Impact of Plant-Based Dietary Fibers on Metabolic Homeostasis in High-Fat Diet Mice via Alterations in the Gut Microbiota and Metabolites.

BACKGROUND: The gut microbiota contributes to metabolic disease, and diet shapes the gut microbiota, emphasizing the need to better understand how diet impacts metabolic disease via gut microbiota alterations. Fiber intake is linked with improvements in metabolic homeostasis in rodents and humans, which is associated with changes in the gut microbiota. However, dietary fiber is extremely heterogeneous, and it is imperative to comprehensively analyze the impact of various plant-based fibers on metabolic homeostasis in an identical setting and compare the impact of alterations in the gut microbiota and bacterially derived metabolites from different fiber sources. OBJECTIVES: The objective of this study was to analyze the impact of different plant-based fibers (pectin, ß-glucan, wheat dextrin, resistant starch, and cellulose as a control) on metabolic homeostasis through alterations in the gut microbiota and its metabolites in high-fat diet (HFD)-fed mice. METHODS: HFD-fed mice were supplemented with 5 different fiber types (pectin, ß-glucan, wheat dextrin, resistant starch, or cellulose as a control) at 10% (wt/wt) for 18 wk (n = 12/group), measuring body weight, adiposity, indirect calorimetry, glucose tolerance, and the gut microbiota and metabolites. RESULTS: Only ß-glucan supplementation during HFD-feeding decreased adiposity and body weight gain and improved glucose tolerance compared with HFD-cellulose, whereas all other fibers had no effect. This was associated with increased energy expenditure and locomotor activity in mice compared with HFD-cellulose. All fibers supplemented into an HFD uniquely shifted the intestinal microbiota and cecal short-chain fatty acids; however, only ß-glucan supplementation increased cecal butyrate concentrations. Lastly, all fibers altered the small-intestinal microbiota and portal bile acid composition. CONCLUSIONS: These findings demonstrate that ß-glucan consumption is a promising dietary strategy for metabolic disease, possibly via increased energy expenditure through alterations in the gut microbiota and bacterial metabolites in mice.

Differential effects of plant-based flours on metabolic homeostasis and the gut microbiota in high-fat fed rats.

BACKGROUND: The gut microbiome is a salient contributor to the development of obesity, and diet is the greatest modifier of the gut microbiome, which highlights the need to better understand how specific diets alter the gut microbiota to impact metabolic disease. Increased dietary fiber intake shifts the gut microbiome and improves energy and glucose homeostasis. Dietary fibers are found in various plant-based flours which vary in fiber composition. However, the comparative efficacy of specific plant-based flours to improve energy homeostasis and the mechanism by which this occurs is not well characterized. METHODS: In experiment 1, obese rats were fed a high fat diet (HFD) supplemented with four different plant-based flours for 12 weeks. Barley flour (BF), oat bran (OB), wheat bran (WB), and Hi-maize amylose (HMA) were incorporated into the HFD at 5% or 10% total fiber content and were compared to a HFD control. For experiment 2, lean, chow-fed rats were switched to HFD supplemented with 10% WB or BF to determine the preventative efficacy of flour supplementation. RESULTS: In experiment 1, 10% BF and 10% WB reduced body weight and adiposity gain and increased cecal butyrate. Gut microbiota analysis of WB and BF treated rats revealed increases in relative abundance of SCFA-producing bacteria. 10% WB and BF were also efficacious in preventing HFD-induced obesity; 10% WB and BF decreased body weight and adiposity, improved glucose tolerance, and reduced inflammatory markers and lipogenic enzyme expression in liver and adipose tissue. These effects were accompanied by alterations in the gut microbiota including increased relative abundance of Lactobacillus and LachnospiraceaeUCG001, along with increased portal taurodeoxycholic acid (TDCA) in 10% WB and BF rats compared to HFD rats. CONCLUSIONS: Therapeutic and preventative supplementation with 10%, but not 5%, WB or BF improves metabolic homeostasis, which is possibly due to gut microbiome-induced alterations. Specifically, these effects are proposed to be due to increased concentrations of intestinal butyrate and circulating TDCA.

Oligofructose improves small intestinal lipid-sensing mechanisms via alterations to the small intestinal microbiota.

