Neuronal cell culture.

Hypothalami were dissected in a saggital plane using the anterior commisure and the oculomotor nerve as neuroanatomical markers from embryonic day 20 chicken embryos (Ross broiler strain). Embryonic day 20 hypothalami are primarily independent of myelination, which begins around embryonic day 14 but occurs primarily after hatch since only 5% myelination is observed in 3-day-old chicks (43). Dissections were conducted in HBSS supplemented with 10 mM HEPES and sodium pyruvate (0.011 μl/ml). Neurons were dissociated in 0.05% trypsin in minimum essential medium for 60 min and then treated with 0.1% soybean trypsin inhibitor in Dulbecco/Vogt modified Eagle's minimal essential medium for 15 min. Neurons were cultured in neurobasal medium supplemented with 2% B-27, 0.25% l-glutamine, and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA) (2 × 10⁶ cells/well) at 37°C for 4 days with 10⁻⁹ M T₃ or 10 ng BDNF (Invitrogen) added 0, 1, 3, 6, or 24 h before harvesting. Culture plates were coated with poly-l-lysine before use. Cells were harvested with 0.05% trypsin, centrifuged, and snap-frozen in liquid nitrogen.

Thyroid hormone manipulations in vivo.

Fertile chicken eggs were injected two times, once on embryonic day 18 and once on embryonic day 19, with 0.1 ml of 10⁻⁷ M T₃, 0.075 g/ml methimazole (MMI; a thyroid hormone synthesis inhibitor), or a similar volume of water in the egg albumen. A hole was drilled through the shell at the bottom of the egg for injection, and then covered with wax until the injection on the next day, with the wax being replaced each time. Hypothalami were then collected on embryonic day 20 as described above for the neuronal cell cultures.

In a second study, male chickens were given a subcutaneous implant of an osmotic minipump (Alzet, model 2001; ALZA, Mountain View, CA) at 29 days of age to continuously release hormone for up to 7 days at a rate of 1.0 μl/h. Cort and T₃ hormones (Sigma Chemicals, St. Louis, MO) were dissolved in solutions of 50% dimethyl sulfoxide and 50% propylene glycol, so as to deliver 600 μg Cort·kg⁻¹·day⁻¹ and 192 μg T₃·kg⁻¹·day⁻¹, respectively. Animals were given ad libitum access to food and water. The light-dark cycle was as follows: 24 h of light for the first 2 days and then 14 h of light and 10 h of dark thereafter. Animals were killed after 72 h of hormone or vehicle treatment, with the hypothalamus being immediately dissected, snap-frozen in liquid nitrogen (n = 4 for group), and stored at −80°C until further processing.

RNA extraction and real-time reverse transcription PCR.

Whole hypothalamic tissue RNA was extracted using RNeasy Midi kits, and RNA from cell cultures was extracted using RNeasy Mini kits (Qiagen, Valencia, CA). RNA was quantified using ultraviolet absorbance (260/280 nm) and with a bioanalyzer (Agilent Technologies, Palo Alto, CA). Superscript III reverse transcriptase (Invitrogen) and an oligo(dT) primer were used to create cDNA from 400 ng of RNA for cell culture and 1 μg of RNA for whole hypothalami experiments. mRNA levels were quantified with Sybr green real-time reverse transcription PCR (qPCR) master mix containing the following: 1 unit of Taq polymerase, 20× SYBR green I, 400 nM fluoroscein, 10 mM dNTPs, 25 mM MgCl₂, 10× PCR buffer, and water.

The qPCR output provided a cycle threshold (C_t) value. The ΔΔC_t value was generated using geNorm software and methods previously described (49). Briefly, the data were first transformed to a ΔC_t value by subtracting the sample C_t value from the sample with the highest expression level to control for amplification efficiency. The ΔΔC_t value was then calculated by normalizing gene expression to two housekeeping genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, with the provided software (URL: http://medgen.ugent.be/~jvdesomp/genorm/).

Primer design, validation, and identification of glucocorticoid and thyroid hormone response elements.

