Dio2 mRNA Levels Research Articles

Photoperiodically driven transcriptome-wide changes in the hypothalamus reveal transcriptional differences between physiologically contrasting seasonal life-history states in migratory songbirds

We investigated time course of photoperiodically driven transcriptional responses in physiologically contrasting seasonal life-history states in migratory blackheaded buntings. Birds exhibiting unstimulated winter phenotype (photosensitive state; responsive to photostimulation) under 6-h short days, and regressed summer phenotype (photorefractory state; unresponsiveness to photostimulation) under 16-h long days, were released into an extended light period up to 22 h of the day. Increased tshβ and dio2, and decreased dio3 mRNA levels in hypothalamus, and low prdx4 and high il1β mRNA levels in blood confirmed photoperiodic induction by hour 18 in photosensitive birds. Further, at hours 10, 14, 18 and 22 of light exposure, the comparison of hypothalamus RNA-Seq results revealed transcriptional differences within and between states. Particularly, we found reduced expression at hour 14 of transthyretin and proopiomelanocortin receptor, and increased expression at hour 18 of apolipoprotein A1 and carbon metabolism related genes in the photosensitive state. Similarly, valine, leucine and isoleucine degradation pathway genes and superoxide dismutase 1 were upregulated, and cocaine- and amphetamine-regulated transcript and gastrin-releasing peptide were downregulated in the photosensitive state. These results show life-history-dependent activation of hypothalamic molecular pathways involved in initiation and maintenance of key biological processes as early as on the first long day.

Decreased expression of the thyroid hormone-inactivating enzyme type 3 deiodinase is associated with lower survival rates in breast cancer

Thyroid hormones (THs) are critical regulators of cellular processes, while changes in their levels impact all the hallmarks of cancer. Disturbed expression of type 3 deiodinase (DIO3), the main TH-inactivating enzyme, occurs in several human neoplasms and has been associated with adverse outcomes. Here, we investigated the patterns of DIO3 expression and its prognostic significance in breast cancer. DIO3 expression was evaluated by immunohistochemistry in a primary cohort of patients with breast cancer and validated in a second cohort using RNA sequencing data from the TCGA database. DNA methylation data were obtained from the same database. DIO3 expression was present in normal and tumoral breast tissue. Low levels of DIO3 expression were associated with increased mortality in the primary cohort. Accordingly, low DIO3 mRNA levels were associated with an increased risk of death in a multivariate model in the validation cohort. DNA methylation analysis revealed that the DIO3 gene promoter is hypermethylated in tumors when compared to normal tissue. In conclusion, DIO3 is expressed in normal and tumoral breast tissue, while decreased expression relates to poor overall survival in breast cancer patients. Finally, loss of DIO3 expression is associated with hypermethylation of the gene promoter and might have therapeutic implications.

1709-P: Increased Adiponectin Levels Are Associated with Impaired Brown Adipose Tissue in Mice

Brown adipose tissue (BAT) thermogenesis affects energy expenditure, as well as glucose and lipid clearance. Thus, making BAT an attractive target in obesity/type-2 diabetes. Adiponectin (Apn) regulates whole-body metabolism and lowered Apn levels are associated with obesity and increased type-2 diabetes risk. Apn has been suggested to be required for maintaining normal body temperature in cold environment, but the role of Apn in BAT functionality is not clear. Here, we have studied BAT in adiponectin overexpressing (Apn-Tg) mice. During unchallenged chow-diet conditions, BAT of Apn-Tg mice was enlarged, fibrotic and more whitish. This phenotype was, as judged by tracer experiments, associated with lower lipid and glucose uptake, decreased de novo lipogenesis and decreased fatty acid oxidation in BAT. High-fat diet (HFD)-induced obesity increased these differences between genotypes. The BAT mRNA expression of thermogenesis markers such as UCP-1 and DIO-2 were down-regulated in BAT of obese, but not lean, Apn-Tg mice. Moreover, Seahorse-based analysis revealed reduced mitochondrial function in Apn-Tg BAT in ex vivo-differentiated brown adipocytes. To test whether increased Apn levels affect cold-induced BAT activation, we exposed Apn-tg and littermate control mice to cold temperature (6°C) for four weeks. Under this setting, the BAT lipid and glucose uptake, and body temperature were similar between genotypes. Accordingly, UCP-1 and DIO-2 mRNA levels were not different between Apn-Tg and WT mice, and highly induced by chronic cold exposure. Nevertheless, Seahorse data from isolated BAT mitochondria from cold-exposed animals still indicate mitochondrial dysfunction in Apn-Tg mice. Thus, our data suggest that increased adiponectin levels are associated with metabolic dysfunction in BAT. We propose that the increased BAT expansion in Apn-Tg mice occurs at the expense of the mitochondrial function. Disclosure B. Chanclon: None. M. Bauza-Thorbrugge: None. A. Benrick: None. I. Wernstedt-Asterholm: None.

