Jovana Kaludjerovic

Adiponectin Is a Negative Regulator of Bone Mineral and Bone Strength in Growing Mice

Obesity is associated with increased bone mineral density (BMD) but the mechanism for this is unclear. Serum levels of the adipokine adiponectin are inversely correlated with obesity, but results from studies on its relationship to bone mass are conflicting. The objective of this study was to compare bone mineral content (BMC), BMD and biomechanical strength properties of femur and lumbar vertebrae in 8- and 16-week old adiponectin transgenic mice (AdTg). These mice exhibit significantly elevated circulating adiponectin but have similar body weights compared to wild-type (WT) littermates that were used as controls. Female AdTg mice displayed significantly lower femur BMC at 8 and 16 weeks of age and femur neck peak load was significantly lower in 8-week old AdTg mice of both genders compared to controls. The peak load from compression testing of an individual lumbar vertebra was significantly lower in female AdTg mice compared to WT at 8 weeks, and this difference persisted at 16 weeks of age. In addition, lumbar vertebrae BMC was significantly lower in 16-week old male AdTg mice compared to WT although vertebra peak load was not different. Serum adiponectin levels were inversely correlated with femur BMC. In summary, elevated circulating adiponectin inhibits the acquisition of bone mass in growing mice and results in decreased biomechanical measures of functional strength that are surrogate measures of susceptibility to fractures. These results support a role for circulating adiponectin as a metabolic link that can explain, at least in part, the positive relationship between obesity and both bone mass and reduced susceptibility to fractures.

Neonatal Exposure to Daidzein, Genistein, or the Combination Modulates Bone Development in Female CD-1 Mice

Neonatal exposure to genistein (GEN), an isoflavone abundant in soy, favorably modulates bone mineral density (BMD) and bone strength in mice at adulthood. The study objective was to determine whether early exposure to a combination of the soy isoflavones daidzein (DAI) and GEN that naturally exists in soy protein-based infant formula results in greater benefits to bone at adulthood than either treatment alone. Male and female CD-1 mice (n = 8-16 pups per group per gender) were randomized to subcutaneous injections of DAI (2 mg x kg body weight(-1) x d(-1)), GEN (5 mg x kg body weight(-1) x d(-1)), DAI+GEN (7 mg x kg body weight(-1) x d(-1)), diethylstilbesterol (DES; positive control) (2 mg x kg body weight(-1) x d(-1)), or control (CON) from postnatal d 1-5 and were studied to 4 mo of age. BMD, biomechanical bone strength, and bone microarchitecture were assessed at the femur and lumbar vertebrae (LV). Females treated with DAI, GEN, DAI+GEN, or DES had greater (P < 0.05) BMD at the LV compared with CON and vertebra in the DAI and DES group were more resistant to compression fractures. Microstructural analyses demonstrated that treatment with DAI and GEN resulted in greater (P < 0.05) trabecular connectivity and trabecular thickness, respectively, than the CON. In conclusion, neonatal exposure to DAI and/or GEN had a positive effect on the skeleton of female mice at adulthood, but, compared with individual treatments, DAI+GEN did not have a greater benefit to bone in females or males.

The Interplay between Estrogen and Fetal Adrenal Cortex

Estrogen is a steroid hormone that regulates embryogenesis, cell proliferation and differentiation, organogenesis, the timing of parturition, and fetal imprinting by carrying chemical messages from glands to cells within tissues or organs in the body. During development, placenta is the primary source of estrogen production but estrogen can only be produced if the fetus or the mother supplies dehydroepiandrosterone (DHEA), the estrogen prohormone. Studies show that the fetal zone of the fetal adrenal cortex supplies 60% of DHEA for placental estrogen production, and that placental estrogen in turn modulates the morphological and functional development of the fetal adrenal cortex. As such, in developed countries where humans are exposed daily to environmental estrogens, there is concern that the development of fetal adrenal cortex, and in turn, placental estrogen production may be disrupted. This paper discusses fetal adrenal gland development, how endogenous estrogen regulates the structure and function of the fetal adrenal cortex, and highlights the potential role that early life exposure to environmental estrogens may have on the development and endocrinology of the fetal adrenal cortex.

Neonatal Administration of Isoflavones Attenuates Deterioration of Bone Tissue in Female but Not Male Mice

