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Phenolic acids, such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA), can be produced from microbiome digestion of polyphenols. Previously it was found that HA and 3-3-PPA facilitate bone formation and suppress bone resorption. However, the mechanism of action by which HA and 3-3-PPA protect bone from degeneration is currently unknown. In this report, we present that HA and 3-3-PPA suppression of bone resorption is able to ameliorate bone loss in an ovariectomy (OVX) osteopenic mouse model though not to the extent of Zoledronic acid (ZA). HA and 3-3-PPA treatments were shown to significantly decrease bone marrow adipocyte-like cell formation and inhibited gene expression of key adipogenesis regulator peroxisome proliferator activated receptor gamma (PPARγ) and lipoprotein lipase (Lpl) in bone from OVX mice. In addition, ChIP experiments showed that the association between PPARγ and Lpl promoter region in preadipocyte-like cells was significantly suppressed following HA or 3-3-PPA treatment. Contrasting HA and 3-3-PPA, ZA significantly increased TRAP activity in the area close to growth plate and significantly suppressed bone cell proliferation. These data suggest that phenolics acids such as HA or 3-3-PPA may prevent bone degeneration after OVX through suppression of inflammatory milieu in the bone., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Osteoclasts derived from hematopoietic stem cells control bone resorption. Identifying novel molecules that can epigenetically regulate osteoclastogenesis is important for developing novel treatments for osteoporosis and other disorders associated with bone deterioration and promoting healthy bone formation. The polycomb group (PcG) protein enhancer of zeste homolog 2 (Ezh2), a histone lysine methyltransferase, is associated with epigenetic regulation of numerous cellular processes, but its involvement in bone cell development and homeostasis is not yet clear. Here, LysM-Cre mice were crossed with Ezh2 flox/flox mice to delete Ezh2 in myeloid cell lineage mature macrophages. Conditional knockout of Ezh2 (CKO) in myeloid cell line resulted in significant increases in postnatal bone growth in the first 6 months of life for both male and female mice. For female mice, optimal bone mass was seen for mice with Ezh2 deleted in both chromosomes in a pair (f/f Cre + ; CKO). For male mice, optimal bone mass was found after deletion of Ezh2 from just one chromosome (f/- Cre + ) with no difference in bone phenotype between f/- Cre + and CKO male mice. In addition to the gender-specific difference in bone phenotype, Ezh2 CKO mice had significantly less macrophages (CD11b+) present in the bone marrow compared with control mice as well as significantly more mature osteoblasts and bone formation biomarkers present (P1NP, osteocalcin). Inflammatory array for protein lysed from bone tissue revealed deletion of Ezh2 decreased inflammatory milieu in both male and female mice compared with controls. Unexpectedly, myeloid cell deletion of Ezh2 also increased the number of mature osteoblast present in the bone. Deletion of Ezh2 also led to an increase in gene expression of osteoclast-suppressive genes IRF8, MafB, and Arg1 due to a decrease in the presence of the suppressive H3K27me3 epigenetic mark. These findings suggest that manipulation of Ezh2 expression may be a viable strategy to combat bone resorptive disorders such as osteoporosis or arthritis., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Studies from both humans and animal models indicated that maternal chronic poor-quality diet, especially a high fat diet (HFD), is significantly associated with reduced bone density and childhood fractures in offspring. When previously studied in a rat model, our data suggested that maternal HFD changes epigenetic marks such as DNA methylation and histone modifications to control osteoblast metabolism. In mouse embryonic and postnatal offspring bone samples, a ChIP-sequencing (ChIP-Seq)-based genome-wide method was used to locate the repressive histone mark H3K27me3 (mediated via the polycomb histone methyltransferase, Ezh2 ) and expressive histone mark H3K27ac ( p300/CBP mediated) throughout the genome. Using isolated mouse embryonic cells from foetal calvaria (osteoblast-like cells), H3K27me3 ChIP-Seq showed that 147 gene bodies and 26 gene promoters in HFD embryotic samples had a greater than twofold increase in H3K27me peaks compared to controls. Among the HFD samples, Pthlh and Col2a1 that are important genes playing roles during chondro- and osteogenesis had significantly enriched levels of H3K27me3. Their decreased mRNA expression was confirmed by real-time PCR and standard ChIP analysis, indicating a strong association with Ezh2 mediated H3K27me3 epigenetic changes. Using embryonic calvaria osteoblastic cells and offspring bone samples, H3K27ac ChIP-Seq analysis showed that osteoblast inhibitor genes Tnfaip3 and Twist1 had significantly enriched peaks of H3K27ac in HFD samples compared to controls. Their increased gene expression and association with H3K27ac were also confirmed by real-time PCR and standard ChIP analysis. These findings indicate that chronic maternal HFD changes histone trimethylation and acetylation epigenetic marks to regulate expression of genes controlling osteoblastogenesis.

Sex steroid deficiency plays critical roles in the pathophysiology of bone as the result of uncertain bone remodeling, i.e., increased bone resorption with equivocal bone formation. We have previously shown that GPR109A, a G protein coupled receptor, controls osteoclastogenesis and bone resorption, where global GPR109A deletion decreased osteoclast bone resorption and increased bone mass. Here, we used global GPR109A gene deletion, ovariectomized (OVX) and orchidectomized (ORX) mouse models to probe the role of GPR109A in gonadectomy-induced bone loss in female and male mice. Six months old GPR109A -/- mice and their wild type littermates were allocated to Sham or gonadectomized groups for six weeks. Using densitometric micro-CT confirmed by peripheral quantitative CT (pQCT) scans on tibia and spine, and three-point bending test on femur ex vivo, we found the bone volume, trabecular number, as well as bone mineral density and content in both trabecular and cortical sites were significantly decreased in wild type OVX and ORX compared with respective Sham groups. While bone mass in both male and female GPR109A -/- Sham groups were significantly higher compared with their respective wild type Sham groups, global GPR109A gene deletion ameliorated gonadectomy-induced bone loss. In GPR109A -/- females, most of bone mass and strength parameters measured by micro-CT, pQCT and three-point bending test were not different between Sham and OVX groups. In wild type but not in GPR109 -/- mice, bone remodeling marker measurements indicated that both bone resorption (Cathepsin K) and bone formation (osteocalcin) markers were increased in gonadectomized mice compared to sham, with the exception of bone specific ALP, which was decreased in gonadectomized mice. Expression of bone resorption markers (Cathepsin K) were significantly lower, but β-catenin expression was higher in GPR109A -/- mice compared with their wild type littermates. Collectively, these data indicate that global GPR109A deletion ameliorates gonadectomy-induced bone loss through suppression of bone resorption., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Tightly regulated and cell-specific NADPH-oxidases (Nox) represent one of the major sources of reactive oxygen species (ROS) signaling molecules that are involved in tissue development and stem cell self-renewal. We have characterized the role of Nox4 in osteo-progenitors during postnatal bone development. Nox4 expression in bone and ROS generation were increased during early osteoblast differentiation and bone development. Stromal osteoblastic cell self-renewal, proliferation and ROS production were significantly lower in samples from whole-body Nox4 knockout mice (Nox4 -/- ) and conditional knockout (CKO) mice with depletion of Nox4 in the limb bud mesenchyme compared with those from control mice (Nox4 fl/fl ), but they were reversed after 9 passages. In both sexes, bone volume, trabecular number and bone mineral density were significantly lower in 3-week old CKO and Nox4 -/- mice compared with Nox4 fl/fl controls. This was reflected in serum levels of bone formation markers alkaline phosphatase (ALP) and procollagen 1 intact N-terminal propeptide (P1NP). However, under-developed bone formation in 3-week old CKO and Nox4 -/- mice quickly caught up to levels of control mice by 6-week of age, remained no different at 13-week of age, and was reversed in 32-week old male mice. Osteoclastogenesis showed no differences among groups, however, CTX1 reflecting osteoclast activity was significantly higher in 3-week old male CKO and Nox4 -/- mice compared with control mice, and significantly lower in 32-week old Nox4 -/- mice compared with control mice. These data suggest that Nox4 expression and ROS signaling in bone and osteoblastic cells coordinately play an important role in osteoblast differentiation, proliferation and maturation., (© 2022. The Author(s).)

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.

