- Email: [email protected]
Uniting GDF15 and GFRAL: Therapeutic Opportunities in Obesity and Beyond
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Review
Uniting GDF15 and GFRAL: Therapeutic Opportunities in Obesity and Beyond Shannon E. Mullican and Shamina M. Rangwala* Growth differentiation factor-15 (GDF15) is a circulating protein that has been implicated in multiple biological processes, including energy homeostasis, body weight regulation, and cachexia driven by cancer and chronic disease. The potential to target GDF15 in the treatment of energy-intake disorders, including obesity and anorexia, is an area of intense investigation, but has been limited by the lack of an identified receptor, signaling mechanism, and target tissue. GDNF family receptor a-like (GFRAL) was recently identified as the neuronal brainstem receptor responsible for mediating the anorectic actions of GDF15. Herein, we provide a brief overview of GDF15 biology with a focus on energy homeostasis, and highlight the implications of the recent receptor identification to this field and beyond. A Brief History of GDF15 Research Twenty years ago, three independent laboratories described a novel member of the TGFb superfamily (see Glossary) and, based on the context of the discoveries, termed the factor ‘placenta BMP’ (PLAB), ‘macrophage inhibitory cytokine-10 (MIC-1) and ‘placental transforming growth factor beta’ (PTGFB) [1–3]. Additional reports of the discovery of this factor followed shortly thereafter, leading to other nomenclature, including nonsteroidal anti-inflammatory drug-activated gene (NAG-1), prostate-derived factor (PDF), and growth differentiation factor 15 (GDF15) [4–6]. For simplicity, we use the terminology GDF15 for the remainder of the discussion. GDF15 is expressed broadly in multiple tissues and is robustly increased in circulation during pregnancy, after tissue injury, and in diverse disease states, including cancer, cardiovascular, and kidney disease, where it often correlates with poor prognosis [7]. These correlations focused the first decade of GDF15 investigation on its assessment as a biomarker in numerous conditions and its potential function in cancer and inflammation [8–12].
Highlights GDF15 is a hormone that is broadly expressed and is considered a biomarker the circulation of which positively correlates with the progression of many diseases. GDF15 administration leads to decreased food intake and body weight in obese rodents and nonhuman primates. GFRAL, a distant relative of receptors for a distinct class of the TGFb superfamily ligands, has recently been identified as the high-affinity receptor binding and mediating the anorectic effects of GDF15. GDF15 and GFRAL are promising therapeutic targets for the treatment of disorders of energy homeostasis, including obesity and anorexia.
The generation of loss- and gain-of-function mouse models provided the first insights into GDF15 biology beyond pregnancy, cancer, and inflammation. Specifically, the observation that mice with increased circulating GDF15 weighed less than mice lacking GDF15 started a decade of work focused on understanding the potential role of GDF15 in disorders of weight maintenance, including anorexia, cancer cachexia, and obesity [13,14]. However, progress in characterizing mechanisms underlying these observations was limited by the uncertainty of the cellular receptor and primary target tissue mediating the various effects of GDF15. Fast forward 20 years after the initial description of GDF15 and four independent pharmaceutical company laboratories have simultaneously described the identification of the GDF15 receptor [15–19]. The identification of GFRAL as the GDF15 receptor has triggered the next era of GDF15 research, which will include not only delineation of the molecular and cellular signaling downstream of GDF15, but also the careful reconsideration of previously reported GDF15 effects in the context of this new family member. As a result, novel pharmacological
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Cardiovascular & Metabolism Therapeutic Area, Janssen Pharmaceuticals, Inc., Spring House, PA 19477, USA
*Correspondence: [email protected] (S.M. Rangwala).
https://doi.org/10.1016/j.tem.2018.05.002 © 2018 Elsevier Ltd. All rights reserved.
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approaches to targeting this pathway for the treatment of obesity, anorexia, and cachexia will result and our understanding of GDF15 physiology beyond weight regulation will expand.
GDF15 Molecular Characteristics and Regulation GDF15 gene structure is conserved across species and comprises two exons separated by one intron of approximately 3 kb [2]. The resulting GDF15 polypeptide includes a signal peptide, a propeptide region, and a mature region at the C terminus, typical of the TGFb superfamily. Divergence within the family is reflected by GDF15 having a relatively low sequence similarity and a unique intrachain disulfide bonding arrangement in the cysteine knot region in the C terminus compared with other members [16]. The precursor GDF15 polypeptide forms a homodimer stabilized through one interchain disulfide bond at the C terminus of the protein. The N-terminal propeptide is proteolytically removed and a mature 25-kDa homodimer is generated and secreted [20]. In addition to the endocrine functions of secreted GDF15, intracellular functions, including modulation of mitochondrial membrane potential and nuclear transcription factor activity, have been reported [21,22]. The cell has multiple mechanisms to rapidly increase and maintain GDF15 levels. Transcription induction occurs in the absence of new protein production, a hallmark of immediate early genes [23]. The Gdf15 promoter has many putative regulatory sites and multiple transcription factors have been implicated in direct regulation upon various stimuli, including p53, EGR-1, Sp1, CHOP, and SMAD2/3 [24–29]. Post-transcriptional stabilization of GDF15 mRNA has been reported [22,30,31]. In addition, unprocessed GDF15 propeptide can be secreted and is proposed to bind the extracellular matrix and form latent stores that can be quickly released upon the appropriate stimulus [32]. Gdf15 is expressed in many tissues, including kidney, liver, muscle, adipose, and placenta, which secretes high levels of GDF15 during pregnancy [1,5,33–35]. In most individuals, circulating GDF15 levels range between 100 and 1200 pg/ml but increase after exercise, tissue injury, and in many disease states, including cancer, cardiovascular, and kidney disease, where they often correlate with weight loss and poor prognosis [23,36–39]. GDF15 levels are predictive of all-cause mortality [40]. Due to these robust correlations, GDF15 serves as a biomarker in multiple diseases and its potential roles in cancer and inflammatory processes have been the focus of many investigations [11,12].
GDF15 and Body-Weight Regulation GDF15 expression and circulating levels correlate with body-weight differences. As with other physiological stresses, circulating GDF15 is elevated in obesity in mice, rats, and humans [41– 43]. The specific tissue source of GDF15 in this context is not clear, although its expression increases in the liver and adipose of obese mice [41]. In contrast to the relatively minor rise in plasma GDF15 in a state of overnutrition, substantial further increases correlate with weight loss in humans [44]. Obese patients were assessed before and 1 year after Roux-en-Y gastric bypass (RYGB) [45]. At baseline, plasma GDF15 was elevated compared with nonobese subjects; however, after RYGB-driven weight loss, GDF15 concentration was even higher. One explanation for these seemingly conflicting observations is that GDF15 could be part of a compensatory mechanism in obesity that limits energy intake during periods of nutrient excess. Furthermore, GDF15 is elevated in patients with anorexia nervosa compared with control subjects and decreases upon realimentation [46]. Anorexia is a component of cancer cachexia and the resulting undernutrition-driven weight loss significantly contributes to an overall decline in advanced disease. Cancer cachexia is a progressive syndrome and patients can be classified into stages according to the degree of depletion of fat and lean mass in combination 2
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Glossary Anorexia: decreased food intake because of diminished appetite or aversion to food. Area postrema (AP): medullary brainstem structure at the base of the fourth ventricle with permeable capillaries allowing exposure to constituent neurons to peptides and hormones of the peripheral circulation. Functions include autonomic regulation of various homeostatic processes, including feeding behavior. Cachexia: disease-associated multiorgan wasting syndrome driving severe weight loss associated with muscle atrophy and appetite suppression in patients not actively trying to lose weight. Central amygdala: forebrain nucleus with a role in physiological and behavioral responses to fear, stress, and drug-related stimuli, including that of noxious agents, via signaling emanating from the brainstem. Chemoreceptor trigger zone (CTZ): anatomical region within the AP implicated in sensing blood-borne drugs or hormones that induce vomiting. Fc portion of immunoglobulin G: tail region of antibody that interacts with cell surface Fc receptors to activate the immune system. Fusion of peptides to a modified nonimmunogenic IgG Fc increases the half-life of the fusion partner in drug discovery efforts. FcRn recycling: neonatal Fc receptor (FcRN) expressed on immune and epithelial cells. Upon cellular uptake, serum albumin and immunoglobulin G bind to FcRN, which transports this cargo back to the plasma membrane, ultimately releasing it extracellularly. This process allows albumin and IgG to bypass lysosomal degradation and has been exploited in drug discovery efforts. Glial cell-derived neurotrophic factor (GDNF) family: subgroup of TGFb superfamily with known roles in neuronal development and function. Signaling is mediated by GDNF family receptors (GFRa1–4) complexed with RET receptor tyrosine kinase. Glucagon-like peptide-1 (GLP-1): peptide hormone made by intestinal
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with the degree of ongoing weight loss [47]. In a study examining GDF15 levels in patients with various stages of cachexia, elevation was measured in patients with early precachexia compared with non-cachectic patients and remained elevated in patients with cachexia and advanced refractory cachexia [37]. Independent analyses demonstrated that patients with cancer experiencing cachexia-driven weight loss have higher levels of plasma GDF15 compared with patients with cancer and a stable weight [14,48,49]. Cachexia and weight loss are serious complications in patients with chronic diseases of the kidney and heart. In a study of patients on dialysis due to chronic kidney disease, higher GDF15 levels were observed in those with low body mass indices (BMI) consistent with findings from a previous cohort with endstage renal failure demonstrating an inverse correlation of plasma GDF15 and BMI [14,50]. Likewise, during an assessment of circulating GDF15 in patients with chronic heart failure (CHF), the highest levels were observed in those with the lowest BMI [51]. While multiple human studies have shown correlations of GDF15 levels with weight loss associated with the cachectic state, causation remains to be proven. In this context, GDF15 is elevated after gastric bypass surgery, which results in significant weight loss, and is not associated with cachexia [45]. Further investigations examining the relationship of circulating levels of GDF15 in scenarios of weight loss in the absence of cachexia, for example with calorie restriction, are warranted. The correlation between weight loss and GDF15 is conserved in rodents and this has enabled interrogation of GDF15 as a potential causal factor impacting body weight. Two independent transgenic models overexpressing GDF15 from birth exhibit lower body weight, fat mass, and associated improvements in glucose tolerance [13,14]. Transgenic overexpression of GDF15 also protected mice from obesity, liver steatosis, and glucose intolerance upon high-fat diet feeding [52,53]. Introduction of GDF15 overexpression in adult livers by hydrodynamic injection of DNA vectors or viral infection decreased food intake and body weight in mice with established obesity [29,41]. Decreases in food intake and body weight associated with xenografted prostate tumor cells transfected with Gdf15 were reversed by blocking GDF15 with an antibody in mice [14]. Conversely, numerous investigators have reported that mice lacking GDF15 gain more weight and have greater fat mass than wild-type animals [54–57]. The potential of pharmacologically administered GDF15 to decrease energy intake and thereby elicit weight loss has been demonstrated in mice, rats and monkeys. Lean mice treated with recombinant GDF15 resulting in circulating levels orders of magnitude above the physiological range, eat less and lose weight [14,16,17,58]. Decreased food intake and body weight is also observed in genetic- and diet-induced rat and mouse models of obesity after administration of GDF15 [14,17,18,41,58]. GDF15 treatment-mediated weight loss in diet-induced obese mice led to metabolic improvements, including enhanced glucose homeostasis and lower serum triglycerides and cholesterol [17,41,58]. These effects translate to higher species because a 6week daily treatment regimen with recombinant human GDF15 in spontaneously obese nonhuman primates reduced food intake, body weight, and plasma triglyceride concentrations and improved glucose tolerance [41]. The physiological mechanism by which GDF15 drives weight loss primarily involves a reduction in total food intake, as has been demonstrated in multiple species as described above. Direct central nervous system (CNS) delivery of GDF15 by intracerebroventricular injection in mice and rats is sufficient to inhibit food intake [18,59]. Lower caloric intake is likely due to increased satiation as opposed to general malaise, because GDF15 treatment resulted in a reduction in meal size with no change in the number of meals or sickness-like behavior (hunched posture, labored movements, or altered breathing) in mice provided with either a standard chow or highfat diet [15,17]. Consistent with less food-exploring behavior, ambulatory activity was reduced
enteroendocrine cells that has many metabolic roles, including modulation of feeding behavior via signaling through the GLP-1 receptor located within the AP and NTS. Hypothalamus: collection of nuclei within the forebrain that coordinate autonomic processes, including homeostatic regulation of sleep, body temperature, thirst, and hunger, and hormonal signaling from the nearby pituitary gland. Leptin: adipose-derived hormone that functions as a long-term mediator of energy balance by suppressing food intake upon binding to the leptin receptor located within the NTS, AP, and hypothalamus. Melanocortin-4 receptor (MC4R): located within the hypothalamus and brainstem, agonists of this receptor inhibit food intake. Nucleus of the solitary tract (NTS): located adjacent to the AP in the brainstem, this is the primary visceral sensory relay station within the brain receiving and responding to stimuli from the respiratory, cardiovascular, and gastrointestinal systems to control homeostatic processes, including feeding behavior. Parabrachial nucleus: neuronal relay center anatomically located between the brainstem and viscerosensory and autonomic centers in the forebrain, including the central amygdala. Functions include taste processing and regulation of feeding. Roux-en-Y gastric bypass (RYGB): weight-loss surgery reducing the size of the stomach restricting the amount of food a patient can eat and bypassing the upper portion of the small intestine, thus decreasing nutrient absorption. Surgery-subsequent changes in gut hormones also contribute to weight loss. Satiation: suppression of ongoing eating; a signal to end a meal. TRAMP mice: model used in oncology research harboring the Transgenic Adenocarcinoma of the Mouse Prostate transgene that develop progressive forms of prostate cancer with distant site metastasis. Transforming growth factor beta (TGFb) superfamily: structurally
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in mice with elevated circulating GDF15 only at the onset of the dark cycle, the time of day when mice typically ingest the largest meals [53,58]. When provided a diet choice, mice treated with GDF15 selectively lowered their fat intake rather than protein or carbohydrate, suggesting that taste preference is a component of GDF15 energy intake regulation [41]. Gastric distension upon the initiation of a meal influences satiation through mechanoreceptors communicating to the CNS through vagal afferent neurons and is determined, in part, by gastric emptying [60]. Exogenously administered GDF15 was reportedly enriched at neuronal beds along the gut and slowed gastric emptying in mice and still effectively reduced food intake in rats after selective vagal deafferentation [18,41]. While a more pleiotropic effect of GDF15 on food preference and gastric motility is intriguing, future studies will be necessary to tease apart the contributions of such changes to the overall effect of GDF15 on body-weight regulation. Feeding-independent mechanisms of GDF15 on body weight are being considered. Reported effects of transgenic overexpression of GDF15 on energy expenditure have been inconsistent, perhaps owing to the lack of normalization of energy expenditure to lean mass, which was proportionally increased in mice treated with GDF15 [52,53,61]. Treatment with recombinant GDF15 had no direct impact on energy expenditure in body-weight matched mice [18]. GDF15 administration led to a shift in fuel preference toward lipid oxidation, which is likely secondary to its effects on anorexia and subsequent weight loss, which results in fat store mobilization [16,41]. Finally, pair-feeding rodents the same amount of food ingested after GDF15 administration was sufficient to elicit the weight loss achieved by treatment, suggesting that additional mechanisms beyond energy intake regulation, such as a direct effect on energy expenditure, are unlikely to contribute to the initial weight loss observed upon GDF15 treatment [14,17,18].
