ESSAY | TOWARD JES 100TH ANNIVERSARY
Memories of our research on the hypothalamo-hypophyseal system
2024 Volume 71 Issue 8 Pages 729-732
Details
2024 Volume 71 Issue 8 Pages 729-732
During the latter half of the Japan Endocrine Society’s hundred-year history, many physiologically active peptides, including hypophysiotropic peptides, were discovered, and their functional analyses progressed. It was a very important era for endocrinology. It is also noteworthy that many of these discoveries were made by Japanese researchers. Fortunately, I was able to spend this exciting time in endocrinology as a researcher.
During this time, I began with cell biological studies of the anterior pituitary gland and became involved in the functional analysis of prolactin releasing peptide (PrRP) and kisspeptin, which were discovered as orphan receptor ligands. In this essay, I would like to describe the history and impressions of these studies and express my views on fostering young researchers who will be responsible for future endocrinology.
Basic biological research often led to many great discoveries. The discovery of hypothalamic factors that control endocrine cells in the anterior pituitary gland also followed such a course. When the nerve fibers projecting to the median eminence of the hypothalamus were cut using the so-called Halasz’s knife, it caused dysfunction of gonadotrophs. In addition, when the anterior pituitary gland was transplanted into the third ventricle, the gonadotrophs on the basal side of the transplant became hypertrophic. It became clear that the hypothalamus is involved in controlling the function of the anterior pituitary gland. Furthermore, Harris in England proved that there is secretion activity of gonadotropin in the hypothalamic extract. Harris’s discovery led to the finding of LHRH (GnRH) by Shirley’s research group in New Orleans. It is a well-known fact in the endocrine society that Japanese researchers Drs. Arimura, Matsuo, and Baba were deeply involved in this discovery.
The story of the discovery of hypothalamic releasing peptides was published as a non-fiction novel and became famous. When I was young, I was given the opportunity to collaborate with Professor Arimura in New Orleans, where the great discovery took place. During this time, I was able to stay at Arimura’s home and experience the passion and romance of Arimura and his wife’s research up close. It is a digression, but shortly after meeting Professor Arimura, I keenly felt my immaturity as a researcher. Despite recognizing my immaturity, Arimura treated me kindly, which is something I will never forget. From Arimura’s attitude, I learned that it is important for leaders to cultivate young researcher with a spirit of tolerance rather than control subordinates with authority.
Soon after the discovery of GnRH, Dr. Fujino of Takeda Pharmaceutical Company found an agonist with strong physiological activity by replacing the leucine of the amino acid sequence of GnRH with D-leucine. This agonist disrupted the hypothalamus-pituitary-gonadal axis and led to the development of Leuprorelin as a specific drug for prostate cancer and other diseases. This demonstrated that the search for neuropeptides also made a significant contribution to pharmaceuticals. For this reason, Fujino established a powerful research group for neuropeptide exploration at Takeda Pharmaceutical’s research institute.
In April 2003, the Human Genome Project was completed, revealing the entire base sequence of the human genome. Surprisingly, the number of genes in the human genome was about 35,000, which was less than expected. Within this human genome, a group of genes existed that encoded seven hydrophobic amino acid sequences.
This group of genes contained many candidate genes for G-protein coupled receptors (GPCRs) that penetrate the hydrophobic cell membrane seven times. Among these, about 100 candidate genes for GPCRs were labeled orphan GPCRs because their ligands were unknown. As a result, the search for unknown ligands for these receptors began. However, these ligands were predicted from gene analysis, and their physiological functions remained completely unknown. Therefore, a so-called reverse pharmacological approach was used to search for them. Takeda Pharmaceutical’s research group was one of the first to embark on this search and discovered prolactin-releasing peptide (PrRP) [1].
PrRP was initially reported as a candidate for a hypophysiotropic factor that stimulates prolactin secretion. However, at that time, it was not known whether PrRP was a true hypophysiotropic factor. Fortunately, shortly after its discovery, I was given the opportunity to study this peptide. Morphological approaches are very effective in exploring the physiological functions of molecules with unknown functions. If PrRP is indeed a hypothalamic hypophysiotropic factor, its nerve fibers should project to the primary plexus of the hypophyseal portal system located in the outer layer of the median eminence.
To confirm this, we first began staining PrRP neurons through immunocytochemistry. For this purpose, a reliable antibody was needed. Fortunately, we were able to use an excellent antibody produced by Dr. Matsumoto of Takeda Pharmaceutical Company. The result of immunocytochemistry was unexpected. PrRP neurons had cell bodies in the hypothalamus and the medulla oblongata, but their nerve fibers did not project to the outer layer of the median eminence of the hypothalamus [2].
This result showed that PrRP was not a hypophysiotropic factor. We were very disappointed at the time, but a new question arose about the true function of PrRP. In response to this, it was found that PrRP projected to the CRH neurons and oxytocin neurons in the hypothalamus. It was also revealed that intraventricular administration of PrRP promoted the secretion of oxytocin and ACTH [3, 4]. Additionally, it was revealed that PrRP nerve cells are activated when rats are subjected to stress. From these findings, it became clear that PrRP is a neural peptide that mediates stress [5-8].
Although PrRP has not yet been applied as a drug, it is expected to be used in future research and development as a substance related to stress. It is also noteworthy that PrRP neurons appear in the area postrema (AP) of adrenalectomized rats [7, 9]. The appearance of PrRP neurons in AP suggests that PrRP may be related to vomiting. I look forward to future research on the relationship between PrRP and vomiting.
