Supplementation Of Mice Research Articles

Metabolic preferences of astrocytes: Functional metabolic mapping reveals butyrate outcompetes acetate.

Disruptions to the gut-brain-axis have been linked to neurodegenerative disorders. Of these disruptions, reductions in the levels of short-chain fatty acids (SCFAs), like butyrate, have been observed in mouse models of Alzheimer's disease (AD). Butyrate supplementation in mice has shown promise in reducing neuroinflammation, amyloid-β accumulation, and enhancing memory. However, the underlying mechanisms remain unclear. To address this, we investigated the impact of butyrate on energy metabolism in mouse brain slices, primary cultures of astrocytes and neurons and in-vivo by dynamic isotope labelling with [U-13C]butyrate and [1,2-13C]acetate to map metabolism via mass spectrometry. Metabolic competition assays in cerebral cortical slices revealed no competition between butyrate and the ketone body, β-hydroxybutyrate, but competition with acetate. Astrocytes favoured butyrate metabolism compared to neurons, suggesting that the astrocytic compartment is the primary site of butyrate metabolism. In-vivo metabolism investigated in the 5xFAD mouse, an AD pathology model, showed no difference in 13C-labelling of TCA cycle metabolites between wild-type and 5xFAD brains, but butyrate metabolism remained elevated compared to acetate in both groups, indicating sustained uptake and metabolism in 5xFAD mice. Overall, these findings highlight the role of astrocytes in butyrate metabolism and the potential use of butyrate as an alternative brain fuel source.

Mitigating lead-induced osteoporosis: The role of butyrate in gut-bone axis restoration

Lead (Pb) is an environmentally widespread bone toxic pollutant, contributes to the development of osteoporosis. Butyric acid, mainly produced by the fermentation of indigestible dietary fiber by gut microbiota, plays a pivotal role in the maintenance of bone homeostasis. However, the effects of butyric acids on the Pb induced osteoporosis have not yet been elucidated. In this study, our results showed that Pb exposure was negatively related to the abundance of butyric acid, in the Pb-exposed population and Pb-exposed mice. Pb exposure caused gut microbiota disorders, resulting in the decline of butyric acid-producing bacteria, such as Butyrivibrio_crossotus, Clostridium_sp._JN9, and the butyrate-producing enzymes through the acetyl-CoA pathway. Moreover, results from the NHANES data suggested that dietary intake of butyrate was associated with a reduced risk of osteoporosis in lead-burdened populations, particularly among men or participants aged 18–60 years. In addition, butyrate supplementation in mice with chronic Pb exposure improved the bone microarchitectures, repaired intestinal damage, upregulated the proportion of Treg cells. Taken together, these results demonstrated that chronic Pb exposure disturbs the gut-bone axis, which can be restored by butyric acid supplement. Our results suggest that butyrate supplementation is a possible therapeutic strategy for lead-induced bone toxicity.

Molecular hydrogen supplementation in mice ameliorates lipopolysaccharide-induced loss of interest.

The objective of this study was to evaluate the potential of hydrogen in preventing and treating psychiatric symptoms, particularly depressed mood and loss of interest, and to explore its underlying mechanisms. A mouse model exhibiting inflammation-derived depressive symptoms was used for the investigation. Institute of Cancer Research mice were subjected to a 7-day intervention of either 30% hydrogen or 40 g per day of air via jelly intake. On the final day, lipopolysaccharide (LPS) was intraperitoneally administered at 5 mg/kg to induce inflammation-related depressive symptoms. Behavioral and biochemical assessments were conducted 24 h post-LPS administration. Following LPS administration, a decrease in spontaneous behavior was observed; however, this effect was mitigated in the group treated with hydrogen. The social interaction test revealed a significant reduction in interactions with unfamiliar mice in the LPS-treated group, whereas the hydrogen-treated group exhibited no such decrease. No significant changes were noted in the forced-swim test for either group. Additionally, the administration of LPS in the hydrogen group did not result in a decrease in zonula occludens-1, a biochemical marker associated with barrier function at the cerebrovascular barrier and expressed in tight junctions. Hydrogen administration demonstrated a preventive effect against the LPS-induced loss of interest, suggesting a potential role in symptom prevention. However, it did not exhibit a suppressive effect on depressive symptoms in this particular model. These findings highlight the nuanced impact of hydrogen in the context of inflammation-induced psychiatric symptoms, indicating potential avenues for further exploration and research.

