**4. Hypothalamic cell models, GT1-7 and rHypoE8, for investigating neuroendocrine mechanisms of the HPG axis**

The hypothalamus is the control center for the HPG axis; however, it has been difficult to study in detail the GnRH neurons as well as the kisspeptin neurons because of the inherent heterogeneity of this brain region. The hypothalamus is composed of a complex network of neurons, and there are different neuronal phenotypes that express a specific complement of neuropeptides, neurotransmitters, and receptors [20]. Immortalized, clonal cell lines represent a relatively homogeneous population of specific neuronal cells and can be used as

experimental models. For the study of the character or the functions of hypothalamic GnRH neurons, we are using GT1-7 cell lines, which have proven to be a valuable GnRH-expressing cell model for GnRH neurons. These cells were created from the hypothalamic tumor cells in a transgenic female mouse that expressed the SV40 T-antigen under the control of the GnRH promoter [21]. GT1-7 cells display neuronal morphology and secrete GnRH; therefore, these cells have become one of the most highly utilized neuronal cell models for studies related to GnRH neurons.

The embryonic rat hypothalamic cell line R8 (rHypoE8) consists of hypothalamic neurons from rat embryonic day 18 hypothalamic primary cultures immortalized by retroviral transfer of SV40 T-antigen. These cells express neuroendocrine markers such as kisspeptin, GnRH, and RF-amide-related peptide-3 (RFRP-3, the mammalian ortholog of the avian gonadotropin-inhibiting hormone, GnIH). Because the expression of kisspeptin or RFRP-3 is functionally altered by physiological neuropeptides, these cells serve as tools for the analysis of the cellular and molecular mechanisms involved in the hypothalamic control of a number of physiological processes [22].

## **5. Effect of kisspeptin on hypothalamic GT1-7 cells**

It is generally agreed that hypothalamic kisspeptin regulates GnRH release from GnRH neurons by kisspeptin binding the Kiss1R that is expressed by GnRH neurons [23]. A previous study by Novaira et al. demonstrated a functional role for kisspeptin in GT1-7 cells, in which they showed that kisspeptin stimulates the expression and secretion of GnRH [24]. Similarly, Terasaka et al. demonstrated the stimulatory effect of kisspeptin on GnRH gene expression, and they also found that this stimulatory effect was antagonized in the presence of bone morphogenetic protein in these cells [25]. In our study using GT1-7 cells, we did not observe any effect of kisspeptin on GnRH expression [26]. Because GT1-7 cells express Kiss1R, we suspected that Kiss1R function was lost or diminished in our GT1-7 cells, probably because of a change in cell character due to cell immortalization or multiple passages. On the other hand, when GT1-7 cells overexpressed Kiss1R after transfection with a Kiss1R expression vector, exogenous Kiss1R was absolutely functional. Furthermore, both extracellular signal-regulated kinase (ERK) and cAMP/protein kinase A (PKA) pathways were activated by kisspeptin in Kiss1R-overexpressing GT1-7 cells. These observations suggested that overexpression of exogenous Kiss1R could lead to activation of the intracellular signaling pathways mediated by kisspeptin stimulation in these cells. It is also noteworthy that, even when GT1-7 cells overexpressed Kiss1R, GnRH expression was not stimulated by kisspeptin [26]. It is still unclear why kisspeptin did not increase GnRH expression in our GT1-7 cells even when Kiss1R was overexpressed; instead, we clearly observed that kisspeptin could stimulate the expression of the GnRH receptor (GnRHR) in GT1-7 cells overexpressing Kiss1R [26] (**Figure 1**). GnRH-producing cells have been reported to respond to GnRH and modify their GnRH expression levels [27]. Furthermore, GnRHRs within GnRH neurons were reported to be involved in the pulsatile secretion of GnRH by an autocrine or paracrine interaction between GnRH and GnRHR [28, 29]. These

observations implied that kisspeptin could affect the function of GnRH neurons by chang-

