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.

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.

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.

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.

Diabetes-associated alterations in the cecal microbiome and metabolome are independent of diet or environment in the UC Davis Type 2 Diabetes Mellitus Rat model.

The composition of the gut microbiome is altered in obesity and type 2 diabetes; however, it is not known whether these alterations are mediated by dietary factors or related to declines in metabolic health. To address this, cecal contents were collected from age-matched, chow-fed male University of California, Davis Type 2 Diabetes Mellitus (UCD-T2DM) rats before the onset of diabetes (prediabetic PD; n = 15), 2 wk recently diabetic (RD; n = 10), 3 mo (D3M; n = 11), and 6 mo (D6M; n = 8) postonset of diabetes. Bacterial species and functional gene counts were assessed by shotgun metagenomic sequencing of bacterial DNA in cecal contents, while metabolites were identified by gas chromatography-quadrupole time-off-flight-mass spectrometry. Metagenomic analysis showed a shift from Firmicutes species in early stages of diabetes (PD + RD) toward an enrichment of Bacteroidetes species in later stages of diabetes (D3M + D6M). In total, 45 bacterial species discriminated early and late stages of diabetes with 25 of these belonging to either Bacteroides or Prevotella genera. Furthermore, 61 bacterial gene clusters discriminated early and later stages of diabetes with elevations of enzymes related to stress response (e.g., glutathione and glutaredoxin) and amino acid, carbohydrate, and bacterial cell wall metabolism. Twenty-five cecal metabolites discriminated early vs. late stages of diabetes, with the largest differences observed in abundances of dehydroabietic acid and phosphate. Alterations in the gut microbiota and cecal metabolome track diabetes progression in UCD-T2DM rats when controlling for diet, age, and housing environment. Results suggest that diabetes-specific host signals impact the ecology and end product metabolites of the gut microbiome when diet is held constant.

NOX4 Deletion in Male Mice Exacerbates the Effect of Ethanol on Trabecular Bone and Osteoblastogenesis.

Chronic alcohol consumption increases bone resorption and decreases bone formation. A major component of ethanol (EtOH) pathology in bone is the generation of excess reactive oxygen species (ROS). The ROS-generating NADPH oxidase-4 (NOX4) is proposed to drive much of the EtOH-induced suppression of bone formation. Here, 13-week-old male wild-type (WT) and NOX4-/- mice were pair fed (PF) a high-fat (35%), Lieber-DeCarli liquid diet with or without EtOH at 30% of their total calories for 12 weeks. Micro-computed tomography analysis demonstrated significant decreases in trabecular bone volume/total volume (BV/TV) percentage and cortical thickness in WT, EtOH-fed mice compared with PF controls. EtOH-fed NOX4-/- mice also displayed decreased trabecular BV/TV and trabecular number compared with PF (P < 0.05). However, NOX4-/- mice were protected against EtOH-induced decreases in cortical thickness (P < 0.05) and decreases in collagen1 and osteocalcin mRNA expression in cortical bone (P < 0.05). In WT and NOX4-/- vertebral bone, EtOH suppressed expression of Wnt signaling components that promote osteoblast maturation. A role for NOX4 in EtOH inhibition of osteoblast differentiation was further demonstrated by protection against EtOH inhibition of osteoblastogenesis in ex vivo bone marrow cultures from NOX4-/-, but not p47phox-/- mice lacking active NADPH oxidase-2. However, bone marrow cultures from NOX4-/- mice formed fewer osteoblastic colonies compared with WT cultures (P < 0.05), suggesting a role for NOX4 in the maintenance of mesenchymal progenitor cell populations. These data suggest that NOX4 deletion is partially protective against EtOH effects on osteoblast differentiation, but may predispose bone to osteogenic impairments.

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.

Infant Formula Feeding Increases Hepatic Cholesterol 7α Hydroxylase (CYP7A1) Expression and Fecal Bile Acid Loss in Neonatal Piglets.