Yersinia infection induces glucose depletion and AMPK-dependent inhibition of pyroptosis in mice.

Nutritional status and pyroptosis are important for host defence against infections. However, the molecular link that integrates nutrient sensing into pyroptosis during microbial infection is unclear. Here, using metabolic profiling, we found that Yersinia pseudotuberculosis infection results in a significant decrease in intracellular glucose levels in macrophages. This leads to activation of the glucose and energy sensor AMPK, which phosphorylates the essential kinase RIPK1 at S321 during caspase-8-mediated pyroptosis. This phosphorylation inhibits RIPK1 activation and thereby restrains pyroptosis. Boosting the AMPK-RIPK1 cascade by glucose deprivation, AMPK agonists, or RIPK1-S321E knockin suppresses pyroptosis, leading to increased susceptibility to Y. pseudotuberculosis infection in mice. Ablation of AMPK in macrophages or glucose supplementation in mice is protective against infection. Thus, we reveal a molecular link between glucose sensing and pyroptosis, and unveil a mechanism by which Y. pseudotuberculosis reduces glucose levels to impact host AMPK activation and limit host pyroptosis to facilitate infection.

Restoration of Lactobacillus johnsonii and Enterococcus faecalis Caused the Elimination of Tritrichomonas sp. in a Model of Antibiotic-Induced Dysbiosis.

Inflammatory bowel disease (IBD) is a multifactorial disease involving the interaction of the gut microbiota, genes, host immunity, and environmental factors. Dysbiosis in IBD is associated with pathobiont proliferation, so targeted antibiotic therapy is a rational strategy. When restoring the microbiota with probiotics, it is necessary to take into account the mutual influence of co-cultivated microorganisms, as the microbiota is a dynamic community of species that mediates homeostasis and physiological processes in the intestine. The aim of our study was to investigate the recovery efficacy of two potential probiotic bacteria, L. johnsonii and E. faecalis, in Muc2-/- mice with impaired mucosal layer. Two approaches were used to determine the efficacy of probiotic supplementation in mice with dysbiosis caused by mucin-2 deficiency: bacterial seeding on selective media and real-time PCR analysis. The recovery time and the type of probiotic bacteria relocated affected only the number of E. faecalis. A significant positive correlation was found between colony-forming unit (CFU) and the amount of E. faecalis DNA in the group that was replanted with probiotic E. faecalis. As for L. johnsonii, it could be restored to its original level even without any additional bacteria supplementation after two weeks. Interestingly, the treatment of mice with L. johnsonii caused a decrease in the amount of E. faecalis. Furthermore, either L. johnsonii or E. faecalis treatment eliminated protozoan overgrowth caused by antibiotic administration.

Targeting the SIRT1 pathway to modulate inflammation in a murine model of Kawasaki Disease vasculitis

Abstract Kawasaki Disease (KD) is an acute febrile systemic vasculitis and the leading cause of acquired heart disease among children. Overactivation of the NLRP3 inflammasome and IL-1b production are critical for the development of cardiovascular lesions during KD. Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, is crucial for responding to cellular stress, and regulates NLRP3 activation as well as clearance of damaged mitochondria in cardiovascular diseases. However, its role in KD pathophysiology remains unknown. Here, we used the Lactobacillus casei cell wall extract (LCWE) murine model of KD to determine the role of SIRT1 on the development of cardiovascular lesions. We show that Sirt1 expression is downregulated in LCWE-injected mice with a significant reduction of nicotinamide, a product resulting from NAD+ degradation by SIRT1. In addition, supplementation of mice with NAD+ precursors significantly reduced the development of LCWE-induced cardiovascular lesions. Furthermore, KD development was less severe in SIRT1 overexpressing mice but was exacerbated with the specific deletion of Sirt1 in vascular smooth muscle cells. Treatment of LCWE-stimulated coronary smooth muscle cells with a SIRT1 activator favored mitophagy by inhibiting ribosomal S6 phosphorylation. These results indicate an impaired NAD+/SIRT1 axis during LCWE-induced KD vasculitis, which could potentially be targeted to reduce cardiovascular lesions in KD.