SAT-448 Limited Adaptive Response by Deiodinase Enzymes in the Perinatal Hypothyroid Cochlea

Thyroid hormone (TH) is essential for cochlear development and normal auditory function. Considering the importance of TH in mediating cochlear development, understanding the degree to which developing tissues can adapt to perturbations in thyroid hormone signaling is extremely important. The deiodinases (Dio2 and Dio3) are enzymes that tightly control TH availability at the tissue level and have been proposed to function as adaptive mechanisms that maintain tissue TH homeostasis. Dio2 converts thyroxine (T4) to a biologically active ligand triiodothyronine (T3); locally amplifying a T3 signal. Conversely, Dio3 inactivates T3 and T4 by converting these iodothyronines to the inactive metabolites diiodothyronine (T2) and reverse T3 (rT3), respectively. During cochlear development, Dio3 expression is high prenatally while Dio2 expression is low. During the first postnatal week, the expression levels of these enzymes invert, resulting in high expression of Dio2 and low expression of Dio3. Together, the deiodinases control the timing of postnatal cochlear remodeling; suggesting a genetic developmental clock controls TH-mediated cochlear development. Considering the role deiodinases as adaptive mechanisms, it is important to understand whether such a developmental clock can be negated during times of developmental thyroid hormone insufficiency. We hypothesize that the perinatal change in deiodinase expression is controlled by a developmental clock rather than environmental clues and therefore have limited capacity function as compensatory mechanisms in response to low TH during development. To test this, timed-pregnant mice were treated with thyroid gland inhibitors to induced hypothyroidism from gestational day 12.5 until pup sacrifice. A parallel set of untreated timed-pregnant mice served as controls. Cochlea were harvest from control and hypothyroid mice at postnatal (P) ages P1, P5, P10 and P15 for qRT-PCR, and P7 for in situ hybridization. In situ hybridization for Dio2 mRNA demonstrated a striking reduction in Dio2 mRNA in hypothyroid animals compared to euthyroid animals. Additionally, we observed a significant effect of age (p<0.001) and an interaction between age and treatment (p=0.0035) on Dio2 mRNA levels analyzed by qRT-PCR. Specifically, Dio2 mRNA levels were similar or reduced in hypothyroid animals compared to controls at P1, P5, and P10. This finding indicates that Dio2 mRNA at these ages did not respond in manner consistent with compensation. However, at P15, Dio2 mRNA levels were increased in hypothyroid animals compared to controls; a finding that is consistent with a compensatory mechanism. Taken together, our results suggest that developmental programs in tissues may be dominant over potential compensatory mechanisms and that developing tissues may be more susceptible to perturbations in tissue TH levels due to a reduced capacity to compensate.

The hypothalamic-pituitary-gonadal axis and thyroid hormone regulation interact to influence seasonal breeding in green anole lizards (Anolis carolinensis)

Reproductive physiology and behavior is mainly regulated by the hypothalamus-pituitary-gonad (HPG) axis, although abnormal thyroid hormone (TH) levels alter HPG axis activity. Seasonally breeding animals, such as green anole lizards (Anolis carolinensis), undergo drastic hormonal and behavioral changes between breeding and non-breeding seasons, with increased sex steroid hormones, larger gonads and increased reproductive behaviors during the breeding compared to non-breeding seasons. Relatively less is known regarding the regulation of gonadal TH in seasonal reproduction. We examined whether the gonadal expression of enzymes involved in TH activation are altered in concert with seasonal reproduction. Type 2 deiodinase (Dio2) mRNA, the TH activating enzyme, was upregulated in breeding compared to non-breeding testes, while type 3 deiodinase (Dio3) mRNA, the TH deactivating enzyme, was upregulated in breeding ovaries. To study the association between the HPG axis and local activation of TH, we manipulated the HPG axis during the non-breeding season by subcutaneously injecting luteinizing hormone (LH) and follicle stimulating hormone (FSH) in male lizards. We found that acute LH and FSH injections induced many aspects of breeding, with increased testes size and testosterone levels. Surprisingly, Dio3 was upregulated in the testes after LH and FSH injections, while Dio2 mRNA levels were unchanged. These results suggest that there might be different roles for local TH activation in developing and maintaining fully mature and functional gonads. Our findings continue to support the role for TH in regulating reproduction.

