538 Proc. Jpn. Acad., Ser. B 95 (2019) [Vol. 95,

Review Translational science: Newly emerging science in biology and medicine — Lessons from translational research on the natriuretic peptide family and leptin —

† By Kazuwa NAKAO*1,

(Edited by Hiroo IMURA, M.J.A.)

Abstract: Translation is the process of turning observations in the laboratory, clinic, and community into interventions that improve the health of individuals and the public, ranging from diagnostics and therapeutics to medical procedures and behavioral changes. Translational research is defined as the effort to traverse a particular step of the translation process for a particular target or disease. Translational science is a newly emerging science, distinct from basic and clinical sciences in biology and medicine, and is a field of investigation focused on understanding the scientific and operational principles underlying each step of the translational process. Advances in translational science will increase the efficacy and safety of translational research in all diagnostic and therapeutic areas. This report examines translational research on novel hormones, the natriuretic peptide family and leptin, which have achieved clinical applications or for which studies are still ongoing, and also emphasizes the lessons that translational science has learned from more than 30 years’ experience in translational research.

Keywords: translational science, translational research, natriuretic peptide family (ANP, BNP, CNP), leptin, animal disease model, rare disease

The original definition of translation refers to 1. Introduction the process of turning observations in the laboratory, Linguistically, the term “translation” refers to clinic, and community into interventions that im- the conversion of one language to another language. prove the health of individuals and the public, The term is also used to refer to the synthesis of ranging from diagnostics and therapeutics to medical proteins from mRNA in biology. Since the last decade procedures and behavioral changes. Translational of the 20th century, the terms “translational research” research is defined as the effort to traverse a and “translational science” have been used to refer to particular step of the translation process for a the process or concept by which we move from particular target or disease. Translational science is discoveries to applications in medicine.1)–3) Because a newly emerging science, distinct from basic and progress in biology was rapidly applied to medicine in clinical sciences in biology and medicine and is a the 20th century, the term “translation” has become field of investigation focused on understanding the widely used in biology and medicine under the scientific and operational principles underlying each proposal of the National Center for Advancing step of the translational process. Translational Science (NCATS), resulting in diver- Despite rapid and remarkable advances in gence from the original conceptual meaning. medical science, especially in basic science, discov- eries in many steps of the translation process are only *1 Medical Innovation Center, Kyoto University Graduate being introduced to clinical applications after a time- School of Medicine, Kyoto, Japan. † consuming process, and only in extremely rare cases. Correspondence should be addressed: K. Nakao, Medical The process has often been likened to a seemingly Innovation Center, Kyoto University Graduate School of Medicine, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan endless trip through a long, dark tunnel. These (e-mail: [email protected]). challenges withstanding, the research field of trans- doi: 10.2183/pjab.95.037 ©2019 The Japan Academy No. 9] Translational science 539 lational science is anticipated to transform discov- Heart eries into clinical applications in medicine and make Atrial Natriuretic Peptide ANP innovative medical technologies a reality. Above all, Brain Natriuretic Peptide BNP advances in translational science will increase the Blood vessel ffi e cacy and safety of translational research in all NO (EDRF) diagnostic, therapeutic, and behavioral applications. C-type Natriuretic Peptide (CNP) Although a multitude of animal models have Adrenomedullin (AM) been developed to emulate various human diseases, Endothelin (ET) there are only a few animal models that mimic Angiotensin II (AII) human disease remarkably accurately, e.g., sponta- Prostacyclin (PGI2) neously hypertensive rats (SHRs)4) and hereditary Thromboxane (TXA2) 5) obese mice, (ob/ob) are very useful for the PTH-related Protein (PTHrP) preclinical steps of translational research on hyper- Fig. 1. Cardiovascular hormones and the cardiovascular system tension and obesity, respectively. Lessons from as an endocrine organ. Members of the natriuretic peptide family research history on SHRs, an excellent animal model are prototype cardiovascular hormones. for hypertension research, led us to investigate the clinical significance of the natriuretic peptide family [atrial natriuretic peptide (ANP), brain natriuretic Leptin peptide (BNP), and C-type natriuretic peptide TNF-α (CNP)] in SHR in the early stages of preclinical Adiponectin research. For translational research on leptin, a Resistin representative adipokine, in addition to obese mouse Adipsin models such as the leptin knockout (KO) ob/ob mice PAI-1 and leptin receptor KO db/db mice, we generated Angiotensinogen leptin transgenic (Tg) “skinny” mice with elevated Free Fatty Acid plasma leptin concentrations similar to those in obese Steroids humans and assessed the functional and therapeutic Fig. 2. Adipocyte-derived factors, adipokines, released from implications of leptin beyond species differences. adipocytes. Adipose tissue is a novel endocrine organ. Leptin is These studies led to establishment of a “proof-of- an adipokine that induces severe obesity when depleted. concept (POC)” that plays a pivotal role in the transitional step from the preclinical stage to clinical Table 1. Translational research on the natriuretic peptide family trials of the translational science spectrum. and leptin achieved or ongoing in Japan This review summarizes the current state of translational research on the natriuretic peptide Application State/Industry Disease and Target family (ANP, BNP, and CNP), which are prototype Achieved/Suntory Congestive heart Therapeutic cardiovascular hormones (Fig. 1), and on leptin, a ANP (Daiichi-Sankyo) failure representative adipokine (Fig. 2 and Table 1). In Diagnostic Achieved/Shionogi Body fluid volume addition, general principles of translational science Heart failure and BNP Diagnostic Achieved/Shionogi are proposed that have accumulated in the course of hypertrophy translational research. Suitable animal disease mod- Ongoing/Chugai CNP Therapeutic Achondroplasia els mimicking human diseases are indispensable in Pharmaceutical the preclinical stage. Target diseases for the initial Therapeutic Achieved/Shionogi Lipodystrophy step of clinical trials could be rare human diseases, Leptin Achieved*/Cosmic Lipodystrophy, Diagnostic and then the target diseases expand from rare human Corporation Adiposity diseases into common human diseases that are *Diagnostic application of leptin for lipodystrophy has been essentially close to rare human diseases in their approved by PMDA after the submission of the manuscript. etiology and pathophysiology.2) fi 2. De nition of translational science and the in the 20th century, and the fields explored by basic translational science spectrum scientists and physicians or clinicians became much 2-1. Definition of translational science. wider, deeper, and more separate. It is very hard for Progress in biology was rapidly applied to medicine medical doctors to be experts in both basic or 540 K. NAKAO [Vol. 95,

smoking cessation. Thus, “translation” refers to a Clinical Science Clinician conceptual and practical multistep process. By contrast, “translational research” is defined by NCATS as the endeavor to traverse a particular Clinician /Scientist step of the translation process for a particular target or disease. In the process from discoveries to medical applications in real-world environments, complexity Basic Science Scientist as well as research and operational challenges increase exponentially. Progress of Medicine Translational science is quite distinct in purpose and operation from translational research. Transla- Fig. 3. Effects of progress in medicine on basic scientists, tional research focuses on the specific case of a target clinician-scientists (translational scientists), and clinicians or or disease, whereas translational science is focused on clinical scientists. a general case that applies to any target or disease. Focus areas of translational science are the common preclinical research and clinical research or medicine causes of inefficiency and failure in translational (Fig. 3). From the last decade of the 20th century, research, such as incorrect predictions of the toxicity the terms “translational research” and “translational or efficacy of new drugs, lack of data interoperability, science” have been used to describe the concept of and ineffective clinical trial recruitment. Because the process of moving from a discovery to clinical these causes are the same across targets, diseases, and practice.1)–3) The term “translation” has been widely therapeutic areas, advances in translational science used in biology and medicine (i.e., biomedicine), will increase the efficacy and safety of translational under a proposal of the NCATS, resulting in research in all diagnostic, therapeutic, and behavioral divergence in the conceptual meaning relative to applications. the previous 30 years. Like any other science, translational science According to the original definition by seeks to elucidate general operative principles in NCATS,3),6) translation is the process of turning order to transform translation from an empirical observations in the laboratory, clinic, and commun- phenomenological process into a predictive science. ity into interventions that improve the health of Thus, translational science is clearly a nascent field. individuals and the public, ranging from diagnostics For historical and cultural reasons, translation has and therapeutics to medical procedures and behav- traditionally been practiced as an empirical craft, ioral changes. This definition is intentionally holistic rather than studied as a science. The limits of with regard to directionality, stage of intervention, empiricism in translation are evident in its persis- development, and modality. Biomedical translation tently high failure rate and cost, which have is not a one-step event, but multistep and recursive. continued to increase despite enormous efforts using “Observations” reflect that translation starts with the the empirical paradigm. In this context, a thought initial perception of phenomena, which must be experiment and a counterfactual are useful. The demonstrated to be reproducible and robust. “Labo- thought experiment is to imagine what drug develop- ratory, clinic, and community” reflects the idea that ment would be like if the general principles of small translation need not start with a basic science molecule target interactions were known, such that observation that subsequently moves towards the the activity of any compound on any target could be clinic and ultimately public health; in fact, until predicted effectively a priori. Failures due to unan- relatively recently, most successful translations began ticipated toxicity and lack of efficacy would decrease with a clinical or public health observation that led to by orders of magnitude. basic discoveries. Thus, translation is bidirectional. Like any other science, translational science will The bidirectionality of translation is particularly advance via research, i.e., translational science significant in translational science. With regards to research that seeks to develop an understanding, “interventions”, translation is modality-agnostic; the technology, theoretical principle, or paradigm that translational process is conceptually similar whether will make the development of any therapeutic its intended result is a small-molecule drug, a intervention more efficient and effective. The aggre- biologic, a device, a medical or surgical procedure, gate study of the results of individual translational or a behavioral change such as diet, exercise, or research projects is one approach to elucidating No. 9] Translational science 541

mechanisms of biology, disease, or behavior. Each stage of the translational spectrum builds upon and informs basic research. 2-2-2. Preclinical research. Preclinical research Basic Science Clinical Science connects basic research and human medicine. During (Basic Research) (Clinical Research) this stage, scientists apply fundamental discoveries made in the laboratory or the clinic to further Bidirectionality of Translation understand the basis of a disease or disorder, and From Bench To Bedside then find ways to treat it. Hypothesis testing is From Bedside To Bench carried out using cell or animal models, samples of human or animal tissues, or computer-assisted Fig. 4. Schematic presentation of translational science, bridging simulations of drugs, devices, or diagnostic interac- basic science and clinical science. Bidirectionality, from bench to tions within living systems. bedside, and, from bedside to bench, is essential in translational 2-2-3. Clinical research. Clinical research science. The significance of bidirectionality in translational research termed “bidirectionality of translation” is noted here. includes clinical trials with human subjects to test intervention safety and effectiveness, behavioral and observational studies, outcomes and health services translational science principles; conversely, individu- research, and testing and refining new technologies. al translational research projects validate transla- The goal of many clinical trials is to obtain tional science principles and lead to their progressive regulatory approval for an intervention. advancement. 2-2-4. Clinical implementation. The clinical The relationship among basic research, transla- implementation stage of translation involves the tional research, and clinical research is expressed adoption of interventions into routine clinical care schematically in Fig. 4. Translational research con- for the general population. This stage also includes sists of basic or preclinical research and clinical implementation research to evaluate clinical trial research. Much translational research is preclinical, results and identify new clinical questions and gaps from target validation to filing of an investigational in care. new drug application. However, a great deal of 2-2-5. Public health. In this stage of translation, clinical research is not translational, but rather researchers study health outcomes at the population focused on advancing the fundamental understand- level to determine the effects of diseases and efforts ing of human physiology and pathophysiology. The to prevent, diagnose, and treat them. Findings help intent of all basic research in the biomedical field is guide scientists working to improve interventions or to understand the normal structure and function of develop new ones. living organisms, along with the characteristics and 2-2-6. Translational blocks. Two obstacles or causes of abnormal structure and function, that is, translational blocks that impede efforts to apply disease. By contrast, the intent of translational science to improve human health in an expeditious research is to ameliorate, via physical or behavioral fashion have been identified.1) The first block intervention, the abnormal structures and functions involves the transfer of new understandings of disease of an organism that are causing, or may lead to, mechanisms gained in the laboratory into the disease. Translational science is among the newest development of new methods for diagnosis, therapy, sciences, emerging about 30 years ago to transform and prevention, as well as their initial testing in science and medicine.1)–3) humans. The second block affects the translation of 2-2. Translational science spectrum. The results from clinical studies into everyday clinical translational science spectrum represents each stage practice and healthcare decision making. A system- of research along the path from the biological basis atic approach to addressing these two translational of health and disease to interventions that improve blocks have broad positive effects on health overall. the health of individuals and the public. The At each juncture, along the continuum from basic spectrum is not linear or unidirectional; each stage biomedical research to clinical research and improved builds upon and informs the others. Patient involve- health, it is imperative that national clinical research ment is a critical feature of all stages in translation.6) enterprises have adequate resources infrastructure. 2-2-1. Basic research. Basic research involves The improved health that the public expects in turn scientific exploration that can reveal fundamental for its investment in clinical research depends on 542 K. NAKAO [Vol. 95, clinical and medical coverage policy decisions which which form a general principle of endocrine regula- will allow the fruits of this research to reach every tion, maintaining homeostasis in the whole body. member of society. Hormones are designated as endogenous, intercellu- The clinical research environment is itself part lar, signal-transducing chemical substances. The of the problem. Increasingly encumbered by rising toxic effects of hormones can be essentially ruled costs, prolonged duration to results, inadequate out when their levels are within the physiological funding, mounting regulatory burdens, fragmented range. These general principles in endocrinology and infrastructure, incompatible databases, and a short- metabolism promoted diagnostic and therapeutic age of both qualified investigators and willing study applications shortly after the discovery of hormones. participants.1) Essentially, the same situation con- The first hormone to be purified, synthesized, and fronts translational research in many developed applied to humans was adrenaline, which was countries, including Japan. purified by Dr. Jokichi Takamine as a cardiotonic or hypertensive substance,7) although it was often 3. Translational research on endocrinology used as a hemostatic drug. Its agonists and antago- and metabolism nists are widely applied to treating not only The discovery and purification of hormones in endocrine diseases but also cardiovascular, respira- the 20th century are summarized in Fig. 5. In the tory, and urological diseases. Thus, it should be kept first half of the 20th century, the sites of hormone in mind that the goals of clinical application go production were thought to be the classical endocrine beyond just validation of the original target disease. organs, or glands. However, in the latter half of the 3-1. Translational research on the natriuretic 20th century, it became clear that almost all organs peptide family. The natriuretic peptide family in the body, including the cardiovascular system and consists of three structurally related peptides: ANP, adipose tissue, produce hormones. In addition to BNP, and CNP. ANP has been identified and endocrine signaling, hormone signals are also trans- isolated from rat and human hearts, whereas BNP mitted in a paracrine or autocrine manner. A wide and CNP were isolated from porcine brain.8) The range of diseases are associated with hormone excess biological actions of natriuretic peptides are medi- or deficiency, signal transduction abnormalities, and ated by the activation of two subtypes of membra- hormone resistance, involving the receptor and post- nous guanylyl cyclase (GC), GC-A and GC-B, receptor molecules. Negative feedback systems, leading to intracellular accumulation of cyclic gua-