Metabolomic and transcriptomic correlative analyses in germ free mice link probiotic-associated metabolites to host intestinal fatty acid metabolism and b-oxidation

While a transcriptomics-led examination of the effects of probiotics offers valuable insights into their influence on host gene regulation, the dynamic interactions between host metabolism and probiotics require an integrated analysis and multiomics approach to provide deeper insight into the underlying molecular mechanisms governing these associations. Here, we developed a high throughput bioinformatic tool ‘metabolome-transcriptome correlation analysis’ (METRCA, in press) that can determine whether a certain gene-metabolite association is up or downregulated and predict whether a metabolite’s or gene’s tissue location impacts its interaction with one another. To test this, we analyzed by LCMS and bulk-RNA sequencing then assessed linkages between serum as well as fecal metabolites and disease- or metabolism-associated genes in the ileum of gnotobiotic mice fed either a tryptophan (trp)-deficient or -suffcient diet then mono-associated with Lactobacillus rhamnosus GG (LGG) or administered PBS (control). We discovered significant associations, mainly in LGG mice fed trp, between metabolites and ileal genes involved in fatty acid metabolism (FAM) and oxidation (FAO). This association was specific and not observed in GF mice gavaged with PBS, monocolonized with R. gnavus, or fed trp- diets, and in SPF mice. Many fecal trp metabolites like indole-3-acetamide and serum trp metabolites like indole acetonitrile were significantly, positively correlated with important intestinal FAM genes Dgat1 and Mgat2. Likewise, numerous fecal metabolites like 5-hydroytrp and several serum trp metabolites like serotonin emerged as positively correlated metabolites with the rate-limiting FAO genes Acaa2 and Cpt2. Serum indoles were more strongly associated with both FAM and FAO genes than fecal indoles. Few fecal and serum metabolites were found negatively correlated with FAM and FAO genes. Since most are positive correlators, these findings suggest a testable hypothesis that LGG- and trp-dependent metabolites increase the intestinal metabolism of dietary lipids, providing a mechanism to previous findings that LGG supplementation in mice protected against diet-induced obesity and fatty liver disease. Analytical approaches like METRCA can thus be used to extract valuable information from metabolite and RNA-Seq datasets, generating novel hypotheses that may discover new interactions between microbe- or nutrient-associated metabolites and host metabolic or disease-related pathways. NIHR01-AT010243 (NG,RF), R01DK119198 (NG), NSF 1754783 (RF)). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Faecal microbial transfer and complex carbohydrates mediate protection against COPD

ObjectiveChronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development...

Intracellular Zn2+ promotes extracellular matrix remodeling in dexamethasone-treated trabecular meshwork.

Given the widespread application of glucocorticoids in ophthalmology, the associated elevation of intraocular pressure (IOP) has long been a vexing concern for clinicians, yet the underlying mechanisms remain inconclusive. Much of the discussion focuses on the extracellular matrix (ECM) of trabecular meshwork (TM). It is widely agreed that glucocorticoids impact the expression of matrix metalloproteinases (MMPs), leading to ECM deposition. Since Zn2+ is vital for MMPs, we explored its role in ECM alterations induced by dexamethasone (DEX). Our study revealed that in human TM cells treated with DEX, the level of intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. This correlated with changes in several Zrt-, Irt-related proteins (ZIPs) and metallothionein. ZIP8 knockdown impaired extracellular Zn2+ uptake, but Zn2+ chelation did not affect ZIP8 expression. Resembling DEX's effects, chelation of Zn2+ decreased MMP2 expression, increased the deposition of ECM proteins, and induced structural disarray of ECM. Conversely, supplementation of exogenous Zn2+ in DEX-treated cells ameliorated these outcomes. Notably, dietary zinc supplementation in mice significantly reduced DEX-induced IOP elevation and collagen content in TM, thereby rescuing the visual function of the mice. These findings underscore zinc's pivotal role in ECM regulation, providing a novel perspective on the pathogenesis of glaucoma.NEW & NOTEWORTHY Our study explores zinc's pivotal role in mitigating extracellular matrix dysregulation in the trabecular meshwork and glucocorticoid-induced ocular hypertension. We found that in human trabecular meshwork cells treated with dexamethasone, intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. Zinc supplementation rescues visual function by modulating extracellular matrix proteins and lowering intraocular pressure, offering a direction for further exploration in glaucoma management.