Myoglobin (Mb) regulates O 2 bioavailability in muscle and heart as the partial pressure of O 2 (Po 2 ) drops with increased tissue workload. Globin proteins also modulate cellular NO pools, "scavenging" NO at higher Po 2 and converting NO 2 - to NO as Po 2 falls. Myoglobin binding of fatty acids may also signal a role in fat metabolism. Interestingly, Mb is expressed in brown adipose tissue (BAT), but its function is unknown. Herein, we present a new conceptual model that proposes links between BAT thermogenic activation, concurrently reduced Po 2 , and NO pools regulated by deoxy/oxy-globin toggling and xanthine oxidoreductase (XOR). We describe the effect of Mb knockout ( Mb -/- ) on BAT phenotype [lipid droplets, mitochondrial markers uncoupling protein 1 (UCP1) and cytochrome C oxidase 4 (Cox4), transcriptomics] in male and female mice fed a high-fat diet (HFD, 45% of energy, ∼13 wk), and examine Mb expression during brown adipocyte differentiation. Interscapular BAT weights did not differ by genotype, but there was a higher prevalence of mid-large sized droplets in Mb -/- . COX4 protein expression was significantly reduced in Mb -/- BAT, and a suite of metabolic/NO/stress/hypoxia transcripts were lower. All of these Mb -/- -associated differences were most apparent in females. The new conceptual model, and results derived from Mb -/- mice, suggest a role for Mb in BAT metabolic regulation, in part through sexually dimorphic systems and NO signaling. This possibility requires further validation in light of significant mouse-to-mouse variability of BAT Mb mRNA and protein abundances in wild-type mice and lower expression relative to muscle and heart. NEW & NOTEWORTHY Myoglobin confers the distinct red color to muscle and heart, serving as an oxygen-binding protein in oxidative fibers. Less attention has been paid to brown fat, a thermogenic tissue that also expresses myoglobin. In a mouse knockout model lacking myoglobin, brown fat had larger fat droplets and lower markers of mitochondrial oxidative metabolism, especially in females. Gene expression patterns suggest a role for myoglobin as an oxygen/nitric oxide-sensor that regulates cellular metabolic and signaling pathways.

Myoglobin (Mb) is a regulator of O 2 bioavailability in type I muscle and heart, at least when tissue O 2 levels drop. Mb also plays a role in regulating cellular nitric oxide (NO) pools. Robust binding of long-chain fatty acids and long-chain acylcarnitines to Mb, and enhanced glucose metabolism in hearts of Mb knockout (KO) mice, suggest additional roles in muscle intermediary metabolism and fuel selection. To evaluate this hypothesis, we measured energy expenditure (EE), respiratory exchange ratio (RER), body weight gain and adiposity, glucose tolerance, and insulin sensitivity in Mb knockout (Mb -/- ) and wild-type (WT) mice challenged with a high-fat diet (HFD, 45% of calories). In males ( n = 10/genotype) and females ( n = 9/genotype) tested at 5-6, 11-12, and 17-18 wk, there were no genotype effects on RER, EE, or food intake. RER and EE during cold (10°C, 72 h), and glucose and insulin tolerance, were not different compared with within-sex WT controls. At ∼18 and ∼19 wk of age, female Mb -/- adiposity was ∼42%-48% higher versus WT females ( P = 0.1). Transcriptomics analyses (whole gastrocnemius, soleus) revealed few consistent changes, with the notable exception of a 20% drop in soleus transferrin receptor (Tfrc) mRNA. Capillarity indices were significantly increased in Mb -/- , specifically in Mb-rich soleus and deep gastrocnemius. The results indicate that Mb loss does not have a major impact on whole body glucose homeostasis, EE, RER, or response to a cold challenge in mice. However, the greater adiposity in female Mb -/- mice indicates a sex-specific effect of Mb KO on fat storage and feed efficiency. NEW & NOTEWORTHY The roles of myoglobin remain to be elaborated. We address sexual dimorphism in terms of outcomes in response to the loss of myoglobin in knockout mice and perform, for the first time, a series of comprehensive metabolic studies under conditions in which fat is mobilized (high-fat diet, cold). The results highlight that myoglobin is not necessary and sufficient for maintaining oxidative metabolism and point to alternative roles for this protein in muscle and heart.

Mechanical stresses associated with physical activity (PA) have beneficial effects on increasing BMD and improving bone quality. However, a high-fat diet (HFD) and obesity tend to have negative effects on bone, by increasing bone marrow adiposity leading to increased excretion of proinflammatory cytokines, which activate RANKL-induced bone resorption. In the current study, whether short-term increased PA via access to voluntary wheel running during early life has persistent and protective effects on HFD-induced bone resorption was investigated. Sixty 4-week-old male C57BL6/J mice were divided into two groups postweaning: without or with PA (access to voluntary running wheel 7-8 km/day) for 4 weeks. After 4 weeks with or without PA, mice were further subdivided into control diet or HFD groups for 8 weeks, and then all animals were switched back to control diet for an additional 4 weeks. Mice from the HFD groups were significantly heavier and obese; however, after 4 weeks of additional control diet their body weights returned to levels of mice on continuous control diet. Using μ-CT and confirmed by pQCT of tibias and spines ex vivo, it was determined that bone volume and trabecular BMD were significantly increased with PA in control diet animals compared with sedentary animals without access to wheels, and such anabolic effects of PA on bone were sustained after ceasing PA in adult mice. Eight weeks of a HFD deteriorated bone development in mice. Unexpectedly, early-life PA did not prevent persistent effects of HFD on deteriorating bone quality; in fact, it exacerbated a HFD-induced inflammation, osteoclastogenesis, and trabecular bone loss in adult mice. In accordance with these data, signal transduction studies revealed that a HFD-induced Ezh2, DNA methyltransferase 3a, and nuclear factor of activated T-cells 1 expression were amplified in nonadherent hematopoietic cells. In conclusion, short-term increased PA in early life is capable of increasing bone mass; however, it alters the HFD-induced bone marrow hematopoietic cell-differentiation program to exacerbate increased bone resorption if PA is halted. © 2021 Arkansas Children's Nutrition Center. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., (© 2021 Arkansas Children's Nutrition Center. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

Saturated fatty acids (SFAs) are implicated in muscle inflammation/cell stress and insulin resistance, but the catalog of factors involved is incomplete. SFA derivatives that accumulate with mismatched FA availability and FA oxidation (FAO) are likely involved, and evidence has emerged that select acylcarnitines should be considered. To understand if excessive long-chain acylcarnitine accumulation and limited FAO associate with lipotoxicity, carnitine palmitoyltransferase 2 knockout C2C12 cells were generated (CPT2 KO). CPT2 KO was confirmed by Western blot, increased palmitoylcarnitine accumulation, and loss of FAO capacity. There was no effect of CPT2 KO on palmitic acid (PA) concentration-dependent increases in media IL-6 or adenylate kinase. PA at 200 and 500 µM did not trigger cell stress responses (phospho-Erk, -JNK, or -p38) above that of vehicle in WT or CPT2 KO cells. In contrast, loss of CPT2 exacerbated PA-induced insulin resistance (acute phospho-Akt; 10 or 100 nM insulin) by as much as ~50-96% compared with WT. Growing cells in carnitine-free media abolished differences between WT and CPT2 KO, but this did not fully rescue PA-induced insulin resistance. The results suggest that PA-induced insulin resistance stems in part from palmitoylcarnitine accumulation, further supporting the hypothesis that select acylcarnitines participate in cell signaling and, when in excess, can compromise cell function. Since carnitine-free conditions could not fully rescue insulin signaling, and CPT2 KO did not alter cell stress responses, the majority of PA-induced "lipotoxicity" in C2C12 myotubes cannot be attributed to palmitoylcarnitine alone.

Background: Neonatal diet impacts many physiological systems and can modify risk for developing metabolic disease and obesity later in life. Less well studied is the effect of postnatal diet (e.g., comparing human milk (HM) or milk formula (MF) feeding) on mitochondrial bioenergetics. Such effects may be most profound in splanchnic tissues that would have early exposure to diet-associated or gut microbe-derived factors., Methods: To address this question, we measured ileal and liver mitochondrial bioenergetics phenotypes in male piglets fed with HM or MF from day 2 to day 21 age. Ileal and liver tissue were processed for mitochondrial respiration (substrate only [pyruvate, malate, glutamate], substrate + ADP, and proton "leak" post-oligomycin; measured by Oroboros methods), mitochondrial DNA (mtDNA) and metabolically-relevant gene expression analyses., Results: No differences between the diet groups were observed in mitochondrial bioenergetics indices in ileal tissue. In contrast, ADP-dependent liver Complex I-linked OXPHOS capacity and Complex I + II-linked OXPHOS capacity were significantly higher in MF animals relative to HM fed piglets. Interestingly, p53, Trap1, and Pparβ transcript abundances were higher in MF-fed relative to HM-fed piglets in the liver. Mitochondrial DNA copy numbers (normalized to nuclear DNA) were similar within-tissue regardless of postnatal diet, and were ~ 2-3 times higher in liver vs. ileal tissue., Conclusion: While mechanisms remain to be identified, the data indicate that neonatal diet can significantly impact liver mitochondrial bioenergetics phenotypes, even in the absence of a change in mtDNA abundance. Since permeabilized liver mitochondrial respiration was increased in MF piglets only in the presence of ADP, it suggests that formula feeding led to a higher ATP turnover. Specific mechanisms and signals involved with neonatal diet-associated differences in liver bioenergetics remain to be elucidated., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Nutritional status during intrauterine and/or early postnatal life has substantial influence on adult offspring health. Along these lines, there is a growing body of evidence illustrating that high fat diet (HFD)-induced maternal obesity can regulate fetal bone development. Thus, we investigated the effects of maternal obesity on both fetal skeletal development and mechanisms linking maternal obesity to osteoblast differentiation in offspring. Embryonic osteogenic calvarial cells (EOCCs) were isolated from fetuses at gestational day 18.5 (E18.5) of HFD-induced obese rat dams. We observed impaired differentiation of EOCCs to mature osteoblasts from HFD obese dams. ChIP-seq-based genome-wide localization of the repressive histone mark H3K27me3 (mediated via the polycomb histone methyltransferase, enhancer of zeste homologue 2 [Ezh2]) showed that this phenotype was associated with increased enrichment of H3K27me3 on the gene of SATB2, a critical transcription factor required for osteoblast differentiation. Knockdown of Ezh2 in EOCCs and ST2 cells increased SATB2 expression; while Ezh2 overexpression in EOCCs and ST2 cells decreased SATB2 expression. These data were consistent with experimental results showing strong association between H3K27me3, Ezh2, and SATB2 in cells from rats and humans. We have further presented that SATB2 mRNA and protein expression were increased in bones, and increased trabecular bone mass from pre-osteoblast specific Ezh2 deletion (Ezh2 flox/flox Osx-Cre + cko) mice compared with those from control Cre + mice. These findings indicate that maternal HFD-induced obesity may be associated with decreasing fetal pre-osteoblastic cell differentiation, under epigenetic control of SATB2 expression via Ezh2-dependent mechanisms., (© 2019 UAMS. The FASEB Journal published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology.)