Following the discovery of PrRP, several novel peptides were identified as orphan receptor ligands by Takeda Pharmaceutical Company. One of them, the endogeans ligand of hOT7T175 (GPR54) was named ‘metastin’ as it was encoded by the metastasis suppressor gene, KiSS-1 [10]. Shortly after the discovery of metastin, a collaborative research project began between Nagoya University and Saitama University. Under the leadership of Dr. Maeda and the proactive research approach of Dr. Tsukamura, it was discovered that metastin (later renamed kisspeptin) played a crucial role in sexual maturation and ovulation via the GnRH nerve [11-13]. Our contribution to the early study of kisspeptin involved elucidating the distribution of kisspeptin nerve fibers and their relationship with GnRH nerves through immunocytochemistry.
In any case, PrRP and kisspeptin, which were discovered as orphan receptor ligands, controlled the function of the anterior pituitary gland through neurons that produce hypophysiotropic factors. This indicates that the function of the anterior pituitary gland is more deeply controlled by the brain.
The discovery of hypophysiotropic factors and brain peptides that control the hypothalamus has revealed that the function of the anterior pituitary gland is more strongly regulated by the central nervous system. However, the mechanisms behind the proliferation and differentiation of pituitary endocrine cells are not yet fully understood. For instance, long-term estrogen administration to rats induces hyperplasia of rat lactotrophs and eventually leads to prolactinoma in vivo. However, estrogen-induced mitoses of lactotrophs are not observed in dissociated lactotrophs in vitro, suggesting the existence of an unknown mechanism.
To study the proliferation and differentiation of pituitary endocrine cells, we established clonal cell lines that produce both growth hormone and prolactin [14]. Using these cell lines, we discovered the multistep differentiation processes of somatotrophs [15-17]. We also identified factors that induce the transdifferentiation of lactotrophs from somatotrophs [18]. However, we have not been able to elucidate the mechanism of prolactin cell mitosis in vitro. It is hypothesized that there is an interaction with adjacent cells and a relationship with microenvironmental factors in normal pituitary tissue. However, the actual situation has not yet been fully elucidated.
Regarding the microenvironmental control of pituitary gland, I have been focusing on folliculo-stellate (FS) cells located within the pituitary gland. These cells reside among the endocrine cells of the anterior lobe and have been considered supporting cells of the anterior pituitary gland [19]. Furthermore, it has been suggested that FS cells show high differentiation ability, such as differentiation into muscle cells, indicating that FS cells may function as stem cells in the pituitary gland [19, 20].
For the study of FS cells, we established a cell line of FS cells [21] and created transgenic mice expressing GFP in FS cells and astrocytes [22]. Using GFP transgenic mice, the genes expressed by FS cells have been comprehensively analyzed, revealing many genes that may act as microenvironmental controls [23, 24]. As this research progresses, it is expected that the actual functional control of anterior pituitary cells, which cannot be explained by hypothalamic factors alone, will be elucidated.
On the other hand, FS cells have been shown to exhibit phagocytosis and act as scavenger cells in tissues. In addition, it has been revealed that FS cells produce amyloid-like colloids, and that clusterin accumulates in these colloids in relation to their scavenger function [19, 25]. In this regard, a new processing mechanism has recently been reported, inducing phagocytosis of abnormal substances occurring extracellularly by scavenger cells through clusterin [26]. This new mechanism for processing extracellular abnormal substances is attracting attention, highlighting the active involvement of FS cells in processing abnormal substances in the anterior pituitary gland and astrocytes in the brain.
Now that I have retired from the front line as a researcher, I am concerned that basic research in Japan is declining. The government has been promoting budget consolidation through competitive funds to enhance research capabilities and incorporating national universities as part of university reform. However, contrary to expectations, Japan’s scientific capabilities are not improving, but rapidly declining. One reason for this may be that researchers have an excessive amount of administrative work. To increase researchers’ motivation for research, it is necessary to secure not only research budgets but also research time. Currently, various evaluations are being conducted to manage researchers, especially university faculty members. However, it seems necessary for administrators to trust researchers and make efforts to redirect researchers back towards research and education.
It is also important to create a forum for exchanges beyond the expertise of young researchers. I recall the neuroendocrine workshop held at the Institute of Endocrinology of Gunma University (now Institute for Molecular and Cellular Regulation). When I started research, Professors Kurosawa, Suzuki, and Wakabayashi at this institute were advancing research in neuroendocrinology and held “neuroendocrine workshops” for several years to disseminate their research methods. This workshop included lectures on Halasz’s cut, immunocytochemistry of hypothalamic factors, and measurement of hormones by radioimmunoassay (RIA). The workshop was attended by more researchers than expected. In particular, participants from different fields such as medicine, science, agriculture and fisheries, and pharmacy gathered at this workshop, and information exchange between different specialties was active. As a result, not only the participants but also the organizers received a lot of stimulation. From this workshop experience, I am convinced that conducting research exchanges in a frank atmosphere that cannot be obtained at academic conferences is very important for the development of science. The Japan Endocrine Society is holding summer seminars to promote such close exchanges, which I think is a very good initiative. Although I have left the research field, I would like to watch over the young researchers who will carry the next hundred years.
Kinji Inoue, PhD.
Honorary Member
Professor Emeritus, Saitama University
E-mail: kininoue@lagoon.ocn.ne.jp
Careers in JES
2014– Honorary Member
2010– Senior Councilor
1991– Councilor
1979– Member
Activities in JES
2005 Chair, 23rd JES Summer Seminar on Endocrinology & Metabolism