Primers were 18–30 nucleotides in length with a melting temperature between 58–64 or 69–72°C and were designed using Primer Express (version 2.0; Applied Biosystems, Foster City, CA). The amplicons were between 100 and 150 bp in length. Forward and reverse primer sequences are listed in Table 1. Glucocorticoid and thyroid hormone response elements were identified using TESS (Transcriptional Element Search System; University of Pennsylvania, URL: http://www.cbil.upenn.edu/tess). Default parameters were used. Sequences were first identified in NCBI having the following annotations: agouti-related protein (AGRP), NM_001031457.1; BDNF, NM_001031616.1; corticotropin-releasing hormone (CRH), XM_418279.2; leptin receptor (LEPR), NM_204323.1; neuropeptide Y (NPY), NM_205473.1; pro-opiomelanocortin (POMC), NM_001031098.1; BDNF tyrosine kinase receptor B (TrkB), NM_205231.1; and TRH, NM_001030383.1. The sequence was blasted to the chicken genome using Ensembl (URL: http://www.ensembl.org), and 2,000 bp of available sequence upstream of the 5′-region of exon 1 for each gene was identified and searched for a glucocorticoid (GRE) or thyroid hormone (TRE) response element sequence. Potential GRE- or TRE-binding sites were scored using the TESS standard parameters and identified to be either a good match, log-likelihood score within one of the best possible score, or a mismatch. All sites with a good match are listed in Table 2.

T₃ and BDNF modulate anabolic/orexigenic and catabolic/anorexigenic gene expression in a complementary manner.

We next assessed whether the different levels of circulating T₃ and hypothalamic BDNF mRNA noted between the fat and lean lines could account for any of the other differences in hypothalamic gene expression. Hypothalamic neuronal cultures were treated with 10⁻⁹ M T₃ or 10 ng BDNF for 0, 1, 3, 6, and 24 h and assayed with qPCR for mRNA levels. Primary hypothalamic neurons were cultured for 4 days, since BDNF mRNA levels initially declined and then stabilized by day 3 in vitro (data not shown), and neurons grown 4 days in vitro have not yet formed synaptic connections with other neurons, implying that results reflect treatment effects on mRNA levels independent of stimulation received from synaptic interactions with other neurons.

Treating hypothalamic neurons with T₃ suppressed expression of BDNF, TrkB, and TRH mRNA, relative to untreated controls (0 h) (Fig. 2, A, C, and E, respectively). Treatment of neurons with 10 ng of BDNF modulated expression of the same genes in the opposite direction, relative to T₃ treatment (Fig. 2, B, D, and F, respectively). β-Actin was not different after either treatment (Fig. 2, M and N). This indicated that T₃ and BDNF regulate expression of some common target genes in an opposite way. Because LEPR is one gene known to modulate many other genes influencing body composition and food intake (21), we determined whether similar effects on LEPR would be seen. T₃ suppressed LEPR gene expression after 1, 3, 6, and 24 h (Fig. 2G), and BDNF increased expression of the LEPR gene after 6 h (Fig. 2H). This suggests that BDNF and T₃ reciprocally modulate expression of a key gene in the hypothalamic obesity gene circuit.

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Fig. 2.

T₃ and BDNF have opposite effects in vitro on hypothalamic genes known to regulate energy homeostasis. A, C, E, G, I, K, and M: T₃ treatment decreased BDNF (A), TrkB (C), TRH (E), LEPR (G), and POMC (I) gene expression and increased expression of AGRP (K), but not β-actin (M), mRNA levels (n = 4–10). B, D, F, H, J, L, and N: BDNF treatment increased gene expression of BDNF (B), TrkB (D), TRH (F), and LEPR (H) relative to control, but not POMC (J), AGRP (L), and β-actin mRNA levels (N) (n = 4–6). Expression levels are normalized to a control value of 100%. *P < 0.05 and **P < 0.01, treatment vs. 0-h control.

Given that T₃ and BDNF reciprocally modulated expression for the catabolic/anorexigenic genes BDNF, TrkB, TRH, and LEPR, we proposed that T₃ would suppress the other catabolic/anorexigenic genes in our gene list, POMC and CRH, and increase the anabolic/orexigenic genes, AGRP and NPY, whereas BDNF would have the opposite effects. Indeed, T₃ suppressed POMC mRNA at 1 and 3 h (Fig. 2I) and increased AGRP expression at 1 and 24 h (Fig. 2K). However, BDNF had no effect on these genes (Fig. 2, J and L, respectively). BDNF and T₃ had no effect on NPY or CRH (data not shown). This suggests that T₃ directly suppresses expression of the catabolic/anorexigenic gene, POMC, and increases expression of the anabolic/orexigenic gene, AGRP. BDNF was able to increase expression of most catabolic/anorexigenic genes directly (BDNF, TrkB, TRH, and LEPR).

We tested whether T₃ was able to modulate these same genes during development, in vivo. The dose of MMI, a thyroid hormone synthesis inhibitor, used suppressed serum T₄ levels to an undetectable level on embryonic day 17, embryonic day 19, and day 1 relative to control in a previous study using a similar injection paradigm (37). We injected T₃ or MMI in developing chicken embryos on embryonic days 18 and 19 and collected hypothalami on embryonic day 20. We observed effects on BDNF, TrkB, and TRH mRNA levels (Fig. 3, A–C, respectively) consistent with those observed in vitro, but, surprisingly, both T₃ and MMI decreased LEPR, increased POMC mRNA levels, and had no effect on AGRP (Fig. 3, D–F, respectively).

