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Hypothalamic regulation of metabolismRole of thyroid hormone and estrogenZhang, Z.

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Citation for published version (APA):Zhang, Z. (2017). Hypothalamic regulation of metabolism: Role of thyroid hormone and estrogen.

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Download date: 19 Aug 2020

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7.1 Hypothalamic regulation of energy metabolism For decades, compelling studies have focused on the hypothalamic control of energy metabolism. Different anatomical structures in the hypothalamus have been demon-strated to regulate distinct aspects of energy homeostasis. Traditionally, peripheral hormones such as thyroid hormone and estrogen are known for their effects on a variety of metabolic organs by direct binding to the parenchymal cells of target tissues. However, in recent years evidence has been accumulating that classic hormones such as thyroid hormone and estrogen also regulate the energy balance, including glucose metabolism, food intake and BAT thermogenesis, by interacting with hypothalamic neuronal circuitries. For example, thyroid hormone modulates pre-autonomic neurons in the PVN thereby regulating liver glucose metabolism and insulin sensitivity via the autonomic outflow [1]. Both thyroid hormone and estradiol act through an AMP-activated protein kinase (AMPK)-sympathetic nervous system (SNS) pathway in the VMH to stimulate BAT thermogenesis [2, 3]. This neuro-modulatory involvement of thyroid hormone and estradiol in the control of energy metabolism potentially provides novel therapeutic possibilities for metabolic diseases. However, most of these hypothalamic effects have been shown in studies using acute interventions lasting from minutes to hours. It is therefore important to take these results a step further and investigate the physiological relevance of these findings in a more chronic setting. In the current thesis we evaluated the chronic metabolic consequence of these neurohormonal effects.

7.2 Thyroid hormone and energy metabolism: acute vs chronic effects

Several studies have explicitly shown that acute administration of T3 in the hypothala-mus results in increased blood glucose, BAT thermogenesis and food intake. Although the acute metabolic effects in these studies are robust, their consequences upon prolonged T3 administration have not been demonstrated. In fact, several studies have shown different effects between acute and chronic T3 treatments either peripherally or centrally [4-6]. We first developed a method that would enable us to chronically administer T3 specifically in the PVN or the VMH of rats. Using slow-releasing T3-containing pellets, we are able to locally deliver T3 to specific hypothalamic nuclei for up to four weeks without affecting other nearby nuclei. We validated this T3-delivery method by: 1) incubating the T3 pellet in serum, demonstrating in vitro the T3 releasing pattern during a prolonged period; 2) measuring the T3 concentration in different brain tissues, giving direct evidence for T3 release in the targeted nucleus as well as the

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7.1 Hypothalamic regulation of energy metabolism For decades, compelling studies have focused on the hypothalamic control of energy metabolism. Different anatomical structures in the hypothalamus have been demon-strated to regulate distinct aspects of energy homeostasis. Traditionally, peripheral hormones such as thyroid hormone and estrogen are known for their effects on a variety of metabolic organs by direct binding to the parenchymal cells of target tissues. However, in recent years evidence has been accumulating that classic hormones such as thyroid hormone and estrogen also regulate the energy balance, including glucose metabolism, food intake and BAT thermogenesis, by interacting with hypothalamic neuronal circuitries. For example, thyroid hormone modulates pre-autonomic neurons in the PVN thereby regulating liver glucose metabolism and insulin sensitivity via the autonomic outflow [1]. Both thyroid hormone and estradiol act through an AMP-activated protein kinase (AMPK)-sympathetic nervous system (SNS) pathway in the VMH to stimulate BAT thermogenesis [2, 3]. This neuro-modulatory involvement of thyroid hormone and estradiol in the control of energy metabolism potentially provides novel therapeutic possibilities for metabolic diseases. However, most of these hypothalamic effects have been shown in studies using acute interventions lasting from minutes to hours. It is therefore important to take these results a step further and investigate the physiological relevance of these findings in a more chronic setting. In the current thesis we evaluated the chronic metabolic consequence of these neurohormonal effects.

7.2 Thyroid hormone and energy metabolism: acute vs chronic effects

Several studies have explicitly shown that acute administration of T3 in the hypothala-mus results in increased blood glucose, BAT thermogenesis and food intake. Although the acute metabolic effects in these studies are robust, their consequences upon prolonged T3 administration have not been demonstrated. In fact, several studies have shown different effects between acute and chronic T3 treatments either peripherally or centrally [4-6]. We first developed a method that would enable us to chronically administer T3 specifically in the PVN or the VMH of rats. Using slow-releasing T3-containing pellets, we are able to locally deliver T3 to specific hypothalamic nuclei for up to four weeks without affecting other nearby nuclei. We validated this T3-delivery method by: 1) incubating the T3 pellet in serum, demonstrating in vitro the T3 releasing pattern during a prolonged period; 2) measuring the T3 concentration in different brain tissues, giving direct evidence for T3 release in the targeted nucleus as well as the

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geographical selectivity of the T3 administration; 3) testing T3-responsive gene expression in the targeted brain area, providing biological evidence for the functional effectiveness of the T3 treatment.