Background: During the postnatal feeding period, formula-fed infants have higher cholesterol synthesis rates and lower circulating cholesterol concentrations than their breastfed counterparts. Although this disparity has been attributed to the uniformly low dietary cholesterol content of typical infant formulas, little is known of the underlying mechanisms associated with this altered cholesterol metabolism phenotype. Objective: We aimed to determine the molecular etiology of diet-associated changes in early-life cholesterol metabolism with the use of a postnatal piglet feeding model. Methods: Two-day-old male and female White-Dutch Landrace piglets were fed either sow milk (Sow group) or dairy-based (Milk group; Similac Advance powder) or soy-based (Soy group; Emfamil Prosobee Lipil powder) infant formulas until day 21. In addition to measuring serum cholesterol concentrations, hepatic and intestinal genes involved in enterohepatic circulation of cholesterol and bile acids were analyzed by real-time reverse-transcriptase polymerase chain reaction and Western blot. Bile acid concentrations were measured by liquid chromatography-mass spectrometry in serum, liver, and feces. Results: Compared with the Sow group, hepatic cholesterol 7α hydroxylase (CYP7A1) protein expression was 3-fold higher in the Milk group (P < 0.05) and expression was 10-fold higher in the Soy group compared with the Milk group (P < 0.05). Likewise, fecal bile acid concentrations were 3-fold higher in the Soy group compared with the Milk group (P < 0.05). Intestinal mRNA expression of fibroblast factor 19 (Fgf19) was reduced in the Milk and Soy groups, corresponding to 54% and 67% decreases compared with the Sow group. In the Soy group, small heterodimer protein (SHP) protein expression was 30% lower compared with the Sow group (P < 0.05). Conclusions: These results indicate that formula feeding leads to increased CYP7A1 protein expression and fecal bile acid loss in neonatal piglets, and this outcome is linked to reduced efficacy in inhibiting CYP7A1 expression through FGF19 and SHP transcriptional repression mechanisms.

Knockout of the Gsta4 Gene in Male Mice Leads to an Altered Pattern of Hepatic Protein Carbonylation and Enhanced Inflammation Following Chronic Consumption of an Ethanol Diet.

BACKGROUND: Glutathione S-transferase A4-4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE). In this study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease by examining the development of liver injury in male wild-type (WT) SV/J mice and SV/J mice lacking functional GSTA4 (GSTA4-/- mice). METHODS: Adult male WT and GSTA4-/- mice were fed chow (N = 10 to 12) or high-fat Lieber-DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18 to 20) for 116 days. At the end of the study, half of the EtOH-fed mice were acutely challenged with an EtOH binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4-HNE adduction and by comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) of purified carbonylated proteins. RESULTS: Chronic EtOH intake significantly increased hepatic 4-HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum alanine aminotransferase. Hepatic infiltration with B cells, T cells, and neutrophils and mRNA expression of pro-inflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)γ was modest in WT mice. However, an EtOH binge increased hepatic necrosis, hepatic cell proliferation, and expression of TNFα mRNA (p < 0.05). EtOH treatment of GSTA4-/- mice increased B-cell infiltration and increased mRNA expression of TNFα and IFNγ and of matrix remodeling markers MMP9, MMP13, and Col1A1 (p < 0.05). GSTA4-/- mice exhibited panlobular rather than periportal distribution of 4-HNE-adducted proteins and increased overall 4-HNE staining after EtOH binge. Comprehensive LC-MS of carbonylated proteins identified 1,022 proteins of which 189 were unique to the GSTA4-/- group. CONCLUSIONS: These data suggest long-term adaptation to EtOH in WT mice does not occur in GSTA4-/- mice. Products of lipid peroxidation appear to play a role in inflammatory responses due to EtOH. And EtOH effects on B-cell infiltration and autoimmune responses may be secondary to formation of carbonyl adducts.

Early Postnatal Diets Affect the Bioregional Small Intestine Microbiome and Ileal Metabolome in Neonatal Pigs.