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

1901 adrenaline 1904“hormone” 1914 thyroxine 1921 insulin 1929 estrone 1935 testosterone 1940 cortisone 1942 ACTH 1953 aldosterone 1959 “radioimmunoassay” 1975 enkephalin 1977 renin 1980 EDRF NO 1984 ANP 1988 BNP 1988 endothelin 1990 CNP 1994 leptin 1999 ghrelin

Fig. 5. Discovery and purification of hormones in the 20th century. After the discovery and purification of hormones in the 20th century, these discoveries have been applied rapidly to clinical practice for diagnostics and therapeutics. No. 9] Translational science 543

Ligands ANP BNP CNP

Receptors Ligand binding Extracellular domain

Protein kinase- Plasma like domain membrane Guanylyl cyclase domain ? Intracellular GTP cGMPGTP cGMP GC-A GC-B Clearance

Fig. 6. Schematic presentation of the natriuretic peptide system, consisting of ANP, BNP, and CNP, and their receptors, GC-A, GC-B, and clearance receptor. The intracellular second messenger is cGMP. nosine monophosphate (cGMP) (Fig. 6).9) The rank failure or cardiac hypertrophy (Fig. 9).13)–15) In order of potency of induction of cGMP production particular, determination of plasma BNP levels has via GC-A is ANP 6 BNP > CNP; for induction; via become a criterion for these conditions in routine GC-B, the order, is CNP > ANP 6 BNP.10) Thus, clinical practice.23) ANP and BNP are both used in ANP and BNP serve as endogenous ligands for the treatment of heart failure.24),25) ANP infusion GC-A, whereas CNP is specific for GC-B. A third therapy (Suntory) was first approved as a drug for natriuretic peptide receptor with no intracellular GC acute congestive heart failure by Pharmaceuticals domain, dubbed the clearance receptor (C-receptor), and Medical Devices Agency (PMDA) in Japan is thought to be engaged in the receptor-mediated (Fig. 10A), and subsequently, BNP injection therapy degradation of natriuretic peptides (Fig. 6).9) was approved by the U.S. Food and Drug Admin- The primary structures of ANP, BNP, and CNP istration (FDA). Currently, ANP infusion therapy in various species are shown in Fig. 7. The primary has reached >30% market share among the drugs amino acid sequence of ANP is highly conserved, given for acute congestive heart failure in Japan whereas BNP differs markedly in molecular size and (Fig. 10B). amino acid sequence among mammals.8) The struc- The posttranslational processing of the human ture of CNP, which has 22 amino acids, is identical in BNP precursor is affected by its O-glycosylation,26) all known mammals (Fig. 7). and plasma ProBNP levels change in human failing The ANP/BNP/GC-A system plays pivotal hearts,27)–30) as well as during renal failure.27),31) roles in the regulation of cardiovascular homeostasis The ANP/BNP/GC-A system in the heart (Fig. 8), as demonstrated by its augmentation in inhibits angiotensin II type 2 receptor-mediated various pathophysiological states such as heart fail- pro-hypertrophic signaling32) and also exerts anti- ure,11)–15) myocardial infarction,16),17) cardiac hyper- hypertrophic effects by inhibiting TRPC6 channel trophy,18),19) and hypertension.20)–22) ANP and BNP activity.33) The ANP/BNP/GC-A system in the are cardiac hormones secreted primarily by the kidney protects podocytes from aldosterone-induced atrium and ventricle of the heart, respectively,15),22) glomerular injury34) and counteracts glomerular and they have strong diuretic, natriuretic, and vaso- injury evoked by aldosterone through p38 mitogen- dilatory activities.11),12),15) Hence, ANP and BNP activated protein kinase inhibition.35) The ANP/ serve as sensitive biochemical markers for heart BNP/GC-A system promotes muscle mitochondrial 544 K. NAKAO [Vol. 95,

ANP 128 α- Rat ANP SLRRSSCFGGR I DRIGAQSGLGCNSFRY 1 28 α - Human ANP SLRRSSCFGGR M DRIGAQSGLGCNSFRY BNP 1 45 Rat BNP SQDSAFRIQERLRLRNSKMAHS S S CFG QKI DRI GAV S R LGC DG LR LF 1 32 Human BNP SPKMVQG S G CFM RKM DRI SSS S G LGC KV LR RH 1 26 Porcine BNP D S G CFG RRL DRI GSL S G LGC NV LR RY 1 35 Bovine BNP ALRGPKMMRD S G CFG RRL DRI GSL S G LGC NV LR RY CNP 122 CNP GLSKGCFGLKLDRIGSMSGLGC

Fig. 7. Primary structures of ANP, BNP, and CNP in various species. BNP exhibits a marked difference in molecular size and amino acid sequence, whereas the structure of CNP is identical in all known mammals.

Suppression of Restenosis Endothelial Regeneration ANP Anti-hypertensive Effect GC-A

GC-A BNP GC-B

Vasodilation GC-B Diuresis GC-A Natriuresis CNP Anti-fibrotic Effect

Anti-hypertensive Effect Promotion of Endochondral Anti-hypervolemic Effect Bone Formation

Fig. 8. Physiological functions of the natriuretic peptide system as an endocrine, paracrine, and autocrine system in the peripheral organs. The natriuretic peptide system is also present in the central nervous system. biogenesis and prevents obesity36) and inhibits brain.8) The half-life of CNP-53 is greater than that hepatic damage and liver fibrosis.37) of CNP.8) Although CNP is the primary natriuretic CNP, the third member of natriuretic peptide peptide in the human brain,8),39) it is also produced family, was first purified from porcine brain.38) The by vascular endothelial cells40)–42) and macro- structure of CNP is identical in all known mammals phages.43) This hormone regulates vascular endothe- (Fig. 7). CNP-53, an endogenous high-molecular lial function and arteriosclerosis via local effects, weight form of CNP, was also isolated from the rather than by acting as a circulating hormone in No. 9] Translational science 545

A B

Concentration of ANP and BNP (pmol/ml)

Lys Met Asp Arg

Ile Ｓｅｒ Ｓｅｒ Ｓｅｒ

Normal Control

Fig. 9. Diagnostic application of ANP and BNP. A: Schematic presentation of an immune-radio-metric assay for BNP. B: Plasma ANP and BNP concentrations in patients with congestive heart failure.

AB Apr-Sep 2006 Japan

furosemide

PDE inhibitor ANP

nitrol 34% nitro- glycerin 12.0% dopamine 15.2% dobutamine 15.4%

Fig. 10. Therapeutic application of ANP and BNP. A: Intravenous drip infusion of ANP improves hemodynamic parameters in patients with congestive heart failure. B: Market share of heart failure drugs in Japan.

peripheral organs.8),44)–46) Substantial amounts of In bone, the CNP/GC-B system stimulates CNP and GC-B are expressed in the vascular wall, endochondral bone formation,2),49)–53) and defects in strongly suggesting the existence of a “vascular this system cause dwarfism2),49),51) with abnormal natriuretic peptide system” (Fig. 8).8),9),39),42),44) The craniofacial growth.54) The CNP/GC-B system in the CNP/GC-B system in the vasculature is implicated bone is also involved in the healing of bone in the regulation of blood pressure and, vascular fractures.55) remodeling and regeneration, implicating it in hyper- The distribution of the natriuretic peptide tension and arteriosclerosis.8),9),47),48) system overlaps with the distribution of the tissue 546 K. NAKAO [Vol. 95,

AB

CNP

CNP -

UD

Fig. 11. Regional distribution and function of the natriuretic peptide system (NPS). A: Regional distribution of the natriuretic peptide system in the human body. B: Antagonistic relationship of the natriuretic peptide system and renin–angiotensin system in the peripheral actions.

reninangiotensin system,8),9),56)–58) prompting us to of each member of the natriuretic peptide family, or examine the functional relationship between the its cognate receptor, have been generated through two (Fig. 11A). We revealed an antagonistic rela- Tg or KO technologies.69)–73) We generated Tg mice tionship between these two systems,8),9) both in their expressing BNP under the control of the serum peripheral and central actions (Fig. 11B).8),9),59)–65) amyloid P (SAP) component promoter, which Furthermore, the natriuretic peptide system has targets hormone expression to the liver.71) BNP-Tg therapeutic implications for vascular regeneration in mice exhibited a 100-fold increase in plasma BNP patients with arteriosclerosis obliterans.48) concentrations, with concomitant elevation of plasma Because of the markedly short stature of CNP- cGMP concentrations. These mice had significantly KO mice, we analyzed food intake and energy lower blood pressure and smaller hearts than non-Tg expenditure of mice generated by crossing CNP- littermates.71) These results indicated that BNP KO mice with a strain with chondrocyte-targeted functions in long-term cardiovascular regulation CNP expression; this rescued marked skeletal and may be useful as a long-term therapeutic agent. dysplasia, allowing us to investigate the significance In addition, proteinuria and renal dysfunction of CNP with minimal influence of skeletal pheno- observed in anti-glomerular basement membrane types. This study unveiled a new role for CNP in nephritis,74) nephrosclerosis induced by subtotal regulating food intake and energy expenditure.66) In nephrectomy,75) and manifestations of diabetic addition, we reported that intracerebroventricular nephropathy76) were ameliorated in BNP-Tg mice administration of CNP suppresses food intake via relative to those in wild-type mice, indicating a activation of the melanocortin system in the possible application of GC-A agonists, ANP or BNP, hypothalamus in mice.67) We also demonstrated in the treatment of renal diseases. that brain-specific GC-B deletion attenuated high We also generated mice bearing targeted dis- fat-induced visceral and hepatic lipid deposition in ruption of the BNP gene.72) At baseline, BNP-KO mice.68) These results clearly indicate that the CNP/ mice did not exhibit any signs of systemic hyper- GC-B system, as a neuropeptide in the brain, is tension or ventricular hypertrophy. However, these involved in negative regulation of food intake and animals developed multifocal fibrotic lesions within energy expenditure. the cardiac ventricle even in the absence of additional 3-2. Mice with genetic alterations in the stresses; these lesions increased in size and number ANP/BNP/GC-A system. Genetically engineered in response to ventricular pressure overload, demon- mice are useful tools for studying the complex strating that BNP is an anti-fibrotic factor acting phenotypic effects of an altered gene in living within the ventricle of the heart as an autocrine/ animals. Mice harboring overexpression or deficiency paracrine regulator to promote ventricular remodel- No. 9] Translational science 547 ing.72) In addition to these cardiovascular manifes- scription factor-A is a common mediator of mechani- tations, BNP-Tg mice exhibited marked skeletal cal stress and neurohumoral stimulation-induced overgrowth via endochondral bone formation.77) cardiac hypertrophic signaling leading to activation Nevertheless, BNP-KO mice did not possess any of BNP gene expression.89) skeletal abnormalities.72) The skeletal overgrowth 3-3. Genetically engineered mice and rats of seen in BNP-Tg mice expressing elevated plasma the CNP/GC-B system. We generated mice with concentrations of BNP was similar to that seen in a targeted disruption of the CNP gene; the resultant cartilage-specific CNP-Tg mice.78) Given that mice CNP-KO mice exhibited markedly short stature due with genetic disruption of the ANP/BNP/GC-A to impaired bone growth.49) Mammalian bones are system do not have any abnormal skeletal pheno- formed through two different mechanisms, endochon- types,72),79) we postulated that the markedly increas- dral ossification and membranous ossification. Most ed circulating levels of BNP (100-fold relative to mammalian bones are formed through endochondral wild-type mice) may cross-react with GC-B to ossification, during which chondrocytes in the growth stimulate endochondral bone growth, even though plate undergo proliferation, hypertrophy, cell death, the affinity of BNP for GC-B is lower than that for and osteoblastic replacement.50) GC-A.9) This interpretation was supported by the The short stature phenotype of CNP-KO mice finding that the skeletal overgrowth observed in resulted from impairment of bone growth through BNP-Tg mice was not abrogated by a genetic endochondral ossification.49) CNP-Tg mice with deficiency of GC-A in BNP-Tg mice.78) targeted overexpression of CNP in the growth plate ANP-Tg mice, which express elevated levels of cartilage exhibited prominent overgrowth of bones circulating ANP under the control of a mouse formed through endochondral ossification.49) GC-B- transthyretin promoter, exhibited decreased arterial KO mice exhibited the same short stature pheno- blood pressure without the induction of diuresis or type90) as CNP-KO mice,49) demonstrating that the natriuresis.69) ANP-KO mice and GC-A-KO mice CNP/GC-B system is a physiologically important exhibited salt-sensitive70) and salt-resistant hyper- stimulator of endochondral bone growth. Dominant- tension,73) respectively. Studies of GC-A-KO mice negative GC-B-Tg rats exhibited blood pressure- implicated GC-A in anti-hypertrophic actions in the independent cardiac hypertrophy, suggesting that heart.79)–84) A more detailed analysis of GC-A GC-B signaling is linked to the control of cardiac performed using conditional KO mice indicated the growth.91) importance of GC-A in vascular endothelial cell- cGMP dependent protein kinase (cGK) has been mediated blood pressure regulation.82)–84) identified as a molecule activated downstream of the As for the regulation of ANP and BNP gene natriuretic peptide family and GC system.92) Mice expression, neuron-restrictive silencer elements with a gene depletion of one subtype of cGK, cGKII- (NRSEs) are located in the 5B-flanking region of the KO mice, have a short stature phenotype secondary BNP gene and the 3B-untranslated region of the ANP to impaired endochondral bone growth,93) similar to gene.85) Neuron-restrictive silencer factor (NRSF) the phenotype of CNP-KO mice.49) We demonstrated can thus repress ANP promoter activity through that cGKII affected endochondral bone growth by binding to NRSE.86) Studies examining mice express- functioning downstream of the CNP/GC-B system, ing dominant-negative NRSF-Tg under the control of by showing that impaired endochondral bone growth an alpha-myosin heavy-chain promoter have demon- observed in cGKII-KO mice could not be rescued by strated that NRSF plays an important role in the targeted overexpression of CNP in the growth plate gene expression of ANP and BNP, as well as in the cartilage.94) progression of cardiac dysfunction and lethal ar- Multiple spontaneous animal models with im- rhythmia associated with heart failure.87) We also pairments in the CNP/GC-B system have been crossed dominant-negative NRSF-Tg mice with GC- identified.95)–98) Two strains of dwarf mice, with A-KO mice to assess the effects of endogenously autosomal recessive mutant genes, named cn/cn95) expressed ANP and BNP during the progression of and short-limbed dwarfism (SLW),96) have sponta- cardiomyopathy, demonstrating that endogenous neous loss-of-function mutations in the GC-B gene. ANP and BNP protect the heart against cardiomyo- Spontaneous mutant mice with a loss-of-function cyte death and the progression of pathological mutation in the CNP gene, named long bone remodeling in a mouse model of dilated cardiomyop- abnormality (lbab) mice, exhibit short stature owing athy and sudden death.88) Myocardin-related tran- to impaired endochondral bone growth,98) and this 548 K. NAKAO [Vol. 95,