In the next step, we investigated the potential metabolic effects upon prolonged T3 administration using this chronic animal model. We demonstrated that administration of T3 in the PVN either for 7 days or 28 days suppressed the HPT-axis leading to lower circulating TH concentrations, mimicking central hypothyroidism. We observed decreased food intake after four weeks T3 administration in the PVN, probably due to the decreased systemic TH levels. Although T3 was shown to stimulate food intake at the hypothalamic level [7], it is difficult to reconcile this effect in our experimental setup, as we would expect decreased body weight following chronic T3 administration in the VMH given the stimulation of BAT thermogenesis in the acute setting as demonstrated by Lopez et al. Peripheral and central administration of TH have been shown to increase WAT lipolysis and BAT thermogenesis [2, 8], both having negative impacts on body weight. Surprisingly, we did not observe changes in either body temperature, locomotor activity, BAT activation or body weight after 7 or 28 days T3 administration in the VMH. These negative effects are unexpected in view of previous findings with acute T3 treatment, indicating probably a more complex mechanism behind the metabolic effects of acute and chronic hypothalamic T3 treatments (Figure 7.1). For example, studies in hamsters indicate that high levels of T3 in the hypothalamus promote an anabolic state, whereas low levels facilitate a catabolic state [9, 10], pointing to the importance of T3 concentration in determining its metabolic effects. Other studies showed that T3 injection into the POA change sleep pattern, effects only seen in hours- but not days- T3 administration [5, 6]. Another potential mechanism for adaption to a local chronic thyrotoxicosis is alterations in T3 transporters and T3 receptors, which are shown to be regulated by T3 per se [11-13]. Although the primary effects of T3 are thought to be mediated through T3 receptors regulating gene transcription, acute effects could be mediated also by non-genomic actions of T3 interacting with membrane kinase pathways such as AMPK [5, 14, 15]. Moreover, the adult hypothalamus still maintains neuronal plasticity [16] and hormones like leptin are able to stimulate neurite growth of NPY and POMC neurons [17]. Hence, it is possible that chronic T3 administration in the hypothalamus induces structural and functional adaptations which may mask the metabolic changes observed during acute T3 administration [18]. It would be interesting to investigate in future what are the different molecular changes that may account for the metabolic differences between acute and chronic intrahypothalamic T3 treatments.

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Figure 7.1 Effects of acute vs chronic intrahypothalamic T3 administration on en-ergy metabolism. REM, rapid eye movement sleep; up arrow: stimulatory effects; “=”: no effect.

7.3 Fat and bone: a role of estrogen in the brain During the last decades, obesity and osteoporosis have become important global health problems. Estrogen has prominent beneficial effects on both body weight and bone density. Different types of fat tissues are innervated with both parasympathetic [19] and sympathetic efferent [20] and white adipose tissue (WAT) metabolism has been shown to be regulated by many hormones, such as leptin and insulin, via the central nervous system [21, 22]. Although several studies also suggest a central regulation of WAT by E2, direct evidence for E2 effects in the brain, especially in the hypothalamus, on WAT metabolism and fat distribution remain sparse. In the current thesis we showed that central E2 administration is able to rescue the ovariectomy induced changes in fat distribution to the same extent as subcutaneous E2. In addition, E2 administration locally into the VMH increases lipolysis in abdominal fat and partly rescuing ovariectomy induced fat distribution. Previous studies using mouse models containing gene muta-tions or inactivation indicate that in the VMH these estrogenic effects on WAT are primarily mediated via the ERα [23]. Together, these data suggest that E2 acts on ERα expressing neurons in the VMH regulating WAT metabolism and body fat distribution (Figure 7.2). Interestingly, there is a sexual dimorphism in the sympathetic innervation of adipose tissue, with more ERα expressing neurons projecting to subcutaneous fat in females than in males [20]. Therefore, it is possible that E2 determines the sexual difference in fat distribution via the VMH.

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geographical selectivity of the T3 administration; 3) testing T3-responsive gene expression in the targeted brain area, providing biological evidence for the functional effectiveness of the T3 treatment.

In the next step, we investigated the potential metabolic effects upon prolonged T3 administration using this chronic animal model. We demonstrated that administration of T3 in the PVN either for 7 days or 28 days suppressed the HPT-axis leading to lower circulating TH concentrations, mimicking central hypothyroidism. We observed decreased food intake after four weeks T3 administration in the PVN, probably due to the decreased systemic TH levels. Although T3 was shown to stimulate food intake at the hypothalamic level [7], it is difficult to reconcile this effect in our experimental setup, as we would expect decreased body weight following chronic T3 administration in the VMH given the stimulation of BAT thermogenesis in the acute setting as demonstrated by Lopez et al. Peripheral and central administration of TH have been shown to increase WAT lipolysis and BAT thermogenesis [2, 8], both having negative impacts on body weight. Surprisingly, we did not observe changes in either body temperature, locomotor activity, BAT activation or body weight after 7 or 28 days T3 administration in the VMH. These negative effects are unexpected in view of previous findings with acute T3 treatment, indicating probably a more complex mechanism behind the metabolic effects of acute and chronic hypothalamic T3 treatments (Figure 7.1). For example, studies in hamsters indicate that high levels of T3 in the hypothalamus promote an anabolic state, whereas low levels facilitate a catabolic state [9, 10], pointing to the importance of T3 concentration in determining its metabolic effects. Other studies showed that T3 injection into the POA change sleep pattern, effects only seen in hours- but not days- T3 administration [5, 6]. Another potential mechanism for adaption to a local chronic thyrotoxicosis is alterations in T3 transporters and T3 receptors, which are shown to be regulated by T3 per se [11-13]. Although the primary effects of T3 are thought to be mediated through T3 receptors regulating gene transcription, acute effects could be mediated also by non-genomic actions of T3 interacting with membrane kinase pathways such as AMPK [5, 14, 15]. Moreover, the adult hypothalamus still maintains neuronal plasticity [16] and hormones like leptin are able to stimulate neurite growth of NPY and POMC neurons [17]. Hence, it is possible that chronic T3 administration in the hypothalamus induces structural and functional adaptations which may mask the metabolic changes observed during acute T3 administration [18]. It would be interesting to investigate in future what are the different molecular changes that may account for the metabolic differences between acute and chronic intrahypothalamic T3 treatments.

