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Obesity reduces bone density associated with activation of PPARγ and suppression of Wnt/β-catenin in rapidly growing male rats.

Chen JR, Lazarenko OP, Wu X, Tong Y, Blackburn ML, Shankar K, Badger TM, Ronis MJ - PLoS ONE (2010)

Bottom Line: This was accompanied by decreases in bone formation, but increases in the bone resorption.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America. chenjinran@uams.edu

ABSTRACT

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.

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Related in: MedlinePlus

Serum from HFD-induced obese rats and an artificial FA mixture suppress osteoblast differentiation.(A), ST2 cells were cultured in 12 well plates. Cells were treated with 2% serum from LFD, MFD or HFD rats, 50 ng/ml Wnt3a, 400 µM FAs and their combination for 7 days in the presence or absence of osteogenic medium. Alkaline phophatase staining was performed. (B), 2% serum from HFD-induced obese rats and an artificial FA mixture significantly decreased TCF/LEF-dependent transcription of a luciferase reporter gene (TOPFLASH) in C2C12 osteoblastic cells compared with cells treated with LFD serum. Luciferase activity in C2C12 cells transfected with a PPRE-luc reporter construct and treated with 2% serum from LFD, MFD or HFD-fed rats, 50 ng/ml Wnt3a, 400 µM FAs and their combination for 24 h. (C), β-catenin gene was knock down using β-catenin siRNA in ST2 cells. After 24 h of β-catenin siRNA, cell proteins were collected and western blot was performed for β-catenin and PPARγ. Bars are expressed as mean ± SEM in triplicates. *, P<0.05, versus control by ANOVA followed by Student-Newman-Keuls post hoc analysis for multiple pairwise comparisons.
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pone-0013704-g007: Serum from HFD-induced obese rats and an artificial FA mixture suppress osteoblast differentiation.(A), ST2 cells were cultured in 12 well plates. Cells were treated with 2% serum from LFD, MFD or HFD rats, 50 ng/ml Wnt3a, 400 µM FAs and their combination for 7 days in the presence or absence of osteogenic medium. Alkaline phophatase staining was performed. (B), 2% serum from HFD-induced obese rats and an artificial FA mixture significantly decreased TCF/LEF-dependent transcription of a luciferase reporter gene (TOPFLASH) in C2C12 osteoblastic cells compared with cells treated with LFD serum. Luciferase activity in C2C12 cells transfected with a PPRE-luc reporter construct and treated with 2% serum from LFD, MFD or HFD-fed rats, 50 ng/ml Wnt3a, 400 µM FAs and their combination for 24 h. (C), β-catenin gene was knock down using β-catenin siRNA in ST2 cells. After 24 h of β-catenin siRNA, cell proteins were collected and western blot was performed for β-catenin and PPARγ. Bars are expressed as mean ± SEM in triplicates. *, P<0.05, versus control by ANOVA followed by Student-Newman-Keuls post hoc analysis for multiple pairwise comparisons.

