Abstract

Translational Research of Novel Hormones
- From Discovery to Clinical Application

Kazuwa Nakao

In order to make proof of concept (POC) in the translational research (TR) for novel hormones, leptin and ghrelin, their physiological and clinical implications have been assessed using animal models of human diseases. Although a multitude of animal models have been developed to emulate various human diseases, there are a few excellent animal models that mimic human diseases remarkably well. The possible presence of species difference between human diseases and animal models should be sensitively concerned and be carefully assessed in comparison with the etiology and pathophysiology of human diseases in TR. In TR of leptin, we focused the target of leptin therapy on generalized lipodystrophic mice model, A-Zip F1 mice, which was the most sensitive to the leptin therapy. The leptin supplementation therapy dramatically improved glucose and lipid metabolism in A-Zip F1 mice, indicating POC for generalized lipodystrophy. We succeeded in demonstrating the long-term efficiency and safety of the leptin supplementation therapy for patients with lipoatrophic diabetes. The leptin therapy was also useful for insulin-deficient diabetic mice with streptozotocin (STZ) administration, genetically non-obese diabetic mice (Akita) and high fat diet-induced obese diabetic mice with a low dose of STZ administration (BMI: around 30). There results indicate that the leptin therapy is useful not only lipodystrophic diabetes but type 1 and type 2 diabetes mellitus. To make POC of ghrelin therapy, we were engaged in developing transgenic mice expressing ghrelin, and have developed several different ghrelin transgenic mice and analysed their phenotypes. It has been possible to establish the efficacy and safety in rare human diseases through studies that began with excellent animal models. These studies provided us with novel treatments for common human diseases, which were explored as adjacent to or in extension of rare human diseases. These lessons teach us the importance of the breakthroughs using excellent animal models and rare human diseases in TR.

The Central Role of Adiponectin and AdipoRs in Obesity
- Linked Diseases

Takashi Kadowaki

Adiponectin is a fat-specific hormone which is the most abundantly expressed adipokine in white adipose tissue (WAT). Adiponectin sensitizes the body to insulin and plasma adiponecin levels are decreased in obesity, which plays a pivotal role in metabolic syndrome and type 2 diabetes - linked to obesity. Adiponectin also directly suppresses atherosclerosis via multiple pathways such as anti-inflammatory action. Adiponectin mediates its biological effects via its plasma membrane receptors, AdipoR1 and AdipoR2. In fact, adiponectin KO mice as well as AdipoR1⁄AdipoR2 double KO mice show insulin resistance and impaired glucose tolerance. In liver, adiponectin activates AMPK and PPARα via AdipoR1 and AdipoR2, respectively, causing suppression of gluconeogenesis and lipogenesis and stimulation fatty acid oxidation. In muscle, adiponectin activates AMPK and PGC-1α via AdipoR1, causing stimulation of glucose uptake and mitochondrial biogenesis. In endothelial cells, adiponectin protects from increased neointimal formation in response to cuff-injury via AdipoR2. Obesity is also accompanied by decreased expressions of adiponectin receptors in liver, muscle and WAT, each which is also causally involved in obesity-linked insulin resistance. Upregulation of plasma adiponectin and adiponectin receptors as well as development of adiponectin receptor agonists may serve as fundamental and versatile therapeutic strategy for obesity-linked morbidities such as metabolic syndrome, type 2 diabetes and atherosclerosis.

Control of Bone Remodeling by Leptin and Neuropeptides

Shu Takeda

Bone mass is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. It has been generally assumed that bone remodeling is mainly controlled by the local environment, i.e., autocrine or paracrine mechanisms. Increasing evidences that neurons and neurotransmitters are intimately involved in bone remodeling shed light on a novel regulatory mechanism for bone homeostasis. We have uncovered that leptin, an adipocyte-derived anorexigenic hormone, regulates bone mass through its receptor located in the central nervous system. Subsequent analysis revealed that this leptin uses sympathetic nervous system to inhibit bone formation and stimulate bone resorption. In addition, many epidemiological studies confirmed the effect of a beta blocker on bone mass or fracture. In search of neuropeptides regulating bone remodeling centrally, we focused on neuromedin U (NMU); NMU is an anorexigenic neuropeptide whose action occurs leptin-independently. We demonstrated that NMU is the novel central mediator of leptin-dependent regulation of bone mass. I will summarize the genetic, molecular and physiological bases for the central control of bone remodeling and discusses the future directions of this new research field, neuroskeletal biology.