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Figure 7.1 Effects of acute vs chronic intrahypothalamic T3 administration on en-ergy metabolism. REM, rapid eye movement sleep; up arrow: stimulatory effects; “=”: no effect.

7.3 Fat and bone: a role of estrogen in the brain During the last decades, obesity and osteoporosis have become important global health problems. Estrogen has prominent beneficial effects on both body weight and bone density. Different types of fat tissues are innervated with both parasympathetic [19] and sympathetic efferent [20] and white adipose tissue (WAT) metabolism has been shown to be regulated by many hormones, such as leptin and insulin, via the central nervous system [21, 22]. Although several studies also suggest a central regulation of WAT by E2, direct evidence for E2 effects in the brain, especially in the hypothalamus, on WAT metabolism and fat distribution remain sparse. In the current thesis we showed that central E2 administration is able to rescue the ovariectomy induced changes in fat distribution to the same extent as subcutaneous E2. In addition, E2 administration locally into the VMH increases lipolysis in abdominal fat and partly rescuing ovariectomy induced fat distribution. Previous studies using mouse models containing gene muta-tions or inactivation indicate that in the VMH these estrogenic effects on WAT are primarily mediated via the ERα [23]. Together, these data suggest that E2 acts on ERα expressing neurons in the VMH regulating WAT metabolism and body fat distribution (Figure 7.2). Interestingly, there is a sexual dimorphism in the sympathetic innervation of adipose tissue, with more ERα expressing neurons projecting to subcutaneous fat in females than in males [20]. Therefore, it is possible that E2 determines the sexual difference in fat distribution via the VMH.

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Figure 7.2 Effects of central E2 on energy metabolism. In addition to the exist-ing knowledge on E2 regulation of energy metabolism (blue line), our study indicates that E2 regulates white adipose tissue but not bone metabolism from the VMH (red solid line). A hypothesis for central E2 regulation of bone remodelling is proposed from the PVN (red and blue dash line).

E2 is also a key regulator for bone remodelling, an effect generally considered to be exerted directly at the level of the bone. In the current thesis we provide the first evidence for central E2 on bone remodelling. As for now it remains unclear which brain areas may mediate these central effects of E2. As mentioned above, with abundant ERα expression, the VMH has been shown to mediate various metabolic effects of E2 [24]. However, chronic E2 administration in the VMH did not change any bone parameters, although fat distribution is significantly changed. This finding is consistent with other genetic mice knockout studies where ERα depletion in the VMH or ventromedial hypothalamus did not show any bone phenotypes [25, 26]. Although the sympathetic nervous system has been demonstrated to mediate the catabolic effects of several hormones on bone metabolism, E2 has an anabolic effect on bone, suggesting that in this case a different neural pathway must be involved. In fact, a parasympathetic regulation of bone remodelling has been reported as interleukin-1 is shown to increase bone mass through a parasympathetic pathway [27]. Therefore, the protective central

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effects of E2 on bone might be mediated via the parasympathetic nervous system. Of note, out data indicate that systemic E2 administration prevent bone loss by reducing bone resorption and bone formation, while intracerebroventricular (ICV) E2 administra-tion primarily reduces bone formation. Thus, another possibility is that there are differential effects by peripheral versus central E2 signalling on bone metabolism. This is supported by the facts that global ERα knockout mice show decreased bone mass while brain specific ERα knock out mice show opposite effects [26, 28, 29]. Similar regulatory patterns on bone have also been shown by other hormones including leptin [30] and serotonin [31, 32].

Our study indicates that E2 does not regulate bone via the VMH; however, several studies suggest that other hypothalamic nuclei, such as the PVN and ARC, may be involved (Figure 7.2). Roepke et al showed that E2 controls bone remodelling via a Gq-coupled membrane receptor (Gq-mER) and that this effect presumably involves the pre-autonomic PVN neurons [33]. In the hypothalamus, ERβ is dominantly expressed in the PVN including the pre-autonomic neurons [34]. Our previous study showed that E2 administration in the PVN increased blood glucose concentrations [35], suggesting a PVN mediated E2 effect on bone. On the other hand, a recent study showed that mice with depletion of ERα in the ARC had increased cortical and trabecular bone mass [26] and E2 treatment enhanced the bone volume, suggesting an inhibitory role of ERα in the ARC. Estrogen has close interaction with POMC/CART neurons in the ARC [36] and CART mediates the leptin control of bone resorption [37]. Future investigations delineating the neural circuits connecting the central action of E2 with the bone will be necessary to test these hypotheses.

