Ciliary Neurotrophic Factor (CNTF) was first characterized as a trophic factor for motor neurons in the ciliary ganglion and spinal cord, leading to its evaluation in humans suffering from motor neuron disease. In these trials, CNTF caused unexpected and substantial weight loss, raising concerns that it might produce cachectic-like effects. Countering this possibility was the suggestion that CNTF was working via a leptin-like mechanism to cause weight loss, based on the findings that CNTF acts via receptors that are not only related to leptin receptors, but also similarly distributed within hypothalamic nuclei involved in feeding. However, although CNTF mimics the ability of leptin to cause fat loss in mice that are obese because of genetic deficiency of leptin (ob͞ob mice), CNTF is also effective in diet-induced obesity models that are more representative of human obesity, and which are resistant to leptin. This discordance again raised the possibility that CNTF might be acting via nonleptin pathways, perhaps more analogous to those activated by cachectic cytokines. Arguing strongly against this possibility, we now show that CNTF can activate hypothalamic leptin-like pathways in diet-induced obesity models unresponsive to leptin, that CNTF improves prediabetic parameters in these models, and that CNTF acts very differently than the prototypical cachectic cytokine, IL-1. Further analyses of hypothalamic signaling reveals that CNTF can suppress food intake without triggering hunger signals or associated stress responses that are otherwise associated with food deprivation; thus, unlike forced dieting, cessation of CNTF treatment does not result in binge overeating and immediate rebound weight gain.

IntroductionInsulin resistance is regarded as the main link between obesity and type 2 diabetes mellitus (1, 2). While multiple hypotheses have been proposed to explain this association, recent discoveries on the regulation of feeding behavior and energy expenditure (3-6) have refocused attention on the potential role of hypothalamic centers in the regulation of both energy homeostasis and insulin action (Figure 1) (7-13). In fact, leptin has potent effects on glucose tolerance and insulin action, which appear to be independent of its modulation of feeding behavior (7,9,13). Leptin exerts its actions on food intake and weight gain partly by activation of the melanocortin pathway in the hypothalamus and in other areas within the central nervous system (14). The pivotal role of melanocortinergic neurons in the regulation of energy balance is supported by solid genetic (15, 16) and pharmacologic (17) evidence.To examine whether the activity of the melanocortin pathway in the hypothalamus modulates insulin action in vivo we used the natural agonist, α-melanocyte-stimulating hormone (α-MSH), and a high-affinity antagonist (SHU9119) of the neural melanocortin receptors type 3 and 4 (MCR3, MCR4) (17) to generate bidirectional variations in the activity of this pathway. Body composition and metabolic parameters were assessed using tracer dilution techniques and pancreatic-insulin clamp studies in conscious rats. MethodsExperimental procedures. Thirty-nine male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Massachusetts, USA) were studied ( Figure 2). Rats were housed in individual cages and subjected to a standard light-dark (0600 to 1800 hours/1800 to 0600 hours) cycle. Three weeks before the in vivo study, rats (at ∼10 weeks of age) were equipped with chronic catheters placed in the third cerebral ventricle (18). Rats were anesthetized with intraperitoneal ketamine (Ketaset; 87 mg/kg) and xylazine (Rompun; 11 mg/kg) and fixed in a stereotaxic apparatus with ear bars and a nose piece set at +5.0 mm. A 26-gauge stainless steel guide cannula (Plastics One Inc., Roanoke, Virginia, USA) was chronically implanted into the third ventricle using the following coordinates from bregma: anterior-posterior; +0.2 mm, dorsal-ventral; -9.0 mm, medial-lateral; 0.0 directly on the midsagittal suture. A 28-gauge dummy cannula was inserted to prevent clogging of the guide cannula. The implant is secured to the skull with Caulk Grip dental cement, and the skin is closed over the implant using wound clips. One week before the study, rats (at ∼12 weeks of age) were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg body weight), and indwelling catheters were inserted in the right internal jugular vein and in the left carotid artery (18-21). The venous catheter was extended to the level of the right atrium, and the arterial catheter was advanced to the level of the aortic arch. All studies were performed in awake, unstressed, chronically catheterized rats. Histological verification of the intracerebrove...