Leptin and the Homeostatic Control of Energy Balance

Jeffrey M. Friedman

The discovery of leptin has led to the elucidation of a robust physiologic system that maintains fat stores at a relatively constant level. Leptin is a peptide hormone secreted by adipose tissue in proportion to its mass. This hormone circulates in blood and acts on the hypothalamus to regulate food intake and energy expenditure. When fat mass falls, plasma leptin levels fall stimulating appetite and suppressing energy expenditure until fat mass is restored. When fat mass increases, leptin levels increase, suppressing appetite until weight is lost. By such a mechanism total energy stores are stably maintained within a relatively narrow range. Recessive mutations in the leptin gene are associated with massive obesity in mice and some humans. Treatment with recombinant leptin markedly reduces food intake and body weight. The low leptin levels in patients with leptin mutations are also associated with multiple abnormalities including infertility, diabetes and immune abnormalities all of which are corrected by leptin treatment. These findings have established important links between energy stores and many other physiologic systems and led to the use of leptin as a treatment for an increasing number of other human conditions including a subset of obesity, some forms of diabetes including lipodystrophy and hypothalamic amennorhea, the cessation of menstruation seen in extremely thin women. Identification of a physiologic system that controls energy balance establishes a biologic basis for obesity and further establishes links between leptin and numerous other physiologic responses. Finally, this research has changed our concept of the cause of obesity from being a defect in willpower to an alteration in hormone signaling.

Phenotypic Analysis of Ghrelin Knockout Mouse

Masayasu Kojima

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor that is synthesized predominantly in the stomach. Previous studies demonstrated that ghrelin stimulates growth hormone release and food intake. These data suggested that antagonism of ghrelin could serve as a useful treatment for eating disorders and obesity. To study the role of endogenous ghrelin in feeding performance further, we generated ghrelin-deficient (ghrl^-⁄-) mice. Unexpectedly, ghrl^-⁄- mice exhibited normal growth, cumulative food intake, reproduction, histological characters, and serum parameters. There were no differences in feeding patterns between ghrl^+⁄+ and ghrl^-⁄- mice. Ghrl^-⁄- mice displayed normal responses to scheduled feedings as seen for ghrl^+⁄+ mice. Memory-related feeding performances of ghrl^-⁄- mice were indistinguishable from ghrl^+⁄+ littermates. These data indicate that ghrelin is not critical for feeding performance. Recently, we found that ghrelin is necessary for maintaining homeostasis of autonomic nervous system, such as cardiovascular functions, gastrointestinal movements and body temperature regulation. In this presentation, I will discuss the new role of ghrelin.

Ghrelin and Gastrointestinal Function

Hidekazu Suzuki, Tatsuhiro Masaoka, Toshihiro Nishizawa,
Juntaro Matsuzaki, Sachiko Suzuki, Eisuke Iwasaki, Yoshimasa Saito,
Kenro Hirata, Hitoshi Tsugawa and Toshifumi Hibi

Ghrelin, a novel acylated endogenous ligand for the growth hormone secretagogue receptor (GHS-R), isolated and identified for the first time in 1999, not only triggers premeal hunger and promotes initiation of food intake, but also stimulates gastric acid secretion and gastrointestinal (GI) motility. Although the ghrelin precursor, preproghrelin mRNA, is expressed ubiquitously, its level of expression is the highest in the A-like cells of the oxyntic mucosa of the stomach. Thus, the stomach is considered as an appetite sensor, which is closely connected with the central nervous system. On the other hand, ghrelin production and secretion would be affected by the lesion of the stomach. H. pylori infection is the most important factor involved in the pathogenesis of gastroduodenal diseases especially in Japan, one of the countries in the world with a considerable number of H. pylori-infected individuals. The colonization of the gastric mucosa by this bacterium evokes gastric mucosal inflammatory cell infiltration extending from the antrum to the corpus, in parallel with the disappearance of parietal cells as well as ghrelin-producing A-like cells and dedifferentiation of mucous neck cells to zymogenic (chief) cells with a suppressed expression of sonic hedgehog gradient. After eradication of H. pylori, mucosal inflammatory cell infiltration is no longer seen, and in the case of younger ages, differentiation of the gastric fundic gland is also completely restored, resulting in normalization of the ghrelin-producing cells. Ghrelin secretion is enhanced under the cholinergic dominant conditions, such as peptic ulcer disease or functional dyspepsia. Although the clinical trial of ghrelin or ghrelin promoting agents for the treatment of functional GI disorders including motility disorders and anorexia are now ongoing, it might be difficult to establish symptomatic benefit with such agents in functional dyspepsia, because of pathophysiological heterogeneity of the patient populations, a lack of well-accepted endpoints, and inconsistent relationships between changes in motor function and symptomatic outcome.

