Cytokine-induced Anorexia Research Articles

Antibodies as pharmacologic tools for studies on the regulation of energy balance

ObjectiveActive immunization in rats may serve several purposes: the production of a disease-like phenotype, the generation of pharmacologic tools, and the development of clinically useful therapies. We selected the melanocortin-4 receptor (MC4R) as a target because its blockade could provide a treatment for anorexia and cachexia. MethodsWe used a sequence of the N-terminal (NT) domain of the MC4R as an antigen. Rats immunized against the NT peptide produced specific MC4R antibodies (Abs) that were purified and characterized in vitro and in vivo. ResultsThe Abs acted as inverse agonists and reduced under basal conditions the production of cyclic adenosine monophosphate in HEK-293 cells expressing the human MC4R. Rats immunized against the NT peptide developed a phenotype consistent with hypothalamic MC4R blockade, i.e., increased food intake and body weight, liver and fat-pad weights, hepatic steatosis, and increased plasma triacylglycerols. With a high-fat diet, plasma insulin levels were significantly increased. In separate experiments an increase in food intake was observed after injection of purified MC4R Abs into the third ventricle. When lipopolysaccharide was administered in NT-immunized rats the reduction of food intake was partly prevented in this model of cytokine-induced anorexia. ConclusionOur results show that active immunization of rats against the MC4R resulted in the generation of specific Abs that stimulated food intake by acting as inverse agonists of the hypothalamic MC4R. Pharmacologically active monoclonal MC4R Abs could be the starting point for the development of novel treatments for patients with anorexia or cachexia.

Central administration of peptide and small molecule MC4 receptor antagonists induce hyperphagia in mice and attenuate cytokine-induced anorexia

We investigated the effect of melanocortin 4 receptor (MC4) antagonists on food intake in mice. Food intake during the light phase was significantly increased by ICV administration of mixed MC3/MC4 antagonists (AgRP and SHU9119) or MC4 selective antagonist peptide [(Cyclo (1–5)[Suc– d–Nal–Arg–Trp–Lys]NH 2] (MBP10) and the small molecule antagonists THP and NBI-30. Both mixed and selective antagonists significantly reversed anorexia induced by ICV administration of the MC4 agonist (c (1–6) HfRWK-NH 2) and the cytokine IL-1β. These findings provide pharmacological evidence that the MC4 receptor mediates the effects of melanocortin agonists and antagonists on food intake in mice, and support the idea that selective small molecule MC4 antagonists may be useful as therapeutics for cachexia.

Involvement of plasma leptin, insulin and free tryptophan in cytokine-induced anorexia

Background: Hypothalamic serotonin, the synthesis of which parallels plasma free tryptophan, contributes to satiety. Plasma free tryptophan, insulin and leptin, all of which can also decrease food intake partly via the hypothalamic serotonergic system, are modulated by cytokines, which decrease food intake. The mechanisms of anorexia induced by cytokines, as related to plasma tryptophan, leptin and insulin, have not been fully determined. Objective: We determined the plasma concentrations of free as well as total tryptophan, leptin and insulin, and correlations to those of food intake and body weight change after cytokines or tryptophan injection. Design: Interleukin-1α and/or tumor necrosis factor-α, or tryptophan was injected subcutaneously into male rats for 2 days. Daily food intake, body weight, carcass adiposity, plasma total as well as free tryptophan, plasma leptin and insulin were measured. Results:Interleukin-1α injection decreased food intake, body weight, carcass adiposity and plasma leptin, but increased plasma free tryptophan and insulin. Tryptophan injection increased both free and total tryptophan, but did not change food intake, body weight, carcass adiposity or leptin. Plasma free tryptophan, but not total tryptophan, was significantly negatively correlated with food intake. There was a negative correlation between plasma insulin and food intake. Conclusions: Increased plasma free tryptophan may contribute to synthesis of brain serotonin but anorexia may be due to stimulation of its release induced by interleukin-1α. Plasma insulin, but not leptin, may partly contribute to anorexia of cytokines.

Plasma leptin, insulin and free tryptophan contribute to cytokine-induced anorexia.

