Central growth hormone action regulates neuroglial and proinflammatory markers in the hypothalamus of male mice.

Growth hormone (GH) action in specific neuronal populations regulates neuroendocrine responses, metabolism, and behavior. However, the potential role of central GH action on glial function is less understood. The present study aims to determine how the hypothalamic expression of several neuroglial markers is affected by central GH action in male mice. The dwarf GH- and insulin-like growth factor-1 (IGF-1)-deficient Ghrhrlit/lit mice showed decreased mRNA expression of Nes (Nestin), Gfap, Iba1, Adgre1 (F4/80), and Tnf (TNFα) in the hypothalamus, compared to wild-type animals. In contrast, transgenic overexpression of GH led to high serum GH and IGF-1 levels, and increased hypothalamic expression of Nes, Gfap, Adgre1, Iba1, and Rax. Hepatocyte-specific GH receptor (GHR) knockout mice, which are characterized by high serum GH levels, but reduced IGF-1 secretion, showed increased mRNA expression of Gfap, Iba1, Tnf, and Sox10, demonstrating that the increase in GH levels alters the hypothalamic expression of glial markers associated with neuroinflammation, independently of IGF-1. Conversely, brain-specific GHR knockout mice showed reduced expression of Gfap, Adgre1, and Vim (vimentin), indicating that brain GHR signaling is necessary to mediate GH-induced changes in the expression of several neuroglial markers. In conclusion, the hypothalamic mRNA levels of several neuroglial markers associated with inflammation are directly modulated by GHR signaling in male mice.

Benfotiamine protects against hypothalamic dysfunction in a STZ-induced model of neurodegeneration in rats.

The neurodegeneration of Alzheimer's disease (AD) affects not only brain structures associate with cognition early in the progression of the disease, but other areas such as the hypothalamus, a region involved in the control of metabolism and appetite. In this context, we evaluated the effects of benfotiamine (BFT), a vitamin B1 analog that is being proposed as a therapeutical approach for AD-related cognitive alterations, which were induced by intracerebroventricular injection of streptozotocin (STZ). In addition to the already described effect of STZ on cognition, we show that this drug also causes metabolic changes which are linked to changes in hypothalamic insulin signaling and orexigenic and anorexigenic circuitries, as well as a decreased cellular integrated stress response. As expected, the supplementation with 150 mg/kg of BFT for 30 days increased blood concentrations of thiamine and its phosphate esters. This led to the prevention of body weight and fat loss in STZ-ICV-treated animals. In addition, we also found an improvement in food consumption, despite hypothalamic gene expression linked to anorexia after STZ exposure. Additionally, decreased apoptosis signaling was observed in the hypothalamus. In in vitro experiments, we noticed a high ability of BFT to increase insulin sensitivity in hypothalamic neurons. Furthermore, we also observed that BFT decreases the mitochondrial unfolded stress response damage by preventing the loss of HSP60 and reversed the mitochondria dysfunction caused by STZ. Taken together, these results suggest that benfotiamine treatment is a potential therapeutic approach in the treatment of hypothalamic dysfunction and metabolic disturbances associated with sporadic AD.

The effect of central growth hormone action on hypoxia ventilatory response in conscious mice.

Growth hormone (GH)-responsive neurons regulate several homeostatic behaviors including metabolism, energy balance, arousal, and stress response. Therefore, it is possible that GH-responsive neurons play a role in other responses such as CO2/H+-dependent breathing behaviors. Here, we investigated whether central GH receptor (GHR) modulates respiratory activity in conscious unrestrained mice. First, we detected clusters of GH-responsive neurons in the tyrosine hydroxylase-expressing cells in the rostroventrolateral medulla (C1 region) and within the locus coeruleus (LC). No significant expression was detected in phox2b-expressing cells in the retrotrapezoid nucleus. Whole body plethysmography revealed a reduction in the tachypneic response to hypoxia (FiO2 = 0.08) without changing baseline breathing and the hypercapnic ventilatory response. Contrary to the physiological findings, we did not find significant differences in the number of fos-activated cells in the nucleus of the solitary tract (NTS), C1, LC and paraventricular nucleus of the hypothalamus (PVH). Our finding suggests a possible secondary role of central GH action in the tachypneic response to hypoxia in conscious mice.

