Molecular backgrounds of age-related osteoporosis from mouse genetics approaches

Backgrounds underlying age-related bone loss can be classified into two categories: systemic abnormality and osteoblast dysfunction. The former includes insufficiency of vitamin D or estrogen, causing a negative balance of calcium metabolism. We propose the contribution of an aging-suppressing gene, klotho, as a novel systemic factor, as a mouse deficient in the klotho gene exhibits multiple aging phenotypes including osteopenia with a low bone turnover. As a factor intrinsic to osteoblasts, we investigated the role of PPARγ, a key regulator of adipocyte differentiation, based on the facts that osteoblasts and adipocytes share a common progenitor. Heterozygous PPARγ-deficient mice exhibited high bone mass by stimulating osteoblastogenesis from bone marrow progenitors, and this effect became prominent with aging, indicating involvement of PPARγ-dependent bone formation in the pathophysiology of age-related bone loss. The local environment of osteoblasts is mainly controlled by cytokines/growth factors, among which insulin-like growth factor-I (IGF-I) is the most possible candidate whose production and activity are decreased with aging. Bone phenotypes of deficient mice of insulin receptor substrates (IRS-1 and IRS-2), essential molecules for intracellular signaling of IGF-I, revealed that IRS-1 is essential to maintain bone turnover by up-regulating anabolic and catabolic functions of osteoblasts, while IRS-2 is needed to keep the predominance of the anabolic function over the catabolic function. A next task ahead of us will be to elucidate the network system of these factors underlying age-related osteoporosis.     

Differential expression of cytokines in subcutaneous and marrow fat of aging C57BL/6J mice

Increasing marrow adipogenesis plays a causative role in the pathogenesis of age-related bone loss that could be associated with high cytokine production. In this study, we characterized the age-related changes in cytokine expression by bone marrow (BM) adipocytes as compared with subcutaneous (SC) fat. BM and SC adipocytes were isolated from young (4 months) and old (24 months) male C57BL/6J. Total proteins were extracted and proteomic analysis of 96 cytokines was performed using a cytokine antibody array. Proteins showing a significant change were grouped according with their known function in bone. We found a significant age-induced difference in the expression of 53 cytokines. As compared with SC adipocytes, aging BM adipocytes showed a more pro-adipogenic, anti-osteoblastogenic and pro-apoptotic phenotype. These data suggest that, with aging, BM adipocytes become significantly more toxic than SC adipocytes. These cytokines, if secreted, could play a role in the pathogenesis of age-related bone loss by affecting other cells within the marrow milieu.     

Mobilization efficiency is critically regulated by fat via marrow PPARδ

The efficiency of mobilization of hematopoietic stem/progenitor cells from bone marrow into the circulation by granulocyte colony-stimulating factor (G-CSF) is extremely varied in humans and mice and a mechanistic explanation for poor mobilizers is lacking. A mechanism of regulating mobilization efficiency by dietary fat was assessed in mice. Compared to a normal diet, a fat-free diet for 2 weeks greatly increased mobilization. The bone marrow mRNA level of peroxisome proliferatoractivated receptorδ (PPARδ), a receptor for lipid mediators, was markedly upregulated by G-CSF in mice fed a normal diet and displayed a strong positive correlation with widely varied mobilization efficiency. It was hypothesized that the bone marrow fat ligand for PPARδ might inhibit mobilization. A PPARδ agonist inhibited mobilization in mice fed a normal diet and enhanced mobilization by a fat-free diet. Mice treated with a PPARδ antagonist and chimeric mice with PPARδ+/- bone marrow showed enhanced mobilization. Immunohistochemical staining and flow cytometry revealed that bone marrow PPARδ expression was enhanced by G-CSF mainly in mature/immature neutrophils. Analysis of bone marrow lipid mediators revealed that G-CSF treatment and a fat-free diet resulted in exhaustion of ω3-polyunsaturated fatty acids such as eicosapentaenoic acid. Eicosapentaenoic acid induced the upregulation of genes downstream of PPARδ, such as Cpt1α and Angptl4, in mature/immature neutrophils in vitro and inhibited enhanced mobilization in mice fed with a fatfree diet in vivo. Treatment of wild-type mice with anti-Angptl4 antibody enhanced mobilization as well as bone marrow vascular permeability. Collectively, PPARδ signaling in mature/immature bone marrow neutrophils induced by dietary fatty acids negatively regulates mobilization, at least partially, via Angptl4 production.     

