Enhanced virulence and waning vaccine-elicited antibodies account for breakthrough infections caused by SARS-CoV-2 delta and beyond.

Here we interrogate the factors responsible for SARS-CoV-2 breakthrough infections in a K18-hACE2 transgenic mouse model. We show that Delta and the closely related Kappa variant cause viral pneumonia and severe lung lesions in K18-hACE2 mice. Human COVID-19 mRNA post-vaccination sera after the 2nd dose are significantly less efficient in neutralizing Delta/Kappa than early 614G virus in vitro and in vivo. By 5 months post-vaccination, ≥50% of donors lack detectable neutralizing antibodies against Delta and Kappa and all mice receiving 5-month post-vaccination sera die after the lethal challenges. Although a 3rd vaccine dose can boost antibody neutralization against Delta in vitro and in vivo, the mean log neutralization titers against the latest Omicron subvariants are 1/3-1/2 of those against the original 614D virus. Our results suggest that enhanced virulence, greater immune evasion, and waning of vaccine-elicited protection account for SARS-CoV-2 variants caused breakthrough infections.

Rats with high aerobic capacity display enhanced transcriptional adaptability and upregulation of bile acid metabolism in response to an acute high-fat diet.

Rats selectively bred for the high intrinsic aerobic capacity runner (HCR) or low aerobic capacity runner (LCR) show pronounced differences in susceptibility for high-fat/high sucrose (HFHS) diet-induced hepatic steatosis and insulin resistance, replicating the protective effect of high aerobic capacity in humans. We have previously shown multiple systemic differences in energy and substrate metabolism that impacts steatosis between HCR and LCR rats. This study aimed to investigate hepatic-specific mechanisms of action via changes in gene transcription. Livers of HCR rats had a greater number of genes that significantly changed in response to 3-day HFHS compared with LCR rats (171 vs. 75 genes: >1.5-fold, p < 0.05). HCR and LCR rats displayed numerous baseline differences in gene expression while on a low-fat control diet (CON). A 3-day HFHS diet resulted in greater expression of genes involved in the conversion of excess acetyl-CoA to cholesterol and bile acid (BA) synthesis compared with the CON diet in HCR, but not LCR rats. These results were associated with higher fecal BA loss and lower serum BA concentrations in HCR rats. Exercise studies in rats and mice also revealed higher hepatic expression of cholesterol and BA synthesis genes. Overall, these results suggest that high aerobic capacity and exercise are associated with upregulated BA synthesis paired with greater fecal excretion of cholesterol and BA, an effect that may play a role in protection against hepatic steatosis in rodents.

Circulating microRNAs Are Associated With Metabolic Markers in Adolescents With Hepatosteatosis.

Background: Altered hepatic microRNA (miRNA) expression may play a role in the development of insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD). Circulating miRNAs could mirror the liver metabolism. Objective: This study aimed to assess the relationship between serum miRNA profile in children with obesity, IR, and NAFLD. Methods: Adolescents with obesity (n = 31) were stratified based on insulin resistance and NAFLD status. One-hundred seventy-nine miRNAs were determined in the serum by quantitative RT-PCR. Differentially expressed miRNAs were compared between groups, and log-transformed levels correlated with metabolic markers and intrahepatic triglyceride. Results: Serum miR-21-5p, -22-3p, -150-5p, and -155-5p levels were higher in children with IR and NAFLD, and their expression levels correlated with hepatic fat and serum triglyceride. In patients with NAFLD, miR-155-5p correlated with ALT (r = 0.68, p<0.01) and AST (r = 0.64, p<0.01) and miR-21-5p and -22-3p levels correlated with plasma adiponectin (r = -0.71 and r = -0.75, respectively, p<0.05) and fibroblast growth factor-21 (r = -0.73 and r = -0.89, respectively, p<0.01). miR-27-3a level was higher in children without IR and NAFLD. Conclusions: Several miRNAs are differentially expressed in children with IR and NAFLD. Determining their mechanistic roles may provide newer diagnostic tools and therapeutic targets for pediatric NAFLD.

Soy Formula Is Not Estrogenic and Does Not Result in Reproductive Toxicity in Male Piglets: Results from a Controlled Feeding Study.