Chronic effects of lead on metamorphosis, development of thyroid gland, and skeletal ossification in Bufo gargarizans

We examined the Pb2+ exposure on tadpoles of Bufo gargarizans from Gosner stage 26–42. Mortality, growth and development, time to metamorphosis, size, and skeletal ossification at metamorphic climax of Bufo gargarizans were examined. Also, histological characteristics of thyroid glands in tadpoles at Gosner stage 33, 38, and 42 as well as transcript levels of thyroid hormone-related genes in the hind-limb, tail, and liver of tadpoles at metamorphic climax were examined. Pb2+ exposure induced mortality in a concentration-dependent manner in Bufo gargarizans larvae. The significant increase in growth and development, percent metamorphosis, size at metamorphic climax, and skeletal ossification were observed at 50 μg Pb2+ L−1; however, exposure to 1000 μg Pb2+ L−1 resulted in the opposite effects in tadpoles. In addition, histological alterations of thyroid gland, such as follicular cell hyperplasia and colloid depletion could be found in 50–1000 μg Pb2+ L−1 treatments. Furthermore, Pb2+ exposure at 1000 μg L−1 resulted in significantly decreased transcript levels of Dio2, TRα and TRβ, and increased transcript levels of Dio3. In contrast, 50 μg Pb2+ L−1 significantly upregulated the mRNA levels of Dio2, TRα, and TRβ, but it reduced the Dio3 expression. These results suggested that Pb2+ might disrupt TH homeostasis in tadpoles by histological alterations of thyroid gland and disturb the transcript levels of Dio2, Dio3, TRα, and TRβ, leading to altered growth and development, as well as percent metamorphosis and skeletal ossification. Further studies are needed to elucidate the underlying mechanisms of low-dose stimulation and high-dose inhibition effects.

Inhibited Metamorphosis and Disruption of Antioxidant Defenses and Thyroid Hormone Systems in Bufo gargarizans Tadpoles Exposed to Copper

Copper is a common aqueous pollutant that is known to cause oxidative stress and disrupt the thyroid axis in amphibians. In the present study, tadpoles of the Chinese toad (Bufo gargarizans) were exposed to 1, 6.4, 32, and 64 μg L−1 of copper from Gosner stages 26 to 42. We aimed to examine the influence of copper on thyroid hormone-responsive and stress-associated gene expression in the hind-limb, tail, and liver of B. gargarizans tadpoles. Exposure to 64 μg L−1 copper decreased percent metamorphosis and increased length of both hind-limb and tail of B. gargarizans tadpoles at Gs 42. In addition, according to real-time PCR results, exposure to 64 μg L−1 copper induced downregulation of Dio2, Dio3, TRα, and TRβ mRNA levels in all tissues examined. We inferred that copper might induce a considerable reduction of TH levels through downregulation of Dio2 and Dio3 mRNA levels in peripheral tissues. Decreased TH levels may then decrease the expressions of TRα and TRβ. Also, HSP, SOD, and PHGPx transcript levels were measured to assess cellular stress which might affect TH signaling and metamorphosis. We found that copper significantly downregulated the level of HSP, SOD, and PHGPx transcripts in the hind-limb and tail. This demonstrates that high concentrations of copper could disrupt the antioxidant system of B. gargarizans tadpoles and increase oxidative damage. Therefore, we conclude that copper could disrupt the antioxidant system and cause thyroid hormone disruption in B. gargarizans tadpoles.

Seasonal alterations in the daily rhythms in hypothalamic expression of genes involved in the photoperiodic transduction and neurosteroid-dependent processes in migratory blackheaded buntings.