Cytokines contribute to anorexia of diseases. Tumor Necrosis Factor (TNF) and/or interleukin-1 (IL-1) stimulate leptin release, but not insulin. Both affect hypothalamus to decrease food intake (FI). Hypothalamic serotonin (5HT) decreases FI. Its synthesis depends on brain availability of precursor, tryptophan (TRP), which depends on plasma free TRP. Purpose is to test involvement of plasma leptin, insulin, TRP, and thus hypothalamic 5HT in cytokine-induced anorexia in rats. In male rats, IL-1alpha (10 mg/kg/d; n=9), TNFalpha (30 mg/kg/d; n=9), Il-1alpha+TNFalpha (10:30 mg/kg/d; n=9), TRP (100 mg/kg/d, n=8) and saline (n=8; Control) were injected sc for 2 days. FI, BW, plasma free and total TRP, leptin and insulin, and body fat were measured. Data analyzed via ANOVA. IL-1alpha and IL-1alpha+TNFalpha vs others decreased FI and BW. TNFalpha and TRP did not change FI and BW. Plasma total TRP was higher in TRP vs IL-1alpha, TNFalpha, and IL-1alpha+TNFalpha. Plasma free TRP was higher in IL-1alpha and IL-1alpha+TNFalpha vs Control. IL-1alpha and IL-1alpha+TNFalpha decreased leptin and body fat. Insulin in Control was lower than others. Data suggest: i) IL-1alpha increases plasma free TRP, but not total TRP, thus increases hypothalamic 5HT synthesis, resulting in anorexia; ii) leptin does not mediate anorexia, but; iii) insulin may contribute to anorexia induced by cytokines.

1998 Curt P. Richter Award. Brain mechanisms in cytokine-induced anorexia.

Our research focuses on the mechanisms underlying cytokine action in the central nervous system (CNS) using an integrative and multidisciplinary strategy organized through supracellular (behavioral analysis by computerized monitoring systems), cellular (extracellular and intracellular neurophysiological recording), and molecular (patch-clamp recording, and DNA, RNA and protein analyses) approaches. An integrative strategy that combines computerized meal pattern analyses with cellular and molecular biology approaches allows the study of underlying brain mechanisms in cytokine- and disease-associated anorexia. This paper presents a comprehensive discussion of our laboratory's previously published data on brain mechanisms involved in cytokine-induced anorexia including the relevance of meal pattern analysis (meal size, meal duration, meal frequency, intermeal intervals), modulation of hypothalamic neuronal activity, molecular processes involving ionic conductances, cytokine-cytokine and cytokine-peptide interactions, and modulation of cytokine and peptide/neuropeptide system components (ligands, endogenous inhibitors, receptor subtypes, signal transduction molecules, intracellular mediators) and cytokine feedback systems.

Cytokine-induced anorexia. Behavioral, cellular, and molecular mechanisms.

Cytokines induce anorexia. Recent issues concerning mechanistic aspects are: (1) Cytokines induce anorexia through different modes of behavioral action, that is, by affecting meal size, meal duration, and meal frequency differentially. Profiles also depend on the concentration or dosage. (2) The interface between the periphery and brain. Specific cytokines may be transported from the periphery to the brain. Cytokines generate mediators that can act on peripheral and/or brain target sites. Cerebrovasculature endothelium can also generate signals to modulate neural activities. Evidence indicates that the proposed vagal afferent signaling requires reassessment. Because of paracrine and autocrine actions, local cytokine production within the brain can induce anorexia. (3) Cytokines act directly on hypothalamic neurons proposed to participate in feeding. (4) Cytokine<-->cytokine and cytokine<-->peptide/neurotransmitter interactions are critical; for example, cytokines interact to induce anorexia synergistically, neuropeptide Y<-->cytokine interactions are antagonist, and cytokine<-->neurotransmitter and cytokine<-->leptin<-->neuropeptide Y<-->CRH-glucocorticoid and other endocrine interactions are important. A leptin receptor is related to gp 130, a signal transducer among interleukin (IL)-6 subfamily receptors; gp 130 and related molecules may be an interface for feeding control in health and disease. Various cytokines upregulate leptin and gp 130. An integrative approach combining computerized meal pattern analyses with cellular and molecular approaches is being used to characterize mechanisms (ligands, receptors, transducing molecules, and intracellular mediators) involved in cytokine-induced anorexia.

