Anti-diabetic Role of Adropin in Streptozotocin Induced Diabetic Rats via Alteration of PI3K/Akt and Insulin Signaling Pathway.

Diabetes mellitus (DM) is a hyperglycemia-related multifactorial condition with an elevated risk of microvascular and microvascular complications associated with this disease. The current experimental study was to examine the antidiabetic activity of streptozotocin (STZ)-induced adropin against diabetic rats by altering the PI3K/Akt and insulin signaling pathways. STZ (60 mg/kg) was used for the induction of DM and rats were divided into different groups and received the adropin (20, 40 and 80 mg/kg) and glibenclamide (10 mg/kg) till 28 days. Body weight, plasma insulin, blood glucose and food intake were estimated, respectively. Biochemical enzymes, carbohydrate enzymes, lipid parameters, AMPK and insulin signalling pathway parameters were estimated. GLUT4 and PPARγ expression were also estimated. Oral administration of adropin significantly (p < 0.001) increased the glycogen, glucose-6-phosphatase dehydrogenase, insulin, hexokinase and belittled the blood glucose level, fructose 1-6-biphosphatase, glucose-6-phosphatase at dose dependent manner. Adropin significantly (p < 0.001) reduced the level of triglyceride, cholesterol, low density lipoprotein, very low density lipoprotein and increased the level of high density lipoprotein at dose dependent manner. Adropin significantly (p < 0.001) activated the Akt, IRS-2, IRS-1, IR, p-AKT and PI3k, which are the key modulator molecules of PI3K/Akt, AMPK and insulin signalling pathway in DM rats. The current experimental study confirms the anti-diabetic effect of adropin on DM rats induced by AMPK and insulin signalling pathway against STZ.

Differential expression of genes associated with hypoxia pathway on bone marrow stem cells in osteoporosis patients with different bone mass index.

BACKGROUND: This study aimed to assess gene expression changes associated with hypoxia pathway on bone marrow stem cells (BMSCs) and explore effects of bone mass index (BMI) on hypoxia pathway of osteoporosis (OP) patients. METHODS: Human BMSCs were isolated from bone marrow. Subjects were divided into healthy control group and OP group which was further divided into BMI <25 OP subgroup and BMI ≥25 OP subgroup. RESULTS: The genes downregulated in OP patients were involved in hypoxia pathway. Furthermore, those genes were even downregulated in OP patients BMI ≥25 subgroup than OP patients BMI <25 subgroup. The genes were expressed in response to decreased oxygen levels, and their functions are related to photoperiodism, positive regulation of myoblast differentiation, and entrainment of circadian clock by gene ontology (GO) analysis. CONCLUSIONS: The expression of genes associated with hypoxia pathway on BMSCs in OP patients are lower than healthy subjects, and the expression of genes related to carbohydrate metabolism are lower in overweight OP patients than in normal weight OP patients. These results need further research.

Prevalence of Vitamin D Deficiency during Second Trimester of Pregnancy in Shanghai China, Risk Factors and Effects on Pregnancy Outcomes.

BACKGROUND: Vitamin D plays important roles in various physiological processes. Vitamin D deficiency is common among pregnant women in some regions, such as China. Our study aimed to determine the prevalence of Vitamin D deficiency during second trimester of pregnancy in Shanghai China, and explore its risk factors and effects on pregnant outcomes. METHODS: Overall, 23100 pregnant women (2013 to 2017, Shanghai, China) were included and vitamin D concentrations were measured at 16 weeks of gestation. Correlations between vitamin D concentrations and participants' general data and maternal and infant outcomes were analyzed by chi square test. Non-conditional multivariate logistic regression analysis was used to screen the independent risk factors for vitamin D deficiency. RESULTS: Vitamin D deficiency was significantly correlated with aging, education level, smoking, dirking, BMI before pregnancy, body weight gain during pregnancy (P<0.01), the use of vitamin D supplement and milk consumption, and older than 30 years, drinking, smoking, BMI before pregnancy> 36, body weight gain during pregnancy< 40g per day, no daily milk consumption, no vitamin D supplement, and education lever below college were independent risk factors for vitamin D deficiency in second trimester of pregnancy. In addition, vitamin D deficiency in second trimester of pregnancy was closely correlated with the occurrence of a serious of adverse maternal and infant outcomes. CONCLUSION: Vitamin D deficiency was still common among women in second trimester of pregnancy in Shanghai China. Vitamin D deficiency was closely correlated with the occurrence of a serious of adverse maternal and infant outcomes.