AB C

Wt mechanism

CNP-Tg

FGFR3-Tg

CNP/FGFR3-Tg

Fig. 12. Clinical implication of the CNP/GC-B system in achondroplasia (Ach). A: A human patient with achondroplasia with a constitutively active mutation of FGFR3. B: Effect of CNP on body length, based on mating of achondroplasia model mice a with CNP-Tg mice. C: Molecular mechanism of the therapeutic effect of CNP in achondroplasia. phenotype can be abrogated by targeted overexpres- in CNP-KO mice, because such abnormalities can sion of CNP in the growth plate cartilage.99) induce neurological damage.54),102) Cartilage-specific CNP-KO mice100) have a 3-4. Clinical application of CNP and its defect in endochondral bone very similar to that of analogs for skeletal dysplasia. To explore the phenotype of systemic CNP-KO mice.49) Cartilage- potential clinical applications of CNP and its specific KO of the GC-B gene100) also results in an analogs, we tested the strong effect of the CNP/ essentially identical phenotype to that of systemic GC-B system on endochondral bone growth in the KO of CNP,90) whereas the disorder of endochondral context of skeletal dysplasia, a group of genetic bone formation in cartilage-specific GC-B-KO mice is disorders characterized by severely impaired bone more severe than that of cartilage-specific CNP-KO growth.103) Achondroplasia (Ach), the most common mice.100) form of skeletal dysplasia, is characterized by short- The blood pressure of endothelium-specific limbed dwarfism, (Fig. 12A) and is caused by a CNP-KO mice is significantly higher than that of constitutively active mutation in fibroblast growth wild-type mice, whereas the blood pressure of smooth factor (FGF) receptor 3 (FGFR3).104) Current muscle cell-specific GC-B-KO mice did not differ therapy for Ach is limited to distraction osteo- from that of wild-type mice.47) This finding indicates genesis,105) an orthopedic procedure; no efficient drug that GC-B in vascular endothelial cells, rather than therapies have yet been developed. vascular smooth muscle cells, is responsible for the We demonstrated that targeted overexpression control of blood pressure. of a CNP transgene in the growth plate cartilage of a To rule out species-related differences in the mouse model of Ach rescues their impaired bone physiological function of the CNP/GC-B system, we growth and short stature phenotype (Fig. 12B).51) generated systemic CNP-KO rats.101) We confirmed To elucidate the molecular mechanism by which an identical short stature phenotype due to defective CNP ameliorates Ach, we examined the effects of endochondral bone formation in CNP-KO rats, CNP on extracellular signal-regulated kinase (ERK) although unlike the mouse CNP-KO model, the signaling. CNP inhibited FGF2-stimulated phospho- knockout rats did not die early.49) Studies are rylation of ERK in a dose-dependent manner through ongoing to clarify the causes and mechanism under- cGMP activation via GC-B (Fig. 12C), ultimately lying the species-related difference underlying early increasing matrix synthesis by chondrocytes.51) death. It might be explained in part by craniofacial To determine whether chronically elevated deformities, including defects in the foramen magna, plasma CNP reaches the growth plate and stimulates No. 9] Translational science 549

AB Gross Appearance Growth Curve

Wt + vehicle

Ach + vehicle

Ach + CNP

After 4-week administration starting at 3-weeks-old

Fig. 13. Therapeutic effect of intravenous infusion of synthetic CNP on achondroplasia (Ach) model mice. A: Gross appearance of achondroplasia model mice treated with CNP by intravenous infusion. B: Growth curves of achondroplasia model mice treated with CNP by intravenous infusion. The growth of CNP-treated achondroplasia model mice caught up with that of wild-type (Wt) mice. endochondral bone formation, we generated CNP-Tg 3-5. Clinical application of CNP and CNP mice that overexpressed CNP in the liver. Circulating analogs for diseases other than skeletal dysplasia. CNP stimulated skeletal growth,106),107) indicating Idiopathic short stature, a common disease of short that CNP injection therapy may be an effective stature phenotype with an unknown etiology, and treatment. bone fracture that heals through endochondral We also demonstrated that systemic and con- ossification,55) are the next likely avenues in which tinuous administration of synthetic CNP is an a therapeutic effect of CNP and its analogs might be effective and safe way to reverse the impaired bone discovered. In addition, CNP and CNP analogs will growth of Ach mice (Fig. 13).108) The efficacy and be useful for treating vascular remodeling and safety of systemic CNP administration in preclinical arteriosclerosis via vascular endothelial cells and studies,108) together with the observation that intra- macrophages, as well as chronic inflammation via venously injected CNP has only a minimal effect on macrophages.2),47),48) Because the CNP/GC-B system blood pressure in humans,109) implies that the is involved in the negative regulation in food intake systemic administration of CNP or CNP analogs and energy expenditure, non-peptide agonists for represents a novel therapeutic strategy for treating GC-B may be used for the treatment of obesity, human skeletal dysplasia, including Ach. One form diabetes mellitus, and steatohepatitis.36),37),66)–68) of human skeletal dysplasia, acromesomelic dysplasia type Maroteaux, is caused by loss-of-function muta- 4. Translational research on leptin tions in the GC-B gene,110) which cannot be treated Leptin, an adipocyte-derived hormone, has been with CNP or CNP analogs.110) These results clearly demonstrated to cause an overt obese phenotype indicate that the CNP/GC-B system is a physiolog- upon deletion in both animals and humans. Leptin ically important enhancer of endochondral bone was originally identified as a hormone absent from growth in humans, suggesting that CNP and CNP hereditary obese (ob/ob) mice.112) This hormone analogs could be applied to multiple types of human plays crucial physiological roles in the regulation of skeletal dysplasia.2),51)–53) energy expenditure and food intake.113)–117) Mice118) A clinical trial of a neutral endopeptidase- and rats119) harboring mutations in leptin receptors resistant CNP analog, aimed at receiving FDA exhibit phenotypes identical to those of ob/ob mice. approval, is now underway in multiple countries The Koletsky rat, an obese sub-strain of SHR that including Japan.111) serves as a model of metabolic syndrome with both 550 K. NAKAO [Vol. 95,

Obesity Lipodystrophy leptin cDNA coding sequence was designed in order to target hormone expression to the liver.142) Over- Abdominal Fat expression of leptin in the liver resulted in complete Ectopic Fat disappearance of both white and brown adipose tissues in mice.141) However, this phenotype did not Adipokines occur when transgene expression was targeted to Insulin Resistance adipose tissue, the endogenous site of leptin produc- tion, using adipocyte-specific promoters.143) The Dyslipidemia hyperleptinemia seen in these transgenic “skinny” Hypertension mice provides a unique experimental system, in which the long-term effects of leptin can be inves- Fig. 14. Comparison of phenotypes between obesity and gener- tigated.132)–135),141),144)–146) Skinny mice exhibit aug- alized lipodystrophy. The two diseases exhibit similar pheno- mented glucose metabolism and increased insulin types in glucose and lipid metabolism, although there is a 141) striking contrast between them in adiposity and levels of sensitivity in both skeletal muscle and liver, adipocyte-derived serum adipokines levels. supporting the idea that leptin acts as an anti- diabetic hormone in vivo.147)–149) These studies sug- gest that leptin could be useful treating diabetes and hypertension and morbid obesity, carries an addi- obesity. tional nonsense mutation in the leptin receptor.120) 4-2. Crossbreeding of transgenic skinny mice In obese animals and patients, plasma or serum with A-ZIP/F-1 mice, a mouse model of severe leptin concentrations are increased in proportion to lipoatrophic diabetes. Generalized lipodystrophy the degree of adiposity,121)–123) indicating that leptin with a systemic deficiency of adipose tissue, is is a satiety signal communicating the size of adipose characterized by severe insulin resistance and hyper- stores to the brain,124)–126) and that leptin resistance triglyceridemia.150) A form of diabetes called lipo- is related to obesity.121),127)–129) Leptin deficiency in atrophic diabetes, eventually develops, although the human patients is associated with morbid obesity precise mechanism by which this paucity of fat and insulin resistance, indicating that leptin plays results in diabetes remains to be elucidated. Plasma a physiological role in both animal models and leptin concentrations are markedly reduced or absent humans.130),131) Leptin is implicated in a number in patients with lipoatrophic diabetes, as well as in of phenotypes observed in obese animal mod- rodent models of this disease.151)–154) Given the els,125),132)–135) especially obesity-related hyperten- antidiabetic action of leptin, its deficiency may play sion,133) abnormal reproduction,132) bone changes,134) a role in the pathogenesis of lipoatrophic diabetes; and Cushing’s syndrome.136) thus, leptin may be useful as a drug to treat Figure 14 compares the phenotypes of obesity lipoatrophic diabetes. and generalized lipodystrophy. The two diseases A mouse model of severe lipoatrophic diabetes have similar phenotypes related to glucose and lipid (A-ZIP/F-1) was generated, by expressing a protein metabolism, although there is a striking contrast that inactivates basic-zipper transcription factors in between them in adiposity and serum adipokine adipose tissue.152) To assess the pathophysiological levels: too high in obesity vs. almost absent in role and therapeutic potential of leptin in lipoatrophic generalized lipodystrophy. This underscores the diabetes, we crossed transgenic skinny (leptin Tg/D) importance of maintaining normal adiposity in order and A-ZIP/F-1 (A-ZIP Tg/D) mice to yield double- to preserve health.2),137),138) transgenic mice (leptin Tg/D: A-ZIP Tg/D) that lack Leptin is also produced by the human placen- adipose tissue and express approximately 10-fold ta139) and choriodecidual tumors.140) The functional higher leptin levels than controls (Fig. 15A).155) significance of leptin in pregnancy and tumors Leptin Tg/D: A-ZIP Tg/D mice were hypophagic remains unclear. relative to A-ZIP Tg/D mice and exhibited decreased 4-1. Generation of Tg mice overexpressing hepatic steatosis. Glucose and insulin tolerance tests leptin. To explore the clinical implications of leptin revealed increased insulin sensitivity and normal in vivo, we generated leptin-Tg “skinny” mice with glucose tolerance in leptin Tg/D: A-ZIP Tg/D mice, elevated plasma leptin concentrations similar to similar to those in leptin Tg/D mice. Pair-feeding those in obese humans.141) A fusion gene consisting experiments demonstrated that the effects of leptin of the human SAP promoter upstream of the mouse were not solely due to decreased food intake. Leptin No. 9] Translational science 551

A B that leptin is therapeutically useful in the treatment 155) (mg/dl) Plasma Glucose (ng/ml) Plasma Insulin of lipoatrophic diabetes. 400 * 25 4-3. Leptin-replacement therapy in Japanese * 300 20 patients with generalized lipodystrophy. We 15 previously reported a novel homozygous mutation 200 of the melanocortin 4 receptor (MC4R) in a Japanese 10 100 * woman with severe obesity (body mass index [BMI] * 5 2 156) * * 62 kg/m ). MC4R mutations have been identified 0 0 in morbidly obese patients in Europe at a relatively +/+ A-ZIPTg/ʶ +/+ A-ZIPTg/ʶ high frequency (3–4%); all of the mutations reported LepTg/ʶ Lep/A-ZIP LepTg/ʶ Lep/A-ZIP to date occur in an autosomal-dominant fashion, 15-week-old male *Pʽ0.005 vs +/+ with the exception of a single unique pedigree in the Fig. 15. Phenotypic effects of cross-mating of generalized lipo- U.K.157),158) Although both parents were heterozygous dystrophy model mice (A-ZIP Tg/D) with leptin-Tg skinny mice /D ff for the mutation, neither exhibited such a severe (Lep Tg ). A: Therapeutic e ects of leptin on plasma glucose / 2 / 2 levels in generalized lipodystrophy model mice. B: Therapeutic obese phenotype (BMI 27 kg m and 26 kg m , pre- effects of leptin on plasma levels of insulin in generalized obese according to WHO criteria and, grade 1 obesity lipodystrophy model mice. Doubly transgenic mice (Lep/A-ZIP) in Japan). Given that genetic backgrounds and had normal glucose metabolism and levels of insulin. lifestyle vary significantly between European and Asian countries, it is necessary to examine the effect of lifestyle on phenotypes resulting from genetic muta- also helped to prevent diabetic nephropathy in tions, as well as treatment efficacy, in each country. generalized lipoatrophic diabetes mice.135) These The first case for leptin replacement therapy results demonstrated that leptin can improve insulin with metreleptin (Shionogi in Japan) was an 11-year- resistance and diabetic manifestations in a mouse old girl with acquired generalized lipodystrophy, model of severe systemic lipodystrophy, indicating seen 6 months after onset (Fig. 16A).159),160) Leptin

A B May 2000 April 2001 Fasting glucose

Leptin

11-year-old girl Leptin Replacement Therapy (days)

75g Oral GTT Glycohemoglobin C Blood Glucose

Before

After 3 months

Time (min) Leptin Therapy (months)

Fig. 16. Dramatic effects of leptin replacement therapy on glucose metabolism in the first case, an 11-year-old girl with acquired generalized lipodystrophy. A: Pictures before and 1 year after onset. B: Fasting plasma glucose normalized within 1 week of starting leptin replacement therapy. C: Oral glucose tolerance test (75 g) and HbA1c concentration also normalized within 3 months. 552 K. NAKAO [Vol. 95,

AB (mg/dl) Fasting glucose (mg/dl) (%) HbA1c (%) 250 n=6 10 200 8 n=6 n=3 n=6 n=5 n=4 n=4 n=2 n=2 150 n=6 n=4 n=4 n=3 6 100 n=5 n=2 n=2 4 50 2 0 0 Baseline 24812182436 Baseline 24812182436 Leptin treatment (months) Leptin treatment (months) CD (mg/dl) Triglyceride (mg/dl)(L) Liver volume (L) 1000 2.0 n=6 n=6 800 1.5 n=6 600 n=5 n=4 n=4 n=3 1.0 n=2 n=2 400 200 n=6 n=5 n=4 n=4 n=3 n=2 n=2 0.5 0 0 Baseline 2 4 8 12 18 24 36Baseline 2 4 8 12 18 24 36 Leptin treatment (months) Leptin treatment (months)

Fig. 17. Chronic leptin replacement therapy improved fasting plasma glucose levels (A), HbA1c (B), plasma triglyceride levels (C), and liver volumes (D). replacement therapy dramatically normalized fasting confirmed by CT imaging (Fig. 17D). By 4 months, glucose levels within 1 week (Fig. 16B), and also six of seven patients were able to discontinue all normalized the 75 g oral glucose tolerance test and antidiabetic drugs, including insulin. The reductions HbA1c within 3 months (Fig. 16C). The dramatic in fasting plasma glucose levels, HbA1c, triglyceride effects of leptin replacement therapy on glucose and levels, and liver volumes in all seven patients were lipid metabolism without any ill effects in the first maintained throughout the therapeutic period with case accelerated clinical studies in Japan. no adverse effects (Fig. 17). The longest period of Four months of leptin replacement therapy leptin replacement therapy had extended beyond 7 was reported to improve glucose and lipid me- years,160) and has now reached 18 years for the first tabolism in lipodystrophy patients in the U.S.161) case. Functional magnetic resonance imaging analy- To elucidate the efficacy, safety, and mechanisms sis clearly demonstrated changes in food-related underlying leptin replacement therapy in Asian brain activity in patients with generalized lipodys- patients with generalized lipodystrophy, we treated trophy before vs. after leptin replacement therapy.162) seven Japanese patients, two acquired and five Leptin replacement therapy was also effective at congenital, with a physiological replacement dose of combating diabetic complications.2),160) The macro- leptin (Fig. 17).159),160) Leptin-replacement therapy albuminuria seen in two patients regressed to micro- significantly improved fasting glucose (mean ’ SE, albuminuria, whereas microalbuminuria in two other 172 ’ 20 to 120 ’ 12 mg/dl; P < 0.05) (Fig. 17A), patients normalized. The creatinine clearance of and triglyceride (mean ’ SE, 700 ’ 272 to 260 ’ patients with glomerular hyperfiltration decreased, 98 mg/dl; P < 0.05) levels (Fig. 17C) within 1 week concomitant, with improved glucose tolerance and normalized HbA1c within 2 months (Fig. 17B). (Fig. 19), consistent with previous findings in mice Leptin replacement therapy decreased insulin resist- models of lipoatrophic diabetes (Fig. 20).135) ance, as demonstrated by the euglycemic clamp We also examined the effects of the leptin method (Fig. 18). Improvement in fatty liver was replacement therapy in a 16-year-old girl with severe also confirmed by changes in computed tomography hypertriglyceridemia (>1,000 mg/ml; approximately (CT) attenuation, and reduction of liver volume was 8,000 mg/ml at the peak) who suffered from repeated No. 9] Translational science 553

Glucose Infusion Rate 7 n=5 6 n=6 n=5 n=4 5 n=7 4 n=7 3 n=7 2 (mg/kg/min) 1 0 Before 1246812 Leptin Treatment (months)

Fig. 18. Leptin replacement therapy gradually improved insulin sensitivity in patients within 4 months examined using a monthly glucose clamp test.

