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Microglia in the mediobasal hypothalamus (MBH) respond to inflammatory stimuli and metabolic perturbations to mediate body composition. This concept is well studied in the context of high fat diet induced obesity (HFDO), yet has not been investigated in the context of cachexia, a devastating metabolic syndrome characterized by anorexia, fatigue, and muscle catabolism. We show that microglia accumulate specifically in the MBH early in pancreatic ductal adenocarcinoma (PDAC)‐associated cachexia and assume an activated morphology. Furthermore, we observe astrogliosis in the MBH and hippocampus concurrent with cachexia initiation. We next show that circulating immune cells resembling macrophages infiltrate the MBH. PDAC‐derived factors induced microglia to express a transcriptional profile in vitro that was distinct from that induced by lipopolysaccharide (LPS). Microglia depletion through CSF1‐R antagonism resulted in accelerated cachexia onset and increased anorexia, fatigue, and muscle catabolism during PDAC. This corresponded with increased hypothalamic–pituitary–adrenal (HPA) axis activation. CSF1‐R antagonism had little effect on inflammatory response in the circulation, liver, or tumor. These findings demonstrate that microglia are protective against PDAC cachexia and provide mechanistic insight into this function., • Microgliosis occurs in the mediobasal hypothalamus early in pancreatic cancer.• Microglia respond to pancreatic cancer‐derived factors and produce arginase‐1.• Microglia depletion worsens cachexia during pancreatic cancer.

When energy balance is altered by aerobic exercise, starvation, and cold exposure, for example, there appears to be coordination of the responses of skeletal muscle, white adipose (WAT), and brown adipose (BAT) tissues. We hypothesized that WAT, BAT, and skeletal muscle may share an integrated regulation by the central nervous system (CNS); specifically, that neurons in brain regions associated with energy balance would possess neuroanatomical connections to permit coordination of multiple, complementary responses in these downstream tissues. To study this, we used trans-neuronal viral retrograde tract tracing, using isogenic strains of pseudorabies virus (PRV) with distinct fluorescent reporters (either eGFP or mRFP), injected pairwise into male rat gastrocnemius, subcutaneous WAT and interscapular BAT, coupled with neurochemical characterization of specific cell populations for cocaine- and amphetamine-related transcript (CART), oxytocin (OX), corticotrophin releasing hormone (CRH) and calcitonin gene-related peptide (CGRP). Cells in the paraventricular (PVN) and parabrachial (PBN) nuclei and brainstem showed dual projections to muscle + WAT, muscle + BAT, and WAT + BAT. Dual PRV-labeled cells were found in parvocellular, magnocellular and descending/pre-autonomic regions of the PVN, and multiple structural divisions of the PBN and brainstem. In most PBN subdivisions, more than 50% of CGRP cells dually projected to muscle + WAT and muscle + BAT. Similarly, 31–68% of CGRP cells projected both to WAT + BAT. However, dual PRV-labeled cells in PVN only occasionally expressed OX or CRH but not CART. These studies reveal for the first time both separate and shared outflow circuitries among skeletal muscle and subcutaneous WAT and BAT.

Whether leptin acts in the paraventricular nucleus (PVN) to increase sympathetic nerve activity (SNA) is unclear, since PVN leptin receptors (LepR) are sparse. We show in rats that PVN leptin slowly increases SNA to muscle and brown adipose tissue, because it induces the expression of its own receptor and synergizes with local glutamatergic neurons. PVN LepR are not expressed in astroglia and rarely in microglia; instead, glutamatergic neurons express LepR, some of which project to a key presympathetic hub, the rostral ventrolateral medulla (RVLM). In PVN slices from mice expressing GCaMP6, leptin excites glutamatergic neurons. LepR are expressed mainly in thyrotropin-releasing hormone (TRH) neurons, some of which project to the RVLM. Injections of TRH into the RVLM and dorsomedial hypothalamus increase SNA, highlighting these nuclei as likely targets. We suggest that this neuropathway becomes important in obesity, in which elevated leptin maintains the hypothalamic pituitary thyroid axis, despite leptin resistance.

