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theses

Plant-based diets are correlated with a decreased risk of developing metabolic syndrome (MetS) and type 2 diabetes (T2D), diseases characterized by which chronic low-grade inflammation. Epidemiological, pre-clinical, and clinical reports indicate that dietary polyphenols, antioxidant plant defense compounds, are protective against chronic metabolic diseases. Despite poor intestinal absorption of parent polyphenols, they are biotransformed by the gut microbiota into more bioavailable metabolites that may benefit host metabolic health. Emerging evidence further demonstrates that metabolic health may benefit from polyphenol-induced changes to the gut bacterial community. The studies described herein investigated mechanisms by which procyanidins (PACs), a class of polyphenols abundant in the skins and seeds of grape berries, may improve the symptoms of MetS and T2D. PAC-rich grape polyphenols (GPs) were extracted from Concord grape and delivered in a complex with soy protein isolate (SPI).The experiment described in Chapter 2 provides evidence that by altering the gut microbial community, GPs alter a BA-intestinal FXR signaling axis to improve glucose metabolism. Briefly, db/db mice, which serve as a genetic model of T2D, were fed low-fat diet (LFD) or LFD supplemented with 1% GPs (LFD-GP) for 4 weeks. Compared to LFD-fed mice, db/db mice fed LFD-GP showed improved glucose tolerance independent of changes in circulating proinflammatory cytokines, anthropometric phenotypes, or genetic markers related to intestinal inflammation and permeability. Rather, GP-supplemented db/db mice had a reduced relative abundance of fecal and cecal bacteria associated with the production of secondary BAs (SBAs), diminished serum levels of SBAs, and increased concentration of the primary BA (PBA) pool. BAs signal to the nuclear receptor farnesoid X receptor (FXR) regulating glucose metabolism, therefore markers of BA-FXR signaling were investigated as a potential mechanism linking polyphenol-induced gut bacterial changes and improved glucose metabolism. While FXR gene expression was unaltered, FXR targets including ceramide biosynthetic enzymes were lower in GP-fed db/db mice, suggesting lower ceramide levels in tissues could contribute to the observed improvement in glucose tolerance. SBAs that were reduced in GP-supplemented db/db mice were shown to act as FXR agonists (increasing FXR-transcribed genes) in ileal organoids derived from wild-type (WT) C57BL6/J male mice. These data suggested that GPs suppressed intestinal FXR activity by reducing gut bacteria responsible for the production of SBAs including taurohyodeoxycholic acid (THDCA), tauro-omega muricholic acid (TωMCA), and omega-muricholic acid (ωMCA), which exhibited FXR-activating capabilities.
In Chapter 3, experiments were conducted to test whether oral gavage of THDCA, could impair glucose metabolism without or with depletion of the gut microbiota. Daily oral gavage of C57BL6/J WT male mice with an antibiotic cocktail or vehicle control in the morning, and THDCA or vehicle control in the evening showed that THDCA did not impair glucose metabolism or promote hepatic gluconeogenesis. Collectively, these in vivo and in vitro data from Chapter 3, suggested that GP-induced suppression of intestinal FXR activity was unrelated to THDCA.
The experiments described in Chapter 4 investigate the tissue-specific requirement of intestinal vs. hepatic FXR for GP-induced improvements in glucose clearance. Male mice (C57BL6 genetic background) with intestine-specific (VKO) or liver-specific (AKO) deletion of FXR, and WT littermate controls were fed HFD or HFD supplemented with 0.5% GPs (HFD-GP) for 12 weeks. HFD-induced hyperglycemia and insulinemia, body weight gain, fat accumulation, and hepatic steatosis were ameliorated in GP-supplemented WT and AKO mice when compared to their HFD-fed controls; however, GP supplementation did not alter the resilience of VKO mice to HFD-induced body weight gain, hepatic steatosis, hyperglycemia, and hyperinsulinemia. GP supplementation differentially altered hepatic BA profiles and signaling in WT, AKO, and VKO strains; however, hepatic expression of inflammatory markers was reduced in all GP-supplemented groups. Analysis of ileal content BAs showed GPs reduced ileal SBA levels in all strains. Gene expression analysis suggested that ileal markers of host-microbe interactions, including toll-like receptors (TLRs) and nod-like receptor pyrin-domain containing (NLRPs) were decreased in GP-supplemented WT and AKO mice while intestinal inflammation was decreased by GPs in all strains. The cecal microbial communities were differentially altered by FXR deletion and by GPs; GPs reduced the abundance of cecal genera associated with LPS-induced inflammation and obesity. Collectively, these in vivo data suggested that GP supplementation improved glucose clearance in association with altered gut microbiota, increased intestinal FXR activity, and blunted host-microbe interactions in an intestinal Fxr-dependent manner. Moreover, GPs may have protected against HFD-induced intestinal and hepatic inflammation in all groups via modulation of the gut microbial communities to reduce pro-inflammatory lipopolysaccharide (LPS)-producing gut bacteria.
To determine whether the GP-induced changes in ileal BAs levels would protect against intestinal inflammation in an intestinal Fxr-dependent manner, ileal gut organoids derived from WT floxed littermates and VKO mice and treated with BAs at concentrations quantified in ileal content of mice. Ileal organoids derived from WT mice were treated with individual BAs and a mixture of the individual BAs at concentrations quantified in ileal content of HFD-fed and HFD-GP-fed WT and VKO mice. The GP-induced BA mixture suppressed the expression of markers related to inflammation, TLRs, and NLRPs in WT ileal organoids, while the mixture of the individual BAs from ileal content of HFD-fed WT mice increased expression of inflammatory, TLR, and NLRP genes. Ileal organoids derived from VKO mice treated with the GP- and HFD-induced ileal BA profile from VKO mice did not alter the expression of inflammatory, TLR, and NLRP markers, which indicated that loss of intestinal Fxr may have blunted BA-induced intestinal inflammation, TLR, and NLRP signaling. To distinguish whether GPs directly activated or suppressed FXR activity, we treated transfected HEK293 cells with individual polyphenol metabolites with and without the synthetic Fxr agonist, GW4064, and measured activity by FXR luciferase reporter assay.
Based on data described in chapters 2 and 3, we hypothesize that supplementation of HFD with GPs can alleviate HFD-induced hyperglycemia, gut dysbiosis, inflammation, and hepatic steatosis at least partially due to a GP-induced gut microbiome, reduction in serum SBAs, and through modulation of intestinal Fxr signaling mechanisms. Data from chapter 4 suggested that GPs may protect against HFD-induced adiposity, weight gain, inflammation, and gut dysbiosis by reducing ileal SBA levels, SBA, and LPS-producing gut bacteria that promote inflammation and impairing intestinal TLR and NLRP signaling mechanisms in an ileal FXR-dependent manner. GPs may indirectly or directly modulate intestinal Fxr signaling to prevent hyperglycemia and promote changes in hepatic Shp signaling to protect against hepatic steatosis.

http://dissertations.umi.com/gsnb.rutgers:12306

Ph.D.

Includes bibliographical references

doi:10.7282/t3-cf10-z276

The author owns the copyright to this work.