Commentary: Parallel Evolution of Molecular Endocrinology as a Journal and a Discipline: Convergence of Interests with the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK/NIH)

Abstract

The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) celebrates in 2010 its 60^th year as an institute of the National Institutes of Health. NIDDK has been fundamental in providing support for research in endocrinology, fostering research to elucidate basic principles of endocrine signaling leading to understanding of diseases and disorders of hormone action. Over this time span, a move to a more molecular level in understanding of the basis of hormone action has emerged and been supported by NIDDK, with many advances finding their way into a new journal, Molecular Endocrinology. The merging of disciplines that has made this possible constitutes a major force for further progress as NIDDK moves forward over the next 60 yr. Together, NIDDK and Molecular Endocrinology have served as catalysts for advancing knowledge in the field, energizing new paradigms that have led to advances in the clinic.

The interests of NIDDK and Molecular Endocrinology are co-evolving, creating both opportunities for support of relevant research and a portal for the publication of the results of this research.

Molecular endocrinology as a discipline developed in response to the conceptual need to understand, at a molecular level, fundamental observations obtained from classical endocrinology. A major driving force came from the development and application of emerging technologies that facilitated probing the underlying mechanisms of action of endocrine signaling pathways. To achieve this unparalleled understanding, it was necessary to integrate many, often disparate, disciplines. Beginning with the tools of the molecular biologist, it became possible to understand processes of gene regulation at this fundamental level. Adding the ability of the cell biologist to visualize location of molecules within cells to the biochemists’ ability to understand the chemical and structural properties and behavior of these molecules, a more thorough picture of the consequences of a signaling pathway could be understood. It became possible to attack an endocrine problem using a multi- or trans-disciplinary approach to develop new understandings of structure, function, and role in disease. These fundamental advances merged in a discipline and journal devoted to molecular endocrinology. This theme has resonated for over the last two decades and is well represented in the pages of Molecular Endocrinology.

As the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) celebrates its 60^th yr as a National Institutes of Health (NIH) institute, the role that NIDDK has played in these developments suggests a synergy that has benefited both the field and the health-related mission of a premier institute of the NIH. NIDDK began in August 1950 as the National Institute of Arthritis and Metabolic Disease. Its mission (http://www.nih.gov/about/almanac/organization/NIDDK.htm) was to continue ongoing investigations in experimental biology and medicine, expanded to include clinical investigation in rheumatic diseases, diabetes, and a number of metabolic, endocrine, and gastrointestinal diseases. The first grant awards were made in November 1950 initiating 60 yr of growth resulting in major advances in basic and clinical science reflected in numerous awards to investigators funded by NIDDK, including Lasker and Nobel prizes. Along the way there have been several name changes, as areas of science shifted and new areas of emphasis emerged. Arthritis moved out of the mission in 1986 becoming part of the new National Institute of Arthritis, Musculoskeletal and Skin Diseases. Thus NIDDK now charted its own course leading up to its current mission of advancing understanding of basic and clinical science in diabetes, digestive, and kidney diseases. From the very beginning an important and comprehensive focus of NIDDK has been in endocrine and metabolic disorders and diabetes. It is thus no surprise that many major advances in these areas supported by NIDDK found their way into a new journal of The Endocrine Society. Indeed, the first volume of Molecular Endocrinology in January 1987 (http://mend.endojournals.org/cgi/issue_pdf/toc_pdf/1/1.pdf) featured many investigators who were at the time major contributors to the mission of NIDDK. As the field and journal evolved, so too did the advances made by investigators supporting the mission of NIDDK. This growth represented a convergence of interests as the field matured and the mission of NIDDK became more focused. MEND thus became the premier journal in the field, and investigators supported by NIDDK became major contributors to both.