Soy infant formula which is fed to over half a million infants per year contains isoflavones such as genistein, which have been shown to be estrogenic at high concentrations. The developing testis is sensitive to estrogens, raising concern that the use of soy formulas may result in male reproductive toxicity. In the current study, male White-Dutch Landrace piglets received either sow milk (Sow), or were provided milk formula (Milk), soy formula (Soy), milk formula supplemented with 17-beta-estradiol (2 mg/kg/d) (M + E2) or supplemented with genistein (84 mg/L of diet; (M + G) from postnatal day 2 until day 21. E2 treatment reduced testis weight (p < 0.05) as percentage of body weight, significantly suppressed serum androgen concentrations, increased tubule area, Germ cell and Sertoli cell numbers (p < 0.05) relative to those of Sow or Milk groups. Soy formula had no such effects relative to Sow or Milk groups. mRNAseq revealed 103 differentially expressed genes in the M + E2 group compared to the Milk group related to endocrine/metabolic disorders. However, little overlap was observed between the other treatment groups. These data suggest soy formula is not estrogenic in the male neonatal piglet and that soy formula does not significantly alter male reproductive development.

Exercise training and diet-induced weight loss increase markers of hepatic bile acid (BA) synthesis and reduce serum total BA concentrations in obese women.

Regular exercise has profound metabolic influence on the liver, but effects on bile acid (BA) metabolism are less well known. BAs are synthesized exclusively in the liver from cholesterol via the rate-limiting enzyme cholesterol 7 alpha-hydroxylase (CYP7A1). BAs contribute to the solubilization and absorption of lipids and serve as important signaling molecules, capable of systemic endocrine function. Circulating BAs increase with obesity and insulin resistance, but effects following exercise and diet-induced weight loss are unknown. To test if improvements in fitness and weight loss as a result of exercise training enhance BA metabolism, we measured serum concentrations of total BAs (conjugated and unconjugated primary and secondary BAs) in sedentary, obese, insulin-resistant women (N = 11) before (PRE) and after (POST) a ∼14-wk exercise and diet-induced weight loss intervention. BAs were measured in serum collected after an overnight fast and during an oral glucose tolerance test (OGTT). Serum fibroblast growth factor 19 (FGF19; a regulator of BA synthesis) and 7-alpha-hydroxy-cholesten-3-one (C4, a marker of CYP7A1 enzymatic activity) also were measured. Using linear mixed-model analyses and the change in V̇O2peak (mL/min/kg) as a covariate, we observed that exercise and weight loss intervention decreased total fasting serum BA by ∼30% (P = 0.001) and increased fasting serum C4 concentrations by 55% (P = 0.004). C4 was significantly correlated with serum total BAs only in the POST condition, whereas serum FGF19 was unchanged. These data indicate that a fitness and weight loss intervention modifies BA metabolism in obese women and suggest that improved metabolic health associates with higher postabsorptive (fasting) BA synthesis. Furthermore, pre- vs. postintervention patterns of serum C4 following an OGTT support the hypothesis that responsiveness of BA synthesis to postprandial inhibition is improved after exercise and weight loss.NEW & NOTEWORTHY Exercise and weight loss in previously sedentary, insulin-resistant women facilitates a significant improvement in insulin sensitivity and fitness that may be linked to changes in bile acid metabolism. Diet-induced weight loss plus exercise-induced increases in fitness promote greater postabsorptive bile acid synthesis while also sensitizing the bile acid metabolic system to feedback inhibition during a glucose challenge when glucose and insulin are elevated.

Markers of branched-chain amino acid catabolism are not affected by exercise training in pregnant women with obesity.

Despite the role of branched-chain amino acids (BCAAs) in physiological processes such as nutrient signaling and protein synthesis, there is ongoing debate about the link between circulating BCAAs and insulin resistance (IR) in various populations. In healthy women, IR mildly increases during pregnancy, whereas both BCAAs and markers of BCAA catabolism decrease, indicating that fetal growth is being prioritized. Exercise reduces IR in nonpregnant adults, but less is known about the effect of exercise during pregnancy in women with obesity on IR and BCAA breakdown. The aim of this study was to determine the effect of a moderate-intensity exercise intervention during pregnancy on maternal circulating BCAAs and markers of BCAA catabolism [short-chain acylcarnitines (ACs)], and their associations with IR. Healthy obese [n = 80, means ± SD; body mass index (BMI): 36.9 ± 5.7 kg/m2] pregnant women were randomized into an exercise (n = 40, aerobic/resistance 3×/wk, ∼13th gestation week until birth) or a nonexercise control (n = 40) group. Blood was collected at 12.2 ± 0.5 and 36.0 ± 0.4 gestation weeks and analyzed for BCAA-derived acylcarnitine concentrations as markers of BCAA breakdown toward oxidative pathways, and glucose and insulin concentrations [updated homeostatic model assessment of IR (HOMA2-IR)]. After adjusting for HOMA2-IR, there were no interaction effects of group by time. In addition, there was a main positive effect of time on HOMA2-IR (12 wk: 2.3 ± 0.2, 36 wk: 3.0 ± 0.2, P = 0.003). A moderate-intensity exercise intervention during pregnancy in women with obesity was not associated with changes in BCAA-derived ACs versus standard of care. The decrease in BCAA-derived ACs throughout gestation could not be explained by IR.NEW & NOTEWORTHY This research showed an increase in insulin resistance (IR) and decrease in branched-chain amino acid catabolism throughout gestation in women with obesity, and addition of a moderate exercise intervention (known to attenuate IR in nonpregnant populations) did not alter these shifts. Findings provide support for metabolic safety of exercise during pregnancy.