Attenuation of Colonic Inflammation by PPARγ in Intestinal Epithelial Cells: Effect on Toll-like Receptor Pathway

The peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor highly expressed in the colon and playing an anti-inflammatory role through inhibition of the NF-κB pathway. Toll-like receptor 4 (TLR4) has been known to mediate LPS-induced cellular signaling through activation of NF-κB pathway in intestinal epithelial cells. The aims of this study were to evaluate attenuation of inflammation by PPARγ in intestinal epithelial cells and to study the possible relation between PPARγ and TLR4. HT-29 human epithelial cells were stimulated with LPS (20 μg/ml) and PPARγ ligand, 15d-PGJ₂ (10 μM), or with LPS (20 μg/ml) alone for 24 hr. COX-2, IL-8, TLR4, and PPARγ mRNA expression was assessed by RT-PCR. IL-8 protein levels and TLR4 protein expression were analyzed by ELISA and Western blot, respectively. To evaluate the action mechanisms of PPARγ ligand, Western blot analysis for IκBα degradation was performed. Costimulation with LPS and PPARγ ligand in comparison to LPS stimulation alone (1) decreased COX-2, IL-8 mRNA expression and IL-8 protein secretion, (2) decreased TLR4 mRNA and protein expression, and (3) decreased PPARγ mRNA expression. PPARγ ligand delayed LPS-induced IκBα degradation. These findings suggest that PPAR-γ ligands suppress inflammation in intestinal epithelial cells. PPARγ and TLR, these two antagonistic signaling pathways in intestinal epithelial cells may be partially cross-linked.     

Combination of ciglitazone, a peroxisome proliferator-activated receptor gamma ligand, and cisplatin enhances the inhibition of growth of human ovarian cancers

Purpose  

We have recently reported that peroxisome proliferator-activated receptor gamma (PPARγ) ligands produce antitumor effects against human ovarian cancer in conjunction with reduction in angiogenesis and induction of apoptosis via regulating prostaglandin (PG) E₂ level. In this study, we investigated the effects of combination of ciglitazone, a PPARγ ligand, and cisplatin, a cytotoxic anti-cancer drug, on growth of ovarian cancer.     

Sirt1 is a regulator of bone mass and a repressor of Sost encoding for sclerostin, a bone formation inhibitor

Sirt1, the mammalian ortholog of the yeast Sir2 (silent information regulator 2), was shown to play an important role in metabolism and in age-associated diseases, but its role in skeletal homeostasis and osteoporosis has yet not been studied. Using 129/Sv mice with a germline mutation in the Sirt1 gene, we demonstrate that Sirt1 haplo-insufficient (Sirt1(+/-)) female mice exhibit a significant reduction in bone mass characterized by decreased bone formation and increased marrow adipogenesis. Importantly, we identify Sost, encoding for sclerostin, a critical inhibitor of bone formation, as a novel target of Sirt1. Using chromatin immunoprecipitation analysis, we reveal that Sirt1 directly and negatively regulates Sost gene expression by deacetylating histone 3 at lysine 9 at the Sost promoter. Sost down-regulation by small interfering RNA and the administration of a sclerostin-neutralizing antibody restore gene expression of osteocalcin and bone sialoprotein as well as mineralized nodule formation in Sirt1(+/-) marrow-derived mesenchymal stem cells induced to osteogenesis. These findings reveal a novel role for Sirt1 in bone as a regulator of bone mass and a repressor of sclerostin, and have potential implications suggesting that Sirt1 is a target for promoting bone formation as an anabolic approach for treatment of osteoporosis.     