The present study investigated seasonal alterations in the daily rhythms of hypothalamic expression of genes involved in the photoperiodic regulation of annual cycles in birds. We measured the 4-hourly mRNA expression of genes involved in the photoperiodic transduction (OPN5, EYA3, CGA, TSHβ, DIO2, DIO3) and neurosteroid-dependent processes (AR, CYP19, ERα, ERβ) in the hypothalamus of migratory blackheaded buntings photoinduced with photosensitive, photostimulated (early and late stimulated) and photorefractory seasonal states. There were significant differences in daily mRNA profiles between the photoperiodic states. Particularly, increased CGA, TSHβ and DIO2 and decreased DIO3 mRNA levels in the early photostimulated state, compared to the photosensitive state, suggest that thyroid hormones have a role in photostimulation in buntings. Similar differences in the expression of genes coding for the aromatase enzyme (CYP19) and receptors for oestrogen (ERα, ERβ) (but not androgen; AR) indicate that there is seasonal alteration in the neuro-oestrogen-mediated functions. Furthermore, peak expression times of CGA, TSHβ and DIO2 genes at hours 14-15 of the day in the early stimulated state indicated molecular regulation of the daily rhythm of photoinducibility in buntings. Most significantly, however, we found an attenuated daily rhythm in thyroid hormone modulatory genes and a switch of peak expression time from day to night in CYP19 mRNA rhythm in the subsequent late photostimulated state, although testicular maturation still persisted. These alterations in daily rhythms may have signalled the initiation of processes underlying other seasonal phenologies in parallel with the gonadal response, such as a manifestation of the night-time flight in buntings. These results show alterations in daily rhythms underlying the transcriptional regulation of the photoperiod-induced seasonal states in migratory blackheaded buntings.

Early Developmental Disruption of Type 2 Deiodinase Pathway in Mouse Skeletal Muscle Does Not Impair Muscle Function.

Myogenesis is positively regulated by thyroid hormone (triiodothyronine [T3]), which is amplified by the type 2 deiodinase (D2) activation of thyroxine to T3. Global inactivation of the Dio2 gene impairs skeletal muscle (SKM) differentiation and regeneration in response to muscle injury. Given that newborn and adult mice with late developmental SKM Dio2 disruption do not develop a significant phenotype, it was hypothesized that D2 plays an early role in this process. This was tested in mice with SKM disruption of Dio2 driven by two early developmental promoters: MYF5 and MYOD. MYF5 myoblasts in culture differentiate normally into myotubes, despite loss of almost all D2 activity. Dio2 mRNA levels in developing SKM obtained from MYF5-D2KO embryos (E18.5) were about 54% of control littermates, but the expression of the T3-responsive genes Myh1 and 7 and Atp2a1 and 2 were not affected. In MYF5-D2KO and MYOD-D2KO neonatal hind-limb muscle, the expression of Myh1 and 7 and Atp2a2 remained unaffected, despite 60-70% loss in D2 activity and/or mRNA. Only in MYOD-D2KO neonatal muscle was there a 40% reduction in Atp2a1 mRNA. Postnatal growth of both mouse models and SKM function as assessed by exercise capacity and measurement of muscle strength were normal. Furthermore, an analysis of the adult soleus revealed no changes in the expression of T3-responsive genes, except for an about 18% increase in MYOD-D2KO SOL Myh7 mRNA. Two mouse models of early developmental disruption of Dio2 in myocyte precursor exhibit no significant SKM phenotype.

17β-Estradiol suppresses visceral adipogenesis and activates brown adipose tissue-specific gene expression.

Both functional ovaries and estrogen replacement therapy (ERT) reduce the risk of type 2 diabetes (T2D). Understanding the mechanisms underlying the antidiabetic effects of 17β-estradiol (E2) may permit the development of a molecular targeting strategy for the treatment of metabolic disease. This study examines how the promotion of insulin sensitivity and weight loss by E2 treatment in high-fat-diet (HFD)-fed mice involve several anti-adipogenic processes in the visceral adipose tissue. Magnetic resonance imaging (MRI) revealed specific reductions in visceral adipose tissue volume in HFD+E2 mice, compared with HFD mice. This loss of adiposity was associated with diminished visceral adipocyte size and reductions in expression of lipogenic genes, adipokines and of the nuclear receptor nr2c2/tr4. Meanwhile, expression levels of adipose triglyceride lipase/pnpla2 and leptin receptor were increased. As mRNA levels of stat3, a transcription factor involved in brown adipose tissue differentiation, were also increased in visceral adipose, the expression of other brown adipose-specific markers was assessed. Both expression and immunohistochemical staining of ucp-1 were increased, and mRNA levels of dio-2, and of adrβ3, a regulator of ucp-1 expression during the thermogenic response, were increased. Furthermore, expression of cpt-1b, a brown adipose-specific gene involved in fatty acid utilization, was also increased. Methylation studies demonstrated that the methylation status of both dio-2 and adrβ3 was significantly reduced. These results show that improved glycemic control and weight loss due to E2 involve anti-adipogenic mechanisms which include suppressed lipogenesis and augmented fatty acid utilization, and in addition, the activation of brown adipose tissue-specific gene expression in association with E2-dependent epigenetic modifications in these genes.