7.4 TRH in the PVN: more than hypophysiotropic Although perhaps best known as the neuroendocrine neurons regulating the HPT axis, the majority of the TRH neurons in the PVN are non-hypophysiotropic [38, 39]. These so-called non-hypophysiotropic TRH neurons send extensive projections to the brainstem and spinal cord to control autonomic responses, as well as to other brain areas important for energy metabolism and cognitive function [38-41]. Triggered by the potent activation of TRH neurons in the PVN during cold exposure [42-44], we investigated the effect of TRH administration in the PVN on glucose metabolism and thermoregulation, two systems critical for coping with a cold challenge. We showed that TRH administra-tion in the PVN largely mimics the behavioural and metabolic responses induced by cold exposure, including increased locomotor activity, glucose production, and energy mobilization (e.g. glucose and lipid) to support BAT thermogenesis (Figure 7.3).

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Figure 7.2 Effects of central E2 on energy metabolism. In addition to the exist-ing knowledge on E2 regulation of energy metabolism (blue line), our study indicates that E2 regulates white adipose tissue but not bone metabolism from the VMH (red solid line). A hypothesis for central E2 regulation of bone remodelling is proposed from the PVN (red and blue dash line).

E2 is also a key regulator for bone remodelling, an effect generally considered to be exerted directly at the level of the bone. In the current thesis we provide the first evidence for central E2 on bone remodelling. As for now it remains unclear which brain areas may mediate these central effects of E2. As mentioned above, with abundant ERα expression, the VMH has been shown to mediate various metabolic effects of E2 [24]. However, chronic E2 administration in the VMH did not change any bone parameters, although fat distribution is significantly changed. This finding is consistent with other genetic mice knockout studies where ERα depletion in the VMH or ventromedial hypothalamus did not show any bone phenotypes [25, 26]. Although the sympathetic nervous system has been demonstrated to mediate the catabolic effects of several hormones on bone metabolism, E2 has an anabolic effect on bone, suggesting that in this case a different neural pathway must be involved. In fact, a parasympathetic regulation of bone remodelling has been reported as interleukin-1 is shown to increase bone mass through a parasympathetic pathway [27]. Therefore, the protective central

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effects of E2 on bone might be mediated via the parasympathetic nervous system. Of note, out data indicate that systemic E2 administration prevent bone loss by reducing bone resorption and bone formation, while intracerebroventricular (ICV) E2 administra-tion primarily reduces bone formation. Thus, another possibility is that there are differential effects by peripheral versus central E2 signalling on bone metabolism. This is supported by the facts that global ERα knockout mice show decreased bone mass while brain specific ERα knock out mice show opposite effects [26, 28, 29]. Similar regulatory patterns on bone have also been shown by other hormones including leptin [30] and serotonin [31, 32].

Our study indicates that E2 does not regulate bone via the VMH; however, several studies suggest that other hypothalamic nuclei, such as the PVN and ARC, may be involved (Figure 7.2). Roepke et al showed that E2 controls bone remodelling via a Gq-coupled membrane receptor (Gq-mER) and that this effect presumably involves the pre-autonomic PVN neurons [33]. In the hypothalamus, ERβ is dominantly expressed in the PVN including the pre-autonomic neurons [34]. Our previous study showed that E2 administration in the PVN increased blood glucose concentrations [35], suggesting a PVN mediated E2 effect on bone. On the other hand, a recent study showed that mice with depletion of ERα in the ARC had increased cortical and trabecular bone mass [26] and E2 treatment enhanced the bone volume, suggesting an inhibitory role of ERα in the ARC. Estrogen has close interaction with POMC/CART neurons in the ARC [36] and CART mediates the leptin control of bone resorption [37]. Future investigations delineating the neural circuits connecting the central action of E2 with the bone will be necessary to test these hypotheses.

7.4 TRH in the PVN: more than hypophysiotropic Although perhaps best known as the neuroendocrine neurons regulating the HPT axis, the majority of the TRH neurons in the PVN are non-hypophysiotropic [38, 39]. These so-called non-hypophysiotropic TRH neurons send extensive projections to the brainstem and spinal cord to control autonomic responses, as well as to other brain areas important for energy metabolism and cognitive function [38-41]. Triggered by the potent activation of TRH neurons in the PVN during cold exposure [42-44], we investigated the effect of TRH administration in the PVN on glucose metabolism and thermoregulation, two systems critical for coping with a cold challenge. We showed that TRH administra-tion in the PVN largely mimics the behavioural and metabolic responses induced by cold exposure, including increased locomotor activity, glucose production, and energy mobilization (e.g. glucose and lipid) to support BAT thermogenesis (Figure 7.3).

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TRH effects on glucose metabolism and thermoregulation were studied in many brain regions, however, confirmation that the release of TRH within the PVN plays a role in these effects was lacking so far. Our study showed that TRH administration in the PVN results in hyperglycaemia and hyperthermia. Interestingly, both the SNS and parasympa-thetic nervous system (PNS) mediate the central TRH effect on glucose production and blood glucose concentration. It is well known that the sympathetic innervation of the liver is a crucial stimulator of glucose production, while the parasympathetic innervation has an inhibitory effect. Curiously, our data suggest that the parasympathetic branch also contributes to the TRH-induced increase in endogenous glucose production.

