GABA Enhances Growth Hormone Expression by Modulating Somatotroph Pit-1 Transcription via Activation of Calmodulin-Dependent Kinases

TL;DR

GABA boosts growth hormone production in mice by activating a specific signaling pathway in pituitary cells.

Contribution

The study identifies a novel mechanism by which GABA modulates growth hormone expression through CaMKK2/CaMKIV signaling.

Findings

GABA increases Pit-1 and GH expression in mice and cultured cells.

GABA's effects are mediated through GABAA receptors and the CaMKK2/CaMKIV pathway.

Inhibiting GABAA receptors or CaMKK2 reduces GABA's stimulatory effects on GH.

Abstract

Background: Gamma-aminobutyric acid (GABA), the principal inhibitory neurotransmitter in the central nervous system (CNS), is also a potent modulator of peripheral endocrine function. We previously demonstrated that dietary GABA supplementation improves growth and fatty acid metabolism in male mice while elevating pituitary growth hormone (GH). However, the mechanisms by which GABA regulates the somatotropic axis remain unclear. Methods: Adolescent mice (3–4 weeks old) were treated with or without GABA in drinking water. Cultured pituitaries and GH3 somatotroph-derived cells were exposed to GABA, Picrotoxin, or STO-609, and protein expression was analyzed by Western blot. Results: GABA treatment increased Pit-1 (POU1F1) protein levels among males in vivo (ctrl: 0.55 ± 0.11; GABA: 1.46 ± 0.16; p = 0.0034) and ex vivo (ctrl: 0.66 ± 0.03; GABA: 1.46 ± 0.14; p = 0.0013), as well as in GH3 cells (ctrl: 1.36 ± 0.12; GABA: 3.05 ± 0.12; p < 0.0001). GH expression was also increased by GABA treatment in ex vivo pituitaries (ctrl: 1.62 ± 0.06; GABA: 1.84 ± 0.01; p = 0.0115) and GH3 cells (ctrl: 0.34 ± 0.08; GABA: 1.35 ± 0.13; p = 0.0006). Mechanistically, GABA, via the GABAA receptor (GABAAR), enhanced CaMKK2 pathway activity, as evidenced by increased phosphorylation of CaMKIV (ctrl: 0.86 ± 0.07; GABA: 1.12 ± 0.07; p = 0.0378) and AKT (ctrl: 0.89 ± 0.08; GABA: 1.75 ± 0.23; p = 0.0122). Inhibition of GABAARs by picrotoxin (PTX) markedly reduced Pit-1 (GABA: 2.73 ± 0.29; GABA + PTX: 1.76 ± 0.21; p = 0.0351) and GH expression (GABA: 0.17 ± 0.02; GABA + PTX: 0.05 ± 0.02; p = 0.0052). Treatment with CaMKK2 inhibitor STO-609 reduced basal Pit-1 (ctrl: 1.76 ± 0.09; STO-609: 1.25 ± 0.12; p = 0.0157) and GH levels (ctrl: 1.18 ± 0.10; STO-609: 0.50 ± 0.04; p = 0.0006). Ghrelin receptor activation by anamorelin (ANA) increased Pit-1 (ctrl: 0.83 ± 0.8; ANA: 1.59 ± 0.28; p = 0.0425) and GH (ctrl: 0.27 ± 0.03; ANA: 0.66 ± 0.16; p = 0.0497) through a CaMKK2-independent pathway but required basal GABAAR activity for maximal effect. Conclusions: These findings identify GABA as a modulator of somatotroph hormone expression through a CaMKK2/CaMKIV-dependent cascade and reveal a previously unrecognized interplay whereby the basal GABAergic tone promotes Pit-1 expression, thereby positively regulating ghrelin receptor signaling. This study provides new insights on the cellular mechanisms behind GABA-induced GH synthesis, which may reveal new strategies for modulating the somatotropic axis and help contextualize the variety of reported physiological and cognitive effects of GABA supplementation.