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Fig. 3.

T₃ reciprocally modulates catabolic/anorexigenic and anabolic/orexigenic genes. A-F: T₃ was injected on embryonic days 18 and 19, and effects on hypothalamic gene expression were assessed on embryonic day 20. T₃ suppressed BDNF (A), TrkB (B), TRH (C), and LEPR (D) gene expression, whereas methimazole (MMI) increased BDNF (A), TrkB (B), and TRH (C), but not LEPR (D), mRNA levels. T₃ and MMI both increased POMC (E) and had no affect on AGRP (F) mRNA (n = 6). *P < 0.05 and **P < 0.01. CNT, control (water).

T₃ and Cort play both overlapping and independent roles in modulating anabolic/orexigenic and catabolic/anorexigenic gene expression.

Although it is known that thyroid hormone and Cort act in opposition to one another, with Cort being considered an anabolic hormone and T₃ a catabolic hormone, they have been shown to have synergistic effects when used in combination. For example, T₃ treatment alone slightly enhanced protein synthesis in muscle, whereas Cort treatment increased protein breakdown, and Cort in conjunction with thyroid hormone enhanced protein breakdown in muscle sixfold (17). Under states of chronic stress, glucocorticoids have been shown to increase food intake, which may be because of a dysregulation of normal mechanisms that modulate food intake (1). When glucocorticoid signaling is no longer effective, increased food intake mimics the food intake phenotype observed when thyroid hormone levels are elevated; however, adiposity is still regulated in opposite directions. Therefore, we wanted to determine which hypothalamic genes known to regulate both food intake and body weight are modulated in the same or different directions after chronic 72-h treatment with Cort, T₃, or Cort plus T₃.

Hormones were delivered via osmotic minipump for 72 h. T₃ decreased expression of the catabolic/anorexigenic neuropeptide genes TRH, BDNF, TrkB, LEPR, and POMC (Fig. 4, A–E). T₃ also increased expression of AGRP, an anabolic/orexigenic neuropeptide (Fig. 4F). Delivering T₃ via osmotic minipump had no effect on NPY or CRH (Fig. 4, G and H), as previously demonstrated in cell culture. This suggests that in vivo T₃ treatment is able to suppress expression of catabolic/anorexigenic genes and increase expression of an anabolic/orexigenic gene. There was no change in β-actin gene expression after any of the treatments (Fig. 4I).

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Fig. 4.

Anabolic/orexigenic and catabolic/anorexigenic gene expression in the hypothalamus after 72 h of hormone treatment. Corticosterone (Cort) and T₃ were administered for 72 h using osmotic minipumps, and then hypothalami were collected. A-H: quantification of hypothalamic BDNF (A), TrkB (B), TRH (C), LEPR (D), POMC (E), AGRP (F), NPY (G), CRH (H), and β-actin (I) mRNA levels (n = 4) by qPCR analysis. *P < 0.05, hormone treatment relative to vehicle control.

We expected to observe the opposite effects as those seen with T₃ with Cort treatment. Surprisingly, for many of the genes, we observed a similar expression pattern as seen after T₃ treatment. For example, we observed that Cort decreased mRNA levels for the catabolic/anorexigenic neuropeptides TRH, LEPR, and POMC (Fig. 4, A, D, and E). On the other hand, opposite to that observed with T₃, Cort increased levels of BDNF mRNA (Fig. 4B). Cort had no effect on TrkB or AGRP (Fig. 4, C and F, respectively), and, unlike T₃, Cort increased mRNA levels for the anabolic/orexigenic neuropeptide NPY and decreased expression for the catabolic/anorexigenic neuropeptide CRH (Fig. 4, G and H).

Delivering Cort in conjunction with T₃ had surprising effects on anabolic/orexigenic and catabolic/anorexigenic gene expression in the hypothalamus. Similar to T₃ treatment alone, T₃ plus Cort decreased expression of TRH and BDNF mRNA (Fig. 4, A and B). Decreased POMC and increased AGRP expression had only a trend toward significance, which is surprising since T₃ or Cort alone was able to decrease POMC mRNA levels, and T₃ alone was able to increase AGRP mRNA levels (Fig. 4, E and F). Surprisingly, simultaneous treatment with Cort and T₃ had no effect on TrkB, LEPR, and NPY (Fig. 4, C, D, and G, respectively). As predicted, Cort treatment, even in conjunction with T₃ delivery, decreased expression of CRH (Fig. 4H).