The Identification of GFRAL The impact of GDF15 on body weight led several independent laboratories to seek the identity of the receptor mediating this biological effect. Its position as a member of the TGFb family led to the hypothesis that it could be signaling via TGFb-family receptor pairs; however, efforts to demonstrate direct binding of GDF15 to any known TGFb receptors did not support this idea [16–18,62–64]. Three independent unbiased screens of thousands of human cell membraneassociated proteins and a biased approach considering orphan members of the glial-cellline-derived neurotrophic factor (GDNF) family in parallel led to the identification of GFRAL as the sole receptor for GDF15 [15–18]. GFRAL was previously an orphan receptor classified as a distant homolog of the GDNF family of receptors [65,66]. GDF15 binds with high affinity to GFRAL, but to no other members of the GDNF family of receptors, including GFRA1, GFRA2, GFRA3,or GFRA4 [15–17]. Similarly, GFRAL only binds GDF15 and not any other GDNFrelated ligands, including neurturin (NRTN), artemin (ARTN), and persephin (PSPN) [18]. GDNF family ligands bind to receptor complexes, including coreceptor RET. GFRAL binds to RET upon GDF15 binding and, once this complex has formed, cell signaling typical to this family, including phosphorylation of RET, AKT, ERK, and PLC-g1, is observed (Figure 1, Key Figure) [16–18]. Importantly, GDF15 effects on food intake and weight loss were abolished in mice lacking GFRAL, confirming the physiological relevance of the ligand–receptor relationship identified in the in vitro systems [15–18]. Highly restricted expression of GFRAL is conserved across species. GFRAL mRNA and protein are limited to the brainstem, specifically in a subset of neurons of the area postrema (AP) in adult mouse, rat, monkey, and humans [15–18]. Expression within the nearby nucleus of the solitary tract (NTS) has also been reported, but these findings are less consistent between independent groups and require further confirmation. More widespread and dynamic CNS Gfral expression in the embryo suggests additional roles unique to development [65]. 4
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related molecules the active moiety of which is a dimer formed through an intermolecular disulfide bond; interact with a conserved family of transmembrane receptors to elicit intracellular signaling and regulate many biological processes.
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Key Figure
Growth Differentiation Factor-15/GDNF Family Receptor a-Like (GDF15/GFRAL) Signaling as a Modulator of Energy Intake and Weight Homeostasis
GFRAL antagonism GFRAL agonism
AMY
HYP
LPBN
Decreased food intake
NTS AP
Increased food intake
P
RET
P
MEK/ERK
P
GDF15
PLC/PKC P
GFRAL
P
P
PI3K/AKT
Figure 1. Peripherally derived GDF15 binds to GRFAL/RET receptor complexes on the cell surface of neurons within the brainstem area postrema (AP), triggering receptor phosphorylation (P) and downstream intracellular signaling. This signaling impinges on yet to be defined neuronal circuitry that may include neurons of the nucleus of the solitary tract (NTS), lateral parabrachial nucleus (LPBN), central amygdala (AMY), and hypothalamus (HYP). The results of this signaling are a decrease in food intake and subsequent weight loss. Therapeutic opportunities targeting this axis include agonism for weight loss in obesity or antagonism in states of decreased energy intake, including anorexia nervosa or disease-related cachexia.
GFRAL localization to the adult brainstem is consistent with previous findings demonstrating that the AP and NTS are required for the anorectic action of GDF15 [59]. GFRAL-positive neurons express RET and, consistent with activation of RET signaling, demonstrated increased phosphorylation of ERK and S6 ribosomal protein upon GDF15 administration [16,18]. Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
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Immediate early gene FOS induction, a marker of neuronal activation, in response to GDF15 treatment has been reported not only in AP GFRAL-expressing neurons, but also in the NTS, hypothalamus, and calcitonin gene-related peptide (CGRP)-positive neurons of the parabrachial nucleus and central amygdala (Figure 1) [14,16,41,57]. Although some findings in these reports are conflicting, particularly around the NTS and hypothalamus, these early data underscore the ability of GDF15 to trigger neuronal activation in centers known to control feeding behavior. Furthermore, GFRAL-deficient mice remain responsive to GLP-1 and leptin, while GDF15-induced weight loss still occurs in rodents lacking receptors for leptin and GLP-1 and the melanocortin-4 receptor, highlighting that the GDF15/GFRAL axis is independent of other pathways well established to control food intake [14–18,28]. GFRAL is required for the anorectic effects of pharmacologically administered GDF15 and initial studies in GFRAL-deficient animals have begun to elucidate the function of this receptor in the face of metabolic challenges that lead to increases in endogenous GDF15. Mice lacking GFRAL are born at expected frequencies and display no overt abnormalities up to 6 months of age [15–18]. Possibly reflective of a role of GFRAL during early development and growth, male GFRAL-deficient mice younger than 12 weeks are reported to be slightly shorter and lighter than littermate counterparts, although this difference is not observed at older ages [15,17]. Upon a nutritional challenge of a 60% high-fat diet, which increases plasma GDF15, mice lacking GFRAL consumed more food and gained a greater amount of fat mass and body weight compared with normal littermates, and this phenotype was more pronounced on a C57Bl/6 genetic background [16,17]. Increased diet-induced obesity upon GFRAL ablation exacerbates glucose intolerance and insulin resistance. The impact of GFRAL signaling in the context of metabolic challenge was further exemplified after treatment of mice with cisplatin, a chemotherapeutic agent known to cause anorexia [16]. Cisplatin treatment elevated circulating GDF15 and mice lacking GFRAL were resistant to treatment-induced anorexia and weight loss. The notion that endogenous GDF15/ GFRAL signaling is integral to metabolic homeostasis in stress conditions is further supported by GDF15 loss-of-function studies. For example, induction of the mitochondrial unfolded protein response (UPRmit), an adaptive stress-response pathway activated by mitochondrial proteotoxic stress, typically limits weight gain and metabolic dysfunction in the context of obesity. GDF15 ablation in a mouse model of UPRmit caused greater weight gain, worsened glucose intolerance and greater hepatic steatosis upon high-fat diet feeding [28].