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1. Introduction

Gamma-aminobutyric acid (GABA) is widely recognized as the primary inhibitory neurotransmitter in the central nervous system (CNS). However, accumulating evidence indicates that GABA also exerts important physiological actions in peripheral tissues and may influence systemic endocrine functions. In the last decade, numerous studies have investigated GABA supplementation and found diverse physiological effects such as regulation of body weight, adipose tissue, pancreatic beta cell mass, and hormone secretion [1,2,3,4,5,6,7]. In addition, dietary GABA supplementation has gained popularity due to reported benefits on stress reduction, sleep quality, cognition, and body composition [8,9,10,11,12,13]. Despite the growing research on the effects of dietary GABA, the cellular mechanisms through which orally administered GABA modulates hormonal pathways are not well understood. The present study focuses on the somatotropic axis, as several reports show that GABA treatment can stimulate GH-producing cells and promote GH secretion [7,14,15,16].

We recently demonstrated that long-term oral GABA supplementation during adolescence elicits robust, sex-dependent effects on growth, metabolism, and hormone secretion. In males, GABA supplementation increased food intake, enhanced linear growth, reduced adiposity, and elevated locomotor activity. These physiological changes were accompanied by significant increases in gastric ghrelin expression, pituitary GH content, and circulating GH concentrations [17]. Ghrelin, a potent GH secretagogue produced primarily in the stomach, plays a central role in regulating appetite, energy balance, and GH release [18,19,20,21]. In contrast, female mice exhibited increased pituitary GH protein without corresponding increases in circulating GH or ghrelin, indicating a sexually dimorphic regulation [17]. These findings raise the possibility that GABA engages distinct pathways—potentially involving gastric ghrelin production, hypothalamic regulation, or pituitary responsiveness—differentially modulating GH synthesis and secretion across sexes. The coordinated elevation of ghrelin and GH in GABA-treated male mice further suggests that GABA may act upstream of the ghrelin–GH axis to promote growth and fatty acid metabolism.

Despite these observations, the cellular and molecular mechanisms through which GABA supplementation enhances GH expression remain unclear. It is important to clarify whether GABA acts directly on GH-producing cells, modulates neural or hormonal inputs to the pituitary, influences peripheral metabolic signals that secondarily regulate GH output, or a combination of these. Elucidating how GABA regulates pituitary GH production not only deepens our understanding of how nutrient-derived amino acids can regulate endocrine function but may also inform the development of therapeutic strategies for modifying the somatotropic axis.

By integrating molecular and endocrine analyses, this study aimed to define how supplemented and/or endogenous GABA signaling engages the ghrelin–GH axis and to uncover the mechanisms through which increasing GABA enhances GH expression, with a focus on upstream regulators and intracellular signaling pathways that mediate this response. It was hypothesized that GABA treatment would upregulate the somatotropic expression of Pit-1, a key transcription factor involved in GH production [22].

2. Materials and Methods

2.1. Experimental Design

Experiments were designed to realize three specific aims. The first aim was to examine whether long-term oral GABA supplementation increases pituitary Pit-1 expression in healthy mice. The second was to assess whether GABA treatment enhances ex vivo pituitary Pit-1 and GH expression in the absence of systemic modulators such as ghrelin or somatostatin. The third was to determine if GABA directly upregulates Pit-1 and GH expression in the somatotropic GH3 cell line independent of inputs from other pituitary cell types and, if so, to elucidate the underlying mechanisms. All experimental procedures were performed in accordance with the Animal Use Protocol #2022-125, which was approved by Animal Care and Veterinary Services at the University of Western Ontario.

2.2. Animals, Oral GABA Treatment, and Pituitary Collection

As described in our previous study [17], 24 recently weaned (3–4 weeks old) C57BL/6J mice (12 controls, 12 treated) were housed in a regulated environment at an ambient temperature of 22 ± 2 °C, humidity of 55 ± 5% and a 12 h light/dark cycle. The study aimed to include all apparently healthy mice within the stated age range and exclude any mice that exhibited signs of illness or did not fall within the age range. No mice were required to be excluded from the study. Mice were randomly allocated into treatment or control groups using a number generator, and those assigned to the treatment group received 2 mg/mL of GABA in their drinking water, which was provided ad libitum, while the control mice received plain drinking water. Animals were monitored daily for overall health, and water was replaced weekly over the 16-week treatment period to maintain GABA stability.

