Grape Polyphenols May Prevent High-Fat Diet-Induced Dampening of the Hypothalamic-Pituitary-Adrenal Axis in Male Mice.

Context : Chronic high-fat diet (HFD) consumption causes obesity associated with retention of bile acids (BAs) that suppress important regulatory axes, such as the hypothalamic-pituitary-adrenal axis (HPAA). HFD impairs nutrient sensing and energy balance due to a dampening of the HPAA and reduced production and peripheral metabolism of corticosterone (CORT). Objective: We assessed whether proanthocyanidin-rich grape polyphenol (GP) extract can prevent HFD-induced energy imbalance and HPAA dysregulation. Methods: Male C57BL6/J mice were fed HFD or HFD supplemented with 0.5% w/w GPs (HFD-GP) for 17 weeks. Results: GP supplementation reduced body weight gain and liver fat while increasing circadian rhythms of energy expenditure and HPAA-regulating hormones, CORT, leptin, and PYY. GP-induced improvements were accompanied by reduced mRNA levels of Il6, Il1b, and Tnfa in ileal or hepatic tissues and lower cecal abundance of Firmicutes, including known BA metabolizers. GP-supplemented mice had lower concentrations of circulating BAs, including hydrophobic and HPAA-inhibiting BAs, but higher cecal levels of taurine-conjugated BAs antagonistic to farnesoid X receptor (FXR). Compared with HFD-fed mice, GP-supplemented mice had increased mRNA levels of hepatic Cyp7a1 and Cyp27a1, suggesting reduced FXR activation and more BA synthesis. GP-supplemented mice also had reduced hepatic Abcc3 and ileal Ibabp and Ostß, indicative of less BA transfer into enterocytes and circulation. Relative to HFD-fed mice, CORT and BA metabolizing enzymes (Akr1d1 and Srd5a1) were increased, and Hsd11b1 was decreased in GP supplemented mice. Conclusion: GPs may attenuate HFD-induced weight gain by improving hormonal control of the HPAA and inducing a BA profile with less cytotoxicity and HPAA inhibition, but greater FXR antagonism.

Bile acid metabolism and signaling: Emerging pharmacological targets of dietary polyphenols.

Beyond their role as emulsifiers of lipophilic compounds, bile acids (BAs) are signaling endocrine molecules that show differential affinity and specificity for a variety of canonical and non-canonical BA receptors. Primary BAs (PBAs) are synthesized in the liver while secondary BAs (SBAs) are gut microbial metabolites of PBA species. PBAs and SBAs signal to BA receptors that regulate downstream pathways of inflammation and energy metabolism. Dysregulation of BA metabolism or signaling has emerged as a feature of chronic disease. Dietary polyphenols are non-nutritive plant-derived compounds associated with decreased risk of metabolic syndrome, type-2 diabetes, hepatobiliary and cardiovascular disease. Evidence suggests that the health promoting effects of dietary polyphenols are linked to their ability to alter the gut microbial community, the BA pool, and BA signaling. In this review we provide an overview of BA metabolism and summarize studies that link the cardiometabolic improvements of dietary polyphenols to their modulation of BA metabolism and signaling pathways, and the gut microbiota. Finally, we discuss approaches and challenges in deciphering cause-effect relationships between dietary polyphenols, BAs, and gut microbes.

Proanthocyanidin B2 derived metabolites may be ligands for bile acid receptors S1PR2, PXR and CAR: an in silico approach.

