Publications by authors named "Michael L Blackburn"

49 Publications

On the potential role of globins in brown adipose tissue: a novel conceptual model and studies in myoglobin knockout mice.

Am J Physiol Endocrinol Metab 2021 May 10. Epub 2021 May 10.

Arkansas Children's Nutrition Center, Little Rock, AR, USA.

Myoglobin (Mb) regulates O bioavailability in muscle and heart as partial pressure of O (pO) drops with increased tissue workload. Globin proteins also modulate cellular NO pools, "scavenging" NO at higher pO and converting NO to NO as pO 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, 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 wildtype mice and lower expression relative to muscle and heart.
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http://dx.doi.org/10.1152/ajpendo.00662.2020DOI Listing
May 2021

Metabolic physiology and skeletal muscle phenotypes in male and female myoglobin knockout mice.

Am J Physiol Endocrinol Metab 2021 May 10. Epub 2021 May 10.

Arkansas Children's Nutrition Center, Little Rock, AR, USA.

Myoglobin (Mb) is a regulator of O bioavailability in type I muscle and heart, at least when tissue O levels drop. Mb also plays a role in regulating cellular 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, suggests 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 wildtype (WT) mice challenged with a high fat diet (HFD, 45% of calories). In males (n=10/genotype) and females (n=9/genotype) aged 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 to within-sex WT controls. At ~18 and ~19 wk of age, female Mb adiposity was ~42-48% higher vs. 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.
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http://dx.doi.org/10.1152/ajpendo.00624.2020DOI Listing
May 2021

Carnitine palmitoyltransferase 2 knockout potentiates palmitate-induced insulin resistance in CC myotubes.

Am J Physiol Endocrinol Metab 2020 08 27;319(2):E265-E275. Epub 2020 May 27.

Arkansas Children's Nutrition Center, Little Rock, Arkansas.

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.
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http://dx.doi.org/10.1152/ajpendo.00515.2019DOI Listing
August 2020

Neonatal diet impacts liver mitochondrial bioenergetics in piglets fed formula or human milk.

BMC Nutr 2020 15;6:13. Epub 2020 Apr 15.

2Arkansas Children's Research Institute, Little Rock, AR USA.

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.
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http://dx.doi.org/10.1186/s40795-020-00338-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158137PMC
April 2020

Maternal regulation of SATB2 in osteo-progeniters impairs skeletal development in offspring.

FASEB J 2020 02 23;34(2):2511-2523. Epub 2019 Dec 23.

Arkansas Children's Nutrition Center, Little Rock, AR, USA.

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 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.
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http://dx.doi.org/10.1096/fj.201901901RDOI Listing
February 2020

3-(3-Hydroxyphenyl)-Propionic Acid (PPA) Suppresses Osteoblastic Cell Senescence to Promote Bone Accretion in Mice.

JBMR Plus 2019 Sep 23;3(9):e10201. Epub 2019 Aug 23.

Arkansas Children's Nutrition Center Little Rock AR USA.

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. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6808226PMC
September 2019

Estradiol and NADPH oxidase crosstalk regulates responses to high fat feeding in female mice.

Exp Biol Med (Maywood) 2019 07 4;244(10):834-845. Epub 2019 Jun 4.

1 Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

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http://dx.doi.org/10.1177/1535370219853563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6643193PMC
July 2019

Blood cytokine patterns suggest a modest inflammation phenotype in subjects with long-chain fatty acid oxidation disorders.

Physiol Rep 2019 03;7(6):e14037

Arkansas Children's Nutrition Center, Little Rock, Arkansas.

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.
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http://dx.doi.org/10.14814/phy2.14037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434073PMC
March 2019

Is palmitate truly proinflammatory? Experimental confounders and context-specificity.

Am J Physiol Endocrinol Metab 2018 11 17;315(5):E780-E794. Epub 2018 Jul 17.

Arkansas Children's Nutrition Center , Little Rock, Arkansas.

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 CC 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 CC 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 CC 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.
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http://dx.doi.org/10.1152/ajpendo.00187.2018DOI Listing
November 2018

Application of an In Vivo Hepatic Triacylglycerol Production Method in the Setting of a High-Fat Diet in Mice.