Circulating miRNA Signatures Associated with Insulin Resistance in Adolescents with Obesity.

PURPOSE: MicroRNAs (miRNAs) are implicated in metabolic changes accompanying progression of obesity, insulin resistance (IR), and metabolic disorders in children. Identifying circulating miRNAs that uniquely associate with these disorders may be useful in early identification and prevention of obesity-related complications. We aimed to identify circulating miRNA signatures that distinguish adolescents with obesity and IR from those with obesity unaccompanied by IR. METHODS: Adolescents (aged 10-17 years) with obesity were recruited from a weight management clinic. Fasting serum samples were obtained from 33 participants. A total of 179 miRNAs were queried by a quantitative RT-PCR-based miRNA focus panel. Differentially expressed miRNAs were compared between groups using Student's t-test or one-way ANOVA analysis, and the association between IR evaluated by homeostatic model assessment model (HOMA-IR > 4) and body mass index (BMI) status was assessed using Pearson's correlation analysis. RESULTS: We found an expression pattern consisting of 12 elevated miRNAs linked to IR in obese adolescents. miR-30d, -221, and -122 were significantly correlated with clinical and biochemical markers of obesity and IR, suggestive of IR in adolescents at risk. CONCLUSION: Specific signatures of circulating miRNAs reflected metabolic phenotypes and predicted the presence of IR in adolescents with obesity, suggesting that miRNA indicators may identify obesity-associated complications in childhood. Further studies will be needed to understand cause versus effect and the mechanisms by which IR status links to changes in blood miRNA profiles.

Early Infant Formula Feeding Impacts Urinary Metabolite Profile at 3 Months of Age.

There is a growing consensus that nutritional programming may persist and influence risk for several chronic diseases in adulthood. In the present study, we used urinary metabolic analysis in assessing diet effects on early-life metabolism. Urine samples from healthy three-month-old infants fed human milk (HM; n = 93), cow's milk-based infant formula [MF; n = 80], or soy protein-based infant formula (SF; n = 76) were analyzed with an untargeted metabolomics approach using GC-TOF MS. PLS-DA and ANOVA analyses were performed using MetaboAnalyst (v4.0). A total of 150 metabolites differed significantly among the feeding groups, including dietary-specific patterns of urinary metabolites of sugars, sugar alcohols, amino acids, and polyphenols. Urinary metabolites may mirror the infant's overall metabolism and serve as a noninvasive tool to examine the neonatal effects of diet on early-infant metabolism.

Fibroblast Growth Factor-21 to Adiponectin Ratio: A Potential Biomarker to Monitor Liver Fat in Children With Obesity.

Background: There is a pressing need for effective and non-invasive biomarkers to track intrahepatic triglyceride (IHTG) in children at-risk for non-alcoholic fatty liver disease (NAFLD), as standard-of-care reference tools, liver biopsy and magnetic resonance imaging (MRI), are impractical to monitor the course disease. Objective: We aimed to examine the association between serum fibroblast growth factor (FGF)-21 to adiponectin ratio (FAR) and IHTG as assessed by MRI in children with obesity. Methods: Serum FGF21 and adiponectin levels and IHTG were measured at two time points (baseline, 6 months) in obese children enrolled in a clinical weight loss program. The association between percent change in FAR and IHTG at final visit was examined using a multiple linear regression model. Results: At baseline, FAR was higher in the subjects with NAFLD (n = 23, 35.8 ± 41.9 pg/ng) than without NAFLD (n = 35, 19.8 ± 13.7 pg/ng; p = 0.042). Forty-eight subjects completed both visits and were divided into IHTG loss (≥1% reduction than baseline), no change (within ±1% change), and gain (≥1% increase than baseline) groups. At 6 months, the percent change in FAR was different among the three groups (p = 0.005). Multiple linear regression showed a positive relationship between percent change in FAR and the final liver fat percent in sex and pubertal stage-similar subjects with NAFLD at baseline (slope coefficient 6.18, 95% CI 1.90-10.47, P = 0.007), but not in those without NAFLD. Conclusions: Higher value in percent increase in FAR is positively associated with higher level of IHTG percent value at 6 months in children with baseline NAFLD. FAR could be a potential biomarker to monitor the changes in IHTG in children with NAFLD.