Mentions: We next examined the ability of serum from the HFD-induced obese rats and of a mixture of FAs to affect osteoblast differentiation. To do this, bone marrow derived mesenchymal stromal ST2 cells were first treated with media containing 2% serum from animals from each diet group for 72 h. Similar to the in vivo data, real-time PCR revealed that β-catenin gene expression was down-regulated by serum from the HFD-induced obese animals compared to serum from LFD controls (P<0.05; Figure 5A). On the other hand, PPARγ gene expression was up-regulated by HFD-induced obese rat serum (P<0.05; Figure 5A). Similar to previous studies with macrophages [19], we examined the effects of FAs (a 2∶1 mixture of palmitate and oleate acids) which are known to be elevated in serum of obese individuals on ST2 cells and found a similar pattern of β-catenin and PPARγ gene expression. After 48 h treatment, β-catenin mRNA was down-regulated in a concentration-dependent manner; whereas, PPARγ was up-regulated by FFAs (P<0.05; Figure 5B). Consistent with real-time data, western blot showed an inverse association between β-catenin and PPARγ. When ST2 cells were treated with FAs, down-regulated β-catenin was also accompanied with up-regulated PPARγ (P<0.05; Figure 5C). On the other hand, when cells were treated with soluble Wnt3a, a well known β-catenin agonist, up-regulated β-catenin was accompanied with down-regulation of PPARγ expression (P<0.05; Figure 5C). To further examine composition and concentration of FAs in serum NEFA from our experimental animals, we used GC-MS after TLC separation (Figure 6A). We found that there were 5 major FAs: palmitic, stearic, oleic, linoleic and arachidonic acid in the ratio of 5∶1∶2∶3∶1 in rat serum and roughly 5-fold higher concentrations in HFD-induced obese rats with palmitate as the most prominent FA compared to either of the other two diet groups (Figure 6B). We next tested whether the mixture of five FAs based on the ratio and concentration of NEFA appearing in serum from obese rats would regulate β-catenin and PPARγ in pre-osteoblasts and observed up-regulated PPARγ and concomitantly down-regulate β-catenin expression (Figure 6C, D). The up-regulation of PPARγ was also found in osteogenic cells, such as calvarial cells isolated from neonatal rat calvaria (data not shown here). ST2 cells were also treated with serum from rats from each diet group or FAs in the presence of either osteogenic (OB) medium or Wnt3a for 8 d. Both serum from the HFD-induced obese rats and FAs suppressed bone specific alkaline phosphatase (ALP) activity as assessed by ALP staining (P<0.05; Figure 7A), indicating that osteoblast differentiation was suppressed. We next explored potential mechanisms by which HFD-induced rat serum and FAs could attenuate pro-osteogenic canonical Wnt signaling. While both serum from HFD-fed rats and FAs significantly increased PPARγ promoter activity as determined by a PPRE-luciferase reporter assay in C2C12 cells (Figure 7B), β-catenin/TCF-mediated transcription was suppressed (P<0.05; Figure 7B). We finally examined whether there is an inter-relation between suppressed β-catenin and activated PPARγ in pre-osteoblasts. C2C12 cells were transfected with siRNA against β-catenin. Silencing of β-catenin in pre-osteoblasts increased expression of endogenous PPARγ (Figure 7C), indicating the possible existence of switch programs in pre-osteoblasts that direct differentiation to either osteoblasts or adipocytes under appropriate stimuli. Finally, we have examined whether the increased expression of PPARγ leads to increased activity and transcriptional regulation of target genes. Sensitive TransAMTM transcription factor ELISA was performed and DNA binding for activated PPARγ transcription factor was analyzed using samples from both in vivo and in vitro. As data depicted in Figure 8A, transcriptional factor abundance was significantly increased in bone from obese animals and in pre-osteoblasts treated with FAs and serum from obese animals. To further detect whether FAs enhances binding of PPARγ and its target genes, ChIP assay was carried out on the mouse aP2 gene (Figure 8B). We used an antibody against mouse PPARγ and subsequent PCR amplification of adjacent PPRE in the enhancer of the murine aP2 (a known target gene for PPARγ) gene. We found that there was a pronounced increase in the binding of PPARγ to the aP2 enhancer in ST2 cells treated with FAs. These data indicated that HFD-induced obesity and FAs not only increase PPARγ expression but also its transcriptional activity.


Obesity reduces bone density associated with activation of PPARγ and suppression of Wnt/β-catenin in rapidly growing male rats.

Chen JR, Lazarenko OP, Wu X, Tong Y, Blackburn ML, Shankar K, Badger TM, Ronis MJ - PLoS ONE (2010)

Serum from HFD-induced obese rats and an artificial FA mixture suppress osteoblast differentiation.(A), ST2 cells were cultured in 12 well plates. Cells were treated with 2% serum from LFD, MFD or HFD rats, 50 ng/ml Wnt3a, 400 µM FAs and their combination for 7 days in the presence or absence of osteogenic medium. Alkaline phophatase staining was performed. (B), 2% serum from HFD-induced obese rats and an artificial FA mixture significantly decreased TCF/LEF-dependent transcription of a luciferase reporter gene (TOPFLASH) in C2C12 osteoblastic cells compared with cells treated with LFD serum. Luciferase activity in C2C12 cells transfected with a PPRE-luc reporter construct and treated with 2% serum from LFD, MFD or HFD-fed rats, 50 ng/ml Wnt3a, 400 µM FAs and their combination for 24 h. (C), β-catenin gene was knock down using β-catenin siRNA in ST2 cells. After 24 h of β-catenin siRNA, cell proteins were collected and western blot was performed for β-catenin and PPARγ. Bars are expressed as mean ± SEM in triplicates. *, P<0.05, versus control by ANOVA followed by Student-Newman-Keuls post hoc analysis for multiple pairwise comparisons.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965663&req=5