Ablation of Growth Hormone Receptor in GABAergic Neurons Leads to Increased Pulsatile Growth Hormone Secretion.

Growth hormone (GH) acts in several hypothalamic neuronal populations to modulate metabolism and the autoregulation of GH secretion via negative-feedback loops. However, few studies have investigated whether GH receptor (GHR) expression in specific neuronal populations is required for the homeostatic control of GH secretion and energy homeostasis. In the present study, we investigated the consequences of the specific GHR ablation in GABAergic (VGAT-expressing) or glutamatergic (VGLUT2-expressing) cells. GHR ablation in GABAergic neurons led to increased GH secretion, lean mass, and body growth in male and female mice. VGAT-specific GHR knockout (KO) male mice also showed increased serum insulin-like growth factor-1, hypothalamic Ghrh, and hepatic Igf1 messenger RNA levels. In contrast, normal GH secretion, but reduced lean body mass, was observed in mice carrying GHR ablation in glutamatergic neurons. GHR ablation in GABAergic cells increased weight loss and led to decreased blood glucose levels during food restriction, whereas VGLUT2-specific GHR KO mice showed blunted feeding response to 2-deoxy-D-glucose both in males and females, and increased relative food intake, oxygen consumption, and serum leptin levels in male mice. Of note, VGLUT2-cre female mice, independently of GHR ablation, exhibited a previously unreported phenotype of mild reduction in body weight without further metabolic alterations. The autoregulation of GH secretion via negative-feedback loops requires GHR expression in GABAergic cells. Furthermore, GHR ablation in GABAergic and glutamatergic neuronal populations leads to distinct metabolic alterations. These findings contribute to the understanding of the neuronal populations responsible for mediating the neuroendocrine and metabolic effects of GH.

Characterization of the metabolic differences between male and female C57BL/6 mice.

AIMS: The present study aims to compare the responses between male and female C57BL/6 mice to multiple metabolic challenges to understand the importance of sex in the control of energy homeostasis. MAIN METHODS: Male and female C57BL/6 mice were subjected to nutritional and hormonal challenges, such as food restriction and refeeding, diet-induced obesity, feeding response to ghrelin and leptin, ghrelin-induced growth hormone secretion, and central responsiveness to ghrelin and leptin. The hypothalamic expression of transcripts that control energy homeostasis was also evaluated. KEY FINDINGS: Male mice lost more weight and lean body mass in response to food restriction, compared to females. During refeeding, males accumulated more body fat and exhibited lower energy expenditure and glycemia, as compared to females. Additionally, female mice exhibited a higher protection against diet-induced obesity and related metabolic imbalances in comparison to males. Low dose ghrelin injection elicited higher food intake and growth hormone secretion in male mice, whereas the acute anorexigenic effect of leptin was more robust in females. However, the sex differences in the feeding responses to ghrelin and leptin were not explained by variations in the central responsiveness to these hormones nor by differences in the fiber density from arcuate nucleus neurons. Female, but not male, mice exhibited compensatory increases in hypothalamic Pomc mRNA levels in response to diet-induced obesity. SIGNIFICANCE: Our findings revealed several sexually differentiated responses to metabolic challenges in C57BL/6 mice, highlighting the importance of taking into account sex differences in metabolic studies.

Vasoactive intestinal peptide exerts an excitatory effect on hypothalamic kisspeptin neurons during estrogen negative feedback.