Postnatal deletion of ß-catenin in osterix-expressing cells is necessary for bone growth and intermittent PTH-induced bone gain.

wnt/ß-catenin signaling has been shown to influence bone homeostasis and is important for parathyroid hormone (PTH)-induced bone gain. To further understand the role of ß-catenin in the early stages of osteoblastic lineage cells for postnatal bone homeostasis and the anabolic actions of PTH on bone, we examined mice with postnatal disruption of ß-catenin in osterix-expressing cells (ß-catenin KO mice) by mating floxed ß-catenin mice with transgenic mice expressing cre under the control of the osterix promoter suppressible by doxycycline. After withdrawal of doxycycline, ß-catenin KO mice developed progressive bone loss, ectopic cartilage formation, accumulation of mesenchymal stromal cells, and bone marrow adiposity. The ß-catenin-defective osteoblasts sorted by flow cytometry from ß-catenin KO mice exhibited decreased EdU incorporation, increased annexin V activity, and profound alterations in gene expression including wnt target genes, osteoclast regulators, and osteoblast markers. A dramatic increase in osteoclasts was observed in both neonatal and postnatal ß-catenin KO mice. Intermittent administration of PTH for 4 weeks significantly increased bone mass in control mice; however, this anabolic effect of PTH was substantially blunted in ß-catenin KO mice. Our data indicate that ß-catenin in osterix-expressing cells is required for postnatal osteoblast differentiation, osteoblast proliferation, and bone resorption, and is essential for the anabolic actions of PTH in bone.

Wnt/ß-catenin signaling in osteoblasts regulates global energy metabolism.

Obesity, diabetes and osteoporosis have become a major public heath burden, and understanding the underlying mechanisms of these pathophysiological process will benefit their treatment. Osteoblast lineage cells in charge of the bone formation have been showed to participate in the whole-body energy metabolism. In this study, we identify that wnt/ß-catenin signaling in osteoblasts could regulate global energy metabolism, including glucose homeostasis, fat accumulation and energy expenditure. Mice lacking ß-catenin specifically in osteoblasts postnatally exhibit decreased bone mass, increased glucose level, decreased insulin production, decreased fat accumulation and increased energy expenditure. Osteocalcin supplement can rescue the impaired glucose balance by improving insulin production but cannot influence the abnormal fat accumulation and energy expenditure. Osteoprotegerin (OPG) overexpression exclusively in osteoblasts in ß-catenin deletion mice can normalize not only the decreased bone mass but also the decreased fat accumulation and increased energy expenditure. The effect of ß-catenin deletion and OPG overexpression in osteoblasts on global energy metabolism had no relation with inguinal fat browning. These results suggest that the regulation of bone on energy metabolism and fat accumulation is not mediated exclusively by osteocalcin. Our findings may provide a new insight into the regulation of bone on fat accumulation and energy metabolism.

ACVR1-Fc suppresses BMP signaling and chondro-osseous differentiation in an in vitro model of Fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare and devastating genetic disease of heterotopic endochondral ossification (HEO), and currently no effective therapies are available for this disease. A recurrent causative heterozygous mutation (c.617 G>A; R206H) for FOP was identified in activin receptor type IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor. This mutation aberrantly activates the BMP-Smad1/5/8 signaling pathway and leads to HEO in FOP patients. Here we report development of a soluble recombinant ACVR1-Fc fusion protein by combining the extracellular domain of human wild type ACVR1 and the Fc portion of human immunoglobulin gamma 1 (IgG1). The ACVR1-Fc fusion protein significantly down-regulated the dysregulated BMP signaling caused by the FOP ACVR1 mutation and effectively suppressed chondro-osseous differentiation in a previously described cellular FOP model, human umbilical vein endothelial cells (HUVECs) that were infected with adenovirus-ACVR1R206H (HUVECR206H). This ACVR1-Fc fusion protein holds great promise for prevention and treatment of HEO in FOP and related diseases.

The Vitamin D Receptor Regulates Tissue Resident Macrophage Response to Injury.