Differential responsiveness of obese (fa/fa) and lean (Fa/Fa) Zucker rats to cytokine-induced anorexia.

Pathophysiological and pharmacological concentrations of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) in the cerebrospinal fluid (CSF) induce anorexia in normal rats. Obesity in humans and rodents is associated with increased TNF-alpha messenger RNA and protein levels in various cell types. This suggests that obese individuals may have differential regulation of cytokine production and dissimilar responsiveness to cytokines. In the present study, we investigated the effects of the intracerebroventricular (ICV) microinfusion of TNF-alpha (50, 100, and 500 ng/rat), IL-1 beta (1.0, 4.0, and 8.0 ng), and TNF-alpha (100 ng) plus IL-1 beta (1.0 ng) on obese (fa/fa) and lean (Fa/Fa) Zucker rats. The results show that: TNF-alpha and IL-1 beta, and the concomitant administration of TNF-alpha and IL-1 beta decreased the short-term (4 hours), nighttime (12 hours), and total daily food intakes in obese and lean rats; IL-1 beta was more potent relative to TNF-alpha; obese rats showed greater responsiveness to IL-1 beta: 8.0 ng IL-1 beta, for example, decreased the 12-hour food intake by 52% in obese and 22% in lean rats. On the other hand, obese and lean rats did not exhibit a significantly different responsiveness to the anorexia induced by 50, 100, or 500 ng TNF-alpha at the 4-hour period; and the concomitant ICV administration of TNF-alpha and IL-1 beta induced anorexia with additive (4-hour period) or synergistic (12-hour and 24-hour periods) effects in obese rats. The effect of TNF-alpha plus IL-1 beta in lean rats was greater than additive for the 12-hour and 24-hour periods. The difference in suppression of total daily food intake by TNF-alpha plus IL-1 beta in obese (-43%) versus lean (-23%) rats was significantly different (p < 0.01). The results show that obese (fa/fa) and lean (Fa/Fa) Zucker rats have differential responsiveness to the ICV microinfusion of two different classes of cytokines.

An approach to study molecular mechanisms involved in cytokine-induced anorexia

Cytokines are released during pathophysiological processes. Cytokines (e.g., interleukin-1β or IL-1β) induce neurological manifestations including anorexia. Here, we show an integrative approach to investigate the cellular and molecular basis of cytokine-induced anorexia. In this approach: (1) the chronic intracerebroventricular (icv) microinfusion (via osmotic minipumps) of cytokines, at doses that will yield estimated pathophysiological concentrations reported in the cerebrospinal fluid, is used. (2) General and computerized behavioral monitoring characterizes the microstructure of behavioral modifications induced by a cytokine, and the time course for cytokine action. (3) Brain regions and subregions (nuclei/areas) from animals exhibiting significant anorexia in response to cytokine(s) are dissected, and RNA and protein are isolated. (4) The profile of cytokine subsystems (ligands, receptors, endogenous inhibitors; for example, IL-1α and β, IL-1 receptor types I and II, and IL-1 receptor antagonist) is characterized in the same brain samples with polymerase chain reaction, sensitive RNase protection assays and immunoblots. (5) The relationship between changes in cytokine subsystems at the molecular level and cytokine-induced anorexia within an animal is determined, and the general profile is analyzed with statistical methods. This approach is also pertinent to study neurotransmitter and neuropeptide profiles, and cytokine-cytokine, cytokine-neuropeptide and cytokine-neurotransmitter interactions in vivo. The results show that this integrative and novel strategy can be used to study the molecular basis of anorexia and other neurological manifestations (e.g., fever, sleep changes) induced by cytokines.