Animals were housed 4 mice per cage with standard environmental enrichment and were maintained on a low-fat Teklad 2018 diet formulated to minimize phytoestrogen exposure from soybean meal and alfalfa [23,24]. At the end of the treatment period, animals were euthanized under deep isoflurane anesthesia, and the pituitary glands of all test mice were dissected under a stereomicroscope using a previously reported protocol with minor modifications [25]. Tissues were immediately frozen in dry ice and stored at −80 °C for future assays. Given the exploratory nature of this study, Western blot experiments were performed using n = 4 independent biological replicates per group, which allows for detection of robust and repeatable treatment-related effects while minimizing animal use in accordance with the “reduction” principle of animal ethics. For in vivo studies, a biological replicate refers to a single whole pituitary extracted from an individual mouse.

2.3. GABA Treatment of Ex Vivo Pituitary Explants

Pituitary glands from 8 male C57BL/6J mice (8 weeks old) were dissected under aseptic conditions and immediately placed individually in 100 mm dishes containing pre-warmed modified Ham’s F-12K culture media containing 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cultures were maintained at 37 °C in a humidified incubator with 5% CO_2_. Four of the dishes were pre-treated with 10 µM GABA in the media. Culture media with and without GABA treatment were changed at 24 and 48 h. At 72 h, pituitaries were snap frozen on dry ice and stored at −80 °C for later processing. Each biological replicate in the ex vivo study refers to a whole cultured pituitary extracted from one individual mouse.

2.4. Cell Culture, Treatments, and ELISA

GH3 and GH1 cells are established rat pituitary-derived somatotroph cell lines that synthesize and secrete GH and respond to regulatory signals such as somatostatin and ghrelin [26,27]. These cell lines are widely used as in vitro models for studying pituitary hormone production, transcriptional regulation, and intracellular signaling pathways [28,29,30].

GH1 cells (ATCC CCL-82) were obtained from ATCC (Manassas, VA, USA) and thawed according to the supplier’s instructions. Cells were plated in 100 mm culture dishes containing Ham’s F-12K with 2.5% FBS and 15% horse serum (HS) at 37 °C and 5% CO_2_. Adherent cells were passaged at 70–80% confluence using 0.25% trypsin–EDTA. Passages 10–15 were used for experiments. Experimental groups were treated with GABA (10 µM), Muscimol (10 µM), or GABA (10 µM) with the GABA_A_R antagonist Picrotoxin (10 µM) in serum-free media for 72 h. Cell culture media was collected, samples were diluted 1:40 or 1:80 and growth hormone (GH) concentration was assessed with the GH ELISA Kit (Invitrogen, Waltham, MA, USA, #KRC5311) following the manufacturer’s protocol using a BioTek 800 TS (Winooski, VT, USA) absorbance reader at the 450 nm wavelength.

To study the cellular mechanisms behind GABA-induced GH expression, GH3 cells (ATCC CCL-82.1) were used due to their widely reported expression of functional GABA receptors and their excitable response to GABA [28,31,32]. Cells from passages 10–15 were thawed and plated in 100 mm culture dishes containing Ham’s F-12K with 2.5% FBS and 15% HS at 37 °C and 5% CO_2_. Treatment groups received GABA (10 µM), Picrotoxin (10 µM), GABA with Picrotoxin, Anamorelin (ANA) (1 µM), Anamorelin with Picrotoxin, or STO-609 (5 µM), a Ca^2+^/calmodulin-dependent protein kinase kinase 2 (CaMKK2) inhibitor. Since GABA_A_Rs in anterior pituitary cells are activated in the low micromolar range, the 10 micromolar GABA dose range has been commonly used in pituitary culture models to elicit receptor-mediated signaling responses [33]. Dosages for Picrotoxin and STO-609 were selected based on previous research showing these concentrations produce reliable effects in blocking GABA_A_R-mediated calcium influx and CaMKK2 activity, respectively [34,35].

