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. 2024 Apr 1;15(2):767-786.
doi: 10.14336/AD.2023.0510.

Altered Osteoblast Metabolism with Aging Results in Lipid Accumulation and Oxidative Stress Mediated Bone Loss

Ananya Nandy  1 , Alison Richards  1 , Santosh Thapa  1 , Alena Akhmetshina  1   2 , Nikita Narayani  1 , Elizabeth Rendina-Ruedy  1   3
Affiliations

Altered Osteoblast Metabolism with Aging Results in Lipid Accumulation and Oxidative Stress Mediated Bone Loss

Ananya Nandy et al. Aging Dis. .

Abstract

Cellular aging is associated with dysfunction of numerous tissues affecting multiple organ systems. A striking example of this is related to age-related bone loss, or osteoporosis, increasing fracture incidence. Interestingly, the two compartments of bone, cortical and cancellous or trabecular, rely on different mechanisms for development and maintenance during 'normal' aging. At a cellular level, the aging process disturbs a multitude of intracellular pathways. In particular, alterations in cellular metabolic functions thereby impacting cellular bioenergetics have been implicated in multiple tissues. Therefore, this study aimed to characterize how metabolic processes were altered in bone forming osteoblasts in aged mice compared to young mice. Metabolic flux analyses demonstrated both stromal cells and mature, matrix secreting osteoblasts from aged mice exhibited mitochondrial dysfunction. This was also accompanied by a lack of adaptability or metabolic flexibility to utilize exogenous substrates compared to osteoblasts cultured from young mice. Additionally, lipid droplets accumulated in both early stromal cells and mature osteoblasts from aged mice, which was further depicted as increased lipid content within the bone cortex of aged mice. Global transcriptomic analysis of the bone further supported these metabolic data as enhanced oxidative stress genes were up-regulated in aged mice, while osteoblast-related genes were down-regulated when compared to the young mice. Collectively, these data suggest that aging results in altered osteoblast metabolic handling of both exogenous and endogenous substrates which could contribute to age-related osteoporosis.

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Conflict of interest statement

Conflict of interest statement

Authors have nothing to declare that could be constructed as potential conflict of interest.

