( Figures S4 B–S4D). Such gut atrophy that resulted from the GF status was not further enhanced by administration of HFD. Moreover, in GF conditions, inulin supplementation did not rescue gut atrophy and enterocyte proliferation as assayed by any of these measurements ( Figures S4 B–S4F). These results suggest that HFD-induced colon atrophy reflects a reduction in gut bacterial loads, and/or key species, and that inulin’s restoration of the former reflects its restoration of the latter. Because GF mice are known to have a number of developmental abnormalities, particularly in regard to the gut-associated immune system (), which is thought to influence development of metabolic syndrome (), we also sought to ablate the microbiota by use of antibiotics. Specifically, we examined the extent to which supplementation of HFD with inulin versus cellulose would promote intestinal mass in mice maintained on a cocktail of oral antibiotics that reduced fecal bacterial loads by about 3 logs ( Figure S4 G). We observed that, analogous to our results with GF mice, antibiotic treatment greatly reduced the ability of inulin to promote microbiota growth and was paralleled by markedly reducing its promotion of colon mass ( Figures 4 B and 4C ).

While mechanisms by which alterations in microbiota alter metabolic phenotype of the host are not well understood, we have shown that both mice and humans with dysglycemia exhibit microbiota that infiltrate the mucus layer to attain a close proximity to host cells (). Herein, we observed that such microbiota encroachment, which was previously observed in mice fed emulsifiers and mice with a genetic deficiency in TLR5, was also observed in response to HFD ( Figures 3 F and 3G). Moreover, such an encroachment was largely reversed by adding 4-fold more fiber as inulin, but not cellulose ( Figures 3 F and 3G). Such a reduction of microbiota encroachment by inulin was associated with restoration of the expression of antimicrobial peptide Reg3γ in whole-colon tissue ( Figure 3 H) or in colon epithelial cells ( Figure S3 G). Altogether, these results are consistent with the notion that inulin’s protection against HFD is mediated by promoting epithelial proliferation and antimicrobial gene expression, which impacts host-microbiota interactions in a manner that may reduce microbiota encroachment and thus LGI.

Obesity and metabolic syndrome are associated with alterations in intestinal microbiota composition in both mice and humans, including an increase in the ratio of Firmicutes to Bacteroidetes and increases in relative abundance of Proteobacteria (). Conversely, inulin is a well-recognized prebiotic known to promote growth of select beneficial bacteria, such as the Bifidobacteria species (). Hence, we next examined the extent to which inulin’s protection against HFD-induced metabolic disease would correlate with microbiota alterations. Relative to chow-fed mice, feeding of the standard HFD (i.e., HFD-50 Cell) resulted in about 10-fold reduction in total fecal bacterial loads as assessed by qPCR, suggesting that, relative to chow, this compositionally defined diet does not support a normal level of bacterial growth ( Figure 3 A). Importantly, such reduction in fecal bacterial load was fully restored by addition of inulin but not cellulose. Moreover, analysis of microbiota composition by 16S sequencing indicated that replacing cellulose with inulin also corrected some of the HFD-induced changes in microbiota that were observed at the phylum level. Specifically, supplementation of HFD with inulin, but not cellulose, ameliorated HFD-induced increases in the Firmicutes/Bacteroidetes ratio and lowered levels of Proteobacteria ( Figures 3 B–3D). Such sequence-based analysis also showed an inulin-induced increase in Bifidobacteria and Akkermansia ( Figurse S3 A and S3B) in accord with previous reports (), although the latter was also observed upon supplementation of HFD with cellulose. Using 16S data to look broadly at microbiota composition by an unbiased method, namely principal component analysis of the UniFrac distance, revealed that addition of inulin, but not cellulose, to the HFD dramatically altered the microbiota composition ( Figures 3 E and S3 C). Such a change in microbiota composition that associated with inulin feeding was driven by enrichment in Bifidobacteriaceae and depletion of Streptococcus, Clostridium, and Enterococcaceae ( Figure S3 D). However, it should be noted that inulin did not restore the microbiota composition of HFD-fed mice toward that of chow-fed mice. Rather, there remained a marked difference in microbiota composition between chow-fed and inulin-HFD-fed mice that was driven by enrichment and depletion of a wide variety of species ( Figure S3 E). Such inulin-induced changes in the microbiota resulted in reduced levels of alpha diversity ( Figure S3 F), which we and others have generally considered a feature of dysbiosis. In contrast, enrichment of HFD with cellulose has a relatively mild impact on microbiota composition.