The medium was refreshed at 24 h and collected at 48 h. To examine acute phosphorylation responses, some dishes were given stimulation with treatment (or vehicle control) 10 min prior to sample collection. After treatment, cells were washed in PBS and mechanically disassociated from the culture dishes prior to homogenization with lysis buffer and further processing. For cell culture experiments, a biological replicate is defined as an individual 100 mm GH3/GH1 culture dish treated and harvested independently.

2.5. Western Blot

Pituitary glands and cultured cells were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer with 0.1% apoprotein, leupeptin and Halt Phosphatase Inhibitor (ThermoFisher, Waltham, MA, USA, #78440). Homogenized samples were incubated in lysis buffer for 30 min then centrifuged at 13,000 rpm for 30 min (pituitaries) or for 5 min (cultured cells) at 4 °C. Total protein content was assessed via Bradford assay (Bio-Rad, Herculer, CA, USA, #5000006). For gel electrophoresis, samples were prepared using 5x sample buffer and loaded to a 10% or 15% SDS-PAGE gel for 2 h at 100 V. Proteins were transferred to a polyvinylidene difluoride membrane for 2 h at 80 V. After transfer, membranes were blocked with 5% bovine serum albumin for 1.5 h before incubation with goat anti-GH (R&D Systems, Minneapolis, MN, USA, #AF1067), mouse anti-GAPDH (Abcam, Cambridge, UK, #ab9482), rabbit anti-Pit-1 (Abcam, #ab273048), rabbit anti-pAKT (Thr308) (Cell Signaling Technology, Danvers, MA, USA, #9275), rabbit anti-CaMKK2 (ThermoFisher, #11549-1-AP) or rabbit anti-pCaMKIV (ThermoFisher, #PA5-37504) antibody overnight at 4 °C using the manufacturer’s recommendations for antibody dilution. The membranes were washed 3 times in Tris Buffered Saline with Tween 20 (TBS-T) and incubated for 1.5 h in complementary horseradish peroxidase-conjugated secondary antibodies at room temperature or overnight at 4 °C. The membranes were incubated for 1–3 min in chemiluminescence substrate (ThermoFisher, #32106) and imaged using the Versa Dock 5000 MP system with Quantity One imaging software V4.6.9 by Bio-Rad. Densitometric analyses were performed using ImageJ V1.54k (Bethesda, MD, USA) open-source software. Due to logistical constraints, treatments and sample processing were required to be performed by the same investigators. Therefore, they were not blinded to the group allocations during the data collection and analysis. To minimize bias, all samples were processed identically and quantified using standard criteria. Protein expression levels on all Western blot membranes were normalized to the GAPDH loading control prior to statistical analysis. Because immunoblot signal varies between membranes, quantification was restricted only to samples run on the same gel, and representative blots are shown.

2.6. Statistical Analysis

Data was visually inspected for normality using Q-Q plots and showed no major deviations from the expected normal distribution. Results were statistically analyzed using GraphPad Prism 10 via unpaired t-tests for Western blots comparing the means of two treatment groups. For the ELISA, which compared four treatment groups against one another, a one-way ANOVA with Tukey’s post hoc test was used to account for multiple comparisons. The threshold for statistical significance was a p-value less than 0.05. All values shown in the figures were reported as means ± standard error of the mean (SEM), effect size (R^2^), and 95% confidence interval (95% CI).

3. Results

3.1. GABA Supplementation Increases Pit-1 Expression in the Pituitary of Male Mice

To investigate the molecular basis by which oral GABA supplementation enhances GH expression, we first assessed its impact on Pit-1, the pituitary-specific transcription factor essential for GH gene regulation [22,36]. Western blot analysis of pituitary tissues revealed a more than two-fold increase in Pit-1 protein expression following oral GABA supplementation in treated male mice (Figure 1A,B). Notably, female GABA-treated mice did not exhibit any change in Pit-1 expression (Figure 1C,D).

