Ciliary Neurotrophic Factor: A Role in Obesity?
Ciliary neurotrophic factor (CNTF) is a neurocytokine expressed by glial cells in peripheral nerves and the central nervous system. CNTF is generally recognized for its function in support and survival of non-neuronal and neuronal cell types. Following a serendipitous finding, CNTF was recently acknowledged for its potential role in the control of obesity.
Key words: ciliary neurotrophic factor, neurocytokine, obesity, energy balance
Cytokines are crucial components in the regulation of immunity, inflammation, tissue repair, cell growth, and other important physiologic processes. Cytokines are typically proteins or peptides secreted by one cell as regulators of neighboring cells. Ciliary neurotrophic factor (CNTF) is one such cytokine that plays a role among several processes within the human body from endogenous neuroprotection to regulation of energy expenditure. CNTF is a pluripotent neurocytokine expressed by glial cells in peripheral nerves and in the central nervous system (CNS). It is implicated in the differentiation and survival of non-neuronal and neuronal cell types, including sensory, sympathetic, ciliary, and motor neurons.1,2 The non-neuronal effects include initiating an acute-phase response in liver cells, maintaining embryonic stem cells in an undifferentiated state, and producing a myotrophic effect on denervated skeletal muscles of mice.
CNTF was initially identified more than 20 years ago for its ability to support the in vitro survival of chick ciliary ganglion neurons at different developmental stages.3 Purified CNTF also proved to support the survival of cultured neurons from certain chick and rodent sensory and sympathetic ganglia. In a later experiment, CNTF was purified from rabbit sciatic nerves and found to have a unique sequence that allowed the cloning of a full-length cDNA for CNTF and the subsequent determination of its primary structure. The crystal structure of human CNTF has been determined; it is dimeric, consisting of a unique antiparallel arrangement of the subunits. The individual subunits contain a double crossover four-helix bundle fold, in which the two helices contain kinks that contribute to its dimer appearance. These findings provide a platform for defining the actions and functions of CNTF at the molecular level within the nervous system.
CNTF, a protein with a molecular weight of 22 kD, is a member of a cytokine family that is structurally and functionally related to leukemia inhibitory factor (LIF), interleukin-6 (IL-6), oncostatin M (OSM), and interleukin-1 (IL-1). This cytokine family is known for its pleiotropic effects and its involvement in cachexia characterized by anorexia, weight loss, and metabolic breakdown. Experimental evidence has shown that these deleterious symptoms cannot be attributed to just one cytokine, however, but rather to interactions among several cytokines. Experimental evidence indicates an increased level of CNTF synthesis in the CNS as a response to CNS injury, trauma, sepsis, and cancer, a set of clinical conditions all characterized by loss of appetite.4
Each member of this distantly related cytokine family acts to initiate receptor signaling by either homo- or heterodimerization of shared [beta] subunits. IL-6 requires the homodimerization of gp130, while CNTF, LIF, and OSM receptor activation depends on heterodimerization between gp130 and leukemia inhibitory factor receptor-[beta] (LIFR[beta]). Although the sequence of CNTF lacks exact homology with other related cytokines, studies have shown that receptor recognition sites of cytokines are organized as exchangeable modules between the cytokines. IL-6 signals via a gp130 homodimer, whereas CNTF and LIF signal by induction of gp130 and LIFR. CNTF also signals via the CNTF receptor (CNTFR[alpha]), which is expressed exclusively within the nervous system and skeletal muscle.5
The binding of CNTF to its receptor CNTFR[alpha] is responsible for the subsequent interaction between CNTF and gp130 and LIFR, which results in the activation of the JAK/STAT pathways.2 CNTF’s possible mechanism of action introduces the possibility that other related molecules, such as a suppositional second ligand for CNTFR[alpha] or LIF, might also play a crucial role in the regulation of food intake and energy expenditure. An important related observation is that in contrast to CNTF- or LIF-deficient animals, mice lacking CNTFR[alpha] or LIFR display deficient neuronal activity, which suggests the presence of additional receptor ligands.2
CNTF was first clinically recognized for its profound effects on amyotrophic lateral sclerosis (ALS), the most experimentally tractable of the neurodegenerative diseases. Administration of CNTF reduces motor neuron cell death, which is characteristic of ALS. During a clinical trial in which recombinant CNTF was used to treat ALS for neurotrophic benefits, researchers serendipitously stumbled on the discovery that the treatment produced severe anorexia and weight loss.6 After a similar outcome was reproduced in experiments using rodents,7 CNTF quickly became associated with its potential effects on metabolic pathways and was recognized as a new target for therapeutic applications for obesity.