Divergence in aerobic capacity impacts bile acid metabolism in young women.

Liver adaptations may be critical for regular exercise and high aerobic capacity to protect against metabolic disease, but mechanisms remain unknown. Bile acids (BAs) synthesized in the liver are bioactive and can putatively modify energy metabolism. Regular exercise influences BA metabolism in rodents, but effects in humans are unknown. This study tested whether female subjects screened for high aerobic capacity (Hi-Fit, n = 19) [peak oxygen consumption (V̇o2peak) ≥45 mL·kg-1·min-1] have increased hepatic BA synthesis and different circulating BA composition compared with those matched for age and body mass with low aerobic capacity (Lo-Fit, n = 19) (V̇o2peak ≤35 mL·kg-1·min-1). Diet patterns, activity level, stool, and blood were collected at baseline before participants received a 1-wk standardized, eucaloric diet. After the 1-wk standardized diet, stool and blood were again collected and an oral glucose tolerance test (OGTT) was performed to assess insulin sensitivity and postprandial BA response. Contrary to our hypothesis, serum 7α-hydroxy-4-cholesten-3-one (C4), a surrogate of BA synthesis, was not different between groups, whereas Hi-Fit women had lower fecal BA concentrations compared with Lo-Fit women. However, Lo-Fit women had a higher and more sustained rise in circulating conjugated BAs during the OGTT. Hi-Fit women showed a significant post-OGTT elevation of the secondary BA, lithocholic acid (a potent TGR5 agonist), in contrast to Lo-Fit women where no response was observed. A 1-wk control diet eliminated most differences in circulating BA species between groups. Overall, the results emphasize the importance of using a standardized diet when evaluating BAs and indicate that regular exercise and aerobic capacity modulate BA metabolism under postprandial conditions.NEW & NOTEWORTHY Women with contrasting exercise and aerobic capacity levels show clear differences in bile acid (BA) metabolism. Women with low aerobic capacity (Lo-Fit) have increased circulating conjugated BAs post oral glucose tolerance test (OGTT), whereas women with high aerobic capacity (Hi-Fit) display a transient increase. Hi-Fit women show an increase in the secondary BA, lithocholic acid, during the OGTT not seen in Lo-Fit women. Differences in circulating BA species between Hi- and Lo-Fit women possibly contribute to differences in insulin sensitivity and energy regulation via different signaling mechanisms.

MicroRNA profiles were altered in neonatal piglet mammary glands following postnatal infant formula feeding.

Postnatal dietary modulation of microRNAs (miRNAs) and effects on miRNA-mRNA interactions in tissues remain unknown. This study aimed to investigate whether dietary factors (formula vs. breastfeeding) affect mammary miRNA expression and to determine if these changes are concurrent with developmental alterations of the mammary gland in neonatal piglets. Female Yorkshire/Duroc piglets were fed sow's milk or cow's milk- or soy-based infant formula (from postnatal day 2 to day 21; n=6/group). Differentially expressed miRNAs were determined using mammary miRNA profiling, followed by miRNA and mRNA expressions characterized by quantitative reverse-transcription polymerase chain reaction. Milk and soy formulas reduced expressions of miR-1, -128, -133a, -193b, -206 and -27a; miRNA down-regulation altered mRNA expressions of genes (e.g., Ccnd1, Tgfb3, Igf1r and Tbx3) that were consistent with enhanced cell proliferation and suppressed apoptotic processes in the developing mammary gland. Interestingly, down-regulation of miR-1, -128 and -27a also correlated with increased mRNA genes such as Hmgcs and Hmgcr encoding cholesterol synthesis in the mammary glands in response to lower circulating cholesterol levels. Infant formula feeding affected mammary miRNA profiles in neonatal piglets, concurrent with increased expression of cell proliferation and cholesterol synthesis genes, suggesting early nutritional modulation of miRNAs may contribute to regulation of proliferative status and cholesterol homeostasis of developing mammary glands during infancy.

