Lack of α2C-Adrenoceptor Results in Contrasting Phenotypes of Long Bones and Vertebra and Prevents the Thyrotoxicosis-Induced Osteopenia

A series of studies have demonstrated that activation of the sympathetic nervous system (SNS) causes osteopenia via β₂-adrenoceptor (β₂-AR) signaling. However, in a recent study, we found an unexpected and generalized phenotype of high bone mass in female mice with chronic sympathetic hyperactivity, due to double gene inactivation of adrenoceptors that negatively regulate norepinephrine release, α_2A-and α_2C-AR (α_2A/2C-AR^-/-). These findings suggest that β₂-AR is not the single adrenoceptor involved in bone turnover regulation and show that α₂-AR signaling may also mediate the SNS actions in the skeleton. In addition, we found that α_2A/2C-AR^-/- animals are resistant to the thyrotoxicosis-induced osteopenia, suggesting that thyroid hormone (TH), when in supraphysiological levels, interacts with the SNS to control bone mass and structure, and that this interaction may also involve α₂-AR signaling. In the present study, to further investigate these hypotheses and to discriminate the roles of α₂-AR subtypes, we have evaluated the bone phenotype of mice with the single gene inactivation of α_2C-AR subtype, which mRNA expression was previously shown to be down regulated by triiodothyronine (T3). A cohort of 30 day-old female α_2CAR^-/- mice and their wild-type (WT) controls were treated with a supraphysiological dose of T3 for 30 or 90 days, which induced a thyrotoxic state in both mouse lineages. The micro-computed tomographic (μCT) analysis showed that α_2C-AR^-/- mice present lower trabecular bone volume (BV/TV) and number (Tb.N), and increased trabecular separation (Tb.Sp) in the femur compared with WT mice; which was accompanied by decreased bone strength (determined by the three-point bending test) in the femur and tibia. The opposite was observed in the vertebra, where α_2C-AR^-/- mice show increased BV/TV, Tb.N and trabecular thickness (Tb.Th), and decreased Tb.Sp, compared with WT animals. In spite of the contrasting bone phenotypes of the femur and L5, thyrotoxicosis negatively regulated most of the micro architectural features of the trabecular bone in both skeletal sites of WT, but not of α_2C-AR^-/- mice. T3 treatment also decreased biomechanical properties (maximum load and ultimate load) in the femur and tibia of WT, but not of knockout mice. The mRNA expression of osteocalcin, a marker of mature osteoblasts, and tartrate-resistant acid phosphatase, which is expressed by osteoclasts and is involved in collagen degradation, was increased by T3 treatment only in WT, and not in α_2C-AR^-/- mice. Altogether, these findings suggest that α_2C-AR subtype mediates the effects of the SNS in the bone in a skeletal site-dependent manner, and that thyrotoxicosis depends on α_2C-AR signaling to promote bone loss, which sustains the hypothesis of a TH-SNS interaction to modulate bone remodeling and structure.     

Effect of in vitro exposure of human serum to 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one on oxidative stress

Increased oxidative stress and impaired antioxidant defense mechanisms are believed to be the important factors contributing to the pathogenesis and progression of diabetes mellitus. In this study, we have reported the effects of the streptozotocin-induced diabetes on the gene expression and the activities of two antioxidant enzymes, manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPx). We also studied the effects of two antioxidants, vitamin C and DL-α-lipoic acid (LA), on the system. Our results showed no significant change in both enzymes activities in diabetic animals compared to controls. Similarly, mRNA and protein profiles of MnSOD showed no change. Though the mRNA expression of GPx did not show any change, Western-blot analysis results demonstrated that protein expression is increased. LA, which is a water- and lipid-soluble antioxidant, decreased the protein expression of MnSOD, though mRNA levels and activities remained unchanged. LA treatment increased the GPx activities in diabetic tissues, significantly, and RT-PCR and Western-blot analysis results demonstrated that this increase in activity is not regulated at the gene level, as both mRNA and protein levels did not change. Supplementing the animals with vitamin C, a powerful water-soluble antioxidant, increased the mRNA expression of MnSOD, though the protein expression and the activity did not change statistically. On the other hand GPx activity increased significantly through post-translational modifications, as both mRNA and protein expressions did not change. These results together with our previous findings about the gene expressions of catalase and Cu–Zn SOD indicate the presence of very intricate control mechanisms regulating the activities of antioxidant enzymes in order to prevent the damaging effects of oxidative stress.     