Hypothalamic kisspeptin neurons are the primary modulators of gonadotropin-releasing hormone (GnRH) neurons. It has been shown that circadian rhythms driven by the suprachiasmatic nucleus (SCN) contribute to GnRH secretion. Kisspeptin neurons are potential targets of SCN neurons due to reciprocal connections with the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) and the arcuate nucleus of the hypothalamus (ARH). Vasoactive intestinal peptide (VIP), a notable SCN neurotransmitter, modulates GnRH secretion depending on serum estradiol levels, aging or time of the day. Considering that kisspeptin neurons may act as interneurons and mediate VIP's effects on the reproductive axis, we investigated the effects of VIP on hypothalamic kisspeptin neurons in female mice during estrogen negative feedback. Our findings indicate that VIP induces a TTX-independent depolarization of approximately 30% of AVPV/PeN kisspeptin neurons in gonad-intact (diestrus) and ovariectomized (OVX) mice. In the ARH, the percentage of kisspeptin neurons that were depolarized by VIP was even higher (approximately 90%). An intracerebroventricular infusion of VIP leds to an increased percentage of kisspeptin neurons expressing the phosphoSer133 cAMP-response-element-binding protein (pCREB) in the AVPV/PeN. On the other hand, pCREB expression in ARH kisspeptin neurons was similar between saline- and VIP-injected mice. Thus, VIP can recruit different signaling pathways to modulate AVPV/PeN or ARH kisspeptin neurons, resulting in distinct cellular responses. The expression of VIP receptors (VPACR) was upregulated in the AVPV/PeN, but not in the ARH, of OVX mice compared to mice on diestrus and estradiol-primed OVX mice. Our findings indicate that VIP directly influences distinct cellular aspects of the AVPV/PeN and ARH kisspeptin neurons during estrogen negative feedback, possibly to influence pulsatile LH secretion.

Growth hormone receptor contributes to the activation of STAT5 in the hypothalamus of pregnant mice.

Growth hormone (GH) receptor (GHR) signaling induces the phosphorylation of the signal transducer and activator of transcription 5 (pSTAT5) in the cells of several tissues including in the hypothalamus. During pregnancy, several STAT5-recruiting hormones (e.g., prolactin, GH and placental lactogens) are highly secreted. However, the precise contribution of GHR signaling to the surge of pSTAT5 immunoreactive neurons that occurs in the hypothalamus of pregnant mice is currently unknown. Thus, the objective of the present study was to determine whether GHR expression in neurons is required for inducing pSTAT5 expression in several hypothalamic nuclei during pregnancy. Initially, we demonstrated that late pregnant C57BL/6 mice (gestational day 14 to 18) exhibited increased pulsatile GH secretion compared to virgin females. Next, we confirmed that neuron-specific GHR ablation robustly reduces hypothalamic Ghr mRNA levels and prevents GH-induced pSTAT5 in the arcuate, paraventricular and ventromedial hypothalamic nuclei. Subsequently, the number of pSTAT5 immunoreactive cells was determined in the hypothalamus of late pregnant mice. Although neuron-specific GHR ablation did not affect the number of pSTAT5 immunoreactive cells in the paraventricular nucleus of the hypothalamus, reduced pSTAT5 expression was observed in the arcuate and ventromedial nuclei of pregnant neuron-specific GHR knockouts, compared to control pregnant mice. In summary, a subset of hypothalamic neurons requires GHR signaling to express pSTAT5 during pregnancy. These findings contribute to the understanding of the endocrine factors that affect the activation of transcription factors in the brain during pregnancy.

TLR4-interactor with leucine-rich repeats (TRIL) is involved in diet-induced hypothalamic inflammation.