Bile acids (BAs) act as signaling molecules via their interactions with various nuclear (FXR, VDR, PXR and CAR) and G-protein coupled (TGR5, M3R, S1PR2) BA receptors. Stimulation of these BA receptors influences several processes, including inflammatory responses and glucose and xenobiotic metabolism. BA profiles and BA receptor activity are deregulated in cardiometabolic diseases; however, dietary polyphenols were shown to alter BA profile and signaling in association with improved metabolic phenotypes. We previously reported that supplementing mice with a proanthocyanidin (PAC)-rich grape polyphenol (GP) extract attenuated symptoms of glucose intolerance in association with changes to BA profiles, BA receptor gene expression, and/or downstream markers of BA receptor activity. Exact mechanisms by which polyphenols modulate BA signaling are not known, but some hypotheses include modulation of the BA profile via changes to gut bacteria, or alteration of ligand-availability via BA sequestration. Herein, we used an in silico approach to investigate putative binding affinities of proanthocyanidin B2 (PACB2) and PACB2 metabolites to nuclear and G-protein coupled BA receptors. Molecular docking and dynamics simulations revealed that certain PACB2 metabolites had stable binding affinities to S1PR2, PXR and CAR, comparable to that of known natural and synthetic BA ligands. These findings suggest PACB2 metabolites may be novel ligands of S1PR2, CAR, and PXR receptors.Communicated by Ramaswamy H. Sarma.

Saturated Fatty Acids and Omega-3 Polyunsaturated Fatty Acids Improve Metabolic Parameters in Ovariectomized Female Mice.

In menopausal and postmenopausal women, the risk for obesity, cardiovascular disease, osteoporosis, and gut dysbiosis are elevated by the depletion of 17ß-estradiol. A diet that is high in omega-6 polyunsaturated fatty acids (PUFAs), particularly linoleic acid (LA), and low in saturated fatty acids (SFAs) found in coconut oil and omega-3 PUFAs may worsen symptoms of estrogen deficiency. To investigate this hypothesis, ovariectomized C57BL/6J and transgenic fat-1 mice, which lower endogenous omega-6 polyunsaturated fatty acids, were treated with either a vehicle or estradiol benzoate (EB) and fed a high-fat diet with a high or low PUFA:SFA ratio for ~15 weeks. EB treatment reversed obesity, glucose intolerance, and bone loss in ovariectomized mice. fat-1 mice fed a 1% LA diet experienced reduced weight gain and adiposity, while those fed a 22.5% LA diet exhibited increased energy expenditure and activity in EB-treated ovariectomized mice. Coconut oil SFAs and omega-3 FAs helped protect against glucose intolerance without EB treatment. Improved insulin sensitivity was observed in wild-type and fat-1 mice fed 1% LA diet with EB treatment, while fat-1 mice fed 22.5% LA diet was protected against insulin resistance without EB treatment. The production of short-chain fatty acids by gut microbial microbiota was linked to omega-3 FAs production and improved energy homeostasis. These findings suggest that a balanced dietary fatty acid profile containing SFAs and a lower ratio of omega-6:omega-3 FAs is more effective in alleviating metabolic disorders during E2 deficiency.

Coconut Oil Saturated Fatty Acids Improved Energy Homeostasis but not Blood Pressure or Cognition in VCD-Treated Female Mice.

Obesity, cardiometabolic disease, cognitive decline, and osteoporosis are symptoms of postmenopause, which can be modeled using 4-vinylcyclohexene diepoxide (VCD)-treated mice to induce ovarian failure and estrogen deficiency combined with high-fat diet (HFD) feeding. The trend of replacing saturated fatty acids (SFAs), for example coconut oil, with seed oils that are high in polyunsaturated fatty acids, specifically linoleic acid (LA), may induce inflammation and gut dysbiosis, and worsen symptoms of estrogen deficiency. To investigate this hypothesis, vehicle (Veh)- or VCD-treated C57BL/6J mice were fed a HFD (45% kcal fat) with a high LA:SFA ratio (22.5%: 8%), referred to as the 22.5% LA diet, or a HFD with a low LA:SFA ratio (1%: 31%), referred to as 1% LA diet, for a period of 23 to 25 weeks. Compared with VCD-treated mice fed the 22.5% LA diet, VCD-treated mice fed the 1% LA diet showed lower weight gain and improved glucose tolerance. However, VCD-treated mice fed the 1% LA diet had higher blood pressure and showed evidence of spatial cognitive impairment. Mice fed the 1% LA or 22.5% LA diets showed gut microbial taxa changes that have been associated with a mix of both beneficial and unfavorable cognitive and metabolic phenotypes. Overall, these data suggest that consuming different types of dietary fat from a variety of sources, without overemphasis on any particular type, is the optimal approach for promoting metabolic health regardless of estrogen status.

