Michael L. Blackburn

Obesity Reduces Bone Density Associated with Activation of PPARγ and Suppression of Wnt/β-Catenin in Rapidly Growing Male Rats

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.

Maternal Obesity Promotes a Proinflammatory Signature in Rat Uterus and Blastocyst

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.

A role for ethanol-induced oxidative stress in controlling lineage commitment of mesenchymal stromal cells through inhibition of Wnt/β-catenin signaling

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 β‐catenin protein expression was inhibited, while glycogen synthase kinase‐3‐β 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 β‐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.

Dietary-induced serum phenolic acids promote bone growth via p38 MAPK/β-catenin canonical Wnt signaling.

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.

Maternal Obesity during Gestation Impairs Fatty Acid Oxidation and Mitochondrial SIRT3 Expression in Rat Offspring at Weaning

In utero exposure to maternal obesity increases the offsprings 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.

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

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.

Ethanol Impairs Estrogen Receptor Signaling Resulting in Accelerated Activation of Senescence Pathways, Whereas Estradiol Attenuates the Effects of Ethanol in Osteoblasts

Epidemiological and animal studies have suggested that chronic alcohol consumption is a major risk factor for osteoporosis. Using bone from cycling female rats infused chronically with ethanol (EtOH) in vivo and osteoblastic cells in vitro, we found that EtOH significantly increased estrogen receptor α (ERα) and β (ERβ) mRNA and ERα protein levels. Treatment with 17β‐estradiol (E2) in vivo and in vitro interfered with these effects of EtOH on bone and osteoblastic cells. ERα agonist propylpyrazoletriol (PPT) and ERβ agonist diarylpropionitrile (DPN) attenuated EtOH‐induced ERα and ERβ gene overexpression, respectively. Similar to the ER antagonist ICI 182780, EtOH blocked nuclear translocation of ERα‐ECFP in the presence of E2 in UMR‐106 osteoblastic cells. EtOH also downregulated ERE‐luc reporter activity. On the other hand, EtOH by itself upregulated some common ERα‐ and ERβ‐mediated genes apparently by an ER‐independent pathway. EtOH also transactivated the luciferase activity of the p21 promoter region independent of additional exogenous ERα, activated p21 and p53, and stimulated senescence‐associated β‐galactosidase activity in rat stromal osteoblasts. E2 treatment attenuated these EtOH actions. We conclude that inhibitory cross‐talk between EtOH and E2 in osteoblasts on ERs, p53/p21, and cell senescence provides a pathophysiologic mechanism underlying bone loss and the protective effects of estrogens in alcohol‐exposed females.

The role of ethanol metabolism in development of alcoholic steatohepatitis in the rat.

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.

Feeding Blueberry Diets in Early Life Prevent Senescence of Osteoblasts and Bone Loss in Ovariectomized Adult Female Rats

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.

Inhibition of NADPH Oxidases Prevents Chronic Ethanol-Induced Bone Loss in Female Rats

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.