Initial concerns were related to the possibility that CNTF’s mode of action is similar to those of cachectic cytokines that induce fever, hepatic acute-phase protein responses, anorexia, weight loss, and muscle wasting.8 Studies have shown that supraphysiologic doses of CNTF resulted in a rapid wasting syndrome characterized by weight loss, breakdown of fat tissue and skeletal muscle protein, and reduction of food and fluid intake. However, other findings showed that although CNTF is closely related to a group of cytokines displaying unfavorable effects, CNTF is unlike prototypical cytokines. When administered at lower doses, CNTF can induce weight loss without causing the typical deleterious effects of other cytokines. IL-1 is a typical cytokine that has often been compared with CNTF to demonstrate how CNTF differs from most cachectic cytokines. CNTF does not induce the muscle wasting, proinflammatory responses, conditioned taste aversion, or corticosterone release seen with doses of IL-1 that cause comparable weight loss.8 In a study involving CNTF administration in both db/db (mutated leptin receptor) and ob/ob (leptin-deficient) mice models, CNTF did not induce toxicity, malaise, illness, or taste aversion.2 These findings support the suggestion that CNTF acts via a leptin-like pathway.
Leptin is an adipocyte-derived cytokine involved in body weight homeostasis.2 Leptin’s discovery in 1994 was a huge breakthrough for obesity research. It was found to act through a negative feedback mechanism that relayed a signal between the body’s fat stores and the hypothalamic networks, thus providing a mechanism for maintaining body weight.4 Leptin is released by fat cells and is found in the blood in proportion to the amount of stored energy.
The comparison of CNTF’s actions to those of leptin is based on the findings that CNTF’s receptors have characteristics similar to those of the leptin receptors, including their distribution within hypothalamic nuclei involved in feeding.8 A plausible explanation for the overlapping biologic activity of leptin and CNTF is that they stimulate common signaling pathways in brain areas involved in the regulation of energy intake and expenditure.2 Several studies have shown that the leptin receptor (OB-Rb) is predominantly expressed in brain regions associated with food intake and energy expenditure, including the arcuate, ventromedial, and paraventricular hypothalamic nuclei. To support the notion that CNTF also targeted hypothalamic satiety centers, in situ hybridization was performed; the results showed that the arcuate and paraventricular nuclei of the mouse hypothalamus express mRNAs for CNTF receptor subunits.2 The physiologic and behavioral effects that CNTF and leptin produce when administered exogenously include weight loss induction and food intake suppression in ob/ob mice, a strain characterized by leptin deficiency.4,9
Recent studies showed that CNTF and leptin negatively regulate the appetite-stimulating signals NPY (neuropeptide Y), AGRP (agouti-related protein), and GABA (gamma-aminobutyric acid) in the arcuate nucleus of the hypothalamus. In addition, they positively regulate CART (cocaine-amphetamine-related transcript) and POMC (pro-opiomelanocortin), which produces the appetite-inhibiting peptide, [alpha]-MSH (melanocyte-stimulating hormone) (Figure 1).4
Daily icv administration of 0.5 µg CNTF in rats was shown to prevent the increase in hypothalamic NPY expression that occurred during food deprivation, and a higher dose of CNTF (5 µg/day) actually suppressed NPY expression.9 In the same study, daily icv injections of leptin also suppressed hypothalamic NPY expression.9 Another interesting finding related to CNTF and leptin’s interaction with NPY involves a system that controls luteinizing hormone (LH) secretion. Both leptin and CNTF have been shown to prevent food deprivation-induced suppression of LH secretion, an effect that is mediated by their suppression of NPY expression.1 This implies that the NPY system is a central target of CNTF and leptin action. These findings support the hypothesis that up-regulation of hypothalamic NPY results in a decrease in pituitary gonadotropin secretion, and that this pathway may act as a communication link between the neural processes involved in reproduction and those that regulate energy balance.4 Other findings have demonstrated that when NPY is centrally infused with CNTF, the anorexic and weight-reducing effects of CNTF are completely reversed.10 This not only reemphasizes the safety and specificity of CNTF but also exhibits CNTF’s interactive position among appetite-stimulating and appetite-suppressing signals.
Administration of leptin has been shown to attenuate the fasting-induced increase in corticosterone secretion, which is consistent with the finding that leptin receptors are found in corticotrophin-releasing hormone (CRH)-containing neurons. Leptin’s effect on food intake may be mediated by altered CRH-mRNA expression in the paraventricular nucleus.11 In contrast with leptin’s effect, CNTF administration does not significantly change the fasting-induced increased levels of corticosterone. Although the CNTF receptor has been found in the paraventricular nucleus,2 these findings indicate that CNTF receptors are not involved in regulation of CRH secretion,12 which suggests that the actions of CNTF and leptin bypass each other at certain points on the pathways involved in food intake control and energy balance regulation. Another discrepancy between the actions of leptin and CNTF is in their effects on hypothalamic AGRP, which is normally increased with food deprivation. In a study in which mice deprived of food for 48 hours showed increased AGRP mRNA expression, the administration of leptin reversed the increase, whereas CNTF administration had no effect on AGRP expression.12 This extensive regulation complex provides a representation of the often concurrent attenuation of appetite-stimulating signals and augmentation of appetite-suppressing signals, thus explaining the resulting decrease in food intake with a consequent decrease in body weight.