Concurrent hypothalamic gene expression under acute and chronic long days: Implications for initiation and maintenance of photoperiodic response in migratory songbirds

Hypothalamic expression of the thyroid hormone (TH) responsive gonadostimulatory (eya3, cga, tshβ, dio2, dio3, gnrh, gnih) and neurosteroid pathway genes (androgen receptor [ar], aromatase [cyp19], estrogen receptor [er] α and β) was examined in photosensitive redheaded buntings exposed to 2 (acute, experiment 1) or 12 (chronic, experiment 2) long days (16L:8D). Experiment 2 also included a photorefractory group. Acute long days caused a significant increase in eya3, cga, tshβ, dio2 and gnrh and decrease in dio3 mRNA levels. eya3, cga and tshβ expressions were unchanged after the chronic long days. We also found increased cyp19, erα and erβ mRNA levels after acute, and increased cyp19 and decreased erβ levels after the chronic long-day exposure. Photorefractory buntings showed expression patterns similar to that in the photosensitive state, except for high gnrh and gnih and low dio3 mRNA levels. Consistent with gene expression patterns, there were changes in fat deposition, body mass, testis size, and plasma levels of testosterone, tri-iodothyronine and thyroxine. These results show concurrent photostimulation of the TH-signalling and neurosteroid pathways, and extend the idea, based on differences in gene expression, that transitions in seasonal photoperiodic states are accomplished at the transcriptional levels in absolute photorefractory species.

Effect of glucocorticoids on the activity, expression and proximal promoter of type II deiodinase in rat brown adipocytes

Triiodothyronine (T3) is important for thermogenesis in brown adipose tissue (BAT). Type II deiodinase (DIO2) produces T3 required for intracellular needs in BAT. Brown adipocytes in culture require T3 for the adrenergic stimulation of DIO2. Glucocorticoids induce adipocyte differentiation (lipogenesis). We investigated the regulation of DIO2 activity, Dio2 mRNA and Dio2 promoter activity by glucocorticoids in primary cultures of rat brown adipocytes using dexamethasone (DEX) and hydrocortisone (HC). DEX and HC regulated the adrenergic stimulation of DIO2 activity in a dose- and time-dependent manner, inhibiting DIO2 activity at short treatment times and large doses (1–10 μM) and stimulating DIO2 at low HC doses (1–100 nM) and longer times (DEX). Insulin depletion reduced DIO2 activity but the response to glucocorticoids remained unchanged. DEX and HC inhibited basal DIO2 activity. DEX had no effect on DIO2 half-life, whereas HC stabilized DIO2 activity. DEX and HC inhibited the adrenergic stimulation of Dio2 mRNA expression (100–10000 nM, 14–96 h), but stabilized Dio2 mRNA, particularly DEX. DEX increased basal Dio2 mRNA levels, possibly through stabilization of Dio2 mRNA. An 807 bp construct of the murine Dio2 proximal promoter showed maximal reporter activity, with the cAMP response element (CRE) essential for transcriptional activity. DEX caused inhibition in most constructs containing the CRE element whereas HC stimulated reporter activity in the 807 bp construct. Glucocorticoids inhibited the adrenergic stimulation of Dio2 at the transcriptional level in brown adipocytes, although DIO2 activity increased with HC, possibly due to stabilization of Dio2 activity and mRNA. The CRE and cEBP elements of the Dio2 promoter seem involved in the regulation by glucocorticoids.

MAPK and SHH pathways modulate type 3 deiodinase expression in papillary thyroid carcinoma.

Type 3 deiodinase (DIO3, D3) is reactivated in human neoplasias. Increased D3 levels in papillary thyroid carcinoma (PTC) have been associated with tumor size and metastatic disease. The objective of this study is to investigate the signaling pathways involved in DIO3 upregulation in PTC. Experiments were performed in human PTC cell lines (K1 and TPC-1 cells) or tumor samples. DIO3 mRNA and activity were evaluated by real-time PCR and ion-exchange column chromatography respectively. Western blot analysis was used to determine the levels of D3 protein. DIO3 gene silencing was performed via siRNA transfection. DIO3 mRNA levels and activity were readily detected in K1 (BRAF(V6) (0) (0E)) and, at lower levels, in TPC-1 (RET/PTC1) cells (P<0.007 and P=0.02 respectively). Similarly, DIO3 mRNA levels were higher in PTC samples harboring the BRAF(V600E) mutation as compared with those with RET/PTC1 rearrangement or negative for these mutations (P<0.001). Specific inhibition of BRAF oncogene (PLX4032, 3 μM), MEK (U0126, 10-20 μM) or p38 (SB203580, 10-20 μM) signaling was associated with decreases in DIO3 expression in K1 and TPC-1 cells. Additionally, the blockage of the sonic hedgehog (SHH) pathway by cyclopamine (10 μM) resulted in markedly decreases in DIO3 mRNA levels. Interestingly, siRNA-mediated DIO3 silencing induced decreases on cyclin D1 expression and partial G1 phase cell cycle arrest, thereby downregulating cell proliferation. In conclusion, sustained activation of the MAPK and SHH pathways modulate the levels of DIO3 expression in PTC. Importantly, DIO3 silencing was associated with decreases in cell proliferation, thus suggesting a D3 role in tumor growth and aggressiveness.