pone-0013704-g007: Serum from HFD-induced obese rats and an artificial FA mixture suppress osteoblast differentiation.(A), ST2 cells were cultured in 12 well plates. Cells were treated with 2% serum from LFD, MFD or HFD rats, 50 ng/ml Wnt3a, 400 µM FAs and their combination for 7 days in the presence or absence of osteogenic medium. Alkaline phophatase staining was performed. (B), 2% serum from HFD-induced obese rats and an artificial FA mixture significantly decreased TCF/LEF-dependent transcription of a luciferase reporter gene (TOPFLASH) in C2C12 osteoblastic cells compared with cells treated with LFD serum. Luciferase activity in C2C12 cells transfected with a PPRE-luc reporter construct and treated with 2% serum from LFD, MFD or HFD-fed rats, 50 ng/ml Wnt3a, 400 µM FAs and their combination for 24 h. (C), β-catenin gene was knock down using β-catenin siRNA in ST2 cells. After 24 h of β-catenin siRNA, cell proteins were collected and western blot was performed for β-catenin and PPARγ. Bars are expressed as mean ± SEM in triplicates. *, P<0.05, versus control by ANOVA followed by Student-Newman-Keuls post hoc analysis for multiple pairwise comparisons.
Mentions: We next examined the ability of serum from the HFD-induced obese rats and of a mixture of FAs to affect osteoblast differentiation. To do this, bone marrow derived mesenchymal stromal ST2 cells were first treated with media containing 2% serum from animals from each diet group for 72 h. Similar to the in vivo data, real-time PCR revealed that β-catenin gene expression was down-regulated by serum from the HFD-induced obese animals compared to serum from LFD controls (P<0.05; Figure 5A). On the other hand, PPARγ gene expression was up-regulated by HFD-induced obese rat serum (P<0.05; Figure 5A). Similar to previous studies with macrophages [19], we examined the effects of FAs (a 2∶1 mixture of palmitate and oleate acids) which are known to be elevated in serum of obese individuals on ST2 cells and found a similar pattern of β-catenin and PPARγ gene expression. After 48 h treatment, β-catenin mRNA was down-regulated in a concentration-dependent manner; whereas, PPARγ was up-regulated by FFAs (P<0.05; Figure 5B). Consistent with real-time data, western blot showed an inverse association between β-catenin and PPARγ. When ST2 cells were treated with FAs, down-regulated β-catenin was also accompanied with up-regulated PPARγ (P<0.05; Figure 5C). On the other hand, when cells were treated with soluble Wnt3a, a well known β-catenin agonist, up-regulated β-catenin was accompanied with down-regulation of PPARγ expression (P<0.05; Figure 5C). To further examine composition and concentration of FAs in serum NEFA from our experimental animals, we used GC-MS after TLC separation (Figure 6A). We found that there were 5 major FAs: palmitic, stearic, oleic, linoleic and arachidonic acid in the ratio of 5∶1∶2∶3∶1 in rat serum and roughly 5-fold higher concentrations in HFD-induced obese rats with palmitate as the most prominent FA compared to either of the other two diet groups (Figure 6B). We next tested whether the mixture of five FAs based on the ratio and concentration of NEFA appearing in serum from obese rats would regulate β-catenin and PPARγ in pre-osteoblasts and observed up-regulated PPARγ and concomitantly down-regulate β-catenin expression (Figure 6C, D). The up-regulation of PPARγ was also found in osteogenic cells, such as calvarial cells isolated from neonatal rat calvaria (data not shown here). ST2 cells were also treated with serum from rats from each diet group or FAs in the presence of either osteogenic (OB) medium or Wnt3a for 8 d. Both serum from the HFD-induced obese rats and FAs suppressed bone specific alkaline phosphatase (ALP) activity as assessed by ALP staining (P<0.05; Figure 7A), indicating that osteoblast differentiation was suppressed. We next explored potential mechanisms by which HFD-induced rat serum and FAs could attenuate pro-osteogenic canonical Wnt signaling. While both serum from HFD-fed rats and FAs significantly increased PPARγ promoter activity as determined by a PPRE-luciferase reporter assay in C2C12 cells (Figure 7B), β-catenin/TCF-mediated transcription was suppressed (P<0.05; Figure 7B). We finally examined whether there is an inter-relation between suppressed β-catenin and activated PPARγ in pre-osteoblasts. C2C12 cells were transfected with siRNA against β-catenin. Silencing of β-catenin in pre-osteoblasts increased expression of endogenous PPARγ (Figure 7C), indicating the possible existence of switch programs in pre-osteoblasts that direct differentiation to either osteoblasts or adipocytes under appropriate stimuli. Finally, we have examined whether the increased expression of PPARγ leads to increased activity and transcriptional regulation of target genes. Sensitive TransAMTM transcription factor ELISA was performed and DNA binding for activated PPARγ transcription factor was analyzed using samples from both in vivo and in vitro. As data depicted in Figure 8A, transcriptional factor abundance was significantly increased in bone from obese animals and in pre-osteoblasts treated with FAs and serum from obese animals. To further detect whether FAs enhances binding of PPARγ and its target genes, ChIP assay was carried out on the mouse aP2 gene (Figure 8B). We used an antibody against mouse PPARγ and subsequent PCR amplification of adjacent PPRE in the enhancer of the murine aP2 (a known target gene for PPARγ) gene. We found that there was a pronounced increase in the binding of PPARγ to the aP2 enhancer in ST2 cells treated with FAs. These data indicated that HFD-induced obesity and FAs not only increase PPARγ expression but also its transcriptional activity.

Bottom Line: This was accompanied by decreases in bone formation, but increases in the bone resorption.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America. chenjinran@uams.edu

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus