GDF15 mediates the effects of metformin on body weight and energy balance.

Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk1,2. More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner3. The molecular mechanisms by which metformin lowers body weight are unknown. Here we show-in two independent randomized controlled clinical trials-that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent.

Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15.

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

Deficiency of GDNF Receptor GFRα1 in Alzheimer's Neurons Results in Neuronal Death.

We have recently developed aged cortical neuron cultures from autopsied human brains with Alzheimer's disease (AD). During the culturing process, we found that glutamatergic cortical neurons from the AD brain lacked a response to glial cell line-derived neurotrophic factor (GDNF), including no axonal regrowth, and were starting to undergo apoptosis. Here we showed that, in cortical neurons from age- and gender-matched cognitively normal control (NC) subjects (NC neurons), GDNF enhanced the expression of GDNF family receptor subtype α1 (GFRα1), but not the other three subtypes (GFRα2, GFRα3, and GFRα4), whereas GDNF failed to induce GFRα1 expression in cortical neurons from the AD brain (AD neurons). The exogenous introduction of GFRα1, but not of its binding partner α1-neural cell adhesion molecule, or RET into AD neurons restored the effect of GDNF on neuronal survival. Moreover, between NC and AD neurons, the AMPA receptor blocker CNQX and the NMDA receptor blocker AP-5 had opposite effects on the GFRα1 expression induced by GDNF. In NC neurons, the presence of glutamate receptors was necessary for GDNF-linked GFRα1 expression, while in AD neurons the absence of glutamate receptors was required for GFRα1 expression by GDNF stimulation. These results suggest that, in AD neurons, specific impairments of GFRα1, which may be linked to glutamatergic neurotransmission, shed light on developing potential therapeutic strategies for AD by upregulation of GFRα1 expression.

Ret signalling integrates a craniofacial muscle module during development.

An appropriate organisation of muscles is crucial for their function, yet it is not known how functionally related muscles are coordinated with each other during development. In this study, we show that the development of a subset of functionally related head muscles in the zebrafish is regulated by Ret tyrosine kinase signalling. Three genes in the Ret pathway (gfra3, artemin2 and ret) are required specifically for the development of muscles attaching to the opercular bone (gill cover), but not other adjacent muscles. In animals lacking Ret or Gfra3 function, myogenic gene expression is reduced in forming opercular muscles, but not in non-opercular muscles derived from the same muscle anlagen. These animals have a normal skeleton with small or missing opercular muscles and tightly closed mouths. Myogenic defects correlate with a highly restricted expression of artn2, gfra3 and ret in mesenchymal cells in and around the forming opercular muscles. ret(+) cells become restricted to the forming opercular muscles and a loss of Ret signalling results in reductions of only these, but not adjacent, muscles, revealing a specific role of Ret in a subset of head muscles. We propose that Ret signalling regulates myogenesis in head muscles in a modular manner and that this is achieved by restricting Ret function to a subset of muscle precursors.

Modulation of visceral hypersensitivity by glial cell line-derived neurotrophic factor family receptor α-3 in colorectal afferents.

Irritable bowel syndrome is characterized by colorectal hypersensitivity and contributed to by sensitized mechanosensitive primary afferents and recruitment of mechanoinsensitive (silent) afferents. Neurotrophic factors are well known to orchestrate dynamic changes in the properties of sensory neurons. Although pain modulation by proteins in the glial cell line-derived neurotrophic factor (GDNF) family has been documented in various pathophysiological states, their role in colorectal hypersensitivity remains unexplored. Therefore, we investigated the involvement of the GDNF family receptor α-3 (GFRα3) signaling in visceral hypersensitivity by quantifying visceromotor responses (VMR) to colorectal distension before and after intracolonic treatment with 2,4,6-trinitrobenzene sulfonic acid (TNBS). Baseline responses to colorectal distension did not differ between C57BL/6 and GFRα3 knockout (KO) mice. Relative to intracolonic saline treatment, TNBS significantly enhanced the VMR to colorectal distension in C57BL/6 mice 2, 7, 10, and 14 days posttreatment, whereas TNBS-induced visceral hypersensitivity was significantly suppressed in GFRα3 KO mice. The proportion of GFRα3 immunopositive thoracolumbar and lumbosacral colorectal dorsal root ganglion neurons was significantly elevated 2 days after TNBS treatment. In single fiber recordings, responses to circumferential stretch of colorectal afferent endings in C57BL/6 mice were significantly increased (sensitized) after exposure to an inflammatory soup, whereas responses to stretch did not sensitize in GFRα3 KO mice. These findings suggest that enhanced GFRα3 signaling in visceral afferents may contribute to development of colorectal hypersensitivity.