**Figure 1.** Schematic summary of the regulation of GnRH in GT1-7 cells. GT1-7 cells express Kiss1R, but endogenous Kiss1R does not respond to kisspeptin. Therefore, we used GT1-7 cells overexpressing Kiss1R. GT1-7 cells overexpressing Kiss1R did not show an increase in GnRH mRNA expression upon kisspeptin treatment. However, kisspeptin increased GnRH receptor expression in these cells. We also found that a γ-subunit-containing GABAA receptor agonist, DS1, as well as histone deacetylase inhibitor trichostatin A, reduces GnRH mRNA expression. Retinoic acid also has an inhibitory

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Although GT1-7 cells endogenously express Kiss1R, kisspeptin does not affect these cells. When GT1-7 cells overexpress Kiss1R, kisspeptin stimulates intracellular signaling pathways and increases GnRHR, but not GnRH expression. To determine the character of kisspeptin neurons in their original, non-transformed state, we used primary cultures of fetal rat brain that contain both GnRH and kisspeptin neurons. GnRH neurons in these cells did not respond to E2, which failed to stimulate GnRH mRNA expression. This observation was consistent with a previous study that revealed a lack of estrogen receptor immunoreactivity in GnRH neurons, raising doubts about the role of E2 in GnRH neuronal function [30]. In contrast, kisspeptin neurons in these primary cultures responded to E2, and Kiss1 mRNA expression was upregulated by E2 [31], suggesting that kisspeptin neurons, but not GnRH neurons, could be a target of E2 in neuronal cells in the fetal brain. GnRH mRNA expression within these primary cultures of fetal rat brain containing GnRH-producing neurons was

**6. Effect of kisspeptin on primary cultures of fetal rat brain**

ing their expression levels of GnRHR.

effect on GnRH expression.

Studies on the Character of Hypothalamic GnRH Neurons and Kisspeptin Neurons Using… http://dx.doi.org/10.5772/intechopen.73128 21

experimental models. For the study of the character or the functions of hypothalamic GnRH neurons, we are using GT1-7 cell lines, which have proven to be a valuable GnRH-expressing cell model for GnRH neurons. These cells were created from the hypothalamic tumor cells in a transgenic female mouse that expressed the SV40 T-antigen under the control of the GnRH promoter [21]. GT1-7 cells display neuronal morphology and secrete GnRH; therefore, these cells have become one of the most highly utilized neuronal cell models for studies related to

The embryonic rat hypothalamic cell line R8 (rHypoE8) consists of hypothalamic neurons from rat embryonic day 18 hypothalamic primary cultures immortalized by retroviral transfer of SV40 T-antigen. These cells express neuroendocrine markers such as kisspeptin, GnRH, and RF-amide-related peptide-3 (RFRP-3, the mammalian ortholog of the avian gonadotropin-inhibiting hormone, GnIH). Because the expression of kisspeptin or RFRP-3 is functionally altered by physiological neuropeptides, these cells serve as tools for the analysis of the cellular and molecular mechanisms involved in the hypothalamic control of a number