Neonatal exposure to soy isoflavones at levels similar to that of infants fed soy protein formula resulted in higher bone mineral density (BMD), improved bone structure, and greater bone strength at young adulthood in female CD-1 mice (1,2). Our objective in this study was to determine whether these improvements in bone quantity and quality at 4 mo of age provide protection against the deterioration of bone tissue that occurs after a decline in endogenous sex steroid production. Male and female CD-1 mice (n = 8-18 pups per group per gender) were randomized to subcutaneous injections of corn oil [negative control (CON)], daidzein + genistein (DG; 7 mg x kg body weight(-1) x d(-1)), or diethylstilbestrol [(DES); positive control, 2 mg x kg body weight(-1) x d(-1)) from postnatal d 1 to 5. At 4 mo of age, mice were ovariectomized (females) or orchidectomized (males) and studied to 8 mo of age. Females treated with DG had higher (P < 0.05) femur and vertebral bone mineral content (BMC) and BMD compared with the CON group. Microstructural analysis revealed that improvements in BMD induced by DG and DES were coupled with greater trabecular thickness at the lumbar spine. Importantly, structural improvements resulted in bones that were more resistant to fracture, as the peak load of the femoral midpoint and lumbar vertebra 2 were higher (P < 0.05) with DG compared with CON. Effects in males were not significant. In conclusion, short-term neonatal exposure to isoflavones provides protection against the deterioration of bone tissue in females but not males after a decline of endogenous sex steroid production.

Klotho expression in osteocytes regulates bone metabolism and controls bone formation

Osteocytes within the mineralized bone matrix control bone remodeling by regulating osteoblast and osteoclast activity. Osteocytes express the aging suppressor Klotho, but the functional role of this protein in skeletal homeostasis is unknown. Here we identify Klotho expression in osteocytes as a potent regulator of bone formation and bone mass. Targeted deletion of Klotho from osteocytes led to a striking increase in bone formation and bone volume coupled with enhanced osteoblast activity, in sharp contrast to what is observed in Klotho hypomorphic (kl/kl) mice. Conversely, overexpression of Klotho in cultured osteoblastic cells inhibited mineralization and osteogenic activity during osteocyte differentiation. Further, the induction of chronic kidney disease with high-turnover renal osteodystrophy led to downregulation of Klotho in bone cells. This appeared to offset the skeletal impact of osteocyte-targeted Klotho deletion. Thus, our findings establish a key role of osteocyte-expressed Klotho in regulating bone metabolism and indicate a new mechanism by which osteocytes control bone formation.

Diethylstilbesterol has gender-specific effects on weight gain and bone development in mice.

Neonatal exposure to diethylstilbesterol (DES) in female mice programs estrogen-sensitive tissues, resulting in greater body weight gain and positive effects on bone architecture at adulthood. Using the CD-1 mouse model, the objective of the present study was to examine how short-term neonatal exposure to DES modulates weight gain as well as bone mineral density (BMD), bone strength, and bone microarchitecture in both males and females at adulthood. Male and female offspring (n =  8–12 pups/treatment/gender) were randomized to DES (2 mg/kg bw/d) or control (corn oil) from postnatal day 1 to 5 (subcutaneous injection, once daily) and sacrificed at 4 mo of age. Body weight was measured weekly, while bone mineral, strength, and microarchitecture were measured at 4 mo of age. DES treatment resulted in significantly higher body weight in females but lower weight in males at 4 mo of age. In DES-treated females, markedly higher BMD of lumbar vertebrae (LV1–LV3) was translated into significantly stronger LV2 that was more resistant to fracture; similar effects were observed at the femur midpoint. At the spine, males had a markedly lower BMD and peak load, suggesting an adverse effect. Microstructural analyses demonstrated that functional changes in femurs, i.e., peak load, were primarily due to modulation of cortical bone. In conclusion, neonatal exposure to DES exerted gender-specific effects on body weight gain and bone health.

Bone-specific gene expression patterns and whole bone tissue of female mice are programmed by early life exposure to soy isoflavones and folic acid

Female mice exposed to soy isoflavones (ISO) during early postnatal life have improved bone outcomes at adulthood. Since long-lasting effects may be mediated by DNA methylation, we hypothesized that providing supplemental folic acid (FA), a methyl donor, during early life, would enhance the positive effect of ISO to bone health. Bone-specific gene expression patterns were studied to understand potential mechanisms. CD-1 dams (n=36) were randomized to adequate or supplemental levels of FA (2 or 8 mg/kg diet) during pregnancy and lactation, and offspring received corn oil or ISO (7 mg/kg body weight/d) from postnatal day 1 to 10. From weaning, pups were fed an adequate FA diet and were studied to 4 months of age. Female offspring exposed to supplemental FA+ISO had higher bone mineral density (BMD), trabecular connectivity and peak load at the lumbar spine compared to females exposed to adequate FA. Female offspring exposed to adequate FA+ISO or supplemental FA had higher (P<.05) BMD and greater resistance to fracture at the lumbar spine and the femur; higher trabecular connectivity at the lumbar spine; and lower expression of DNA methyltransferase 3a (Dnmt3a) and neuropeptide Y (NPY) in the femur compared to mice exposed to adequate FA. In addition, only mice exposed to adequate FA+ISO had microstructural improvements at the femur neck and higher serum osteoprotegrin (OPG) and insulin growth factor-I (IGF-I). In summary, exposure to supplemental FA did not enhance the positive effect of ISO in bone. However, exposure to adequate FA+ISO or supplemental FA improved bone at least in part by suppressing Dnmt3a and NPY.

Early life exposure to genistein and daidzein disrupts structural development of reproductive organs in female mice.