Urinary Albumin (mg/day) 900 8 (%)

7 HbA1c 600 6 300 5

0 4 Leptin Treatment Before 12 34 (months) 205 138 62 70 CCr (ml/min) 170

Fig. 19. Leptin replacement therapy improved microalbuminuria and also improved the glomerular hyperfiltration rate in a patient with diabetic nephropathy complicated with generalized lipodystrophy. episodes of acute pancreatitis. After the initiation of higher in Japan, a country where the prevalence of leptin therapy, her triglyceride levels normalized and obesity is relatively low, than in countries with a high she did not have any additional episodes of acute prevalence of obesity.2),160) The change in satiety pancreatitis.2),160) These results clearly demonstrate sense due to the leptin replacement therapy could be the striking efficacy and safety of long-term leptin- recognized within a few days in adult patients, replacement therapy in patients with generalized although it was less clear in children.159),160) lipodystrophy.2),160) Although these results are im- Chronic leptin replacement therapy for general- pressive, it is important to remember that the efficacy ized lipodystrophy patients induced no significant of leptin-replacement therapy appears to be much changes in systolic and diastolic blood pressures 554 K. NAKAO [Vol. 95,

Development of diabetic nephropathy leptin Prevention of diabetic nephropathy A-ZIP/F-1 mice leptin Reversal of diabetic nephropathy

Fig. 20. Schematic presentation of the effects of leptin replacement therapy in generalized lipodystrophy model mice on prevention for diabetic nephropathy in mice mated with leptin-Tg skinny mice, and its amelioration after onset of diabetic nephropathy.

Case 1 Case 4 Bone Mineral Case 1 Case 4 Blood Pressure Case 2 Case 5 Density Case 2 Case 5 Case 3 Case 6 ) (mmHg) 2 Case 3 160 1.5 140 Systolic BP 120 1.0 100 80 60 0.5 40 20 Diastolic BP 0 0 Bone mineraldensity (g/cm 0481218243036 Before 2 4 8 12 18 24 36 Leptin Treatment (months) Leptin Treatment (months)

Fig. 21. Chronic leptin replacement therapy did not induce any Fig. 22. Chronic leptin replacement therapy did not induce any fi significant changes in systolic or diastolic blood pressure in signi cant changes in bone mineral density in patients with patients with generalized lipodystrophy. generalized lipodystrophy.

(Fig. 21), or bone mineral density (Fig. 22). Because trophy (Case 1, BMI: 16.9 kg/m2, leptin: 6.5 ng/ml, the generalized lipodystrophy patients examined in Case 2, BMI: 17.1 kg/m2, leptin: 3.5 ng/ml) in clinical research were younger than 50 years, the Japan,166) suggesting that the efficacy of leptin effects of chronic leptin replacement therapy on blood therapy in partial lipodystrophy depends on BMI pressure and bone mineral density should be followed and plasma leptin concentrations. BMI and plasma up carefully. leptin concentrations are known to reflect the degree 4-4. Leptin therapy for partial lipodystro- of adiposity.121)–123) A highly sensitive leptin assay phy. Leptin therapy for partial lipodystrophy has method (Cosmic Corporation) was recently approved been approved only in Japan.163) The first use of as a diagnostic for lipodystrophy by PMDA in Japan leptin therapy for partial lipodystrophy, a 28-year- (Table 1). No neutralizing antibodies were reported old woman (BMI: 21.3 kg/m2, leptin: 2.1 ng/ml) with in these patients, but the appearance of neutralizing familial partial lipodystrophy, was effective and safe antibodies should be kept in mind as a possible for more than 10 years (Fig. 23); recently, the patient consequence of chronic leptin administration with was able to have a baby, which she had strongly metreleptin in partial lipodystrophy. desired. In addition, patients with atypical partial 4-5. Leptin therapy for more prevalent forms lipodystrophy have been reported in Japan, as well of diabetes. To assess the therapeutic potential as in other countries.164)–166) Leptin therapy was also of leptin therapy in insulin-deficient diabetes, we effective and safe in two cases of partial lipodys- generated diabetic animal models by treating wild- No. 9] Translational science 555

A C

B

Fig. 23. Therapeutic effects of leptin therapy on partial lipodystrophy. A: MRI (T1) image of a 28-year-old female patient with familial partial lipodystrophy disclosed presence of the adipose tissue in the head region, bone marrow and sole and loss of the subcutaneous adipose tissue in both upper and lower extremities. B: Leptin therapy improved glucose tolerance, and HbA1c decreased from 6.7% to 5.3%, for 2 months in a patient with familial partial lipodystrophy. C: Lateral view of a patient with partial lipodystrophy like Cushing’s syndrome. type and leptin-Tg mice with a relatively low dose with increased adiposity,169) generated using a of streptozotocin (STZ) (180 g/g body weight).167) combination of a low-dose (STZ120 g/g body weight) Plasma insulin concentrations were reduced (<0.10 and a high-fat diet (HFD; 45% of energy as fat) ng/ml), resulting in severe hyperglycemia in both (STZ/HFD). In STZ/HFD mice, continuous infusion wild-type and leptin-Tg mice 2 weeks after STZ of leptin (20 ng/g body weight/hour) reduced food treatment. Leptin-Tg mice were more sensitive to intake and body weight gain, and also improved exogenously administered insulin than wild-type glucose and lipid metabolism which enhancing insulin mice. STZ-treated leptin-Tg mice became normogly- sensitivity. Leptin therapy also decreased triglyceride cemic at doses of insulin that did not improve content in both liver and skeletal muscle. These hyperglycemia in STZ-treated wild-type mice. To results indicate a beneficial effect of leptin therapy determine whether combination therapy with leptin for type 2 diabetes mellitus with increased adiposity, and insulin is beneficial for insulin-deficient diabetes, which corresponds to BMI in the range of 2530 we also examined the effect of chronic co-admin- kg/m2, pre-obese according to WHO criteria or istration of leptin and insulin in STZ-treated wild- grade 1 obesity in Japan.169) type mice. The results revealed that sub-threshold Our previous and ongoing studies on leptin-Tg doses of insulin that do not affect glucose homeo- skinny mice and other mouse animal models, have stasis, in combination with leptin, are effective at revealed pleiotropic actions of leptin in the improving diabetes in STZ-treated wild-type mice. regulation of energy homeostasis and food in- We also demonstrated that leptin therapy is useful take,132)–135),141),144),145),170) and suggested that leptin for the treatment of diabetic complications and might be clinically useful as a therapy for a increases longevity in insulin-deficient diabetic variety of diseases, particularly diabetes melli- mice.168) These results indicate that leptin therapy tus,144),155),159),160),168) depression with obesity,171) may be used as an adjunct to insulin therapy in and steatohepatitis.172),173) Leptin-Tg skinny mouse insulin-deficient diabetes.167),168) could be a useful model for studying the long-term We also investigated the therapeutic usefulness effects of leptin action in vivo and evaluating the of leptin in a mouse model of type 2 diabetes mellitus clinical implications of the therapy.2) 556 K. NAKAO [Vol. 95,

To further assess the target validation of Treatment of congestive heart failure by drip infusion diabetes mellitus beyond species-specificdifferences, of ANP, as performed in Japan corresponds to the rat models for obesity and general lipodystrophy “Clinical Implementation” step on the translational were established via N-ethyl-N-nitrosourea muta- science spectrum. genesis of the leptin gene174) and the seipin gene,175) The POC of CNP therapy for Ach is based on respectively. Steatohepatitis in ob/ob mice was the observation that endochondral bone formation in aggravated by pioglitazone and rosiglitazone treat- the growth plate is disrupted in CNP-KO mice and ment, but improved in leptin-KO obese rats, being accelerated in CNP-Tg mice.2),49) Disrupted endo- consistent with the beneficial effects of these com- chondral bone formation in an Ach model mouse is pounds on steatohepatitis in humans.174) Thus, improved by cross-mating with CNP-Tg mice or by mouse disease models are not always the suitable CNP infusion.51)–53),107) CNP therapy for patients for studying human disease. with Ach corresponds to the “Clinical Research” step 4-6. Drugs for leptin resistance. The on the translational science spectrum. prevalence of obesity and its associated lifestyle- The POC of leptin-replacement therapy for related diseases, including metabolic syndrome, is patients with lipoatrophic diabetes or generalized increasing worldwide. Hyperleptinemia and central and partial lipodystrophy is derived from basic and leptin resistance or insufficiency of leptin function are preclinical research using animal models, including the main features of obesity, suggesting that leptin mice2),138),155) and rats,174) which emulate human sensitizers represent promising target molecules for generalized lipodystrophy remarkably well. In addi- drug discovery and treatment of obesity. Amylin and tion, the serum leptin concentration is markedly glucagon-like peptide-1 (GLP-1) agonists are pep- reduced or almost absent in patients with generalized tide-nature leptin sensitizers.176),177) The naturally lipodystrophy, and low to normal in patients with occurring phytochemicals celastrol and withaferin partial lipodystrophy. Leptin replacement therapy is A, both identified in thunder god vine, are leptin effective and safe for patients with generalized and sensitizers.178)–180) Leptin-responsive cell lines from partial lipodystrophy. Leptin replacement therapy adult mouse hypothalamus are good tools for study- was approved for the first time by the PMDA in ing the molecular mechanism of leptin sensitizers.181) 2014. Therefore, leptin replacement therapy corre- sponds to the “Clinical Implementation” step on the 5. Translational science lessons from translational science spectrum. translational research on the In medicine or clinical practice, patients are natriuretic peptide family genetically heterogeneous, whereas the experimental and leptin animals used in basic research and preclinical ANP and BNP concentrations increase in research are strains, i.e., genetically homogeneous. parallel with the severity of congestive heart failure, The differences in genetic background between suggesting a diagnostic and therapeutic application disease model animals and human patients is critical in the context of this disease.2),8),11)–15) The increase for the evaluation and interpretation of results in plasma BNP concentration in patients with severe regarding physiological and pharmacological func- congestive heart failure is three orders of magnitude tions obtained from basic and clinical research. In higher than normal plasma,2),8),13)–15) and the source basic research, lower animals such as yeast, C. ele- of plasma BNP is the ventricle of the heart.2),8),13) gans, Drosophila, and zebrafish are used as model Therefore, the measurement of plasma BNP concen- animals. The distribution and primary structures of tration is an indispensable diagnostic method in protein and peptide hormones, their receptors, and clinical practice, corresponding to the “Public Health” signal transduction molecules, as well as the final step on the translational science spectrum. Because products of biosynthesis of non-protein, or non- the half-life of plasma BNP is longer than that of peptide hormones, differ significantly between lower ANP,2),8),13) BNP is administered by the bolus animals and humans. In particular, the primary injection method in the U.S.A.; ANP is administered structure and tissue distribution of BNP and post- by the drip infusion method in Japan.24) For treat- translational processing of the BNP precursor exhibit ment of congestive heart failure2),8),24),25) and renal marked species-differences (Fig. 7).2),8),39) BNP was failure without unstable hemodynamic changes, drip first discovered in the porcine brain,8),38) but very infusion of ANP and BNP is much safer than bolus little BNP is detectable in the brains of rodents and injection of BNP, which is impossible to adjust. humans.2),8),39) Thus, on the “Basic Research” and No. 9] Translational science 557