3.2. Ex Vivo GABA Treatment Elevates Pit-1 and GH in Isolated Pituitaries

GABA regulates the production/secretion of various hormones [5,17,37] and Pit-1 expression may be influenced by circulating hormones and metabolic factors [36]. Therefore, we next examined whether GABA acts directly on pituitary tissues independent of systemic factors. Freshly dissected pituitaries from young male mice were cultured with or without GABA, eliminating peripheral hormonal, metabolic, and neural inputs. Males were exclusively used because in vivo results showed a strong effect of GABA on Pit-1 expression only in males (Figure 1). Moreover, previous investigations demonstrated more pronounced increases in pituitary and circulating GH compared with females [17,38]. Western blot analysis revealed that GABA treatment produced a robust increase in Pit-1 expression (Figure 2A,B) with a similar effect magnitude as the in vivo treatments. GH protein expression was also modestly increased among GABA-treated samples compared with untreated pituitaries (Figure 2C).

3.3. GABA Directly Increases GH and Pit-1 in GH3 Cells via GABAARs

To determine whether GABA increased Pit-1 and GH by directly stimulating somatotrophs, we used GH3 cells, a well-established somatotroph-derived pituitary cell line. Immunoblot assays showed that GABA treatment resulted in a robust and significant increase in Pit-1 and GH protein levels (Figure 3). It should be noted that the effect of GABA on GH expression in this cell line is much more pronounced than the modest increases observed in ex vivo pituitaries (Figure 2C) and our previous in vivo studies [17]. To assess receptor involvement, GABA-treated cells were co-treated with the GABA_A_R channel inhibitor picrotoxin. Blocking GABA_A_R activity markedly attenuated the GABA-induced increases in Pit-1 and GH (Figure 4).

3.4. Endogenous GABA Tone Sustains Basal GH Secretion in GH1 Cells

Previous studies established that pituitary somatotrophs express glutamate decarboxylase (GAD) and GABA receptors, meaning that somatotrophs are endowed with an intrinsic autocrine/paracrine GABAergic system [30,32,39]. Prior studies also reported that GABA administration increased circulating GH in rodents and humans [14,15,17], indicating that GABA can promote GH secretion. To verify this effect in vitro, we measured GH concentrations in the culture media of GH1 cells using ELISA. GABA_A_R inhibition with picrotoxin significantly reduced GH concentrations in the media, whereas exogenous GABA or the GABA_A_R agonist muscimol did not significantly alter GH compared with controls (Appendix A Figure A1).

3.5. GABA Increases Pit-1 and GH via a Ca2+/CaMKK2/CaMKIV-Dependent Cascade

Activation of GABA_A_Rs in GH-producing cells has been shown to cause membrane depolarization and promote Ca^2+^ influx [28,32,40]. To test whether GABA increases Pit-1 and GH via a Ca^2+^-dependent signaling cascade, we treated GH3 cells with GABA and assessed Ca^2+^-regulated calmodulin (CaM)-dependent signaling proteins. Western blot analysis showed no significant effect on calmodulin-dependent Protein Kinase Kinase 2 (CaMKK2) expression but did reveal a modest yet statistically significant elevation in phosphorylated calcium/calmodulin-dependent protein kinase IV (pCaMKIV) and phosphorylated AKT (pAKT) (Figure 5). In contrast, inhibition of GABA_A_Rs with picrotoxin reduced CaMKK2 levels by nearly half in GABA-treated cells and modestly lowered pCaMKIV expression compared to controls (Figure 6). Furthermore, the inhibition of CaMKK2 activity with STO-609 moderately decreased Pit-1 and lowered GH expression by more than half (Figure 7), resembling the effect size observed with picrotoxin (Figure 4C).

3.6. Ghrelin Receptor Activation Stimulates Pit-1 and GH via a CaMKK2-Independent Pathway but Requires Basal GABA Tone

Given that oral GABA supplementation increased gastric ghrelin expression in vivo [17], we evaluated whether ghrelin receptor (GHS-R) activation enhances GH expression and if this effect involves CaMKK2/CaMKIV activation. Treating GH3 cells with the ghrelin receptor agonist anamorelin significantly increased Pit-1 and GH protein (Figure 8B,C), confirming other reports that ghrelin receptor (GHS-R) signaling stimulates the Pit-1–GH axis [18,27,36]. However, anamorelin did not alter CaMKK2 or pCaMKIV protein expression, suggesting a CaMKK2-independent mechanism. Notably, co-treatment with picrotoxin blunted anamorelin’s stimulatory effects and reduced CaMKK2 as well as pCaMKIV expression (Figure 9).

