Cecilia H. A. Gouveia

Marked Disturbance of Calcium Homeostasis in Mice With Targeted Disruption of the Trpv6 Calcium Channel Gene

We report the phenotype of mice with targeted disruption of the Trpv6 (Trpv6 KO) epithelial calcium channel. The mice exhibit disordered Ca2+ homeostasis, including defective intestinal Ca2+ absorption, increased urinary Ca2+ excretion, decreased BMD, deficient weight gain, and reduced fertility. Although our Trpv6 KO affects the closely adjacent EphB6 gene, the phenotype reported here is not related to EphB6 dysfunction.

Effects of thyroid hormone administration and estrogen deficiency on bone mass of female rats

To investigate the effects of thyroxine (T4) administration on bone mass, five 81‐day‐old female rats were treated with T4 (25 μg of T4/100 g of body weight [bw]/day), and bone mineral density (BMD) was measured by dual‐energy X‐ray absorptiometry (DXA) 28 days later. The BMD values for the total skeleton, femoral, and tibial subsegments were lower than in controls (p ≤ 0.05). The lumbar spine (L2–L5) was not significantly affected by T4 treatment. Next, thirty‐seven 211 ± 1.5 (mean ± SEM)‐day‐old female rats were divided into six groups as follows: (1) control; (2) ovariectomized (OVX); (3) 1xT4 (∼1.0 μg of T4/100 g of bw/day; approximately physiological replacement dose); (4) OVX+1xT4; (5) 2xT4 (∼2.0 μg of T4/100 g of bw/day); (6) OVX+2xT4. DXA scans were performed at days 0 and 85. Control rats showed a generalized BMD increase, as opposed to a decrease in OVX rats. The trabecular bone volume of the fifth lumbar vertebra was also lower in OVX rats than in controls (p < 0.05). The 1xT4 treatment had no effect on BMD of intact rats, while treatment with 2xT4 impaired the expected BMD increase. Unexpectedly, the OVX+1xT4 group presented a generalized BMD increase that was significant for the total skeleton, L2–L5, and femoral subsegments (p < 0.05), comparable to controls. Treating OVX animals with 2xT4 did not potentiate the osteopenic effects of estrogen deficiency, nor did it reverse the osteopenic effects of OVX. In conclusion, treatment with high doses of T4 caused BMD to decrease substantially, particularly at the femur, whereas near physiological doses of T4 prevented bone loss associated with OVX, and regardless of bone type (trabecular or cortical), the skeleton site seems to be a more important determinant of the effects of thyroid hormone on bone mass.

The Thyroid Hormone Receptor β‐Specific Agonist GC‐1 Selectively Affects the Bone Development of Hypothyroid Rats

We investigated the effects of GC‐1, a TRβ‐selective thyromimetic, on bone development of hypothyroid rats. Whereas T3 reverted the IGF‐I deficiency and the skeletal defects caused by hypothyroidism, GC‐1 had no effect on serum IGF‐I or on IGF‐I protein expression in the epiphyseal growth plate of the femur, but induced selective effects on bone development. Our findings indicate that T3 exerts some essential effects on bone development that are mediated by TRβ1.

β1 Adrenergic receptor is key to cold- and diet-induced thermogenesis in mice

Brown adipose tissue (BAT) is predominantly regulated by the sympathetic nervous system (SNS) and the adrenergic receptor signaling pathway. Knowing that a mouse with triple β-receptor knockout (KO) is cold intolerant and obese, we evaluated the independent role played by the β(1) isoform in energy homeostasis. First, the 30  min i.v. infusion of norepinephrine (NE) or the β(1) selective agonist dobutamine (DB) resulted in similar interscapular BAT (iBAT) thermal response in WT mice. Secondly, mice with targeted disruption of the β(1) gene (KO of β(1) adrenergic receptor (β(1)KO)) developed hypothermia during cold exposure and exhibited decreased iBAT thermal response to NE or DB infusion. Thirdly, when placed on a high-fat diet (HFD; 40% fat) for 5 weeks, β(1)KO mice were more susceptible to obesity than WT controls and failed to develop diet-induced thermogenesis as assessed by BAT Ucp1 mRNA levels and oxygen consumption. Furthermore, β(1)KO mice exhibited fasting hyperglycemia and more intense glucose intolerance, hypercholesterolemia, and hypertriglyceridemia when placed on the HFD, developing marked non-alcoholic steatohepatitis. In conclusion, the β(1) signaling pathway mediates most of the SNS stimulation of adaptive thermogenesis.