CNP Therapy Leptin Therapy Selection and analysis of appropriate animal disease models emulating human diseases are essen- Achondroplasia Lipodystrophy tial for successful clinical applications. To this end, selection of suitable animal models must be based on multifaceted clinical experience and substantial Idiopathic Dwarfism Diabetes Mellitus knowledge of the target human diseases. The original Bone Fracture Metabolic Syndrome research targets may well be rare diseases. More importantly, one can hope and try to expand the Fig. 24. Clinical applications, from rare diseases to common scope of the research into common diseases with the diseases, originating in translational research on CNP and “ ” leptin. aid of clinical wisdom . Clinical wisdom may be derived from multiple bedside experiences and sub- stantial knowledge in human physiology and patho- physiology. “Preclinical Research” steps of the translational Clinical investigators or physician-scientists (or science spectrum, translational scientists should clinician-scientists) who are familiar with both basic carefully consider the possible existence of species- science and clinical science, which is bidirectional, related differences, from the standpoints of both should become authentic translational scientists. efficacy and safety. There is no question that close and precise collabo- In translational research on ANP as a drug for ration among basic scientists, translational scientists, congestive heart failure,2),8),24) and on ANP and BNP and clinical scientists is essential for advancing as diagnostics for congestive heart failure,2),8),13)–15) translational science, not only in Japan, but all over the disease target of our translational research was the world. a common disease, whereas the POC of CNP therapy was established for Ach, and that of leptin replace- 6. Voyage into the uncharted seas of ment therapy was established for patients with translational science lipodystrophy, both of which are rare diseases with The author was born and grew up in a rural, a very limited number of patients (Fig. 24). It is very doctorless village environment and enjoyed insect likely that drugs that are efficacious and safe for the collecting and had a vague dream of becoming a treatment of rare human diseases will provide us medical doctor or scholar. A famous historical with novel treatments for common human diseases, person from the author’s hometown, Mr. Kunimichi which are adjacent to or extensions of rare human Kitagaki, was a patriot at the end of the Edo period, diseases in both etiology and pathophysiology. became the Governor of Kyoto, and founded the Lessons from the research history of SHRs as a Lake Biwa Canal in 1890, which enabled Japan to useful animal model for essential hypertension, in build its first hydroelectric power plant. As a medical which almost all antihypertensive drugs for human student at Kyoto University, the author visited the essential hypertension were known to be efficacious, late Professor Osamu Hayaishi, a great biochemist, and from the history of clinical application of with my classmate (later, Professor Shu Narumiya, blockade of the renin-angiotensin system originating who became Professor, Department of Pharmacol- from relatively rare hypertension, renovascular hy- ogy, Kyoto University), and had the chance to be pertension, to widespread cardiovascular diseases introduced to basic research on biochemistry. In his including essential hypertension, congestive heart laboratory, during the summer vacation and after failure and renal diseases2) should be kept in mind as school, we learned the pleasure of basic research. concrete examples of translational research. Common We obtained valuable experience similar to that of features of translational research provide us with students on an MD-PhD course, although there was important principles for translational science via:1) no such course at that time in Japan. Later, after suitable disease animal models in the preclinical entering the Graduate Course of Internal Medicine phase, 2) rare human diseases in the clinical phase at Kyoto University, headed by the outstanding of translational research, and 3) the subsequent endocrinologist Professor Hiroo Imura, my MD-PhD widespread clinical applications to common human thesis work focused on the clinical significance of diseases, beyond the original target validation, that endogenous opioid peptides such as O-endorphin, are adjacent to or extensions and share the mecha- enkephalins, and leumorphin.182)–188) My research nistic implications of rare human diseases. into opioid peptides involved strict but excellent 558 K. NAKAO [Vol. 95, supervision from late Professor Shousaku Numa, a pointed out by Professors J. L. Goldstein and M. S. great molecular biologist. These fortune encounters Brown, in reference to the critical state of research in with three excellent mentors in biochemistry, basic the U.S. at the start of the 21st century.189) We are science, and internal medicine, and clinical science, currently facing the same situation in Japan. respectively, enabled the author to become a bidirec- Without persistent efforts towards clinical ap- tional physician-scientist. The past 35 years, has seen plications, discoveries can be wasted at any step in my engagement in translational research on novel the translational process. If a translational scientist hormones, especially the natriuretic peptide family fails in achieving a clinical application for one target, and leptin, as a translational scientist bridging basic he or she could use that failure to their advantage at or preclinical research and clinical application. My the next opportunity, or when examining a different enthusiasm for the achievement of translational target. Translational science created by experienced research and science might originate in part from translational scientists who are bidirectional and my dream and respect in childhood. have an excellent eye for judgement in translational The author’s research career has involved close research, is unlike basic studies done for no clinical collaboration with Professors Kenji Kangawa and purpose by clinicians or casual remarks on the clinical Hisayuki Matsuo of Miyazaki Medical College (later application of discoveries by basic scientists. of National Cardiovascular Research Center, Osaka), who isolated the natriuretic peptide family. Further 7. Conclusion collaborations have been with Professor Hirofumi Translational science is a newly emerging Yasue and his colleagues at the Department of science, distinct from basic and clinical sciences in Cardiology, Kumamoto University Graduate School biomedicine. Its field of investigation encompasses of Medicine, especially in the clinical applications the scientific and operational principles underlying of ANP and BNP. To promote research and the each step of the translational process. Advances in exchange of knowledge about preclinical and clinical translational science increase the efficacy and safety studies on these cardiovascular hormones, we of translational research in all diagnostic, therapeu- founded “The Society of Cardiovascular Endocrinol- tic, and behavioral applications. This review summa- ogy and Metabolism (CVEM)” in 1996. In addition rizes the current state of translational research on to collaborating with the late Dr. Nobuo Yoshida of novel hormones, the natriuretic peptide family and Shionogi on the diagnostic applications of ANP and leptin, which has been achieved for clinical applica- BNP for congestive heart failure, working with young tion or are in still ongoing in Japan. The importance PhD staffs at Suntory (later, Daiichi-Sankyo) has of translational science is emphasized, as we have developed ANP and BNP as drugs for congestive learned, from experiences in translational research. heart failure. Current collaborations with Chugai In order to advance translational science, it is Pharmaceutical are looking into the clinical applica- essential that we train authentic translational tions of CNP. Translational scientists should be scientists, who are bidirectional, “from bench to familiar with scientists in the pharmaceutical indus- bedside” or “from discovery to application” and “from try and not miss chance encounters with those in bedside to bench” or “from medical practice to both the academia and industry. laboratory”, and also have an excellent eye for Medical applications of diagnostics, therapeu- judgement in translational research, with under- tics, and behavioral changes, “needs” should be standing and support from academia, the pharma- accomplished by clinical applications of discoveries ceutical industry, and society. “seeds”. Medical research is effort in vain without clinical application. As a translational physician- Acknowledgements scientist, it is not possible to forget the personal sense The author is deeply grateful to all of the of accomplishment that was felt when we succeeded collaborators in the Department of Medicine and in developing diagnostic and therapeutic applications Clinical Science, Medical Innovation Center, EBM of the natriuretic peptide family and leptin. Research Center, and Translational Research Center, It must be hoped that young, talented medical Kyoto University Graduate School of Medicine, and students, including MD-PhD students, will seek to the Hormone Foundation of Japan, especially Pro- become translational physician-scientists, who might fessors Hiroo Imura, Hisayuki Matsuo, Kenji otherwise become an endangered species “bewitched, Kangawa, and Shu Narumiya for valuable advice bothered, and bewildered-but still beloved”,as and encouragement. The author is also deeply No. 9] Translational science 559 grateful to patients and their families for their 13) Mukoyama, M., Nakao, K., Saito, Y., Ogawa, Y., understanding and participation in translational Hosoda, K., Suga, S. et al. (1990) Human brain natriuretic peptide, a novel cardiac hormone. research. Lancet 335, 801–802. This work was mainly supported by a Grant- 14) Mukoyama, M., Nakao, K., Saito, Y., Ogawa, Y., in-Aid for Scientific Research from the Japanese Hosoda, K., Suga, S. et al. (1990) Increased Ministry of Education, Culture, Sports, Science, and human brain natriuretic peptide in congestive – Technology. This work was also supported in part by heart failure. N. Engl. J. Med. 323, 757 758. 15) Mukoyama, M., Nakao, K., Hosoda, K., Suga, S., grants from Smoking Research Foundation and The Saito, Y., Ogawa, Y. et al. (1991) Brain natriu- Hormone Station of Japan. The author declares no retic peptide as a novel cardiac hormone in other conflicts of interest. humans. Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and References brain natriuretic peptide. J. Clin. Invest. 87, 1402–1412. 1) Sung, N.S., Crowley, W.F. Jr., Genel, M., Salber, P., 16) Morita, E., Yasue, H., Yoshimura, M., Ogawa, H., Sandy, L., Sherwood, L.M. et al. (2003) Central Jougasaki, M., Matsumura, T. et al. (1993) challenges facing the national clinical research Increased plasma levels of brain natriuretic pep- enterprise. JAMA 289, 1278–1287. tide in patients with acute myocardial infarction. 2) Nakao, K., Yasoda, A., Ebihara, K., Hosoda, K. and Circulation 88,82–91. Mukoyama, M. (2009) Translational research of 17) Kawakami, R., Saito, Y., Kishimoto, I., Harada, M., novel hormones: Lessons from animal models and Kuwahara, K., Takahashi, N. et al. (2004) Over- rare human diseases for common human diseases. expression of brain natriuretic peptide facilitates J. Mol. Med. 87, 1029–1039. neutrophil infiltration and cardiac matrix metal- 3) Austin, P.C. (2018) Translating translation. Nat. loproteinase-9 expression after acute myocardial Rev. Drug Discov. 17, 455–456. infarction. Circulation 110, 3306–3312. 4) Okamoto, K. and Aoki, K. (1963) Development of a 18) Arai, H., Nakao, K., Saito, Y., Morii, N., Sugawara, strain of spontaneously hypertensive rats. Jpn. A., Yamada, T. et al. (1988) Augmented expres- Circ. J. 27, 282–293. sion of atrial natriuretic polypeptide gene in 5) Ingalls, A.M., Dickie, M.M. and Snell, G.D. (1950) ventricles of spontaneously hypertensive rats Obese, a new mutation in the house mouse. J. (SHR) and SHR-stroke prone. Circ. Res. 62, Hered. 41, 317–318. 926–930. 6) https://ncats.nih.gov/translation. 19) Yasue, H., Yoshimura, M., Sumida, H., Kikuta, K., 7) Takamine, J. (1902) The blood-pressure raising Kugiyama, K., Jougasaki, M. et al. (1994) Local- principle in the suprarenal gland. J. Am. Med. ization and mechanism of secretion of B-type Assoc. 38, 153–155. natriuretic peptide in comparison with those of 8) Nakao, K., Ogawa, Y., Suga, S. and Imura, H. A-type natriuretic peptide in normal subjects and (1992) Molecular biology and biochemistry of the patients with heart failure. Circulation 90, 195– natriuretic peptide system. I: Natriuretic peptides. 203. J. Hypertens. 10, 907–912. 20) Sugawara, A., Nakao, K., Sakamoto, M., Morii, N., 9) Nakao, K., Ogawa, Y., Suga, S. and Imura, H. Yamada, T., Itoh, H. et al. (1985) Plasma (1992) Molecular biology and biochemistry of the concentration of atrial natriuretic polypeptide in natriuretic peptide system. II: Natriuretic peptide essential hypertension. Lancet 2, 1426–1427. receptors. J. Hypertens. 10, 1111–1114. 21) Itoh, H., Nakao, K., Mukoyama, M., Yamada, T., 10) Suga, S., Nakao, K., Hosoda, K., Mukoyama, M., Hosoda, K., Shirakami, G. et al. (1989) Chronic Ogawa, Y., Shirakami, G. et al. (1992) Receptor blockade of endogenous atrial natriuretic polypep- selectivity of natriuretic peptide family, atrial tide (ANP) by monoclonal antibody against ANP natriuretic peptide, brain natriuretic peptide, and accelerates the development of hypertension in C-type natriuretic peptide. Endocrinology 130, spontaneously hypertensive and deoxycorticoster- 229–239. one acetate-salt-hypertensive rats. J. Clin. Invest. 11) Sugawara, A., Nakao, K., Morii, N., Yamada, T., 84, 145–154. Itoh, H., Shiono, S. et al. (1988) Synthesis of atrial 22) Ogawa, Y., Nakao, K., Mukoyama, M., Hosoda, K., natriuretic polypeptide in human failing hearts. Shirakami, G., Arai, H. et al. (1991) Natriuretic Evidence for altered processing of atrial natriu- peptides as cardiac hormones in normotensive and retic polypeptide precursor and augmented syn- spontaneously hypertensive rats. The ventricle is thesis of O-human ANP. J. Clin. Invest. 81, 1962– a major site of synthesis and secretion of brain 1970. natriuretic peptide. Circ. Res. 69, 491–500. 12) Saito, Y., Nakao, K., Arai, H., Nishimura, K., 23) Kawai, M., Yoshimura, M., Harada, M., Mizuno, Y., Okumura, K., Obata, K. et al. (1989) Augmented Hiramitsu, S., Shimizu, M. et al. (2013) Determi- expression of atrial natriuretic polypeptide gene nation of the B-type natriuretic peptide level as in ventricle of human failing heart. J. Clin. Invest. a criterion for abnormalities in Japanese individ- 83, 298–305. uals in routine clinical practice: The J-ABS multi- 560 K. NAKAO [Vol. 95,

center study (Japan Abnormal BNP Standard). 34) Ogawa, Y., Mukoyama, M., Yokoi, H., Kasahara, Intern. Med. 52, 171–177. M., Mori, K., Kato, Y. et al. (2012) Natriuretic 24) Saito, Y., Nakao, K., Nishimura, K., Sugawara, A., peptide receptor guanylyl cyclase-A protects Okumura, K., Obata, K. et al. (1987) Clinical podocytes from aldosterone-induced glomerular application of atrial natriuretic polypeptide in injury. J. Am. Soc. Nephrol. 23, 1198–1209. patients with congestive heart failure: Beneficial 35) Kato, Y., Mori, K., Kasahara, M., Osaki, K., Ishii, effects on left ventricular function. Circulation 76, A., Mori, K.P. et al. (2017) Natriuretic peptide 115–124. receptor guanylyl cyclase-A pathway counteracts 25) Yoshimura, M., Yasue, H., Morita, E., Sakaino, N., glomerular injury evoked by aldosterone through Jougasaki, M., Kurose, M. et al. (1991) Hemody- p38 mitogen-activated protein kinase inhibition. namic, renal, and hormonal responses to brain Sci. Rep. 7, 46624. natriuretic peptide infusion in patients with 36) Miyashita, K., Itoh, H., Tsujimoto, H., Tamura, N., congestive heart failure. Circulation 84, 1581– Fukunaga, Y., Sone, M. et al. (2009) Natriuretic 1588. peptides/cGMP/cGMP-dependent protein kinase 26) Nishikimi, T., Nakagawa, Y., Minamino, N., Ikeda, cascades promote muscle mitochondrial biogenesis M., Tabei, K., Fujishima, A. et al. (2015) Pro-B- and prevent obesity. Diabetes 58, 2880–2892. type natriuretic peptide is cleaved intracellularly: 37) Sonoyama, T., Tamura, N., Miyashita, K., Park, K., Impact of distance between O-glycosylation and Oyamada, N., Taura, D. et al. (2009) Inhibition cleavage sites. Am. J. Physiol. Regul. Integr. of hepatic damage and liver fibrosis by brain Comp. Physiol. 309, R639–R649. natriuretic peptide. FEBS Lett. 583, 2067–2070. 27) Nishikimi, T., Ikeda, M., Takeda, Y., Ishimitsu, T., 38) Sudoh, T., Minamino, N., Kangawa, K. and Matsuo, Shibasaki, I., Fukuda, H. et al. (2012) The effect H. (1990) C-type natriuretic peptide (CNP): A of glycosylation on plasma N-terminal proBNP-76 new member of natriuretic peptide family identi- levels in patients with heart or renal failure. Heart fied in porcine brain. Biochem. Biophys. Res. 98, 152–161. Commun. 168, 863–870. 28) Nishikimi, T., Okamoto, H., Nakamura, M., Ogawa, 39) Komatsu, Y., Nakao, K., Suga, S., Ogawa, Y., N., Horii, K., Nagata, K. et al. (2013) Direct Mukoyama, M., Arai, H. et al. (1991) C-type immunochemiluminescent assay for proBNP and natriuretic peptide (CNP) in rats and humans. total BNP in human plasma proBNP and total Endocrinology 129, 1104–1106. BNP levels in normal and heart failure. PLoS One 40) Suga, S., Nakao, K., Itoh, H., Komatsu, Y., Ogawa, 8, e53233. Y., Hama, N. et al. (1992) Endothelial production 29) Nishikimi, T., Kuwahara, K., Nakagawa, Y., of C-type natriuretic peptide and its marked Kangawa, K., Minamino, N. and Nakao, K. augmentation by transforming growth factor-O. (2013) Complexity of molecular forms of B-type Possible existence of “vascular natriuretic peptide natriuretic peptide in heart failure. Heart 99, 677– system”. J. Clin. Invest. 90, 1145–1149. 679. 41) Suga, S., Itoh, H., Komatsu, Y., Ogawa, Y., Hama, 30) Nakagawa, Y., Nishikimi, T., Kuwahara, K., N., Yoshimasa, T. et al. (1993) Cytokine-induced Fujishima, A., Oka, S., Tsutamoto, T. et al. C-type natriuretic peptide (CNP) secretion from (2017) MiR30-GALNT1/2 Axis-mediated glyco- vascular endothelial cells–evidence for CNP as a sylation contributes to the increased secretion of novel autocrine/paracrine regulator from endo- inactive human prohormone for brain natriuretic thelial cells. Endocrinology 133, 3038–3041. peptide (proBNP) from failing hearts. J. Am. 42) Komatsu, Y., Nakao, K., Itoh, H., Suga, S., Ogawa, Heart Assoc. 6, e003601. Y. and Imura, H. (1992) Vascular natriuretic 31) Nakagawa, Y., Nishikimi, T., Kuwahara, K., peptide. Lancet 340, 622. Yasuno, S., Kinoshita, H., Kuwabara, Y. et al. 43) Kubo, A., Isumi, Y., Ishizaka, Y., Tomoda, Y., (2014) The effects of super-flux (high perform- Kangawa, K., Dohi, K. et al. (2001) C-type ance) dialyzer on plasma glycosylated pro-B-type natriuretic peptide is synthesized and secreted natriuretic peptide (proBNP) and glycosylated from leukemia cell lines, peripheral blood cells, N-Terminal proBNP in end-stage renal disease and peritoneal macrophages. Exp. Hematol. 29, patients on dialysis. PLoS One 9, e92314. 609–615. 32) Li, Y., Saito, Y., Kuwahara, K., Rong, X., 44) Komatsu, Y., Itoh, H., Suga, S., Ogawa, Y., Hama, Kishimoto, I., Harada, H. et al. (2009) Natriuretic N., Kishimoto, I. et al. (1996) Regulation of peptide receptor/guanylyl cyclase-A inhibits an- endothelial production of C-type natriuretic pep- giotensin II type 2 receptor-mediated pro-hyper- tide in coculture with vascular smooth muscle trophic signaling in the heart. Endocrinology 150, cells. Role of the vascular natriuretic peptide 3759–3765. system in vascular growth inhibition. Circ. Res. 33) Kinoshita, H., Kuwahara, K., Nishida, M., Jian, Z., 78, 606–614. Rong, X., Kiyonaka, S. et al. (2010) Inhibition of 45) Doi, K., Itoh, H., Komatsu, Y., Igaki, T., Chun, TRPC6 channel activity contributes to the anti- T.H., Takaya, K. et al. (1996) Vascular endothe- hypertrophic effects of natriuretic peptides-gua- lial growth factor suppresses C-type natriuretic nylyl cyclase-A signaling in the heart. Circ. Res. peptide secretion. Hypertension 27, 811–815. 106, 1849–1860. 46) Hama, N., Itoh, H., Shirakami, G., Suga, S., No. 9] Translational science 561