Effects of nitrate on metamorphosis, thyroid and iodothyronine deiodinases expression in Bufo gargarizans larvae

Chinese toad (Bufo gargarizans) tadpoles were exposed to nitrate (10, 50 and 100mg/L NO3–N) from the beginning of the larval period through metamorphic climax. We examined the effects of chronic nitrate exposure on metamorphosis, mortality, body size and thyroid gland. In addition, thyroid hormone (TH) levels, type II iodothyronine deiodinase (Dio2) and type III iodothyronine deiodinase (Dio3) mRNA levels were also measured to assess disruption of TH synthesis. Results showed that significant metamorphic delay and mortality increased were caused in larvae exposed to 100mg/L NO3–N. The larvae exposed to 100mg/L NO3–N clearly exhibited a greater reduction in thyroxine (T4) and 3,5,3′-triiodothyronine (T3) levels. Moreover, treatment with NO3–N induced down-regulation of Dio2 mRNA levels and up-regulation of Dio3 mRNA levels, reflecting the disruption of thyroid endocrine. It seems that increased mass and body size may be correlated with prolonged metamorphosis. Interestingly, we observed an exception that exposure to 100mg/L NO3–N did not exhibit remarkable alterations of thyroid gland size. Compared with control groups, 100mg/L NO3–N caused partial colloid depletion in the thyroid gland follicles. These results suggest that nitrate can act as a chemical stressor inducing retardation in development and metamorphosis. Therefore, we concluded that the presence of high concentrations nitrate can influence the growth, decline the survival, impair TH synthesis and induce metamorphosis retardation of B. gargarizans larvae.

Leptin intake in suckling rats restores altered T3 levels and markers of adipose tissue sympathetic drive and function caused by gestational calorie restriction

Maternal calorie restriction during gestation in rats has been associated with altered white adipose tissue (WAT) sympathetic innervation and function in offspring. Here, we aimed to investigate whether supplementation with oral leptin (a breast milk component) throughout the lactation period may revert the aforementioned adverse programming effects. Three groups of male and female rats were studied at the postnatal day 25: the offspring of control dams, the offspring of 20% calorie-restricted dams during pregnancy (CR) and CR rats supplemented with physiological doses of leptin throughout lactation (CR-Leptin). Tyrosine hydroxylase (TH) levels and its immunoreactive area, and mRNA expression levels of lipid metabolism-related genes and of deiodinase iodothyronine type II (Dio2) were determined in WAT. Triiodothyronine (T3) levels were determined in the blood. In CR males, leptin treatment restored the decreased TH levels and its immunoreactive area in WAT, and partially normalized expression levels of genes related to lipolysis and fatty acid oxidation (adipose triglyceride lipase, hormone-sensitive lipase, carnitine palmitoyltransferase 1b and peroxisome proliferator-activated receptor gamma coactivator 1-alpha). Leptin treatment also reverted the decreased T3 plasma levels and WAT lipoprotein lipase mRNA levels occurring in CR males and females, and the decreased Dio2 mRNA levels in CR females. Leptin supplementation throughout the lactation period reverts the malprogrammed effects on WAT structure and function induced by undernutrition during pregnancy. These findings support the relevance of the intake of leptin during lactation, bearing clear characteristics of essential nutrient, and provide a strategy to treat and/or prevent the programmed trend to obesity acquired by inadequate fetal nutrition.