Influence of estrogen deficiency and replacement on T-cell populations in rat lymphoid tissues and organs

Estrogen deficiency following ovariectomy or menopause results in bone loss. Although evidence strongly suggests that the immune system is involved in the pathogenesis of estrogen-deficient osteoporosis, it is not clear what role, if any, the T-lymphocyte plays in this process. Therefore, we examined the distribution of T-cell subsets in lymphoid organs and tissues, under varying estrogenic states in the rat. Six-month-old female Sprague-Dawley rats, ovariectomized (Ovx) and sham-operated, were randomized 5 d post-surgery into six groups to receive the following treatments: (A) sham/placebo; (B) sham/low-dose E₂; (C) sham/high-dose E₂; (D) Ovx/placebo; (E) Ovx/low-dose E₂; (F) Ovx/high-dose E₂. Half of the treated rats (groups A–F) were sacrificed on d 14; the remainder on d 28. Following euthanasia, mononuclear cells were isolated from the thymus, peripheral blood, spleen, lymph node and bone marrow, and were labeled for flow cytometric analysis using mouse anti-rat monoclonal antibodies directed against CD5, CD4, and CD8 antigenic markers. In the thymus, ovariectomy caused a dramatic increase and E₂ treatment caused a dose-dependent decrease in weight that was proportional to the number of thymocytes. In the bone marrow, ovariectomy caused a significant reduction in the percentage of all T-cell subsets examined and this effect persisted throughout the duration of the study. Estrogen replacement therapy at the low-dose reversed the effects of ovariectomy and high-dose E₂ treatment caused an increase in T-cell subsets in both the sham and Ovx groups, an effect that was more pronounced at d 14 compared with d 28. Although the percentages of some T-cell subsets in the other lymphoid organs/tissues were altered by ovariectomy or E₂ treatment at d 0 and 14, all these changes had normalized by d 28 except for CD5 and CD4 cells in peripheral blood. In summary, with the exception of T-lymphocytes in the bone marrow, the effects of varying estrogenic states on T-cells were variable and transient. The influence of estrogen status on bone marrow T-lymphocytes suggests that these cells may play a role in mediating the effects of estrogen on bone turnover and warrant additional studies focusing on the functional role of T-cells in the bone marrow compartment.     

PPARβ/δ Protects Against Experimental Colitis Through a Ligand-Independent Mechanism

Peroxisome proliferator-activated receptors (PPARs) β/δ and γ have overlapping roles in the negative regulation of inflammatory response genes. Ligand activation of PPARγ protects against experimental colitis in mice. PPARβ/δ can negatively regulate inflammation and is highly expressed in the epithelial cells of the colon, therefore PPARβ/δ may also have a role in experimental colitis. In these studies, colitis was induced by dextran sodium sulfate (DSS) treatment in wild-type and PPARβ/δ-null mice, with and without the PPARβ/δ specific ligand GW0742. PPARβ/δ-null mice exhibited increased sensitivity to DSS-induced colitis, as shown by marked differences in body weight loss, colon length, colonic morphology, myeloperoxidase activity and increased expression of mRNAs encoding the inflammatory markers interferon γ, tumor necrosis factor-α, and interleukin-6 compared to similarly treated wild-type mice. Interestingly, these differences were not affected by ligand activation of PPARβ/δ in either genotype. These studies demonstrate that PPARβ/δ expression in the colonic epithelium inhibits inflammation and protects against DSS-induced colitis through a ligand-independent mechanism.     

Age-related resistance of skeletal muscle-derived progenitor cells to SPARC may explain a shift from myogenesis to adipogenesis

Aging causes phenotypic changes in skeletal muscle progenitor cells (SMPCs) that lead to the loss of myogenicity and adipogenesis. Secreted protein acidic and rich in cysteine (SPARC), which is secreted from SMPCs, stimulates myogenesis and inhibits adipogenesis. The present study aimed to examine whether changes in SPARC expression, its signaling pathway, or both are involved in age-related phenotypic changes in SMPCs. SPARC expression levels were comparable in SMPCs derived from young and old rats. However, when SPARC expression was reduced by a SPARC-specific siRNA, SMPCs from young rats showed reduced myogenesis and increased adipogenesis. In striking contrast, old rats showed little changes in these functions. Recombinant SPARC was effective in inhibiting adipogenesis and promoting myogenesis of SMPCs from young rats but had no effect on SMPCs from old rats when endogenous SPARC levels were reduced by the SPARC-siRNA. Further, the level of integrin α5, a subunit of the putative SPARC receptor, was decreased in SMPCs from old rats, and its inhibition in SMPCs from young rats by siRNA reduced adipogenesis in response to SPARC. These results suggest that, although SPARC plays a role in regulating SMPC function, SMPCs become refractory to the action of SPARC with age. Our data may explain an age-related shift from myogenesis to adipogenesis, associated with sarcopenia.     