Enhanced anti-fatigue and exercise performances due to Huáng qí polysaccharide supplementation in mice

In modern society, people have busy and competitive lives, with different stresses causing fatigue and overwork, which may ultimately harm their health. We investigated the beneficial effects of Astragalus polysaccharide (rAPS) on fatigue and exercise performance. Forty male Institute of Cancer Research (ICR) mice were randomly divided into four groups according to body weight (BW): control group (distilled water), 1-fold dose group (0.70 g rAPS/kg BW/day), 2-fold dose group (1.40 g rAPS/kg BW/day) and a 5-fold dose group (3.50 g rAPS/kg BW/day). In terms of exercise performance, rAPS supplementation significantly improved grip strength performance and time to exhaust-loaded swim times when compared with controls (p < 0.05). Moreover, rAPS supplementation significantly reduced fatigue-related biochemical parameters, including blood lactate and BUN levels, while significantly improve muscle strength and swimming endurance times t(p < 0.05). Hence, we recommend that rAPS elicit antifatigue and performance-enhancing health benefits.

High dose NAD+ boosting as pan-sirtuin activation increases atherosclerotic plaques and systemic inflammation in Apoe knockout mice

Abstract Background NAD+ (nicotinamide adenine diphosphate) is a cosubstrate of sirtuins (SIRT) that are activated upon caloric restriction. The nuclear SIRT1 and SIRT6 exert protective effects on atherothrombosis through immunometabolic pathways. By supplementing its precursors, an increase in the NAD+ pool has been reported to extend life span in mice. However, supplementing NAD+ precursors may also stimulate inflammation depending on the context. Nicotinamide riboside (NR) is a precursor of NAD+, whose supplementation has been used to combat metabolic syndromes and liver steatosis. Notably, the effects of pan-sirtuin activation using NR on atherosclerosis remain unknown. Purpose We hypothesized that NR supplementation confers atheroprotection by activating the NAD+ sirtuin axis. We aimed to test this hypothesis by investigating the effects of NR supplementation on plaque formation using an atherosclerosis-prone mouse model. Methods 8-week-old male apolipoprotein E (ApoE) knockout (KO) mice were fed for 12 weeks a high-cholesterol diet (HCD, 1.25 wt%) supplemented with three NR doses: 0, 1.2, and 2.4 g/kg diet. The groups were designated as NR-, NR+, and NR++, respectively (n=14/group). After overnight fasting, aortae, liver, and blood were collected. Cytokines were analyzed using a multiplex ELISA. Triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels were determined by colorimetry and the Friedewald equation. Cross sections of the aortic root were stained with Oil-Red O. Results High-dose (HD) NR supplementation increased plaque thickness in NR++ ApoE KO mice, compared with NR+ mice (Figure 1A and B). In parallel, plasma expression levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 as well as TG and LDL-C levels were increased in NR++ mice (Figure 1C-F) suggesting that NR supplementation induced systemic inflammation and disturbed lipid metabolism. Analyses of liver lysates showed that expression levels of SIRT1 were decreased and CD38 increased in NR++ mice. Full length PARP1 remained unchanged, while both cleaved PARP1 and total PARylation in liver decreased. NAD+ concentrations in liver and plasma remained unchanged. These data suggest that an increase in hepatic CD38 might consume more NAD+ in the atherosclerotic environment and could be the culprits for worsening atherosclerotic progression and systemic inflammation (Figure 2). Conclusions HD NR supplementation increased plaque thickness, as well as IL-6 and LDL-C plasma levels, moderate NR dose had neutral effects. Elevated hepatic expression levels of CD38 and decreased SIRT1 may at least in part explain the atherosclerotic phenotype. Thus, HD NR supplementation in mice does not decrease but worsen atherosclerosis and induce systemic inflammation. A cautious approach should be considered when applying NAD+ boosting in patients with atherosclerotic disease.