Phenolic acids (PAs) are metabolites derived from polyphenolic compounds found in fruits and vegetables resulting from the actions of gut bacteria. Previously, we reported that the levels of seven individual PAs were found to be at least 10 times higher in the serum of rats fed a blueberry (BB)-containing diet compared to those fed a control diet. We have characterized the effects of one such BB-associated serum PA, 3-(3-hydroxyphenyl)-propionic acid (PPA), on senescence signaling and promotion of mesenchymal stem cell differentiation toward osteoblasts, while suppressing adipogenesis in the stem cells. To better understand the mechanistic actions of PPA on bone formation in vivo, we administered four doses of PPA (0.1, 0.5, 1, and 5 mg/kg/day; daily i.p.) to 1-month-old female C57BL6/J mice for 30 days. We did not observe significant effects of PPA on cortical bone; however, there were significantly higher bone volume and trabecular thickness and increased osteoblastic cell number, but decreased osteoclastic cell number in PPA-treated groups compared to controls. These morphological and cellular outcomes of bone were reflected in changes of bone formation markers in serum and bone marrow plasma. PPA treatment reduced senescence signaling as evaluated by senescence-associated β-galactosidase activity, PPARγ, p53, and p21 expression in bone. In conclusion, PPA is capable of altering the mesenchymal stem cell differentiation program and bone cell senescence. This raises the possibility that BB-rich diets promote bone growth through increasing systemic PAs, a question that merits additional investigation. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research., (© 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.)

Excessive cellular accumulation or exposure to lipids such as long-chain acylcarnitines (LCACs), ceramides, and others is implicated in cell stress and inflammation. Such a situation might manifest when there is a significant mismatch between long-chain fatty acid (LCFA) availability versus storage and oxidative utilization; for example, in cardiac ischemia, increased LCACs may contribute to tissue cell stress and infarct damage. Perturbed LCFAβ-oxidation is also seen in fatty acid oxidation disorders (FAODs). FAODs typically manifest with fasting- or stress-induced symptoms, and patients can manage many symptoms through control of diet and physical activity. However, episodic clinical events involving cardiac and skeletal muscle myopathies are common and can present without an obvious molecular trigger. We have speculated that systemic or tissue-specific lipotoxicity and activation of inflammation pathways contribute to long-chain FAOD pathophysiology. With this in mind, we characterized inflammatory phenotype (14 blood plasma cytokines) in resting, overnight-fasted (~10 h), or exercise-challenged subjects with clinically well-controlled long-chain FAODs (n = 12; 10 long-chain 3-hydroxyacyl-CoA dehydrogenase [LCHAD]; 2 carnitine palmitoyltransferase 2 [CPT2]) compared to healthy controls (n = 12). Across experimental conditions, concentrations of three cytokines were modestly but significantly increased in FAOD (IFNγ, IL-8, and MDC), and plasma levels of IL-10 (considered an inflammation-dampening cytokine) were significantly decreased. These novel results indicate that while asymptomatic FAOD patients do not display gross body-wide inflammation even after moderate exercise, β-oxidation deficiencies might be associated with chronic and subtle activation of "sterile inflammation." Further studies are warranted to determine if inflammation is more apparent in poorly controlled long-chain FAOD or when long-chain FAOD-associated symptoms are present., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Based primarily on cell culture results, saturated fatty acids (SFAs) are proposed to promote inflammation and contribute to metabolic dysfunction through Toll-like receptor activation. Studies are often complicated by a requirement for carriers (e.g., BSA) or solvents (e.g., ethanol) to increase SFA solubility. To ascertain whether these factors influence interpretations of SFA-associated inflammation activity, we measured responses of RAW264.7 monocyte/macrophages and C 2 C 12 myotubes to various BSA, ethanol, and cyclodextrin (alternative FA carrier) conditions. Fatty acid-free, low-endotoxin BSA preparations (0.33% to 2% wt/vol) activated whereas 0.5-1.0% ethanol inhibited RAW264.7 TNFα release. Ethanol modestly increased IL-6 secretion in C 2 C 12 myotubes. Cyclodextrins (0.3-6.0 mM) were tested as alternative carriers of palmitate, but their usefulness was limited due to toxicity and solubility issues. Using a lower-inflammation BSA source and no ethanol, ∼24-h sodium palmitate treatment (≤600 µM) failed to trigger RAW264.7 TNFα release and, in fact, significantly dampened BSA-induced inflammation by >50%. In C 2 C 12 myotubes, only high palmitate concentrations (500-600 µM) elicited IL-6 secretion (>2.5-fold increase). Acute palmitate (200 or 500 µM) treatment did not activate MAP kinase pathways above that of fresh BSA-containing media alone in either cell type. These results highlight the importance of experimental conditions in studies exploring SFA inflammation effects. The limited (or even anti-inflammatory) effects of palmitate that we observed indicate that immunomodulatory effects of SFAs are context-specific. Thus, caution is needed when interpreting the literature related to putative proinflammatory effects of SFA.

Nutritional status during intrauterine and early postnatal life impacts the risk of chronic diseases; however, evidence for an association between early-life dietary factors and bone health in adults is limited. Soy protein isolate (SPI) may be one such dietary factor that promotes bone accretion during early life with persistent effects into adulthood. In the present study, we fed postnatal day (PND) 24 weanling female rats an SPI diet for 30 d [short-term SPI (ST-SPI)], and on PND 55, we switched SPI diet to control Cas diet until age 6 mo. Rats then underwent either ovariectomy (OVX) or sham surgery and thereafter either continued to be fed an SPI diet or control diet for 1 or 3 wk. We showed significantly increased bone mass in 30-d SPI-fed young rats compared with controls. OVX-induced bone loss was associated with increased osteoblastic cell senescence. On the one hand, both long-term SPI (continuous SPI diet throughout life) and ST-SPI diet only in early life protected against 1 wk post-OVX-associated bone loss. On the other hand, long-term SPI diet diminished the loss of total, trabecular, and cortical bone mineral density, whereas ST-SPI diet only reduced cortical bone mineral density loss 3 wk post-OVX. Persistent and protective effects of SPI diets on OVX-induced bone loss were associated with down-regulation of the caveolin-1/p53-mediated senescence pathway in bone. We recapitulated these results in cell cultures. Reprogramming of cellular senescence signaling by SPI-associated isoflavones in osteoblastic cells may explain the persistent effects of SPI on bone. These results suggest that OVX-induced bone loss, in part, is a result of increased osteoblastic cell senescence, and that ST-SPI diet early in life has modest but persistent programming effects on bone formation to prevent OVX-induced bone loss in adult female rats.-Chen, J.-R., Lazarenko, O. P., Blackburn, M. L., Shankar, K. Dietary factors during early life program bone formation in female rats., (© FASEB.)