Targeting the GDF15/GFRAL Axis – Therapeutic Opportunities GFRAL Agonism Activating GFRAL to reduce food intake and body weight holds promise as an antiobesity strategy. The most straightforward approach is delivery of the natural ligand, GDF15. However, the circulating half-life of native GDF15 is approximately 3 h in mice and nonhuman primates, limiting the dosing strategy that could be applied therapeutically [41]. Despite this limitation, as described above, daily administration of native GDF15 to obese nonhuman primates efficaciously lowered body weight and improved metabolic parameters [41]. Pharmacokinetic properties of native proteins can be enhanced by increasing molecular size or harnessing the FcRn-recycling mechanism, both of which can be achieved through fusion with human serum albumin (HSA) or the Fc portion of immunoglobulin G (IgG) [67]. Fusion with HSA or Fc does not hinder GDF15-mediated anorexia and weight loss, as demonstrated in lean and obese rodents, and extends GDF15 half-life from hours to days in monkeys [15,17,41]. A single administration of an HSA-GDF15 fusion was sufficient to decrease food intake and drive and maintain weight loss in obese nonhuman primates for 4 weeks [17]. Repeated weekly administration of an Fc-GDF15 fusion for 6 weeks to obese primates decreased food intake and body weight, resulting in lower plasma insulin and triglycerides and improved glucose homeostasis [41]. 6
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GDF15/GFRAL Antagonism Prevention of cisplatin-induced weight loss upon GFRAL deletion suggests that inhibitors of the GDF15/GFRAL pathway could be harnessed to limit the anorexia and/or cachexia elicited in chronic disease, cancer, or with chemotherapy [16]. Indeed, administration of an antibody specifically targeting GDF15 reversed weight loss and restored muscle and fat tissue mass in multiple human xenograft tumor-induced mouse models of cancer cachexia [14,37]. GDF11 is a known driver of cancer cachexia by both promoting anorexia and driving muscle breakdown. Recently, delivery of an antibody targeting GDF15 was demonstrated to block the anorectic effects of GDF11 but not its impact on muscle cell integrity [29]. Furthermore, administration of an anti-GDF15 antibody blocked the effects of both endogenous and pharmacologically administered GDF15 in obese mice,resulting in greater food intake and weight gain compared with control animals [41]. Antagonizing this pathway is not limited to GDF15 targeting because the anorectic effects of Fc-GDF15 in rats were prevented with co-administration of an antiGFRAL antibody [15]. GDF15 and GFRAL beyond Weight Homeostasis GDF15 is robustly induced in many tissues upon diverse stimuli, suggesting a key role in a variety of contexts, and has led to the hypothesis that weight-independent functions exist. Understanding function beyond weight maintenance is important not only for appreciating the full spectrum of benefits achievable by targeting this axis, but also for recognizing potential risks. This effort has been met by several challenges. For example, findings from studies examining the cellular consequences of GDF15 exposure are proposed to be both advantageous and deleterious depending on context and are often difficult to interpret, given the uncertainty of protein quality and purity, as exemplified in a recent report of contamination of commercial sources of GDF15 with TGFb [9,68]. Furthermore, studies have been mechanistically limited by the lack of understanding around GDF15-induced cell signaling, particularly the receptors involved. The identification of GFRAL/RET as the receptors downstream of GDF15 should expand the means to explore the pathway further. Notably, in line with the tissue expression pattern in multiple species, and in stark contrast to its ligand GDF15, no human or rodent cell line has been identified that endogenously expresses Gfral [17,18]. This makes it challenging to study the cellular signaling mediated by GFRAL and difficult to interpret the many previous observations of GDF15 effects in cell culture experiments. GDF15 is unlikely to have additional receptors to explain the reported in vitro effects because multiple efforts were unable to demonstrate binding to any TGFb receptor pair and GFRAL was the only confirmed positive GDF15-binding hit from exhaustive unbiased screens of all known human transmembrane proteins [16–18,62]. Finally, consideration that some of the effects of GDF15 knockdown or overexpression in cells could be due to intracellular functions is warranted [21,22]. One area demanding further exploration is the potential immunomodulatory role of GDF15. An anti-inflammatory and tissue-protective role for GDF15 is supported by the observation that mice lacking GDF15 are more susceptible to lipopolysaccharide-induced organ damage, neuronal injury, lethal myocardial infarction from carotid artery ligation, and renal damage in models of diabetes and collagen-induced pulmonary thromboembolism [69–74]. GDF15 overexpression reduces atherosclerotic plaque size and arthritis disease severity in rodent models [7]. Conversely, lack of GD15 in mice is reported to be protective in atherosclerosis and led to nonsteroidal anti-inflammatory drug (NSAID) chemoprevention resistance [56,75,76]. The immunomodulatory nature of GDF15 is also hypothesized to underlie the pleotropic roles described in tumorigenesis and cancer progression [7,77]. There have been many reports of GDF15 effects in cancer cells and rodent models, and the results are conflicting. For example, loss and gain of function of GDF15 in the TRAMP mouse model Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
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of prostate cancer suggests that GDF15 is protective against tumorigenesis, but that it promotes metastasis during later stages of the disease [78]. Nonetheless, the correlation of elevated levels of GDF15 and poor prognosis in human cancer underscores the need to understand the potential risks of long-term elevated circulating GDF15. While circulating GDF15 positively correlates with all-cause mortality in humans, transgenic overexpression in mice resulted in an increase in life span, suggesting a protective function for GDF15 [40,79]. Importantly, no increase in tumor formation or other pathology was reported in mice transduced with AAV-GDF15 for up to 1 year [41]. In addition, while levels of GDF15 increase in many cancers, GDF15 expression is also positively regulated by tumor suppressor pathways and cancer treatments (chemotherapy, surgery, and radiation) known to halt disease progression [10,78]. The discovery of GFRAL as the receptor for this seemingly pleotropic signaling molecule will enable further investigation of the various reported effects of GDF15 on the immune system and tumor formation and progression. In cachexia of cancer and chronic disease, weight loss and wasting are due to a combination of anorexia and direct effects on muscle [80]. As outlined above, GDF15 is produced by many tumor types and drives weight loss in rodent cancer models; therefore, a potential GDF15mediated effect on muscle atrophy has been considered. Indeed, mice bearing GDF15expressing tumor xenografts lose both fat and muscle mass, which can be reversed by driving weight regain with an antibody blocking GDF15 function [37]. However, obese mice treated with or overexpressing GDF15 lose a greater proportion of fat mass compared with lean mass, in a ratio that is typical of other anorectic agents, leading to a body composition resembling that of lean animals [41,58]. Consistent with GDF15 specifically driving the anorectic arm of cachexia, antibody neutralization of GDF15 prevented feeding inhibition but not muscle atrophy in a GDF11-overexpression cachexia model [29]. These data, coupled with the finding that GFRAL is not expressed in muscle, demonstrate that GDF15 is unlikely to have a direct effect on muscle that causes wasting or loss of lean mass that is more than expected from other weight-loss modalities. Based on the localization of GFRAL to the AP, a region that includes the chemoreceptor trigger zone, and the correlation between circulating levels and emesis during secondtrimester pregnancies, it is logical to hypothesize that GDF15 signaling could induce nausea, malaise, and vomiting [81]. Single nucleotide polymorphisms located in a noncoding region of GDF15 show some association with hyperemesis gravidarum, a syndrome of severe nausea during pregnancy [82]. In this context, it has been proposed that GDF15 decreases food intake and thereby limits exposure to potentially noxious stimuli as a fetal protective mechanism, especially during the vulnerable first trimester of pregnancy, although it is more likely that any survival benefit to the fetus due to mitigation of such a risk may be entirely outweighed by a maternal negative energy balance [81,83]. GDF15 levels also increase after exposure to cisplatin treatment, a cancer therapy often associated with nausea and emesis [84]. These data support GDF15 antagonism as a novel antiemetic strategy, while suggesting that gastrointestinal (GI) adverse events are to be expected from GDF15 pharmacotherapy. Rodents are a nonemetic species and surrogate experiments, such as conditioned taste aversion or pica tests, upon GDF15 treatment have not yet been reported. Furthermore, in nonhuman primate weight-loss studies specifically designed to monitor for signs of nausea, malaise, and vomiting, no GI-related events were reported after GDF15 treatment [17,41]. Future studies characterizing GDF15/GFRAL circuitry will be important to determine whether this pathway has an obligatory role in the proemetic function of the AP. Importantly, leptin and pancreatic polypeptide provide precedence for signals that influence food intake via the AP without causing emesis [85–88]. 8
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Concluding Remarks and Future Perspectives
Outstanding Questions
Evidence demonstrating that circulating GDF15 controls food intake and body weight has accumulated over the past two decades, driving exploration of the potential of targeting this molecule in pathological states of overnutrition (obesity) and undernutrition (anorexia/cachexia) (Figure 1). The recent finding that GFRAL, a receptor that is exclusively located to neurons of the AP, is necessary for GDF15-mediated anorexia has begun to outline a novel circuit controlling energy homeostasis. Despite sharing characteristics with other endocrine factors that control food intake (expressed in peripheral metabolic tissues, targeting neurons in the brain, and increased after RYGB), GDF15 appears to be unique from several perspectives. For example, its regulation favors increased and sustained circulating levels under many conditions, including broad tissue expression. In addition, unlike transient postprandial anorectic signals, GDF15 levels do not increase after meal ingestion, similar to leptin, a well-known homeostatic regulator of food intake and body weight. However, the importance of leptin is most pronounced in the absence of signaling, as exemplified in the extreme hyperphagia and obesity observed in ob/ob and db/db mice; while leptin treatment in wild-type animals only has minor effects. By contrast, GDF15/GFRAL physiology is most impactful in times of excess signaling; GDF15 treatment has robust weight-loss effects in mice and primates, but its deletion results in only minor phenotypes under nonstressed physiological conditions. It will be of interest to the field to overlay these observations with a detailed molecular and cellular characterization of the GDF15/GFRAL pathway. In doing so, future studies will expand pharmacological approaches to, and implications of, targeting this pathway in obesity and other diseases (see Outstanding Questions).
What are the characteristics of GFRAL-positive neurons in the area postrema? What functional changes are elicited in these cells upon GDF15 binding? What is the neuronal circuitry associated with these cells? Does GDF15/GFRAL signaling impact gastric motility, energy expenditure, or nausea and/or emesis, and do these mechanisms contribute significantly to weight loss in this context? Will neutralizing GDF15/GFRAL signaling lessen or reverse the anorexia contributing to cancer cachexia in humans? Will this be sufficient to drive beneficial weight gain in patients? Are there functions of GDF15 independent of anorexia and weight loss? Are all GDF15 functions mediated through GFRAL?
Acknowledgments We thank James Leonard, Vedrana Stojanovic-Susulic, Eve Frank, and Mark Erion for careful reading of this manuscript, Songmao Zheng for helpful discussion, and Susan Leibowitz Basile (BasilPix) for figure artwork. We apologize to researchers whose relevant studies were not discussed due to space limitations.
References 1. Hromas, R. et al. (1997) PLAB, a novel placental bone morphogenetic protein. Biochim. Biophys. Acta 1354, 40–44 2. Bootcov, M.R. et al. (1997) MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc. Natl. Acad. Sci. U. S. A. 94, 11514–11519 3. Lawton, L.N. et al. (1997) Identification of a novel member of the TGF-beta superfamily highly expressed in human placenta. Gene 203, 17–26 4. Baek, S.J. et al. (2001) Cyclooxygenase inhibitors regulate the expression of a TGF-beta superfamily member that has proapoptotic and antitumorigenic activities. Mol. Pharmacol. 59, 901– 908
11. Bauskin, A.R. et al. (2006) Role of macrophage inhibitory cytokine-1 in tumorigenesis and diagnosis of cancer. Cancer Res. 66, 4983–4986 12. Mimeault, M. and Batra, S.K. (2010) Divergent molecular mechanisms underlying the pleiotropic functions of macrophage inhibitory cytokine-1 in cancer. J. Cell. Physiol. 224, 626–635 13. Baek, S.J. et al. (2006) Nonsteroidal anti-inflammatory drug-activated gene-1 over expression in transgenic mice suppresses intestinal neoplasia. Gastroenterology 131, 1553–1560 14. Johnen, H. et al. (2007) Tumor-induced anorexia and weight loss are mediated by the TGF-beta superfamily cytokine MIC-1. Nat. Med. 13, 1333–1340
5. Paralkar, V.M. et al. (1998) Cloning and characterization of a novel member of the transforming growth factor-beta/bone morphogenetic protein family. J. Biol. Chem. 273, 13760–13767
15. Emmerson, P.J. et al. (2017) The metabolic effects of GDF15 are mediated by the orphan receptor GFRAL. Nat. Med. 23, 1215– 1219
6. Bottner, M. et al. (1999) Expression of a novel member of the TGF-beta superfamily, growth/differentiation factor-15/macrophage-inhibiting cytokine-1 (GDF-15/MIC-1) in adult rat tissues. Cell Tissue Res. 297, 103–110
16. Hsu, J.Y. et al. (2017) Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15. Nature 550, 255–259