Obesity and high-fat diet (HFD) consumption result in hypothalamic inflammation and metabolic dysfunction. While the TLR4 activation by dietary fats is a well-characterized pathway involved in the neuronal and glial inflammation, the role of its accessory proteins in diet-induced hypothalamic inflammation remains unknown. Here, we demonstrate that the knockdown of TLR4-interactor with leucine-rich repeats (Tril), a functional component of TLR4, resulted in reduced hypothalamic inflammation, increased whole-body energy expenditure, improved the systemic glucose tolerance and protection from diet-induced obesity. The POMC-specific knockdown of Tril resulted in decreased body fat, decreased white adipose tissue inflammation and a trend toward increased leptin signaling in POMC neurons. Thus, Tril was identified as a new component of the complex mechanisms that promote hypothalamic dysfunction in experimental obesity and its inhibition in the hypothalamus may represent a novel target for obesity treatment.

Leptin Receptor Expression in GABAergic Cells is Not Sufficient to Normalize Metabolism and Reproduction in Mice.

Previous studies indicate that leptin receptor (LepR) expression in GABAergic neurons is necessary for the biological effects of leptin. However, it is not clear whether LepR expression only in GABAergic neurons is sufficient to prevent the metabolic and neuroendocrine imbalances caused by LepR deficiency. In the present study, we produced mice that express the LepR exclusively in GABAergic cells (LepRVGAT mice) and compared them with wild-type (LepR+/+) and LepR-deficient (LepRNull/Null) mice. Although LepRVGAT mice showed a pronounced reduction in body weight and fat mass, as compared with LepRNull/Null mice, male and female LepRVGAT mice exhibited an obese phenotype relative to LepR+/+ mice. Food intake was normalized in LepRVGAT mice; however, LepRVGAT mice still exhibited lower energy expenditure in both sexes and reduced ambulatory activity in the females, compared with LepR+/+ mice. The acute anorexigenic effect of leptin and hedonic feeding were normalized in LepRVGAT mice despite the hyperleptinemia they present. Although LepRVGAT mice showed improved glucose homeostasis compared with LepRNull/Null mice, both male and female LepRVGAT mice exhibited insulin resistance. In contrast, LepR expression only in GABAergic cells was sufficient to normalize the density of agouti-related peptide (AgRP) and α-MSH immunoreactive fibers in the paraventricular nucleus of the hypothalamus. However, LepRVGAT mice exhibited reproductive dysfunctions, including subfertility in males and alterations in the estrous cycle of females. Taken together, our findings indicate that LepR expression in GABAergic cells, although critical to the physiology of leptin, is insufficient to normalize several metabolic aspects and the reproductive function in mice.

Rolling out physical exercise and energy homeostasis: Focus on hypothalamic circuitries.

Energy balance is the fine regulation of energy expenditure and energy intake. Negative energy balance causes body weight loss, while positive energy balance promotes weight gain. Modern societies offer a maladapted way of life, where easy access to palatable foods and the lack of opportunities to perform physical activity are considered the roots of the obesity pandemic. Physical exercise increases energy expenditure and, consequently, is supposed to promote weight loss. Paradoxically, physical exercise acutely drives anorexigenic-like effects, but the mechanisms are still poorly understood. Using an evolutionary background, this review aims to highlight the potential involvement of the melanocortin system and other hypothalamic neural circuitries regulating energy balance during and after physical exercise. The physiological significance of these changes will be explored, and possible signalling agents will be addressed. The knowledge discussed here might be important for clarifying obesity aetiology as well as new therapeutic approaches for body weight loss.

Characterization of the onset of leptin effects on the regulation of energy balance.