Cachexia, a devastating wasting syndrome characterized by severe weight loss with specific losses of muscle and adipose tissue, is driven by reduced food intake, increased energy expenditure, excess catabolism, and inflammation. Cachexia is associated with poor prognosis and high mortality and frequently occurs in patients with cancer, chronic kidney disease, infection, and many other illnesses. There is no effective treatment for this condition. Hypothalamic melanocortins have a potent and long-lasting inhibitory effect on feeding and anabolism, and pathophysiological processes increase melanocortin signaling tone, leading to anorexia, metabolic changes, and eventual cachexia. We used 3 rat models of anorexia and cachexia (LPS, methylcholanthrene sarcoma, and 5/6 subtotal nephrectomy) to evaluate efficacy of TCMCB07, a synthetic antagonist of the melanocortin-4 receptor. Our data show that peripheral treatment using TCMCB07 with intraperitoneal, subcutaneous, and oral administration increased food intake and body weight and preserved fat mass and lean mass during cachexia and LPS-induced anorexia. Furthermore, administration of TCMCB07 diminished hypothalamic inflammatory gene expression in cancer cachexia. These results suggest that peripheral TCMCB07 treatment effectively inhibits central melanocortin signaling and therefore stimulates appetite and enhances anabolism, indicating that TCMCB07 is a promising drug candidate for treating cachexia.

Objective Activation of hypothalamic agouti-related peptide expressing (AgRP)+ve neurons during energy deficit is a negative valence signal, rapidly activating food-seeking behaviors. This study examined the roles of melanocortin-3 receptors (MC3Rs) coexpressed in a subpopulation of AgRP+ve neurons. Methods AgRP-MC3R mice expressing MC3Rs selectively in AgRP+ve neurons were generated by crossing AgRP-IRES-Cre mice with LoxTBMc3r mice containing a "loxP-STOP-loxP" sequence in the 5' untranslated region. Body weight, body composition, and feeding behavior were assessed during ad libitum and time-restricted feeding conditions. Results In females, food intake of AgRP-IRES-Cre+ve (n = 7) or AgRP-IRES-Cre-ve (n = 9) mice was not significantly different; these mice were therefore pooled to form the "control" group. Female AgRP-MC3R mice exhibited lower food intake (25.4 ± 2.4 kJ/12 h; n = 6) compared with controls (35.3 ± 1.8 kJ/12 h; n = 16) and LoxTBMc3r mice (32.1 ± 2.1 kJ/12 h; n = 9) in the active phase during the dark period. Food intake during the rest phase (lights on) when mice consume less food (9-10 kJ) was normal between genotypes. Body weight and composition of AgRP-MC3R and LoxTBMc3r mice were similar, suggesting compensatory mechanisms for reduced calorie intake. Remarkably, AgRP-MC3R mice continued to consume less food during refeeding after fasting and time-restricted feeding. Conclusions MC3Rs expressed on AgRP+ve neurons appear to exert a strong inhibitory signal on hypothalamic networks governing feeding behavior.

Muscle atrophy occurs during chronic diseases, resulting in diminished quality of life and compromised treatment outcomes. There is a high demand for therapeutics that increase muscle mass while abrogating the need for special dietary and exercise requirements. Therefore, we developed an efficient nanomedicine approach capable of increasing muscle mass. Methods: The therapy is based on nanoparticle-mediated delivery of follistatin messenger RNA (mRNA) to the liver after subcutaneous administration. The delivered mRNA directs hepatic cellular machinery to produce follistatin, a glycoprotein that increases lean mass through inhibition of negative regulators of muscle mass (myostatin and activin A). These factors are elevated in numerous disease states, thereby providing a target for therapeutic intervention. Results: Animal studies validated that mRNA-loaded nanoparticles enter systemic circulation following subcutaneous injection, accumulate and internalize in the liver, where the mRNA is translated into follistatin. Follistatin serum levels were elevated for 72 h post injection and efficiently reduced activin A and myostatin serum concentrations. After eight weeks of repeated injections, the lean mass of mice in the treatment group was ~10% higher when compared to that of the controls. Conclusion: Based on the obtained results demonstrating an increased muscle mass as well as restricted fat accumulation, this nanoplatform might be a milestone in the development of mRNA technologies and the treatment of muscle wasting disorders.