7. Breit, S.N. et al. (2011) The TGF-beta superfamily cytokine, MIC1/GDF15: a pleotrophic cytokine with roles in inflammation, cancer and metabolism. Growth Factors 29, 187–195 8. Adela, R. and Banerjee, S.K. (2015) GDF-15 as a target and biomarker for diabetes and cardiovascular diseases: a translational prospective. J. Diabetes Res. 2015, 490842 9. Corre, J. et al. (2013) Concise review: growth differentiation factor 15 in pathology: a clinical role? Stem Cells Transl. Med. 2, 946– 952 10. Wang, X. et al. (2013) The diverse roles of nonsteroidal antiinflammatory drug activated gene (NAG-1/GDF15) in cancer. Biochem. Pharmacol. 85, 597–606
17. Mullican, S.E. et al. (2017) GFRAL is the receptor for GDF15 and the ligand promotes weight loss in mice and nonhuman primates. Nat. Med. 23, 1150–1157 18. Yang, L. et al. (2017) GFRAL is the receptor for GDF15 and is required for the anti-obesity effects of the ligand. Nat. Med. 23, 1158–1166 19. Breit, S.N. et al. (2017) Targeting obesity and cachexia: identification of the GFRAL receptor-MIC-1/GDF15 pathway. Trends Mol. Med. 23, 1065–1067 20. Bauskin, A.R. et al. (2000) The propeptide of macrophage inhibitory cytokine (MIC-1), a TGF-beta superfamily member, acts as a quality control determinant for correctly folded MIC-1. EMBO J. 19, 2212–2220
Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
9
TEM 1330 No. of Pages 11
21. Min, K.W. et al. (2016) NAG-1/GDF15 accumulates in the nucleus and modulates transcriptional regulation of the Smad pathway. Oncogene 35, 377–388
43. Kempf, T. et al. (2012) Growth differentiation factor 15 predicts future insulin resistance and impaired glucose control in obese nondiabetic individuals: results from the XENDOS trial. Eur. J. Endocrinol. 167, 671–678
22. Zhang, X. et al. (2017) GL-V9 induced upregulation and mitochondrial localization of NAG-1 associates with ROS generation and cell death in hepatocellular carcinoma cells. Free Radic. Biol. Med. 112, 49–59
44. Tsai, V.W. et al. (2012) Anorexia/cachexia of chronic diseases: a role for the TGF-beta family cytokine MIC-1/GDF15. J. Cachexia Sarcopenia Muscle 3, 239–243
23. Zimmers, T.A. et al. (2005) Growth differentiation factor-15/macrophage inhibitory cytokine-1 induction after kidney and lung injury. Shock 23, 543–548
45. Vila, G. et al. (2011) The relationship between insulin resistance and the cardiovascular biomarker growth differentiation factor-15 in obese patients. Clin. Chem. 57, 309–316
24. Baek, S.J. et al. (2001) Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter. Basal transcription is mediated by Sp1 and Sp3. J. Biol. Chem. 276, 33384–33392
46. Dostalova, I. et al. (2010) Association of macrophage inhibitory cytokine-1 with nutritional status, body composition and bone mineral density in patients with anorexia nervosa: the influence of partial realimentation. Nutr. Metab. 7, 34
25. Osada, M. et al. (2007) A p53-type response element in the GDF15 promoter confers high specificity for p53 activation. Biochem. Biophys. Res. Commun. 354, 913–918
47. Fearon, K. et al. (2011) Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 12, 489–495
26. Lim, J.H. et al. (2007) NAG-1 up-regulation mediated by EGR-1 and p53 is critical for quercetin-induced apoptosis in HCT116 colon carcinoma cells. Apoptosis 12, 411–421
48. Lerner, L. et al. (2015) Plasma growth differentiation factor 15 is associated with weight loss and mortality in cancer patients. J. Cachexia Sarcopenia Muscle 6, 317–324
27. Han, M. et al. (2017) CXXC4 activates apoptosis through up-regulating GDF15 in gastric cancer. Oncotarget 8, 103557–103567
49. Lerner, L. et al. (2016) Growth differentiating factor-15 (GDF-15): A potential biomarker and therapeutic target for cancer-associated weight loss. Oncol. Lett. 12, 4219–4223
28. Chung, H.K. et al. (2017) Growth differentiation factor 15 is a myomitokine governing systemic energy homeostasis. J. Cell Biol. 216, 149–165
50. Breit, S.N. et al. (2012) Macrophage inhibitory cytokine-1 (MIC-1/ GDF15) and mortality in end-stage renal disease. Nephrol. Dial. Transplant. 27, 70–75
29. Jones, J.E. et al. (2018) Supraphysiologic administration of GDF11 induces cachexia in part by upregulating GDF15. Cell Rep. 22, 1522–1530
51. Kempf, T. et al. (2007) Growth-differentiation factor-15 improves risk stratification in ST-segment elevation myocardial infarction. Eur. Heart J. 28, 2858–2865
30. Yoshioka, H. et al. (2008) Nonsteroidal anti-inflammatory drugactivated gene (NAG-1/GDF15) expression is increased by the histone deacetylase inhibitor trichostatin A. J. Biol. Chem. 283, 33129–33137
52. Chrysovergis, K. et al. (2014) NAG-1/GDF-15 prevents obesity by increasing thermogenesis, lipolysis and oxidative metabolism. Int. J. Obes. 38, 1555–1564 53. Macia, L. et al. (2012) Macrophage inhibitory cytokine 1 (MIC-1/ GDF15) decreases food intake, body weight and improves glucose tolerance in mice on normal & obesogenic diets. PLoS One 7, e34868
31. Martinez, J.M. et al. (2006) Drug-induced expression of nonsteroidal anti-inflammatory drug-activated gene/macrophage inhibitory cytokine-1/prostate-derived factor, a putative tumor suppressor, inhibits tumor growth. J. Pharmacol. Exp. Ther. 318, 899–906
54. Strelau, J. et al. (2009) Progressive postnatal motoneuron loss in mice lacking GDF-15. J. Neurosci. 29, 13640–13648
32. Bauskin, A.R. et al. (2010) The TGF-beta superfamily cytokine MIC-1/GDF15: secretory mechanisms facilitate creation of latent stromal stores. J. Interferon Cytokine Res. 30, 389–397
55. Casanovas, G. et al. (2013) The murine growth differentiation factor 15 is not essential for systemic iron homeostasis in phlebotomized mice. Haematologica 98, 444–447
33. Fairlie, W.D. et al. (1999) MIC-1 is a novel TGF-beta superfamily cytokine associated with macrophage activation. J. Leukoc. Biol. 65, 2–5
56. Bonaterra, G.A. et al. (2012) Growth differentiation factor-15 deficiency inhibits atherosclerosis progression by regulating interleukin-6-dependent inflammatory response to vascular injury. J. Am. Heart Assoc. 1, e002550
34. Moore, A.G. et al. (2000) The transforming growth factor-ss superfamily cytokine macrophage inhibitory cytokine-1 is present in high concentrations in the serum of pregnant women. J. Clin. Endocrinol. Metab. 85, 4781–4788 35. Ding, Q. et al. (2009) Identification of macrophage inhibitory cytokine-1 in adipose tissue and its secretion as an adipokine by human adipocytes. Endocrinology 150, 1688–1696 36. Kleinert, M. et al. (2018) Exercise increases circulating GDF15 in humans. Mol. Metab. 9, 187–191
57. Tsai, V.W. et al. (2013) TGF-b superfamily cytokine MIC-1/GDF15 is a physiological appetite and body weight regulator. PLoS One 8, e55174 58. Tsai, V.W. et al. (2017) Treatment with the TGF-b superfamily cytokine MIC-1/GDF15 reduces the adiposity and corrects the metabolic dysfunction of mice with diet-induced obesity. Int. J. Obes. 42, 561–571
37. Lerner, L. et al. (2016) MAP3K11/GDF15 axis is a critical driver of cancer cachexia. J. Cachexia Sarcopenia Muscle 7, 467–482
59. Tsai, V.W. et al. (2014) The anorectic actions of the TGFbeta cytokine MIC-1/GDF15 require an intact brainstem area postrema and nucleus of the solitary tract. PLoS One 9, e100370
38. Nair, V. et al. (2017) Growth differentiation factor-15 and risk of CKD progression. J. Am. Soc. Nephrol. 28, 2233–2240
60. Janssen, P. et al. (2011) Review article: the role of gastric motility in the control of food intake. Aliment. Pharmacol. Ther. 33, 880–894
39. Kempf, T. et al. (2007) Prognostic utility of growth differentiation factor-15 in patients with chronic heart failure. J. Am. Coll. Cardiol. 50, 1054–1060
61. Tschop, M.H. et al. (2011) A guide to analysis of mouse energy metabolism. Nat. Methods 9, 57–63
40. Wiklund, F.E. et al. (2010) Macrophage inhibitory cytokine-1 (MIC-1/GDF15): a new marker of all-cause mortality. Aging Cell 9, 1057–1064 41. Xiong, Y. et al. (2017) Long-acting MIC-1/GDF15 molecules to treat obesity: evidence from mice to monkeys. Sci. Transl. Med. 9, eaan8732 42. Dostalova, I. et al. (2009) Increased serum concentrations of macrophage inhibitory cytokine-1 in patients with obesity and type 2 diabetes mellitus: the influence of very low calorie diet. Eur. J. Endocrinol. 161, 397–404
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62. Unsicker, K. et al. (2013) The multiple facets of the TGF-beta family cytokine growth/differentiation factor-15/macrophage inhibitory cytokine-1. Cytokine Growth Factor Rev. 24, 373–384 63. Artz, A. et al. (2016) GDF-15 inhibits integrin activation and mouse neutrophil recruitment through the ALK-5/TGF-betaRII heterodimer. Blood 128, 529–541 64. Liu, D.D. et al. (2016) Growth differentiation factor-15 promotes glutamate release in medial prefrontal cortex of mice through upregulation of T-type calcium channels. Sci. Rep. 6, 28653 65. Li, Z. et al. (2005) Identification, expression and functional characterization of the GRAL gene. J. Neurochem. 95, 361–376
Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
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66. Hatinen, T. et al. (2007) Loss of neurturin in frog–comparative genomics study of GDNF family ligand-receptor pairs. Mol. Cell. Neurosci. 34, 155–167
77. Vanhara, P. et al. (2012) Growth/differentiation factor-15: prostate cancer suppressor or promoter? Prostate Cancer Prostatic Dis. 15, 320–328
67. van Witteloostuijn, S.B. et al. (2016) Half-life extension of biopharmaceuticals using chemical methods: alternatives to PEGylation. ChemMedChem 11, 2474–2495
78. Husaini, Y. et al. (2015) Macrophage inhibitory cytokine-1 (MIC?1/ GDF15) gene deletion promotes cancer growth in TRAMP prostate cancer prone mice. PLoS One 10, e0115189
68. Olsen, O.E. et al. (2017) TGF-beta contamination of purified recombinant GDF15. PLoS One 12, e0187349
79. Wang, X. et al. (2014) hNAG-1 increases lifespan by regulating energy metabolism and insulin/IGF-1/mTOR signaling. Aging 6, 690–704
69. Wang, X. et al. (2015) Growth/differentiation factor-15 and its role in peripheral nervous system lesion and regeneration. Cell Tissue Res. 362, 317–330 70. Machado, V. et al. (2016) Growth/differentiation factor-15 deficiency compromises dopaminergic neuron survival and microglial response in the 6-hydroxydopamine mouse model of Parkinson’s disease. Neurobiol. Dis. 88, 1–15 71. Abulizi, P. et al. (2017) Growth differentiation factor-15 deficiency augments inflammatory response and exacerbates septic heart and renal injury induced by lipopolysaccharide. Sci. Rep. 7, 1037 72. Kempf, T. et al. (2011) GDF-15 is an inhibitor of leukocyte integrin activation required for survival after myocardial infarction in mice. Nat. Med. 17, 581–588
80. Fearon, K.C. et al. (2012) Cancer cachexia: mediators, signaling, and metabolic pathways. Cell Metab. 16, 153–166 81. O’Rahilly, S. (2017) GDF15-from biomarker to allostatic hormone. Cell Metab. 26, 807–808 82. Fejzo, M.S. et al. (2018) Placenta and appetite genes GDF15 and IGFBP7 are associated with hyperemesis gravidarum. Nat. Commun. 9, 1178 83. Abu-Saad, K. and Fraser, D. (2010) Maternal nutrition and birth outcomes. Epidemiol. Rev. 32, 5–25 84. Altena, R. et al. (2015) Growth differentiation factor 15 (GDF-15) plasma levels increase during bleomycin- and cisplatin-based treatment of testicular cancer patients and relate to endothelial damage. PLoS One 10, e0115372
73. Mazagova, M. et al. (2013) Genetic deletion of growth differentiation factor 15 augments renal damage in both type 1 and type 2 models of diabetes. Am. J. Physiol. Renal Physiol. 305, F1249– F1264
85. Huo, L. et al. (2007) Leptin and the control of food intake: neurons in the nucleus of the solitary tract are activated by both gastric distension and leptin. Endocrinology 148, 2189–2197
74. Rossaint, J. et al. (2013) GDF-15 prevents platelet integrin activation and thrombus formation. J. Thromb. Haemost. 11, 335– 344
86. Cuenco, J. et al. (2017) Degradation paradigm of the gut hormone, pancreatic polypeptide, by hepatic and renal peptidases. Endocrinology 158, 1755–1765
75. Zimmers, T.A. et al. (2010) Loss of GDF-15 abolishes sulindac chemoprevention in the ApcMin/+ mouse model of intestinal cancer. J. Cancer Res. Clin. Oncol. 136, 571–576
87. Woods, S.C. et al. (2006) Pancreatic signals controlling food intake; insulin, glucagon and amylin. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361, 1219–1235
76. de Jager, S.C. et al. (2011) Growth differentiation factor 15 deficiency protects against atherosclerosis by attenuating CCR2-mediated macrophage chemotaxis. J. Exp. Med. 208, 217–225
88. Paz-Filho, G. et al. (2015) Leptin treatment: facts and expectations. Metabolism 64, 146–156
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Review
Uniting GDF15 and GFRAL: Therapeutic Opportunities in Obesity and Beyond Shannon E. Mullican and Shamina M. Rangwala* Growth differentiation factor-15 (GDF15) is a circulating protein that has been implicated in multiple biological processes, including energy homeostasis, body weight regulation, and cachexia driven by cancer and chronic disease. The potential to target GDF15 in the treatment of energy-intake disorders, including obesity and anorexia, is an area of intense investigation, but has been limited by the lack of an identified receptor, signaling mechanism, and target tissue. GDNF family receptor a-like (GFRAL) was recently identified as the neuronal brainstem receptor responsible for mediating the anorectic actions of GDF15. Herein, we provide a brief overview of GDF15 biology with a focus on energy homeostasis, and highlight the implications of the recent receptor identification to this field and beyond. A Brief History of GDF15 Research Twenty years ago, three independent laboratories described a novel member of the TGFb superfamily (see Glossary) and, based on the context of the discoveries, termed the factor ‘placenta BMP’ (PLAB), ‘macrophage inhibitory cytokine-10 (MIC-1) and ‘placental transforming growth factor beta’ (PTGFB) [1–3]. Additional reports of the discovery of this factor followed shortly thereafter, leading to other nomenclature, including nonsteroidal anti-inflammatory drug-activated gene (NAG-1), prostate-derived factor (PDF), and growth differentiation factor 15 (GDF15) [4–6]. For simplicity, we use the terminology GDF15 for the remainder of the discussion. GDF15 is expressed broadly in multiple tissues and is robustly increased in circulation during pregnancy, after tissue injury, and in diverse disease states, including cancer, cardiovascular, and kidney disease, where it often correlates with poor prognosis [7]. These correlations focused the first decade of GDF15 investigation on its assessment as a biomarker in numerous conditions and its potential function in cancer and inflammation [8–12].