Leptin is a hormone required for the regulation of body weight in adult animals. However, during the postnatal period, leptin is mostly involved in developmental processes. Because the precise moment at which leptin starts to exert its metabolic effects is not well characterized, our objective was to identify the approximate onset of leptin effects on the regulation of energy balance. We observed that male Lepob/ob mice started to exhibit increased body fat mass from postnatal day 13 (P13), whereas in females, the increase in adiposity began on P20. Daily leptin injections from P10 to P22 did not reduce the weight gain of WT mice. However, an acute leptin injection induced an anorexigenic response in 10-day-old C57BL/6 mice but not in 7-day-old mice. An age-dependent increase in the number of leptin receptor-expressing neurons and leptin-induced pSTAT3 cells was observed in the hypothalamus of P7, P10 and P16 mice. Leptin deficiency started to modulate the hypothalamic expression of transcripts involved in the regulation of metabolism between P7 and P12. Additionally, fasting-induced hypothalamic responses were prevented by leptin replacement in 10-day-old mice. Finally, 12-day-old males and females showed similar developmental timing of axonal projections of arcuate nucleus neurons in both WT and Lepob/ob mice. In summary, we provided a detailed characterization of the onset of leptin's effects on the regulation of energy balance. These findings contribute to the understanding of leptin functions during development.

Deletion of growth hormone receptor in hypothalamic neurons affects the adaptation capacity to aerobic exercise.

The hypothalamus mediates important exercise-induced metabolic adaptations, possibly via hormonal signals. Hypothalamic leptin receptor (LepR)- and steroidogenic factor 1 (SF1)-expressing neurons are directly responsive to growth hormone (GH) and deletion of GH receptor (GHR) in these cells impairs neuroendocrine responses during situations of metabolic stress. In the present study, we determined whether GHR ablation in LepR- or SF1-expressing cells modifies acute and chronic metabolic adaptations to exercise. Male mice carrying deletion of GHR in LepR- or SF1-expressing cells were submitted to 8 weeks of treadmill running training. Changes in aerobic performance and exercise-induced metabolic adaptations were determined. Mice carrying GHR deletion in LepR cells showed increased aerobic performance after 8 weeks of treadmill training, whereas GHR ablation in SF1 cells prevented improvement in running capacity. Trained mice carrying GHR ablation in SF1 cells exhibited increased fat mass and reduced cross-sectional area of the gastrocnemius muscle. In contrast, deletion of GHR in LepR cells reduced fat mass and increased gastrocnemius muscle hypertrophy, energy expenditure and voluntary locomotor activity in trained mice. Although glucose tolerance was not significantly affected by targeted deletions, glycemia before and immediately after maximum running tests was altered by GHR ablation. In conclusion, GHR signaling in hypothalamic neurons regulates the adaptation capacity to aerobic exercise in a cell-specific manner. These findings suggest that GH may represent a hormonal cue that informs specific hypothalamic neurons to produce exercise-induced acute and chronic metabolic adaptations.

Distribution of growth hormone-responsive cells in the brain of rats and mice.

A growth hormone (GH) injection is able to induce the phosphorylated form of the signal transducer and activator of transcription 5 (pSTAT5) in a large number of cells throughout the mouse brain. The present study had the objective to map the distribution of GH-responsive cells in the brain of rats that received an intracerebroventricular injection of GH and compare it to the pattern found in mice. We observed that rats and mice exhibited a similar distribution of GH-induced pSTAT5 in the majority of areas of the telencephalon, hypothalamus and brainstem. However, rats exhibited a higher density of GH-responsive cells than mice in the horizontal limb of the diagonal band of Broca (HDB), supraoptic and suprachiasmatic nuclei, whereas mice displayed more GH-responsive cells than rats in the hippocampus, lateral hypothalamic area and dorsal motor nucleus of the vagus (DMX). Since both HDB and DMX contain acetylcholine-producing neurons, pSTAT5 was co-localized with choline acetyltransferase in GH-injected animals. We found that 50.0 ± 4.5% of cholinergic neurons in the rat HDB coexpressed GH-induced pSTAT5, whereas very few co-localizations were observed in the mouse HDB. In contrast, rats displayed fewer cholinergic neurons responsive to GH in the DMX at the level of the area postrema. In summary, pSTAT5 can be used as a marker of GH-responsive cells in the rat brain. Although rats and mice exhibit a relatively similar distribution of GH-responsive neurons, some species-specific differences exist, as exemplified for the responsiveness to GH in distinct populations of cholinergic neurons.