One-Carbon Metabolites Promote Systemic Inflammation, Gut Dysbiosis, and a Myeloid Lineage Differentiation Bias

TET methylcytosine dioxygenase 2 ( TET2) loss-of-function mutations induce a pre-malignant state known as clonal hematopoiesis of indeterminate potential (CHIP). CHIP occurs in approximately 10% of people over 65 years of age and confers a 10-fold greater risk of developing hematological malignancy. Several environmental factors, including radiation, sleep deprivation, atherosclerosis, and diet, have been associated with the expansion of pre-malignant clones in CHIP patients. It has previously been shown that hematopoietic Tet2 deficiency in mice triggers a pro-inflammatory state with increased intestinal permeability, gut bacterial translocation, and accelerated clonal expansion. Gut microbes themselves can exert an influence on myeloid leukemia progression through synthesis of compounds including short-chain fatty acids (SCFAs), which promote intestinal barrier integrity. Dietary levels of one-carbon metabolites and cofactors have been found to alter gut microbial composition, affecting SCFA production and intestinal permeability, in disease-free adults. Given the connection between diet, SCFAs and gut permeability, we sought to determine the impact of dietary one-carbon metabolites on gut microbial composition and function in CHIP progression. We performed competitive bone marrow transplantation assays with Tet2+/- cells in mice supplemented with altered one-carbon metabolites: a control diet, high or low methionine diets, high or low folate diets, and high or low vitamin B12 supplementation. Mice were treated for a total of 7 months before timed sacrifice. Altered supplementation of the one carbon metabolites tested did not influence the competitiveness of Tet2-deficient cells in peripheral blood, spleen or bone marrow, however, high vitamin B12 levels promoted a myeloid differentiation bias in the spleen and bone marrow. Plasma cytokine analysis also showed that high vitamin B12 supplementation caused increased circulating inflammatory cytokines (IL-1b, IL-23, IL-27, CCL3/4, CXCL2). Based on this strong inflammatory phenotype, we performed scRNA-sequencing of splenic Tet2+/- CD11b+ cells. These data confirmed a neutrophilic cell expansion in the spleens of vitamin B12-treated mice along with increased expression of innate inflammatory genes S100a8/9 and Lyz2 as well as IL-1 b and Cxcl2 in the neutrophil clusters. Gut microbial-dependent inflammation is known to drive TET2-dependent myeloproliferation, thus we collected fecal samples before timed sacrifice and performed shotgun sequencing. Vitamin B12 supplementation decreased alpha diversity and the composition of genera that produce butyrate, an SCFA known to promote intestinal barrier integrity. Metagenomic analysis revealed increased amino acid metabolism and fatty acid biosynthesis in the gut microbiome of vitamin B12-supplemented mice, along with decreased butanoate metabolism, suggesting an alteration in microbial function that could influence SCFA content. To test whether these alterations in SCFA-producers led to increased gut permeability, we performed fluorescence in situ hybridization (FISH) against bacterial ribosomal 16S in livers from our mouse cohorts and observed increased levels of bacterial dissemination in mice with vitamin B12 supplementation. Our findings suggest that vitamin B12 supplementation in mice exacerbates the myeloid lineage differentiation bias of Tet2+/- hematopoiesis, possibly due to decreased gut barrier integrity which contributes to a heightened innate inflammatory response. This study highlights the potential for micronutrients of one-carbon metabolism to influence CHIP progression through maintenance of proper gut microbial homeostasis.

Probiotic Potential of Bacillus sp. 62A Isolated from a Marine Extreme Environment.

Antimicrobial resistance is an important health concern globally, and probiotics are considered an alternative to minimize it. The present study examined the in vitro probiotic characteristics and in vivo immunomodulatory potential of Bacillus sp. 62A - an extremophile bacterium. Bacillus sp. 62A was evaluated in vitro for its cytotoxicity, hemolytic activity, antibiotic susceptibility, and resistance to gastrointestinal conditions (bile salts, low pH, and intestinal adherence). Additionally, the immunomodulatory effect of Bacillus sp. 62A was studied in mice. The animals were supplemented daily with phosphate-buffered saline (control) and Bacillus sp. 62A at 1 × 108 colony forming units (CFU). Samples were taken on days 5 and 10. Isolated splenocytes were challenged with Escherichia coli for immunological analyses and immune-related gene expression. Serum and feces were collected for IgA and IgG determination. Bacillus sp. 62A did not show cytotoxicity, hemolytic activity, or resistance to antibiotics. Furthermore, the bacterium has autoaggregation and intestinal adhesion capacities and grows in the presence of bile salts and low pH. Bacillus supplementation in mice improved respiratory burst activity, nitric oxide production, and IL-1β and IL-6 gene expressions, mainly at 10days. After E. coli challenge, Bacillus supplementation in mice induced an anti-inflammatory response through a decrease in immunological parameters and an increase in IL-10 gene expression. Moreover, serum IgA and IgG and fecal IgG augmented in supplemented mice. In conclusion, Bacillus sp. 62A has biosafe and immunomodulatory probiotic potential.