Several studies have demonstrated that TRH administration in the preoptic area of the POA, which is particularly known for its involvement in thermoregulation, induces hyperthermia [45-47]; however, cold exposure was shown to decrease TRH mRNA in the POA [48]. On the other hand, cold increased TRH peptide [44] and TRH mRNA expression specifically in the PVN (not in the DMH and LH) [43, 49]. Both TRH receptors and TRH-immunoreactive terminals have been identified in the PVN, providing an anatomic basis for a neural circuit involving TRH signalling within the PVN. During cold exposure, thermosensitive neurons in POA receive temperature information from peripheral inputs and project this information directly to the brainstem as well as many other hypothalam-ic nuclei including the PVN [50, 51]. Importantly, thermosensitive neurons are also found in the PVN, which may play an important role in cold induced activation of TRH neurons [52]. Although it is not clear which neurons in the PVN mediate the TRH effects, it is tempting to hypothesize that cold-induced TRH release acts on TRH or other pre-autonomic motor neurons in the PVN thereby stimulating glucose production and thermogenesis (Figure 7.3). Therefore, it is interesting to investigate in future experi-ments which neurons, e.g. TRH- or non-TRH-neurons in the PVN, are activated or inhibited by TRH release in the PVN.

The PVN has been implicated in both stimulatory [53-55] and inhibitory effects on thermoregulation [56-59]. Various studies using different stimuli show opposite results on BAT thermogenesis [60]. Our study provides evidence for a stimulatory role of TRH in the PVN in thermoregulation. Interestingly, a recent study demonstrated that cortico-tropin-releasing hormone (CRH) neurons, but not TRH neurons, in the PVN mediate an inhibitory effect on body temperature [61]. In contrast to TRH, CRH injection in the PVN fails to stimulate sympathetic nerve activity to BAT [62]. Therefore, it is likely that distinct neuronal populations in the PVN control different aspects of thermoregulation, i.e., CRH neurons inhibiting and TRH neurons stimulating thermogenesis.

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Figure 7.3 Schematic model for the role of TRH release in the PVN during cold de-fence

Both proper heat production and conservation are critical for survival during cold exposure. TRH administration in the PVN stimulates BAT thermogenesis and increases body temperature. However, heat preservation via vasoconstriction is also important for thermoregulation during cold exposure. TRH has been reported to increase cutaneous vasoconstriction [46]. Thus, it is likely that in addition to the increased thermogenesis, TRH in the PVN also reduces heat loss together contributing to an increased body temperature. Although our data provide solid evidence to link TRH in the PVN with the cold-defence response, it would be interesting to confirm the critical role of TRH in cold defence by blocking TRH signalling in the PVN during cold exposure. In addition, many other neurochemical signals in the PVN also play essential role in the cold-defence reaction including leptin, CRH and oxytocin. To investigate how TRH possibly interacts with these signalling pathways in defending cold will be another interesting topic in the future.

BAT has been recently shown to exist in adult humans and can be activated upon cold exposure [63, 64]. Since BAT is able to generate heat by dissipating energy substrates (e.g. lipid and glucose) and dysfunctional BAT activation has been associated with obesity, it is a promising target to combat obesity, diabetes and other metabolic diseases [65, 66]. Cold is the major activator for BAT recruitment and thermogenesis [67, 68], however, chronic or repeated cold exposure may not be suitable for obesity treatment.

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TRH effects on glucose metabolism and thermoregulation were studied in many brain regions, however, confirmation that the release of TRH within the PVN plays a role in these effects was lacking so far. Our study showed that TRH administration in the PVN results in hyperglycaemia and hyperthermia. Interestingly, both the SNS and parasympa-thetic nervous system (PNS) mediate the central TRH effect on glucose production and blood glucose concentration. It is well known that the sympathetic innervation of the liver is a crucial stimulator of glucose production, while the parasympathetic innervation has an inhibitory effect. Curiously, our data suggest that the parasympathetic branch also contributes to the TRH-induced increase in endogenous glucose production.

Several studies have demonstrated that TRH administration in the preoptic area of the POA, which is particularly known for its involvement in thermoregulation, induces hyperthermia [45-47]; however, cold exposure was shown to decrease TRH mRNA in the POA [48]. On the other hand, cold increased TRH peptide [44] and TRH mRNA expression specifically in the PVN (not in the DMH and LH) [43, 49]. Both TRH receptors and TRH-immunoreactive terminals have been identified in the PVN, providing an anatomic basis for a neural circuit involving TRH signalling within the PVN. During cold exposure, thermosensitive neurons in POA receive temperature information from peripheral inputs and project this information directly to the brainstem as well as many other hypothalam-ic nuclei including the PVN [50, 51]. Importantly, thermosensitive neurons are also found in the PVN, which may play an important role in cold induced activation of TRH neurons [52]. Although it is not clear which neurons in the PVN mediate the TRH effects, it is tempting to hypothesize that cold-induced TRH release acts on TRH or other pre-autonomic motor neurons in the PVN thereby stimulating glucose production and thermogenesis (Figure 7.3). Therefore, it is interesting to investigate in future experi-ments which neurons, e.g. TRH- or non-TRH-neurons in the PVN, are activated or inhibited by TRH release in the PVN.