Komatsu, Y., Yoshimasa, T. et al. (1994) Detec- Biochem. Biophys. Res. Commun. 145, 196–203. tion of C-type natriuretic peptide in human 59) Katsuura, G., Nakamura, M., Inouye, K., Kono, M., circulation and marked increase of plasma CNP Nakao, K. and Imura, H. (1986) Regulatory role level in septic shock patients. Biochem. Biophys. of atrial natriuretic polypeptide in water drinking Res. Commun. 198, 1177–1182. in rats. Eur. J. Pharmacol. 121, 285–287. 47) Nakao, K., Kuwahara, K., Nishikimi, T., Nakagawa, 60) Yamada, T., Nakao, K., Morii, N., Itoh, H., Shiono, Y., Kinoshita, H., Minami, T. et al. (2017) S., Sakamoto, M. et al. (1986) Central effect of Endothelium-derived C-type natriuretic peptide atrial natriuretic polypeptide on angiotensin II- contributes to blood pressure regulation by main- stimulated vasopressin secretion in conscious rats. taining endothelial integrity. Hypertension 69, Eur. J. Pharmacol. 125, 453–456. 286–296. 61) Shirakami, G., Nakao, K., Yamada, T., Itoh, H., 48) Yamahara, K., Itoh, H., Chun, T.H., Ogawam, Y., Mori, K., Kangawa, K. et al. (1988) Inhibitory Yamashita, J., Sawada, N. et al. (2003) Signifi- effect of brain natriuretic peptide on central cance and therapeutic potential of the natriuretic angiotensin II-stimulated pressor response in con- peptides/cGMP/cGMP-dependent protein kinase scious rats. Neurosci. Lett. 91,77–83. pathway in vascular regeneration. Proc. Natl. 62) Shirakami, G., Itoh, H., Suga, S., Komatsu, Y., Acad. Sci. U.S.A. 100, 3404–3409. Hama, N., Mori, K. et al. (1993) Central action of 49) Chusho, H., Tamura, N., Ogawa, Y., Yasoda, A., C-type natriuretic peptide on vasopressin secre- Suda, M., Miyazawa, T. et al. (2001) Dwarfism tion in conscious rats. Neurosci. Lett. 159,25–28. and early death in mice lacking C-type natriuretic 63) Itoh, H., Nakao, K., Katsuura, G., Morii, N., Shiono, peptide. Proc. Natl. Acad. Sci. U.S.A. 98, 4016– S., Sakamoto, M. et al. (1986) Centrally infused 4021. atrial natriuretic polypeptide attenuates exagger- 50) Kronenberg, H.M. (2003) Developmental regulation ated salt appetite in spontaneously hypertensive of the growth plate. Nature 423, 332–336. rats. Circ. Res. 59, 342–347. 51) Yasoda, A., Komatsu, Y., Chusho, H., Miyazawa, 64) Itoh, H., Nakao, K., Morii, N., Yamada, T., Shiono, T., Ozasa, A., Miura, M. et al. (2004) Over- S., Sakamoto, M. et al. (1986) Central action of expression of CNP in chondrocytes rescues achon- atrial natriuretic polypeptide on blood pressure in droplasia through a MAPK-dependent pathway. conscious rats. Brain Res. Bull. 16, 745–749. Nat. Med. 10,80–86. 65) Itoh, H., Nakao, K., Yamada, T., Morii, N., Shiono, 52) Yasoda, A. and Nakao, K. (2010) Genetic basis for S., Sugawara, A. et al. (1988) Brain renin- skeletal disease. CNP therapy for achondroplasia. angiotensin. Central control of secretion of atrial Clin. Calcium 20, 1212–1218. natriuretic factor from the heart. Hypertension 53) Yasoda, A. and Nakao, K. (2015) Translational 11, I57–I61. research of the activation of the C-type natriuretic 66) Inuzuka, M., Tamura, N., Yamada, N., Katsuura, peptide (CNP)-guanylyl cyclase-B pathway for G., Oyamada, N., Taura, D. et al. (2010) C-type skeletal dysplasia. In Innovative Medicine: Basic natriuretic peptide as a new regulator of food Research and Development (eds. Nakao, K., intake and energy expenditure. Endocrinology Minato, N. and Uemoto, S.). Springer, Tokyo, 151, 3633–3642. pp. 177–186. 67) Yamada-Goto, N., Katsuura, G., Ebihara, K., 54) Nakao, K., Okubo, Y., Yasoda, A., Koyama, N., Inuzuka, M., Ochi, Y., Yamashita, Y. et al. Osawa, K., Isobe, Y. et al. (2013) The Effects of (2013) Intracerebroventricular administration of C-type natriuretic peptide on craniofacial skeleto- C-type natriuretic peptide suppresses food intake genesis. J. Dent. Res. 92,58–64. via activation of the melanocortin system in mice. 55) Kondo, E., Yasoda, A., Fujii, T., Nakao, K., Diabetes 62, 1500–1504. Yamashita, Y., Ueda-Sakane, Y. et al. (2015) 68) Yamashita, Y., Yamada-Goto, N., Katsuura, G., Increased bone turnover and possible accelerated Ochi, Y., Kanai, Y., Miyazaki, Y. et al. (2016) fracture healing in a murine model with an Brain-specific natriuretic peptide receptor-B dele- increased circulating C-type natriuretic peptide. tion attenuates high-fat diet-induced visceral and Endocrinology 156, 2518–2529. hepatic lipid deposition in mice. Peptides 81,38– 56) Morii, N., Nakao, K., Sugawara, A., Sakamoto, M., 50. Suda, M., Shimokura, M. et al. (1985) Occurrence 69) Steinhelper, M.E., Cochrane, K.L. and Field, L.J. of atrial natriuretic polypeptide in brain. Bio- (1990) Hypotension in transgenic mice expressing chem. Biophys. Res. Commun. 127, 413–419. atrial natriuretic factor fusion genes. Hyperten- 57) Kawata, M., Nakao, K., Morii, N., Kiso, Y., sion 16, 301–307. Yamashita, H., Imura, H. et al. (1985) Atrial 70) John, S.W., Krege, J.H., Oliver, P.M., Hagaman, natriuretic polypeptide: Topographical distribu- J.R., Hodgin, J.B., Pang, S.C. et al. (1995) tion in the rat brain by radioimmunoassay and Genetic decreases in atrial natriuretic peptide immunohistochemistry. Neuroscience 16, 521– and salt-sensitive hypertension. Science 267, 679– 546. 681. 58) Morii, N., Nakao, K., Itoh, H., Shiono, S., Yamada, 71) Ogawa, Y., Itoh, H., Tamura, N., Suga, S., T., Sugawara, A. et al. (1987) Atrial natriuretic Yoshimasa, T., Uehira, M. et al. (1994) Molecular polypeptide in spinal cord and autonomic ganglia. cloning of the complementary DNA and gene 562 K. NAKAO [Vol. 95,

that encode mouse brain natriuretic peptide and 83) Holtwick, R., van Eickels, M., Skryabin, B.V., Baba, generation of transgenic mice that overexpress the H.A., Bubikat, A., Begrow, F. et al. (2003) brain natriuretic peptide gene. J. Clin. Invest. 93, Pressure-independent cardiac hypertrophy in mice 1911–1921. with cardiomyocyte-restricted inactivation of the 72) Tamura, N., Ogawa, Y., Chusho, H., Nakamura, K., atrial natriuretic peptide receptor guanylyl cy- Nakao, K., Suda, M. et al. (2000) Cardiac fibrosis clase-A. J. Clin. Invest. 111, 1399–1407. in mice lacking brain natriuretic peptide. Proc. 84) Sabrane, K., Kruse, M.N., Fabritz, L., Zetsche, B., Natl. Acad. Sci. U.S.A. 97, 4239–4244. Mitko, D., Skryabin, B.V. et al. (2005) Vascular 73) Lopez, M.J., Wong, S.K., Kishimoto, I., Dubois, S., endothelium is critically involved in the hypo- Mach, V., Friesen, J. et al. (1995) Salt-resistant tensive and hypovolemic actions of atrial natriu- hypertension in mice lacking the guanylyl cyclase- retic peptide. J. Clin. Invest. 115, 1666–1674. A receptor for atrial natriuretic peptide. Nature 8, 85) Tamura, N., Ogawa, Y., Yasoda, A., Itoh, H., Saito, 65–68. Y. and Nakao, K. (1996) Two cardiac natriuretic 74) Suganami, T., Mukoyama, M., Sugawara, A., Mori, peptide genes (atrial natriuretic peptide and brain K., Nagae, T., Kasahara, M. et al. (2001) Over- natriuretic peptide) are organized in tandem in expression of brain natriuretic peptide in mice the mouse and human genomes. J. Mol. Cell. ameliorates immune-mediated renal injury. J. Cardiol. 28, 1811–1815. Am. Soc. Nephrol. 12, 2652–2663. 86) Kuwahara, K., Saito, Y., Ogawa, E., Takahashi, N., 75) Kasahara, M., Mukoyama, M., Sugawara, A., Nakagawa, Y., Naruse, Y. et al. (2001) The Makino, H., Suganami, T., Ogawa, Y. et al. neuron-restrictive silencer element-neuron-restric- (2000) Ameliorated glomerular injury in mice tive silencer factor system regulates basal and overexpressing brain natriuretic peptide with endothelin 1-inducible atrial natriuretic peptide renal ablation. J. Am. Soc. Nephrol. 11, 1691– gene expression in ventricular myocytes. Mol. 1701. Cell. Biol. 21, 2085–2097. 76) Makino, H., Mukoyama, M., Mori, K., Suganami, 87) Kuwahara, K., Saito, Y., Takano, M., Arai, Y., T., Kasahara, M., Yahata, K. et al. (2006) Yasuno, S., Nakagawa, Y. et al. (2003) NRSF Transgenic overexpression of brain natriuretic regulates the fetal cardiac gene program and peptide prevents the progression of diabetic maintains normal cardiac structure and function. nephropathy in mice. Diabetologia 49, 2514– EMBO J. 22, 6310–6321. 2524. 88) Yasuno, S., Usami, S., Kuwahara, K., Nakanishi, 77) Suda, M., Ogawa, Y., Tanaka, K., Tamura, N., M., Arai, Y., Kinoshita, H. et al. (2009) Endog- Yasoda, A., Takigawa, T. et al. (1998) Skeletal enous cardiac natriuretic peptides protect the overgrowth in transgenic mice that overexpress heart in a mouse model of dilated cardiomyopathy brain natriuretic peptide. Proc. Natl. Acad. Sci. and sudden death. Am. J. Physiol. Heart Circ. U.S.A. 95, 2337–2342. Physiol. 296, H1804–H1810. 78) Chusho, H., Ogawa, Y., Tamura, N., Suda, M., 89) Kuwahara, K., Kinoshita, H., Kuwabara, Y., Yasoda, A., Miyazawa, T. et al. (2000) Genetic Nakagawa, Y., Usami, S., Minami, T. et al. models reveal that brain natriuretic peptide can (2010) Myocardin-related transcription factor A signal through different tissue-specific receptor- is a common mediator of mechanical stress- and mediated pathways. Endocrinology 141, 3807– neurohumoral stimulation-induced cardiac hyper- 3813. trophic signaling leading to activation of brain 79) Oliver, P.M., Fox, J.E., Kim, R., Rockman, H.A., natriuretic peptide gene expression. Mol. Cell. Kim, H.S., Reddick, R.L. et al. (1997) Hyper- Biol. 30, 4134–4148. tension, cardiac hypertrophy, and sudden death in 90) Tamura, N., Doolittle, L.K., Hammer, R.E., mice lacking natriuretic peptide receptor A. Proc. Shelton, J.M., Richardson, J.A. and Garbers, Natl. Acad. Sci. U.S.A. 94, 14730–14735. D.L. (2004) Critical roles of the guanylyl cyclase 80) Knowles, J.W., Esposito, G., Mao, L., Hagaman, B receptor in endochondral ossification and J.R., Fox, J.E., Smithies, O. et al. (2001) development of female reproductive organs. Proc. Pressure-independent enhancement of cardiac Natl. Acad. Sci. U.S.A. 101, 17300–17305. hypertrophy in natriuretic peptide receptor A- 91) Langenickel, T.H., Buttgereit, J., Pagel- deficient mice. J. Clin. Invest. 107, 975–984. Langenickel, I., Lindner, M., Monti, J., 81) Kishimoto, I., Rossi, K. and Garbers, D.L. (2001) A Beuerlein, K. et al. (2006) Cardiac hypertrophy genetic model provides evidence that the receptor in transgenic rats expressing a dominant-negative for atrial natriuretic peptide (guanylyl cyclase-A) mutant of the natriuretic peptide receptor B. inhibits cardiac ventricular myocyte hypertrophy. Proc. Natl. Acad. Sci. U.S.A. 103, 4735–4740. Proc. Natl. Acad. Sci. U.S.A. 98, 2703–2706. 92) Feil, R., Lohmann, S.M., de Jonge, H., Walter, U. 82) Holtwick, R., Gotthardt, M., Skryabin, B., and Hofmann, F. (2003) Cyclic GMP-dependent Steinmetz, M., Potthast, R., Zetsche, B. et al. protein kinases and the cardiovascular system: (2002) Smooth muscle-selective deletion of gua- Insights from genetically modified mice. Circ. Res. nylyl cyclase-A prevents the acute but not chronic 93, 907–916. effects of ANP on blood pressure. Proc. Natl. 93) Pfeifer, A., Aszodi, A., Seidler, U., Ruth, P., Acad. Sci. U.S.A. 99, 7142–7147. Hofmann, F. and Fassler, R. (1996) Intestinal No. 9] Translational science 563