The microbial metabolite desaminotyrosine enhances T-cell priming and cancer immunotherapy with immune checkpoint inhibitors

Inter-individual differences in response to immune checkpoint inhibitors (ICI) remain a major challenge in cancer treatment. The composition of the gut microbiome has been associated with differential ICI outcome, but the underlying molecular mechanisms remain unclear, and therapeutic modulation challenging. We established an invivo model to treat C57Bl/6j mice with the type-I interferon (IFN-I)-modulating, bacterial-derived metabolite desaminotyrosine (DAT) to improve ICI therapy. Broad spectrum antibiotics were used to mimic gut microbial dysbiosis and associated ICI resistance. We utilized genetic mouse models to address the role of host IFN-I in DAT-modulated antitumour immunity. Changes in gut microbiota were assessed using 16S-rRNA sequencing analyses. We found that oral supplementation of mice with the microbial metabolite DAT delays tumour growth and promotes ICI immunotherapy with anti-CTLA-4 or anti-PD-1. DAT-enhanced antitumour immunity was associated with more activated T cells and natural killer cells in the tumour microenvironment and was dependent on host IFN-I signalling. Consistent with this, DAT potently enhanced expansion of antigen-specific T cells following vaccination with an IFN-I-inducing adjuvant. DAT supplementation in mice compensated for the negative effects of broad-spectrum antibiotic-induced dysbiosis on anti-CTLA-4-mediated antitumour immunity. Oral administration of DAT altered the gut microbial composition in mice with increased abundance of bacterial taxa that are associated with beneficial response to ICI immunotherapy. We introduce the therapeutic use of an IFN-I-modulating bacterial-derived metabolite to overcome resistance to ICI. This approach is a promising strategy particularly for patients with a history of broad-spectrum antibiotic use and associated loss of gut microbial diversity. Melanoma Research Alliance, Deutsche Forschungsgemeinschaft, German Cancer Aid, Wilhelm Sander Foundation, Novartis Foundation.

Krill oil treatment ameliorates lipid metabolism imbalance in chronic unpredicted mild stress-induced depression-like behavior in mice.

The pathology of depression involves various factors including the interaction between genes and the environment. The deficiency of n-3 polyunsaturated fatty acids (n-3 PUFAs) in the brain and depressive symptoms are closely related. Krill oil contains abundant amounts of n-3 PUFAs incorporated in phosphatidylcholine. However, the effect of krill oil treatment on depression-like behaviors induced by chronic stress and its molecular mechanism in the brain remain poorly understood. Here, we used a chronic unpredictable mild stress (CUMS) model to evaluate the effect of krill oil on depression-like behaviors and explored its molecular mechanism through lipid metabolomics and mRNA profiles in the whole brain. We observed that CUMS-induced depression-like behaviors were ameliorated by krill oil supplementation in mice. The metabolism of glycerophospholipids and sphingolipids was disrupted by CUMS treatment, which were ameliorated after krill oil supplementation. Further analysis found that differently expressed genes after krill oil supplementation were mainly enriched in the membrane structures and neuroactive ligand-receptor interaction pathway, which may be responsible for the amelioration of CUMS-induced depression-like behaviors. Altogether, our results uncovered the relationship between lipid metabolism and CUMS, and provided new strategies for the prevention and treatment of depression.