Background Cachexia is a complex metabolic and behavioural syndrome lacking effective therapies. Pancreatic ductal adenocarcinoma (PDAC) is one of the most important conditions associated with cachexia, with >80% of PDAC patients suffering from the condition. To establish the cardinal features of a murine model of PDAC-associated cachexia, we characterized the effects of implanting a pancreatic tumour cell line from a syngeneic C57BL/6 KRASG12D P53R172H Pdx-Cre+/+ (KPC) mouse. Methods Male and female C57BL/6 mice were inoculated subcutaneously, intraperitoneally, or orthotopically with KPC tumour cells. We performed rigorous phenotypic, metabolic, and behavioural analysis of animals over the course of tumour development. Results All routes of administration produced rapidly growing tumours histologically consistent with moderate to poorly differentiated PDAC. The phenotype of this model was dependent on route of administration, with orthotopic and intraperitoneal implantation inducing more severe cachexia than subcutaneous implantation. KPC tumour growth decreased food intake, decreased adiposity and lean body mass, and decreased locomotor activity. Muscle catabolism was observed in both skeletal and cardiac muscles, but the dominant catabolic pathway differed between these tissues. The wasting syndrome in this model was accompanied by hypothalamic inflammation, progressively decreasing brown and white adipose tissue uncoupling protein 1 (Ucp1) expression, and increased peripheral inflammation. Haematological and endocrine abnormalities included neutrophil-dominant leukocytosis and anaemia, and decreased serum testosterone. Conclusions Syngeneic KPC allografts are a robust model for studying cachexia, which recapitulate key features of the PDAC disease process and induce a wide array of cachexia manifestations. This model is therefore ideally suited for future studies exploring the physiological systems involved in cachexia and for preclinical studies of novel therapies.

Objective Appetitive responses to weight loss are mediated by a nutrient-sensing neural network comprised of melanocortin neurons. The role of neural melanocortin-3 receptors (MC3R) in mediating these responses is enigmatic. Mc3r knockout mice exhibit a paradoxical phenotype of obesity and reduced feeding-related behaviors in situations of nutrient scarcity. Here we examined whether MC3Rs expressed in mesolimbic neurons regulate feeding-related motivational responses. Methods Interactions between Mc3r genotype, cognitive function and energy balance on food self-administration were assessed using operant conditioning with fixed- and progressive ratio (FR1/PR1) settings. Inhibition of Mc3r transcription by a loxP-flanked transcriptional blocker (TB) in C57BL/6JN mice (Mc3rTB/TB) was reversed in mesolimbic neurons using DAT-Cre (DAT-MC3R). Results Caloric restriction (CR) caused 10–15% weight loss and increased motivation to acquire food rewards during training sessions. c-Fos-expression in the nucleus accumbens was increased 1 h following food presentation. While exhibiting weight loss, total food self-administration, enhanced motivation to self-administer food rewards in training sessions held during CR and c-Fos-activation in the nucleus accumbens following re-feeding were all markedly attenuated in Mc3rTB/TB mice. In contrast, cognitive abilities were normal in Mc3rTB/TB mice. Total food self-administration during FR1 sessions was not rescued in DAT-MC3R mice, however enhanced motivational responses to self-administer food rewards in PR1 conditions were restored. The nutrient-partitioning phenotype observed with Mc3r-deficiency was not rescued in DAT-MC3R mice. Conclusions Mesolimbic MC3Rs mediate enhanced motivational responses during CR. However, they are insufficient to restore normal caloric loading when food is presented during CR and do not affect metabolic conditions altering nutrient partitioning., Graphical abstract, Highlights • Food-related motivational responses in mice increase with caloric restriction (CR). • Melanocortin-3 receptors (MC3R) are required for food-related motivational responses. • MC3Rs role in food-related motivational responses depends on metabolic condition. • Mesolimbic MC3Rs increase food-related motivational responses during CR.

Peak bone mass, one of the most important predictors for fracture risk later in life, is attained during puberty and adolescence and influenced by neonatal and pubertal sex-specific gonadal hormones and GH-IGF-I secretion patterns. This study examined the effects of brief neonatal estrogen (NE) exposure on growth and skeletal development in C57BL/6J mice. A single injection of 100-μg estradiol or vehicle was administered on the first day of life. Growth parameters were monitored and skeletal phenotyping performed at 16 weeks in female mice and at 4 and 16 weeks in male mice. NE exposure negatively impacted adult femoral length in both sexes, but adult body weight, areal bone density, and bone strength in female mice were unaffected. In contrast, somatic growth was attenuated in estrogen-exposed male mice throughout the study period. At the prepubertal time point, the estrogen-exposed males exhibited higher bone mineral density, cortical volume, and cortical thickness compared with controls. However, by the time of peak bone mass acquisition, the early skeletal findings had reversed; estrogen-exposed mice had lower bone density with reduced cross-sectional area, cortical volume, and cortical thickness, resulting in cortical bones that were less resistant to fracture. NE exposure also resulted in reduced testicular volume and lower circulating IGF-I. Male mice exposed to estrogen on the first day of life experience age-dependent changes in skeletal development. Prepubertal animals experience greater endocortical bone acquisition as a result of estrogen exposure. However, by adulthood, continued developmental changes result in overall reduced skeletal integrity.