Whereas the similarities between CNTF and leptin have spawned great interest in their possible overlapping mechanisms, it is their differences that have researchers extremely curious about the possible implications that CNTF’s actions could have for pharmaceutical applications for obesity in the near future. Having two well-researched mechanisms (i.e., those of CNTF and leptin) that are very similar but that possess unique aspects will make it easier to discover the overall neuronal pathway responsible for food intake and energy expenditure. The premature excitement about leptin’s potential as an obesity treatment was quickly quelled after the finding that leptin resistance is very common in diet-induced obese rodent models.4 This type of obesity is readily compared with the ever-rising obesity epidemic found in humans, which is attributed to a self-inflicted lifestyle rather than an unpreventable genetic disorder. In the case of leptin resistance, even the administration of supraphysiologic doses of leptin is largely ineffective in reducing body weight in clinically obese patients.13 CNTF appears to be more efficacious than leptin because body weight was reduced in conditions in which leptin was ineffective, most often in leptin-resistant models. Whereas leptin has been successful in normalizing the obese phenotype in ob/ob (leptin-deficient) rodent models, CNTF has done the same in ob/ob, db/db, MC-4 receptor-deficient mice, and diet-induced obese mice.2,7-9 CNTF appears to activate hypothalamic pathways that are downstream of leptin in diet-induced obese models (more representative of human obesity) that are unresponsive to leptin treatment. CNTF is also noted for its ability to cause weight loss without post-treatment overeating and immediate rebound weight gain. Experimental evidence suggests these results are due to CNTF’s ability to reduce food intake without triggering the typical hunger signals associated with stress responses. This suggests the possible modification of a set body weight point encoded by the brain.8
After CNTF proved to have a profound effect on appetite and energy expenditure, it became a promising prospect as a leptin-like cytokine. After a chance discovery within an ALS clinical trial, CNTF was quickly recognized by the pharmaceutical industry as a hopeful candidate for a much anticipated obesity drug. By early 2000, a re-engineered analog of CNTF, named AXOKINE®, produced by traditional biotechnology processes, was already in the first stages of clinical trials at Regeneron Pharmaceuticals. In March of 2000, investigators initiated a Phase-II dose-ranging trial to study the safety and efficacy of AXOKINE.11 By July of 2001, a press release announced that Regeneron had initiated a Phase-III clinical program of AXOKINE treatment for obesity. On April 14th, 2003, another press release announced the initial results of its Phase-II study of AXOKINE. The study involved a 12-week trial testing AXOKINE on overweight and obese people with type 2 diabetes at doses 1 µg/kg and 0.5 µg . kg^sup -1^ . day^sup -1^. At the end of the trial period, subjects who were treated with the 1 µg/kg dose of AXOKINE lost 6.5 pounds on average, whereas those treated with placebo and dietary counseling lost only 2.5 pounds. There were also improvements in blood glucose and other metabolic parameters seen in the AXOKINE-treated patients (unpublished data, 3/31/2003, available at: http://www.regeneron.com/investor/press_detail.asp?v_c_id=169).
Although the initial results of the AXOKINE clinical trials appear promising, the most recent finding indicates an adverse side effect was reported by many of the participants. From the phase-II results, approximately one third of the 1 µg/kg AXOKINE-treated subjects developed antibodies to AXOKINE. In the phase-III study involving overweight and obese non-diabetic participants, approximately half of the AXOKINE-treated subjects developed AXOKINE-neutralizing antibodies after 12 weeks. The higher incidence of antibody development in non-diabetic subjects will be explored further throughout the phase-III study. Further weight loss appeared to be limited in those people who had developed antibodies (unpublished data, 3/31/2003; available at: http://www.regeneron.com/investor/press_detail.asp?v_c_id=169). AXOKINE, a modified version of natural CNTF, could be sensitizing the immune system to produce the neutralizing antibodies as a result of the amino acid modifications, because of impurities in the formulation being used, or through direct CNTF stimulation of the immune system, etc. It is likely that the cause for the immunogenicity of AXOKINE will have to be identified and eliminated before it is approved as an antiobesity therapeutic agent.
The discovery that linked CNTF with decreased food intake and weight loss promises a potential therapeutic application for obesity. However, there is still much more to be learned about its mechanisms of action and how it is integrated into the neural pathways that control energy expenditure and food intake. The growing obesity problem is no longer believed to be predominantly a genetic mystery but is becoming more accepted as an issue of unhealthy lifestyle choices. Although the discovery of leptin was a landmark for metabolic research, there are many obstacles yet to be overcome because of leptin resistance, which is a characteristic of diet-induced obesity. The leptin-like effects of CNTF do not appear to be hindered in models characterized by leptin resistance, a finding that is encouraging enthusiasm for finding a treatment for diet-induced obesity.
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This review was prepared by Emily Duff, B.S., and Clifton A. Baile, Ph.D., Departments of Foods & Nutrition and Animal & Dairy Science, 444 Animal Science Complex, University of Georgia, Athens, GA 30602.
Copyright International Life Sciences Institute and Nutrition Foundation Dec 2003
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