High-fat (HF) diets typically promote diet-induced obesity (DIO) and metabolic dysfunction (i.e., insulin resistance, hypertriglyceridemia, and hepatic steatosis). Dysfunction of triacylglycerol (TAG) metabolism may contribute to the development of hepatic steatosis, via increased de novo lipogenesis or repackaging of circulating nonesterified fatty acids (NEFAs). Hepatic TAG production (HTP) rate can be assessed through injecting mice with nonionic detergents that inhibit tissue lipoprotein lipase. Potential confounding effects of detergent-based HTP tests (HTPTs) used in longitudinal studies-including the impact on food intake, energy balance, and weight gain-have not been reported. To examine this, male C57BL/6J mice were fed a 10% or 60% kcal diet. After 4 weeks, the mice underwent an HTPT via poloxamer 407 intraperitoneal injections (1000 mg/kg). Weight gain, energy intake, and postabsorptive TAG levels normalized 7-10 days post-HTPT. The post-HTPT recovery of body weight and energy intake suggest that, in metabolic phenotyping studies, any additional sample collection should occur at least 7-10 days after the HTPT to reduce confounding effects. Diet-specific effects on HTP were also observed: HF-fed mice had reduced HTP, plasma TAG, and NEFA levels compared to controls. In conclusion, the current study highlights the procedural and physiological complexities associated with studying lipid metabolism using a HTPT in the DIO mouse model., Competing Interests: H.I. is a former employee of XOMA (US) Corporation. All other authors declare that they have no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Nutritional status during intrauterine and early postnatal life impacts the risk of chronic diseases, presumably via epigenetic mechanisms. However, evidence on the impact of gestational events on regulation of embryonic bone cell fate is sparse. We investigated the effects of maternal obesity on fetal osteoblast development in both rodents and humans. Female rats were fed control or an obesogenic high-fat diet (HFD) for 12 weeks and mated with male rats fed control diets, and respective maternal diets were continued during pregnancy. Embryonic rat osteogenic calvarial cells (EOCCs) were taken from gestational day 18.5 fetuses from control and HFD dams. EOCCs from HFD obese dams showed increases in p53/p21-mediated cell senescence signaling but decreased glucose metabolism. Decreased aerobic glycolysis in HFD-EOCCs was associated with decreased osteoblastic cell differentiation and proliferation. Umbilical cord human mesenchymal stem cells (MSCs) from 24 pregnant women (12 obese and 12 lean) along with placentas were collected upon delivery. The umbilical cord MSCs of obese mothers displayed less potential toward osteoblastogenesis and more towards adipogenesis. Human MSCs and placenta from obese mothers also exhibited increased cell senescence signaling, whereas MSCs showed decreased glucose metabolism and insulin resistance. Finally, we showed that overexpression of p53 linked increased cell senescence signaling and decreased glucose metabolism in fetal osteo-progenitors from obese rats and humans. These findings suggest programming of fetal preosteoblastic cell senescence signaling and glucose metabolism by maternal obesity.

This data file describes the bioinformatics analysis of uterine RNA-seq data comparing genome wide effects of feeding soy protein isolate compared to casein to ovariectomized female rats age 64 days relative to treatment of casein fed rats with 5 μg/kg/d estradiol and relative to rats treated with estradiol and also fed soy protein isolate. Complete raw data files were deposited in the gene Expression Omnibus (GEO) at NCBI (http:/www.ncbi.nlm.nih.gov.geo/) under the GEO accession number GEO: GSE69819. Data presented here incudes a summary of the differential expression analysis with top 30 genes up- and down-regulated by soy protein isolate (SPI), estradiol (E2) and SPI+E2. Additional functional annotation analysis of KEGG pathways is also presented for each treatment, together with networks of interaction between those pathways. Further interpretation and discussion of this data can be found in the article "Uterine responses to feeding soy protein isolate and treatment with 17β-estradiol differ in ovariectomized female rats" Ronis et al. (2016) [1].

There are concerns regarding reproductive toxicity from consumption of soy foods, including an increased risk of endometriosis and endometrial cancer, as a result of phytoestrogen consumption. In this study, female rats were fed AIN-93G diets made with casein (CAS) or soy protein isolate (SPI) from postnatal day (PND) 30, ovariectomized on PND 50 and infused with 5 μg/kg/d 17β-estradiol (E2) or vehicle. E2 increased uterine wet weight (P<0.05). RNAseq analysis revealed that E2 significantly altered expression of 1991 uterine genes (P<0.05). SPI feeding had no effect on uterine weight and altered expression of far fewer genes than E2 at 152 genes (P<0.05). Overlap between E2 and SPI genes was limited to 67 genes. Functional annotation analysis indicated significant differences in uterine biological processes affected by E2 and SPI and little evidence for recruitment of estrogen receptor (ER)α to the promoters of ER-responsive genes after SPI feeding. The major E2 up-regulated uterine pathways were carcinogenesis and extracellular matrix organization, whereas SPI feeding up-regulated uterine peroxisome proliferator activated receptor (PPAR) signaling and fatty acid metabolism. The combination of E2 and SPI resulted in significant regulation of 504 fewer genes relative to E2 alone. The ability of E2 to induce uterine proliferation in response to the carcinogen dimethybenz(a)anthracene (DMBA) as measured by expression of PCNA and Ki67 mRNA was suppressed by feeding SPI (P<0.05). These data suggest that SPI is a selective estrogen receptor modulator (SERM) interacting with a small sub-set of E2-regulated genes and is anti-estrogenic in the presence of endogenous estrogens., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Chronic consumption by experimental animals of a typical Western diet high in saturated fats and cholesterol during postnatal life has been demonstrated to impair skeletal development. However, the underlying mechanism by which high-fat, energy-dense diets affect bone-forming cell phenotypes is poorly understood. Here, we show that male weanling rats fed a diet containing 45% fat and 0.5% cholesterol made with casein (HF-Cas) for 6 weeks displayed lower bone mineral density and strength compared with those of AIN-93G-fed dietary controls. Substitution of casein with soy protein isolate (SPI) in the high-fat diet (HF-SPI) prevented these effects. The bone-sparing effects of SPI were associated with prevention of HF-Cas-induced osteoblast senescence pathways through suppression of the p53/p21 signaling pathways. HF-Cas-fed rats had increased caveolin-1 and down-regulated Sirt1, leading to activations of peroxisome proliferator-activated receptor γ (PPARγ) and p53/p21, whereas rats fed HF-SPI suppressed caveolin-1 and activated Sirt1 to deacetylate PPARγ and p53 in bone. Treatment of osteoblastic cells with nonesterified free fatty acid (NEFA) increased cell senescence signaling pathways. Isoflavones significantly blocked activations of senescence-associated β-galactosidase and PPARγ/p53/p21 by NEFA. Finally, replicative senescent osteoblastic cells and bone marrow mesenchymal ST2 cells exhibited behavior similar to that of cells treated with NEFA and in vivo bone cells in rats fed the HF-Cas diet. These results suggest that (1) high concentrations of NEFA occurring with HF intake are mediators of osteoblast cell senescence leading to impairment of bone development and acquisition and (2) the molecular mechanisms underlying the SPI-protective effects involve isoflavone-induced inhibition of osteoblastic cell senescence to prevent HF-induced bone impairments.

Differences in trace element composition and bioavailability between breast milk and infant formulas may affect metal homeostasis in neonates. However, there is a paucity of controlled studies in this area. Here, piglets were fed soy infant formula (soy), cow's milk formula (milk), or were allowed to suckle from the sow from PND2 to PND21. Serum iron concentrations were higher in formula-fed compared to breastfed piglets (P < 0.05). Serum zinc values were higher in milk compared to breastfed or soy groups (P < 0.05). Zinc transporter Zip4 mRNA was elevated in small intestine of the soy compared to breastfed group (P < 0.05). Transporter Znt1 mRNA was greater in small intestine of both formula-fed groups and in liver of the milk compared to the breastfed group (P < 0.05). Metallothionein Mt1 mRNA expression was higher in small intestine and liver of milk compared to breastfed and soy groups (P < 0.05). In liver, metallothionein protein levels and protein bound zinc were also highly elevated in the milk compared to other groups (P < 0.05). mRNA encoding the hepatic zinc-regulated gene Gclc was higher in the milk than soy group (P < 0.05). ChIP assay revealed increased binding of the zinc-regulated transcription factor MTF1 to the promoters of hepatic Mt3 and Gclc genes in the milk compared to the soy group. These data provide evidence that trace element status differs in breastfed, milk-fed, and soy-fed piglets and that despite similar levels of dietary supplementation, allows strong causal inference that significant differences in serum zinc after cow's milk formula compared to soy formula consumption result in compensatory changes in expression of zinc transporters, binding proteins, and zinc-regulated genes., (© 2014 by the Society for Experimental Biology and Medicine.)