Exercise training changes IL-10/TNF-α ratio in the skeletal muscle of post-MI rats

Heart failure (HF) is associated with changes in the skeletal muscle (SM) which might be a consequence of the unbalanced local expression of pro- (TNF-α) and anti- (IL-10) inflammatory cytokines, leading to inflammation-induced myopathy, and SM wasting. This local effect of HF on SM may, on the other hand, contribute to systemic inflammation, as this tissue actively secretes cytokines. Since increasing evidence points out to an anti-inflammatory effect of exercise training, the goal of the present study was to investigate its effect in rats with HF after post-myocardial infarction (MI), with special regard to the expression of TNF-α and IL-10 in the soleus and extensor digitorum longus (EDL), muscles with different fiber composition. Wistar rats underwent left thoracotomy with ligation of the left coronary artery, and were randomly assigned to either a sedentary (Sham-operated and MI sedentary) or trained (Sham-operated and MI trained) group. Animals in the trained groups ran on a treadmill (0% grade at 13–20 m/min) for 60 min/day, 5 days/week, for 8–10 weeks. The training protocol was able to reverse the changes induced by MI, decreasing TNF-α protein (26%, P < 0.05) and mRNA (58%, P < 0.05) levels in the soleus, when compared with the sedentary MI group. Training also increased soleus IL-10 expression (2.6-fold, P < 0.001) in post-MI HF rats. As a consequence, the IL-10/TNF-α ratio was increased. This “anti-inflammatory effect” was more pronounced in the soleus than in the EDL, suggesting a fiber composition dependent response.     

Spatial and temporal segregation in dung beetles (Coleoptera: Scarabaeinae) in the Cerrado of Central Brazil

Biodiversity and Conservation - In dung beetles, spatial and temporal segregation is determined by the niche and evolutionary history of the species and is mediated by competition. Different...     

Effects of creatine supplementation on muscle wasting and glucose homeostasis in rats treated with dexamethasone

We aimed to investigate the possible role of creatine (CR) supplementation in counteracting dexamethasone-induced muscle wasting and insulin resistance in rats. Also, we examined whether CR intake would modulate molecular pathways involved in muscle remodeling and insulin signaling. Animals were randomly divided into four groups: (1) dexamethasone (DEX); (2) control pair-fed (CON-PF); (3) dexamethasone plus CR (DEX-CR); and (4) CR pair-fed (CR-PF). Dexamethasone (5 mg/kg/day) and CR (5 g/kg/day) were given via drinking water for 7 days. Plantaris and extensor digitorum longus (EDL) muscles were removed for analysis. Plantaris and EDL muscle mass were significantly reduced in the DEX-CR and DEX groups when compared with the CON-PF and CR-PF groups (P < 0.05). Dexamethasone significantly decreased phospho-Ser⁴⁷³-Akt protein levels compared to the CON-PF group (P < 0.05) and CR supplementation aggravated this response (P < 0.001). Serum glucose was significantly increased in the DEX group when compared with the CON-PF group (DEX 7.8 ± 0.6 vs. CON-PF 5.2 ± 0.5 mmol/l; P < 0.05). CR supplementation significantly exacerbated hyperglycemia in the dexamethasone-treated animals (DEX-CR 15.1 ± 2.4 mmol/l; P < 0.05 vs. others). Dexamethasone reduced GLUT-4 translocation when compared with the CON-PF and CR-PF (P < 0.05) groups and this response was aggravated by CR supplementation (P < 0.05 vs. others). In conclusion, supplementation with CR resulted in increased insulin resistance and did not attenuate muscle wasting in rats treated with dexamethasone. Given the contrast with the results of human studies that have shown benefits of CR supplementation on muscle atrophy and insulin sensitivity, we suggest caution when extrapolating this animal data to human subjects.     

Reference gene for primary culture of prostate cancer cells

Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.