Figures

Figure 1.
Figure 1.
Age-related changes in bone microarchitecture. (A-G) micro computed tomography (µCT) analysis of trabecular bone; trabecular bone of tibia of 2 months or 22 months old male mice. (A) Representative 3D micro CT image of trabecular bone. (B) Percentage bone volume over total volume (Tb. BV/TV; %). (C) Connection density (Tb. Conn Dens; mm-3). (D) Trabecular number (Tb. N; mm-1). (E) Trabecular spacing (Tb.Sp;mm). (F) Trabecular thickness (Tb. Th;mm). (G) Trabecular structure model index (Tb.SMI;0= plates, 3= rods). (H-M) micro computed tomography (µCT) analysis of cortical bone; (H) Representative 3D micro CT image of cortical bone. (I) Cortical bone area (Ct.Ar;mm2 ). (J) Total cross-sectional area (Crosssectional;mm2). (K) Thickness (Ct. Th;mm). (L) Tissue mineral density (Ct.TMD;mgHA/cm3) (M) Percentage porosity (Ct.Porosity; %). Each dot represents data from individual animal where N=5 and data are mean ± standard error of mean. t tests or non-parametric Mann-Whitney tests were done accordingly after testing normal distribution using Shapiro-Wilk normality test to determine significance between two groups where *, p <0.05, **, p <0.01, ***, p <0.001, ****, p <0.0001.
Figure 2.
Figure 2.
Effect of aging on cellular metabolic profile. (A) Representative confocal image of undifferentiated stromal cells on 0th day and (C) fully differentiated osteoblasts on 8th day of ex vivo differentiation of 2 months or 22 months old mice immunostained for Runx2 along with mounting with DAPI. Panel 1 shows monochrome images of nuclei staining by Runx2 whereas panel 2 shows merged image (Runx2 in red and DAPI in blue). (B, D) Quantification of percentage of Runx2 positive cells in the 2 months (open) and (D) 22 months (light gray) old mice on (B) 0th and (D) 8th day of differentiation. Data are mean ± standard deviation (SD) where percentage of Runx2 positive cells were counted from independent images captured in 5 different field of view of coverslip (n=5) with pooled BMSCs obtained from 6 mice (N=6) in each age group. t tests or non-parametric Mann-Whitney tests were done accordingly after testing normal distribution using Shapiro-Wilk normality test to determine significance between two groups where *, p <0.05, **, p <0.01, ***, p <0.001, ****, p <0.0001. (E-G) Percentage of glycolytic (shaded) versus oxidative phosphorylation (white) measured by Seahorse ATP rate assay in absence of any external nutrients during ex vivo osteoblastogenesis of stromal cells harvested from 2 months or 22 months old mice in (E) stromal cells on 0th day before addition of differentiation media. (F) cells midway in their osteoblastogenesis process on 3rd day of differentiation. (G) matured osteoblast on 8th day of differentiation. (H) Percentage glycolysis and oxidative phosphorylation measured in stromal cells on 0th day of ex vivo differentiation harvested from 2 months or 22 months old male mice by Seahorse ATP rate assay done in presence (with pattern in shaded or open) (Complete media) or absence (without pattern in shaded or open) (Basal media) of additional external nutrients i.e., in complete or basal media respectively. Data are mean ± standard error of mean of data normalized to cell counts per well with data from minimum 17 wells per group (n=17) done from pooled BMSCs obtained from 6 mice (N=6) in each age group. t tests or non-parametric Mann-Whitney tests were done accordingly after testing normal distribution using Shapiro-Wilk normality test to determine significance between two groups where *, p <0.05, **, p <0.01, ***, p <0.001, ****, p <0.0001.
Figure 3.
Figure 3.
Effect of aging on flexibility and dependency towards endogenous and exogenous nutrients. (A) Dependency (open) and flexibility (lighter gray shaded) of cells from 2 months or 22 months old mice towards three basic endogenous nutrients glucose/Glc (carbohydrate), glutamine/Gln (amino acid) and fatty acid/FA (lipid) in absence of any external nutrients during ex vivo osteoblastogenesis (0th, 3rd and 8th day of differentiation) measured by Seahorse Mitoflex assay where data are mean ± standard error of mean of data normalized to cell counts per well with data from minimum 21 wells per group (n=21) done from pooled BMSCs obtained from 6 mice(N=6) in each age group. (B-E) ATP production and Proton leak measured by Seahorse Mitostress assay from 2 months(open) or 22 months (gray) old mice in presence (with pattern) (Complete media) or absence (without pattern) (Basal media) of additional external nutrients (B), (C) in stromal cells on 0th day of ex vivo differentiation. (D), (E) in matured osteoblasts on 8th day of differentiation. Data are mean ± standard error of mean of data normalized to cell counts per well with data from minimum 21 wells per group (n=21) done from pooled BMSCs obtained from 6 mice (N=6) in each age group. t tests or non-parametric Mann-Whitney tests were done accordingly after testing normal distribution using Shapiro-Wilk normality test to determine significance between two groups where *, p <0.05, **, p <0.01, ***, p <0.001, ****, p <0.0001.
Figure 4.
Figure 4.