Highlights GDF15 is a hormone that is broadly expressed and is considered a biomarker the circulation of which positively correlates with the progression of many diseases. GDF15 administration leads to decreased food intake and body weight in obese rodents and nonhuman primates. GFRAL, a distant relative of receptors for a distinct class of the TGFb superfamily ligands, has recently been identified as the high-affinity receptor binding and mediating the anorectic effects of GDF15. GDF15 and GFRAL are promising therapeutic targets for the treatment of disorders of energy homeostasis, including obesity and anorexia.
The generation of loss- and gain-of-function mouse models provided the first insights into GDF15 biology beyond pregnancy, cancer, and inflammation. Specifically, the observation that mice with increased circulating GDF15 weighed less than mice lacking GDF15 started a decade of work focused on understanding the potential role of GDF15 in disorders of weight maintenance, including anorexia, cancer cachexia, and obesity [13,14]. However, progress in characterizing mechanisms underlying these observations was limited by the uncertainty of the cellular receptor and primary target tissue mediating the various effects of GDF15. Fast forward 20 years after the initial description of GDF15 and four independent pharmaceutical company laboratories have simultaneously described the identification of the GDF15 receptor [15–19]. The identification of GFRAL as the GDF15 receptor has triggered the next era of GDF15 research, which will include not only delineation of the molecular and cellular signaling downstream of GDF15, but also the careful reconsideration of previously reported GDF15 effects in the context of this new family member. As a result, novel pharmacological
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Cardiovascular & Metabolism Therapeutic Area, Janssen Pharmaceuticals, Inc., Spring House, PA 19477, USA
*Correspondence: [email protected] (S.M. Rangwala).
https://doi.org/10.1016/j.tem.2018.05.002 © 2018 Elsevier Ltd. All rights reserved.
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approaches to targeting this pathway for the treatment of obesity, anorexia, and cachexia will result and our understanding of GDF15 physiology beyond weight regulation will expand.
GDF15 Molecular Characteristics and Regulation GDF15 gene structure is conserved across species and comprises two exons separated by one intron of approximately 3 kb [2]. The resulting GDF15 polypeptide includes a signal peptide, a propeptide region, and a mature region at the C terminus, typical of the TGFb superfamily. Divergence within the family is reflected by GDF15 having a relatively low sequence similarity and a unique intrachain disulfide bonding arrangement in the cysteine knot region in the C terminus compared with other members [16]. The precursor GDF15 polypeptide forms a homodimer stabilized through one interchain disulfide bond at the C terminus of the protein. The N-terminal propeptide is proteolytically removed and a mature 25-kDa homodimer is generated and secreted [20]. In addition to the endocrine functions of secreted GDF15, intracellular functions, including modulation of mitochondrial membrane potential and nuclear transcription factor activity, have been reported [21,22]. The cell has multiple mechanisms to rapidly increase and maintain GDF15 levels. Transcription induction occurs in the absence of new protein production, a hallmark of immediate early genes [23]. The Gdf15 promoter has many putative regulatory sites and multiple transcription factors have been implicated in direct regulation upon various stimuli, including p53, EGR-1, Sp1, CHOP, and SMAD2/3 [24–29]. Post-transcriptional stabilization of GDF15 mRNA has been reported [22,30,31]. In addition, unprocessed GDF15 propeptide can be secreted and is proposed to bind the extracellular matrix and form latent stores that can be quickly released upon the appropriate stimulus [32]. Gdf15 is expressed in many tissues, including kidney, liver, muscle, adipose, and placenta, which secretes high levels of GDF15 during pregnancy [1,5,33–35]. In most individuals, circulating GDF15 levels range between 100 and 1200 pg/ml but increase after exercise, tissue injury, and in many disease states, including cancer, cardiovascular, and kidney disease, where they often correlate with weight loss and poor prognosis [23,36–39]. GDF15 levels are predictive of all-cause mortality [40]. Due to these robust correlations, GDF15 serves as a biomarker in multiple diseases and its potential roles in cancer and inflammatory processes have been the focus of many investigations [11,12].
GDF15 and Body-Weight Regulation GDF15 expression and circulating levels correlate with body-weight differences. As with other physiological stresses, circulating GDF15 is elevated in obesity in mice, rats, and humans [41– 43]. The specific tissue source of GDF15 in this context is not clear, although its expression increases in the liver and adipose of obese mice [41]. In contrast to the relatively minor rise in plasma GDF15 in a state of overnutrition, substantial further increases correlate with weight loss in humans [44]. Obese patients were assessed before and 1 year after Roux-en-Y gastric bypass (RYGB) [45]. At baseline, plasma GDF15 was elevated compared with nonobese subjects; however, after RYGB-driven weight loss, GDF15 concentration was even higher. One explanation for these seemingly conflicting observations is that GDF15 could be part of a compensatory mechanism in obesity that limits energy intake during periods of nutrient excess. Furthermore, GDF15 is elevated in patients with anorexia nervosa compared with control subjects and decreases upon realimentation [46]. Anorexia is a component of cancer cachexia and the resulting undernutrition-driven weight loss significantly contributes to an overall decline in advanced disease. Cancer cachexia is a progressive syndrome and patients can be classified into stages according to the degree of depletion of fat and lean mass in combination 2
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Glossary Anorexia: decreased food intake because of diminished appetite or aversion to food. Area postrema (AP): medullary brainstem structure at the base of the fourth ventricle with permeable capillaries allowing exposure to constituent neurons to peptides and hormones of the peripheral circulation. Functions include autonomic regulation of various homeostatic processes, including feeding behavior. Cachexia: disease-associated multiorgan wasting syndrome driving severe weight loss associated with muscle atrophy and appetite suppression in patients not actively trying to lose weight. Central amygdala: forebrain nucleus with a role in physiological and behavioral responses to fear, stress, and drug-related stimuli, including that of noxious agents, via signaling emanating from the brainstem. Chemoreceptor trigger zone (CTZ): anatomical region within the AP implicated in sensing blood-borne drugs or hormones that induce vomiting. Fc portion of immunoglobulin G: tail region of antibody that interacts with cell surface Fc receptors to activate the immune system. Fusion of peptides to a modified nonimmunogenic IgG Fc increases the half-life of the fusion partner in drug discovery efforts. FcRn recycling: neonatal Fc receptor (FcRN) expressed on immune and epithelial cells. Upon cellular uptake, serum albumin and immunoglobulin G bind to FcRN, which transports this cargo back to the plasma membrane, ultimately releasing it extracellularly. This process allows albumin and IgG to bypass lysosomal degradation and has been exploited in drug discovery efforts. Glial cell-derived neurotrophic factor (GDNF) family: subgroup of TGFb superfamily with known roles in neuronal development and function. Signaling is mediated by GDNF family receptors (GFRa1–4) complexed with RET receptor tyrosine kinase. Glucagon-like peptide-1 (GLP-1): peptide hormone made by intestinal
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with the degree of ongoing weight loss [47]. In a study examining GDF15 levels in patients with various stages of cachexia, elevation was measured in patients with early precachexia compared with non-cachectic patients and remained elevated in patients with cachexia and advanced refractory cachexia [37]. Independent analyses demonstrated that patients with cancer experiencing cachexia-driven weight loss have higher levels of plasma GDF15 compared with patients with cancer and a stable weight [14,48,49]. Cachexia and weight loss are serious complications in patients with chronic diseases of the kidney and heart. In a study of patients on dialysis due to chronic kidney disease, higher GDF15 levels were observed in those with low body mass indices (BMI) consistent with findings from a previous cohort with endstage renal failure demonstrating an inverse correlation of plasma GDF15 and BMI [14,50]. Likewise, during an assessment of circulating GDF15 in patients with chronic heart failure (CHF), the highest levels were observed in those with the lowest BMI [51]. While multiple human studies have shown correlations of GDF15 levels with weight loss associated with the cachectic state, causation remains to be proven. In this context, GDF15 is elevated after gastric bypass surgery, which results in significant weight loss, and is not associated with cachexia [45]. Further investigations examining the relationship of circulating levels of GDF15 in scenarios of weight loss in the absence of cachexia, for example with calorie restriction, are warranted. The correlation between weight loss and GDF15 is conserved in rodents and this has enabled interrogation of GDF15 as a potential causal factor impacting body weight. Two independent transgenic models overexpressing GDF15 from birth exhibit lower body weight, fat mass, and associated improvements in glucose tolerance [13,14]. Transgenic overexpression of GDF15 also protected mice from obesity, liver steatosis, and glucose intolerance upon high-fat diet feeding [52,53]. Introduction of GDF15 overexpression in adult livers by hydrodynamic injection of DNA vectors or viral infection decreased food intake and body weight in mice with established obesity [29,41]. Decreases in food intake and body weight associated with xenografted prostate tumor cells transfected with Gdf15 were reversed by blocking GDF15 with an antibody in mice [14]. Conversely, numerous investigators have reported that mice lacking GDF15 gain more weight and have greater fat mass than wild-type animals [54–57]. The potential of pharmacologically administered GDF15 to decrease energy intake and thereby elicit weight loss has been demonstrated in mice, rats and monkeys. Lean mice treated with recombinant GDF15 resulting in circulating levels orders of magnitude above the physiological range, eat less and lose weight [14,16,17,58]. Decreased food intake and body weight is also observed in genetic- and diet-induced rat and mouse models of obesity after administration of GDF15 [14,17,18,41,58]. GDF15 treatment-mediated weight loss in diet-induced obese mice led to metabolic improvements, including enhanced glucose homeostasis and lower serum triglycerides and cholesterol [17,41,58]. These effects translate to higher species because a 6week daily treatment regimen with recombinant human GDF15 in spontaneously obese nonhuman primates reduced food intake, body weight, and plasma triglyceride concentrations and improved glucose tolerance [41]. The physiological mechanism by which GDF15 drives weight loss primarily involves a reduction in total food intake, as has been demonstrated in multiple species as described above. Direct central nervous system (CNS) delivery of GDF15 by intracerebroventricular injection in mice and rats is sufficient to inhibit food intake [18,59]. Lower caloric intake is likely due to increased satiation as opposed to general malaise, because GDF15 treatment resulted in a reduction in meal size with no change in the number of meals or sickness-like behavior (hunched posture, labored movements, or altered breathing) in mice provided with either a standard chow or highfat diet [15,17]. Consistent with less food-exploring behavior, ambulatory activity was reduced
enteroendocrine cells that has many metabolic roles, including modulation of feeding behavior via signaling through the GLP-1 receptor located within the AP and NTS. Hypothalamus: collection of nuclei within the forebrain that coordinate autonomic processes, including homeostatic regulation of sleep, body temperature, thirst, and hunger, and hormonal signaling from the nearby pituitary gland. Leptin: adipose-derived hormone that functions as a long-term mediator of energy balance by suppressing food intake upon binding to the leptin receptor located within the NTS, AP, and hypothalamus. Melanocortin-4 receptor (MC4R): located within the hypothalamus and brainstem, agonists of this receptor inhibit food intake. Nucleus of the solitary tract (NTS): located adjacent to the AP in the brainstem, this is the primary visceral sensory relay station within the brain receiving and responding to stimuli from the respiratory, cardiovascular, and gastrointestinal systems to control homeostatic processes, including feeding behavior. Parabrachial nucleus: neuronal relay center anatomically located between the brainstem and viscerosensory and autonomic centers in the forebrain, including the central amygdala. Functions include taste processing and regulation of feeding. Roux-en-Y gastric bypass (RYGB): weight-loss surgery reducing the size of the stomach restricting the amount of food a patient can eat and bypassing the upper portion of the small intestine, thus decreasing nutrient absorption. Surgery-subsequent changes in gut hormones also contribute to weight loss. Satiation: suppression of ongoing eating; a signal to end a meal. TRAMP mice: model used in oncology research harboring the Transgenic Adenocarcinoma of the Mouse Prostate transgene that develop progressive forms of prostate cancer with distant site metastasis. Transforming growth factor beta (TGFb) superfamily: structurally
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in mice with elevated circulating GDF15 only at the onset of the dark cycle, the time of day when mice typically ingest the largest meals [53,58]. When provided a diet choice, mice treated with GDF15 selectively lowered their fat intake rather than protein or carbohydrate, suggesting that taste preference is a component of GDF15 energy intake regulation [41]. Gastric distension upon the initiation of a meal influences satiation through mechanoreceptors communicating to the CNS through vagal afferent neurons and is determined, in part, by gastric emptying [60]. Exogenously administered GDF15 was reportedly enriched at neuronal beds along the gut and slowed gastric emptying in mice and still effectively reduced food intake in rats after selective vagal deafferentation [18,41]. While a more pleiotropic effect of GDF15 on food preference and gastric motility is intriguing, future studies will be necessary to tease apart the contributions of such changes to the overall effect of GDF15 on body-weight regulation. Feeding-independent mechanisms of GDF15 on body weight are being considered. Reported effects of transgenic overexpression of GDF15 on energy expenditure have been inconsistent, perhaps owing to the lack of normalization of energy expenditure to lean mass, which was proportionally increased in mice treated with GDF15 [52,53,61]. Treatment with recombinant GDF15 had no direct impact on energy expenditure in body-weight matched mice [18]. GDF15 administration led to a shift in fuel preference toward lipid oxidation, which is likely secondary to its effects on anorexia and subsequent weight loss, which results in fat store mobilization [16,41]. Finally, pair-feeding rodents the same amount of food ingested after GDF15 administration was sufficient to elicit the weight loss achieved by treatment, suggesting that additional mechanisms beyond energy intake regulation, such as a direct effect on energy expenditure, are unlikely to contribute to the initial weight loss observed upon GDF15 treatment [14,17,18].