Thyroid hormone receptor-β-selective agonist GC-24 spares skeletal muscle type I to II fiber shift

Triiodothyronine (T3) is known to play a key role in the function of several tissues/organs via the thyroid hormone receptor isoforms alpha (TRα) and beta (TRβ). We have investigated the effects of GC-24, a novel synthetic TRβ-selective compound, on skeletal muscle fiber-type determination, cross-sectional area, and gene expression in rat skeletal muscles. For fiber typing, cross sections of soleus and extensor digitorum longus (EDL) muscles were stained for myosin ATPase activity at various pHs. Serum T3, T4, and cholesterol levels were also determined. Analysis of highly T3-responsive genes, viz., myosin heavy chain IIa (MHCIIa) and sarcoendoplasmic reticulum adenosine triphosphatase (SERCA1), was performed by quantitative real-time polymerase chain reaction. Equimolar doses of T3 and GC-24 had a similar cholesterol-lowering effect. T3, but not GC-24, decreased fiber type I and increased fiber type II abundance in soleus and EDL muscles. Conversely, in EDL, both T3 and GC-24 decreased the mean cross-sectional area of type I fibers. MHCIIa gene expression was reduced (approximately 50%) by T3 and unchanged by GC-24. SERCA1 gene expression was strongly induced by T3 (approximately 20-fold) and mildly induced by GC-24 (approximately two-fold). These results show that GC-24 does not significantly alter the composition of skeletal muscle fiber type and further strengthens the putative use of GC compounds as therapeutic agents.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton.

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To study this, pregnant mice were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved up to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocytes, in addition to a slight delay in endochondral and intramembranous ossification. This suggests that up to E16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly ( approximately 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast, D2 mRNA expression increased significantly by E18.5 and markedly ( approximately 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development.

A TRβ-selective agonist confers resistance to diet-induced obesity

Thyroid hormone receptor beta (TRbeta also listed as THRB on the MGI Database)-selective agonists activate brown adipose tissue (BAT) thermogenesis, while only minimally affecting cardiac activity or lean body mass. Here, we tested the hypothesis that daily administration of the TRbeta agonist GC-24 prevents the metabolic alterations associated with a hypercaloric diet. Rats were placed on a high-fat diet and after a month exhibited increased body weight (BW) and adiposity, fasting hyperglycemia and glucose intolerance, increased plasma levels of triglycerides, cholesterol, nonesterified fatty acids and interleukin-6. While GC-24 administration to these animals did not affect food ingestion or modified the progression of BW gain, it did increase energy expenditure, eliminating the increase in adiposity without causing cardiac hypertrophy. Fasting hyperglycemia remained unchanged, but treatment with GC-24 improved glucose tolerance by increasing insulin sensitivity, and also normalized plasma triglyceride levels. Plasma cholesterol levels were only partially normalized and liver cholesterol content remained high in the GC-24-treated animals. Gene expression in liver, skeletal muscle, and white adipose tissue was only minimally affected by treatment with GC-24, with the main target being BAT. In conclusion, during high-fat feeding treatment with the TRbeta-selective agonist, GC-24 only partially improves metabolic control probably as a result of accelerating the resting metabolic rate.

The monocarboxylate transporter 8 and L-type amino acid transporters 1 and 2 are expressed in mouse skeletons and in osteoblastic MC3T3-E1 cells.