The PVN has been implicated in both stimulatory [53-55] and inhibitory effects on thermoregulation [56-59]. Various studies using different stimuli show opposite results on BAT thermogenesis [60]. Our study provides evidence for a stimulatory role of TRH in the PVN in thermoregulation. Interestingly, a recent study demonstrated that cortico-tropin-releasing hormone (CRH) neurons, but not TRH neurons, in the PVN mediate an inhibitory effect on body temperature [61]. In contrast to TRH, CRH injection in the PVN fails to stimulate sympathetic nerve activity to BAT [62]. Therefore, it is likely that distinct neuronal populations in the PVN control different aspects of thermoregulation, i.e., CRH neurons inhibiting and TRH neurons stimulating thermogenesis.

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Figure 7.3 Schematic model for the role of TRH release in the PVN during cold de-fence

Both proper heat production and conservation are critical for survival during cold exposure. TRH administration in the PVN stimulates BAT thermogenesis and increases body temperature. However, heat preservation via vasoconstriction is also important for thermoregulation during cold exposure. TRH has been reported to increase cutaneous vasoconstriction [46]. Thus, it is likely that in addition to the increased thermogenesis, TRH in the PVN also reduces heat loss together contributing to an increased body temperature. Although our data provide solid evidence to link TRH in the PVN with the cold-defence response, it would be interesting to confirm the critical role of TRH in cold defence by blocking TRH signalling in the PVN during cold exposure. In addition, many other neurochemical signals in the PVN also play essential role in the cold-defence reaction including leptin, CRH and oxytocin. To investigate how TRH possibly interacts with these signalling pathways in defending cold will be another interesting topic in the future.

BAT has been recently shown to exist in adult humans and can be activated upon cold exposure [63, 64]. Since BAT is able to generate heat by dissipating energy substrates (e.g. lipid and glucose) and dysfunctional BAT activation has been associated with obesity, it is a promising target to combat obesity, diabetes and other metabolic diseases [65, 66]. Cold is the major activator for BAT recruitment and thermogenesis [67, 68], however, chronic or repeated cold exposure may not be suitable for obesity treatment.

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Therefore, many studies have been dedicated to identify novel pharmacological activators of BAT. However, so far only a few molecules have been found to be able to activate BAT in humans, including bile acid [69] and β3-adrenergic receptor agonists [70]. We and others have shown a substantial stimulatory effect of TRH on BAT in rodents. Subsequently, we investigated whether TRH activates BAT in humans as well using 18FDG-PET-CT technique. We found that TRH administration activates BAT in cold-exposed men without changes in systemic thyroid hormones at an early stage, providing the first evidence that TRH induces BAT thermogenesis in humans. As suggested by the animal studies, centrally the TRH effect on BAT in human is probably mediated by the hypothalamus and the SNS. However, future studies should address the mechanism of systemic TRH-induced BAT activation in humans. Of note, in contrast to the studies using β3-adrenergic receptor agonists [70, 71], TRH activates BAT without significant changes in the sympatho-vagal balance, heart rate or blood pressure, suggesting a specific effect of TRH on only the sympathetic input to BAT. Although the total amount of BAT in adults is not large, studies have indicated that BAT is able to dissipate significant amounts of energy into heat upon full activation [64]. However, achieving this metabolic benefits requires constant stimulation of BAT. Future studies are needed to investigate pharma-cological mechanisms of TRH on BAT that allow for a permanent activation while circumventing the influence on the HPT axis.

7.5 Significance of the study Our studies investigated the metabolic effects of thyroid hormone, estradiol and TRH in the hypothalamus. For the thyroid hormone study, we developed an animal model for chronic intrahypothalamic T3 administration. Using this animal model, we demonstrated unexpected results in view of the metabolic effects observed previously in acute studies. The present results point to more complex mechanisms when translating results from studies in an acute setting to a chronic situation. In the estradiol study, we showed a direct rescuing effects of E2 in the VMH on fat distribution, results that may point to a potential target for future anti-obesity treatments. This study also indicated a central role of E2 in bone metabolism, highlighting a neural mechanism in hormone replace-ment therapy of osteoporosis. Our TRH study in rat indicated a novel role for TRH release in the PVN in cold defence. In addition, the stimulatory effects of TRH on BAT also apply to humans. Together, these findings indicate TRH to be a future pharmacolog-ical candidate for obesity treatment by modulating BAT activation.

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[1] L.P. Klieverik, S.F. Janssen, A. van Riel, E. Foppen, P.H. Bisschop, M.J. Serlie, A. Boelen, M.T. Ackermans, H.P. Sauerwein, E. Fliers, A. Kalsbeek, Thyroid hormone modulates glucose production via a sympathetic pathway from the hypothalamic paraventricular nucleus to the liver, Proc Natl Acad Sci U S A 106(14) (2009) 5966-71.

[2] M. Lopez, L. Varela, M.J. Vazquez, S. Rodriguez-Cuenca, C.R. Gonzalez, V.R. Velagapudi, D.A. Morgan, E. Schoenmakers, K. Agassandian, R. Lage, P.B. Martinez de Morentin, S. Tovar, R. Nogueiras, D. Carling, C. Lelliott, R. Gallego, M. Oresic, K. Chatterjee, A.K. Saha, K. Rahmouni, C. Dieguez, A. Vidal-Puig, Hypothalamic AMPK and fatty acid metabolism mediate thyroid regulation of energy balance, Nat Med 16(9) (2010) 1001-8.