Moderate calorie restriction during gestation programs offspring for lower BAT thermogenic capacity driven by thyroid and sympathetic signaling

Maternal calorie restriction during pregnancy programs offspring for later overweight and metabolic disturbances. Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis and has recently emerged as a very likely target for human obesity therapy. Here we aimed to assess whether the detrimental effects of undernutrition during gestation could be related to impaired thermogenic capacity in BAT and to investigate the potential mechanisms involved. Offspring of control and 20% calorie-restricted rats (days 1-12 of pregnancy) (CR) were studied at the age of 25 days. Protein levels of uncoupling protein 1 (UCP1) and tyrosine hydroxylase (TyrOH); mRNA levels of lipoprotein lipase (LPL), carnitine palmitoyltransferase 1 (CPT1) and deiodinase iodothyronine type II (DIO2) in BAT; and blood parameters including thyroid hormones, were determined. The response to 24-h cold exposure was also studied by measuring body temperature changes over time, and final BAT UCP1 levels. Compared with controls, CR animals displayed in BAT lower UCP1 and TyrOH protein levels and lower LPL and CPT1 mRNA levels; they also showed lower triiodothyronine (T3) plasma levels. CR males, but not females, revealed lower DIO2 mRNA levels than controls. When exposed to cold, CR rats experienced a transient decline in body temperature, but the values were reestablished after 24 h, despite having lower UCP1 levels than controls. These results suggest that BAT thermogenic capacity is diminished in CR animals, involving impaired BAT sympathetic innervation and thyroid hormone signaling. These alterations make animals more sensitive to cold and may contribute to long-term outcomes of gestational calorie restriction in promoting obesity and related metabolic alterations.

Overexpression of suppressor of cytokine signaling 3 in the arcuate nucleus of juvenile Phodopus sungorus alters seasonal body weight changes

The profound seasonal cycle in body weight exhibited by the Djungarian hamster (Phodopus sungorus) is associated with the development of hypothalamic leptin resistance during long day photoperiod (LD, 16:8h light dark cycle), when body weight is elevated relative to short day photoperiod (SD, 8:16h light dark cycle). We previously have shown that this seasonal change in physiology is associated with higher levels of mRNA for the potent inhibitor of leptin signaling, suppressor of cytokine signaling-3 (SOCS3), in the arcuate nucleus (ARC) of LD hamsters relative to hamsters in SD. The alteration in SOCS3 gene expression preceded the body weight change suggesting that SOCS3 might be the molecular switch of seasonal body weight changes. To functionally characterize the role of SOCS3 in seasonal body weight regulation, we injected SOCS3 expressing recombinant adeno-associated virus type-2 (rAAV2-SOCS3) constructs into the ARC of leptin sensitive SD hamsters immediately after weaning. Hamsters that received rAAV2 expressing enhanced green fluorescent protein (rAAV2-EGFP) served as controls. ARC-directed SOCS3 overexpression led to a significant increase in body weight over a period of 12 weeks without fully restoring the LD phenotype. This increase was partially due to elevated brown and white adipose tissue mass. Gene expression of pro-opiomelanocortin was increased while thyroid hormone converting enzyme DIO3 mRNA levels were reduced in SD hamsters with SOCS3 overexpression. In conclusion, our data suggest that ARC-directed SOCS3 overexpression partially overcomes the profound seasonal body weight cycle exhibited by the hamster which is associated with altered pro-opiomelanocortin and DIO3 gene expression.

Fetal and Neonatal Iron Deficiency Reduces Thyroid Hormone-Responsive Gene mRNA Levels in the Neonatal Rat Hippocampus and Cerebral Cortex

Copper (Cu), iron (Fe), and thyroid hormone (TH) deficiencies produce similar defects in late brain development including hypomyelination of axons and impaired synapse formation and function, suggesting that these micronutrient deficiencies share a common mechanism contributing to these derangements. We previously demonstrated that fetal/neonatal Cu and Fe deficiencies lower circulating TH concentrations in neonatal rats. Fe deficiency also reduces whole-brain T(3) content, suggesting impaired TH action in the developing Fe-deficient brain. We hypothesized that fetal/neonatal Cu and Fe deficiencies will produce mild or moderate TH deficiencies and will impair TH-responsive gene expression in the neonatal cerebral cortex and hippocampus. To test this hypothesis, we rendered pregnant Sprague Dawley rats Cu-, Fe-, or TH-deficient from early gestation through postnatal d 10 (P10). Mild and moderate TH deficiencies were induced by 1 and 3 ppm propylthiouracil treatment, respectively. Cu deficiency did not significantly alter serum or tissue TH concentrations or TH-responsive brain mRNA expression. Fe deficiency significantly lowered P10 serum total T(3) (45%), serum total T(4) (52%), whole brain T(3) (14%), and hippocampal T(3) (18%) concentrations, producing a mild TH deficiency similar to 1 ppm propylthiouracil treatment. Fe deficiency lowered Pvalb, Enpp6, and Mbp mRNA levels in the P10 hippocampus. Fe deficiency also altered Hairless, Dbm, and Dio2 mRNA levels in the P10 cerebral cortex. These results suggest that some of the brain defects associated with Fe deficiency may be mediated through altered thyroidal status and the concomitant alterations in TH-responsive gene transcription.