4. Discussion

4.1. GABA Directly Increases Pit-1 and GH Expression in Somatotrophs

Our in vivo and ex vivo data demonstrated that GABA treatment elevated Pit-1 and GH protein levels in pituitaries of male mice, indicating that GABA’s effect is not dependent on systemic hormonal or metabolic inputs. This direct action was likewise observed in GH3 cells, where GABA robustly increased Pit-1 and GH. Notably, co-treatment with picrotoxin attenuated the effects of GABA treatment, establishing GABA_A_R activation as an upstream driver of the Pit-1–GH axis. Results also showed that picrotoxin reduced CaMKK2 and pCaMKIV protein levels, indicating that GABA_A_R signaling contributes to upstream Ca^2+^-dependent pathways that promote Pit-1 and GH expression.

4.2. CaMKK2 Signaling Links GABAAR–Ca2+ Influx to Pit-1 Transcriptional Competence

Western blot analyses showed that GABA treatment in GH3 cells increased the phosphorylation of CaMKIV, consistent with activation of the Ca^2+^/CaM/CaMKK2 pathway in response to Ca^2+^ influx. Remarkably, STO-609 lowered Pit-1 and GH protein expression, positioning CaMKK2 as an essential conduit between GABA_A_R-evoked Ca^2+^ entry and Pit-1 expression in somatotrophs. Activated CaMKK2 is a key Ca^2+^/calmodulin-dependent kinase that phosphorylates CaMKIV [41], linking GABA_A_R-induced membrane depolarization and Ca^2+^ signaling with transcriptional programs. For example, CaMKIV has been shown to phosphorylate cyclic AMP response element binding protein (CREB) in various cell types [42,43,44]. There is evidence in several pituitary cell types, including somatotrophs, that pCREB can interact with CRE binding sites on the Pit-1 promoter, enhancing Pit-1 protein transcription or function [45]. A study on bovine anterior pituitary cells found that inhibition of the cAMP/PKA/CREB pathway by short-chain fatty acids diminished GH and PRL gene transcription [46]. In addition, CaMKIV is reported to phosphorylate CREB-binding protein (CBP) [47,48], which acts as a cofactor of Pit-1 to enhance its activities on the GH promoter independently of CREB activation [49,50].

GABA also increased AKT phosphorylation in GH3 cells, suggesting activation of Ca^2+^/CaM/PI3K signaling downstream of Ca^2+^ influx and/or autocrine GH signaling [51]. Notably, recent work using GH3 cell models showed that inhibition of AKT phosphorylation diminished Pit-1 expression [52]. AKT activation likely works in parallel with CaMKK2/CaMKIV signaling to enhance transcriptional competence, facilitating Pit-1–dependent GH synthesis. Moreover, CaMKK2 can also cross-talk with AKT/AMPK nodes tied to metabolic homeostasis [53], suggesting that calcium signals may integrate transcriptional control of GH synthesis with broader metabolic regulation in somatotrophs.

4.3. Endogenous GABA Tone Sustains Basal GH Output and Primes Responsiveness to GH Secretagogues

ELISA experiments using GH1 cells showed that blocking GABA_A_Rs with picrotoxin significantly reduced GH concentrations in culture media, whereas acute GABA or muscimol did not increase GH, suggesting that the intrinsic autocrine GABA tone may already hold secretion near a functional maximum under these conditions. Prior human and animal studies have reported that oral or systemic GABA can elevate circulating GH [12,15,16,17], albeit with context-dependent variability linked to site of action and neuroendocrine milieu. These observations align with evidence that pituitary somatotrophs harbor a self-contained GABAergic system. Multiple components of the GABA system, including GAD65/67, vesicular GABA transporter (VGAT), and GABA receptor subunits, have been reported in anterior pituitary cells. This indicates autocrine/paracrine GABAergic regulation of somatotroph cell function [30,39,54].