P27-021-23 Fetal Cognitive Neurodevelopment by the Maternal Gut Microbiome Remodeling by β-Glucan Supplementation in Mice

P27-021-23 Fetal Cognitive Neurodevelopment by the Maternal Gut Microbiome Remodeling by β-Glucan Supplementation in Mice

Lactobacillus salivarius and Lactobacillus gasseri supplementation reduces stress-induced sugar craving in mice.

Increased intake of sweets or sugar craving may occur in response to chronic stress representing a risk factor for development of eating disorders and obesity. However, no safe treatment of stress-induced sugar craving is available. In this study we analysed effects of two Lactobacillus strains on food and sucrose intake in mice before and during their exposure to a chronic mild stress (CMS). C57Bl6 mice were gavaged daily for 27days with a mix of L. salivarius (LS) LS7892 and L. gasseri (LG) LG6410 strains or with 0.9% NaCl as a control. Following 10days of gavage, mice were individually placed into the Modular Phenotypic cages, and after 7days of acclimation were exposed to a CMS model for 10days. Food, water and 2% sucrose intakes as well as meal pattern were monitored. Anxiety and depressive-like behaviour were analysed by standard tests. Exposure of mice to CMS was accompanied by increased size of sucrose intake in the control group likely reflecting the stress-induced sugar craving. A consistent, about 20% lower total sucrose intake, was observed in the Lactobacilli-treated group during stress which was mainly due to a reduced number of intakes. Lactobacilli treatment also modified the meal pattern before and during the CMS, showing a decrease of meal number and an increase of meal size with a tendency of reduced total daily food intake. Mild anti-depressive behavioural effects of the Lactobacilli mix were also present. Supplementation of mice with LS LS7892 and LG LG6410 decreases sugar consumption suggesting a potential utility of these strains against stress-induced sugar craving.

Vitamin E Induces Liver Iron Depletion and Alters Iron Regulation in Mice

BackgroundVitamin E (vit E) is an essential nutrient that functions as a lipophilic antioxidant and is used clinically to treat nonalcoholic fatty liver disease, where it suppresses oxidative damage and impedes the progression of steatosis and fibrosis. Mice lacking a critical liver iron–trafficking protein also manifest steatosis because of iron-mediated oxidative damage and are protected from liver disease by oral vit E supplements. ObjectivesWe aimed to examine the role of dietary vit E supplementation in modulating iron-sensing regulatory systems and nonheme iron levels in mouse liver. MethodsC57Bl/6 male mice, aged 6 wk, were fed purified diets containing normal amounts of iron and either control (45 mg/kg) or elevated (450 mg/kg) levels of 2R-α-tocopherol (vit E) for 18 d. Mouse plasma and liver were analyzed for nonheme iron, levels and activity of iron homeostatic proteins, and markers of oxidative stress. We compared means ± SD for iron and oxidative stress parameters between mice fed the control diet and those fed the vit E diet. ResultsThe Vit E–fed mice exhibited lower levels of liver nonheme iron (38% reduction, P < 0.0001) and ferritin (74% reduction, P < 0.01) than control-fed mice. The levels of liver mRNA for transferrin receptor 1 and divalent metal transporter 1 were reduced to 42% and 57% of the control, respectively. The mRNA levels for targets of nuclear factor erythroid 2–related factor (Nrf2), a major regulator of the oxidative stress response and iron-responsive genes, were also suppressed in vit E livers. Hepcidin, an iron regulatory hormone, levels were lower in the plasma (P < 0.05), and ferroportin (FPN), the iron exporter regulated by hepcidin, was expressed at higher levels in the liver (P < 0.05). ConclusionsOral vit E supplementation in mice can lead to depletion of liver iron stores by suppressing the iron- and redox-sensing transcription factor Nrf2, leading to enhanced iron efflux through liver FPN. Iron depletion may indirectly enhance the antioxidative effects of vit E.

Gut Microbiota-Derived Trimethylamine N-Oxide Contributes to Abdominal Aortic Aneurysm Through Inflammatory and Apoptotic Mechanisms.