Background: Breastfeeding is known to be protective against gastrointestinal disorders and may modify gut development. Although the gut microbiome has been implicated, little is known about how early diet affects the small intestine microbiome.Objective: We hypothesized that disparate early diets would promote unique microbial profiles in the small intestines of neonatal pigs.Methods: Male and female 2-d-old White Dutch Landrace pigs were either sow fed or provided dairy (Similac Advance powder; Ross Products Abbott Laboratories) or soy (Enfamil Prosobee Lipil powder; Mead Johnson Nutritionals) infant formulas until day 21. Bacterial ecology was assessed in the contents of the small intestine through the use of 16S ribosomal RNA sequencing. α-Diversity, ß-diversity, and differential abundances of operational taxonomic units were assessed by ANOVA, permutational ANOVA, and negative binomial regression, respectively. Ileum tissue metabolomics were measured by LC-mass spectrometry and assessed by weighted correlation network analysis.Results: Greater α-diversity was observed in the duodena of sow-fed compared with formula-fed neonatal pigs (P < 0.05). No differences were observed in the ilea. Firmicutes represented the most abundant phylum across all diets in duodena (78.8%, 80.1%, and 53.4% relative abundance in sow, dairy, and soy groups, respectively), followed by Proteobacteria in sow (12.2%) and dairy (12.4%) groups and Cyanobacteria in soy-fed (36.2%) pigs. In contrast to those in the duodenum, Proteobacteria was the dominant phylum in the ileum, with >60% relative abundance in all of the groups. In the duodenum, 77 genera were altered by diet, followed by 48 in the jejunum and 19 in the ileum. Metabolomics analyses revealed associations between ileum tissue metabolites (e.g., acylcarnitines, 3-aminoisobutyric acid) and diet-responsive microbial genera.Conclusions: These results indicate that the neonatal diet has regional effects on the small intestine microbiome in pigs, with the most pronounced effects occurring in the duodena. Regional effects may be important factors when considering gut tissue metabolism and development in the postnatal period.

Partial Protection by Dietary Antioxidants Against Ethanol-Induced Osteopenia and Changes in Bone Morphology in Female Mice.

BACKGROUND: Chronic alcohol consumption leads to increased fracture risk and an elevated risk of osteoporosis by decreasing bone accrual through increasing osteoclast activity and decreasing osteoblast activity. We have shown that this mechanism involves the generation of reactive oxygen species (ROS) produced by NADPH oxidases. It was hypothesized that different dietary antioxidants, N-acetyl cysteine (NAC; 1.2 mg/kg/d), and α-tocopherol (Vit.E; 60 mg/kg/d) would be able to attenuate the NADPH oxidase-mediated ROS effects on bone due to chronic alcohol intake. METHODS: To study the effects of these antioxidants, female mice received a Lieber-DeCarli liquid diet containing ethanol (EtOH) with or without additional antioxidant for 8 weeks. RESULTS: Tibias displayed decreased cortical bone mineral density in both the EtOH and EtOH + antioxidant groups compared to pair-fed (PF) and PF + antioxidant groups (p < 0.05). However, there was significant protection from trabecular bone loss in mice fed either antioxidant (p < 0.05). Microcomputed tomography analysis demonstrated a significant decrease in bone volume (bone volume/tissue volume) and trabecular number (p < 0.05), along with a significant increase in trabecular separation in the EtOH compared to PF (p < 0.05). In contrast, the EtOH + NAC and EtOH + Vit.E did not statistically differ from their respective PF controls. Ex vivo histologic sections of tibias were stained for nitrotyrosine, an indicator of intracellular damage by ROS, and tibias from mice fed EtOH exhibited significantly more staining than PF controls. EtOH treatment significantly increased the number of marrow adipocytes per mm as well as mRNA expression of aP2, an adipocyte marker in bone. Only NAC was able to reduce the number of marrow adipocytes to PF levels. EtOH-fed mice exhibited reduced bone length (p < 0.05) and had a reduced number of proliferating chondrocytes within the growth plate. NAC and Vit.E prevented this (p < 0.05). CONCLUSIONS: These data show that alcohol's pathological effects on bone extend beyond decreasing bone mass and suggest a partial protective effect of the dietary antioxidants NAC and Vit.E at these doses with regard to alcohol effects on bone turnover and bone morphology.

p47phox-Nox2-dependent ROS Signaling Inhibits Early Bone Development in Mice but Protects against Skeletal Aging.