Corrigendum: Cannabidiol-treated ovariectomized mice show improved glucose, energy, and bone metabolism with a bloom in Lactobacillus.

[This corrects the article DOI: 10.3389/fphar.2022.900667.].

Cannabidiol-Treated Ovariectomized Mice Show Improved Glucose, Energy, and Bone Metabolism With a Bloom in Lactobacillus.

Loss of ovarian 17ß-estradiol (E2) in postmenopause is associated with gut dysbiosis, inflammation, and increased risk of cardiometabolic disease and osteoporosis. The risk-benefit profile of hormone replacement therapy is not favorable in postmenopausal women therefore better treatment options are needed. Cannabidiol (CBD), a non-psychotropic phytocannabinoid extracted from hemp, has shown pharmacological activities suggesting it has therapeutic value for postmenopause, which can be modeled in ovariectomized (OVX) mice. We evaluated the efficacy of cannabidiol (25 mg/kg) administered perorally to OVX and sham surgery mice for 18 weeks. Compared to VEH-treated OVX mice, CBD-treated OVX mice had improved oral glucose tolerance, increased energy expenditure, improved whole body areal bone mineral density (aBMD) and bone mineral content as well as increased femoral bone volume fraction, trabecular thickness, and volumetric bone mineral density. Compared to VEH-treated OVX mice, CBD-treated OVX mice had increased relative abundance of fecal Lactobacillus species and several gene expression changes in the intestine and femur consistent with reduced inflammation and less bone resorption. These data provide preclinical evidence supporting further investigation of CBD as a therapeutic for postmenopause-related disorders.

Impact of grape polyphenols on Akkermansia muciniphila and the gut barrier.

A healthy gastrointestinal tract functions as a highly selective barrier, allowing the absorption of nutrients and metabolites while preventing gut bacteria and other xenobiotic compounds from entering host circulation and tissues. The intestinal epithelium and intestinal mucus provide a physical first line of defense against resident microbes, pathogens and xenotoxic compounds. Prior studies have indicated that the gut microbe Akkermansia muciniphila, a mucin-metabolizer, can stimulate intestinal mucin thickness to improve gut barrier integrity. Grape polyphenol (GP) extracts rich in B-type proanthocyanidin (PAC) compounds have been found to increase the relative abundance of A. muciniphila, suggesting that PACs alter the gut microbiota to support a healthy gut barrier. To further investigate the effect of GPs on the gut barrier and A. muciniphila, male C57BL/6 mice were fed a high-fat diet (HFD) or low-fat diet (LFD) with or without 1% GPs (HFD-GP, LFD-GP) for 12 weeks. Compared to the mice fed unsupplemented diets, GP-supplemented mice showed increased relative abundance of fecal and cecal A. muciniphila, a reduction in total bacteria, a diminished colon mucus layer and increased fecal mucus content. GP supplementation also reduced the presence of goblet cells regardless of dietary fat. Compared to the HFD group, ileal gene expression of lipopolysaccharide (LPS)-binding protein (Lbp), an acute-phase protein that promotes pro-inflammatory cytokine expression, was reduced in the HFD-GP group, suggesting reduced LPS in circulation. Despite depletion of the colonic mucus layer, markers of inflammation (Ifng, Il1b, Tnfa, and Nos2) were similar among the four groups, with the exception that ileal Il6 mRNA levels were lower in the LFD-GP group compared to the LFD group. Our findings suggest that the GP-induced increase in A. muciniphila promotes redistribution of the intestinal mucus layer to the intestinal lumen, and that the GP-induced decrease in total bacteria results in a less inflammatory intestinal milieu.