Large-scale human and mechanistic mouse studies indicate a strong relationship between the microbiome-dependent metabolite trimethylamine N-oxide (TMAO) and several cardiometabolic diseases. This study aims to investigate the role of TMAO in the pathogenesis of abdominal aortic aneurysm (AAA) and target its parent microbes as a potential pharmacological intervention. TMAO and choline metabolites were examined in plasma samples, with associated clinical data, from 2 independent patient cohorts (N=2129 total). Mice were fed a high-choline diet and underwent 2 murine AAA models, angiotensin II infusion in low-density lipoprotein receptor-deficient (Ldlr-/-) mice or topical porcine pancreatic elastase in C57BL/6J mice. Gut microbial production of TMAO was inhibited through broad-spectrum antibiotics, targeted inhibition of the gut microbial choline TMA lyase (CutC/D) with fluoromethylcholine, or the use of mice genetically deficient in flavin monooxygenase 3 (Fmo3-/-). Finally, RNA sequencing of in vitro human vascular smooth muscle cells and in vivo mouse aortas was used to investigate how TMAO affects AAA. Elevated TMAO was associated with increased AAA incidence and growth in both patient cohorts studied. Dietary choline supplementation augmented plasma TMAO and aortic diameter in both mouse models of AAA, which was suppressed with poorly absorbed oral broad-spectrum antibiotics. Treatment with fluoromethylcholine ablated TMAO production, attenuated choline-augmented aneurysm initiation, and halted progression of an established aneurysm model. In addition, Fmo3-/- mice had reduced plasma TMAO and aortic diameters and were protected from AAA rupture compared with wild-type mice. RNA sequencing and functional analyses revealed choline supplementation in mice or TMAO treatment of human vascular smooth muscle cells-augmented gene pathways associated with the endoplasmic reticulum stress response, specifically the endoplasmic reticulum stress kinase PERK. These results define a role for gut microbiota-generated TMAO in AAA formation through upregulation of endoplasmic reticulum stress-related pathways in the aortic wall. In addition, inhibition of microbiome-derived TMAO may serve as a novel therapeutic approach for AAA treatment where none currently exist.

Ketohexokinase-C regulates global protein acetylation to decrease carnitine palmitoyltransferase 1a-mediated fatty acid oxidation

The consumption of sugar and a high-fat diet (HFD) promotes the development of obesity and metabolic dysfunction. Despite their well-known synergy, the mechanisms by which sugar worsens the outcomes associated with a HFD are largely elusive. Six-week-old, male, C57Bl/6J mice were fed either chow or a HFD and were provided with regular, fructose- or glucose-sweetened water. Moreover, cultured AML12 hepatocytes were engineered to overexpress ketohexokinase-C (KHK-C) using a lentivirus vector, while CRISPR-Cas9 was used to knockdown CPT1α. The cell culture experiments were complemented with invivo studies using mice with hepatic overexpression of KHK-C and in mice with liver-specific CPT1α knockout. We used comprehensive metabolomics, electron microscopy, mitochondrial substrate phenotyping, proteomics and acetylome analysis to investigate underlying mechanisms. Fructose supplementation in mice fed normal chow and fructose or glucose supplementation in mice fed a HFD increase KHK-C, an enzyme that catalyzes the first step of fructolysis. Elevated KHK-C is associated with an increase in lipogenic proteins, such as ACLY, without affecting their mRNA expression. An increase in KHK-C also correlates with acetylation of CPT1α at K508, and lower CPT1α protein invivo. Invitro, KHK-C overexpression lowers CPT1α and increases triglyceride accumulation. The effects of KHK-C are, in part, replicated by a knockdown of CPT1α. An increase in KHK-C correlates negatively with CPT1α protein levels in mice fed sugar and a HFD, but also in genetically obese db/db and lipodystrophic FIRKO mice. Mechanistically, overexpression of KHK-C invitro increases global protein acetylation and decreases levels of the major cytoplasmic deacetylase, SIRT2. KHK-C-induced acetylation is a novel mechanism by which dietary fructose augments lipogenesis and decreases fatty acid oxidation to promote the development of metabolic complications. Fructose is a highly lipogenic nutrient whose negative consequences have been largely attributed to increased de novo lipogenesis. Herein, we show that fructose upregulates ketohexokinase, which in turn modifies global protein acetylation, including acetylation of CPT1a, to decrease fatty acid oxidation. Our findings broaden the impact of dietary sugar beyond its lipogenic role and have implications on drug development aimed at reducing the harmful effects attributed to sugar metabolism.