Increased Type 3 Deiodinase Expression in Papillary Thyroid Carcinoma

Thyroid hormone regulates a wide range of cellular activities, including the balance between cell proliferation and differentiation. The thyroid-hormone-inactivating type 3 deiodinase (DIO3, D3) has been shown to be reactivated in human neoplasias. Here, we evaluated DIO3 expression in human papillary thyroid carcinoma (PTC). Tumor and surrounding normal thyroid tissue were collected from 26 unselected patients with PTC. Clinical data were retrospectively reviewed in medical records. DIO3 mRNA levels were measured by real-time polymerase chain reaction and D3 activity by paper-descendent chromatography. Studies of DIO3 gene regulation were performed in a human PTC-derived cell line (K1 cells). BRAF(V600E) mutation was identified in DNA from paraffin-embedded tissues by direct sequencing. Immunohistochemistry analyses were performed using a specific human D3 antibody. Increased D3 activity was detected in all 26 PTC samples analyzed as compared with adjacent thyroid tissue. The augmentations in D3 activity were paralleled by increased DIO3 mRNA levels (approximately fivefold). In PTC-derived cells, DIO3 transcripts were further upregulated by the transforming growth factor β1 (TGFβ1). Interestingly, preincubation with mitogen-activated protein kinase (MAPK) cascade inhibitors U0126 (ERK pathway) and SB203580 (p38 pathway) decreased DIO3 mRNA levels and blocked the TGFβ1-induced increase in DIO3 transcripts, suggesting that D3 induction might be mediated through the MAPK signaling pathway. Accordingly, DIO3 mRNA and activity levels were significantly higher in BRAF(V600E)-mutated samples (p=0.001). Increased D3 activity was correlated with tumor size (r=0.68, p=0.003), and associated with lymph node (p=0.03) or distant metastasis (p=0.006) at diagnosis. Conversely, decreased levels of the thyroid-hormone-activating type 2 deiodinase (DIO2) gene were observed in PTC, which might contribute to further decreases in intracellular thyroid hormone levels. Increased D3 expression was also observed in follicular thyroid carcinoma but not in medullary or anaplastic thyroid carcinoma samples. These results indicate that the malignant transformation of thyroid follicular cell toward PTC promotes opposite changes in DIO3 and DIO2 expression by pretranscriptional mechanisms. The association between increased levels of D3 activity and advanced disease further supports a role for intracellular triiodothyronine concentration on the thyroid tumor cell proliferation or/and dedifferentiation.

Novel polymorphisms within the Dlk1-Dio3 imprinted locus in rat: a putative genetic basis for strain-specific allelic gene expression

The imprinted iodothyronine deiodinase-III (Dio3) thyroid hormone metabolizing gene exhibits paternal expression in most fetal tissues, yet exhibits aberrant, maternal expression in the hippocampus in F1 offspring of Sprague Dawley (SD) × Brown Norway (BN) rats. The maternal hippocampal expression is associated with lower Dio3 mRNA levels specifically in the hippocampus. Here, we tested the hypothesis that genetic polymorphisms between the SD and BN parent strains cause this aberrant allelic Dio3 expression and contribute to behavioral sequelae of higher thyroid hormone levels locally in the hippocampus, including anxiety-related behavior. We mapped and sequenced the Dio3 gene and several previously unmapped regions in the Dlk1-Dio3 locus that could regulate imprinting of the Dio3 gene. In the Dio3 promoter we identified four novel polymorphisms between the BN and SD strains. Next we took advantage of the fact that the Long Evans (LE) strain exhibits identical polymorphisms as the SD strain in the region 5' and including the Dio3 gene. By reciprocally crossing LE and BN strains we tested the relationship among Dio3 promoter region polymorphisms and Dio3 mRNA expression in the hippocampus. Aberrant strain-specific hippocampal Dio3 allelic expression replicated in the LE-BN reciprocal crosses, suggesting that hippocampal-specific imprinting of the Dio3 gene is not the result of a unique genetic or epigenetic characteristic of the SD rat strain, or a unique epistatic interaction between SD and BN. To our knowledge no other studies have reported a genetic × epigenetic interaction of genetic origin in the brain.