secretory defects and dwarfism in mice lacking transgenic mice. Am. J. Physiol. Endocrinol. cGMP-dependent protein kinase II. Science 274, Metab. 297, E1339–E1348. 2082–2086. 107) Yasoda, A., Kitamura, H., Fujii, T., Kondo, E., 94) Miyazawa, T., Ogawa, Y., Chusho, H., Yasoda, A., Murao, N., Miura, M. et al. (2009) Systemic Tamura, N., Komatsu, Y. et al. (2002) Cyclic administration of C-type natriuretic peptide as a GMP-dependent protein kinase II plays a critical novel therapeutic strategy for skeletal dysplasias. role in C-type natriuretic peptide-mediated endo- Endocrinology 150, 3138–3144. chondral ossification. Endocrinology 143, 3604– 108) Fujii, T., Komatsu, Y., Yasoda, A., Kondo, E., 3610. Yoshioka, T., Nambu, T. et al. (2010) Circulating 95) Tsuji, T. and Kunieda, T. (2005) A loss-of-function C-type natriuretic peptide (CNP) rescues chon- mutation in natriuretic peptide receptor 2 (Npr2) drodysplastic CNP knockout mice from their gene is responsible for disproportionate dwarfism impaired skeletal growth and early death. Endo- in cn/cn mouse. J. Biol. Chem. 280, 14288–14292. crinology 151, 4381–4388. 96) Sogawa, C., Tsuji, T., Shinkai, Y., Katayama, K. 109) Igaki, T., Itoh, H., Suga, S.I., Hama, N., Ogawa, Y., and Kunieda, T. (2007) Short-limbed dwarfism: Komatsu, Y. et al. (1998) Effects of intravenously slw is a new allele of Npr2 causing chondrodys- administered C-type natriuretic peptide in hu- plasia. J. Hered. 98, 575–580. mans: Comparison with atrial natriuretic peptide. 97) Tsuji, T., Kondo, E., Yasoda, A., Inamoto, M., Hypertens. Res. 21,7–13. Kiyosu, C., Nakao, K. et al. (2008) Hypomorphic 110) Bartels, C.F., Bukulmez, H., Padayatti, P., Rhee, mutation in mouse Nppc gene causes retarded D.K., van Ravenswaaij-Arts, C., Pauli, R.M. et al. bone growth due to impaired endochondral (2004) Mutations in the transmembrane natriu- ossification. Biochem. Biophys. Res. Commun. retic peptide receptor NPR-B impair skeletal 376, 186–190. growth and cause acromesomelic dysplasia, type 98) Jiao, Y., Yan, J., Jiao, F., Yang, H., Donahue, L.R., Maroteaux. Am. J. Hum. Genet. 75,27–34. Li, X. et al. (2007) A single nucleotide mutation in 111) Wendt, D.J., Dvorak-Ewell, M., Bullens, S., Lorget, Nppc is associated with a long bone abnormality F., Bell, S.M., Peng, J. et al. (2015) Neutral in lbab mice. BMC Genet. 8, 16. endopeptidase-resistant C-type natriuretic pep- 99) Kondo, E., Yasoda, A., Tsuji, T., Fujii, T., Miura, tide variant represents a new therapeutic ap- M., Kanamoto, N. et al. (2012) Skeletal analysis proach for treatment of fibroblast growth factor of the long bone abnormality (lbab/lbab) mouse, receptor 3-related dwarfism. J. Pharmacol. Exp. a novel chondrodysplastic C-type natriuretic Ther. 353, 132–149. peptide mutant. Calcif. Tissue Int. 90, 307–318. 112) Zhang, Y., Proenca, R., Maffei, M., Barone, M., 100) Nakao, K., Osawa, K., Yasoda, A., Yamanaka, S., Leopold, L. and Friedman, J.M. (1994) Positional Fujii, T., Kondo, E. et al. (2015) The local CNP/ cloning of the mouse obese gene and its human GC-B system. In growth plate is responsible for homologue. Nature 372, 425–432. physiological endochondral bone growth. Sci. Rep. 113) Friedman, J.M. and Halaas, J.L. (1998) Leptin and 5, 10554. the regulation of body weight in mammals. Nature 101) Fujii, T., Hirota, K., Yasoda, A., Takizawa, A., 395, 763–770. Morozumi, N., Nakamura, R. et al. (2018) Rats 114) Pelleymounter, M.A., Cullen, M.J., Baker, M.B., deficient C-type natriuretic peptide suffer from Hecht, R., Winters, D., Boone, T. et al. (1995) impaired skeletal growth without early death. Effects of the obese gene product on body weight PLoS One 22, e0194812. regulation in ob/ob mice. Science 269, 540–543. 102) Yamanaka, S., Nakao, K., Koyama, N., Isobe, Y., 115) Halaas, J.L., Gajiwala, K.S., Maffei, M., Cohen, Ueda, Y., Kanai, Y. et al. (2017) Circulatory CNP S.L., Chait, B.T., Rabinowitz, D. et al. (1995) Rescues Craniofacial Hypoplasia in Achondropla- Weight-reducing effects of the plasma protein sia. J. Dent. Res. 96, 1526–1534. encoded by the obese gene. Science 269, 543–546. 103) Superti-Furga, A., Bonafe, L. and Rimoin, D.L. 116) Campfield, L.A., Smith, F.J., Guisez, Y., Devos, (2001) Molecular-pathogenetic classification of R. and Burn, P. (1995) Recombinant mouse OB genetic disorders of the skeleton. Am. J. Med. protein: Evidence for a peripheral signal linking Genet. 106, 282–293. adiposity and central neural networks. Science 104) Rousseau, F., Bonaventure, J., Legeai-Mallet, L., 269, 546–549. Pelet, A., Rozet, J.M., Maroteaux, P. et al. (1994) 117) Satoh, N., Ogawa, Y., Katsuura, G., Numata, Y., Mutations in the gene encoding fibroblast growth Tsuji, T., Hayase, M. et al. (1999) Sympathetic factor receptor-3 in achondroplasia. Nature 371, activation of leptin via the ventromedial hypo- 252–254. thalamus: Leptin-induced increase in catechol- 105) Cattaneo, R., Villa, A., Catagni, M. and Tentori, amine secretion. Diabetes 48, 1787–1793. L. (1988) Limb lengthening in achondroplasia by 118) Tartaglia, L.A., Dembski, M., Weng, X., Deng, N., Ilizarov’s method. Int. Orthop. 12, 173–179. Culpepper, J., Devos, R. et al. (1995) Identifica- 106) Kake, T., Kitamura, H., Adachi, Y., Yoshioka, T., tion and expression cloning of a leptin receptor, Watanabe, T., Matsushita, H. et al. (2009) OB-R. Cell 83, 1263–1271. Chronically elevated plasma C-type natriuretic 119) Ogawa, Y., Masuzaki, H., Isse, N., Okazaki, T., peptide (CNP) level stimulates skeletal growth in Mori, K., Shigemoto, M. et al. (1995) Molecular 564 K. NAKAO [Vol. 95,

cloning of rat obese cDNA and augmented gene obesity. Nat. Genet. 18, 213–215. expression in genetically obese Zucker fatty (fa/ 132) Yura, S., Ogawa, Y., Sagawa, N., Masuzaki, H., fa) rats. J. Clin. Invest. 96, 1647–1652. Itoh, H., Ebihara, K. et al. (2000) Accelerated 120) Takaya, K., Ogawa, Y., Hiraoka, J., Hosoda, K., puberty and late-onset hypothalamic hypogonad- Yamori, Y., Nakao, K. et al. (1996) Nonsense ism in female transgenic skinny mice overexpress- mutation of leptin receptor in the obese sponta- ing leptin. J. Clin. Invest. 105, 749–755. neously hypertensive Koletsky rat. Nat. Genet. 133) Aizawa-Abe, M., Ogawa, Y., Masuzaki, H., Ebihara, 14, 130–131. K., Satoh, N., Iwai, H. et al. (2000) Pathophysio- 121) Frederich, R.C., Hamann, A., Anderson, S., logical role of leptin in obesity-related hyper- Lollmann, B., Lowell, B.B. and Flier, J.S. (1995) tension. J. Clin. Invest. 105, 1243–1252. Leptin levels reflect body lipid content in mice: 134) Elefteriou, F., Takeda, S., Ebihara, K., Magre, J., Evidence for diet-induced resistance to leptin Patano, N., Kim, C.A. et al. (2004) Serum leptin action. Nat. Med. 1, 1311–1314. level is a regulator of bone mass. Proc. Natl. Acad. 122) Maffei, M., Halaas, J., Ravussin, E., Pratley, R.E., Sci. U.S.A. 101, 3258–3263. Lee, G.H., Zhang, Y. et al. (1995) Leptin levels 135) Suganami, T., Mukoyama, M., Mori, K., Yokoi, H., in human and rodent: Measurement of plasma Koshikawa, M., Sawai, K. et al. (2005) Prevention leptin and ob RNA in obese and weight-reduced and reversal of renal injury by leptin in a new subjects. Nat. Med. 1, 1155–1161. mouse model of diabetic nephropathy. FASEB J. 123) Considine, R.V., Sinha, M.K., Heiman, M.L., 19, 127–129. Kriauciunas, A., Stephens, T.W., Nyce, M.R. et al. 136) Masuzaki, H., Ogawa, Y., Hosoda, K., Miyawaki, (1996) Serum immunoreactive-leptin concentra- T., Hanaoka, I., Hiraoka, J. et al. (1997) tions in normal-weight and obese humans. N. Glucocorticoid regulation of leptin synthesis and Engl. J. Med. 334, 292–295. secretion in humans: Elevated plasma leptin levels 124) Isse, N., Ogawa, Y., Tamura, N., Masuzaki, H., in Cushing’s syndrome. J. Clin. Endocrinol. Mori, K., Okazaki, T. et al. (1995) Structural Metab. 82, 2542–2547. organization and chromosomal assignment of the 137) Nakao, K. (2009) Adiposcience and adipotoxicity. human obese gene. J. Biol. Chem. 270, 27728– Nat. Clin. Pract. Endocrinol. Metab. 5, 63. 27733. 138) Ebihara, K. and Nakao, K. (2015) Translational 125) Satoh, N., Ogawa, Y., Katsuura, G., Tsuji, T., research of leptin in lipodystrophy and its related Masuzaki, H., Hiraoka, J. et al. (1997) Pathophy- diseases. In Innovative Medicine: Basic Research siological significance of the obese gene product, and Development (eds. Nakao, K., Minato, N. and leptin, in ventromedial hypothalamus (VMH)- Uemoto, S.). Springer, Tokyo, pp. 165–175. lesioned rats: Evidence for loss of its satiety effect 139) Masuzaki, H., Ogawa, Y., Sagawa, N., Hosoda, K., in VMH-lesioned rats. Endocrinology 138, 947– Matsumoto, T., Mise, H. et al. (1997) Nonadipose 954. tissue production of leptin: Leptin as a novel 126) Imagawa, K., Numata, Y., Katsuura, G., Sakaguchi, placenta-derived hormone in humans. Nat. Med. I., Morita, A., Kikuoka, S. et al. (1998) Structure- 3, 1029–1033. function studies of human leptin. J. Biol. Chem. 140) Sagawa, N., Mori, T., Masuzaki, H., Ogawa, Y. and 273, 35245–35249. Nakao, K. (1997) Leptin production by hydatidi- 127) Caro, J.F., Kolaczynski, J.W., Nyce, M.R., form mole. Lancet 350, 1518–1519. Ohannesian, J.P., Opentanova, I., Goldman, 141) Ogawa, Y., Masuzaki, H., Hosoda, K., Aizawa-Abe, W.H. et al. (1996) Decreased cerebrospinal-fluid/ M., Suga, J., Suda, M. et al. (1999) Increased serum leptin ratio in obesity: A possible mechan- glucose metabolism and insulin sensitivity in ism for leptin resistance. Lancet 348, 159–161. transgenic skinny mice overexpressing leptin. 128) Halaas, J.L., Boozer, C., Blair-West, J., Fidahusein, Diabetes 48, 1822–1829. N., Denton, D.A. and Friedman, J.M. (1997) 142) Masuzaki, H., Ogawa, Y., Isse, N., Satoh, N., Physiological response to long-term peripheral Okazaki, T., Shigemoto, M. et al. (1995) Human and central leptin infusion in lean and obese mice. obese gene expression. Adipocyte-specific expres- Proc. Natl. Acad. Sci. U.S.A. 94, 8878–8883. sion and regional differences in the adipose tissue. 129) Tanaka, T., Masuzaki, H., Yasue, S., Ebihara, K., Diabetes 44, 855–858. Shiuchi, T., Ishii, T. et al. (2007) Central 143) Ioffe, E., Moon, B., Connolly, E. and Friedman, J.M. melanocortin signaling restores skeletal muscle (1998) Abnormal regulation of the leptin gene in AMP-activated protein kinase phosphorylation in the pathogenesis of obesity. Proc. Natl. Acad. Sci. mice fed a high-fat diet. Cell Metab. 5, 395–402. U.S.A. 95, 11852–11857. 130) Montague, C.T., Farooqi, I.S., Whitehead, J.P., 144) Masuzaki, H., Ogawa, Y., Aizawa-Abe, M., Hosoda, Soos, M.A., Rau, H., Wareham, N.J. et al. (1997) K., Suga, J., Ebihara, K. et al. (1999) Glucose Congenital leptin deficiency is associated with metabolism and insulin sensitivity in transgenic severe early-onset obesity in humans. Nature 387, mice overexpressing leptin with lethal yellow 903–908. agouti mutation: Usefulness of leptin for the 131) Strobel, A., Issad, T., Camoin, L., Ozata, M. and treatment of obesity-associated diabetes. Diabetes Strosberg, A.D. (1998) A leptin missense muta- 48, 1615–1622. tion associated with hypogonadism and morbid 145) Tanaka, T., Hidaka, S., Masuzaki, H., Yasue, S., No. 9] Translational science 565