It is generally agreed that hypothalamic kisspeptin regulates GnRH release from GnRH neurons by kisspeptin binding the Kiss1R that is expressed by GnRH neurons [23]. A previous study by Novaira et al. demonstrated a functional role for kisspeptin in GT1-7 cells, in which they showed that kisspeptin stimulates the expression and secretion of GnRH [24]. Similarly, Terasaka et al. demonstrated the stimulatory effect of kisspeptin on GnRH gene expression, and they also found that this stimulatory effect was antagonized in the presence of bone morphogenetic protein in these cells [25]. In our study using GT1-7 cells, we did not observe any effect of kisspeptin on GnRH expression [26]. Because GT1-7 cells express Kiss1R, we suspected that Kiss1R function was lost or diminished in our GT1-7 cells, probably because of a change in cell character due to cell immortalization or multiple passages. On the other hand, when GT1-7 cells overexpressed Kiss1R after transfection with a Kiss1R expression vector, exogenous Kiss1R was absolutely functional. Furthermore, both extracellular signal-regulated kinase (ERK) and cAMP/protein kinase A (PKA) pathways were activated by kisspeptin in Kiss1R-overexpressing GT1-7 cells. These observations suggested that overexpression of exogenous Kiss1R could lead to activation of the intracellular signaling pathways mediated by kisspeptin stimulation in these cells. It is also noteworthy that, even when GT1-7 cells overexpressed Kiss1R, GnRH expression was not stimulated by kisspeptin [26]. It is still unclear why kisspeptin did not increase GnRH expression in our GT1-7 cells even when Kiss1R was overexpressed; instead, we clearly observed that kisspeptin could stimulate the expression of the GnRH receptor (GnRHR) in GT1-7 cells overexpressing Kiss1R [26] (**Figure 1**). GnRH-producing cells have been reported to respond to GnRH and modify their GnRH expression levels [27]. Furthermore, GnRHRs within GnRH neurons were reported to be involved in the pulsatile secretion of GnRH by an autocrine or paracrine interaction between GnRH and GnRHR [28, 29]. These

GnRH neurons.

20 Hypothalamus in Health and Diseases

of physiological processes [22].

**5. Effect of kisspeptin on hypothalamic GT1-7 cells**

**Figure 1.** Schematic summary of the regulation of GnRH in GT1-7 cells. GT1-7 cells express Kiss1R, but endogenous Kiss1R does not respond to kisspeptin. Therefore, we used GT1-7 cells overexpressing Kiss1R. GT1-7 cells overexpressing Kiss1R did not show an increase in GnRH mRNA expression upon kisspeptin treatment. However, kisspeptin increased GnRH receptor expression in these cells. We also found that a γ-subunit-containing GABAA receptor agonist, DS1, as well as histone deacetylase inhibitor trichostatin A, reduces GnRH mRNA expression. Retinoic acid also has an inhibitory effect on GnRH expression.

observations implied that kisspeptin could affect the function of GnRH neurons by changing their expression levels of GnRHR.

## **6. Effect of kisspeptin on primary cultures of fetal rat brain**

Although GT1-7 cells endogenously express Kiss1R, kisspeptin does not affect these cells. When GT1-7 cells overexpress Kiss1R, kisspeptin stimulates intracellular signaling pathways and increases GnRHR, but not GnRH expression. To determine the character of kisspeptin neurons in their original, non-transformed state, we used primary cultures of fetal rat brain that contain both GnRH and kisspeptin neurons. GnRH neurons in these cells did not respond to E2, which failed to stimulate GnRH mRNA expression. This observation was consistent with a previous study that revealed a lack of estrogen receptor immunoreactivity in GnRH neurons, raising doubts about the role of E2 in GnRH neuronal function [30]. In contrast, kisspeptin neurons in these primary cultures responded to E2, and Kiss1 mRNA expression was upregulated by E2 [31], suggesting that kisspeptin neurons, but not GnRH neurons, could be a target of E2 in neuronal cells in the fetal brain. GnRH mRNA expression within these primary cultures of fetal rat brain containing GnRH-producing neurons was

**8. DS1, a δ-subunit-containing GABAA receptor agonist, reduces GnRH mRNA expression and increases that of GnRHR in GT1-7 cells**

with the expression of their GnRHR within the cell (**Figure 1**).

**rHypoE8 cells and GT1-7 cells**

by changing kisspeptin expression.