Bone remodeling is age-dependently regulated and changes dramatically during the course of development. Progressive accumulation of reactive oxygen species (ROS) has been suspected to be the leading cause of many inflammatory and degenerative diseases, as well as an important factor underlying many effects of aging. In contrast, how reduced ROS signaling regulates inflammation and remodeling in bone remains unknown. Here, we utilized a p47(phox) knock-out mouse model, in which an essential cytosolic co-activator of Nox2 is lost, to characterize bone metabolism at 6 weeks and 2 years of age. Compared with their age-matched wild type controls, loss of Nox2 function in p47(phox-/-) mice resulted in age-related switch of bone mass and strength. Differences in bone mass were associated with increased bone formation in 6-week-old p47(phox-/-) mice but decreased in 2-year-old p47(phox-/-) mice. Despite decreases in ROS generation in bone marrow cells and p47(phox)-Nox2 signaling in osteoblastic cells, 2-year-old p47(phox-/-) mice showed increased senescence-associated secretory phenotype in bone compared with their wild type controls. These in vivo findings were mechanistically recapitulated in ex vivo cell culture of primary fetal calvarial cells from p47(phox-/-) mice. These cells showed accelerated cell senescence pathway accompanied by increased inflammation. These data indicate that the observed age-related switch of bone mass in p47(phox)-deficient mice occurs through an increased inflammatory milieu in bone and that p47(phox)-Nox2-dependent physiological ROS signaling suppresses inflammation in aging.

Reactive Oxygen Species Differentially Regulate Bone Turnover in an Age-Specific Manner in Catalase Transgenic Female Mice.

Chronic ethyl alcohol (EtOH) consumption results in reactive oxygen species (ROS) generation in bone and osteopenia due to increased bone resorption and reduced bone formation. In this study, transgenic C57Bl/6J mice overexpressing human catalase (TgCAT) were used to test whether limiting excess hydrogen peroxide would protect against EtOH-mediated bone loss. Micro-computed tomography analysis of the skeletons of 6-week-old female chow-fed TgCAT mice revealed a high bone mass phenotype with increased cortical bone area and thickness as well as significantly increased trabecular bone volume (P < 0.05). Six-week-old wild-type (WT) and TgCAT female mice were chow fed or pair fed (PF) liquid diets with or without EtOH, approximately 30% of calories, for 8 weeks. Pair feeding of WT had no demonstrable effect on the skeleton; however, EtOH feeding of WT mice significantly reduced cortical and trabecular bone parameters along with bone strength compared with PF controls (P < 0.05). In contrast, EtOH feeding of TgCAT mice had no effect on trabecular bone compared with PF controls. At 14 weeks of age, there was significantly less trabecular bone and cortical cross-sectional area in TgCAT mice than WT mice (P < 0.05), suggesting impaired normal bone accrual with age. TgCAT mice expressed less collagen1α and higher sclerostin mRNA (P < 0.05), suggesting decreased bone formation in TgCAT mice. In conclusion, catalase overexpression resulted in greater bone mass than in WT mice at 6 weeks and lower bone mass at 14 weeks. EtOH feeding induced significant reductions in bone architecture and strength in WT mice, but TgCAT mice were partially protected. These data implicate ROS signaling in the regulation of bone turnover in an age-dependent manner, and indicate that excess hydrogen peroxide generation contributes to alcohol-induced osteopenia.

Increased 4-hydroxynonenal protein adducts in male GSTA4-4/PPAR-α double knockout mice enhance injury during early stages of alcoholic liver disease.