Tumor Necrosis Factor α and Interleukin-1ß Acutely Inhibit AgRP Neurons in the Arcuate Nucleus of the Hypothalamus.

Obesity-associated low-grade inflammation favors weight gain, whereas systemic infection frequently leads to anorexia. Thus, inflammatory signals can either induce positive or negative energy balance. In this study, we used whole-cell patch-clamp to investigate the acute effects of three important proinflammatory cytokines, tumor necrosis factor α (TNF-α), interleukin-6, and interleukin-1ß (IL-1ß) on the membrane excitability of agouti-related peptide (AgRP)- or proopiomelanocortin (POMC)-producing neurons. We found that both TNF-α and IL-1ß acutely inhibited the activity of 35-42% of AgRP-producing neurons, whereas very few POMC neurons were depolarized by TNF-α. Interleukin-6 induced no acute changes in the activity of AgRP or POMC neurons. Our findings indicate that the effect of TNF-α and IL-1ß, especially on the activity of AgRP-producing neurons, may contribute to inflammation-induced anorexia observed during acute inflammatory conditions.

Cholinergic neurons in the hypothalamus and dorsal motor nucleus of the vagus are directly responsive to growth hormone.

AIMS: Cholinergic neurons are distributed in brain areas containing growth hormone (GH)-responsive cells. We determined if cholinergic neurons are directly responsive to GH and the metabolic consequences of deleting the GH receptor (GHR) specifically in choline acetyltransferase (ChAT)-expressing cells. MAIN METHODS: Mice received an acute injection of GH to detect neurons co-expressing ChAT and phosphorylated STAT5 (pSTAT5), a well-established marker of GH-responsive cells. For the physiological studies, mice carrying ablation of GHR exclusively in ChAT-expressing cells were produced and possible changes in energy and glucose homeostasis were determined when consuming regular chow or high-fat diet (HFD). KEY FINDINGS: The majority of cholinergic neurons in the arcuate nucleus (60%) and dorsomedial nucleus (84%) of the hypothalamus are directly responsive to GH. Approximately 34% of pre-ganglionic parasympathetic neurons in the dorsal motor nucleus of the vagus also exhibited GH-induced pSTAT5. GH-induced pSTAT5 in these ChAT neurons was absent in GHR ChAT knockout mice. Mice carrying ChAT-specific GHR deletion, either in chow or HFD, did not exhibit significant changes in body weight, body adiposity, lean body mass, food intake, energy expenditure, respiratory quotient, ambulatory activity, serum leptin levels, glucose tolerance, insulin sensitivity and metabolic responses to 2-deoxy-d-glucose. However, GHR deletion in ChAT neurons caused decreased hypothalamic Pomc mRNA levels in HFD mice. SIGNIFICANCE: Cholinergic neurons that regulate the metabolism are directly responsive to GH, although GHR signaling in these cells is not required for energy and glucose homeostasis. Thus, the physiological importance of GH action on cholinergic neurons still needs to be identified.

Prolonged fasting induces long-lasting metabolic consequences in mice.

To endure prolonged fasting, animals undergo important acute physiological adjustments. However, whether severe fasting also leads to long-term metabolic adaptations is largely unknown. Forty-eight-hour fasting caused a pronounced weight loss in adult C57BL/6 male mice. Seven days of refeeding increased body adiposity to levels above baseline, whereas fasting-induced reductions in lean body mass and energy expenditure were not fully recovered. Respiratory exchange ratio and locomotor activity also remained altered. A fasting/refeeding cycle led to persistent suppression of Pomc mRNA levels and significant changes in the expression of histone deacetylases and DNA methyltransferases in the hypothalamus. Additionally, histone acetylation in the ventromedial nucleus of the hypothalamus was reduced by prolonged fasting and remained suppressed after refeeding. Mice subjected to 48-h fasting 30 days earlier exhibited higher body weight and fat mass compared to aged-matched animals that were never food-deprived. Furthermore, a previous fasting experience altered the changes in body weight, lean mass, energy expenditure and locomotor activity induced by a second cycle of fasting and refeeding. Notably, when acutely exposed to high-palatable/high-fat diet, mice that went through cumulative fasting episodes presented higher calorie intake and reduced energy expenditure and fat oxidation, compared to mice that had never been subjected to fasting. When chronically exposed to high-fat diet, mice that experienced cumulative fasting episodes showed higher gain of body and fat mass and reduced energy expenditure and calorie intake. In summary, cumulative episodes of prolonged fasting lead to hypothalamic epigenetic changes and long-lasting metabolic adaptations in mice.