Although GABA is classically regarded as the principal inhibitory neurotransmitter in the adult CNS, developmental and tissue-specific contexts reveal that GABA_A_R activation can cause membrane depolarization in numerous types of endocrine cells [3,40,55], thereby promoting Ca^2+^ influx [32], hormone secretion and downstream transcriptional responses. The stimulatory effect of GABA in anterior pituitary cells is attributed to the higher expression of the NKCC1 transporter, which imports chloride into the cell, resulting in a relatively greater intracellular chloride concentration [28].

4.4. Ghrelin Receptor Activation Stimulates Pit-1 and GH via a CaMKK2-Independent Route

We observed that the ghrelin receptor agonist anamorelin increased Pit-1 and GH without altering CaMKK2 or pCaMKIV, indicating a CaMKK2-independent mechanism. Nevertheless, picrotoxin blunted anamorelin’s effects and lowered CaMKK2–CaMKIV signaling, suggesting that basal GABA_A_R activity creates a permissive CaMKK2-dependent environment that maintains Pit-1 transcriptional competence and allows maximal ghrelin responsiveness. This interpretation aligns with the known biology of the ghrelin receptor GHS-R1a, which is widely expressed in the hypothalamus and pituitary, activating the somatotropic axis peripherally while exerting orexigenic effects centrally [56].

4.5. Developing a Mechanistic Framework

Taken together, our data support a developing model in which GABA_A_R-induced depolarization triggers Ca^2+^ influx via voltage-gated calcium channels in pituitary somatotrophs, as others have reported [28,32,40,57]. This influx triggers the Ca^2+^/CaM/CaMKK2 signaling pathway, which stimulates phosphorylated kinase pCaMKIV to activate specific transcription factors such as CREB [43,44,58] and CBP [47,48], ultimately promoting Pit-1/GH expression [46,49,59,60]. The basal autocrine GABAergic tone simultaneously primes the pituitary for maximal responsiveness to additional regulators such as ghrelin, which elevates GH via CaMKK2/CaMKIV-independent pathways yet requires GABA_A_R activity for full effect. Moreover, the canonical pathway for pituitary GH synthesis via hypothalamic growth hormone-releasing hormone stimulation involves the activation of CREB, albeit through cAMP/PKA signaling [29]. Given that pCaMKIV can also phosphorylate CREB [43,44,58], it is plausible to reason that the effects observed in the present study may converge on shared regulatory pathways. Future work should focus on elucidating the specific signaling cascades that link GABA-induced CaMKK2/CaMKIV activation with Pit-1 expression.

4.6. Physiological Relevance

Using in vivo and ex vivo murine models and GH-secreting cell lines, the present study provides mechanistic insight into GABA’s role in regulating Pit-1 and GH expression. However, the translational relevance of these results to GABA supplementation in humans remains a field of active investigation. Human studies have reported that the administration of GABA caused a transient elevation of circulating GH compared to placebo [15,61], suggesting that exogenous GABA can influence the somatotropic axis in humans. In addition, prolonged daily GABA supplementation combined with whey protein has been associated with higher blood GH concentrations and increases in fat-free mass, implying potential endocrine/anabolic effects in men [12]. Despite these findings, the magnitude and consistency of GH responses vary and the physiological mechanisms by which GABA increases circulating GH in humans are not well-defined. Our work provides a mechanistic framework that may help interpret human responses to nutritional GABA, but direct validation using human pituitary tissue or stem cell-derived somatotrophs is necessary to determine how well this GABA/CaMKK2 pathway is conserved across species.

Under physiological conditions, GABA concentrations in the blood are relatively low compared to what is typically used in cell culture experiments. However, it should be noted that GABA is synthesized within the anterior pituitary and is present in somatotrophs, where it can act in an autocrine and/or paracrine manner [39,54], suggesting that local GABA concentrations may promote receptor signaling independent of circulating GABA levels. Moreover, functional studies demonstrate that pituitary GABA receptors are activated in the low micromolar range, with robust responses observed at around 10 µM [28,33]. Therefore, micromolar GABA exposure was deemed appropriate for probing receptor-mediated signaling in GH3 cells and ex vivo pituitary tissue. However, it should not be interpreted as directly reflecting physiological GABA concentrations in the pituitary.