IntroductionInsulin resistance is regarded as the main link between obesity and type 2 diabetes mellitus (1, 2). While multiple hypotheses have been proposed to explain this association, recent discoveries on the regulation of feeding behavior and energy expenditure (3-6) have refocused attention on the potential role of hypothalamic centers in the regulation of both energy homeostasis and insulin action (Figure 1) (7-13). In fact, leptin has potent effects on glucose tolerance and insulin action, which appear to be independent of its modulation of feeding behavior (7,9,13). Leptin exerts its actions on food intake and weight gain partly by activation of the melanocortin pathway in the hypothalamus and in other areas within the central nervous system (14). The pivotal role of melanocortinergic neurons in the regulation of energy balance is supported by solid genetic (15, 16) and pharmacologic (17) evidence.To examine whether the activity of the melanocortin pathway in the hypothalamus modulates insulin action in vivo we used the natural agonist, α-melanocyte-stimulating hormone (α-MSH), and a high-affinity antagonist (SHU9119) of the neural melanocortin receptors type 3 and 4 (MCR3, MCR4) (17) to generate bidirectional variations in the activity of this pathway. Body composition and metabolic parameters were assessed using tracer dilution techniques and pancreatic-insulin clamp studies in conscious rats. MethodsExperimental procedures. Thirty-nine male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Massachusetts, USA) were studied ( Figure 2). Rats were housed in individual cages and subjected to a standard light-dark (0600 to 1800 hours/1800 to 0600 hours) cycle. Three weeks before the in vivo study, rats (at ∼10 weeks of age) were equipped with chronic catheters placed in the third cerebral ventricle (18). Rats were anesthetized with intraperitoneal ketamine (Ketaset; 87 mg/kg) and xylazine (Rompun; 11 mg/kg) and fixed in a stereotaxic apparatus with ear bars and a nose piece set at +5.0 mm. A 26-gauge stainless steel guide cannula (Plastics One Inc., Roanoke, Virginia, USA) was chronically implanted into the third ventricle using the following coordinates from bregma: anterior-posterior; +0.2 mm, dorsal-ventral; -9.0 mm, medial-lateral; 0.0 directly on the midsagittal suture. A 28-gauge dummy cannula was inserted to prevent clogging of the guide cannula. The implant is secured to the skull with Caulk Grip dental cement, and the skin is closed over the implant using wound clips. One week before the study, rats (at ∼12 weeks of age) were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg body weight), and indwelling catheters were inserted in the right internal jugular vein and in the left carotid artery (18-21). The venous catheter was extended to the level of the right atrium, and the arterial catheter was advanced to the level of the aortic arch. All studies were performed in awake, unstressed, chronically catheterized rats. Histological verification of the intracerebrove...

Peroxynitrite (ONOO؊ ) has been shown in studies on vascular relaxation and guanylate cyclase activation to react with glutathione (GSH), generating an intermediate product that promotes a time-dependent production of nitric oxide (NO). In this study, reactions of ONOO ؊ with GSH produced a new substance, which was characterized by liquid chromatography, ultraviolet spectroscopy, and electrospray tandem mass spectrometry. The mass spectrometric data provided evidence that the product of this reaction was S-nitroglutathione (GSNO 2 ) and that S-nitrosoglutathione (GSNO) was not a detectable product of this reaction. Further evidence was obtained by comparison of the spectral and chromatographic properties with synthetic standards prepared by reaction of GSH with nitrosonium or nitronium borofluorates. Both the synthetic and ONOO ؊ /GSH-derived GSNO 2 generated a protonated ion, GSNO 2 H ؉ , at m/z 353, which was unusually resistant to decomposition under collision activation, and no fragmentation was observed at collision energy of 25 eV. In contrast, an ion at m/z 337 (GSNOH ؉ ), generated from the synthetic GSNO, readily fragmented with the abundant loss of NO at 9 eV. Reactions of ONOO ؊ with GSH resulted in the generation of NO, which was detected by the head space/ NO-chemiluminescence analyzer method. The generation of NO was inhibited by the presence of glucose and/or CO 2 in the buffers employed. Synthetic GSNO 2 spontaneously generated NO in a manner that was not significantly altered by glucose or CO 2 . Thus, ONOO ؊ reacts with GSH to form GSNO 2 , and GSNO 2 decomposes in a manner that generates NO.Exposure of vascular tissue to peroxynitrite (ONOO Ϫ ) 1 results in a prolonged relaxation (1) that appears to be mediated through a glutathione (GSH)-dependent regeneration of NO (2). Peroxynitrite has also been observed to stimulate guanylate cyclase activity in a thiol-dependent manner in vascular endothelial and smooth muscle preparations (3, 4). Whereas the reaction of ONOO Ϫ with GSH has been reported to form small amounts of S-nitroso-GSH (GSNO) (3, 5), our previous studies detected a different product of this reaction, which was isolated and demonstrated to possess potent vascular relaxant activity (2). Examination of the reaction of nitrogen dioxide (NO 2 ) with GSH detected the formation of what appears to be the same product as that observed in the reaction with ONOO Ϫ (6). Because the biologically active metabolite of these reactions co-migrated on HPLC with a the product of a reaction between nitrosonium borofluorate (NO 2 BF 4 ) and GSH, the vascular relaxant detected was suggested to be a nitrated product of GSH (GSNO 2 ) (6). Thus, additional studies are needed to identify the biologically active substances derived from the reaction of GSH with ONOO Ϫ . Peroxynitrite is also known to undergo additional reactions in the presence of physiological buffered systems and GSH. One of the first observed actions of ONOO Ϫ on thiols was that it caused oxidation reactions, and an analysis of products o...

Leptin, the product of the Obese (Lep) gene, orchestrates behavioral and metabolic responses to nutrient intake. Here, we demonstrate tissue-specific autoregulation of Lep. Moderate increases in circulating leptin considerably decreased Lep expression in adipose tissue and induced lep expression in skeletal muscle, a tissue that normally does not express this gene. Changes in nutrient availability resulted in rapid alterations in Lep autoregulation. These findings demonstrate negative feedback regulation of Lep in fat, and indicate that leptin secretion can function as a vehicle of 'cross-talk' between adipose tissue and skeletal muscle, leading to tissue-specific modulation of the 'leptin signal'.