Short-term exposure to air pollution (PM2.5) induces hypothalamic inflammation, and long-term leads to leptin resistance and obesity via Tlr4/Ikbke in mice.

A previous study demonstrated that a high-fat diet (HFD), administered for one-three-days, induces hypothalamic inflammation before obesity's established, and the long term affects leptin signaling/action due to inflammation. We investigate whether exposure to particulate matter of a diameter of ≤2.5 µm (PM2.5) in mice fed with a chow diet leads to similar metabolic effects caused by high-fat feeding. Compared to the filtered air group (FA), one-day-exposure-PM2.5 did not affect adiposity. However, five-days-exposure-PM2.5 increased hypothalamic microglia density, toll-like-receptor-4 (Tlr4), and the inhibitor-NF-kappa-B-kinase-epsilon (Ikbke) expression. Concurrently, fat mass, food intake (FI), and ucp1 expression in brown adipose tissue were also increased. Besides, decreased hypothalamic STAT3-phosphorylation and Pomc expression were found after twelve-weeks-exposure-PM2.5. These were accompanied by increased FI and lower energy expenditure (EE), leading to obesity, along with increased leptin and insulin levels and HOMA. Mechanistically, the deletion of Tlr4 or knockdown of the Ikbke gene in the hypothalamus was sufficient to reverse the metabolic outcomes of twelve-weeks-exposure-PM2.5. These data demonstrated that short-term exposure-PM2.5 increases hypothalamic inflammation, similar to a HFD. Long-term exposure-PM2.5 is even worse, leading to leptin resistance, hyperphagia, and decreased EE. These effects are most likely due to chronic hypothalamic inflammation, which is regulated by Tlr4 and Ikbke signaling.

Tyrosine Hydroxylase Neurons Regulate Growth Hormone Secretion via Short-Loop Negative Feedback.

Classical studies suggest that growth hormone (GH) secretion is controlled by negative-feedback loops mediated by GH-releasing hormone (GHRH)- or somatostatin-expressing neurons. Catecholamines are known to alter GH secretion and neurons expressing TH are located in several brain areas containing GH-responsive cells. However, whether TH-expressing neurons are required to regulate GH secretion via negative-feedback mechanisms is unknown. In the present study, we showed that between 50% and 90% of TH-expressing neurons in the periventricular, paraventricular, and arcuate hypothalamic nuclei and locus ceruleus of mice exhibited STAT5 phosphorylation (pSTAT5) after an acute GH injection. Ablation of GH receptor (GHR) from TH cells or in the entire brain markedly increased GH pulse secretion and body growth in both male and female mice. In contrast, GHR ablation in cells that express the dopamine transporter (DAT) or dopamine ß-hydroxylase (DBH; marker of noradrenergic/adrenergic cells) did not affect body growth. Nevertheless, less than 50% of TH-expressing neurons in the hypothalamus were found to express DAT. Ablation of GHR in TH cells increased the hypothalamic expression of Ghrh mRNA, although very few GHRH neurons were found to coexpress TH- and GH-induced pSTAT5. In summary, TH neurons that do not express DAT or DBH are required for the autoregulation of GH secretion via a negative-feedback loop. Our findings revealed a critical and previously unidentified group of catecholaminergic interneurons that are apt to sense changes in GH levels and regulate the somatotropic axis in mice.SIGNIFICANCE STATEMENT Textbooks indicate until now that the pulsatile pattern of growth hormone (GH) secretion is primarily controlled by GH-releasing hormone and somatostatin neurons. The regulation of GH secretion relies on the ability of these cells to sense changes in circulating GH levels to adjust pituitary GH secretion within a narrow physiological range. However, our study identifies a specific population of tyrosine hydroxylase-expressing neurons that is critical to autoregulate GH secretion via a negative-feedback loop. The lack of this mechanism in transgenic mice results in aberrant GH secretion and body growth. Since GH plays a key role in cell proliferation, body growth, and metabolism, our findings provide a major advance to understand how the brain regulates the somatotropic axis.