Pharmacological but not physiological GDF15 suppresses feeding and the motivation to exercise.

Growing evidence supports that pharmacological application of growth differentiation factor 15 (GDF15) suppresses appetite but also promotes sickness-like behaviors in rodents via GDNF family receptor α-like (GFRAL)-dependent mechanisms. Conversely, the endogenous regulation of GDF15 and its physiological effects on energy homeostasis and behavior remain elusive. Here we show, in four independent human studies that prolonged endurance exercise increases circulating GDF15 to levels otherwise only observed in pathophysiological conditions. This exercise-induced increase can be recapitulated in mice and is accompanied by increased Gdf15 expression in the liver, skeletal muscle, and heart muscle. However, whereas pharmacological GDF15 inhibits appetite and suppresses voluntary running activity via GFRAL, the physiological induction of GDF15 by exercise does not. In summary, exercise-induced circulating GDF15 correlates with the duration of endurance exercise. Yet, higher GDF15 levels after exercise are not sufficient to evoke canonical pharmacological GDF15 effects on appetite or responsible for diminishing exercise motivation.

Regionalized loss of parvalbumin interneurons in the cerebral cortex of mice with deficits in GFRalpha1 signaling.

Inhibitory interneurons are crucially important for cerebral cortex function and behavior. The mechanisms controlling inhibitory interneuron diversification and allocation to distinct cortical areas remain poorly understood. GDNF (glial cell line-derived neurotrophic factor) and its receptor GFRalpha1 have been implicated in the development of GABAergic precursors but, because of the early lethality of null mutants, their roles in postnatal maturation and function of cortical interneurons are unknown. "cis-only" mutant mice lack GFRalpha1 only in cells that do not express the RET signaling receptor subunit and survive to adulthood. At birth, both null mutants and cis-only mice showed a specific loss of GABAergic interneurons in rostro- and caudolateral cortical regions but not in more medial areas. Unexpectedly, the adult cortex of cis-only mice displayed a complete loss of parvalbumin (PV)-expressing GABAergic interneurons in discrete regions (PV holes) interspersed among areas of normal PV cell density. PV holes predominantly occurred in the visual and frontal cortices, and their size could be affected by neuronal activity. Consistent with deficits in cortical inhibitory activity, these mice showed enhanced cortical excitability, increased sensitivity to epileptic seizure, and increased social behavior. We propose that GFRalpha1 signaling guides the development of a subset of PV-expressing GABAergic interneurons populating discrete regions of the cerebral cortex and may thus contribute to the diversification and allocation of specific cortical interneuron subtypes.

The neurotrophic effects of glial cell line-derived neurotrophic factor on spinal motoneurons are restricted to fusimotor subtypes.

Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha1 (GFRalpha1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRalpha1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFRalpha1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFRalpha1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.

Development of satellite glia in mouse sympathetic ganglia: GDNF and GFR alpha 1 are not essential.