Nutrients 2016 Dec 28;9(1). Epub 2016 Dec 28.

Arkansas Children's Nutrition Center, Little Rock, AR 72202, USA.

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.
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http://dx.doi.org/10.3390/nu9010016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295060PMC
December 2016

Maternal Obesity Programs Senescence Signaling and Glucose Metabolism in Osteo-Progenitors From Rat and Human.

Endocrinology 2016 Nov 21;157(11):4172-4183. Epub 2016 Sep 21.

Arkansas Children's Nutrition Center (J.-R.C., O.P.L., M.L.B., T.M.B., A.A., K.S.), Department of Pediatrics (J.-R.C., O.P.L., M.L.B., R.E.F., T.M.B., A.A., K.S.), University of Arkansas for Medical Sciences, and Arkansas Children's Hospital Research Institute (S.R., R.E.F.), Little Rock, Arkansas 72202.

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.
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http://dx.doi.org/10.1210/en.2016-1408DOI Listing
November 2016

Dietary factors during early life program bone formation in female rats.

FASEB J 2017 01 12;31(1):376-387. Epub 2016 Oct 12.

Arkansas Children's Nutrition Center, Little Rock, Arkansas, USA; and.

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.
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http://dx.doi.org/10.1096/fj.201600703RDOI Listing
January 2017

RNA-sequencing data analysis of uterus in ovariectomized rats fed with soy protein isolate, 17β-estradiol and casein.

Data Brief 2016 Jun 21;7:1491-6. Epub 2016 Apr 21.

Arkansas Children's Nutrition Center, Little Rock, AR, United States.

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].
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http://dx.doi.org/10.1016/j.dib.2016.04.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857400PMC
June 2016

Uterine responses to feeding soy protein isolate and treatment with 17β-estradiol differ in ovariectomized female rats.

Toxicol Appl Pharmacol 2016 Apr 3;297:68-80. Epub 2016 Mar 3.

Arkansas Children's Nutrition Center, University of Arkansas for Medical Sciences, Little Rock, AR 72202, United States; Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72202, United States.

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.
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http://dx.doi.org/10.1016/j.taap.2016.02.019DOI Listing
April 2016

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

J Biol Chem 2015 Jun 28;290(23):14692-704. Epub 2015 Apr 28.

From the Arkansas Children's Nutrition Center and the Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202.

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.
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http://dx.doi.org/10.1074/jbc.M114.633461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505535PMC
June 2015

Soy protein isolate inhibits high-fat diet-induced senescence pathways in osteoblasts to maintain bone acquisition in male rats.

Endocrinology 2015 Feb 9;156(2):475-87. Epub 2014 Dec 9.

Arkansas Children's Nutrition Center (J.-R.C., O.P.L, M.L.B., T.M.B., M.J.J.R.), Little Rock, Arkansas 72202; and Department of Pediatrics (J.-R.C., O.P.L, M.L.B., T.M.B., M.J.J.R.), University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202.

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.
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http://dx.doi.org/10.1210/en.2014-1427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4298323PMC
February 2015

In utero exposure to prepregnancy maternal obesity and postweaning high-fat diet impair regulators of mitochondrial dynamics in rat placenta and offspring.

Physiol Genomics 2014 Dec 21;46(23):841-50. Epub 2014 Oct 21.

Arkansas Children's Nutrition Center, Little Rock, Arkansas; and Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas

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.
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http://dx.doi.org/10.1152/physiolgenomics.00059.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254938PMC
December 2014

Trace element status and zinc homeostasis differ in breast and formula-fed piglets.

Exp Biol Med (Maywood) 2015 Jan 1;240(1):58-66. Epub 2014 Sep 1.

Arkansas Children's Nutrition Center, Little Rock, AR 72202, USA Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

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.
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http://dx.doi.org/10.1177/1535370214547162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935178PMC
January 2015

Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways.

FASEB J 2014 Jul 9;28(7):3134-45. Epub 2014 Apr 9.

Arkansas Children's Nutrition Center, Little Rock, Arkansas, USA; Department of Pediatrics and

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.
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http://dx.doi.org/10.1096/fj.13-243659DOI Listing
July 2014

Influence of fat/carbohydrate ratio on progression of fatty liver disease and on development of osteopenia in male rats fed alcohol via total enteral nutrition (TEN).