Neuroendocrinology

The isolation and identification of key signaling molecules in the hypothalamus and pituitary by Roger Guillemin, Andrew Schally, and colleagues, supported by NIDDK, and other institutes of the NIH, proved the long-held belief that neuropeptides constituted the major means for integration of neuroendocrine regulation in the hypothalamus and pituitary. The Vale laboratory was then able to identify new peptides and establish the key regulatory connections between the cortex, hypothalamus, and pituitary and to define the feedback loops of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes (e.g. Ref. 1). The rapid adoption of the new tools of molecular biology to advance discovery in endocrinology was nowhere more evident then the pioneering work of Mellon and co-workers (2) who exploited findings in the field of oncogenesis by using the T-antigen oncogene and targeting it to specific endocrine cells in transgenic mice. The net result was derivation of immortalized cell lines specific to (e.g.) anterior pituitary cells with the cells maintaining their differentiated functions. Using this same approach the Mellon laboratory was able to develop the first cultures of immortalized hypothalamic neuroendocrine cells. These studies, and many more exploiting the concept, have served to energize work by providing new tools for in vitro study of endocrine cell function. By exploiting these, and other tools of molecular and cellular biology, it became possible to understand the regulation of peptide production and secretion leading to the discovery of the physiological implications of many hormones and peptides, such as the role(s) that splice products, derived from the parent proopiomelanocortin, played in subsequent physiology of feeding and body composition. After the isolation and cloning of the receptors and mapping of their tissue-specific localization, it became possible to focus on specific molecules, as was done by the Cone laboratory (3) in linking individual peptides to specific receptors and signaling networks. Indeed, the localization of the newly identified melanocortin-4 receptor (MC4-R) as a target of proopiomelanocortin peptides from the arcuate nucleus and the identification of the hypothalamic nuclei harboring receptors for the peptide showed how neuropeptide receptors could elicit pharmacologically distinct but important effects. This work, along with the discovery of leptin and its receptor (4), formed the foundation of our current understanding of regulation of energy homeostasis by the brain. These studies illustrate how fields such as neuroanatomy, cell biology, and molecular biology, when integrated, can shed light on a fundamental central question in molecular endocrinology. Endocrine cells and tissues became models for basic molecular and developmental investigations due, in large part, to the availability of robust antibodies, gene probes, and defined cell lines. Laboratories such as those of Mellon (2) and Rosenfeld (5) used these tools to make fundamental discoveries in organogenesis of endocrine organs and mechanisms of tissue-specific gene regulation. Indeed, such an integrated approach has been central to determining the origin of endocrine organs, themselves. The Parker laboratory (6), using the tools of molecular biology combined with that of physiology, showed that deletion of a singular hormone receptor, steroidogenic factor-1, resulted in agenesis of steroid-producing organs, including the adrenal cortex. Thus, the end target of the hypothalamic-pituitary-adrenal axis, the adrenal gland itself, depended on the timely expression of a hormone receptor.