The proportion of pregnant women who are obese at conception continues to rise. Compelling evidence suggests the intrauterine environment is an important determinant of offspring health. Maternal obesity and unhealthy diets are shown to promote metabolic programming in the offspring. Mitochondria are maternally inherited, and we have previously shown impaired mitochondrial function in rat offspring exposed to maternal obesity in utero. Mitochondrial health is maintained by mitochondrial dynamics, or the processes of fusion and fission, which serve to repair damaged mitochondria, remove irreparable mitochondria, and maintain mitochondrial morphology. An imbalance between fusion and fission has been associated with obesity, insulin resistance, and reproduction complications. In the present study, we examined the influence of maternal obesity and postweaning high-fat diet (HFD) on key regulators of mitochondrial fusion and fission in rat offspring at important developmental milestones which included postnatal day (PND)35 (2 wk HFD) and PND130 (∼16 wk HFD). Our results indicate HFD-fed offspring had reduced mRNA expression of presenilin-associated rhomboid-like (PARL), optic atrophy (OPA)1, mitofusin (Mfn)1, Mfn2, fission (Fis)1, and nuclear respiratory factor (Nrf)1 at PND35, while OPA1 and Mfn2 remained decreased at PND130. Putative transcriptional regulators of mitochondrial dynamics were reduced in rat placenta and offspring liver and skeletal muscle [peroxisome proliferator-activated receptor gamma coactivator (PGC1)α, PGC1β, and estrogen-related receptor (ERR)α], consistent with indirect calorimetry findings revealing reduced energy expenditure and impaired fat utilization. Overall, maternal obesity detrimentally alters mitochondrial targets that may contribute to impaired mitochondrial health and increased obesity susceptibility in later life., (Copyright © 2014 the American Physiological Society.)

It has been suggested that the beneficial effects of soy protein isolate (SPI) on bone quality are due to either stimulation of estrogenic signaling via isoflavones or through a novel and as yet uncharacterized nonestrogenic pathway. In our study, SPI-fed rat serum inhibited the osteoblastic cell senescence pathway. This effect was accompanied by stimulation of cell differentiation, proliferation, and significant restoration of replicative senescent bone marrow mesenchymal ST2 cells (passaged 30 times). These effects were reproduced in bone from 5-wk-old intact and 10-wk-old ovariectomized female rats fed SPI diets. Caveolin-1 and p53 expression was decreased in bone in SPI-fed, but not in 17β-estradiol (E2)-treated rats. In cell culture studies, membranous caveolin-1 and nuclear p53 expression was greater in replicative senescent ST2 cell cultures than in earlier passaged cells. SPI-fed rat serum significantly down-regulated both caveolin-1 and p53 in senescent and nonsenescent cells. Replicative senescent ST2 cells exhibited a strong association among caveolin-1, p53, and mouse double minute 2 homologue (mdm2), which was inhibited by SPI-fed rat serum. Overexpression of caveolin-1 in ST2 cells resulted in increased expression of p53 and p21, whereas, knockdown of caveolin-1 using shRNA led to increases in mdm2 and eliminated SPI-fed rat serum's effects on p53 and p21 expression. In contrast, manipulation of caveolin-1 expression did not affect the actions of E2 or isoflavones on p53 expression in either ST2 or OB6 cells. These results suggest that caveolin-1 is a mediator of nonestrogenic SPI effects on bone cells.-Zhang, J., Lazarenko, O. P., Blackburn, M. L., Badger, T. M., Ronis, M. J. J., Chen, J.-R. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways., (© FASEB.)

Alcohol abuse is associated with the development of fatty liver disease and also with significant osteopenia in both genders. In this study, we examined ethanol-induced pathology in response to diets with differing fat/carbohydrate ratios. Male Sprague-Dawley rats were fed intragastrically with isocaloric liquid diets. Dietary fat content was either 5% (high carbohydrate, HC) or 45% (high fat, HF), with or without ethanol (12-13 g/kg/day). After 14, 28, or 65 days, livers were harvested and analyzed. In addition, bone morphology was analyzed after 65 days. HC rats gained more weight and had larger fat pads than HF rats with or without ethanol. Steatosis developed in HC + ethanol (HC + EtOH) compared to HF + ethanol (HF + EtOH) rats, accompanied by increased fatty acid (FA) synthesis and increased nuclear carbohydrate response element binding protein (ChREBP) (p < 0.05), but in the absence of effects on hepatic silent mating type information regulation 2 homolog (SIRT-1) or nuclear sterol regulatory binding element protein (SREBP-1c). Ethanol reduced serum leptin (p < 0.05) but not adiponectin. Over time, HC rats developed fatty liver independent of ethanol. FA degradation was significantly elevated by ethanol in both HC and HF groups (p < 0.05). HF + EtOH rats had increased oxidative stress from 28 days, increased necrosis compared to HF controls and higher expression of cytochromes P450, CYP2E1, and CYP4A1 compared to HC + EtOH rats (p < 0.05). In contrast, HC + EtOH rats had no significant increase in oxidative stress until day 65 with no observed increase in necrosis. Unlike liver pathology, no dietary differences were observed on ethanol-induced osteopenia in HC compared to HF groups. These data demonstrate that interactions between diet composition and alcohol are complex, dependent on the length of exposure, and are an important influence in development of fatty liver injury. Importantly, it appears that diet composition does not affect alcohol-associated skeletal toxicity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The risk of obesity in adulthood is subject to programming beginning at conception. In animal models, exposure to maternal obesity and high fat diets influences the risk of obesity in the offspring. Among other long-term changes, offspring from obese rats develop hyperinsulinemia, hepatic steatosis, and lipogenic gene expression in the liver at weaning. However, the precise underlying mechanisms leading to metabolic dysregulation in the offspring remains unclear. Using a rat model of overfeeding-induced obesity, we previously demonstrated that exposure to maternal obesity from pre-conception to birth, is sufficient to program increased obesity risk in the offspring. Offspring of obese rat dams gain greater body weight and fat mass when fed high fat diet (HFD) as compared to lean dam. Since, disruptions of diurnal circadian rhythm are known to detrimentally impact metabolically active tissues such as liver, we examined the hypothesis that maternal obesity leads to perturbations of core clock components and thus energy metabolism in offspring liver. Offspring from lean and obese dams were examined at post-natal day 35, following a short (2 wk) HFD challenge. Hepatic mRNA expression of circadian (CLOCK, BMAL1, REV-ERBα, CRY, PER) and metabolic (PPARα, SIRT1) genes were strongly suppressed in offspring exposed to both maternal obesity and HFD. Using a mathematical model, we identified two distinct biological mechanisms that modulate PPARα mRNA expression: i) decreased mRNA synthesis rates; and ii) increased non-specific mRNA degradation rate. Moreover, our findings demonstrate that changes in PPARα transcription were associated with epigenomic alterations in H3K4me3 and H3K27me3 histone marks near the PPARα transcription start site. Our findings indicated that offspring from obese rat dams have detrimental alternations to circadian machinery that may contribute to impaired liver metabolism in response to HFD, specifically via reduced PPARα expression prior to obesity development.

A blueberry (BB)-supplemented diet has been previously shown to significantly stimulate bone formation in rapidly growing male and female rodents. Phenolic acids (PAs) are metabolites derived from polyphenols found in fruits and vegetables as a result of the actions of gut bacteria, and they were found in the serum of rats fed BB-containing diet. We conducted in vitro studies with PAs and demonstrated stimulation of osteoblast differentiation and proliferation. On the other hand, adipogenesis was inhibited. To more fully understand the mechanistic actions of PAs on bone formation, we administered hippuric acid, one of the major metabolites found in animal circulation after BB consumption, to prepubertal female mice for 2 weeks. We found that hippuric acid was able to stimulate bone-forming gene expression but suppress PPARγ expression, leading to increased bone mass dose-dependently. Cellular signaling studies further suggested that the skeletal effects of PAs appeared to be mediated through activation of G-protein-coupled receptor 109A and downstream p38 MAP kinase and osterix. In conclusion, PAs are capable of altering the mesenchymal stem cell differentiation program and merit investigation as potential dietary therapeutic alternatives to drugs for degenerative bone disorders. © 2014 American Society for Bone and Mineral Research., (© 2014 American Society for Bone and Mineral Research.)

In both rodents and humans, excessive consumption of a typical Western diet high in saturated fats and cholesterol is known to result in disruption of energy metabolism and development of obesity and insulin resistance. However, how these high-fat, energy-dense diets affect bone development, morphology, and modeling is poorly understood. Here we show that male weanling rats fed a high-fat (HF) diet containing 45% fat and 0.5% cholesterol made with casein (HF-Cas) for 6 wk displayed a significant increase in bone marrow adiposity and insulin resistance. Substitution of casein with soy protein isolate (SPI) in the HF diet (HF-SPI) prevented these effects. Maintenance of bone quantity in the SPI-fed rats was associated with increased undercarboxylated osteocalcin secretion and altered JNK/IRS1/Akt insulin signaling in osteoblasts. The HF-Cas group had significantly greater serum nonesterified free fatty acid (NEFA) concentrations than controls, whereas the HF-SPI prevented this increase. In vitro treatment of osteoblasts or mesenchymal stromal ST2 cells with NEFAs significantly decreased insulin signaling. An isoflavone mixture similar to that found in serum of HF-SPI rats significantly increased in vitro osteoblast proliferation and blocked significantly reduced NEFA-induced insulin resistance. Finally, insulin/IGF1 was able to increase both osteoblast activity and differentiation in a set of in vitro studies. These results suggest that high-fat feeding may disrupt bone development and modeling; high concentrations of NEFAs and insulin resistance occurring with high fat intake are mediators of reduced osteoblast activity and differentiation; diets high in soy protein may help prevent high dietary fat-induced bone impairments; and the molecular mechanisms underlying the SPI-protective effects involve isoflavone-induced normalization of insulin signaling in bone.