[3] P.B. Martinez de Morentin, I. Gonzalez-Garcia, L. Martins, R. Lage, D. Fernandez-Mallo, N. Martinez-Sanchez, F. Ruiz-Pino, J. Liu, D.A. Morgan, L. Pinilla, R. Gallego, A.K. Saha, A. Kalsbeek, E. Fliers, P.H. Bisschop, C. Dieguez, R. Nogueiras, K. Rahmouni, M. Tena-Sempere, M. Lopez, Estradiol Regulates Brown Adipose Tissue Thermogenesis via Hypothalamic AMPK, Cell metabolism (2014).

[4] K. Andersson, P. Eneroth, The effects of acute and chronic treatment with triiodothyronine and thyroxine on the hypothalamic and telencephalic catecholamine nerve terminal systems of the hypophysectomized male rat. , Neuroendocrinology 40(5) (1985) 398-408.

[5] J.V. Martin, P.F. Giannopoulos, S.X. Moffett, T.D. James, Effects of acute microinjections of thyroid hormone to the preoptic region of euthyroid adult male rats on sleep and motor activity, Brain Res 1516 (2013) 45-54.

[6] S.X. Moffett, P.F. Giannopoulos, T.D. James, J.V. Martin, Effects of acute microinjections of thyroid hormone to the preoptic region of hypothyroid adult male rats on sleep, motor activity and body temperature, Brain Res 1516 (2013) 55-65.

[7] A. Amin, W.S. Dhillo, K.G. Murphy, The central effects of thyroid hormones on appetite, Journal of thyroid research 2011 (2011) 306510.

[8] L.P. Klieverik, C.P. Coomans, E. Endert, H.P. Sauerwein, L.M. Havekes, P.J. Voshol, P.C. Rensen, J.A. Romijn, A. Kalsbeek, E. Fliers, Thyroid hormone effects on whole-body energy homeostasis and tissue-specific fatty acid uptake in vivo, Endocrinology 150(12) (2009) 5639-48.

[9] M. Murphy, F.J. Ebling, The role of hypothalamic tri-iodothyronine availability in seasonal regulation of energy balance and body weight, Journal of thyroid research 2011 (2011) 387562.

[10] P. Barrett, F.J. Ebling, S. Schuhler, D. Wilson, A.W. Ross, A. Warner, P. Jethwa, A. Boelen, T.J. Visser, D.M. Ozanne, Z.A. Archer, J.G. Mercer, P.J. Morgan, Hypothalamic thyroid hormone catabolism acts as a gatekeeper for the seasonal control of body weight and reproduction, Endocrinology 148(8) (2007) 3608-17.

[11] L. Mebis, Y. Debaveye, B. Ellger, S. Derde, E.J. Ververs, L. Langouche, V.M. Darras, E. Fliers, T.J. Visser, G. Van den Berghe, Changes in the central component of the hypothalamus-pituitary-thyroid axis in a rabbit model of prolonged critical illness, Critical care 13(5) (2009) R147.

[12] L.P. Capelo, E.H. Beber, T.L. Fonseca, C.H. Gouveia, The monocarboxylate transporter 8 and L-type amino acid transporters 1 and 2 are expressed in mouse skeletons and in osteoblastic MC3T3-E1 cells, Thyroid 19(2) (2009) 171-80.

[13] M.A. Pou, J. Bismuth, J. Gharbi-Chihi, J. Torresani, Triiodothyronine (T3)-induced down-regulation of the nuclear T3 receptor in mouse preadipocyte cell lines, Endocrinology 119(5) (1986) 2360-7.

[14] S.Y. Cheng, J.L. Leonard, P.J. Davis, Molecular aspects of thyroid hormone actions, Endocrine reviews 31(2) (2010) 139-70.

[15] I. Irrcher, D.R. Walkinshaw, T.E. Sheehan, D.A. Hood, Thyroid hormone (T3) rapidly activates p38 and AMPK in skeletal muscle in vivo, Journal of applied physiology 104(1) (2008) 178-85.

[16] M.V. Kokoeva, H. Yin, J.S. Flier, Evidence for constitutive neural cell proliferation in the adult murine hypothalamus, The Journal of comparative neurology 505(2) (2007) 209-20.

[17] S.G. Bouret, S.J. Draper, R.B. Simerly, Trophic action of leptin on hypothalamic neurons that regulate feeding, Science 304(5667) (2004) 108-110.

7

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Therefore, many studies have been dedicated to identify novel pharmacological activators of BAT. However, so far only a few molecules have been found to be able to activate BAT in humans, including bile acid [69] and β3-adrenergic receptor agonists [70]. We and others have shown a substantial stimulatory effect of TRH on BAT in rodents. Subsequently, we investigated whether TRH activates BAT in humans as well using 18FDG-PET-CT technique. We found that TRH administration activates BAT in cold-exposed men without changes in systemic thyroid hormones at an early stage, providing the first evidence that TRH induces BAT thermogenesis in humans. As suggested by the animal studies, centrally the TRH effect on BAT in human is probably mediated by the hypothalamus and the SNS. However, future studies should address the mechanism of systemic TRH-induced BAT activation in humans. Of note, in contrast to the studies using β3-adrenergic receptor agonists [70, 71], TRH activates BAT without significant changes in the sympatho-vagal balance, heart rate or blood pressure, suggesting a specific effect of TRH on only the sympathetic input to BAT. Although the total amount of BAT in adults is not large, studies have indicated that BAT is able to dissipate significant amounts of energy into heat upon full activation [64]. However, achieving this metabolic benefits requires constant stimulation of BAT. Future studies are needed to investigate pharma-cological mechanisms of TRH on BAT that allow for a permanent activation while circumventing the influence on the HPT axis.