Growth Hormone Receptor Deletion Reduces the Density of Axonal Projections from Hypothalamic Arcuate Nucleus Neurons.

The arcuate nucleus (ARH) is an important hypothalamic area for the homeostatic control of feeding and other metabolic functions. In the ARH, proopiomelanocortin- (POMC) and agouti-related peptide (AgRP)-expressing neurons play a key role in the central regulation of metabolism. These neurons are influenced by circulating factors, such as leptin and growth hormone (GH). The objective of the present study was to determine whether a direct action of GH on ARH neurons regulates the density of POMC and AgRP axonal projections to major postsynaptic targets. We studied POMC and AgRP axonal projections to the hypothalamic paraventricular (PVH), lateral (LHA) and dorsomedial (DMH) nuclei in leptin receptor (LepR)-deficient mice (Leprdb/db), GH-deficient mice (Ghrhrlit/lit) and in mice carrying specific ablations of GH receptor (GHR) either in LepR- or AgRP-expressing cells. Leprdb/db mice presented reduction in the density of POMC innervation to the PVH compared to wild-type and Ghrhrlit/lit mice. Additionally, both Leprdb/db and Ghrhrlit/lit mice showed reduced AgRP fiber density in the PVH, LHA and DMH. LepR GHR knockout mice showed decreased density of POMC innervation in the PVH and DMH, compared to control mice, whereas a reduction in the density of AgRP innervation was observed in all areas analyzed. Conversely, AgRP-specific ablation of GHR led to a significant reduction in AgRP projections to the PVH, LHA and DMH, without affecting POMC innervation. Our findings indicate that GH has direct trophic effects on the formation of POMC and AgRP axonal projections and provide additional evidence that GH regulates hypothalamic neurocircuits controlling energy homeostasis.

STAT5 ablation in AgRP neurons increases female adiposity and blunts food restriction adaptations.

AgRP neurons are important players in the control of energy homeostasis and are responsive to several hormones. In addition, STAT5 signalling in the brain, which is activated by metabolic hormones and growth factors, modulates food intake, body fat and glucose homeostasis. Given that, and the absence of studies that describe STAT5 function in AgRP cells, the present study investigated the metabolic effects of Stat5a/b gene ablation in these neurons. We observed that STAT5 signalling in AgRP neurons regulates body fat in female mice. However, male and female STAT5-knockout mice did not exhibit altered food intake, energy expenditure or glucose homeostasis compared to control mice. The counter-regulatory response or glucoprivic hyperphagia induced by 2-deoxy-d-glucose treatment were also not affected by AgRP-specific STAT5 ablation. However, under 60% food restriction, AgRP STAT5-knockout mice had a blunted upregulation of hypothalamic Agrp mRNA expression and corticosterone serum levels compared to control mice, suggesting a possible role for STAT5 in AgRP neurons for neuroendocrine adaptations to food restriction. Interestingly, ad libitum fed knockout male mice had reduced Pomc and Ucp-1 mRNA expression compared to control group. Taken together, these results suggest that STAT5 signalling in AgRP neurons regulates body adiposity in female mice, as well as some neuroendocrine adaptations to food restriction.