Previous studies have demonstrated that weanling rats fed AIN-93G semi-purified diets supplemented with 10% whole blueberry (BB) powder for two weeks beginning on postnatal day 21 (PND21) significantly increased bone formation at PND35. However, the minimal level of dietary BB needed to produce these effects is, as yet, unknown. The current study examined the effects of three different levels of BB diet supplementation (1, 3, and 5%) for 35 days beginning on PND25 on bone quality, and osteoclastic bone resorption in female rats. Peripheral quantitative CT scan (pQCT) of tibia, demonstrated that bone mineral density (BMD) and content (BMC) were dose-dependently increased in BB-fed rats compared to controls (P<0.05). Significantly increased bone mass after feeding 5% BB extracts was also observed in a TEN (total enteral nutrition) rat model in which daily caloric and food intake was precisely controlled. Expression of RANKL (receptor activator of nuclear factor-κB ligand) a protein essential for osteoclast formation was dose-dependently decreased in the femur of BB animals. In addition, expression of PPARγ (peroxisome proliferator-activated receptor γ) which regulates bone marrow adipogenesis was suppressed in BB diet rats compared to non-BB diet controls. Finally, a set of in vitro cell cultures revealed that the inhibitory effect of BB diet rat serum on RANKL expression was more profound in mesenchymal stromal cells compared to its effect on mature osteoblasts, pre-adipocytes and osteocytes. These results suggest that inhibition of bone resorption may contribute to increased bone mass during early development after BB consumption.

Ovariectomy (OVX)-induced bone loss has been linked to increased bone turnover and higher bone matrix collagen degradation as the result of osteoclast activation. However, the role of degraded collagen matrix in the fate of resident bone-forming cells is unclear. In this report, we show that OVX-induced bone loss is associated with profound decreases in collagen 1 and Sirt1. This was accompanied by increases in expression and activity of the senescence marker collagenase and expression of p16/p21 in bone. Feeding a diet supplemented with blueberries (BB) to pre-pubertal rats throughout development or only prior to puberty [postnatal day 21 (PND21) to PND34] prevents OVX-induced effects on expression of these molecules at PND68. In order to provide more evidence and gain a better understanding on the association between bone collagen matrix and resident bone cell fate, in vitro studies on the cellular senescence pathway using primary calvarial cells and three cell lines (ST2 cells, OB6, and MLO-Y4) were conducted. We found that senescence was inhibited by collagen in a dose-response manner. Treatment of cells with serum from OVX rats accelerated osteoblastic cell senescence pathways, but serum from BB-fed OVX rats had no effect. In the presence of low collagen or treatment with OVX rat serum, ST2 cells exhibited higher potential to differentiate into adipocytes. Finally, we demonstrated that bone cell senescence is associated with decreased Sirt1 expression and activated p53, p16, and p21. These results suggest that (1) a significant prevention of OVX-induced bone cell senescence from adult rats can occur after only 14 days consumption of a BB-containing diet immediately prior to puberty, and (2) the molecular mechanisms underlying this effect involves, at least in part, prevention of collagen degradation.

Soy foods have been suggested to have both positive health benefits and potentially adverse effects as a result of their content of phytoestrogens. However, studies on the estrogenicity of soy foods are lacking. Here we directly compared the effects of soy protein isolate (SPI), the protein in soy infant formula, with those of 17β-estradiol (E2), on global gene expression profiles and morphology in the female rat mammary gland. Rats were fed AIN-93G diets containing casein or SPI beginning on postnatal d 30. Rats were ovariectomized on postnatal d 50 and treated with 5 μg/kg/d E2 or vehicle for 14 d. Microarray analysis revealed that E2 treatment altered expression of 780 genes more than or equal to 2-fold (P < 0.05), whereas SPI feeding altered expression of only 53 genes more than or equal to 2-fold. Moreover, the groups had only 10 genes in common to increase more than or equal to 2-fold. The combination of SPI feeding and E2 altered expression of 422 genes and reversed E2 effects on many mRNAs, including those involved in the c-myc signaling pathway, cyclin D1, and Ki67. ERα binding to its response element on the Tie-2/Tek and progesterone receptor promoters was increased by E2, but not SPI, and this promoter binding was suppressed by the combination of E2 + SPI for the Tie-2/Tek promoter but increased for the progesterone receptor promoter (P < 0.05). SPI reduced the ratio of epithelial to fat pad area and E2 + SPI reduced both epithelial and fat pad area (P < 0.05). These data suggest that SPI is only minimally estrogenic in the rat mammary gland even in the absence of endogenous estrogens.

The rat placentation site is distinctly organized into interacting zones, the so-called labyrinth, junctional, and metrial gland compartments. These zones house unique cell populations equipped to undertake myriad prescribed functions including transport, hormonal responses, and immune interactions. Although much is known about the genesis of these cell types and specific markers that characterize each zone, a detailed global overview of gene expression in the three zones is absent. In this report, we used massively parallel sequencing (RNA-seq) to assess mRNA expression profiles and generated transcriptomic maps for each zone of the late-gestation rat placentation site (18.5 d postcoitum). Analysis of expression profiles revealed that each compartment expressed a unique signature, characterized by biological processes specific to the zone. Transport and vasculature-related processes predominated in the labyrinth, hormone secretion in the junctional, and immune interactions in the metrial gland. Furthermore, our analysis identified approximately 4000 differentially expressed genes within the zones. Using k-means clustering, we identified transcription factors with highest expression in either labyrinth, junctional, or metrial gland. Direct interaction (pathway) analysis revealed unique transcription factor networks operating in each compartment. The site-specific expression of 27 transcription factors in the three zones was ascertained via quantitative PCR and protein expression of six transcription factors was confirmed by immunohistochemistry. Finally, we elucidated the expression of key developmentally important families (Sox, GATA, Fox, Wnt, Tead, and IGF/IGFBP) in the placentation site to reveal novel expression of these several factors. The present dataset provides a novel resource to understand zonal gene expression and function in the placenta.

Epidemiological studies show that maternal obesity during intrauterine and early postnatal life increases the risk of low bone mass and fracture later in life. Here, we show that bone development is inhibited in gestational embryonic day 18.5 (E18.5) embryos from rat dams made obese by feeding a high-fat diet (HFD). Moreover, fetal rat osteogenic calvarial cells (FOCCs) from these obese dams have significantly less potential to develop into mature osteoblasts compared to cells from AIN-93G diet-fed controls. Profiling of transcriptional genes for osteogenesis revealed a profound decrease in the homeodomain-containing factor A10 (HoxA10) in FOCCs from fetuses of HFD-induced obese dams. Significant methylation of the HoxA10 promoter was found in those FOCCs, as well as in mouse ST2 cells treated with a mixture of free fatty acids similar to that found in serum from HFD-induced obese rats. This was accompanied by lower expression of osteogenic markers, but higher levels of PPARγ. Control FOCCs depleted of the HoxA10 gene (shRNA) ex vivo behave similarly to cells from fetuses of obese dams; conversely, overexpression of HoxA10 gene in FOCCs from HFD rats exhibit the same phenotype as controls. Treatment of FOCCs from control rats or of ST2 cells with an artificial mixture of free fatty acids significantly down-regulated HoxA10 protein expression, and cells exhibited adipocyte-like properties. These results suggest that maternal obesity impairs fetal skeletal development through down-regulation of the HoxA10 gene, which may lead to an increase in the prevalence of low bone mass in the offspring later in life.

Background: Previous reports suggest that beneficial effects of soy on bone quality are due to the estrogenic actions of isoflavone phytochemicals associated with the protein. However, mechanistic studies comparing the effects of soy diet and estrogens on bone, particularly in rapidly growing animals are lacking., Methodology and Principal Findings: We studied the effects of short term feeding of soy protein isolate (SPI) on bone in comparison to the effects of 17β-estradiol (E2) in pre-pubertal rats. Female rats were weaned to one of 4 treatments: 1) a control casein-based diet (CAS); 2) CAS with subcutaneous E2 (10 µg/kg/d) (CAS+E2); 3) a SPI-containing diet (SPI); or 4) SPI with subcutaneous E2 (SPI) or SPI with 10 µg/kg/d E2 (SPI+E2) for 14 days beginning on postnatal day 20. SPI increased while E2 decreased bone turnover compared to CAS. In contrast, both treatments decreased serum sclerostin levels. Microarray analysis of RNA isolated from bone revealed 652 genes regulated by SPI, 491 genes regulated by E2, and 266 genes regulated by both SPI diet and E2 compared to CAS. The expression of caveolin-1, a protein localized in the cell membrane, was down-regulated (p<0.05) in rats fed SPI, but not by E2 compared to rats fed casein. Down-regulation of caveolin-1 by SPI was associated with increased BMP2, Smad and Runx2 expression in bone and osteoblasts (p<0.05)., Conclusions/significance: These results suggest SPI and E2 have different effects on bone turnover prior to puberty. Approximately half of the genes are regulated in the same direction by E2 or SPI, but in combination, SPI blocks the estrogen effects and returns the profile towards control levels. In addition, there are E2 specific and SPI-specific gene changes related to regulation of bone formation.