The phenotypic development of satellite cells in mouse sympathetic ganglia was examined by localizing the transcription factors, Sox10 and Phox2b, the neuronal marker, tyrosine hydroxylase (TH), and brain-derived fatty acid binding protein (B-FABP), which identifies glial precursors and mature glia. In E10.5 mice, most cells in the sympathetic chain expressed both Sox10 and Phox2b, with a minority of cells expressing Sox10 only or Phox2b only. In E11.5 mice, the majority of cells expressed Sox10 only or Phox2b only. B-FABP was colocalized with Sox10 in satellite glial precursors, which were located on the periphery of the ganglion. There was no overlap between B-FABP and Phox2b or B-FABP and TH. During subsequent development, the number of B-FABP+ cells increased and they became more common deep within the ganglion. In E12.5 and E18.5 mice, there was no overlap between Sox10 and Phox2b, and 98% of Sox10 cells were also B-FABP+. Satellite glial precursors in E11.5-E15.5 mice also expressed the GDNF-binding molecule, GFRalpha1. B-FABP immunoreactive cells did not express Ret or NCAM, two potential signaling molecules for GDNF/GFRalpha1. In E12.5 and E18.5 mice lacking GFRalpha1 or GDNF, the development of B-FABP immunoreactive satellite cells was normal, and hence neither GDNF or GFRalpha1 are essential for the development of satellite glia in sympathetic ganglia.

Deficient nonpeptidergic epidermis innervation and reduced inflammatory pain in glial cell line-derived neurotrophic factor family receptor alpha2 knock-out mice.

Most unmyelinated nociceptive neurons that mediate pain and temperature sensation from the skin bind isolectin B4 (IB4)-lectin and express Ret, the common signaling component of glial cell line-derived neurotrophic factor (GDNF) family. One of these factors, neurturin, is expressed in the epidermis, whereas its GDNF family receptor alpha2 (GFRalpha2) is expressed in the majority of unmyelinated Ret-positive sensory neurons. However, the physiological roles of endogenous neurturin signaling in primary sensory neurons are poorly understood. Here, we show that the vast majority (approximately 85%) of IB4 binding and P2X3 purinoreceptor-positive neurons, but virtually none of the calcitonin gene-related peptide (CGRP) or vanilloid receptor transient receptor potential vanilloid 1-positive neurons in mouse dorsal root ganglion (DRG) express GFRalpha2. In GFRalpha2 knock-out (KO) mice, the IB4-binding and P2X3-positive DRG neurons were present but reduced in size, consistent with normal number but reduced caliber of unmyelinated axons in a cutaneous nerve. Strikingly, nonpeptidergic (CGRP-negative) free nerve endings in footpad epidermis were >70% fewer in GFRalpha2-KO mice than in their wild-type littermates. In contrast, the density of CGRP-positive epidermal innervation remained unaffected. In the formalin test, the KO mice showed a normal acute response but a markedly attenuated persistent phase, indicating a deficit in inflammatory pain response. Behavioral responses of GFRalpha2-KO mice to innocuous warm and noxious heat were not blunted; the mice were actually markedly hypersensitive to noxious cold in tail immersion test. Overall, our results indicate a critical role for endogenous GFRalpha2 signaling in maintaining the size and terminal innervation of the nonpeptidergic class of cutaneous nociceptors in vivo.

Downregulations of TRPM8 expression and membrane trafficking in dorsal root ganglion mediate the attenuation of cold hyperalgesia in CCI rats induced by GFRα3 knockdown.