**9. Kisspeptin expression is induced by glucagon-like peptide-1 in** 

As described above, we used GT1-7 cells as a model for GnRH-producing neurons; however, GT1-7 cells also express the Kiss1 gene, which encodes kisspeptin [40]. rHypoE8 cells, another hypothalamic model that was developed from rat embryonic hypothalamic primary cultures, express the Kiss1 gene, and they also express the GnRH gene [22]. Because both rHypoE8 and GT1-7 are immortalized cell lines derived from heterogeneous hypothalamic cell populations, they express several types of neuropeptides. Using these hypothalamic cell models, we found that Kiss1 mRNA was regulated by several metabolic factors. Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone produced by the small intestine and colon in response to food intake [41]. GLP-1 is also expressed in the central nervous system, and its expression in the brain is altered during fasting or feeding [42], suggesting that GLP-1 plays a role as a satiety factor. We found that GLP-1 increased the expression of Kiss1 mRNA in rHypoE8 cells as well as GT1-7 cells [43]. Moreover, leptin, which is an anorexigenic factor that is released from adipocytes, can also stimulate Kiss1 mRNA expression in these cells (**Figure 3**). These observations suggest that the levels of metabolic factors such as GLP-1 or leptin, which change during a state of starvation or negative energy balance, can critically influence the HPG axis

It is well documented that the neurotransmitter γ-aminobutyric acid (GABA) can modulate the activity of GnRH neurons. GnRH neurons possess functional GABAA receptors [34], and GABAergic neurons establish synapses with GnRH neurons [35]. GABA neurons predominantly exert their inhibitory effect on GnRH neurons in rodents and sheep. GT1-7 cells also express functional GABAA receptors [36]. GABAA receptors are multimeric proteins that are composed of five subunits drawn from a repertoire of several homologous protein groups (α1-6, β1-3, γ1-3, δ, ε, θ, and π); the majority of GABAA receptors in the central nervous system are composed of α, β, and γ subunits, and less abundant populations of GABA<sup>A</sup> receptor contain the δ subunit [37]. DS1, an α4β3δ GABAA receptor agonist, reduces GnRH mRNA expression in GT1-7 cells, although DS1 can exert a stimulatory effect on signal transduction systems, such as ERK and cAMP/PKA [38]. The δ-subunit-containing α4β3δ GABA<sup>A</sup> receptor was found in extra-synaptic sites and is known to control neuronal excitability [39]. Interestingly, although GnRH mRNA expression was decreased, GnRHR expression within GT1-7 cells was significantly increased by DS1 stimulation [38] (**Figure 1**). At present, it is still unknown why δ-subunit-containing GABAA receptor agonism decreases the production of GnRH in spite of increasing GnRHR expression. We currently speculate that GABA could modulate GnRH-producing neurons through δ-containing GABAA receptors and deplete their GnRH content by modulating gene expression and secretory function in association

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**Figure 2.** Schematic summary of the regulation of Kiss1 mRNA and GnRH mRNA expression in primary cultures of fetal rat brain and the proposed interaction between kisspeptin neurons and GnRH neurons. In experiments using primary cultures of fetal rat neuronal cells, Kiss1 mRNA, but not GnRH mRNA expression, was upregulated by estradiol (E2). GnRH mRNA expression was clearly increased by treatment with kisspeptin. GnRH stimulation increased the expression of both Kiss1 and GnRH mRNAs, and kisspeptin itself was found to increase the expression of the Kiss1 gene. We postulate that GnRH neurons reversibly interact with kisspeptin neurons and also form an autocrine interaction with kisspeptin neurons.

clearly increased by treatment with kisspeptin. Therefore, we could conclude that kisspeptin can stimulate GnRH synthesis in GnRH-expressing neurons in vivo. However, kisspeptin increased GnRH mRNA expression only up to about 1.5-fold [31]. In addition, we have found that GnRH stimulation increased the expression of the Kiss1 gene as well as that of the GnRH gene and also found that kisspeptin itself increased the expression of the Kiss1 gene. We postulate that GnRH neurons reversibly interact with kisspeptin neurons and also form an autocrine interaction with kisspeptin neurons (**Figure 2**).