In the U.S. formula feeding remains more popular than breast-feeding. In the current study, neonatal piglets were breast fed and compared with those fed commercially available milk-based formula (milk) or soy-based formula (soy) from postnatal day 2 (PND2) until death at PND21 (the usual age of weaning). Liver weights were greater in formula-fed piglets (P<0.05) than in breast-fed piglets (P<0.05). Affymetrix array analysis revealed significant differences in hepatic gene expression signatures between piglets fed breast milk or formula, as well as between piglets fed milk or soy. In males, expression of 346 hepatic genes differed between formula-fed and breast-fed piglets, and soy-fed differed from milk-fed piglets in 277 genes. Furthermore, gene expression profiles of males differed from females, even when the same diet was consumed. Serum cholesterol was lower in piglets fed formula relative to breast-fed piglets (P<0.05), and this was associated with elevations in mRNA encoding cholesterol 7α-hydroxylase (CYP7A1). Consistent with the human literature, breast-fed piglets had lower hepatic iron accumulation than formula-fed piglets. Hepcidin, a major regulator of hepatic iron trafficking, was elevated in piglets fed formula relative to breast-fed piglets (P<0.05). Female piglets fed soy formula had increased expression of CYP3A enzymes (P<0.05), and soy formula feeding decreased expression of several hepatic genes considered estrogen inducible. These data suggest that: 1) gene expression profiles in neonates differ significantly depending on the diet consumed, 2) hepatic iron storage and cholesterol metabolism clearly differ between breast and formula feeding in piglets, 3) there is no evidence that soy is estrogenic in neonatal pig liver.

Maternal obesity at conception increases the risk of offspring obesity, thus propagating an intergenerational vicious cycle. Male offspring born to obese dams are hyperresponsive to high fat-diets, gaining greater body weight, fat mass, and additional metabolic sequelae compared to lean controls. In this report, we identify the impact of maternal obesity before conception, on the embryo, and intrauterine milieu during the periimplantation period. We conducted global transcriptomic profiling in the uterus and periimplantation blastocyst, gene/protein expression analyses of inflammatory pathways in conjunction with endocrine and metabolic characterization in the dams at implantation. Uterine gene expression profiles of lean and obese dams revealed distinct signatures for genes regulating inflammation and lipid metabolism. Both pathway and gene-set enrichment analysis revealed uterine nuclear factor-κB and c-Jun N-terminal kinase signaling to be up-regulated in the uterus of obese dams, which was confirmed via immunoblotting. Obese uteri also evidenced an inflammatory secretome with higher chemokine mRNA abundance (CCL2, CCL5, CCL7, and CxCL10) and related regulators (TLR2, CD14, and Ccr1). Increased inflammation in the uterus was associated with ectopic lipid accumulation and expression of lipid metabolic genes. Gene expression in sex-identified male periimplantation blastocyst at day postcoitum 4.5 was clearly influenced by maternal obesity (359 transcripts, ±1.4-fold), including changes in developmental and epigenetic regulators. Akin to the uterus, nuclear factor-κB-regulated proinflammatory genes (CCL4 and CCL5) increased and expression of antioxidant (GPx3) and mitochondrial (TFAM and NRF1) genes decreased in the obese embryos. Our results suggest that ectopic lipid and inflammation may link maternal obesity to increased predisposition of offspring to obesity later in life.

Previous in vitro data suggest that ethanol (EtOH) activates NADPH oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption observed in vivo after EtOH exposure. In a rat model in which cycling females were infused intragastrically with EtOH-containing liquid diets, EtOH significantly decreased bone formation and stimulated osteoblast-dependent osteoclast differentiation. These effects were reversed by exogenous 17-β-estradiol coadministration. Moreover, coadministration of N-acetyl cysteine (NAC), an antioxidant, or diphenylene iodonium (DPI), a specific Nox inhibitor, also abolished chronic EtOH-associated bone loss. EtOH treatment up-regulated mRNA levels of Nox1, 2, 4, and the receptor activator of nuclear factor-κB ligand (RANKL), an essential factor for differentiation of osteoclasts in bone. Protein levels of Nox4, a major Nox isoform expressed in nonphagocytic cells, was also up-regulated by EtOH in bone. 17-β-Estradiol, NAC, and DPI were able to normalize EtOH-induced up-regulation of Nox and RANKL. In vitro experiments demonstrated that EtOH directly up-regulated Nox expression in osteoblasts. Pretreatment of osteoblasts with DPI eliminated EtOH-induced RANKL promoter activity. Furthermore, EtOH induced RANKL gene expression, and RANKL promoter activation in osteoblasts was ROS-dependent. These data suggest that inhibition of Nox expression and activity may be critical for prevention of chronic EtOH-induced osteoblast-dependent bone loss.

Background: Appropriate nutrition during early development is essential for maximal bone mass accretion; however, linkage between early nutrition, childhood bone mass, peak bone mass in adulthood, and prevention of bone loss later in life has not been studied., Methodology and Principal Findings: In this report, we show that feeding a high quality diet supplemented with blueberries (BB) to pre-pubertal rats throughout development or only between postnatal day 20 (PND20) and PND34 prevented ovariectomy (OVX)-induced bone loss in adult life. This protective effect of BB is due to suppression of osteoblastic cell senescence associated with acute loss of myosin expression after OVX. Early exposure of pre-osteoblasts to serum from BB-fed rats was found to consistently increase myosin expression. This led to maintenance osteoblastic cell development and differentiation and delay of cellular entrance into senescence through regulation of the Runx2 gene. High bone turnover after OVX results in insufficient collagenous matrix support for new osteoblasts and their precursors to express myosin and other cytoskeletal elements required for osteoblast activity and differentiation., Conclusions/significance: These results indicate: 1) a significant prevention of OVX-induced bone loss from adult rats can occur with only 14 days consumption of a BB-containing diet immediately prior to puberty; and 2) the molecular mechanisms underlying these effects involves increased myosin production which stimulates osteoblast differentiation and reduces mesenchymal stromal cell senescence.

In utero exposure to maternal obesity increases the offspring's risk of obesity in later life. We have also previously reported that offspring of obese rat dams develop hepatic steatosis, mild hyperinsulinemia, and a lipogenic gene signature in the liver at postnatal day (PND)21. In the current study, we examined systemic and hepatic adaptations in male Sprague-Dawley offspring from lean and obese dams at PND21. Indirect calorimetry revealed decreases in energy expenditure (p<0.001) and increases in RER values (p<0.001), which were further exacerbated by high fat diet (45% kcals from fat) consumption indicating an impaired ability to utilize fatty acids in offspring of obese dams as analyzed by PRCF. Mitochondrial function is known to be associated with fatty acid oxidation (FAO) in the liver. Several markers of hepatic mitochondrial function were reduced in offspring of obese dams. These included SIRT3 mRNA (p = 0.012) and mitochondrial protein content (p = 0.002), electron transport chain complexes (II, III, and ATPase), and fasting PGC-1α mRNA expression (p<0.001). Moreover, hepatic LCAD, a SIRT3 target, was not only reduced 2-fold (p<0.001) but was also hyperacetylated in offspring of obese dams (p<0.005) suggesting decreased hepatic FAO. In conclusion, exposure to maternal obesity contributes to early perturbations in whole body and liver energy metabolism. Mitochondrial dysfunction may be an underlying event that reduces hepatic fatty acid oxidation and precedes the development of detrimental obesity associated co-morbidities such as insulin resistance and NAFLD.

Diet and nutritional status are critical factors that influences bone development. In this report we demonstrate that a mixture of phenolic acids found in the serum of young rats fed blueberries (BB) significantly stimulated osteoblast differentiation, resulting in significantly increased bone mass. Greater bone formation in BB diet-fed animals was associated with increases in osteoblast progenitors and osteoblast differentiation and reduced osteoclastogenesis. Blockade of p38 phosphorylation eliminated effects of BB on activation of Wnt signaling in preosteoblasts. Knocking down β-catenin expression also blocked the ability of serum from BB diet-fed rats to stimulate osteoblast differentiation in vitro. Based on our in vivo and in vitro data, we propose that the underlying mechanisms of these powerful bone-promoting effects occur through β-catenin activation and the nuclear accumulation and transactivation of TCF/LEF gene transcription in bone and in osteoblasts. These results indicate stimulation of molecular events leading to osteoblast differentiation triggered by P38 MAP kinase (MAPK)/β-catenin canonical Wnt signaling results in significant increases in bone growth in young rats consuming BB-supplemented diets. Liquid chromatography/mass spectrometry (LC/MS) characterization of the serum after BB feeding revealed a mixture of simple phenolic acids that may provide a basis for developing a new treatment to increase peak bone mass and delay degenerative bone disorders such as osteoporosis., (© 2010 American Society for Bone and Mineral Research.)

