A probiotic mix partially protects against castration-induced bone loss in male mice

Studies in postmenopausal women and ovariectomized mice show that the probiotic mix Lacticaseibacillus paracasei DSM13434, Lactiplantibacillus plantarum DSM 15312 and DSM 15313 (L. Mix) can protect from bone loss caused by sex steroid deficiency. Whether probiotic bacteria can protect bone also in sex steroid-deficient males is less studied. We used the orchiectomized mouse as a model for age-dependent bone loss caused by decreasing sex hormone levels in males. We treated 10-week-old male mice with either vehicle (veh) or L. Mix for 6 weeks, starting 2 weeks before orchiectomy (orx) or sham surgery. Importantly, mice treated with L. Mix had a general increase in total body bone mineral density (BMD) and lean mass (P ≤ 0.05) compared with veh-treated mice. Detailed computer tomography analysis of dissected bones showed increased trabecular BMD of the distal metaphyseal region of the femur in L. Mix compared to veh-treated orx mice (+8.0%, P ≤ 0.05). In the vertebra, L. Mix treatment increased trabecular bone volume fraction BV/TV (+11.5%, P ≤ 0.05) compared to veh in orx mice. Also, L. Mix increased the levels of short-chain fatty acids (SCFAs) such as propionate and acetate and important intermediates in SCFA synthesis such as succinate and lactate in the cecal content of male mice. In conclusion, L. Mix treatment resulted in a general increase in BMD in adult male mice and prevented trabecular bone loss in femur and vertebra of orx mice. These bone protective effects of L. Mix were associated with increased levels of SCFAs in the cecal content of male mice

Dehydroepiandrosterone Supplementation Results in Varying Tissue-specific Levels of Dihydrotestosterone in Male Mice

Dehydroepiandrosterone (DHEA), an adrenal androgen precursor, can be metabolized in target tissues into active sex steroids. It has been proposed that DHEA supplementation might result in restoration of physiological local sex steroid levels, but knowledge on the effect of DHEA treatment on local sex steroid levels in multiple tissues is lacking. To determine the effects of DHEA on tissue-specific levels of sex steroids, we treated orchiectomized (ORX) male mice with DHEA for 3 weeks and compared them with vehicle-treated ORX mice and gonadal intact mice. Intra-tissue levels of sex steroids were analyzed in reproductive organs (seminal vesicles, prostate, m. levator ani), major body compartments (white adipose tissue, skeletal muscle, and brain), adrenals, liver, and serum using a sensitive and validated gas chromatography-mass spectrometry method. DHEA treatment restored levels of both testosterone (T) and dihydrotestosterone (DHT) to approximately physiological levels in male reproductive organs. In contrast, this treatment did not increase DHT levels in skeletal muscle or brain. In the liver, DHEA treatment substantially increased levels of T (at least 4-fold) and DHT (+536%, P < 0.01) compared with vehicle-treated ORX mice. In conclusion, we provide a comprehensive map of the effect of DHEA treatment on intra-tissue sex steroid levels in ORX mice with a restoration of physiological levels of androgens in male reproductive organs while DHT levels were not restored in the skeletal muscle or brain. This, and the unexpected supraphysiological androgen levels in the liver, may be a cause for concern considering the uncontrolled use of DHEA

Comprehensive sex steroid profiling in multiple tissues reveals novel insights in sex steroid distribution in male mice

A comprehensive atlas of sex steroid distribution in multiple tissues is currently lacking, and how circulating and tissue sex steroid levels correlate remains unknown. Here, we adapted and validated a gas chromatography tandem mass spectrometry method for simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), androstenedione, progesterone (Prog), estradiol, and estrone in mouse tissues. We then mapped the sex steroid pattern in 10 different endocrine, reproductive, and major body compartment tissues and serum of gonadal intact and orchiectomized (ORX) male mice. In gonadal intact males, high levels of DHT were observed in reproductive tissues, but also in white adipose tissue (WAT). A major part of the total body reservoir of androgens (T and DHT) and Prog was found in WAT. Serum levels of androgens and Prog were strongly correlated with corresponding levels in the brain while only modestly correlated with corresponding levels in WAT. After orchiectomy, the levels of the active androgens T and DHT decreased markedly while Prog levels in male reproductive tissues increased slightly. In ORX mice, Prog was by far the most abundant sex steroid, and, again, WAT constituted the major reservoir of Prog in the body. In conclusion, we present a comprehensive atlas of tissue and serum concentrations of sex hormones in male mice, revealing novel insights in sex steroid distribution. Brain sex steroid levels are well reflected by serum levels and WAT constitutes a large reservoir of sex steroids in male mice. In addition, Prog is the most abundant sex hormone in ORX mice

Androgen receptor SUMOylation regulates bone mass in male mice

The crucial effects of androgens on the male skeleton are at least partly mediated via the androgen receptor (AR). In addition to hormone binding, the AR activity is regulated by post-translational modifications, including SUMOylation. SUMOylation is a reversible modification in which Small Ubiquitin-related MOdifier proteins (SUMOs) are attached to the AR and thereby regulate the activity of the AR and change its interactions with other proteins.To elucidate the importance of SUMOylation of AR for male bone metabolism, we used a mouse model devoid of the two AR SUMOylation sites (ARSUM−; K381R and K500R are substituted). Six-month-old male ARSUM− mice displayed significantly reduced trabecular bone volume fraction in the distal metaphyseal region of femur compared with wild type (WT) mice (BV/TV, −19.1 ± 4.9%, P In conclusion, mice devoid of AR SUMOylation have reduced trabecular bone mass as a result of reduced bone formation. We propose that therapies enhancing AR SUMOylation might result in bone-specific anabolic effects with minimal adverse effects in other tissues.</p