BDNF and T₃ affected hypothalamic gene expression in a complementary fashion.

Our results show that T₃ was able to increase anabolic/orexigenic gene expression (AGRP) and decrease catabolic/anorexigenic gene expression (BDNF, TrkB, TRH, LEPR, and POMC) both in vivo and in vitro. BDNF was able to directly modulate catabolic/anorexigenic genes in vitro in the opposite direction as T₃, with the exception of POMC and AGRP. However, it has been previously shown that leptin can suppress AGRP and increase POMC gene expression (25), and, since T₃ and BDNF reciprocally modulated LEPR gene expression, there may be indirect modulation of AGRP and POMC gene expression via LEPR. We propose a model based on our in vivo and in vitro data showing reciprocal regulation of the hypothalamic obesity gene circuit by T₃, BDNF, and Cort (Fig. 5). Collectively, our results suggest that levels of BDNF, T₃, and Cort may explain how some individuals are more susceptible to develop an obese phenotype without altering food intake, as observed in the euthyroid morbidly obese patients with moderately elevated T₃ levels (34).

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Fig. 5.

Model for BDNF, T₃, and Cort interactions modulating the hypothalamic obesity gene network. We propose that elevated systemic T₃ levels decrease BDNF, TRH, LEPR, and POMC mRNA levels but increase AGRP gene expression in the hypothalamus. In contrast, we propose that BDNF reciprocally modulates expression of the same genes by increasing BDNF, TRH, and LEPR mRNA levels. BDNF may also indirectly modulate POMC and AGRP mRNA levels by first increasing LEPR or TRH expression. In addition, we propose that systemic glucocorticoids (Cort) can increase hypothalamic expression of AGRP and BDNF and decrease expression of LEPR, POMC, and TRH. These effects culminate in a neuropeptide/endocrine homeostatic mechanism that contributes to the regulation of metabolism and energy expenditure.

Our results from the divergently selected fat and lean chicken lines parallel the cell culture and osmotic minipump data, suggesting that BDNF or T₃ levels may affect hypothalamic gene expression during the development of adiposity. Corresponding with predictions presented in our model, plasma T₃ was increased and BDNF expression decreased in the fat line relative to the lean line before and at the onset of adiposity divergence (weeks 1 and 3). An interaction between BDNF and T₃ was also demonstrated in vitro. Hypothalamic TRH mRNA levels were lower in the fat line at week 3, suggesting that increased BDNF expression in the lean line could increase expression of TRH, or increased T₃ in the fat line could inhibit expression of TRH, similar to the in vitro data. Finally, hypothalamic BDNF mRNA levels continued to increase at week 5 in the lean line relative to the fat line, which corresponds to an increase in TrkB and LEPR gene expression. In vitro, BDNF increased TrkB and LEPR mRNA levels. Our findings support a novel pathway that contributes to hypothalamic control of constitutive differences in the capacity for body fat deposition.

Complex effects of Cort and thyroid hormone on hypothalamic gene expression.

Cort and T₃ had overlapping effects on hypothalamic mRNA levels for genes known to modulate food intake and body weight, specifically TRH, LEPR, and POMC. We also found that Cort increased and T₃ decreased BDNF gene expression. Some genes were only regulated by one hormone; Cort increased NPY mRNA levels, whereas T₃ had no effect; T₃ decreased TrkB mRNA, whereas Cort had no effect on TrkB expression; and T₃ increased AGRP mRNA, whereas Cort had no effect. These effects agree with increased Cort levels resulting in anabolic/orexigenic activity by increasing expression of the anabolic/orexigenic gene NPY. On the other hand, increased T₃ levels generally increase catabolic activity, which would explain the downregulation of the anabolic/orexigenic gene AGRP.

Glucocorticoids and thyroid hormones are known to modulate adipose mass and food intake. Increased glucocorticoid levels decrease insulin levels, leptin levels, and POMC mRNA levels but increase AGRP and NPY mRNA in the hypothalamus (33). Thyroid hormone increases leptin hormone and NPY mRNA levels and decreases CART and POMC mRNA (20). As predicted, we found that Cort decreased POMC levels and increased NPY levels, whereas T₃ treatment decreased POMC. Surprisingly, Cort had no effect on AGRP mRNA levels, and T₃ had no effect on NPY mRNA levels. These unexpected differences may be a species-specific phenomenon or attributable to the duration of the treatment.