Cannabidiol Decreases Intestinal Inflammation in the Ovariectomized Murine Model of Postmenopause.

Cannabidiol (CBD) (25 mg/kg peroral) treatment was shown to improve metabolic outcomes in ovariectomized (OVX) mice deficient in 17ß-estradiol (E2). Herein, CBD effects on intestinal and hepatic bile acids (BAs) and inflammation were investigated. Following RNA sequencing of colon tissues from vehicle (VEH)- or CBD-treated sham surgery (SS) or OVX mice (n = 4 per group), differentially expressed genes (DEGs) were sorted in ShinyGO. Inflammatory response and bile secretion pathways were further analyzed. Colon content and hepatic BAs were quantified by LC-MS (n = 8-10 samples/group). Gut organoids were treated with CBD (100, 250, 500 µM) with or without TNFα and lipopolysaccharide (LPS) followed by mRNA extraction and qPCR to assess CBD-induced changes to inflammatory markers. The expression of 78 out of 114 inflammatory response pathway genes were reduced in CBD-treated OVX mice relative to vehicle (VEH)-treated OVX mice. In contrast, 63 of 111 inflammatory response pathway genes were increased in CBD-treated sham surgery (SS) mice compared to VEH-treated SS group and 71 of 121 genes were increased due to ovariectomy. CBD did not alter BA profiles in colon content or liver. CBD repressed Tnf and Nos2 expression in intestinal organoids in a dose-dependent manner. In conclusion, CBD suppressed colonic inflammatory gene expression in E2-deficient mice but was pro-inflammatory in E2-sufficient mice suggesting CBD activity in the intestine is E2-dependent.

Grape Polyphenols Attenuate Diet-Induced Obesity and Hepatic Steatosis in Mice in Association With Reduced Butyrate and Increased Markers of Intestinal Carbohydrate Oxidation.

A Western Diet (WD) low in fiber but high in fats and sugars contributes to obesity and non-alcoholic fatty liver disease (NAFLD). Supplementation with grape polyphenols (GPs) rich in B-type proanthocyanidins (PACs) can attenuate symptoms of cardiometabolic disease and alter the gut microbiota and its metabolites. We hypothesized that GP-mediated metabolic improvements would correlate with altered microbial metabolites such as short chain fatty acids (SCFAs). To more closely mimic a WD, C57BL/6J male mice were fed a low-fiber diet high in sucrose and butterfat along with 20% sucrose water to represent sugary beverages. This WD was supplemented with 1% GPs (WD-GP) to investigate the impact of GPs on energy balance, SCFA profile, and intestinal metabolism. Compared to WD-fed mice, the WD-GP group had higher lean mass along with lower fat mass, body weight, and hepatic steatosis despite consuming more calories from sucrose water. Indirect and direct calorimetry revealed that reduced adiposity in GP-supplemented mice was likely due to their greater energy expenditure, which resulted in lower energy efficiency compared to WD-fed mice. GP-supplemented mice had higher abundance of Akkermansia muciniphila, a gut microbe reported to increase energy expenditure. Short chain fatty acid measurements in colon content revealed that GP-supplemented mice had lower concentrations of butyrate, a major energy substrate of the distal intestine, and reduced valerate, a putrefactive SCFA. GP-supplementation also resulted in a lower acetate:propionate ratio suggesting reduced hepatic lipogenesis. Considering the higher sucrose consumption and reduced butyrate levels in GP-supplemented mice, we hypothesized that enterocytes would metabolize glucose and fructose as a replacement energy source. Ileal mRNA levels of glucose transporter-2 (GLUT2, SLC2A2) were increased indicating higher glucose and fructose uptake. Expression of ketohexokinase (KHK) was increased in ileum tissue suggesting increased fructolysis. A GP-induced increase in intestinal carbohydrate oxidation was supported by: (1) increased gene expression of duodenal pyruvate dehydrogenase (PDH), (2) a decreased ratio of lactate dehydrogenase a (LDHa): LDHb in jejunum and colon tissues, and (3) decreased duodenal and colonic lactate concentrations. These data indicate that GPs protect against WD-induced obesity and hepatic steatosis by diminishing portal delivery of lipogenic butyrate and sugars due to their increased intestinal utilization.