Xenometabolite signatures in the UC Davis type 2 diabetes mellitus rat model revealed using a metabolomics platform enriched with microbe-derived metabolites.

The gut microbiome has the potential to create or modify xenometabolites (i.e., nonhost-derived metabolites) through de novo synthesis or modification of exogenous and endogenous compounds. While there are isolated examples of xenometabolites influencing host health and disease, wide-scale characterization of these metabolites remains limited. We developed a metabolomics platform ("XenoScan") using liquid chromatography-mass spectrometry to characterize a range of known and suspected xenometabolites and their derivatives. This assay currently applies authentic standards for 190 molecules, enriched for metabolites of microbial origin. As a proof-of-principle, we characterized the cecal content xenometabolomics profile in adult male lean Sprague-Dawley (LSD) and University of California, Davis type 2 diabetes mellitus (UCD-T2DM) rats at different stages of diabetes. These results were correlated to specific bacterial species generated via shotgun metagenomic sequencing. UCD-T2DM rats had a unique xenometabolite profile compared with LSD rats, regardless of diabetes status, suggesting that at least some of the variation is associated with host genetics. Furthermore, modeling approaches revealed that several xenometabolites discriminated UCD-T2DM rats at early stages of diabetes versus those at 3 mo postdiabetes onset. Several xenometabolite hubs correlated with specific bacterial species in both LSD and UCD-T2DM rats. For example, indole-3-propionic acid negatively correlated with species within the Oscillibacter genus in UCD-T2DM rats considered to be prediabetic or recently diagnosed diabetic, in contrast to gluconic acid and trimethylamine, which were positively correlated with Oscillibacter species. The application of a xenometabolite-enriched metabolomics assay in relevant milieus will enable rapid identification of a wide variety of gut-derived metabolites, their derivatives, and their potential biochemical origins of xenometabolites in relationship to host gastrointestinal microbial ecology.NEW & NOTEWORTHY We debut a liquid chromatography-mass spectrometry (LC/MS) platform called the XenoScan, which is a metabolomics platform for xenometabolites (nonself-originating metabolites). This assay has 190 in-house standards with the majority enriched for microbe-derived metabolites. As a proof-of-principle, we used the XenoScan to discriminate genetic differences from cecal samples associated with different rat lineages, in addition to characterizing diabetes progression in rat model of type 2 diabetes. Complementing microbial sequencing data with xenometabolites uncovered novel microbial metabolism in targeted organisms.

Net release and uptake of xenometabolites across intestinal, hepatic, muscle, and renal tissue beds in healthy conscious pigs.

Xenometabolites from microbial and plant sources are thought to confer beneficial as well as deleterious effects on host physiology. Studies determining absorption and tissue uptake of xenometabolites are limited. We utilized a conscious catheterized pig model to evaluate interorgan flux of annotated known and suspected xenometabolites, derivatives, and bile acids. Female pigs (n = 12, 2-3 mo old, 25.6 ± 2.2 kg) had surgically implanted catheters across portal-drained viscera (PDV), splanchnic compartment (SPL), liver, kidney, and hindquarter muscle. Overnight-fasted arterial and venous plasma was collected simultaneously in a conscious state and stored at -80°C. Thawed samples were analyzed by liquid chromatography-mass spectrometry. Plasma flow was determined with para-aminohippuric acid dilution technology and used to calculate net organ balance for each metabolite. Significant organ uptake or release was determined if net balance differed from zero. A total of 48 metabolites were identified in plasma, and 31 of these had at least one tissue with a significant net release or uptake. All bile acids, indole-3-acetic acid, indole-3-arylic acid, and hydrocinnamic acid were released from the intestine and taken up by the liver. Indole-3-carboxaldehyde, p-cresol glucuronide, 4-hydroxyphenyllactic acid, dodecanendioic acid, and phenylacetylglycine were also released from the intestines. Liver or kidney uptake was noted for indole-3-acetylglycine, p-cresol glucuronide, atrolactic acid, and dodecanedioic acid. Indole-3-carboxaldehyde, atrolactic acid, and dodecanedioic acids showed net release from skeletal muscle. The results confirm gastrointestinal origins for several known xenometabolites in an in vivo overnight-fasted conscious pig model, whereas nongut net release of other putative xenometabolites suggests a more complex metabolism.NEW & NOTEWORTHY Xenometabolites from microbe origins influence host health and disease, but absorption and tissue uptake of these metabolites remain speculative. Results herein are the first to demonstrate in vivo organ uptake and release of these metabolites. We used a conscious catheterized pig model to confirm gastrointestinal origins for several xenometabolites (e.g., indolic compounds, 4-hydroxyphenyllactic acid, dodecanendioic acid, and phenylacetylgycine). Liver and kidney were major sites for xenometabolite uptake, likely highlighting liver conjugation metabolism and renal excretion.