BACKGROUND: Upper small intestinal dietary lipids activate a gut-brain axis regulating energy homeostasis. The prebiotic, oligofructose (OFS) improves body weight and adiposity during metabolic dysregulation but the exact mechanisms remain unknown. This study examines whether alterations to the small intestinal microbiota following OFS treatment improve small intestinal lipid-sensing to regulate food intake in high fat (HF)-fed rats. RESULTS: In rats fed a HF diet for 4 weeks, OFS supplementation decreased food intake and meal size within 2 days, and reduced body weight and adiposity after 6 weeks. Acute (3 day) OFS treatment restored small intestinal lipid-induced satiation during HF-feeding, and was associated with increased small intestinal CD36 expression, portal GLP-1 levels and hindbrain neuronal activation following a small intestinal lipid infusion. Transplant of the small intestinal microbiota from acute OFS treated donors into HF-fed rats also restored lipid-sensing mechanisms to lower food intake. 16S rRNA gene sequencing revealed that both long and short-term OFS altered the small intestinal microbiota, increasing Bifidobacterium relative abundance. Small intestinal administration of Bifidobacterium pseudolongum to HF-fed rats improved small intestinal lipid-sensing to decrease food intake. CONCLUSION: OFS supplementation rapidly modulates the small intestinal gut microbiota, which mediates improvements in small intestinal lipid sensing mechanisms that control food intake to improve energy homeostasis. Video Abstract.

Oligofructose restores postprandial short-chain fatty acid levels during high-fat feeding.

OBJECTIVE: Obesity is associated with consumption of a Western diet low in dietary fiber, while prebiotics reduce body weight. Fiber induces short-chain fatty acid (SCFA) production, and SCFA administration is beneficial to host metabolic homeostasis. However, the role of endogenous SCFA signaling in the development of obesity is contentious. Therefore, the primary objective of this study is to evaluate the postprandial time course of SCFA production and uptake in healthy (chow-fed), Western diet-fed (high-fat diet [HFD]) obese, and oligofructose-treated HFD-fed (HFD + OFS) rats. METHODS: Male Sprague-Dawley rats were maintained on chow or HFD for 5 weeks, with or without supplementation of 10% OFS for 3 weeks. SCFAs were measured in the ileum, cecum, colon, portal vein, and vena cava at 0, 2, 4, 6, and 8 hours postprandially. RESULTS: Postprandial cecal and portal vein SCFAs were decreased in obese rats compared with lean chow controls, whereas no differences were observed in fasting SCFA concentrations. OFS supplementation increased SCFA levels in the cecum and portal vein during obesity. Butyrate levels were positively associated with portal glucagon-like peptide 1 and adiposity and with Roseburia relative abundance. CONCLUSIONS: The current study demonstrates that obesity is associated with reduced SCFA production, and that OFS supplementation increases SCFA levels. Additionally, postprandial butyrate production appears to be beneficial to host energy homeostasis.

RAC1 nitration at Y32 IS involved in the endothelial barrier disruption associated with lipopolysaccharide-mediated acute lung injury.

Acute lung injury (ALI), a devastating illness induced by systemic inflammation e.g., sepsis or local lung inflammation e.g., COVID-19 mediated severe pneumonia, has an unacceptably high mortality and has no effective therapy. ALI is associated with increased pulmonary microvascular hyperpermeability and alveolar flooding. The small Rho GTPases, RhoA and Rac1 are central regulators of vascular permeability through cytoskeleton rearrangements. RhoA and Rac1 have opposing functional outcome: RhoA induces an endothelial contractile phenotype and barrier disruption, while Rac1 stabilizes endothelial junctions and increases barrier integrity. In ALI, RhoA activity is increased while Rac1 activity is reduced. We have shown that the activation of RhoA in lipopolysaccharide (LPS)-mediated ALI, is dependent, at least in part, on a single nitration event at tyrosine (Y)34. Thus, the purpose of this study was to determine if the inhibition of Rac1 is also dependent on its nitration. Our data show that Rac1 inhibition by LPS is associated with its nitration that mass spectrometry identified as Y32, within the switch I region adjacent to the nucleotide-binding site. Using a molecular modeling approach, we designed a nitration shielding peptide for Rac1, designated NipR2 (nitration inhibitor peptide for the Rho GTPases 2), which attenuated the LPS-induced nitration of Rac1 at Y32, preserves Rac1 activity and attenuates the LPS-mediated disruption of the endothelial barrier in human lung microvascular endothelial cells (HLMVEC). Using a murine model of ALI induced by intratracheal installation of LPS we found that NipR2 successfully prevented Rac1 nitration and Rac1 inhibition, and more importantly attenuated pulmonary inflammation, reduced lung injury and prevented the loss of lung function. Together, our data identify a new post-translational mechanism of Rac1 inhibition through its nitration at Y32. As NipR2 also reduces sepsis induced ALI in the mouse lung, we conclude that Rac1 nitration is a therapeutic target in ALI.