Polyphenol-induced improvements in glucose metabolism are associated with bile acid signaling to intestinal farnesoid X receptor.

INTRODUCTION: Bile acid (BA) biotransformation by gut bacteria impacts BA profile and signaling to nuclear receptors, such as the farnesoid X receptor (FXR) regulating glucose metabolism. Altered BA-FXR signaling was therefore investigated as a potential mechanism linking polyphenol-induced gut bacterial changes and improved glucose metabolism. RESEARCH DESIGN AND METHODS: Diabetic db/db were fed low-fat diet (LFD) or LFD supplemented with a proanthocyanidin-rich extract of grape polyphenols (LFD-GP) for 4 weeks. Metabolic phenotypes, serum BAs, gut microbiota composition, and gene expression markers relevant to gut barrier and glucose metabolism were assessed. Gut organoids were used to investigate effects of individual BAs on ileal FXR activity. RESULTS: Compared with LFD-fed controls, GP supplemented db/db mice showed improved glucose metabolism, decreased relative abundance of gut bacteria associated with production of secondary BAs (SBAs), and depleted serum levels of SBAs taurohyodeoxycholic acid (THDCA), ω-muricholic acid (ωMCA), and tauro-ω-muricholic acid (TωMCA). Serum levels of primary BAs (PBAs) increased, consistent with higher gene expression of PBA synthesis enzyme Cyp7a1. GP-induced BA changes associated with FXR inhibition as evidenced by reduced expression of FXR-responsive genes Shp, Fgf15, and Fabp6 in ileum tissue as well as hepatic Shp, which negatively regulates PBA synthesis. GP treatment did not affect expression of hepatic Fxr or expression of Abcb11, Slc51b, and Obp2a genes controlling BA transport. Ceramide biosynthesis genes Smpd3, Sptlc2, and Cers4 were decreased in liver and intestine suggesting lower tissue ceramides levels may contribute to improved glucose metabolism. THDCA, ωMCA, and TωMCA behaved as FXR agonists in ileal organoid experiments; therefore, their depletion in serum of GP-supplemented db/db and wild type (WT) mice was consistent with FXR inhibition. CONCLUSION: These data suggest that by altering the gut microbiota, GPs modify BA-FXR signaling pathways to promote glucoregulation.

Grape proanthocyanidin-induced intestinal bloom of Akkermansia muciniphila is dependent on its baseline abundance and precedes activation of host genes related to metabolic health.

We previously showed that C57BL/6J mice fed high-fat diet (HFD) supplemented with 1% grape polyphenols (GP) for 12 weeks developed a bloom of Akkermansia muciniphila with attenuated metabolic syndrome symptoms. Here we investigated early timing of GP-induced effects and the responsible class of grape polyphenols. Mice were fed HFD, low-fat diet (LFD) or formulations supplemented with GP (HFD-GP, LFD-GP) for 14 days. Mice fed HFD-GP, but not LFD-GP, showed improved oral glucose tolerance compared to controls. A. muciniphila bloom occurred earlier in mice fed LFD-GP than HFD-GP; however, timing was dependent on baseline A. muciniphila levels rather than dietary fat. Mice gavaged for 10 days with GP extract (GPE) or grape proanthocyanidins (PACs), each delivering 360 mg PACs/kg body weight, induced a bloom of fecal and cecal A. muciniphila, the rate of which depended on initial A. muciniphila abundance. Grape PACs were sufficient to induce a bloom of A. muciniphila independent of specific intestinal gene expression changes. Gut microbial community analysis and in vitro inhibition of A. muciniphila by GPE or PACs suggest that the A. muciniphila bloom in vivo occurs via indirect mechanisms.