Potential role of gut microbiota, the proto-oncogene PIKE (Agap2) and cytochrome P450 CYP2W1 in promotion of liver cancer by alcoholic and nonalcoholic fatty liver disease and protection by dietary soy protein.

We have previously demonstrated promotion of diethylnitrosamine (DEN) initiated liver tumorigenesis after feeding diets high in fat or ethanol (EtOH) to male mice. This was accompanied by hepatic induction of the proto-oncogene PIKE (Agap2). Switch of dietary protein from casein to soy protein isolate (SPI) significantly reduced tumor formation in these models. We have linked EtOH consumption in mice to microbial dysbiosis. Adoptive transfer studies demonstrate that microbiota from mice fed ethanol can induce hepatic steatosis in the absence of ethanol suggesting that microbiota or the microbial metabolome play key roles in development of fatty liver disease. Feeding SPI significantly changed gut bacteria in mice increasing alpha diversity (P < 0.05) and levels of Clostidiales spp. Feeding soy formula to piglets also resulted in significant changes in microbiota, the pattern of bile acid metabolites and in inhibition of the intestinal-hepatic FXR/FGF19-SHP pathway which has been linked to both steatosis and hepatocyte proliferation. Moreover, feeding SPI also resulted in induction of hepatic PPARα signaling and inhibition of PIKE mRNA expression coincident with inhibition of steatosis and cancer prevention. Feeding studies in the DEN model with differing dietary fats demonstrated tumor promotion specific to the saturated fat, cocoa butter relative to diets containing olive oil or corn oil associated with microbial dysbiosis including dramatic increases in Lachnospiraceae particularly from the genus Coprococcus. Immunohistochemical analysis demonstrated that tumors from EtOH-fed mice and patients with alcohol-associated HCC also expressed high levels of a novel cytochrome P450 enzyme CYP2W1. Additional adoptive transfer experiments and studies in knockout mice are required to determine the exact relationship between soy effects on the microbiota, expression of PIKE, CYP2W1, PPARα activation and prevention of tumorigenesis.

Neonatal diet alters fecal microbiota and metabolome profiles at different ages in infants fed breast milk or formula.

BACKGROUND: Neonatal diet has a large influence on child health and might modulate changes in fecal microbiota and metabolites. OBJECTIVES: The aim is to investigate fecal microbiota and metabolites at different ages in infants who were breastfed (BF), received dairy-based milk formula (MF), or received soy-based formula (SF). METHODS: Fecal samples were collected at 3 (n = 16, 12, and 14, respectively), 6 (n = 20, 19, and 15, respectively), 9 (n = 12, 11, and 12, respectively), and 12 mo (n = 14, 14, and 15, respectively) for BF, MF, and SF infants. Infants that breastfed until 9 mo and switched to formula were considered as no longer breastfeeding at 12 mo. Microbiota data were obtained using 16S ribosomal RNA sequencing. Untargeted metabolomics was conducted using a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. The data were analyzed using R (version 3.6.0) within the RStudio (version 1.1.463) platform. RESULTS: At 3, 6, and 9 mo of age BF infants had the lowest α-diversity, SF infants had the highest diversity, and MF was intermediate. Bifidobacterium was 2.6- to 5-fold lower in SF relative to BF infants through 1 y of life. An unidentified genus from Ruminococcaceae higher in the SF (2%) than in the MF (0.4%) and BF (0.08%) infants at 3 mo of age was observed. In BF infants higher levels of butyric acid, d-sphingosine, kynurenic acid, indole-3-lactic acid, indole-3-acetic acid, and betaine were observed than in MF and SF infants. At 3 mo Ruminococcaceae was positively correlated to azelaic, gentisic, isocitric, sebacic, and syringic acids. At 6 mo Oscillospira was negatively correlated with 3-hydroxybutyric-acid, hydroxy-hydrocinnamic acid, and betaine whereas Bifidobacterium was negatively associated with 5-hydroxytryptamine. At 12 mo of age, Lachnospiraceae was negatively associated with hydroxyphenyllactic acid. CONCLUSIONS: Infant diet has a large impact on the fecal microbiome and metabolome in the first year of life.This study was registered at clinicaltrials.gov as NCT00616395.