Background: It is well established that excessive consumption of a high fat diet (HFD) results in obesity; however, the consequences of obesity on postnatal skeletal development have not been well studied., Methodology and Principal Findings: Total enteral nutrition (TEN) was used to feed postnatal day 27 male rats intragastrically with a high 45% fat diet (HFD) for four weeks to induce obesity. Fat mass was increased compared to rats fed TEN diets containing 25% fat (medium fat diet, MFD) or a chow diet (low fat diet, LFD) fed ad libitum with matched body weight gains. Serum leptin and total non-esterified fatty acids (NEFA) were elevated in HFD rats, which also had reduced bone mass compared to LFD-fed animals. This was accompanied by decreases in bone formation, but increases in the bone resorption. Bone marrow adiposity and expression of adipogenic genes, PPARγ and aP2 were increased, whereas osteoblastogenic markers osteocalcin and Runx2 were decreased, in bone in HFD rats compared to LFD controls. The diversion of stromal cell differentiation in response to HFD stemmed from down-regulation of the key canonical Wnt signaling molecule β-catenin protein and reciprocal up-regulation of nuclear PPARγ expression in bone. In a set of in vitro studies using pluripotent ST2 bone marrow mesenchymal stromal cells treated with serum from rats on the different diets or using the free fatty acid composition of NEFA quantified in rat serum from HFD-fed animals by GC-MS, we were able to recapitulate our in vivo findings., Conclusions/significance: These observations strongly suggest that increased NEFA in serum from rats made obese by HFD-feeding impaired bone formation due to stimulation of bone marrow adipogenesis. These effects of obesity on bone in early life may result in impaired attainment of peak bone mass and therefore increase the prevalence of osteoporosis later on in life.

The mechanisms by which chronic ethanol intake induces bone loss remain unclear. In females, the skeletal response to ethanol varies depending on physiologic status (e.g., cycling, pregnancy, or lactation). Ethanol-induced oxidative stress appears to be a key event leading to skeletal toxicity. In this study, ethanol-containing liquid diets were fed to postlactational female Sprague-Dawley rats intragastrically for 4 weeks beginning at weaning. Ethanol consumption decreased bone mineral density (BMD) compared with control animals during this period of bone rebuilding following the end of lactation. Coadministration of the antioxidant N-acetylcysteine (NAC) was able to block bone loss and downregulation of the bone-formation markers alkaline phosphatase and osteocalcin in serum and gene expression in bone. Real-time array analysis of total RNA isolated from bone tissue revealed that the majority of Wnt signaling components were downregulated by chronic ethanol infusion. Real-time PCR confirmed downregulated gene expression in a subset of the Wnt signaling components by ethanol. However, the Wnt antagonist DKK1 was upregulated by ethanol. The key canonical Wnt signaling molecule beta-catenin protein expression was inhibited, while glycogen synthase kinase-3-beta was dephosphorylated by ethanol in bone and preosteoblastic cells. These actions of ethanol were blocked by NAC. Ethanol treatment inactivated TCF/LEF gene transcription, eliminated beta-catenin nuclear translocation in osteoblasts, and reciprocally suppressed osteoblastogenesis and enhanced adipogenesis. These effects of ethanol on lineage commitment of mesenchymal stem cells were eliminated by NAC pretreatment. These observations are consistent with the hypothesis that ethanol inhibits bone formation through stimulation of oxidative stress to suppress Wnt signaling., ((c) 2010 American Society for Bone and Mineral Research.)

The importance of ethanol metabolism in the development of alcoholic liver disease remains controversial. The present study examined the effects of selective inhibition of the cytochrome P450 enzyme CYP2E1 compared with the inhibition of overall ethanol metabolism on the development of alcoholic steatohepatitis. Adult male Sprague-Dawley rats were fed via total enteral nutrition for 45 days with or without 10-12g/kg/d ethanol. Some groups were given 200mg/kg/d of the CYP2E1 inhibitor diallyl sulfide (DAS). Other groups were treated with 164mg/kg/d of the alcohol dehydrogenase (ADH) inhibitor 4-methylpyrazole (4-MP) and dosed at 2-3g/kg/d ethanol to maintain similar average urine ethanol concentrations. Liver pathology scores and levels of apoptosis were elevated by ethanol (P<.05) but did not differ significantly on cotreatment with DAS or 4-MP. However, liver triglycerides were lower when ethanol-fed rats were treated with DAS or 4-MP (P<.05). Serum alanine aminotransferase values were significantly lower in ethanol-fed 4-MP-treated rats indicating reduced necrosis. Hepatic oxidative stress and the endoplasmic reticulum (ER) stress marker tribbles-related protein 3 were increased after ethanol (P<.05); further increased by DAS but partly attenuated by 4-MP. Both DAS and 4-MP reversed ethanol increases in the cytokine, tumor necrosis factor-alpha (TNF-alpha), and the chemokine CXCL-2 (P<.05). However, neither inhibitors prevented ethanol suppression of interleukins IL-4 or IL-12. Moreover, neither inhibitors prevented ethanol increases in tumor growth factor-beta mRNA. Ethanol and DAS additively induced hepatic hyperplasia (P<.05). These data suggest that a significant proportion of hepatic injury after ethanol exposure is independent of alcohol metabolism. Ethanol metabolism by CYP2E1 may be linked in part to triglyceride accumulation, to induction of TNF-alpha, and to chemokine production. Ethanol metabolism by ADH may be linked in part to oxidative and ER stress and necrotic injury., (2010 Elsevier Inc. All rights reserved.)

Relatively few studies have examined the effects of formula feeding relative to breast-feeding on bone in the neonate. Using peripheral quantitative CT scan and histomorphometric analysis, we demonstrated that neonatal piglets fed with soy-based formula (SF) and cow milk-based formula (MF) for 21 or 35 d had greater bone mineral density and content than breast-fed piglets (BF) (P < 0.05). Osteoblast numbers and bone formation rate at postnatal d 35 were greater in SF compared with other groups (P < 0.05), whereas osteoclast numbers were lower in both MF and SF groups than in the BF group (P < 0.05). Osteoblastogenesis was greater in ex vivo bone marrow cell cultures from SF than in MF or BF piglets (P < 0.05). Bone formation markers in serum were greater, whereas bone resorption markers were lower in the MF- and SF-fed groups than in the BF group (P < 0.05). Bone morphogenic protein (BMP) 2 and alkaline phosphatase mRNAs were upregulated in the MF and SF groups compared with the BF group (P < 0.05), whereas receptor activator of NF-kappaB ligand was downregulated (P < 0.05). Extracellular signal-regulated kinase, p38, Smad1/5/8 phosphorylation, and runt-related transcription factor 2 expression were greater in bone from the MF and SF groups compared with the BF group (P < 0.05). In vitro studies showed that 2.5% serum from SF- or MF-fed piglets was able to stimulate osteoblast differentiation but not in the presence of the BMP blocker noggin. Therefore, formula feeding promoted bone growth compared with BF. SF piglets had the highest bone volume over tissue volume. This suggests that SF-fed piglets may have the best quality bone. The anabolic effects of SF on bone appear to be mediated through enhanced BMP signaling.

The process of chondrogenesis can be mimicked in vitro by insulin treatment of mouse ATDC5 chondroprogenitor cells. To identify novel factors that are involved in the control of chondrogenesis, we carried out a large-scale screening through retroviral insertion mutagenesis and isolated a fast-growing ATDC5 clone incapable of chondrogenic differentiation. Inverse-PCR analysis of this clone revealed that the retroviral DNA was inserted into the promoter region of mouse Id2 (inhibitor of DNA-binding protein 2) gene. This retroviral insertion increased Id2 protein levels to twice those found in normal ATDC5 cells. To investigate whether an elevated level of Id2 protein was responsible for inhibition of chondrogenic differentiation, ATDC5 cells were infected with a retrovirus to stably express Id2. ATDC5 cells expressing ectopic Id2 exhibited signs of de-differentiation, such as rapid growth, and insulin failed to induce expression of Sox9 (Sry-type high-mobility-group box 9) or matrix genes such as type II collagen (COL2) in these cells. When endogenous Id2 was knocked down by siRNA (small interfering RNA) in ATDC5 cells, expression of Sox9 and COL2 was increased and chondrogenic differentiation was accelerated. To examine how Id2 is expressed in chondrocytes in vivo, we carried out immunostaining of E16.5 mouse embryos and found that Id2 is expressed in articular chondrocytes and proliferating chondrocytes, but barely detectable in hypertrophic chondrocytes. Our results suggest that proper expression of Id2 is important to achieving a fine balance between growth and differentiation during chondrogenesis.