To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male 129/SvJ mice (wild-type, WT) and glutathione S-transferase A4-4-null (GSTA4-/-) mice for 40 days. GSTA4-/- mice were crossed with peroxisome proliferator-activated receptor-α-null mice (PPAR-α-/-), and the effects of EtOH in the resulting double knockout (dKO) mice were compared with the other strains. EtOH increased lipid peroxidation in all except WT mice (P < 0.05). Increased steatosis and mRNA expression of the inflammatory markers CXCL2, tumor necrosis factor-α (TNF-α), and α-smooth muscle actin (α-SMA) were observed in EtOH GSTA4-/- compared with EtOH WT mice (P < 0.05). EtOH PPAR-α-/- mice had increased steatosis, serum alanine aminotransferase (ALT), and hepatic CD3+ T cell populations and elevated mRNA encoding CD14, CXCL2, TNF-α, IL-6, CD138, transforming growth factor-ß, platelet-derived growth factor receptor-ß (PDGFR-ß), matrix metalloproteinase (MMP)-9, MMP-13, α-SMA, and collagen type 1 compared with EtOH WT mice. EtOH-fed dKO mice displayed elevation of periportal hepatic 4-hydroxynonenal adducts and serum antibodies against malondialdehyde adducts compared with EtOH feeding of GSTA4-/-, PPAR-α-/-, and WT mice (P < 0.05). ALT was higher in EtOH dKO mice compared with all other groups (P < 0.001). EtOH-fed dKO mice displayed elevated mRNAs for TNF-α and CD14, histological evidence of fibrosis, and increased PDGFR, MMP-9, and MMP-13 mRNAs compared with the EtOH GSTA4-/- or EtOH PPAR-α-/- genotype (P < 0.05). These findings demonstrate the central role lipid peroxidation plays in mediating progression of alcohol-induced necroinflammatory liver injury, stellate cell activation, matrix remodeling, and fibrosis.

Loss of functional NADPH oxidase 2 protects against alcohol-induced bone resorption in female p47phox-/- mice.

BACKGROUND: In bone, NADPH oxidase (NOX)-derived reactive oxygen species (ROS) superoxide and/or hydrogen peroxide are an important stimulus for osteoclast differentiation and activity. Previously, we have demonstrated that chronic ethanol (EtOH) consumption generates excess NOX-dependent ROS in osteoblasts, which functions to stimulate nuclear factor kappa-ß receptor ligand (RANKL)-RANK signaling, thus increasing osteoclastogenesis and activity. This activity can be blocked by co-administration of EtOH with the pan-NOX inhibitor diphenylene idonium (DPI). METHODS: To test whether EtOH-induced bone loss is dependent on a functional NOX2 enzyme, 6-week-old female C57BL/6J-Ncf1/p47phox(-/-) (p47phox KO) and wild-type (WT) mice were pair-fed EtOH diets for 40 days. Bone loss was assessed by 3-point bending, micro-computed tomography and static histomorphometric analysis. Additionally, ST2 cultured cells were co-treated with EtOH and NOX inhibitors, DPI, gliotoxin, and plumbagin, after which changes in ROS production, and in RANKL and NOX mRNA expression were analyzed. RESULTS: In WT mice, EtOH treatment significantly reduced bone density and mechanical strength, and increased total osteoclast number and activity. In EtOH-treated p47phox KO mice, bone density and mechanical strength were completely preserved. EtOH p47phox KO mice had no changes in osteoclast numbers or activity, and no elevations in serum CTX or RANKL gene expression (p < 0.05). In both WT and p47phox KO mice, EtOH feeding reduced biochemical markers of bone formation (p < 0.05). In vitro EtOH exposure of ST2 cells increased ROS, which was blocked by pretreating with DPI or the NOX2 inhibitor gliotoxin. EtOH-induced RANKL and NOX2 gene expression were inhibited by the NOX4-specific inhibitor plumbagin. CONCLUSIONS: These data suggest that NOX2-derived ROS is necessary for EtOH-induced bone resorption. In osteoblasts, NOX2 and NOX4 appear to work in tandem to increase RANKL expression, whereas EtOH-mediated inhibition of bone formation occurs via a NOX2-independent mechanism.

Female mice lacking p47phox have altered adipose tissue gene expression and are protected against high fat-induced obesity.