Growth Factor and Cytokine Signaling

Signaling by growth factors, hormones, cytokines, and dietary/environmental factors represents an arm of endocrinology that has evolved in complexity and detail over the past more than 25 yr. The fundamental observation that the insulin receptor is a receptor tyrosine kinase and that intracellular signaling involves the insulin receptor-signaling family of factors and a vast and interconnected intracellular signaling cascade led to a quantum leap in our understanding of how insulin actions in tissues and cells occur such that a delicate balance in the cell to maintain glucose homeostasis is effected (7). Often it is dysfunction in a signaling pathway that leads to disease. The search for the meaning of dysfunction in signaling and role in disease has been a major focus of NIDDK during the past 60 yr. The application of the tools of genetics, molecular biology, and physiology ultimately led to the identification and cloning of leptin, a hormone released by fat cells with profound and still not fully understood effects both centrally and peripherally (4). At first only studied in the context of obesity, the identification of receptors for leptin and their localization in areas of the brain, further linked leptin to food intake, body composition, and reproduction (4,8). At the same time pioneering work from the Baird laboratory (9) demonstrated that growth factors play broader roles that even included circulating to distant sites. Such was the case for one of the members of the fibroblast growth factor family (FGF), FGF-2, which circulated as a hormone. Moreover, an equally diverse class of FGF receptors was shown to mediate the responses of specific FGFs, leading to the testing of new hypotheses for their roles in regulation of physiological functions. Building on this knowledge, the Kliewer laboratory (10) subsequently demonstrated that another member of the FGF family, FGF 15, circulated in the gastrointestinal system as part of a signaling loop to maintain a tight control of bile acid synthesis. Indeed, coupling to nuclear hormone receptor pathways in liver for bile acid synthesis showed the remarkable degree of cross talk between cell-signaling pathways required for the integration of complex physiological functions. These studies illustrate how understanding signaling through insulin, leptin, growth factor, and other receptors requires trans-disciplinary approaches to solve complex problems in endocrine physiology. By taking a molecular path to understanding hormone action, it has become possible to develop a thorough understanding of how cells respond to numerous inputs, each input representing a point for potential therapeutic intervention and representative of the goal of NIDDK to better define the pathology, implications, and potential treatments for diabetes and other metabolic diseases.

Nuclear Receptors (NRs) and Transcriptional Regulation

The NR superfamily further illustrates the progress possible through a layering of disciplines. The exciting realization that signaling through steroid receptors led to changes in gene expression opened a new, more molecular, window on endocrine physiology. When steroid receptors were subsequently found to constitute a definitive family of ligand-activated transcription factors linked by common structural motifs, an explosion of new understanding resulted, later commemorated with a series of retrospective papers on their cloning from pioneers in the field and highlighted in the pages of Molecular Endocrinology (11,12,13,14,15). Still, the initial observation of homology, expanded to include orphan NRs, necessitated further advances to translate protein structure into understanding of function. Fletterick and colleagues (16) applied the tools of the structural biologist to a cloned steroid receptor, the thyroid receptor, revealing structural domains in the receptor that led directly to insight into function. Definitive three-dimensional structure of a NR ligand-binding domain (LBD) occupied by ligand emerged and energized the field, presenting new insights into how the linear structure of the receptor folds to make room for the ligand and how the individual domains of the receptor then assume new conformations that foster signaling. These insights ultimately led Rastinejad and co-workers (17) to provide the structure of a full-length receptor coupled to a coactivator with bound ligand with the entire complex bound to DNA, further demonstrating the power of a multidisciplinary approach that has helped to reconcile both pharmacological and physiological observations earlier made in the absence of definitive structural information. Taking this a step further, McDonnell et al. (18) used biochemical, molecular, and chemical methods to show that different compounds demonstrating agonist, antagonist, and partial agonist activities against the estrogen receptor elicited different responses based on how they shaped the receptor. By folding the receptor differentially, the actions of the receptor could be modulated, with the ICI compound 182,164, resulting in complete antagonism and tamoxifen and raloxifene giving partial agonist activities, although with different effects in different tissues. The merging of disciplines and the ability to get at molecular mechanisms of action clearly foster studies that led to therapeutic applications. One important insight that emerged was that of differential actions of receptor domains as for the C-terminal activation domain, constituting the last of 12 α-helices (H12) in the LBD and folded against the LBD upon ligand binding giving profound effects on transactivation. The exciting observation that nuclear accessory proteins bound to H12 mediated the actions led to even deeper insights into hormone action. Indeed, at about the same time, O'Malley and colleagues (19) revealed the existence of a class of NR transcriptional coactivators referred to as steroid receptor coactivators-1, -2, -3, or the p160 family of coactivators. Nearly simultaneously, two laboratories (20,21) reported a class of NR corepressors, silencing mediator of retinoid and thyroid hormone receptor and nuclear receptor corepressor. Coregulators, soon joined by many others, including key regulatable ones such as peroxisome proliferator-activated coactivator-1α/β, paved the way for answering fundamental questions in endocrinology, including how hormone action can stimulate or repress gene expression and affect metabolism (22). These and other coregulators have proved to be involved not only in mediating NR action. Once opened up, this seminal finding led to a long series of observations about the nature of NR coregulators, such as those of Stallcup and co-workers (23), who showed complex and preferential binding strategies for members of the p160 family of coactivators with several of the steroid receptors through discrete receptor interaction domains. Together these observations, many again contributing to advances in the mission of NIDDK, not only added to the complexity of NR action but began to point toward unified mechanisms of action.