Anorexia induced by chronic central administration of cytokines at estimated pathophysiological concentrations

Interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-α (TNF-α) induce acute anorexia by direct action in the central nervous system (CNS) at estimated pathophysiological concentrations reported in the cerebrospinal fluid (CSF). Cytokine-induced anorexia may also participate in the long-term anorexia observed during disease. Here, we studied the effects of chronic intracerebroventricular (ICV) microinfusion (through osmotic minipumps) of various cytokines on feeding and drinking in rats. The results show: IL-1β decreased nighttime feeding dose-dependently (with 0.5, l.0, 2.0, and 8.0 ng/24 h, n ≥ 8/group). The decrease of feeding and corresponding decrease of body weight persisted during the 7-day infusion. Total daily food intake decrease was less prominent relative to the nighttime decrease because daytime food intake slightly increased. Feeding and body weight increased toward baseline following the end of the IL-1β infusion. ICV microinfusion of heat-inactivated IL-1β or IL-1β plus IL-1 receptor antagonist had no effect, suggesting specificity of action of IL-1β. Water intake did not decrease in any IL-1β-β-treated group, suggesting specificity on feeding. Chronic ICV administration of TNF-α (20, 100, or 300 ng/24 h), IL-6 (100 ng/24 h), or IL-8 (20 ng/24 h) was significantly less effective than IL-1β in inducing behavioral modifications. The results suggest that IL-1β, at doses that yield estimated pathophysiological concentrations in the CSF, is capable of inducing long-term anorexia and this effect may participate in the anorexia observed during chronic disease.

Anorexia Induced by Chronic Central Administration of Cytokines at Estimated Pathophysiological Concentrations

Interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-α (TNF-α) induce acute anorexia by direct action in the central nervous system (CNS) at estimated pathophysiological concentrations reported in the cerebrospinal fluid (CSF). Cytokine-induced anorexia may also participate in the long-term anorexia observed during disease. Here, we studied the effects of chronic intracerebroventricular (ICV) microinfusion (through osmotic minipumps) of various cytokines on feeding and drinking in rats. The results show: IL-1β decreased nighttime feeding dose-dependently (with 0.5, l.0, 2.0, and 8.0 ng/24 h, n ≥ 8/group). The decrease of feeding and corresponding decrease of body weight persisted during the 7-day infusion. Total daily food intake decrease was less prominent relative to the nighttime decrease because daytime food intake slightly increased. Feeding and body weight increased toward baseline following the end of the IL-1β infusion. ICV microinfusion of heat-inactivated IL-1β or IL-1β plus IL-1 receptor antagonist had no effect, suggesting specificity of action of IL-1β. Water intake did not decrease in any IL-1β-β-treated group, suggesting specificity on feeding. Chronic ICV administration of TNF-α (20, 100, or 300 ng/24 h), IL-6 (100 ng/24 h), or IL-8 (20 ng/24 h) was significantly less effective than IL-1β in inducing behavioral modifications. The results suggest that IL-1β, at doses that yield estimated pathophysiological concentrations in the CSF, is capable of inducing long-term anorexia and this effect may participate in the anorexia observed during chronic disease.

Cytokine action in the nervous system at pathophysiological versus pharmacological concentrations.

Cytokines induce a variety of neurological actions.15 Cytokine action in the nervous system may present different profiles depending on the cytokine concentration or dosage. Pathophysiological and pharmacological analyses of cytokine neuroactivity have been assessed with an integrative approach organized through neurological → behavioral → cellular → molecular → genetical levels. Cytokine-induced anorexia represents an example of this integrative approach.

Effect of (n-3) polyunsaturated fatty acidson cytokine production and their biologic function

Cytokines are important biologic mediators with tightly regulated production. Overproduction contributes to pathogenesis of acute and chronic inflammatory, autoimmune, atherosclerotic, and neoplastic diseases. Animal and human studies have shown that production of cytokines can be reduced by long-chain (n-3) polyunsaturated fatty acids (PUFA). This, in turn, results in reduction of the severity of certain autoimmune, inflammatory, and atherosclerotic diseases and reduces cytokine-induced anorexia. Because these cytokines are also involved in control of the host defense, substantial reduction in their production could impair normal immune response. In addition, increased intake of (n-3) PUFAs without adequate antioxidant protection could result in increased free radical formation and lipid peroxidation, leading to a reduction in T cell-mediated function, natural killer cell activity, and macrophage cytotoxicity. These risks associated with the intake of (n-3) PUFAs may be minimized without compromising its beneficial effects by the intake of appropriate levels of antioxidants such as vitamin E.