In mice, exposure to isoflavones (ISO), abundant in soy infant formula, during the first 5 d of life alters structural and functional development of reproductive organs. Effects of longer exposures are unknown. The study objective was to evaluate whether exposure to a combination of daidzein and genistein in the first 10 compared to 5 d of life results in greater adverse effects on ovarian and uterine structure in adult mice. Thirteen litters of 8–12 pups were cross-fostered and randomized to corn oil or ISO (2 mg daidzein + 5 mg genistein/kg body weight/d) for the first 5 or 10 d of life. The 10-d protocol mimicked the period when infants are fed soy protein formula (SPF) but avoids the time when suckling pups can consume mothers diet. Body and organ weights, and histology of ovaries and uteri were analyzed. There were no differences in the ovary or uterus weight, number of ovarian follicles, number of multiple oocyte follicles, or percent of ovarian cysts with 5 or 10 d ISO intervention compared to respective controls. The 10-d ISO group had higher body weights from 6 d to 4 mo of age and a higher percent of hyperplasia in the oviduct than the respective control. Lower number of ovarian corpus lutea and a higher incidence of abnormal changes were reported in the uteri of both ISO groups compared to their respective controls. Five and 10-d exposure to ISO had similar long-lasting adverse effects on the structure of ovaries and uterus in adult mice. Only the 10-d ISO exposure resulted in greater body weight gain at adulthood.

Circulating isoflavonoid levels in CD-1 mice: effect of oral versus subcutaneous delivery and frequency of administration

The CD-1 mouse is a commonly used animal model to understand the biological effects of early-life exposure to soy isoflavones in infants. Most studies using CD-1 mice have administered isoflavones by daily subcutaneous injection, while infants receive oral feeds every few hours. The study objectives were to compare the total serum levels of genistein (GEN), daidzein (DAI) and the DAI metabolites equol and O-desmethyl-angolensin (O-DMA), after subcutaneous injection and oral dosing and to determine if frequency of oral administration results in different circulating levels of isoflavones using the CD-1 mouse model. From postnatal days 1 to 5, pups randomly received corn oil or soy isoflavones (total daily dose, 0.010 mg DAI+0.025 mg GEN) by subcutaneous injection once a day, orally once a day or orally every 4 hours. On postnatal day 5, 1 h posttreatment, mice were killed and serum was collected. Mice treated with soy isoflavones had higher (P<.05) serum GEN (female: 1895-3391 ng/ml and male: 483-578 ng/ml) and DAI (female: 850-1580 ng/ml and male: 248-322 ng/ml) concentrations versus control (5-20 ng/ml) mice, regardless of route or frequency of administration, and were similar among dosing strategies. Total serum concentrations of GEN and DAI were higher (P<.05) among females (GEN: 2714 ± 393 ng/ml and DAI: 1205 ± 164 ng/ml) than males (GEN: 521 ± 439 ng/ml and DAI: 288 ± 184 ng/ml) across treatment groups. Serum equol and O-DMA concentrations were negligible (<3 ng/ml) across groups. In conclusion, different routes of delivery and frequency of administration resulted in similar total serum levels of GEN, DAI¸ equol or O-DMA.

Klotho expression in long bones regulates FGF23 production during renal failure

Circulating levels of bone‐derived fibroblast growth factor 23 (FGF23) increase early during acute and chronic kidney disease and are associated with adverse outcomes. Membrane‐bound Klotho acts as a permissive coreceptor for FGF23, and its expression was recently found in osteoblasts/osteocytes. We hypothesized that Klotho in bone cells is part of an autocrine feedback loop that regulates FGF23 expression during renal failure. Thus, we induced renal failure in mice with targeted deletion of Klotho in long bones. Uremic wild‐type (KLfl/fl) and knockout (Prx1‐Cre;KLfl/fl) mice both responded with reduced body weight, kidney atrophy, hyperphosphatemia, and increased bone turnover. Importantly, long bones of Prx1‐Cre;KLfl/fl mice but not their axial skeleton failed to increase FGF23 expression as observed in uremic KLfl/fl mice. Consequently, Prx1‐Cre;KLfl/fl mice had significantly lower serum FGF23 and parathyroid hormone levels, and higher renal 1‐α‐hydroxylase expression, serum 1,25‐dihydroxyvitamin D, and calcium levels than KLfl/fl mice. These results were confirmed in two independent models of renal failure, adenine diet induced and 5/6 nephrectomy. Moreover, FGF23‐treated bone cells required Klotho to increase FGF23 mRNA and ERK phosphorylation. In summary, our novel findings show that Klotho in bone is crucial for inducing FGF23 production upon renal failure. We propose the presence of an autocrine feedback loop in which Klotho senses the need for FGF23.—Kaludjerovic, J., Komaba, H., Sato, T., Erben, R. G., Baron, R., Olauson, H., Larsson, T. E., Lanske, B. Klotho expression in long bones regulates FGF23 production during renal failure. FASEB J. 31, 2050–2064 (2017). www.fasebj.org