The promotion of adiposity by diets lacking fermentable fiber, irrespective of fat content, correlates with a marked loss of colon mass, quantifiable by weighing the organ, that occurs within a few days of the change in diet (). Such loss of colon length and mass ( Figures 2 A and 2B ) largely reflects a reduction in colon cross-sectional area, which is proportional to crypt length ( Figures 2 D and 2E). We hypothesize that such HFD-induced intestinal atrophy contributes to the low-grade inflammation (LGI) and metabolic syndrome-like phenotype that eventuates from HFD consumption. In accord with this notion, the reductions in colon mass and crypt length that resulted from switching from chow to HFD were fully restored by enrichment of the diet with inulin, but not cellulose, thus correlating with inulin’s protection against HFD-induced metabolic syndrome. Such promotion of colon mass by inulin enrichment correlated with increased enterocyte proliferation, as measured by the number of BrdU-positive cells per crypt in the proximal colon section ( Figures 2 F and 2G). A similar pattern of results was yielded from immunostaining for PCNA ( Figure S2 A). We hypothesize that inulin’s ability to promote enterocyte growth may have broad impacts upon a range of cell types. Accordingly, we observed that inulin, but not cellulose, increased the level of Paneth cells in the ileum, as assessed by quantitating the number of lysozyme-expressing cells per crypt by immunostaining ( Figures 2 H and 2I). Inulin, but not cellulose, also increased colon expression of tight junction proteins Occludin and Claudin-2 ( Figure 2 C), although such changes did not correlate with a significant change in overall gut permeability to fluorescein isothiocyanate-dextran (data not shown). In contrast to the colon, neither a generalized change in tissue growth ( Figures S2 B and S2C) nor changes in tight junction proteins ( Figure S2 D) were observed in the small intestine in response to HFD or inulin. However, enrichment of HFD with inulin, but not cellulose, did result in a significant increase in GLP1-expressing L cells ( Figures S2 E and S2F), which can be envisaged to contribute to inulin’s impact upon glucose metabolism. Thus, inulin, but not cellulose, promotes enterocyte proliferation, which correlates with protection against HFD-induced metabolic syndrome.

(H and I) The Paneth cells in ileum (n = 5) were stained for lysozyme (H), and the number of Paneth cells per crypt was counted (I). Scale bars, 50 μm. Data are expressed as mean ± SEM. Statistical significance was assessed by unpaired Student’s t test.p < 0.05;p < 0.01. See also Figure S2

A widely used mouse model of DIO compares mice fed a grain-based, poorly defined rodent chow with mice fed a compositionally defined obesogenic purified diet, commonly referred to as a “high-fat diet” (HFD), which is typically 35% fat by weight (60% by calories) and contains 5% cellulose as a source of fiber. This is in contrast to chows that typically contain 5% fat by weight (10%–15% by calories) and 15%–25% fiber, coming from diverse sources. Relative to chow-fed mice, this obesogenic diet results in a marked increase in adiposity associated with numerous features of metabolic syndrome. Moreover, in addition to its high-fat content, its relatively low fiber content, particularly its lack of fermentable fiber, drives the metabolic syndrome phenotype induced by this diet (). Accordingly, manipulating the fiber content of this diet, particularly adding fermentable fiber, ameliorates the severity of metabolic syndrome, although the extent of such amelioration is highly variable across different studies (). Hence, we first sought to establish a well-controlled experimental platform whereby supplementation of an obesogenic diet with fiber would result in a strong degree of protection against indices of metabolic syndrome that would be amenable to mechanistic study. We found that, relative to the “standard” HFD, a diet comprised of 20% inulin (w/w) reduced weight gain and markedly attenuated HFD-induced adiposity, as assessed by amount of epididymal, mesenteric, and subcutaneous fat ( Figures 1 A and 1B ). This reduction in adiposity was accompanied by a reduction in adipocyte size ( Figures 1 C and S1 A). In contrast, no changes were found in brown adipose tissue adipocyte size ( Figure S1 B), and expression of mRNA of UCP1 (data not shown) was similar between mice fed inulin or cellulose-enriched diets, suggesting that fiber content did not affect thermogenesis of brown adipose tissue. Dietary enrichment with inulin did not significantly impact serum levels of free fatty acids or triglycerides ( Figures S1 D and S1E), but markedly lowered levels of cholesterol, which were increased by HFD relative to chow ( Figure S1 F). Supplementation of the HFD with inulin also largely prevented dysglycemia, as assessed by measuring blood glucose after administration of glucose ( Figures 1 D and 1E), or in a fasted state and following injection of insulin ( Figures 1 F and 1G), although the statistical significance of differences following insulin administration varied depending upon metric used for the analysis ( Figures 1 H and S1 G). Inulin also reduced hepatosteatosis, as assessed by staining liver sections with hematoxylin and eosin (H&E) ( Figure S1 C) or oil red O ( Figures 1 I and 1J), which otherwise resulted from exposure to the standard HFD (HFD-50 Cell). Inulin’s ability to reduce indices of metabolic syndrome was associated with a modest but significant reduction in food consumption, based on both food weight and calorie content ( Figures S1 H and S1I). Such reduction in food consumption was not likely a simple “bulking effect,” as food consumption was not significantly impacted by cellulose. Moreover, enriching HFD with cellulose (HFD-200 Cell), which is not easily fermentable by mice, only very modestly reduced adiposity and dysglycemia ( Figures 1 and S1 ). Together, these results suggest that comparing phenotypes of mice being fed HFDs comprised of about 20% cellulose or inulin provides a tractable means to study how fermentable fiber is impacting DIO.