Effect of the Pro12Ala polymorphism of the PPARg2 gene on serum adiponectin changes

The Pro12Ala polymorphism of the peroxisome proliferator-activated receptor gamma 2 (PPARγ2) gene and adiponectin, a protein secreted from adipose tissue, have been associated with insulin sensitivity. The present study demonstrates that in Finnish servicemen who were on a high-caloric diet for 6 mo only subjects with the Ala 12 allele of PPARγ2 had a significant increase in adiponectin levels with weight loss induced by heavy exercise. This study demonstrates an interaction of genetic and environmental factors in the regulation of serum adiponectin concentrations. Department of Medicine, University of Kuopio, Finland.     

Effect of rosiglitazone on capillary density and angiogenesis in adipose tissue of normoglycaemic humans in a randomised controlled trial

Aims/hypothesis  

Recent reports of decreased capillary density in the adipose tissue of obese individuals suggest that an imbalance of angiogenesis and adipogenesis may, in part, underlie insulin resistance. This study aimed to determine whether the insulin-sensitising peroxisome proliferator-activated receptor γ (PPARγ) activator rosiglitazone affects adipose tissue vascularisation in normal humans.     

Modulation of insulin resistance in ovariectomized rats by endurance exercise training and estrogen replacement

Estrogen is known to play a role in fat metabolism, but its role in carbohydrate metabolism remains controversial. We investigated alterations in carbohydrate and fat metabolism after prolonged estrogen deprivation by determining body weight, food intake, visceral fat content, serum lipids, glucose tolerance, and insulin action on glucose transport activity in isolated soleus and extensor digitorum longus muscles. In addition, effects of endurance exercise training with or without estrogen replacement on metabolic alterations occurring under estrogen deficiency were examined. Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (SHAM). The OVX rats remained sedentary, received 5 μg of 17β-estradiol (E₂), performed exercise training (ET), or underwent both estrogen treatment and exercise training (E₂ + ET) for 12 weeks. Compared with SHAM, OVX animals had greater final body weights, visceral fat content, and serum levels of total and low-density lipoprotein cholesterol (P < .05). Exercise training and E₂ significantly reduced body weights (6% and 25%), visceral fat (37% and 51%), and low-density lipoprotein cholesterol level (19% and 26%). Exercise training alone improved whole-body glucose tolerance (29%), which was enhanced to the greatest extent (51%) in the ET rats that also received E₂. Insulin-stimulated glucose transport activity in OVX group was lower than that in SHAM by 29% to 44% (P < .05). Exercise training and E₂ corrected the diminished insulin action on skeletal muscle glucose transport in OVX animals, which was partly due to elevated glucose transporter-4 protein expression. These findings indicate that 12 weeks of ovariectomy caused metabolic alterations mimicking features of the insulin resistance syndrome. Furthermore, these metabolic disturbances were attenuated by ET or E₂, whereas the beneficial interactive effects of ET and E₂ on these defects were not apparent.     

Validation of noninvasive quantification of bone marrow fat volume with microCT in aging rats

Marrow fat infiltration is one of the hallmarks of age-related bone loss. This fat infiltration has been quantified by invasive and noninvasive methods. However, the validity of the noninvasive methods has not been correlated with a gold standard. In this study we aim to validate the usefulness of marrow fat quantification by correlating microCT (μCT) images with histology analysis. Fat volume (FV) and bone volume (BV) of distal femora of young (4 months) and old (27 months) Louvain/c (LOU) rats (n = 22) were quantified by histology and compared with μCT images analyzed by an image analysis software (SliceOMatic).We found that for SliceOMatic/μCT the intra-rater reliability for duplicate measurements was 0.94 (p < 0.001) and the inter-rater reliability for FV/BV ratio in young and old rats was 98% and 99% respectively. Both methods showed a significant increase (~ 2 fold) in the FV/BV ratio in the old rats as compared with their young counterparts (p < 0.001). A significantly higher correlation (r2 = 0.85) in the old rats was found between our noninvasive method and histology. Furthermore, our noninvasive method showed good agreement with histology.In conclusion, noninvasive quantification of FV/BV ratio using an image analysis software is as reliable as histology for identifying age related marrow fat changes with high inter and intra-rater reliability. These findings provide a new noninvasive method for quantifying marrow fat, which is useful and can be tested not only in animals but also in human studies.     