Ligand-dependent actions of the vitamin D receptor (VDR) play a pleiotropic role in the regulation of innate and adaptive immunity. The liganded VDR is required for recruitment of macrophages during the inflammatory phase of cutaneous wound healing. Although the number of macrophages in the granulation tissue 2 days after wounding is markedly reduced in VDR knockout (KO) compared with wild-type mice, VDR ablation does not alter macrophage polarization. Parabiosis studies demonstrate that circulatory chimerism with wild-type mice is unable to rescue the macrophage defect in the wounds of VDR KO mice and reveal that wound macrophages are of local origin, regardless of VDR status. Wound cytokine analyses demonstrated a decrease in macrophage colony-stimulating factor (M-CSF) protein levels in VDR KO mice. Consistent with this, induction of M-CSF gene expression by TGFß and 1,25-dihydroxyvitamin D was impaired in dermal fibroblasts isolated from VDR KO mice. Because M-CSF is important for macrophage self-renewal, studies were performed to evaluate the response of tissue resident macrophages to this cytokine. A decrease in M-CSF induced proliferation and cyclin D1 expression was observed in peritoneal resident macrophages isolated from VDR KO mice, suggesting an intrinsic macrophage abnormality. Consistent with this, wound-healing assays in mice with macrophage-specific VDR ablation demonstrate that a normal wound microenvironment cannot compensate for the absence of the VDR in macrophages and thus confirm a critical role for the macrophage VDR in the inflammatory response to injury.

Adipocytes exert lipotoxic effects on osteoblast through activating hypoxia signaling pathway in vitro.

This study aimed to investigate the effect of adipocytes on osteoblastic bone formation in vitro. The differentiated and undifferentiated 3T3-L1 cells were co-cultured with primary calvarial osteoblasts. At 48 h, proliferated osteoblasts decreased significantly after co-culture with differentiated preadipocytes as compared to those co-cultured with undifferentiated preadipocytes; at 7 days, the expressions of bone formation-related genes decreased in osteoblasts co-cultured with differentiated preadipocytes; at 14 days, osteoblasts mineralization also decreased significantly after co-culture with differentiated preadipocytes. To further determine whether the decreased proliferation and mineralization were related to the hypoxia signaling pathway, the expressions of hypoxia related-genes were detected. Results showed the expressions of these genes significantly increased after co-culture with differentiated preadipocytes. NF-κB and IL-6 expressions were also up-regulated in osteoblasts co-cultured with differentiated preadipocytes. Osteoblasts from Hif1α(f/f) mice showed increased proliferation and mineralization after co-culture with adipocytes transfected with adenoviral-cre, accompanied by up-regulated expressions of bone formation-related genes and down-regulated expressions of NF-κB and IL-6. These results demonstrated that adipocytes exert a negative effect on the proliferation and mineralization of osteoblasts via up-regulating the expressions of hypoxia-related genes, and NF-κB and IL-6 may impair the osteoblastic bone formation.

Estrogen-mimicking isoflavone genistein prevents bone loss in a rat model of obstructive sleep apnea-hypopnea syndrome.

OBJECTIVE: Intermittent hypoxia was introduced to mimic obstructive sleep apnea-hypopnea syndrome (OSAHS) in rats. Then, bone mass, bone strength and bone turnover were evaluated, and the influence of genistein on bone mass reduction was investigated in these rats. METHODS: OSAHS animal model was established via chronic intermittent hypoxia, and genistein (2.5 mg/kg/day) was used to treat OSAHS rats. The bone mineral density (BMD), bone Histomorphometric indicators, bone biomechanics and expressions of genes related to bone formation and resorption (Runx2, Col I, ALP, Osteocalcin, OPG, RANKL and TRAP-5b) were measured after treatment. RESULTS: The BMD in OSAHS+OVX group was significantly lower than that in OVX group (P<0.05). The BMD in OSAHS+OVX+Genistein group was markedly increased when compared with OSAHS+OVX group (P<0.05), accompanied by partial improvement of the OSAHS induced damage to the lumbar biomechanics. In OSAHS+OVX group, the expressions of Runx2, Col I, ALP and Osteocalcin were significantly reduced when compared with OVX group, and rats in OSAHS+OVX+Genistein group had significantly higher expressions of Runx2, Col I, ALP and Osteocalcin and reduced TRAP-5b expression as compared to OSAHS+OVX group (P<0.05). CONCLUSIONS: Genistein can improve the reduction in bone mass and bone strength due to OSAHS in OVX rats, which may be attributed to the increase in bone formation and inhibition of bone resorption. Our findings suggest that genistein may be used to treat and prevent osteoporosis in postmenopausal women with OSAHS.

Icariin induces osteoblast proliferation, differentiation and mineralization through estrogen receptor-mediated ERK and JNK signal activation.