Infant Formula Feeding Changes the Proliferative Status in Piglet Neonatal Mammary Glands Independently of Estrogen Signaling.

BACKGROUND: Soy infant formula contains isoflavones, which are able to bind to and activate estrogen receptor (ER) pathways. The mammary gland is sensitive to estrogens, raising concern that the use of soy formulas may promote premature development. OBJECTIVE: We aimed to determine if soy formula feeding increases mammary gland proliferation and differentiation in comparison to other infant postnatal diets. METHODS: White-Dutch Landrace piglets aged 2 d received either sow milk (Sow), or were provided milk formula (Milk), soy formula (Soy), milk formula supplemented with 17-beta-estradiol (2 mg/(kg·d); M + E2), or milk formula supplemented with genistein (84 mg/L of diet; M + G) until day 21. Mammary gland proliferation and differentiation was assessed by histology, and real-time RT-PCR confirmation of differentially expressed genes identified by microarray analysis. RESULTS: Mammary terminal end bud numbers were 19-31% greater in the Milk, Soy, and M + G groups relative to the Sow and M + E2, P <0.05. Microarray analysis identified differentially expressed genes between each formula-fed group relative to the Sow (±1.7-fold, P <0.05). Real-time RT-PCR confirmed 2- to 4-fold increases in mRNA transcripts of genes involved in cell proliferation, insulin-like growth factor 1 (IGF1), fibroblast growth factor 10 (FGF10), and fibroblast growth factor 18 (FGF18), in all groups relative to the Sow, P <0.05. In contrast, genes involved in cell differentiation and ductal morphogenesis, angiotensin II receptor type 2 (AGTR2), microtubule associated protein 1b (MAP1B), and kinesin family member 26b (KIF26B), were significantly upregulated by 2-, 4-, and 13-fold, respectively, in the M + E2 group. Additionally, mRNA expression of ER-specific gene targets, progesterone receptor (PGR), was increased by 12-fold, and amphiregulin (AREG) and Ras-like estrogen regulated growth inhibitor (RERG) expression by 1.5-fold in the M + E2 group, P <0.05. In the soy and M + G groups, mRNA expressions of fatty acid synthesis genes were increased 2- to 4-fold. CONCLUSIONS: Our data indicate soy formula feeding does not promote ER-signaling in the piglet mammary gland. Infant formula feeding (milk- or soy-based) may initiate proliferative pathways independently of estrogenic signaling.

Obesity leads to distinct metabolomic signatures in follicular fluid of women undergoing in vitro fertilization.

Although obesity negatively influences the metabolic homeostasis of cells within a broad range of tissues, its impact on oocyte metabolism is not fully understood. Prior evidence suggests that obesity increases expression of oocyte genes associated with inflammation, oxidative stress, and lipid metabolism; however, the metabolic impact of these genetic differences is not known. To address this gap, we conducted an exploratory assessment of the follicular fluid (FF) metabolome in eight overweight/obese (OW) and nine normal-weight (NW) women undergoing in vitro fertilization. FF and serum were collected and analyzed by untargeted metabolomics using gas chromatography-quadrupole time-of-flight mass spectrometry and charged-surface hybrid column-electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Untargeted metabolomics identified obesity-associated changes in FF metabolites related to oxidative stress/antioxidant capacity, xenometabolism/amino acid biosynthesis, and lipid metabolism. Discriminant FF metabolites included elevated uric acid, isothreonic acid, one unknown primary metabolite, and six unknown complex lipids in OW compared with NW women. Conversely, 2-ketoglucose dimethylacetal, aminomalonate, two unknown primary metabolites, and two unknown complex lipids were decreased in FF of OW relative to NW women. Indole-3-propionic acid (IPA), a bacteria-derived metabolite, was also decreased in both FF and serum of OW women ( P < 0.05). The significant correlation between antioxidant IPA in serum and FF ( R = 0.95, P < 0.0001) suggests a potential serum biomarker of FF antioxidant status or reflection of the gut metabolism interaction with the follicle. These results suggest that obesity has important consequences for the follicular environment during the preconception period, a window of time that may be important for lifestyle interventions to ameliorate obesity-associated risk factors.

Concentrations of Purine Metabolites Are Elevated in Fluids from Adults and Infants and in Livers from Mice Fed Diets Depleted of Bovine Milk Exosomes and their RNA Cargos.