Minokoshi, Y., Ebihara, K. et al. (2005) Skeletal cortin-4 receptor mutations are a frequent and muscle AMP-activated protein kinase phospho- heterogeneous cause of morbid obesity. J. Clin. rylation parallels metabolic phenotype in leptin Invest. 106, 253–262. transgenic mice under dietary modification. 158) Farooqi, I.S., Yeo, G.S., Keogh, J.M., Aminian, S., Diabetes 54, 2365–2374. Jebb, S.A., Butler, G. et al. (2000) Dominant and 146) Masuda, S., Tanaka, T., Masuzaki, H., Nakao, K. recessive inheritance of morbid obesity associated and Taguchi, S. (2014) Overexpression of leptin with melanocortin 4 receptor deficiency. J. Clin. reduces the ratio of glycolytic to oxidative Invest. 106, 271–279. enzymatic activities without changing muscle 159) Ebihara, K., Masuzaki, H. and Nakao, K. (2004) fiber types in mouse skeletal muscle. Biol. Pharm. Long-term leptin-replacement therapy for lipo- Bull. 37, 169–173. atrophic diabetes. N. Engl. J. Med. 351, 615–616. 147) Kamohara, S., Burcelin, R., Halaas, J.L., Friedman, 160) Ebihara, K., Kusakabe, T., Hirata, M., Masuzaki, J.M. and Charron, M.J. (1997) Acute stimulation H., Miyanaga, F., Kobayashi, N. et al. (2007) of glucose metabolism in mice by leptin treatment. Efficacy and safety of leptin-replacement therapy Nature 389, 374–377. and possible mechanisms of leptin actions in 148) Liu, L., Karkanias, G.B., Morales, J.C., Hawkins, patients with generalized lipodystrophy. J. Clin. M., Barzilai, N., Wang, J. et al. (1998) Intra- Endocrinol. Metab. 92, 532–541. cerebroventricular leptin regulates hepatic but 161) Oral, E.A., Simha, V., Ruiz, E., Andewelt, A., not peripheral glucose fluxes. J. Biol. Chem. 273, Premkumar, A., Snell, P. et al. (2002) Leptin- 31160–31167. replacement therapy for lipodystrophy. N. Engl. 149) Cusin, I., Zakrzewska, K.E., Boss, O., Muzzin, P., J. Med. 346, 570–578. Giacobino, J.P., Ricquier, D. et al. (1998) Chronic 162) Aotani, D., Ebihara, K., Sawamoto, N., Kusakabe, central leptin infusion enhances insulin-stimulated T., Aizawa-Abe, M., Kataoka, S. et al. (2012) glucose metabolism and favors the expression of Functional magnetic resonance imaging analysis uncoupling proteins. Diabetes 47, 1014–1019. of food-related brain activity in patients with 150) Goldstein, B.J. (1994) Syndrome of extreme insulin lipodystrophy undergoing leptin replacement resistance. In Joslin’s Diabetes Mellitus, 13th ed. therapy. J. Clin. Endocrinol. Metab. 97, 3663– (eds. Joslin, E.P., Kahn, C.R. and Weir, G.C.). 3671. Lea & Febiger, Philadelphia. 163) Brown, R.J., Araujo-Vilar, D., Cheung, P.T., 151) Andreelli, F., Hanaire-Broutin, H., Laville, M., Dunger, D., Garg, A., Jack, M. et al. (2016) The Tauber, J.P., Riou, J.P. and Thivolet, C. (2000) diagnosis and management of lipodystrophy Normal reproductive function in leptin-deficient syndromes: A multi-society practice guideline. patients with lipoatropic diabetes. J. Clin. Endo- J. Clin. Endocrinol. Metab. 101, 4500–4511. crinol. Metab. 85, 715–719. 164) Iwanishi, M., Ebihara, K., Kuskabe, T., Chen, W., 152) Pardini, V.C., Victoria, I.M., Rocha, S.M., Andrade, Ito, J., Masuzaki, H. et al. (2009) Clinical D.G., Rocha, A.M., Pieroni, F.B. et al. (1998) characteristics and efficacy of pioglitazone in a Leptin levels, O-cell function, and insulin sensitiv- Japanese diabetic patient with an unusual type of ity in families with congenital and acquired familial partial lipodystrophy. Metabolism 58, generalized lipoatropic diabetes. J. Clin. Endocri- 1681–1687. nol. Metab. 83, 503–508. 165) Iwanishi, M., Ebihara, K., Kusakabe, T., Harada, S., 153) Moitra, J., Mason, M.M., Olive, M., Krylov, D., Ito-Kobayashi, J., Tsuji, A. et al. (2012) Prema- Gavrilova, O., Marcus-Samuels, B. et al. (1998) ture atherosclerosis in a Japanese diabetic patient Life without white fat: A transgenic mouse. Genes with atypical familial partial lipodystrophy and Dev. 12, 3168–3181. hypertriglyceridemia. Intern. Med. 51, 2573– 154) Shimomura, I., Hammer, R.E., Richardson, J.A., 2579. Ikemoto, S., Bashmakov, Y., Goldstein, J.L. et al. 166) Shibata, Y., Nakatsukasa, A., Eguchi, J., (1998) Insulin resistance and diabetes mellitus in Miyamoto, S., Masuda, Y., Awazawa, M. et al. transgenic mice expressing nuclear SREBP-1c in (2018) Acquired partial lipoatrophy as graft- adipose tissue: Model for congenital generalized versus-host disease and treatment with metrelep- lipodystrophy. Genes Dev. 12, 3182–3194. tin: Two case reports. J. Med. Case Reports 12, 155) Ebihara, K., Ogawa, Y., Masuzaki, H., Shintani, 368–372. M., Miyanaga, F., Aizawa-Abe, M. et al. (2001) 167) Miyanaga, F., Ogawa, Y., Ebihara, K., Hidaka, S., Transgenic overexpression of leptin rescues insulin Tanaka, T., Hayashi, S. et al. (2003) Leptin as resistance and diabetes in a mouse model of an adjunct of insulin therapy in insulin-deficient lipoatrophic diabetes. Diabetes 50, 1440–1448. diabetes. Diabetologia 46, 1329–1337. 156) Kobayashi, H., Ogawa, Y., Shintani, M., Ebihara, 168) Naito, M., Fujikura, J., Ebihara, K., Miyanaga, F., K., Shimodahira, M., Iwakura, T. et al. (2002) A Yokoi, H., Kusakabe, T. et al. (2011) Therapeutic Novel homozygous missense mutation of melano- impact of leptin on diabetes, diabetic complica- cortin-4 receptor (MC4R) in a Japanese woman tions, and longevity in insulin-deficient diabetic with severe obesity. Diabetes 51, 243–246. mice. Diabetes 60, 2265–2273. 157) Vaisse, C., Clement, K., Durand, E., Hercberg, S., 169) Kusakabe, T., Tanioka, H., Ebihara, K., Hirata, M., Guy-Grand, B. and Froguel, P. (2000) Melano- Miyamoto, L., Miyanaga, F. et al. (2009) Benefi- 566 K. NAKAO [Vol. 95,

cial effects of leptin on glycaemic and lipid control 179) Lee, J., Lie, J., Feng, X., Salazar Hernandez, M.A., in a mouse model of type 2 diabetes with increas- Mucka, P., Choi, J.W. et al. (2016) Withaferin A ed adiposity induced by streptozotocin and a high- is a leptin sensitizer with strong antidiabetic fat diet. Diabetologia 52, 675–683. properties in mice. Nat. Med. 22, 1023–1032. 170) Shimizu, Y., Son, C., Aotani, D., Nomura, H., 180) Chellappa, K., Perron, I.J., Naidoo, N. and Baur, Hikida, T., Hosoda, K. et al. (2017) Role of leptin J.A. (2019) The leptin sensitizer celastrol reduces in conditioned place preference to high-fat diet in age-associated obesity and modulates behavioral leptin-deficient ob/ob mice. Neurosci. Lett. 640, rhythm. Aging Cell 18, e12874. 60–63. 181) Iwakura, H., Dote, K., Bando, M., Koyama, H., 171) Yamada, N., Katsuura, G., Ochi, Y., Ebihara, K., Hosoda, K., Kangawa, K. et al. (2016) Establish- Kusakabe, T., Hosoda, K. et al. (2011) Impaired ment of leptin-responsive cell lines from adult CNS leptin action is implicated in depression mouse hypothalamus. PLoS One 11, e0148639. associated with obesity. Endocrinology 152, 182) Nakao, K., Nakai, Y., Oki, S., Horii, K. and Imura, 2634–2643. H. (1978) Presence of immunoreactive O-endor- 172) Miyamoto, L., Ebihara, K., Kusakabe, T., Aotani, phin in normal human plasma: A concomitant D., Yamamoto-Kataoka, S., Sakai, T. et al. (2012) release of O-endorphin with adrenocorticotropin Leptin activates hepatic 5B AMP-Activated Pro- after metyrapone administration. J. Clin. Invest. tein Kinase through sympathetic nervous system 62, 1395–1398. and ,1 adrenergic receptor: A potential mechan- 183) Nakao, K., Nakai, Y., Jingami, H., Oki, S., Fukata, ism for improvement of fatty liver in lipodys- J. and Imura, H. (1979) Substantial rise of plasma trophy by leptin. J. Biol. Chem. 287, 40441– O-endorphin levels after insulin-induced hypogly- 40447. cemia in human subjects. J. Clin. Endocrinol. 173) Yamamoto-Kataoka, S., Ebihara, K., Aizawa-Abe, Metab. 49, 838–841. M., Nishio, M., Kusakabe, T., Yamamoto, Y. 184) Nakao, K., Oki, S., Tanaka, I., Horii, K., Nakai, Y., et al. (2015) Leptin improves fatty liver independ- Furui, T. et al. (1980) Immunoreactive O-endor- ently of insulin sensitization and appetite sup- phin and adrenocorticotropin in human cerebro- pression in hepatocyte-specific pten-deficient mice spinal fluid. J. Clin. Invest. 66, 1383–1390. with insulin hypersensitivity. Horm. Metab. Res. 185) Yoshimasa, T., Nakao, K., Ohtsuki, H., Li, S. and 47, 168–175. Imura, H. (1982) Methionine-enkephalin and 174) Aizawa-Abe, M., Ebihara, K., Ebihara, C., leucine-enkephalin in human sympathoadrenal Mashimo, T., Takizawa, A., Tomita, T. et al. system and pheochromocytoma. J. Clin. Invest. (2013) Generation of leptin deficient Lepmkyo/ 69, 643–650. Lepmkyo rats and identification of leptin respon- 186) Yoshimasa, T., Nakao, K., Li, S., Ikeda, Y., Suda, sive genes in the liver. Physiol. Genomics 45, 786– M., Sakamoto, M. et al. (1983) Plasma methi- 793. onine-enkephalin and leucine-enkephalin in nor- 175) Ebihara, C., Ebihara, K., Aizawa-Abe, M., mal subjects and patients with pheochromocyto- Mashimo, T., Tomita, T., Zhao, M. et al. (2015) ma. J. Clin. Endocrinol. Metab. 57, 706–712. Seipin is necessary for normal brain development 187) Nakao, K., Suda, M., Sakamoto, M., Yoshimasa, T., and spermatogenesis in addition to adipogesis. Morii, N., Ikeda, Y. et al. (1983) Leumorphin is Hum. Mol. Genet. 24, 4238–4249. a novel endogenous opioid peptide derived from 176) Kusakabe, T., Ebihara, K., Sakai, T., Miyamoto, L., preproenkephalin B. Biochem. Biophys. Res. Aotani, D., Yamamoto, Y. et al. (2012) Amylin Commun. 117, 695–701. improves the effect of leptin on insulin sensitivity 188) Imura, H., Nakao, K., Yanaihara, N., Katsuura, G. in leptin-resistant diet-induced obese mice. Am. J. and Nakamura, M. (1986) Potent action of Physiol. Endocrinol. Metab. 302, E924–E931. leumorphin on consummatory behaviors in rats: 177) Sakai, T., Kusakabe, T., Ebihara, K., Aotani, D., Comparison with other opioid peptides. Neuro- Yamamoto-Kataoka, S., Zhao, M. et al. (2014) endocrinology 44, 142–148. Leptin restores the insulinotropic effect of exena- 189) Goldstein, J.L. and Brown, M.S. (1997) The clinical tide in a mouse model of type 2 diabetes with investigator: bewitched, bothered, and bewil- increased adiposity induced by streptozotocin and dered-but still beloved. J. Clin. Invest. 99, 2803– high-fat diet. Am. J. Physiol. Endocrinol. Metab. 2812. 307, E712–E719. 178) Liu, J., Lee, J., Salazar Hernandez, M.A., Mazitschek, R. and Ozcan, U. (2015) Treatment (Received May 13, 2019; accepted Aug. 30, 2019) of obesity with celastrol. Cell 161, 999–1011.

Added in proof: A paper of Phase II clinical trial of CNP analog on patients with achondroplasia presenting its safety and efficacy has been published after the submission of manuscript (N. Engl. J. Med. 2019, 381,25–35). No. 9] Translational science 567

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Kazuwa Nakao was born in Hyogo Prefecture in 1948 and entered Kyoto University School of Medicine in 1967. During his time at the School of Medicine, he had the chance to be introduced to basic research in biochemistry in the laboratory of late Professor Osamu Hayaishi and learned the pleasure of basic research and gained valuable experience similar to MD-PhD students. He graduated from Kyoto University Graduate School of Medicine in 1973 and entered the Graduate Course of Internal Medicine at Kyoto University headed by Professor Hiroo Imura and did his MD-PhD thesis work on the clinical significance of an opioid peptide, O-endorphin. During this research on opioid peptides, he also received excellent supervision from late Professor Shosaku Numa, an outstanding molecular biologist. These fortune encounters with three excellent mentors in biochemistry, internal medicine, and molecular biology enabled him to become a bidirectional translational physician-scientist, that is, “from bench to bedside” or “from bedside to bench”. He became Professor of Medicine at Kyoto University Graduate School of Medicine in 1992 and retired in 2013. He has performed pioneering translational studies on the natriuretic peptide family (atrial natriuretic peptide, brain natriuretic peptide, and C- type natriuretic peptide) and on leptin. He has led the development of clinical applications of the natriuretic peptide family and leptin as both diagnostics and therapeutics, and created translational science to promote translational research and trained young translational scientists in Japan. He served as President of the Japan Society of Internal Medicine, Chairman of the Board of Directors of the Japan Endocrine Society, Japan Society of the Study for Obesity, Japan Society of Cardiovascular Endocrinology and Metabolism (CVEM), and Japan Society of Molecular Medicine. He has received Awards from the Japan Endocrine Society, Japan Society of the Study for Obesity, Takamine Award of CVEM, Japan Medical Association Award, Takeda Medical Award and also Medal with Purple Ribbon. He is now Professor Emeritus at Kyoto University, and is working as a researcher in the Medical Innovation Center, Kyoto University Graduate School of Medicine, and the President of certified NPO “The Hormone Station of Japan”. He is an Honorary Citizen of Yabu-City, Hyogo Prefecture.