Icariin, the main active flavonoid glucoside isolated from Herba epimedii (HEF), is an anabolic agent in bone that has been reported to prevent bone loss in ovariectomized rats and postmenopausal women. However, the molecular mechanism for this anabolic action of Icariin remain largely unknown. Here, we found that Icariin could promote MC3T3-E1 osteoblastic cell proliferation and reduce cell apoptosis, associated with increased mRNA levels of positive regulators of cell cycle gene Cyclin E and proliferating cell nuclear antigen (PCNA), decreaed mRNA level of negative regulator gene, Cyclin-dependent kinase 4 inhibitor B (Cdkn2B), and reduced caspase-3 activity. Icariin also enhanced MC3T3-E1 cell differentiation and mineralization demonstrated by increased the expression of differentiation markers, alkaline phosphatase (ALP) and collagen type I (Col I), and bone nodule formation via Alizarin red S staining. To characterize the underlying mechanisms, we examined the effect of Icariin on mitogen-activated protein kinase (MAPK) signaling. Icariin treatment rapidly induced extracellular signal-regulated kinase (ERK) and c-Jun N terminal kinase (JNK) activation but showed no effect on activation of p38 kinase. Furthermore, Icariin-mediated effects on osteoblasts were dramatically attenuated by treatment with specific inhibitors of MAPKs, U0126 (ERK inhibitor) and SP600125 (JNK inhibitor). Interestingly, treatment of osteoblasts with estrogen receptor antagonist ICI182780 attenuated Icariin-mediated effect of proliferation and mineralization, associated with suppression of ERK and JNK phosphorylation. These observations provide a potential mechanism of anabolic actions of Icariin involving ERK and JNK pathway by estrogen receptor.

Activation of a non-cAMP/PKA signaling pathway downstream of the PTH/PTHrP receptor is essential for a sustained hypophosphatemic response to PTH infusion in male mice.

PTH increases urinary Pi excretion by reducing expression of two renal cotransporters [NaPi-IIa (Npt2a) and NaPi-IIc (Npt2c)]. In contrast to acute transporter regulation that is cAMP/protein kinase A dependent, long-term effects require phospholipase C (PLC) signaling by the PTH/PTHrP receptor (PPR). To determine whether the latter pathway regulates Pi through Npt2a and/or Npt2c, wild-type mice (Wt) and animals expressing a mutant PPR incapable of PLC activation (DD) were tested in the absence of one (Npt2a(-/-) or Npt2c(-/-)) or both phosphate transporters (2a/2c-dko). PTH infusion for 8 days caused a rapid and persistent decrease in serum Pi in Wt mice, whereas serum Pi in DD mice fell only transiently for the first 2 days. Consistent with these findings, fractional Pi excretion index was increased initially in both animals, but this increase persisted only when the PPR Wt was present. The hypophosphatemic response to PTH infusion was impaired only slightly in PPR Wt/Npt2c(-/-) or DD/Npt2c(-/-) mice. Despite lower baselines, PTH infusion in PPR Wt/Npt2a(-/-) mice decreased serum Pi further, an effect that was attenuated in DD/Npt2a(-/-) mice. Continuous PTH had no effect on serum Pi in 2a/2c-dko mice. PTH administration increased serum 1,25 dihydroxyvitamin D3 levels in Wt and DD mice and increased levels above the elevated baseline with ablation of either but not of both transporters. Continuous PTH elevated serum fibroblast growth factor 23 and blood Ca(2+) equivalently in all groups of mice. Our data indicate that PLC signaling at the PPR contributes to the long-term effect of PTH on Pi homeostasis but not to the regulation of 1,25 dihydroxyvitamin D3, fibroblast growth factor 23, or blood Ca(2+).

Loss of wnt/ß-catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes.

Wnt signaling is essential for osteogenesis and also functions as an adipogenic switch, but it is not known if interrupting wnt signaling via knockout of ß-catenin from osteoblasts would cause bone marrow adiposity. Here, we determined whether postnatal deletion of ß-catenin in preosteoblasts, through conditional cre expression driven by the osterix promoter, causes bone marrow adiposity. Postnatal disruption of ß-catenin in the preosteoblasts led to extensive bone marrow adiposity and low bone mass in adult mice. In cultured bone marrow-derived cells isolated from the knockout mice, adipogenic differentiation was dramatically increased, whereas osteogenic differentiation was significantly decreased. As myoblasts, in the absence of wnt/ß-catenin signaling, can be reprogrammed into the adipocyte lineage, we sought to determine whether the increased adipogenesis we observed partly resulted from a cell-fate shift of preosteoblasts that had to express osterix (lineage-committed early osteoblasts), from the osteoblastic to the adipocyte lineage. Using lineage tracing both in vivo and in vitro we showed that the loss of ß-catenin from preosteoblasts caused a cell-fate shift of these cells from osteoblasts to adipocytes, a shift that may at least partly contribute to the bone marrow adiposity and low bone mass in the knockout mice. These novel findings indicate that wnt/ß-catenin signaling exerts control over the fate of lineage-committed early osteoblasts, with respect to their differentiation into osteoblastic versus adipocytic populations in bone, and thus offers potential insight into the origin of bone marrow adiposity.