Melanocortin neurons conserve body mass in hyper- or hypo-caloric conditions by conveying signals from nutrient sensors into areas of the brain governing appetite and metabolism. In mice, melanocortin-3 receptor (MC3R) deletion alters nutrient partitioning independently of hyperphagia, promoting accumulation of fat over muscle mass. Enhanced rhythms in insulin and insulin-responsive metabolic genes during hypocaloric feeding suggest partial insulin resistance and enhanced lipogenesis. However, exactly where and how MC3Rs affect metabolic control to alter nutrient partitioning is not known. The behavioral phenotypes exhibited by MC3R-deficient mice suggest a contextual role in appetite control. Homeostatic responses to hypocaloric conditioning involving increased expression of appetite-stimulating (orexigenic) neuropeptides, binge-feeding, food anticipatory activity, entrainment to nutrient availability and enhanced feeding-related motivational responses are compromised with MC3R-deficiency. Rescuing Mc3r transcription in hypothalamic and limbic neurons improves appetitive responses during hypocaloric conditioning, suggesting orexigenic functions. Rescuing hypothalamic MC3Rs also restores responses of fasting-responsive hypothalamic orexigenic neurons in hypocaloric conditions, suggesting actions that sensitize fasting-responsive neurons to signals from nutrient sensors. MC3R signaling in ventromedial hypothalamic SF1(+ve) neurons improves metabolic control, but does not restore appetitive responses or nutrient partitioning. In summary, desensitization of fasting-responsive orexigenic neurons may underlie attenuated appetitive responses of MC3R-deficient mice in hypocaloric situations. The orexigenic actions of hypothalamic and limbic MC3Rs are contextual, and do not affect feeding behavior in ad libitum conditions or nutrient partitioning. Further studies are needed to identify the specific location(s) of MC3Rs controlling appetitive responses and partitioning of nutrients between fat and lean tissues.

Fatigue is the most common symptom related to cytotoxic chemotherapeutic treatment of cancer. Peripheral inflammation associated with cytotoxic chemotherapy is likely a causal factor of fatigue. The neural mechanisms by which cytotoxic chemotherapy associated inflammation induces fatigue behavior are not known. This lack of knowledge hinders development of interventions to reduce or prevent this disabling symptom. Infection induced fatigue/lethargy in rodents is mediated by suppression of hypothalamic orexin activity. Orexin is critical for maintaining wakefulness and motivated behavior. Though there are differences between infection and cytotoxic chemotherapy in some symptoms, both induce peripheral inflammation and fatigue. Based on these similarities we hypothesized that cytotoxic chemotherapy induces fatigue by disrupting orexin neuron activity. We found that a single dose of a cytotoxic chemotherapy cocktail (cyclophosphamide, adriamycin, 5-fluorouracil—CAF) induced fatigue/lethargy in mice and rats as evidenced by a significant decline in voluntary locomotor activity measured by telemetry. CAF induced inflammatory gene expression—IL-1R1 (p

Purpose/Objectives: The disease-associated wasting condition cachexia is a common complication of pancreatic ductal adenocarcinoma (PDAC) that impacts quality of life and portends poor survival. Undernutrition is a major driver of wasting in PDAC, yet cachexia remains refractory to nutritional supplementation. By modifying nutritional challenges at different stages of cachexia development, we sought to understand the relative contributions of undernutrition and metabolic reprogramming on adipose and skeletal muscle wasting. Materials/Methods: Adult mice received orthotopic PDAC tumor injections (KrasG12D; p53R172H/+; Pdx1-cre) or sham injections. Mice were fasted or food restricted at different times during tumor growth in a series of 2x2 factorial studies. Anthropometrics, voluntary wheel running activity, and body composition were measured throughout study. Blood glucose and ketones were measured by glucometer and ketometer. Ketogenic potential was assessed using octanoate challenge after fasting. Liver mRNA levels were measured using qPCR. Hepatic IL-6 signaling was assessed using qPCR and ELISA for plasma IL-6. Results: PDAC mice maintained food intake and wheel running for 8 days after tumor injection, before progressive decline. Loss of adipose mass in PDAC mice occurred only in the context of decreased nutrition, whereas loss of lean mass preceded decreases in food intake. Overnight fasting mitigated differences in fat mass between groups, whereas the effect of fasting on skeletal muscle loss was similar in PDAC and control mice. Fasting blood glucose and ketogenic potential were lower in PDAC mice, suggesting impaired hepatic response to metabolic stress. In the liver, glycolytic genes were increased, whereas gluconeogenic and ketogenic genes were decreased in PDAC mice. Wasting and hepatic reprogramming occurred independently of hepatic IL-6 signaling. Conclusions: Undernutrition drives PDAC-associated adipose wasting, yet muscle wasting occurs prior to changes in food intake and in the absence of systemic inflammation. Hepatic macronutrient partitioning and metabolic gene expression are altered in PDAC, implicating impaired adaptive responses to negative energy balance in PDAC-associated cachexia. Citation Format: Heike Mendez, Xinxia Zhu, Brennan Olsen, Daniel L. Marks, Aaron J. Grossberg. Impaired adaptation to negative energy balance in pancreatic cancer-associated wasting [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C19.