Activation of the mechanosensitive Ca2+ channel TRPV4 induces endothelial barrier permeability via the disruption of mitochondrial bioenergetics.

Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS), a refractory lung disease with an unacceptable high mortality rate. Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The specific mechanisms involved in VILI-induced pulmonary capillary leakage, a key pathologic feature of VILI are still far from resolved. The mechanoreceptor, transient receptor potential cation channel subfamily V member 4, TRPV4 plays a key role in the development of VILI through unresolved mechanism. Endothelial nitric oxide synthase (eNOS) uncoupling plays an important role in sepsis-mediated ARDS so in this study we investigated whether there is a role for eNOS uncoupling in the barrier disruption associated with TRPV4 activation during VILI. Our data indicate that the TRPV4 agonist, 4α-Phorbol 12,13-didecanoate (4αPDD) induces pulmonary arterial endothelial cell (EC) barrier disruption through the disruption of mitochondrial bioenergetics. Mechanistically, this occurs via the mitochondrial redistribution of uncoupled eNOS secondary to a PKC-dependent phosphorylation of eNOS at Threonine 495 (T495). A specific decoy peptide to prevent T495 phosphorylation reduced eNOS uncoupling and mitochondrial redistribution and preserved PAEC barrier function under 4αPDD challenge. Further, our eNOS decoy peptide was able to preserve lung vascular integrity in a mouse model of VILI. Thus, we have revealed a functional link between TRPV4 activation, PKC-dependent eNOS phosphorylation at T495, and EC barrier permeability. Reducing pT495-eNOS could be a new therapeutic approach for the prevention of VILI.

LPS-induced Acute Lung Injury Involves NF-κB-mediated Downregulation of SOX18.

One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.

Redox regulation of epidermal growth factor receptor signaling during the development of pulmonary hypertension.

The development of pulmonary hypertension (PH) involves the uncontrolled proliferation of pulmonary smooth muscle cells via increased growth factor receptor signaling. However, the role of epidermal growth factor receptor (EGFR) signaling is controversial, as humans with advanced PH exhibit no changes in EGFR protein levels and purpose of the present study was to determine whether there are post-translational mechanisms that enhance EGFR signaling in PH. The EGFR inhibitor, gefinitib, significantly attenuated EGFR signaling and prevented the development of PH in monocrotaline (MCT)-exposed rats, confirming the contribution of EGFR activation in MCT induced PH. There was an early MCT-mediated increase in hydrogen peroxide, which correlated with the binding of the active metabolite of MCT, monocrotaline pyrrole, to catalase Cys377, disrupting its multimeric structure. This early oxidative stress was responsible for the oxidation of EGFR and the formation of sodium dodecyl sulfate (SDS) stable EGFR dimers through dityrosine cross-linking. These cross-linked dimers exhibited increased EGFR autophosphorylation and signaling. The activation of EGFR signaling did not correlate with pp60(src) dependent Y845 phosphorylation or EGFR ligand expression. Importantly, the analysis of patients with advanced PH revealed the same enhancement of EGFR autophosphorylation and covalent dimer formation in pulmonary arteries, while total EGFR protein levels were unchanged. As in the MCT exposed rat model, the activation of EGFR in human samples was independent of pp60(src) phosphorylation site and ligand expression. This study provides a novel molecular mechanism of oxidative stress stimulated covalent EGFR dimerization via tyrosine dimerization that contributes into development of PH.

Asymmetric Dimethylarginine Stimulates Akt1 Phosphorylation via Heat Shock Protein 70-Facilitated Carboxyl-Terminal Modulator Protein Degradation in Pulmonary Arterial Endothelial Cells.

Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial nitric oxide synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at threonine (T) 308 and serine (S) 473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells to ADMA enhanced Akt1 phosphorylation at both threonine 308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog activity, or membrane Akt1 levels. Heat shock protein (Hsp) 90 plays a pivotal role in maintaining Akt1 activity, and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but, surprisingly, overexpression of a dominant-negative Hsp90 mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of dominant-negative Hsp90 increased Hsp70 levels, and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, carboxyl-terminal modulator protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMP protein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo, and that increasing CTMP levels may be an avenue to treat pulmonary hypertension.

The sexual dimorphism associated with pulmonary hypertension corresponds to a fibrotic phenotype.