The current study was designed to determine if the NADPH-oxidase NOX2 plays a role in development of obesity after high fat feeding. Wild-type (WT) mice and mice lacking the essential cytosolic NOX2 system component p47(phox) (P47KO mice) were fed AIN-93G diets or high-fat diets (HFD) containing 45% fat and 0.5% cholesterol for 13 wk from weaning. Fat mass was increased to a similar degree by HFD in males of both genotypes (P < 0.05). However, female P47KO-HFD mice had no increase in adiposity or adipocyte size relative to female WT-HFD mice. Resistance to HFD-driven obesity in P47KO females was associated with increased expression of hepatic TFAM and UCP-2 mRNA, markers of mitochondrial number and uncoupling, and increased expression of hepatic mitochondrial respiratory complexes and whole body energy expenditure in response to HFD. Microarray analysis revealed significantly lower expression of mRNA encoding genes linked to energy metabolism, adipocyte differentiation (PPARγ), and fatty acid uptake (CD36, lipoprotein lipase), in fat pads from female P47KO-HFD mice compared with WT-HFD females. Moreover, differentiation of preadipocytes ex vivo was suppressed more by 17ß-estradiol in cells from P47KO compared with cells from WT females in conjunction with overexpression of mRNA for Pref-1 (P < 0.05). HFD mice of both sexes were resistant to the development of hyperglycemia and hepatic steatosis (P < 0.05) and had reduced serum triglycerides, leptin, and adiponectin relative to WT-HFD mice (P < 0.05). These data suggest that NOX2 is an important regulator of metabolic homeostasis and diet-induced obesity.

Vitamin D supplementation protects against bone loss associated with chronic alcohol administration in female mice.

Chronic alcohol abuse results in decreased bone mineral density (BMD), which can lead to increased fracture risk. In contrast, low levels of alcohol have been associated with increased BMD in epidemiological studies. Alcohol's toxic skeletal effects have been suggested to involve impaired vitamin D/calcium homeostasis. Therefore, dietary vitamin D supplementation may be beneficial in reducing bone loss associated with chronic alcohol consumption. Six-week-old female C57BL/6J mice were pair-fed ethanol (EtOH)-containing liquid diets (10 or 36% total calories) for 78 days. EtOH exposure at 10% calories had no effects on any measured bone or serum parameter. EtOH consumption at 36% of calories reduced BMD and bone strength (P<0.05), decreased osteoblastogenesis, increased osteoclastogenesis, suppressed 1,25-hydroxyvitamin D3 [1,25(OH)2D3] serum concentrations (P<0.05), and increased apoptosis in bone cells compared with pair-fed controls. In a second study, female mice were pair-fed 30% EtOH diets with or without dietary supplementation with vitamin D3 (cholecalciferol; VitD) for 40 days. VitD supplementation in the EtOH diet protected against cortical bone loss, normalized alcohol-induced hypocalcaemia, and suppressed EtOH-induced expression of receptor of nuclear factor-κB ligand mRNA in bone. In vitro, pretreatment of 1,25(OH)2D3 in osteoblastic cells inhibited EtOH-induced apoptosis. In EtOH/VitD mice circulating 1,25(OH)2D3 was lower compared with mice receiving EtOH alone (P<0.05), suggesting increased sensitivity to feedback control of VitD metabolism in the kidney. These findings suggest dietary VitD supplementation may prevent skeletal toxicity in chronic drinkers by normalizing calcium homeostasis, preventing apoptosis, and suppressing EtOH-induced increases in bone resorption.

Identification of a mammalian mitochondrial porphyrin transporter.

The movement of anionic porphyrins (for example, haem) across intracellular membranes is crucial to many biological processes, but their mitochondrial translocation and coordination with haem biosynthesis is not understood. Transport of porphyrins into isolated mitochondria is energy-dependent, as expected for the movement of anions into a negatively charged environment. ATP-binding cassette transporters actively facilitate the transmembrane movement of substances. We found that the mitochondrial ATP-binding cassette transporter ABCB6 is upregulated (messenger RNA and protein in human and mouse cells) by elevation of cellular porphyrins and postulated that ABCB6 has a function in porphyrin transport. We also predicted that ABCB6 is functionally linked to haem biosynthesis, because its mRNA is found in both human bone marrow and CD71+ early erythroid cells (by database searching), and because our results show that ABCB6 is highly expressed in human fetal liver, and Abcb6 in mouse embryonic liver. Here we demonstrate that ABCB6 is uniquely located in the outer mitochondrial membrane and is required for mitochondrial porphyrin uptake. After ABCB6 is upregulated in response to increased intracellular porphyrin, mitochondrial porphyrin uptake activates de novo porphyrin biosynthesis. This process is blocked when the Abcb6 gene is silenced. Our results challenge previous assumptions about the intracellular movement of porphyrins and the factors controlling haem biosynthesis.

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.