BACKGROUND: Cold hyperalgesia is an intractable sensory abnormality commonly seen in peripheral neuropathies. Although glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3) is required for the formation of pathological cold pain has been revealed, potential transduction mechanism is poorly elucidated. We have previously demonstrated the contribution of enhanced activity of transient receptor potential melastatin 8 (TRPM8) to cold hyperalgesia in neuropathic pain using a rat model of chronic constriction injury (CCI) to the sciatic nerve. Recently, the enhancement of TRPM8 activity is attributed to the increased TRPM8 plasma membrane trafficking. In addition, TRPM8 can be sensitized by the activation of GFRα3, leading to increased cold responses in vivo. The aim of this study was to investigate whether GFRα3 could influence cold hyperalgesia of CCI rats via modulating TRPM8 expression and plasma membrane trafficking in dorsal root ganglion (DRG). METHODS: Mechanical allodynia, cold and heat hyperalgesia were measured on 1day before CCI and the 1st, 4th, 7th, 10th and 14th day after CCI. TRPM8 total expression and membrane trafficking as well as GFRα3 expression in DRG were detected by immunofluorescence and western blot. Furthermore, GFRα3 small interfering RNA (siRNA) was intrathecally administrated to reduce GFRα3 expression in DRG, and the effects of GFRα3 knockdown on CCI-induced behavioral sensitization as well as TRPM8 total expression and membrane trafficking in both mRNA and protein levels were investigated, and the change in coexpression of TRPM8 with GFRα3 was also evaluated. Then, the effect of GFRα3 activation with artemin on pain behavior of CCI rats pretreated with the selective TRPM8 antagonist RQ-00203078 was observed. RESULTS: Here we found that TRPM8 total expression and plasma membrane trafficking as well as GFRα3 expression in DRG were initially increased on the 4th day after CCI, and maintained at the peak level from the 10th to the 14th day, which entirely conformed with the induction and maintenance of behavioral-reflex facilitation following CCI. The coexpression of TRPM8 with GFRα3, which was mainly located in peptidergic C-fibers DRG neurons, was also increased after CCI. Downregulation of GFRα3 protein in DRG attenuated CCI-induced cold hyperalgesia without affecting mechanical allodynia and heat hyperalgesia, and reduced the upregulations of TRPM8 total expression and plasma membrane trafficking as well as coexpression of TRPM8 with GFRα3 induced by CCI. Additionally, the inhibition of TRPM8 abolished the influence of GFRα3 activation on cold hyperalgesia after CCI. CONCLUSION: Our results demonstrate that GFRα3 knockdown specially inhibits cold hyperalgesia following CCI via decreasing the expression level and plasma membrane trafficking of TRPM8 in DRG. GFRα3 and its downstream mediator, TRPM8, represent a new analgesia axis which can be further exploited in sensitized cold reflex under the condition of chronic pain.

Cis and trans RET signaling control the survival and central projection growth of rapidly adapting mechanoreceptors.

RET can be activated in cis or trans by its co-receptors and ligands in vitro, but the physiological roles of trans signaling are unclear. Rapidly adapting (RA) mechanoreceptors in dorsal root ganglia (DRGs) express Ret and the co-receptor Gfrα2 and depend on Ret for survival and central projection growth. Here, we show that Ret and Gfrα2 null mice display comparable early central projection deficits, but Gfrα2 null RA mechanoreceptors recover later. Loss of Gfrα1, the co-receptor implicated in activating RET in trans, causes no significant central projection or cell survival deficit, but Gfrα1;Gfrα2 double nulls phenocopy Ret nulls. Finally, we demonstrate that GFRα1 produced by neighboring DRG neurons activates RET in RA mechanoreceptors. Taken together, our results suggest that trans and cis RET signaling could function in the same developmental process and that the availability of both forms of activation likely enhances but not diversifies outcomes of RET signaling.

Different requirements for GFRα2-signaling in three populations of cutaneous sensory neurons.

Many primary sensory neurons in mouse dorsal root ganglia (DRG) express one or several GFRα's, the ligand-binding receptors of the GDNF family, and their common signaling receptor Ret. GFRα2, the principal receptor for neurturin, is expressed in most of the small nonpeptidergic DRG neurons, but also in some large DRG neurons that start to express Ret earlier. Previously, GFRα2 has been shown to be crucial for the soma size of small nonpeptidergic nociceptors and for their target innervation of glabrous epidermis. However, little is known about this receptor in other Ret-expressing DRG neuron populations. Here we have investigated two populations of Ret-positive low-threshold mechanoreceptors that innervate different types of hair follicles on mouse back skin: the small C-LTMRs and the large Aß-LTMRs. Using GFRα2-KO mice and immunohistochemistry we found that, similar to the nonpeptidergic nociceptors, GFRα2 controls the cell size but not the survival of both C-LTMRs and Aß-LTMRs. In contrast to the nonpeptidergic neurons, GFRα2 is not required for the target innervation of C-LTMRs and Aß-LTMRs in the back skin. These results suggest that different factors drive target innervation in these three populations of neurons. In addition, the observation that the large Ret-positive DRG neurons lack GFRα2 immunoreactivity in mature animals suggests that these neurons switch their GFRα signaling pathways during postnatal development.