The ‘Omics Revolution

Application of new high-throughput methods for obtaining information about gene sequence, protein structure, identification of ligands, and of key metabolites, embodied in genomics, proteomics, and metabolomics, has revolutionized fields such as molecular endocrinology, enabling further insights but now at an integrated systems level. The ability to accrue large amounts of data often required new approaches to computational biology to integrate the data and required the layering of still other disciplines on top of molecular endocrinology. For hormones at the molecular level, these approaches have shaken the prevailing dogma that the up- or down-regulation of expression of genes in response to hormone signals is a simple, on or off response to a signal. Intriguing evidence now suggests that signals that lead to changes in gene expression involve multiple binding sites in chromatin for transcription factor(s), such as the NRs. The full magnitude of this observation required the addition of new technologies to the toolkit of the molecular endocrinologist in the form of chromatin immunoprecipitation (ChIP) coupled to microarray (ChIP-chip) and more recently to the extremely powerful addition of high-throughput massively parallel sequencing (ChIP-seq). Brown and colleagues (24) applied first ChIP-chip and then ChIP-seq to the question of estrogen receptor binding in the genome, arriving at the observation that large numbers of sites could be identified in estrogen-responsive MCF-7 cells at which the estrogen receptor bound-across the entire genome. Now duplicated for several additional NRs, as well as for other transcription factors, the resulting concept has been embodied as the “cistrome” or the full complement of binding sites for a given factor in a specific cell. The fact that binding occurs throughout the genome, including intergenic, intronic, as well as promoter-specific sites, creates enormous challenges to understanding of the physiological implications for such widespread binding. An important corollary finding was that often other transcription factors are found in close proximity to the NR on chromatin and acting as cooperating or pioneering factors, adding an additional layer of regulation that must be explored and explained (25). Thus, the molecular endocrinologist must reach out to the computational and systems biologist to form larger, trans-disciplinary teams to address these very complex problems in endocrinology. Again, the disease implications relevant to NIDDK suggest potential new explanations for mechanisms of disease as well as potential points for therapeutic interventions.

The convergence of the journal Molecular Endocrinology and the interests of NIDDK in an ‘omics integration of signaling moved to a new level with the development of a strategic alliance between Molecular Endocrinology and the NIDDK-supported Nuclear Receptor Signaling Atlas (NURSA) consortium. NURSA was designed to bring together, in one accessible web portal, information about the NR superfamily, coregulators, ligands, and downstream targets (26). Centered on a genomics and proteomics explanation of signaling through NRs, NURSA has been focused on how this information can be used to develop a system-wide understanding of the structure, function, and role in disease of NRs and associated coregulators. A web portal (www.nursa.org) serves as the focal point for data accrual, display, and access, and a reciprocal curation agreement between Molecular Endocrinology and NURSA makes it easy to access published data and online data resources catalyzing greater access to information for journal submitters and readers. The discovery-based studies of NURSA investigators have led Molecular Endocrinology to provide a new venue for resource-based studies beginning with NURSA genomic and proteomic profiling work and presented as a series of discovery-based papers that serve as important and highly cited resources to the community (27,28,29). This convergence of interests and novel presentation provides an outlet for discovery-based science that leads to new hypothesis generation and testing.