Maintaining good hearing: Calorie restriction,Sirt3, and glutathione

Reducing calorie intake extends the lifespan of a variety of experimental models and delays progression of age-related hearing loss (AHL). AHL is a common feature of aging and is characterized by age-related decline of hearing associated with loss of sensory hair cells, spiral ganglion neurons, and/or stria vascularis degeneration in the cochlea. Sirtuins are a family of NAD⁺-dependent enzymes that regulate lifespan in lower organisms and have emerged as broad regulators of cellular fate. Our recent study indicated that mitochondrial Sirt3, a member of the sirtuin family, mediates the anti-aging effects of calorie restriction (CR) on AHL in mice. Interestingly, we also found that weight loss alone may not be sufficient for maintaining normal hearing. How does CR slow the progression of AHL through regulation of Sirt3? Here we review the evidence that during CR, Sirt3 slows the progression of AHL by promoting the glutathione-mediated mitochondrial antioxidant defense system in mice. A significant reduction in food consumption in one's daily life may not be a desirable and realistic option for most people. Therefore, identification/discovery of compounds that induce the activation of SIRT3 or glutathione reductase, or that increase mitochondrial glutathione levels has potential for maintaining good hearing through mimicking the anti-aging effects of CR in human inner ear cells.     

Reciprocal regulation by estradiol 17-beta of ezrin and cadherin-catenin complexes in pituitary GH3 cells

The antiestrogen, ICI 182780, and estradiol-17β (E₂) regulate cadherin-mediated cell adhesion in pituitary GH₃ cells. Using a cDNA expression array to screen for E₂-regulated genes that are associated with the cytoskeleton, we observed that E₂ stimulated ezrin gene expression and confirmed that ezrin gene expression is regulated pretranslationally by ICI 182780 versus E₂. E₂ increased ezrin protein levels in whole-cell lysates and in the cytoskeletal-associated, detergent-insoluble fraction. Confocal microscopy revealed that ezrin was associated with free apical membranes of E₂-treated cells. E₂ decreased N-cadherin and β-catenin levels and induced a redistribution of p120^ctn to the cytoplasm. In GH₃ transfectants overexpressing E-cadherin, E₂ had no effect on adhesiveness or on E-cadherin and p120^ctn distribution, but increased levels of active ezrin. Ezrin was concentrated at free and apical membranes. These studies provide the first demonstration of the regulation of ezrin by E₂ and show that the ER signaling pathway coordinately regulates two cytoskeletal-associated protein complexes, with mutually exclusive cellular distributions, in a reciprocal manner. These findings indicate that E₂ enriches the cell membrane with ezrin-membrane protein complexes by both increasing ezrin expression and by enlarging the relative area of nonadhesive membrane to which ezrin is targeted.     

Impact of estradiol on parametrial adipose tissue function: evidence for establishment of a new set point of leptin sensitivity in control of energy metabolism in female rat

Estradiol has been implicated in the regulation of food intake; however, its effect seems to be exerted in a bimodal fashion. We examined whether a single im injection of estradiol valerate (E₂V), lastingly effective, could induce changes in parametrial fat function that further induce a new set point of leptin sensitivity in the female rat. E₂V induced severe anorexia and loss of body weight between d 4 and 12 posttreatment. E₂V rats recovered normal food intake and departing body weights on wk 2 and 3 posttreatment, respectively; however, they did not reach body weights of control rats. On d 61 post-treatment, we found that unfasting E₂V, vs control, rats displayed increased E₂ and leptin circulating levels; reduced plasma tumor necrosis factor-α (TNF-α) concentrations; similar circulating levels of glucose, insulin, and triglyceride; and lower parametrial fat mass containing a higher number of adipocytes that, although normal in size, in vitro released more leptin. Metabolic responses to fasting indicated that unlike control animals, E₂V rats did not decrease triglyceride circulating levels, and that both groups decreased plasma glucose, leptin, and insulin, but not TNF-α, levels. High glucose load experiments indicated that E₂V animals displayed a better insulin sensitivity than control rats; did not significantly increase circulating leptin concentrations as control rats did; and, unlike control, significantly decreased plasma triglyceride levels. Our data strongly support a potent acute anorectic effect of E₂ and that, after several weeks, E₂ modified parametrial fat function and insulin sensitivity, protecting the organism against future unfavorable metabolic conditions.