Discovery of Novel Bioactive Peptides, with Special Reference to Ghrelin

Kenji Kangawa

A complex network of cell-cell communication system by peptide hormones works for maintaining the mammalian homeostatic balance. To further clarify the intricate mechanisms of the regulation, we have been searching for still unidentified bioactive peptides. In the course of these studies, we discovered 3 natriuretic peptides, ANP (1984), BNP (1988) and CNP (1990), in mammalian heart and brain, and adrenomedullin (1993) in human pheochromocytoma. These studies elucidated new regulatory mechanisms of cardiovascular system, and also brought their therapeutic potentials on cardiovascular diseases. In 1999, we discovered ghrelin, a novel GH-releasing peptide, from rat stomach. Ghrelin has a marvelous structure modified by fatty acid, n-octanoic acid, which is essential to its activity. Ghrelin is primarily produced in distinct endocrine cells, X⁄A-like cells, in the stomach. Ghrelin-producing neurons are also present in the hypothalamic arcuate nucleus, a region that regulates GH release and food intake. In fact, ghrelin stimulates feeding when administered centrally and peripherally. Ghrelin secretion is up regulated under conditions of negative energy balance, whereas it is down regulated under conditions of positive energy balance. Ghrelin is the first neuro-enteric peptide that acts as a starvation-signaling molecule in the periphery. The recent studies indicate that the gastric vagal afferent is the major pathway conveying ghrelin′s signals for starvation and GH secretion to the brain. Therefore, beside the stimulatory effect of GH release, ghrelin is also involved in the stimulation of feeding and the regulation of energy metabolism. Moreover, ghrelin has positive cardiovascular effects. The administration of ghrelin improves cardiac structure and function, and attenuates the development of cardiac cachexia in rats with heart failure. In clinical trial, repeated administration of ghrelin improves left ventricular structure and function, exercise capacity, and muscle wasting in heart failure patients. Thus, ghrelin has multifaceted roles in the cardiovascular and metabolic systems, and also has therapeutic potentials in various diseases by GH-dependent and independent mechanisms.

Energy Metabolism and Aging: Significance of Hormonal Regulation

Hiroshi Itoh

Derangement of energy metabolism results in obesity, diabetes and metabolic syndrome, which contribute to the blunting of the increase of our life span. In contrast, caloric restriction (CR) is demonstrated to extend life span with the decrease of age-related diseases, including neoplasms, cardiovascular diseases, dementia and diabetes in wide range of living organisms including the monkey. CR achieves low plasma glucose and insulin levels and elevates insulin sensitivity. Thus, aging can be understood in the context of energy metabolism (“metabo-aging”) We have demonstrated that vasoactive hormones (angiotensin II and natriuretic peptides) regulate not only oxygen supply by regulating vascular tone and remodeling⁄regeneration, but also oxygen usage⁄energy expenditure by controlling mitochondrial biogenesis. Mitochondrial function is recognized to be crucial for glucose⁄lipid metabolism and generation for oxidative stress, which is the determining factor for aging. Guts and adipose tissues are responsible for energy “capture” (food digestion and absorption) and energy storage, respectively. Ghrelin and leptin, the newly-discovered hormones produced in guts and adipose tissues, respectively, have been demonstrated to be critically involved in energy homeostasis by modulating food intake and energy expenditure. By regulating autonomic neural activities, these novel hormones are expected to exert further more actions, which can affect metabo-aging.