Although female predominance in the development of all types of pulmonary hypertension (PH) is well established, many clinical studies have confirmed that females have better prognosis and higher survival rate than males. There is no clear explanation of why sex influences the pathogenesis and progression of PH. Using a rat angioproliferative model of PH, which closely resembles the primary pathological changes observed in humans, we evaluated the role of sex in the development and progression of PH. Female rats had a more pronounced increase in medial thickness in the small pulmonary arteries. However, the infiltration of small pulmonary arteries by inflammatory cells was found only in male rats, and this corresponded to increased myeloperoxidase activity and abundant adventitial and medial fibrosis that were not present in female rats. Although the level of right ventricle (RV) peak systolic pressure was similar in both groups, the survival rate in male rats was significantly lower. Moreover, male rats presented with a more pronounced increase in RV thickness that correlated with diffuse RV fibrosis and significantly impaired right cardiac function. The reduction in fibrosis in female rats correlated with increased expression of caveolin-1 and reduced endothelial nitric oxide synthase-derived superoxide. We conclude that, in the pathogenesis of PH, female sex is associated with greater remodeling of the pulmonary arteries but greater survival. Conversely, in males, the development of pulmonary and cardiac fibrosis leads to early and severe RV failure, and this may be an important reason for the lower survival rate among males.

Endothelial nitric oxide synthase deficient mice are protected from lipopolysaccharide induced acute lung injury.

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS-/-) are protected against ALI. In both wild-type and eNOS-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr34. Finally, we found that the reduction in NOS uncoupling in eNOS-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.

Lipopolysaccharide-induced lung injury involves the nitration-mediated activation of RhoA.

Acute lung injury (ALI) is characterized by increased endothelial hyperpermeability. Protein nitration is involved in the endothelial barrier dysfunction in LPS-exposed mice. However, the nitrated proteins involved in this process have not been identified. The activation of the small GTPase RhoA is a critical event in the barrier disruption associated with LPS. Thus, in this study we evaluated the possible role of RhoA nitration in this process. Mass spectroscopy identified a single nitration site, located at Tyr(34) in RhoA. Tyr(34) is located within the switch I region adjacent to the nucleotide-binding site. Utilizing this structure, we developed a peptide designated NipR1 (nitration inhibitory peptide for RhoA 1) to shield Tyr(34) against nitration. TAT-fused NipR1 attenuated RhoA nitration and barrier disruption in LPS-challenged human lung microvascular endothelial cells. Further, treatment of mice with NipR1 attenuated vessel leakage and inflammatory cell infiltration and preserved lung function in a mouse model of ALI. Molecular dynamics simulations suggested that the mechanism by which Tyr(34) nitration stimulates RhoA activity was through a decrease in GDP binding to the protein caused by a conformational change within a region of Switch I, mimicking the conformational shift observed when RhoA is bound to a guanine nucleotide exchange factor. Stopped flow kinetic analysis was used to confirm this prediction. Thus, we have identified a new mechanism of nitration-mediated RhoA activation involved in LPS-mediated endothelial barrier dysfunction and show the potential utility of "shielding" peptides to prevent RhoA nitration in the management of ALI.

Endothelin-1 stimulates catalase activity through the PKCδ-mediated phosphorylation of serine 167.

Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H2O2) levels. These changes correlated with an increase in serine-phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be protein kinase Cδ (PKCδ) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H2O2 compared to the wild-type enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist tezosentan. S167 is located on the dimeric interface, suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel filtration to examine the multimeric structure of recombinant wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKCδ was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity.

Berry fruits modulated endothelial cell migration and angiogenesis via phosphoinositide-3 kinase/protein kinase B pathway in vitro in endothelial cells.

Polyphenolic-rich berry fruits are known to activate redox-sensitive cellular signaling molecules such as phosphatidylinositol-3-kinase (PI3 kinase)/kinase B (Akt), resulting in a cascade of downstream signaling pathways. This study investigated the ability of strawberry (SB), wild blueberry (WBB), and cranberry (CB) extracts to induce the activation of PI3 kinase/Akt signaling in vitro in human umbilical endothelial cells (HUVECs) and whether this activation would enhance cell migration and angiogenesis. Anthocyanin profiles of the extracts were characterized using HPLC-ESI/MS, and Akt activation was investigated using the Alpha Screen SureFire assay. The total anthocyanin contents of SB, WBB, and CB extracts were 81.7, 82.5, and 83.0 mg/100 g fresh weight, respectively. SB, WBB, and CB extracts activated Akt in a dose-dependent manner via PI3 kinase and induced cell migration and angiogenesis in vitro in HUVECs. The results from this study suggest that polyphenolics in berry fruits may play a role in promoting vascular health.