Background: Humans and mice absorb bovine milk exosomes and their RNA cargos. Objectives: The objectives of this study were to determine whether milk exosome- and RNA-depleted (ERD) and exosome- and RNA-sufficient (ERS) diets alter the concentrations of purine metabolites in mouse livers, and to determine whether diets depleted of bovine milk alter the plasma concentration and urine excretion of purine metabolites in adults and infants, respectively. Methods: C57BL/6 mice were fed ERD (providing 2% of the microRNA cargos compared with ERS) and ERS diets starting at age 3 wk; livers were collected at age 7 wk. Plasma and 24-h urine samples were collected from healthy adults who consumed (DCs) or avoided (DAs) dairy products. Spot urine samples were collected from healthy infants fed human milk (HM), milk formula (MF), or soy formula (SF) at age 3 mo. Purine metabolites were analyzed in liver, plasma, and urine; mRNAs and microRNAs were analyzed in the livers of female mice. Results: We found that 9 hepatic purine metabolites in ERD-fed mice were 1.76 ± 0.43 times the concentrations in ERS-fed mice (P < 0.05). Plasma concentrations and urine excretion of purine metabolites in DAs was ≤1.62 ± 0.45 times the concentrations in DCs (P < 0.05). The excretion of 13 purine metabolites in urine from SF infants was ≤175 ± 39 times the excretion in HM and MF infants (P < 0.05). mRNA expression of 5'-nucleotidase, cytosolic IIIB, and adenosine deaminase in mice fed ERD was 0.64 ± 0.52 and 0.60 ± 0.28 times the expression in mice fed ERS, respectively. Conclusion: Diets depleted of bovine-milk exosomes and RNA cargos caused increases in hepatic purine metabolites in mice, and in plasma and urine from human adults and infants, compared with exosome-sufficient controls. These findings are important, because purines play a role in intermediary metabolism and cell signaling.

Modulating Sterol Concentrations in Infant Formula Influences Cholesterol Absorption and Synthesis in the Neonatal Piglet.

Formula-fed infants present higher cholesterol synthesis rates and lower circulating cholesterol during the postnatal feeding period compared to breast-fed infants, though the mechanisms underlying this phenotype are not fully understood. Typical infant formulas contain vegetable-based fats, inherently including phytosterols (PS), which are structurally similar to cholesterol and may interfere with their absorption. A seven-day old piglets model was used to test the inhibitory effects of PS on cholesterol absorption during postnatal feeding. Following feeding for 21 days with milk-based formulas containing PS and cholesterol levels resembling those in formulas or human-milk, apparent cholesterol digestibility was analyzed in ileal digesta, and cholesterol, PS, and cholesterol synthesis markers were analyzed in plasma and liver samples. Ileal cholesterol digestibility content was increased in the piglets fed low PS formulas and the rate of the hepatic cholesterol synthesis, as determined by the lathosterol-to-cholesterol ratios (L:C), was decreased in the piglets fed LP-formulas and corresponded to reduced nuclear expression of SREBP2 relative to those fed HP-formulas. These results are consistent with the hypothesis that PS in formula can inhibit cholesterol absorption and enhance cholesterol synthesis. Whether or not this leads to entrainment of cholesterol synthesis later in life via early programming awaits further research.

Maternal High-Fat Diet Programs Offspring Liver Steatosis in a Sexually Dimorphic Manner in Association with Changes in Gut Microbial Ecology in Mice.

The contributions of maternal diet and obesity in shaping offspring microbiome remain unclear. Here we employed a mouse model of maternal diet-induced obesity via high-fat diet feeding (HFD, 45% fat calories) for 12 wk prior to conception on offspring gut microbial ecology. Male and female offspring were provided access to control or HFD from weaning until 17 wk of age. Maternal HFD-associated programming was sexually dimorphic, with male offspring from HFD dams showing hyper-responsive weight gain to postnatal HFD. Likewise, microbiome analysis of offspring cecal contents showed differences in α-diversity, ß-diversity and higher Firmicutes in male compared to female mice. Weight gain in offspring was significantly associated with abundance of Lachnospiraceae and Clostridiaceae families and Adlercreutzia, Coprococcus and Lactococcus genera. Sex differences in metagenomic pathways relating to lipid metabolism, bile acid biosynthesis and immune response were also observed. HFD-fed male offspring from HFD dams also showed worse hepatic pathology, increased pro-inflammatory cytokines, altered expression of bile acid regulators (Cyp7a1, Cyp8b1 and Cyp39a1) and serum bile acid concentrations. These findings suggest that maternal HFD alters gut microbiota composition and weight gain of offspring in a sexually dimorphic manner, coincident with fatty liver and a pro-inflammatory state in male offspring.