BACKGROUND Several plasma membrane transporters have been shown to mediate the cellular influx and/or efflux of iodothyronines, including the sodium-independent organic anion co-transporting polypeptide 1 (OATP1), the sodium taurocholate co-transporting polypeptide (NTCP), the L-type amino acid transporter 1 (LAT1) and 2 (LAT2), and the monocarboxylate transporter 8 (MCT8). The aim of this study was to investigate if the mRNAs of these transporters were expressed and regulated by thyroid hormone (TH) in mouse calvaria-derived osteoblastic MC3T3-E1 cells and in the fetal and postnatal bones of mice. METHODS The mRNA expression of the iodothyronine transporters was investigated with real-time polymerase chain reaction analysis in euthyroid and hypothyroid fetuses and litters of mice and in MC3T3-E1 cells treated with increasing doses of triiodothyronine (T(3); 10(-10) to 10(-6) M) or with 10(-8) M T(3) for 1-9 days. RESULTS MCT8, LAT1, and LAT2 mRNAs were detected in fetal and postnatal femurs and in MC3T3-E1 cells, while OATP1 and NTCP mRNAs were not. LAT1 and LAT2 mRNAs were not affected by TH status in vivo or in vitro or by the stage of bone development or osteoblast maturation (analyzed by the expression of osteocalcin and alkaline phosphatase, which are key markers of osteoblastic differentiation). In contrast, the femoral mRNA expression of MCT8 decreased significantly during post-natal development, whereas MCT8 mRNA expression increased as MC3T3-E1 cells differentiated. We also showed that MCT8 mRNA was up-regulated in the femur of hypothyroid animals, and that it was down-regulated by treatment with T(3) in MC3T3-E1 cells. CONCLUSIONS This is the first study to demonstrate the mRNA expression of LAT1, LAT2, and MCT8 in the bone tissue of mice and in osteoblast-like cells. In addition, the pattern of MCT8 expression observed in vivo and in vitro suggests that MCT8 may be important to modulate TH effects on osteoblast differentiation and on bone development and metabolism.

Double disruption of α2A- and α2C -adrenoceptors results in sympathetic hyperactivity and high-bone-mass phenotype

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via β2‐adrenoceptor (β2‐AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, α2A‐AR and α2C‐AR (α2A/α2C‐ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In α2A/α2C‐ARKO versus wild‐type (WT) mice, micro–computed tomographic (µCT) analysis showed increased, better connected, and more plate‐shaped trabeculae in the femur and vertebra and increased cortical thickness in the vertebra, whereas biomechanical analysis showed increased tibial and femoral strength. Tibial mRNA expression of tartrate‐resistant acid phosphatase (TRACP) and receptor activator of NF‐κB (RANK), which are osteoclast‐related factors, was lower in knockout (KO) mice. Plasma leptin and brain mRNA levels of cocaine amphetamine–regulated transcript (CART), which are factors that centrally affect bone turnover, and serum levels of estradiol were similar between mice strains. Tibial β2‐AR mRNA expression also was similar in KO and WT littermates, whereas α2A‐, α2B‐ and α2C‐AR mRNAs were detected in the tibia of WT mice and in osteoblast‐like MC3T3‐E1 cells. By immunohistochemistry, we detected α2A‐, α2B‐, α2C‐ and β2‐ARs in osteoblasts, osteoclasts, and chondrocytes of 18.5‐day‐old mouse fetuses and 35‐day‐old mice. Finally, we showed that isolated osteoclasts in culture are responsive to the selective α2‐AR agonist clonidine and to the nonspecific α‐AR antagonist phentolamine. These findings suggest that β2‐AR is not the single adrenoceptor involved in bone turnover regulation and show that α2‐AR signaling also may mediate the SNS actions in the skeleton.

S-nitrosoglutathione reductase–dependent PPARγ denitrosylation participates in MSC-derived adipogenesis and osteogenesis

Bone marrow-derived mesenchymal stem cells (MSCs) are a common precursor of both adipocytes and osteoblasts. While it is appreciated that PPARγ regulates the balance between adipogenesis and osteogenesis, the roles of additional regulators of this process remain controversial. Here, we show that MSCs isolated from mice lacking S-nitrosoglutathione reductase, a denitrosylase that regulates protein S-nitrosylation, exhibited decreased adipogenesis and increased osteoblastogenesis compared with WT MSCs. Consistent with this cellular phenotype, S-nitrosoglutathione reductase-deficient mice were smaller, with reduced fat mass and increased bone formation that was accompanied by elevated bone resorption. WT and S-nitrosoglutathione reductase-deficient MSCs exhibited equivalent PPARγ expression; however, S-nitrosylation of PPARγ was elevated in S-nitrosoglutathione reductase-deficient MSCs, diminishing binding to its downstream target fatty acid-binding protein 4 (FABP4). We further identified Cys 139 of PPARγ as an S-nitrosylation site and demonstrated that S-nitrosylation of PPARγ inhibits its transcriptional activity, suggesting a feedback regulation of PPARγ transcriptional activity by NO-mediated S-nitrosylation. Together, these results reveal that S-nitrosoglutathione reductase-dependent modification of PPARγ alters the balance between adipocyte and osteoblast differentiation and provides checkpoint regulation of the lineage bifurcation of these 2 lineages. Moreover, these findings provide pathophysiological and therapeutic insights regarding MSC participation in adipogenesis and osteogenesis.