7.5 Significance of the study Our studies investigated the metabolic effects of thyroid hormone, estradiol and TRH in the hypothalamus. For the thyroid hormone study, we developed an animal model for chronic intrahypothalamic T3 administration. Using this animal model, we demonstrated unexpected results in view of the metabolic effects observed previously in acute studies. The present results point to more complex mechanisms when translating results from studies in an acute setting to a chronic situation. In the estradiol study, we showed a direct rescuing effects of E2 in the VMH on fat distribution, results that may point to a potential target for future anti-obesity treatments. This study also indicated a central role of E2 in bone metabolism, highlighting a neural mechanism in hormone replace-ment therapy of osteoporosis. Our TRH study in rat indicated a novel role for TRH release in the PVN in cold defence. In addition, the stimulatory effects of TRH on BAT also apply to humans. Together, these findings indicate TRH to be a future pharmacolog-ical candidate for obesity treatment by modulating BAT activation.

General discussion

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[18] M. Murphy, P.H. Jethwa, A. Warner, P. Barrett, K.N. Nilaweera, J.M. Brameld, F.J. Ebling, Effects of manipulating hypothalamic triiodothyronine concentrations on seasonal body weight and torpor cycles in Siberian hamsters, Endocrinology 153(1) (2012) 101-12.

[19] F. Kreier, E. Fliers, P.J. Voshol, C.G. Van Eden, L.M. Havekes, A. Kalsbeek, C.L. Van Heijningen, A.A. Sluiter, T.C. Mettenleiter, J.A. Romijn, H.P. Sauerwein, R.M. Buijs, Selective parasympathetic innervation of subcutaneous and intra-abdominal fat--functional implications, J Clin Invest 110(9) (2002) 1243-50.

[20] E.S. Adler, J.H. Hollis, I.J. Clarke, D.R. Grattan, B.J. Oldfield, Neurochemical characterization and sexual dimorphism of projections from the brain to abdominal and subcutaneous white adipose tissue in the rat, J Neurosci 32(45) (2012) 15913-21.

[21] W. Zeng, R.M. Pirzgalska, M.M. Pereira, N. Kubasova, A. Barateiro, E. Seixas, Y.H. Lu, A. Kozlova, H. Voss, G.G. Martins, J.M. Friedman, A.I. Domingos, Sympathetic Neuro-adipose Connections Mediate Leptin-Driven Lipolysis, Cell 163(1) (2015) 84-94.

[22] A. Kleinridders, H.A. Ferris, W. Cai, C.R. Kahn, Insulin action in brain regulates systemic metabolism and brain function, Diabetes 63(7) (2014) 2232-43.

[23] Y. Xu, T.P. Nedungadi, L. Zhu, N. Sobhani, B.G. Irani, K.E. Davis, X. Zhang, F. Zou, L.M. Gent, L.D. Hahner, S.A. Khan, C.F. Elias, J.K. Elmquist, D.J. Clegg, Distinct hypothalamic neurons mediate estrogenic effects on energy homeostasis and reproduction, Cell metabolism 14(4) (2011) 453-65.

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[26] H.H. Farman, S.H. Windahl, L. Westberg, H. Isaksson, E. Egecioglu, E. Schele, H. Ryberg, J.O. Jansson, J. Tuukkanen, A. Koskela, S.K. Xie, L. Hahner, J. Zehr, D.J. Clegg, M.K. Lagerquist, C. Ohlsson, Female Mice Lacking Estrogen Receptor-alpha in Hypothalamic Proopiomelanocortin (POMC) Neurons Display Enhanced Estrogenic Response on Cortical Bone Mass, Endocrinology 157(8) (2016) 3242-52.

[27] A. Bajayo, A. Bar, A. Denes, M. Bachar, V. Kram, M. Attar-Namdar, A. Zallone, K.J. Kovacs, R. Yirmiya, I. Bab, Skeletal parasympathetic innervation communicates central IL-1 signals regulating bone mass accrual, Proc Natl Acad Sci U S A 109(38) (2012) 15455-60.

[28] E.P. Smith, B. Specker, K.S. Korach, Recent experimental and clinical findings in the skeleton associated with loss of estrogen hormone or estrogen receptor activity, The Journal of steroid biochemistry and molecular biology 118(4-5) (2010) 264-72.

[29] C. Ohlsson, C. Engdahl, A.E. Borjesson, S.H. Windahl, E. Studer, L. Westberg, E. Eriksson, A. Koskela, J. Tuukkanen, A. Krust, P. Chambon, H. Carlsten, M.K. Lagerquist, Estrogen receptor-alpha expression in neuronal cells affects bone mass, Proc Natl Acad Sci U S A 109(3) (2012) 983-8.

[30] V. Cirmanova, M. Bayer, L. Starka, K. Zajickova, The effect of leptin on bone: an evolving concept of action, Physiological research / Academia Scientiarum Bohemoslovaca 57 Suppl 1 (2008) S143-51.

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