The Identification of GFRAL The impact of GDF15 on body weight led several independent laboratories to seek the identity of the receptor mediating this biological effect. Its position as a member of the TGFb family led to the hypothesis that it could be signaling via TGFb-family receptor pairs; however, efforts to demonstrate direct binding of GDF15 to any known TGFb receptors did not support this idea [16–18,62–64]. Three independent unbiased screens of thousands of human cell membraneassociated proteins and a biased approach considering orphan members of the glial-cellline-derived neurotrophic factor (GDNF) family in parallel led to the identification of GFRAL as the sole receptor for GDF15 [15–18]. GFRAL was previously an orphan receptor classified as a distant homolog of the GDNF family of receptors [65,66]. GDF15 binds with high affinity to GFRAL, but to no other members of the GDNF family of receptors, including GFRA1, GFRA2, GFRA3,or GFRA4 [15–17]. Similarly, GFRAL only binds GDF15 and not any other GDNFrelated ligands, including neurturin (NRTN), artemin (ARTN), and persephin (PSPN) [18]. GDNF family ligands bind to receptor complexes, including coreceptor RET. GFRAL binds to RET upon GDF15 binding and, once this complex has formed, cell signaling typical to this family, including phosphorylation of RET, AKT, ERK, and PLC-g1, is observed (Figure 1, Key Figure) [16–18]. Importantly, GDF15 effects on food intake and weight loss were abolished in mice lacking GFRAL, confirming the physiological relevance of the ligand–receptor relationship identified in the in vitro systems [15–18]. Highly restricted expression of GFRAL is conserved across species. GFRAL mRNA and protein are limited to the brainstem, specifically in a subset of neurons of the area postrema (AP) in adult mouse, rat, monkey, and humans [15–18]. Expression within the nearby nucleus of the solitary tract (NTS) has also been reported, but these findings are less consistent between independent groups and require further confirmation. More widespread and dynamic CNS Gfral expression in the embryo suggests additional roles unique to development [65]. 4
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related molecules the active moiety of which is a dimer formed through an intermolecular disulfide bond; interact with a conserved family of transmembrane receptors to elicit intracellular signaling and regulate many biological processes.
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Key Figure
Growth Differentiation Factor-15/GDNF Family Receptor a-Like (GDF15/GFRAL) Signaling as a Modulator of Energy Intake and Weight Homeostasis
GFRAL antagonism GFRAL agonism
AMY
HYP
LPBN
Decreased food intake
NTS AP
Increased food intake
P
RET
P
MEK/ERK
P
GDF15
PLC/PKC P
GFRAL
P
P
PI3K/AKT
Figure 1. Peripherally derived GDF15 binds to GRFAL/RET receptor complexes on the cell surface of neurons within the brainstem area postrema (AP), triggering receptor phosphorylation (P) and downstream intracellular signaling. This signaling impinges on yet to be defined neuronal circuitry that may include neurons of the nucleus of the solitary tract (NTS), lateral parabrachial nucleus (LPBN), central amygdala (AMY), and hypothalamus (HYP). The results of this signaling are a decrease in food intake and subsequent weight loss. Therapeutic opportunities targeting this axis include agonism for weight loss in obesity or antagonism in states of decreased energy intake, including anorexia nervosa or disease-related cachexia.
GFRAL localization to the adult brainstem is consistent with previous findings demonstrating that the AP and NTS are required for the anorectic action of GDF15 [59]. GFRAL-positive neurons express RET and, consistent with activation of RET signaling, demonstrated increased phosphorylation of ERK and S6 ribosomal protein upon GDF15 administration [16,18]. Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
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Immediate early gene FOS induction, a marker of neuronal activation, in response to GDF15 treatment has been reported not only in AP GFRAL-expressing neurons, but also in the NTS, hypothalamus, and calcitonin gene-related peptide (CGRP)-positive neurons of the parabrachial nucleus and central amygdala (Figure 1) [14,16,41,57]. Although some findings in these reports are conflicting, particularly around the NTS and hypothalamus, these early data underscore the ability of GDF15 to trigger neuronal activation in centers known to control feeding behavior. Furthermore, GFRAL-deficient mice remain responsive to GLP-1 and leptin, while GDF15-induced weight loss still occurs in rodents lacking receptors for leptin and GLP-1 and the melanocortin-4 receptor, highlighting that the GDF15/GFRAL axis is independent of other pathways well established to control food intake [14–18,28]. GFRAL is required for the anorectic effects of pharmacologically administered GDF15 and initial studies in GFRAL-deficient animals have begun to elucidate the function of this receptor in the face of metabolic challenges that lead to increases in endogenous GDF15. Mice lacking GFRAL are born at expected frequencies and display no overt abnormalities up to 6 months of age [15–18]. Possibly reflective of a role of GFRAL during early development and growth, male GFRAL-deficient mice younger than 12 weeks are reported to be slightly shorter and lighter than littermate counterparts, although this difference is not observed at older ages [15,17]. Upon a nutritional challenge of a 60% high-fat diet, which increases plasma GDF15, mice lacking GFRAL consumed more food and gained a greater amount of fat mass and body weight compared with normal littermates, and this phenotype was more pronounced on a C57Bl/6 genetic background [16,17]. Increased diet-induced obesity upon GFRAL ablation exacerbates glucose intolerance and insulin resistance. The impact of GFRAL signaling in the context of metabolic challenge was further exemplified after treatment of mice with cisplatin, a chemotherapeutic agent known to cause anorexia [16]. Cisplatin treatment elevated circulating GDF15 and mice lacking GFRAL were resistant to treatment-induced anorexia and weight loss. The notion that endogenous GDF15/ GFRAL signaling is integral to metabolic homeostasis in stress conditions is further supported by GDF15 loss-of-function studies. For example, induction of the mitochondrial unfolded protein response (UPRmit), an adaptive stress-response pathway activated by mitochondrial proteotoxic stress, typically limits weight gain and metabolic dysfunction in the context of obesity. GDF15 ablation in a mouse model of UPRmit caused greater weight gain, worsened glucose intolerance and greater hepatic steatosis upon high-fat diet feeding [28].