Inducible Wnt16 inactivation: WNT16 regulates cortical bone thickness in adult mice

Substantial progress has been made in the therapeutic reduction of vertebral fracture risk in patients with osteoporosis, but non-vertebral fracture risk has been improved only marginally. Human genetic studies demonstrate that the WNT16 locus is a major determinant of cortical bone thickness and non-vertebral fracture risk and mouse models with life-long Wnt16 inactivation revealed that WNT16 is a key regulator of cortical thickness. These studies, however, could not exclude that the effect of Wnt16 inactivation on cortical thickness might be caused by early developmental andor growth effects. To determine the effect of WNT16 specifically on adult cortical bone homeostasis, Wnt16 was conditionally ablated in young adult and old mice through tamoxifen-inducible Cre-mediated recombination using CAG-Cre-ER; Wnt16(flox/flox) (Cre-Wnt16(flox/flox)) mice. First, 10-week-old Cre-Wnt16(flox/flox) and Wnt16(flox/flox) littermate control mice were treated with tamoxifen. Four weeks later, Wnt16 mRNA levels in cortical bone were reduced and cortical thickness in femur was decreased in Cre-Wnt16(flox/flox) mice compared to Wnt16(flox/flox) mice. Then, inactivation of Wnt16 in 47-week-old mice (evaluated four weeks later) resulted in a reduction of Wnt16 mRNA levels, cortical thickness and cortical bone strength with no effect on trabecular bone volume fraction. Mechanistic studies demonstrated that the reduced cortical bone thickness was caused by a combination of increased bone resorption and reduced periosteal bone formation. In conclusion, WNT16 is a crucial regulator of cortical bone thickness in young adult and old mice. We propose that new treatment strategies targeting the adult regulation of WNT16 might be useful to reduce fracture risk at cortical bone sites

Androgen receptor signaling mechanisms in bone

Osteoporosis is a common age-related disease that increases the risk of fractures. Androgens are crucial for bone health in males. Although a substantial part of the effects of androgens on the skeleton is mediated via conversion of testosterone to estradiol, direct effects of androgens on the androgen receptor (AR) also contribute to male bone homeostasis. The aim of this thesis is to increase the knowledge about the significance of the AR for bone metabolism to potentially identify bone-specific AR signaling pathways. The thesis is based on studies using several different mouse models with altered AR signaling. In Paper I, we demonstrated that inactivation of the AR in immature osteoblast-lineage cells reduces trabecular but not cortical bone mass. Since antiandrogens are frequently used in the treatment of men with prostate cancer, we investigated the possible skeletal side effects of the recently approved antiandrogen drug enzalutamide (Paper II). Although this drug effectively reduced the weights of androgen-sensitive reproductive tissues, bone mass was reduced moderately and only in the axial skeleton. To determine the importance of the AR for pubertal and adult bone metabolism, avoiding confounding developmental effects, we inactivated the AR in pre-pubertal as well as in young adult male mice (Paper III). We demonstrated that adult AR expression is crucial for tra-becular and cortical bone mass maintenance while pubertal AR expression is crucial for normal fat mass homeostasis in adult male mice. The AR activity is regulated by post-translational modifications, including AR SUMOylation. In Paper IV, we demonstrated that AR SUMOylation regulates bone mass but not the weights of androgen-responsive reproductive tissues, suggesting that therapies targeting AR SUMOylation might result in bone-specific anabolic effects with minimal adverse effects in other tissues. The findings in this thesis contribute with important knowledge for the development of new treatment options for men with osteoporosis and safer endocrine treatments, with minimal skeletal side effects, for men with prostate cancer

Characterization and propagation from meteorological to groundwater drought in different aquifers with multiple timescales

Study region: Zhanjiang City District, southwest of Guangdong Province, China, was selected as the case study area owing to its relatively frequent occurrence of droughts and groundwater overexploitation. Study focus: This study presents a useful framework to characterize the propagation from meteorological drought (MD) to groundwater drought (GD) in different aquifers from the perspective of multiple timescales. Two standardized drought indices, i.e., standardized groundwater index and standardized precipitation index with different timescales (1–48 months), were used to examine the propagation time from MD to GD in different aquifers. Three clusters, i.e., natural(P)-, natural+human(P + H)-, and human(H)-induced drought, were considered for revealing the potential driving mechanism of propagation. Four characteristic mechanisms of lag, pooling, lengthening, and attenuation were used to analyze the propagation process from MD to GD, among which the lag characteristic, showing the propagation time, was discussed corresponding to varied clusters. New hydrological insights for the region: Results indicate the following: (1) The propagation time in different aquifers varied; that is, in the shallow aquifer, the average propagation time showed 1 month for the P cluster and 3 months for the P + H cluster, while the propagation time in the middle and deep aquifers were found to be longer than that in the shallow layer though much uncertainty remained for the former. (2) The influences from MD obtained the decreasing order of P > P + H>H. The propagation time of the P + H cluster was longer than that of the P cluster, suggesting the negative influence of human activities. This study can provide additional information for policymakers involved in groundwater management during a drought