Alcohol 2014 Mar 1;48(2):133-44. Epub 2014 Feb 1.

Arkansas Children's Nutrition Center, Little Rock, AR, USA; Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, USA; Department of Physiology & Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

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.
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http://dx.doi.org/10.1016/j.alcohol.2013.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970237PMC
March 2014

High fat diet and in utero exposure to maternal obesity disrupts circadian rhythm and leads to metabolic programming of liver in rat offspring.

PLoS One 2014 9;9(1):e84209. Epub 2014 Jan 9.

Arkansas Children's Nutrition Center, Little Rock, Arkansas, United States of America ; Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084209PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3886966PMC
September 2014

Feeding blueberry diets to young rats dose-dependently inhibits bone resorption through suppression of RANKL in stromal cells.

PLoS One 2013 6;8(8):e70438. Epub 2013 Aug 6.

Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070438PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3735613PMC
April 2014

Diet-derived phenolic acids regulate osteoblast and adipocyte lineage commitment and differentiation in young mice.

J Bone Miner Res 2014 ;29(5):1043-53

Arkansas Children's Nutrition Center, University of Arkansas for Medical Sciences, Little Rock, AR, USA; Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

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.
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http://dx.doi.org/10.1002/jbmr.2034DOI Listing
December 2014

Soy protein isolates prevent loss of bone quantity associated with obesity in rats through regulation of insulin signaling in osteoblasts.

FASEB J 2013 Sep 17;27(9):3514-23. Epub 2013 Jun 17.

Arkansas Children's Nutrition Center, 15 Children's Way, Slot 512-20B, Little Rock, AR 72202, USA.

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.
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http://dx.doi.org/10.1096/fj.12-226464DOI Listing
September 2013

Mammary gland morphology and gene expression differ in female rats treated with 17β-estradiol or fed soy protein isolate.

Endocrinology 2012 Dec 1;153(12):6021-32. Epub 2012 Oct 1.

Arkansas Children's Nutrition Center, Little Rock, AR 72202, USA.

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.
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http://dx.doi.org/10.1210/en.2012-1591DOI Listing
December 2012

Blueberry consumption prevents loss of collagen in bone matrix and inhibits senescence pathways in osteoblastic cells.

Age (Dordr) 2013 Jun 4;35(3):807-20. Epub 2012 May 4.

Arkansas Children's Nutrition Center, Slot 512-20B 15 Children's Way, Little Rock, AR 72202, USA.

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.
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http://dx.doi.org/10.1007/s11357-012-9412-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636388PMC
June 2013

Differential effects of short term feeding of a soy protein isolate diet and estrogen treatment on bone in the pre-pubertal rat.

PLoS One 2012 20;7(4):e35736. Epub 2012 Apr 20.

Arkansas Children's Nutrition Center, Little Rock, Arkansas, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035736PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335011PMC
August 2012

RNA-seq analysis of the functional compartments within the rat placentation site.

Endocrinology 2012 Apr 21;153(4):1999-2011. Epub 2012 Feb 21.

Arkansas Children's Nutrition Center, 15 Children's Way, Slot 512-20B, Little Rock, Arkansas 72202, USA.

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.
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http://dx.doi.org/10.1210/en.2011-1833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393303PMC
April 2012

Inhibition of fetal bone development through epigenetic down-regulation of HoxA10 in obese rats fed high-fat diet.

FASEB J 2012 Mar 30;26(3):1131-41. Epub 2011 Nov 30.

Arkansas Children's Nutrition Center, 15 Children's Way, Slot 512-20B, Little Rock, AR 72202, USA.

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.
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http://dx.doi.org/10.1096/fj.11-197822DOI Listing
March 2012

Formula feeding alters hepatic gene expression signature, iron and cholesterol homeostasis in the neonatal pig.

Physiol Genomics 2011 Dec 27;43(23):1281-93. Epub 2011 Sep 27.

Arkansas Children's Nutrition Center and Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72202, USA.

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.
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http://dx.doi.org/10.1152/physiolgenomics.00055.2011DOI Listing
December 2011