Although our mechanistic model suggests a GABA-CaMKK2-Pit-1 pathway regulating GH expression in rodent somatotrophs, important species-specific differences in somatotropic axis regulation must be considered when relating these findings to human physiology. Specifically, neuroendocrine control of GH secretion differs between rodents and humans. For example, rodents exhibit more pronounced sexually dimorphic GH pulse profiles, with males having larger pulses of GH secretion followed by low periods, while female rodents secrete GH in a more continuous fashion, with higher baseline levels of GH between pulses. In humans, GH secretion is also pulsatile and sexually dimorphic but is more heavily influenced by other factors such as age, sleep, and metabolic state. Similar to female mice, women also have higher mean daily GH production rates than men [62,63].

4.7. Sex-Specific Differences

The present study showed that in vivo GABA supplementation increased Pit-1 expression in male mice but not females. Therefore, mechanistic experiments focused on male pituitary explants and GH3 cells, a somatotroph cell line derived from a female rat. Despite lacking male chromosome-linked genes, GH3 cells responded robustly to GABA, suggesting that the sex differences observed in vivo are mediated by sex hormones rather than Y-linked genes. Indeed, estrogens and androgens are known to regulate GH secretion, Pit-1 transcriptional activity, and hypothalamic control of the somatotropic axis [64,65,66]. In addition, rodent GH secretory patterns are strongly sexually dimorphic, with males exhibiting high-amplitude intermittent pulses and females more continuous secretion. Interestingly, there is evidence of sex-specific differences in GH responses to GABAergic stimulation [38] as well as differences in GH responses by females during different stages of the menstrual cycle [67]. Moreover, female sex hormones modulate GABA synthesis and receptor expression [68,69]. These observations imply that the proposed GABA–Pit-1 mechanism may not fully generalize to females. Future studies are warranted to examine whether modulation of gonadal steroids or intrinsic GABA tone alters somatotroph responsiveness in females.

4.8. Study Limitations

Results from this study must be interpreted considering several limitations. First, differences in somatotropic axis regulation between species constrain direct translation of these findings to human physiology. Secondly, the focus of this work was to elucidate the mechanisms behind GABA-induced Pit-1 expression and in vivo GABA supplementation caused elevation of Pit-1 expression in males, but not females. Therefore, only male mice were used in the ex vivo experiments. Third, sample sizes for Western blot data were modest (n = 4 biological replicates per treatment), which restricts the statistical power of the analyses, particularly when the effect size is small. Similarly, the ELISA data on GH secretion is preliminary, and additional dose–response or functional secretion studies are necessary to further validate these observations. Another limitation is the exclusive use of western blot results to make inferences about transcriptional regulation. Future research may employ qPCR or reporter assays to more directly measure the effects of GABA treatment on the transcription of Pit-1/GH and the associated calmodulin-dependent kinases.

Lastly, the mechanistic conclusions in this study rely primarily on pharmacological interventions that may have off-target actions. For example, picrotoxin is a non-competitive GABA_A_R blocker that may affect other ligand-gated ion channels [70]. Moreover, picrotoxin also inhibits GABA_C_Rs (a sub-type of GABA_A_Rs), which was useful for broadly inhibiting GABA_A_R activity in this study. However, it also limits investigators from distinguishing between the involvement of different GABA_A_R sub-types [71]. Likewise, STO-609 is widely used to inhibit CaMKK2 signaling, but it can exert inhibitory effects on other kinases [72]. To better establish pathway causality, genetic loss-of-function approaches such as siRNA knockdown or targeted deletions will be essential in future investigations.

5. Conclusions

Together, our findings demonstrate that GABA enhances Pit-1 and GH expression in male pituitary somatotrophs through activation of GABA_A_R and Ca^2+^/CaM-dependent signaling pathways, partially supporting our initial hypothesis. These results identify a pituitary-intrinsic mechanism by which GABA critically regulates somatotroph function and provide a framework that may help explain the GH responses to GABA supplementation. Beyond mechanistic insight, our findings help contextualize the numerous reported peripheral and central effects of GABA supplementation through the lens of endocrine regulation.