A synergetic role of 1,25-dihydroxyvitamin D(3) in 17ß-estradial induced-proliferation and differentiation of osteoblastic MC3T3-E1 cells.

Although the effect of 17ß-estradial, a polyphenolic phytoestrogen, on bone cell function has been studied in numerous cell models, the synergetic role of 1, 25-dihydroxyvitamin D(3) on 17ß-estradial induced-proliferation and differentiation of osteoblastic cells, and the underlying mechanism are obscure. Here, we investigated the in vitro effect of 17ß-estradial on cell proliferation and osteoblastic maturation in MC3T3-E1 cells. 17ß-estradial could promote the proliferation and viability of MC3T3-E1 cells, associated with upregulation of cyclin E and proliferation cell nuclear antigen (PCNA) mRNA expression, and downregulation of cyclin-dependent kinase inhibitor 2b (Cdkn2b) mRNA expression. Moreover, 17ß-estradial also could stimulate osteoblastic differentiation and bone formation as assessed by alkaline phosphatase (ALP) and Alizarin Red S staining, through induction of the expression of osteoblastic markers, including ALP, osteopontin and type I collagen in MC3T3-E1 cells. However, 1,25-dihydroxyvitamin D(3) treatment alone showed no effect on proliferation and differentiation of MC3T3-E1 cells, but could coordinately augment effects of 17ß-estradial on MC3T3-E1 cells. The mechanism conducted demonstrated that 17ß-estradial activated ERK1/2 but not JNK and p38, and U0126, an ERK1/2 pathway inhibitor, significantly downregulated vitamin D receptor expression induced by 17ß-estradial in MC3T3-E1 cells. Thus, our data demonstrated a synergistical role of 1,25-dihydroxyvitamin D(3) and 17ß-estradial in proliferation and differentiation of osteoblasts, and this coordinated regulation might depend on the upregulation of vitamin D receptor in osteoblasts by 17ß-estradial. Moreover, during the process of vitamin D receptor upregulation by 17ß-estradial, ERK1/2 signaling is involved.

[Effects of alendronate on bone mineral density, cytokines and indices of bone metabolism in postmenopausal osteoporotic patients].

OBJECTIVE: To observe the effects of alendronate on bone mineral density (BMD), cytokines and indices of bone metabolism in postmenopausal osteoporotic (PMO) patients. METHODS: 185 PMO patients, aged from 55 to 60 years, were randomized into three groups. All of them received a kind of calcium preparation, 1.0 g/d. Group A, 69 patients, received alendronate, 10 mg/d; group B, 66 patients, received tibolone 1.25 mg/d. The remaining patients, group C, received calcium preparation only. 20 women of 55 - 60 (57.4 +/- 3.5) years old were taken as normal controls. Dual-energy X-ray absorptiometry and measurement of a series of biochemical indices were carried out before and after medication at 24th and 48th week. RESULTS: After medication, BMD increased in alendronate and tibolone group. The increment in spine BMD was 2.53% and 3.65% (P < 0.05) and that in nondominant proximal femur BMD was 7.17% and 3.01% (P < 0.001). In the tibolone group, the levels of estradiol (E(2)) increased rapidly (P < 0.01), but those of IL-6, TNFalpha and type I collagen cross-linked N-telopeptides (NTX) decreased (P < 0.01). In the alendronate group, no change of levels of E(2) happened, while levels of alkaline phosphatase (ALP) and osteocalcin (BGP) increased (P < 0.05) and levels of NTX decreased (P < 0.05). There was no change of the levels of other parameters. In the calcium preparation group and control group, the levels of BMD, E(2), ALP, BGP and insulin-like growth factor I decreased (P < 0.05), while those of IL-6, TNFalpha and NTX increased (P < 0.05). CONCLUSION: Alendronate can significantly improve BMD as tibolone do. Thus it plays an important role in the treatment of osteoporosis. However calcium tablet can not prevent the loss of bone.