Effect of aging on lipid storage and metabolism in osteoblasts. (A) Representative confocal image of undifferentiated stromal cells on 0th day and fully differentiated osteoblasts on 8th day of ex vivo differentiation of 2 months or 22 months old mice. Cellular lipid droplets were stained with BODIPY 493/503 (green in merged panel) and nuclei were stained with DAPI (blue in merged panel). Panel 1 and 2 show monochrome images of lipid droplet and nuclei staining respectively whereas panel 3 shows merged image. Quantification of (B) the intensity of BODIPY 493/503 per lipid droplet in 2 months (open circle) or 22 months (gray closed circle) where each dot represents intensity of one lipid droplet. Data are mean ± standard error of mean where intensity of each lipid droplets were measured from independent images captured in 5 or 6 different field of view of the coverslip (n=5/6) with pooled BMSCs obtained from 6 mice (N=6) in each age group.t tests were done assuming normal distribution since, data points were more than 40, to determine significance between two groups where *,p <0.05, **,p <0.01, ***,p <0.001, ****, p <0.0001 (C) number of lipid droplets per cell in the two groups on 0th and 8th day of differentiation. Data are mean ± standard deviation (SD) where lipid droplets per cell were counted from independent images captured in 5 or 6 different field of view (n=5/6) from pooled BMSCs obtained from 6 mice (N=6) in each age group. The number of lipid droplets counted from each image were divided by number of cells (number of DAPI positive nucleus) in that image to get lipid droplets per cell. t tests or non-parametric Mann-Whitney tests were done accordingly after testing normal distribution using Shapiro-Wilk normality test to determine significance between two groups where *, p <0.05, **, p <0.01, ***, p <0.001, ****, p <0.0001. (D) Thin layer chromatogram of lipid harvested from BODIPY 558/568 C12 (red fluorescent fatty acid) labelled stromal, or ex vivo differentiated matured osteoblast cells from 2 months or 22 months old mice, along with the chromatogram of BODIPY 558/568 C12 fatty acid ran as the standard. The bands below fatty acid are degraded products of the labelled fatty acid (E) Densitometric quantification of fatty acid normalized to origin. Data are mean ± standard deviation done from 3 wells (n=3) with pooled BMSCs obtained from 6 mice in each age group (N=6). t tests or non-parametric Mann-Whitney tests were done accordingly after testing normal distribution using Shapiro-Wilk normality test to determine significance between two groups where *, p <0.05, **, p <0.01, ***, p <0.001, ****, p <0.0001.
Figure 5.
Figure 5.
Lipid profile in bone cortex from young or aged mice. (A) Thin layer chromatogram (TLC) of lipid harvested from flushed tibia (tibia without any bone marrow) from 2 months or 22 months old mice. Standards include triglycerides (TG), fatty acids (FA), cholesterol (Ch), and cholesteryl esters (CE). (B-D) Densitometric quantification of the lipid species from 2 months (open circle) and 22 months (gray closed circle) old mice from the chromatogram (B) triglyceride, (C) cholesteryl ester (D) fatty acid normalized to bone weight. Each dot represents data from individual animal where N=6 animals in each group and data are mean ± standard error of mean. t tests or non-parametric Mann-Whitney tests were done accordingly after testing normal distribution using Shapiro-Wilk normality test to determine significance between two groups where *, p <0.05, **, p <0.01, ***, p <0.001, ****, p <0.0001.
Figure 6.
Figure 6.
Transcriptional profiling and analysis of differentially expressed genes in bones with aging. (A) Correlation heatmap using 25% variant gene. (B) Volcano plot showing differentially expressed genes (FDR adjusted p <0.05 in red circles) in flushed femur of 2 months versus 22 months old mice in pairwise comparison. (C) Top enriched differentially expressed pathways based on functional enrichment analysis (D) log2 fold change of top 10 differentially expressed genes.
Figure 7.
Figure 7.
Aging alters expression of genes involved in metabolic processes. (A) Distribution of differentially expressed genes belonging to bone formation or metabolic pathways. (B) Fold change expression of genes involved in glucose metabolism, (C) Lipid metabolism (LD or lipid droplets), (D) Lipid peroxidation mitigation.
Figure 8.
Figure 8.
Image depicting metabolic alterations occurring in osteoblasts contributing to age-related bone loss. Genes upregulated in bones of aged mice compared to young mice are shown in red whereas those downregulated in green. The pathways upregulated are shown in red and downregulated in green dashed line arrows whereas one step reactions are shown with solid line arrows. Blue circle: Nuclei; Green circles: Lipid droplet.
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References

    1. Weilbaecher KN, Guise TA, McCauley LK (2011). Cancer to bone: a fatal attraction. Nat Rev Cancer, 11:411-425. - PMC - PubMed
    1. Zaidi M (2007). Skeletal remodeling in health and disease. Nat Med, 13:791-801. - PubMed
    1. Iqbal J, Sun L, Zaidi M (2009). Coupling bone degradation to formation. Nat Med, 15:729-731. - PubMed
    1. Etich J, Lessmeier L, Rehberg M, Sill H, Zaucke F, Netzer C, et al. (2020). Osteogenesis imperfecta-pathophysiology and therapeutic options. Mol Cell Pediatr, 7:9. - PMC - PubMed
    1. Garibaldi N, Contento BM, Babini G, Morini J, Siciliani S, Biggiogera M, et al. (2021). Targeting cellular stress in vitro improves osteoblast homeostasis, matrix collagen content and mineralization in two murine models of osteogenesis imperfecta. Matrix Biol, 98:1-20. - PMC - PubMed