Discussion

The central goal of this study was to elucidate the mechanism whereby enrichment of an obesogenic HFD with inulin suppresses adiposity and its associated parameters of metabolic syndrome. We hypothesized that bacterial metabolism of inulin, specifically generation of SCFAs, would promote enterocyte proliferation and thus broadly fortify innate mucosal defense, leading to reduced bacterial encroachment. Subsequently, this would reduce LGI, which promotes numerous events that promote and define the metabolic syndrome. Our results supported some aspects of this hypothesis, particularly the notion that inulin restores the HFD-induced loss of enterocyte proliferation, reduces microbiota encroachment, and protects against metabolic syndrome in a microbiota-dependent manner. However, our data did not support a major role for SCFAs in inulin’s restoration of colonic health or amelioration of metabolic syndrome. Rather, our results indicate that inulin restores gut health and protects against metabolic syndrome in a manner that correlates with, and is dependent upon, microbiota-dependent induction of IL-22 expression. We now hypothesize that such inulin-induced IL-22 expression promotes colon health in a manner that reduces microbiota encroachment by fortifying the epithelium via promoting crypt regeneration and increasing expression of antibacterial proteins. These results add to understanding of pathophysiologic mechanisms that underlie DIO and provide a previously unappreciated means by which fermentable fibers might promote health.

Chassaing et al., 2015b Chassaing B.

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Gewirtz A.T. Colonic microbiota encroachment correlates with dysglycemia in humans. Our previous study revealed that HFD resulted in a loss of gut mass, and that such gut atrophy was not caused by dietary fat content per se but, rather, by its lack of fermentable fiber (). Herein, we observed that gut atrophy correlated with reduced enterocyte proliferation and, moreover, microbiota encroachment, all of which were reversed by enrichment of HFD with inulin, but not with additional insoluble fiber, cellulose. Interestingly, such microbiota encroachment was inversely proportional to total bacterial loads in the gut. Specifically, relative to chow-fed mice, HFD administration resulted in bacterial infiltration into the mucus layer despite an approximate 10-fold reduction in fecal bacteria. Inulin supplementation of HFD restored bacterial loads and resulted in some changes in gut microbiota composition that have been associated with health. Specifically, inulin enrichment of HFD restored the Firmicutes/Bacteroidetes ratio, which is associated with, and is thought to contribute to, leanness; reduced levels of Proteobacteria, which have been proposed to promote an array of chronic inflammatory diseases; and increased levels of Bifidobacteria, which are depleted in inflammatory diseases and have been used as probiotics to ameliorate such diseases. While there are a myriad of potential means by which these and other inulin-induced changes in microbiota might ameliorate metabolic disease, we hypothesize that reducing microbiota encroachment plays a central role in this process. We envision that such reduced microbiota encroachment should reduce inflammatory signaling, which is thought to impair a range of metabolic signaling pathways including those involved in glycemic control and satiety signaling, such as insulin and leptin, respectively. Thus, we propose that inulin-induced fortification of the mucosa enhances both insulin and satiety signaling in an intertwined manner to ameliorate metabolic syndrome. In accord with our hypothesis, we recently reported that microbiota encroachment is a feature of metabolic syndrome in humans (), wherein it may play a role in driving this low-grade chronic inflammatory disorder. While reduced microbiota encroachment can be imagined to result in reduced expression of IL-22 and Reg3γ, we propose that, in fact, inulin-induced microbiotas resulted in a relatively stable host-microbiota equilibrium state. We speculate that inulin-induced IL-22-dependent fortification of the mucosa likely involves increased barrier function but further studies are needed to determine the extent to which this involves exclusion of bacteria themselves and/or their products.