Thyroid hormone and glucocorticoids modulate adiposity in the opposite direction, but both have been shown to increase food intake. Thus we wanted to determine how increased levels of both Cort plus T₃ would modulate gene expression patterns. This allows elucidation for whether T₃ or Cort predominantly regulates gene expression for anabolic/orexigenic and catabolic/anorexigenic genes, perhaps providing an explanation for the opposite effects observed on body weight or similar effects on food intake. BDNF mRNA levels were regulated by both T₃ and Cort, with T₃ inhibiting and Cort increasing expression levels. When treating with T₃ plus Cort, BDNF mRNA levels resembled the inhibitory pattern observed after treating with T₃ alone. NPY mRNA levels were increased after Cort treatment, not changed after T₃ treatment alone, and Cort plus T₃ ablated the increased gene expression. This suggests that T₃ is the primary regulator for BDNF gene expression and that T₃ can also block the increased expression of NPY mRNA observed when Cort was added alone. On the other hand, CRH gene expression was decreased when either Cort was added alone and when Cort was added with T₃, as predicted.

Last, identification of putative transcription factor binding sites revealed that BDNF, TrkB, NPY, AGRP, TRH, and LEPR have sites that may function as a dual TRE/GRE. However, the functionality of these sites is currently unknown. If the sites are active, it is possible that, if the GRE site induces transcription, then T₃ may inhibit transcription by competitively binding to the site, or perhaps binding with a higher affinity. TRH and LEPR are least likely to have a functional GRE/TRE since Cort treatment alone had no effect on TRH mRNA and since Cort plus T₃ only partially decreased LEPR gene expression. It must be noted that the functionality of these elements remains to be confirmed.

Paradoxically, T₃ decreased POMC gene expression despite the lack of TRE sites within 2,000 bp upstream of exon 1. It is also possible that T₃ could increase expression of POMC mRNA indirectly by increasing expression of another, unknown, protein. However, we observed that POMC mRNA levels increased after treating primary neuronal cultures for 1 and 3 h, suggesting direct effects on POMC gene expression by T₃. The most plausible explanation is that regulatory TREs are located elsewhere in the POMC gene. It is uncertain why MMI-treated embryos would increase POMC expression relative to the observed decrease in expression observed later in life, as demonstrated by osmotic minipump treatment. The different experimental paradigms used to evaluate thyroid hormone effects on gene expression in the hypothalamus are suggestive of direct effects; however, the link between direct effects suggested by cell culture relative to possible indirect effects in the osmotic minipump experiment should be interpreted conservatively. Moreover, technical considerations limited our ability to test multiple doses and durations of thyroid hormone treatment in the minipump experiment. Nonetheless, effects of thyroid hormone on hypothalamic gene expression were demonstrated in vivo.

Potential role for regulating energy balance.

During the development of adiposity, it is plausible that interactions between T₃ and Cort program an initial set point within the hypothalamus to establish long-term levels of body fat stores, possibly by regulating the maturation of homeostatic signaling circuitry (e.g., neural, hormonal, or metabolic). For example, in the fat and lean animals, one mechanism altered during the development of adiposity could be initially signaled by T₃ or BDNF and secondarily by metabolic parameters, such as glucose utilization (10, 30, 45), lipogenesis (42), or fuel partitioning (31). Currently, the rate of neuropeptide release in response to T₃ or BDNF is unknown and may modulate neural circuitry more than protein or mRNA production. Furthermore, interactions between BDNF and LEPR signaling may modulate fat deposition and metabolism. In the mouse, leptin increased hypothalamic BDNF mRNA levels (23), and here we show that BDNF increased hypothalamic LEPR mRNA. In the fat line relative to the lean line, liver leptin levels are reportedly increased 1.7-fold at 15 wk of age (9). However, a controversy about the validity of this identified chicken leptin sequence exists (13), suggesting that the results from this previous report should be interpreted cautiously. Regardless, the avian LEPR has been sequenced accurately (40). Thus BDNF and LEPR levels may modulate energy expenditure and energy balance to influence adipose deposition by affecting hypothalamic signaling networks (Fig. 5). Therefore, T₃ acts in a reciprocal manner to inhibit BDNF, TrkB, LEPR, and even POMC to compensate for these changes in energy expenditure, thereby establishing a feedback loop that is enforced further by increasing AGRP expression. We propose that BDNF and T₃ levels may serve as a fine tipping point for determining how the hypothalamic obesity circuitry develops, along with interactions between glucocorticoids and thyroid hormone. Elevated levels of one hormone may not necessarily modulate gene expression in the same manner as it would if the other hormone is concurrently elevated. Here we begin to elucidate the underpinnings for how hormone interactions may modulate anabolic/orexigenic and catabolic/anorexigenic gene expression in the hypothalamus.