Integrative Physiology

Returning full circle to endocrine physiology, application of molecular approaches has catalyzed the search for answers to old questions. The emerging understanding that tissues not classically defined as endocrine function in fact, if not name, as endocrine organs is one such area. When the tools of molecular endocrinology were applied to adipose tissue, it became clear that a host of important hormones and cytokines are released physiologically and temporally, in response to both endocrine and environmental signals; these contribute to normal function and, when dysregulated, contribute to the development and progression of obesity, diabetes, and other metabolic diseases (30). Work from the Spiegelman laboratory (31) showed that the inappropriate expression of the cytokine TNF-α played a role in the dysregulation of adipose metabolism seen in obesity. The link to pathology occurred when it became clear that elevated levels of TNF-α contributed to the development of peripheral insulin resistance seen in mouse models of type 2 diabetes. Additional scrutiny of the murine fat cell led to the finding that a novel adipose hormone, resistin, accumulates in obesity and contributes to peripheral insulin resistance, particularly in skeletal muscle (32). These studies were dependent on the extension of ‘omics approaches to molecular endocrinology, so that it became possible to ask what genes in a given tissue were up-or down-regulated under a given set of physiological or pathological conditions. When the role of other adipocytokines is added, the role of the fat cell as an endocrine organ has emerged as a major advance of the last 20 yr. In a further development, whereas the central role of the adipocyte in maintenance of energy balances initially focused attention almost solely on white adipose tissue, the puzzling finding that animals and humans have significant amounts of a second fat cell, in the form of brown adipose tissue, was left unexplained. Whereas the former is genetically and functionally sculpted for energy storage and utilization, the latter has a more basic function: generation of heat using fat as the substrate. This ability to burn energy makes it well suited as a net consumer of energy stores in the form of fat. Spiegelman and co-workers (30) molecularly dissected these fat cells to better understand how the brown adipocyte is derived; they found a transcription factor, PRDM16, to be a primary factor responsible for brown fat tissue specification. Specifically, expression of PRDM16 at key times in development triggered differentiation of cells in the muscle cell lineage to develop into brown fat cells. It was possible to take precursors and expose them to PRDM16 and stimulate the appearance of brown fat cells. The challenge now is to identify factors that may trigger PRDM16 expression and thus convert more precursors into brown fat and thereby stimulate greater energy expenditure. In an environment with great caloric excess, the translational implications would be enormous. Another major contributor to both insulin sensitivity and balance in energy expenditure is skeletal muscle, in which fiber types dictate use of glucose (type II) or fat (type I) as substrate and classified as fast-twitch and slow-twitch fiber (33). Evans and co-workers (34) elegantly showed that exercise training coupled with administration of a ligand for the peroxisome proliferator-activated receptor-δ isoform of the peroxisome proliferator-activated receptor subfamily of NRs resulted in fiber type switching to type I and a mouse that showed extreme exercise endurance. When probed at the molecular level, it became clear that expression of a key metabolic enzyme, AMP-activated protein kinase, was intimately involved and could be chemically regulated independent of hormone action, again pointing toward a potential therapeutic target for regulation of energy balance and body composition.

Summary and Conclusions

The layering of disciplines, as exemplified by the marriage of the tools of molecular biology with those of classical endocrinology melded together to form molecular endocrinology as a discipline through which fundamental understanding of mechanisms of cellular signaling could be appreciated. Subsequent overlay of disciplines as diverse as structural and cellular biology together with the advent of the ‘omics revolution has opened new paths to understanding of the role of hormone action in physiology and disease. Ultimately, it is the goal of NIDDK to bring fundamental understandings of disease mechanisms forward as a platform from which true translational findings can be used to advance treatments in the clinic. It could be said that during its first 60 yr NIDDK served a crucial role in defining the nature of the problem, and that as classical endocrinology matured and evolved, NIDDK has helped to chart a course for the future as we seek to pinpoint both underlying disease mechanisms and new therapeutic targets. NIDDK is poised to support the next 60 yr, and beyond, in the quest to improve scientific understanding and subsequent health outcomes.