Increased apoptosis of parasympathetic but not enteric neurons in mice lacking GFRalpha2.

Enteric neurons, unlike sympathetic and sensory neurons that require target-derived neurotrophins for survival, do not undergo classical caspase-3-mediated programmed cell death (PCD) during normal development. Whether parasympathetic neurons in the pancreas, which originate from a subpopulation of enteric nervous system (ENS) precursors, or other parasympathetic neurons undergo PCD during normal mammalian development is unknown. In GFRalpha2-deficient mice, many submandibular and intrapancreatic parasympathetic neurons are missing but whether this is due to increased neuronal death is unclear. Here we show that activated caspase-3 and PGP9.5 doubly positive neurons are present in wild-type mouse pancreas between embryonic day E15 and birth. Thus, in contrast to ENS neurons, intrapancreatic neurons undergo PCD via apoptosis during normal development. We also show that, in GFRalpha2-deficient mice, most intrapancreatic neurons are lost during this late fetal period, which coincides with a period of increased apoptosis of the neurons. Since the percentage of BrdU and Phox2b doubly positive cells in the fetal pancreas and the number of intrapancreatic neurons at E15 were similar between the genotypes, impaired precursor proliferation and migration are unlikely to contribute to the loss of intrapancreatic neurons in GFRalpha2-KO mice. Caspase-3-positive neurons were also found in GFRalpha2-deficient submandibular ganglia around birth, suggesting that parasympathetic neurons depend on limited supply of (presumably target-derived neurturin) signaling via GFRalpha2 for survival.

Role of glial cell-line derived neurotropic factor family receptor alpha2 in the actions of the glucagon-like peptides on the murine intestine.

The intestinal glucagon-like peptides GLP-1 and GLP-2 inhibit intestinal motility, whereas GLP-2 also stimulates growth of the intestinal mucosa. However, the mechanisms of action of these peptides in the intestine remain poorly characterized. To determine the role of the enteric nervous system in the actions of GLP-1 and GLP-2 on the intestine, the glial cell line-derived neurotropic factor family receptor alpha(2) (GFRalpha2) knockout (KO) mouse was employed. The mice exhibited decreased cholinergic staining, as well as reduced mRNA transcripts for substance P-ergic excitatory motoneurons in the enteric nervous system (ENS) (P < 0.05). Examination of parameters of intestinal growth (including small and large intestinal weight and small intestinal villus height, crypt depth, and crypt cell proliferation) demonstrated no differences between wild-type and KO mice in either basal or GLP-2-stimulated mucosal growth. Nonetheless, KO mice exhibited reduced numbers of synaptophysin-positive enteroendocrine cells (P < 0.05), as well as a markedly impaired basal gastrointestinal (GI) transit rate (P < 0.05). Furthermore, acute administration of GLP-1 and GLP-2 significantly inhibited transit rates in wild-type mice (P < 0.05-0.01) but had no effect in GFRalpha2 KO mice. Despite these changes, expression of mRNA transcripts for the GLP receptors was not reduced in the ENS of KO animals, suggesting that GLP-1 and -2 modulate intestinal transit through enhancement of inhibitory input to cholinergic/substance P-ergic excitatory motoneurons. Together, these findings demonstrate a role for GFRalpha2-expressing enteric neurons in the downstream signaling of the glucagon-like peptides to inhibit GI motility, but not in intestinal growth.