Targeting the GDF15/GFRAL Axis – Therapeutic Opportunities GFRAL Agonism Activating GFRAL to reduce food intake and body weight holds promise as an antiobesity strategy. The most straightforward approach is delivery of the natural ligand, GDF15. However, the circulating half-life of native GDF15 is approximately 3 h in mice and nonhuman primates, limiting the dosing strategy that could be applied therapeutically [41]. Despite this limitation, as described above, daily administration of native GDF15 to obese nonhuman primates efficaciously lowered body weight and improved metabolic parameters [41]. Pharmacokinetic properties of native proteins can be enhanced by increasing molecular size or harnessing the FcRn-recycling mechanism, both of which can be achieved through fusion with human serum albumin (HSA) or the Fc portion of immunoglobulin G (IgG) [67]. Fusion with HSA or Fc does not hinder GDF15-mediated anorexia and weight loss, as demonstrated in lean and obese rodents, and extends GDF15 half-life from hours to days in monkeys [15,17,41]. A single administration of an HSA-GDF15 fusion was sufficient to decrease food intake and drive and maintain weight loss in obese nonhuman primates for 4 weeks [17]. Repeated weekly administration of an Fc-GDF15 fusion for 6 weeks to obese primates decreased food intake and body weight, resulting in lower plasma insulin and triglycerides and improved glucose homeostasis [41]. 6
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GDF15/GFRAL Antagonism Prevention of cisplatin-induced weight loss upon GFRAL deletion suggests that inhibitors of the GDF15/GFRAL pathway could be harnessed to limit the anorexia and/or cachexia elicited in chronic disease, cancer, or with chemotherapy [16]. Indeed, administration of an antibody specifically targeting GDF15 reversed weight loss and restored muscle and fat tissue mass in multiple human xenograft tumor-induced mouse models of cancer cachexia [14,37]. GDF11 is a known driver of cancer cachexia by both promoting anorexia and driving muscle breakdown. Recently, delivery of an antibody targeting GDF15 was demonstrated to block the anorectic effects of GDF11 but not its impact on muscle cell integrity [29]. Furthermore, administration of an anti-GDF15 antibody blocked the effects of both endogenous and pharmacologically administered GDF15 in obese mice,resulting in greater food intake and weight gain compared with control animals [41]. Antagonizing this pathway is not limited to GDF15 targeting because the anorectic effects of Fc-GDF15 in rats were prevented with co-administration of an antiGFRAL antibody [15]. GDF15 and GFRAL beyond Weight Homeostasis GDF15 is robustly induced in many tissues upon diverse stimuli, suggesting a key role in a variety of contexts, and has led to the hypothesis that weight-independent functions exist. Understanding function beyond weight maintenance is important not only for appreciating the full spectrum of benefits achievable by targeting this axis, but also for recognizing potential risks. This effort has been met by several challenges. For example, findings from studies examining the cellular consequences of GDF15 exposure are proposed to be both advantageous and deleterious depending on context and are often difficult to interpret, given the uncertainty of protein quality and purity, as exemplified in a recent report of contamination of commercial sources of GDF15 with TGFb [9,68]. Furthermore, studies have been mechanistically limited by the lack of understanding around GDF15-induced cell signaling, particularly the receptors involved. The identification of GFRAL/RET as the receptors downstream of GDF15 should expand the means to explore the pathway further. Notably, in line with the tissue expression pattern in multiple species, and in stark contrast to its ligand GDF15, no human or rodent cell line has been identified that endogenously expresses Gfral [17,18]. This makes it challenging to study the cellular signaling mediated by GFRAL and difficult to interpret the many previous observations of GDF15 effects in cell culture experiments. GDF15 is unlikely to have additional receptors to explain the reported in vitro effects because multiple efforts were unable to demonstrate binding to any TGFb receptor pair and GFRAL was the only confirmed positive GDF15-binding hit from exhaustive unbiased screens of all known human transmembrane proteins [16–18,62]. Finally, consideration that some of the effects of GDF15 knockdown or overexpression in cells could be due to intracellular functions is warranted [21,22]. One area demanding further exploration is the potential immunomodulatory role of GDF15. An anti-inflammatory and tissue-protective role for GDF15 is supported by the observation that mice lacking GDF15 are more susceptible to lipopolysaccharide-induced organ damage, neuronal injury, lethal myocardial infarction from carotid artery ligation, and renal damage in models of diabetes and collagen-induced pulmonary thromboembolism [69–74]. GDF15 overexpression reduces atherosclerotic plaque size and arthritis disease severity in rodent models [7]. Conversely, lack of GD15 in mice is reported to be protective in atherosclerosis and led to nonsteroidal anti-inflammatory drug (NSAID) chemoprevention resistance [56,75,76]. The immunomodulatory nature of GDF15 is also hypothesized to underlie the pleotropic roles described in tumorigenesis and cancer progression [7,77]. There have been many reports of GDF15 effects in cancer cells and rodent models, and the results are conflicting. For example, loss and gain of function of GDF15 in the TRAMP mouse model Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
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of prostate cancer suggests that GDF15 is protective against tumorigenesis, but that it promotes metastasis during later stages of the disease [78]. Nonetheless, the correlation of elevated levels of GDF15 and poor prognosis in human cancer underscores the need to understand the potential risks of long-term elevated circulating GDF15. While circulating GDF15 positively correlates with all-cause mortality in humans, transgenic overexpression in mice resulted in an increase in life span, suggesting a protective function for GDF15 [40,79]. Importantly, no increase in tumor formation or other pathology was reported in mice transduced with AAV-GDF15 for up to 1 year [41]. In addition, while levels of GDF15 increase in many cancers, GDF15 expression is also positively regulated by tumor suppressor pathways and cancer treatments (chemotherapy, surgery, and radiation) known to halt disease progression [10,78]. The discovery of GFRAL as the receptor for this seemingly pleotropic signaling molecule will enable further investigation of the various reported effects of GDF15 on the immune system and tumor formation and progression. In cachexia of cancer and chronic disease, weight loss and wasting are due to a combination of anorexia and direct effects on muscle [80]. As outlined above, GDF15 is produced by many tumor types and drives weight loss in rodent cancer models; therefore, a potential GDF15mediated effect on muscle atrophy has been considered. Indeed, mice bearing GDF15expressing tumor xenografts lose both fat and muscle mass, which can be reversed by driving weight regain with an antibody blocking GDF15 function [37]. However, obese mice treated with or overexpressing GDF15 lose a greater proportion of fat mass compared with lean mass, in a ratio that is typical of other anorectic agents, leading to a body composition resembling that of lean animals [41,58]. Consistent with GDF15 specifically driving the anorectic arm of cachexia, antibody neutralization of GDF15 prevented feeding inhibition but not muscle atrophy in a GDF11-overexpression cachexia model [29]. These data, coupled with the finding that GFRAL is not expressed in muscle, demonstrate that GDF15 is unlikely to have a direct effect on muscle that causes wasting or loss of lean mass that is more than expected from other weight-loss modalities. Based on the localization of GFRAL to the AP, a region that includes the chemoreceptor trigger zone, and the correlation between circulating levels and emesis during secondtrimester pregnancies, it is logical to hypothesize that GDF15 signaling could induce nausea, malaise, and vomiting [81]. Single nucleotide polymorphisms located in a noncoding region of GDF15 show some association with hyperemesis gravidarum, a syndrome of severe nausea during pregnancy [82]. In this context, it has been proposed that GDF15 decreases food intake and thereby limits exposure to potentially noxious stimuli as a fetal protective mechanism, especially during the vulnerable first trimester of pregnancy, although it is more likely that any survival benefit to the fetus due to mitigation of such a risk may be entirely outweighed by a maternal negative energy balance [81,83]. GDF15 levels also increase after exposure to cisplatin treatment, a cancer therapy often associated with nausea and emesis [84]. These data support GDF15 antagonism as a novel antiemetic strategy, while suggesting that gastrointestinal (GI) adverse events are to be expected from GDF15 pharmacotherapy. Rodents are a nonemetic species and surrogate experiments, such as conditioned taste aversion or pica tests, upon GDF15 treatment have not yet been reported. Furthermore, in nonhuman primate weight-loss studies specifically designed to monitor for signs of nausea, malaise, and vomiting, no GI-related events were reported after GDF15 treatment [17,41]. Future studies characterizing GDF15/GFRAL circuitry will be important to determine whether this pathway has an obligatory role in the proemetic function of the AP. Importantly, leptin and pancreatic polypeptide provide precedence for signals that influence food intake via the AP without causing emesis [85–88]. 8
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Concluding Remarks and Future Perspectives
Outstanding Questions
Evidence demonstrating that circulating GDF15 controls food intake and body weight has accumulated over the past two decades, driving exploration of the potential of targeting this molecule in pathological states of overnutrition (obesity) and undernutrition (anorexia/cachexia) (Figure 1). The recent finding that GFRAL, a receptor that is exclusively located to neurons of the AP, is necessary for GDF15-mediated anorexia has begun to outline a novel circuit controlling energy homeostasis. Despite sharing characteristics with other endocrine factors that control food intake (expressed in peripheral metabolic tissues, targeting neurons in the brain, and increased after RYGB), GDF15 appears to be unique from several perspectives. For example, its regulation favors increased and sustained circulating levels under many conditions, including broad tissue expression. In addition, unlike transient postprandial anorectic signals, GDF15 levels do not increase after meal ingestion, similar to leptin, a well-known homeostatic regulator of food intake and body weight. However, the importance of leptin is most pronounced in the absence of signaling, as exemplified in the extreme hyperphagia and obesity observed in ob/ob and db/db mice; while leptin treatment in wild-type animals only has minor effects. By contrast, GDF15/GFRAL physiology is most impactful in times of excess signaling; GDF15 treatment has robust weight-loss effects in mice and primates, but its deletion results in only minor phenotypes under nonstressed physiological conditions. It will be of interest to the field to overlay these observations with a detailed molecular and cellular characterization of the GDF15/GFRAL pathway. In doing so, future studies will expand pharmacological approaches to, and implications of, targeting this pathway in obesity and other diseases (see Outstanding Questions).
What are the characteristics of GFRAL-positive neurons in the area postrema? What functional changes are elicited in these cells upon GDF15 binding? What is the neuronal circuitry associated with these cells? Does GDF15/GFRAL signaling impact gastric motility, energy expenditure, or nausea and/or emesis, and do these mechanisms contribute significantly to weight loss in this context? Will neutralizing GDF15/GFRAL signaling lessen or reverse the anorexia contributing to cancer cachexia in humans? Will this be sufficient to drive beneficial weight gain in patients? Are there functions of GDF15 independent of anorexia and weight loss? Are all GDF15 functions mediated through GFRAL?
Acknowledgments We thank James Leonard, Vedrana Stojanovic-Susulic, Eve Frank, and Mark Erion for careful reading of this manuscript, Songmao Zheng for helpful discussion, and Susan Leibowitz Basile (BasilPix) for figure artwork. We apologize to researchers whose relevant studies were not discussed due to space limitations.
References 1. Hromas, R. et al. (1997) PLAB, a novel placental bone morphogenetic protein. Biochim. Biophys. Acta 1354, 40–44 2. Bootcov, M.R. et al. (1997) MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc. Natl. Acad. Sci. U. S. A. 94, 11514–11519 3. Lawton, L.N. et al. (1997) Identification of a novel member of the TGF-beta superfamily highly expressed in human placenta. Gene 203, 17–26 4. Baek, S.J. et al. (2001) Cyclooxygenase inhibitors regulate the expression of a TGF-beta superfamily member that has proapoptotic and antitumorigenic activities. Mol. Pharmacol. 59, 901– 908