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Klaus S. Effects of dietary inulin on bacterial growth, short-chain fatty acid production and hepatic lipid metabolism in gnotobiotic mice. While our observation that soluble fiber improves parameters of metabolic syndrome via a microbiota-dependent process is generally in accord with most published work in this area, we note thatrecently reported that such fibers improved glycemic control, even in GF mice. While this apparent discrepancy between their and our results could reflect differences in specific fibers, source, and/or mouse strains utilized, we speculate that the absolute absence of bacteria in GF mice makes it very difficult for them to digest these fibers, resulting in reduced appetite and, consequently, alterations in glycemic control by mechanisms that would not be relevant in mice with a microbiota. Such apparent inability to digest inulin-enriched diets was not seen in antibiotic-treated mice, perhaps reflecting that a minimal microbiota is sufficient to at least partially digest this fiber () and, in any case, arguing that the inability of inulin to suppress adiposity or improve glycemic control in antibiotic-treated mice reflects an essential role for the microbiota in mediating inulin’s amelioration of HFD-induced metabolic syndrome.

Brooks et al. (2017) Brooks L.

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et al. Fermentable carbohydrate stimulates FFAR2-dependent colonic PYY cell expansion to increase satiety. That inulin’s restoration of gut bacterial loads correlated with a microbiota-dependent increase in fecal SCFA levels supported our presumption that SCFAs would be pivotal in mediating inulin’s beneficial effects. Yet suppressing SCFA production to levels approaching or below baseline levels in the cecum, which is the most sensitive locale to measure changes in SCFAs, resulted in only a modest loss of glucose tolerance and did not ameliorate inulin’s ability to suppress adiposity and fasting glucose levels. Nor did loss of the free fatty acid receptor GPR43 impact upon inulin’s ability to ameliorate HFD-induced dysglycemia or adiposity. This latter result is an apparent contradiction with a recent publication from, which reported that inulin suppressed HFD-induced weight gain and adiposity in WT, but not GPR43KO, mice. The reason for this discrepancy is unclear but given that our study used a higher level of inulin, which appears to have a stronger suppression of adiposity, we speculate that some portion of inulin’s beneficial metabolic effects might be mediated by SCFAs acting via GPR43, but that SCFA-independent effects may mask the need for this receptor. We also note that our approaches to manipulate SCFA levels have significant limitations. Indeed, the specificity of β acids is not well defined, and administration of SCFAs in drinking water may not reach the colon in adequate levels. Thus, further work is needed in this area to better define the role of SCFAs in protecting against metabolic syndrome. Specific needs include determining what metabolites are produced when SCFA production is blocked and discern how they impact the host. The need for such studies notwithstanding, our results indicate that SCFAs may not be a limiting factor in the microbiota-dependent ability of inulin to protect against HFD-induced metabolic syndrome.

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et al. Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes. It should be noted that, although inulin restored total gut bacterial loads, it did not restore microbiota composition toward that of chow-fed mice but, rather, resulted in a microbiota composition seemingly equally distinct from chow- and HFD-fed mice, at least as assessed by unbiased metrics such as UniFrac analysis. While such altered microbiota composition did not result in an obvious phenotypic difference relative to chow-fed mice, we recently reported that exposure of mice fed inulin-enriched diets, irrespective of fat content, exhibited very severe colitis upon challenge with dextran sodium sulfate (DSS), a chemical colitogen (). This observation highlights the potential risk in promoting bacterial growth in the gut, particularly in inducing a microbiota composition whose functional attributes are not well defined. Moreover, it underscores the need to define specific mechanisms whereby inulin impacts microbiota to protect against metabolic disease. That inulin’s protection against HFD-induced metabolic syndrome correlated with IL-22 induction and is IL-22 dependent, combined with the observation that recombinant IL-22 ameliorates HFD-induced obesity, indicates that IL-22 is one such potential mechanism (). This notion is in accord with recent work that direct IL-22 administration protects against HFD-induced metabolic syndrome and the observation that IL-22 receptor KO mice potentiated disease in this model. Yet interestingly, IL-22KO mice were not found to share this latter phenotype (). Our results were in accord with this observation in that we did not observe a difference in the extent of standard HFD-induced metabolic disease between IL-22 and WT mice, but it should be noted that, although maintained in the same facility in similar conditions, KO mice had not been bred with the WT control mice so we do not view our comparison to be a rigorous confirmation of this seemingly enigmatic result.