11. Bauskin, A.R. et al. (2006) Role of macrophage inhibitory cytokine-1 in tumorigenesis and diagnosis of cancer. Cancer Res. 66, 4983–4986 12. Mimeault, M. and Batra, S.K. (2010) Divergent molecular mechanisms underlying the pleiotropic functions of macrophage inhibitory cytokine-1 in cancer. J. Cell. Physiol. 224, 626–635 13. Baek, S.J. et al. (2006) Nonsteroidal anti-inflammatory drug-activated gene-1 over expression in transgenic mice suppresses intestinal neoplasia. Gastroenterology 131, 1553–1560 14. Johnen, H. et al. (2007) Tumor-induced anorexia and weight loss are mediated by the TGF-beta superfamily cytokine MIC-1. Nat. Med. 13, 1333–1340
5. Paralkar, V.M. et al. (1998) Cloning and characterization of a novel member of the transforming growth factor-beta/bone morphogenetic protein family. J. Biol. Chem. 273, 13760–13767
15. Emmerson, P.J. et al. (2017) The metabolic effects of GDF15 are mediated by the orphan receptor GFRAL. Nat. Med. 23, 1215– 1219
6. Bottner, M. et al. (1999) Expression of a novel member of the TGF-beta superfamily, growth/differentiation factor-15/macrophage-inhibiting cytokine-1 (GDF-15/MIC-1) in adult rat tissues. Cell Tissue Res. 297, 103–110
16. Hsu, J.Y. et al. (2017) Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15. Nature 550, 255–259
7. Breit, S.N. et al. (2011) The TGF-beta superfamily cytokine, MIC1/GDF15: a pleotrophic cytokine with roles in inflammation, cancer and metabolism. Growth Factors 29, 187–195 8. Adela, R. and Banerjee, S.K. (2015) GDF-15 as a target and biomarker for diabetes and cardiovascular diseases: a translational prospective. J. Diabetes Res. 2015, 490842 9. Corre, J. et al. (2013) Concise review: growth differentiation factor 15 in pathology: a clinical role? Stem Cells Transl. Med. 2, 946– 952 10. Wang, X. et al. (2013) The diverse roles of nonsteroidal antiinflammatory drug activated gene (NAG-1/GDF15) in cancer. Biochem. Pharmacol. 85, 597–606
17. Mullican, S.E. et al. (2017) GFRAL is the receptor for GDF15 and the ligand promotes weight loss in mice and nonhuman primates. Nat. Med. 23, 1150–1157 18. Yang, L. et al. (2017) GFRAL is the receptor for GDF15 and is required for the anti-obesity effects of the ligand. Nat. Med. 23, 1158–1166 19. Breit, S.N. et al. (2017) Targeting obesity and cachexia: identification of the GFRAL receptor-MIC-1/GDF15 pathway. Trends Mol. Med. 23, 1065–1067 20. Bauskin, A.R. et al. (2000) The propeptide of macrophage inhibitory cytokine (MIC-1), a TGF-beta superfamily member, acts as a quality control determinant for correctly folded MIC-1. EMBO J. 19, 2212–2220
Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
9
TEM 1330 No. of Pages 11
21. Min, K.W. et al. (2016) NAG-1/GDF15 accumulates in the nucleus and modulates transcriptional regulation of the Smad pathway. Oncogene 35, 377–388
43. Kempf, T. et al. (2012) Growth differentiation factor 15 predicts future insulin resistance and impaired glucose control in obese nondiabetic individuals: results from the XENDOS trial. Eur. J. Endocrinol. 167, 671–678
22. Zhang, X. et al. (2017) GL-V9 induced upregulation and mitochondrial localization of NAG-1 associates with ROS generation and cell death in hepatocellular carcinoma cells. Free Radic. Biol. Med. 112, 49–59
44. Tsai, V.W. et al. (2012) Anorexia/cachexia of chronic diseases: a role for the TGF-beta family cytokine MIC-1/GDF15. J. Cachexia Sarcopenia Muscle 3, 239–243
23. Zimmers, T.A. et al. (2005) Growth differentiation factor-15/macrophage inhibitory cytokine-1 induction after kidney and lung injury. Shock 23, 543–548
45. Vila, G. et al. (2011) The relationship between insulin resistance and the cardiovascular biomarker growth differentiation factor-15 in obese patients. Clin. Chem. 57, 309–316
24. Baek, S.J. et al. (2001) Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter. Basal transcription is mediated by Sp1 and Sp3. J. Biol. Chem. 276, 33384–33392
46. Dostalova, I. et al. (2010) Association of macrophage inhibitory cytokine-1 with nutritional status, body composition and bone mineral density in patients with anorexia nervosa: the influence of partial realimentation. Nutr. Metab. 7, 34
25. Osada, M. et al. (2007) A p53-type response element in the GDF15 promoter confers high specificity for p53 activation. Biochem. Biophys. Res. Commun. 354, 913–918
47. Fearon, K. et al. (2011) Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 12, 489–495
26. Lim, J.H. et al. (2007) NAG-1 up-regulation mediated by EGR-1 and p53 is critical for quercetin-induced apoptosis in HCT116 colon carcinoma cells. Apoptosis 12, 411–421
48. Lerner, L. et al. (2015) Plasma growth differentiation factor 15 is associated with weight loss and mortality in cancer patients. J. Cachexia Sarcopenia Muscle 6, 317–324
27. Han, M. et al. (2017) CXXC4 activates apoptosis through up-regulating GDF15 in gastric cancer. Oncotarget 8, 103557–103567
49. Lerner, L. et al. (2016) Growth differentiating factor-15 (GDF-15): A potential biomarker and therapeutic target for cancer-associated weight loss. Oncol. Lett. 12, 4219–4223
28. Chung, H.K. et al. (2017) Growth differentiation factor 15 is a myomitokine governing systemic energy homeostasis. J. Cell Biol. 216, 149–165
50. Breit, S.N. et al. (2012) Macrophage inhibitory cytokine-1 (MIC-1/ GDF15) and mortality in end-stage renal disease. Nephrol. Dial. Transplant. 27, 70–75
29. Jones, J.E. et al. (2018) Supraphysiologic administration of GDF11 induces cachexia in part by upregulating GDF15. Cell Rep. 22, 1522–1530
51. Kempf, T. et al. (2007) Growth-differentiation factor-15 improves risk stratification in ST-segment elevation myocardial infarction. Eur. Heart J. 28, 2858–2865
30. Yoshioka, H. et al. (2008) Nonsteroidal anti-inflammatory drugactivated gene (NAG-1/GDF15) expression is increased by the histone deacetylase inhibitor trichostatin A. J. Biol. Chem. 283, 33129–33137
52. Chrysovergis, K. et al. (2014) NAG-1/GDF-15 prevents obesity by increasing thermogenesis, lipolysis and oxidative metabolism. Int. J. Obes. 38, 1555–1564 53. Macia, L. et al. (2012) Macrophage inhibitory cytokine 1 (MIC-1/ GDF15) decreases food intake, body weight and improves glucose tolerance in mice on normal & obesogenic diets. PLoS One 7, e34868
31. Martinez, J.M. et al. (2006) Drug-induced expression of nonsteroidal anti-inflammatory drug-activated gene/macrophage inhibitory cytokine-1/prostate-derived factor, a putative tumor suppressor, inhibits tumor growth. J. Pharmacol. Exp. Ther. 318, 899–906
54. Strelau, J. et al. (2009) Progressive postnatal motoneuron loss in mice lacking GDF-15. J. Neurosci. 29, 13640–13648
32. Bauskin, A.R. et al. (2010) The TGF-beta superfamily cytokine MIC-1/GDF15: secretory mechanisms facilitate creation of latent stromal stores. J. Interferon Cytokine Res. 30, 389–397
55. Casanovas, G. et al. (2013) The murine growth differentiation factor 15 is not essential for systemic iron homeostasis in phlebotomized mice. Haematologica 98, 444–447
33. Fairlie, W.D. et al. (1999) MIC-1 is a novel TGF-beta superfamily cytokine associated with macrophage activation. J. Leukoc. Biol. 65, 2–5
56. Bonaterra, G.A. et al. (2012) Growth differentiation factor-15 deficiency inhibits atherosclerosis progression by regulating interleukin-6-dependent inflammatory response to vascular injury. J. Am. Heart Assoc. 1, e002550
34. Moore, A.G. et al. (2000) The transforming growth factor-ss superfamily cytokine macrophage inhibitory cytokine-1 is present in high concentrations in the serum of pregnant women. J. Clin. Endocrinol. Metab. 85, 4781–4788 35. Ding, Q. et al. (2009) Identification of macrophage inhibitory cytokine-1 in adipose tissue and its secretion as an adipokine by human adipocytes. Endocrinology 150, 1688–1696 36. Kleinert, M. et al. (2018) Exercise increases circulating GDF15 in humans. Mol. Metab. 9, 187–191
57. Tsai, V.W. et al. (2013) TGF-b superfamily cytokine MIC-1/GDF15 is a physiological appetite and body weight regulator. PLoS One 8, e55174 58. Tsai, V.W. et al. (2017) Treatment with the TGF-b superfamily cytokine MIC-1/GDF15 reduces the adiposity and corrects the metabolic dysfunction of mice with diet-induced obesity. Int. J. Obes. 42, 561–571
37. Lerner, L. et al. (2016) MAP3K11/GDF15 axis is a critical driver of cancer cachexia. J. Cachexia Sarcopenia Muscle 7, 467–482
59. Tsai, V.W. et al. (2014) The anorectic actions of the TGFbeta cytokine MIC-1/GDF15 require an intact brainstem area postrema and nucleus of the solitary tract. PLoS One 9, e100370
38. Nair, V. et al. (2017) Growth differentiation factor-15 and risk of CKD progression. J. Am. Soc. Nephrol. 28, 2233–2240
60. Janssen, P. et al. (2011) Review article: the role of gastric motility in the control of food intake. Aliment. Pharmacol. Ther. 33, 880–894
39. Kempf, T. et al. (2007) Prognostic utility of growth differentiation factor-15 in patients with chronic heart failure. J. Am. Coll. Cardiol. 50, 1054–1060
61. Tschop, M.H. et al. (2011) A guide to analysis of mouse energy metabolism. Nat. Methods 9, 57–63
40. Wiklund, F.E. et al. (2010) Macrophage inhibitory cytokine-1 (MIC-1/GDF15): a new marker of all-cause mortality. Aging Cell 9, 1057–1064 41. Xiong, Y. et al. (2017) Long-acting MIC-1/GDF15 molecules to treat obesity: evidence from mice to monkeys. Sci. Transl. Med. 9, eaan8732 42. Dostalova, I. et al. (2009) Increased serum concentrations of macrophage inhibitory cytokine-1 in patients with obesity and type 2 diabetes mellitus: the influence of very low calorie diet. Eur. J. Endocrinol. 161, 397–404
10
62. Unsicker, K. et al. (2013) The multiple facets of the TGF-beta family cytokine growth/differentiation factor-15/macrophage inhibitory cytokine-1. Cytokine Growth Factor Rev. 24, 373–384 63. Artz, A. et al. (2016) GDF-15 inhibits integrin activation and mouse neutrophil recruitment through the ALK-5/TGF-betaRII heterodimer. Blood 128, 529–541 64. Liu, D.D. et al. (2016) Growth differentiation factor-15 promotes glutamate release in medial prefrontal cortex of mice through upregulation of T-type calcium channels. Sci. Rep. 6, 28653 65. Li, Z. et al. (2005) Identification, expression and functional characterization of the GRAL gene. J. Neurochem. 95, 361–376
Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
TEM 1330 No. of Pages 11
66. Hatinen, T. et al. (2007) Loss of neurturin in frog–comparative genomics study of GDNF family ligand-receptor pairs. Mol. Cell. Neurosci. 34, 155–167
77. Vanhara, P. et al. (2012) Growth/differentiation factor-15: prostate cancer suppressor or promoter? Prostate Cancer Prostatic Dis. 15, 320–328
67. van Witteloostuijn, S.B. et al. (2016) Half-life extension of biopharmaceuticals using chemical methods: alternatives to PEGylation. ChemMedChem 11, 2474–2495
78. Husaini, Y. et al. (2015) Macrophage inhibitory cytokine-1 (MIC?1/ GDF15) gene deletion promotes cancer growth in TRAMP prostate cancer prone mice. PLoS One 10, e0115189
68. Olsen, O.E. et al. (2017) TGF-beta contamination of purified recombinant GDF15. PLoS One 12, e0187349
79. Wang, X. et al. (2014) hNAG-1 increases lifespan by regulating energy metabolism and insulin/IGF-1/mTOR signaling. Aging 6, 690–704
69. Wang, X. et al. (2015) Growth/differentiation factor-15 and its role in peripheral nervous system lesion and regeneration. Cell Tissue Res. 362, 317–330 70. Machado, V. et al. (2016) Growth/differentiation factor-15 deficiency compromises dopaminergic neuron survival and microglial response in the 6-hydroxydopamine mouse model of Parkinson’s disease. Neurobiol. Dis. 88, 1–15 71. Abulizi, P. et al. (2017) Growth differentiation factor-15 deficiency augments inflammatory response and exacerbates septic heart and renal injury induced by lipopolysaccharide. Sci. Rep. 7, 1037 72. Kempf, T. et al. (2011) GDF-15 is an inhibitor of leukocyte integrin activation required for survival after myocardial infarction in mice. Nat. Med. 17, 581–588
80. Fearon, K.C. et al. (2012) Cancer cachexia: mediators, signaling, and metabolic pathways. Cell Metab. 16, 153–166 81. O’Rahilly, S. (2017) GDF15-from biomarker to allostatic hormone. Cell Metab. 26, 807–808 82. Fejzo, M.S. et al. (2018) Placenta and appetite genes GDF15 and IGFBP7 are associated with hyperemesis gravidarum. Nat. Commun. 9, 1178 83. Abu-Saad, K. and Fraser, D. (2010) Maternal nutrition and birth outcomes. Epidemiol. Rev. 32, 5–25 84. Altena, R. et al. (2015) Growth differentiation factor 15 (GDF-15) plasma levels increase during bleomycin- and cisplatin-based treatment of testicular cancer patients and relate to endothelial damage. PLoS One 10, e0115372
73. Mazagova, M. et al. (2013) Genetic deletion of growth differentiation factor 15 augments renal damage in both type 1 and type 2 models of diabetes. Am. J. Physiol. Renal Physiol. 305, F1249– F1264
85. Huo, L. et al. (2007) Leptin and the control of food intake: neurons in the nucleus of the solitary tract are activated by both gastric distension and leptin. Endocrinology 148, 2189–2197
74. Rossaint, J. et al. (2013) GDF-15 prevents platelet integrin activation and thrombus formation. J. Thromb. Haemost. 11, 335– 344
86. Cuenco, J. et al. (2017) Degradation paradigm of the gut hormone, pancreatic polypeptide, by hepatic and renal peptidases. Endocrinology 158, 1755–1765
75. Zimmers, T.A. et al. (2010) Loss of GDF-15 abolishes sulindac chemoprevention in the ApcMin/+ mouse model of intestinal cancer. J. Cancer Res. Clin. Oncol. 136, 571–576
87. Woods, S.C. et al. (2006) Pancreatic signals controlling food intake; insulin, glucagon and amylin. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361, 1219–1235
76. de Jager, S.C. et al. (2011) Growth differentiation factor 15 deficiency protects against atherosclerosis by attenuating CCR2-mediated macrophage chemotaxis. J. Exp. Med. 208, 217–225
88. Paz-Filho, G. et al. (2015) Leptin treatment: facts and expectations. Metabolism 64, 146–156
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