Adipose tissue inflammation is a cause of obesity-related metabolic disease. Natural killer (NK) cells are an understudied cell type in the context of obesity. The goal of this study was to determine the phenotype of human adipose tissue NK cells.We used flow cytometry phenotyping to study adipose tissue and peripheral blood NK cells from obese and lean humans.Human adipose tissue NK cells, relative to peripheral blood NK cells, express increased levels of activation markers. Adipose tissue NK cells also demonstrate an activated phenotype in obese relative to lean subjects, with increased expression of the activating receptor NKG2D.These data are the first detailed phenotypic characterization of human adipose tissue NK cells, and suggest a role for NK cells in adipose tissue inflammation in obesity.

Hyperglycemia worsens stroke, yet rigorous glycemic control does not improve neurologic outcome. An alternative is to target downstream molecular mediator(s) triggered by hyperglycemia but independent of prevailing glycemia. Soluble epoxide hydrolase (sEH) is a potential mediator of injury via its metabolism of neuroprotective epoxyeicosatrienoic acids (EETs). We tested whether hyperglycemia exacerbates cerebral injury by upregulating sEH and decreasing brain EET levels. Type 1 diabetes mellitus was modeled by streptozotocin (STZ;50 mg/kg per day intraperitoneally, 5 days) in male mice. At 4 weeks, STZ-treated and control mice underwent 45-minute middle cerebral artery occlusion (MCAO) with or without sEH blockade by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB;1 mg/kg intraperitoneally daily for 6 days before MCAO). The STZ-treated mice had increased sEH mRNA expression in cerebral vessels and decreased EET concentrations in brain. There was no difference in cortical perfusion between groups. The STZ-treated mice sustained larger brain infarct than controls. Pretreatment with t-AUCB eliminated the difference in infarct size and EETs concentration between STZ-treated mice and controls, without altering glycemia. We conclude that type 1 diabetes mellitus upregulates sEH mRNA and decreases concentrations of neuroprotective EETs within the brain, leading to worse stroke outcome. The data indicate that sEH antagonism may be beneficial in the setting of hyperglycemic stroke.

Cachexia is a wasting condition defined by skeletal muscle atrophy in the setting of systemic inflammation. To explore the site at which inflammatory mediators act to produce atrophy in vivo, we utilized mice with a conditional deletion of the inflammatory adaptor protein myeloid differentiation factor 88 (MyD88). Although whole-body MyD88-knockout (wbMyD88KO) mice resist skeletal muscle atrophy in response to LPS, muscle-specific deletion of MyD88 is not protective. Furthermore, selective reexpression of MyD88 in the muscle of wbMyD88KO mice via electroporation fails to restore atrophy gene induction by LPS. To evaluate the role of glucocorticoids as the inflammation-induced mediator of atrophy in vivo, we generated mice with targeted deletion of the glucocorticoid receptor in muscle (mGRKO mice). Muscle-specific deletion of the glucocorticoid receptor affords a 71% protection against LPS-induced atrophy compared to control animals. Furthermore, mGRKO mice exhibit 77% less skeletal muscle atrophy than control animals in response to tumor growth. These data demonstrate that glucocorticoids are a major determinant of inflammation-induced atrophy in vivo and play a critical role in the pathogenesis of endotoxemic and cancer cachexia.—Braun, T. P., Grossberg, A. J., Krasnow, S. M., Levasseur, P. R., Szumowski, M., Zhu, X. X., Maxson, J. E., Knoll, J. G., Barnes, A. P., and Marks, D. L. Cancer- and endotoxin-induced cachexia require intact glucocorticoid signaling in skeletal muscle.

Melanocortin-3 receptors (MC3R) have a contextual role in appetite control that is amplified with hypocaloric conditioning. C57BL/6J (B6) mice subjected to hypocaloric feeding schedules (HFS) exhibit compulsive behavioral responses involving food anticipatory activity (FAA) and caloric loading following food access. These homeostatic responses to calorie-poor environs are attenuated in B6 mice in which Mc3r transcription is suppressed by a lox-stop-lox sequence in the 5’UTR (Mc3rTB/TB ). Here, we report that optimization of caloric loading in B6 mice subject to HFS, characterized by increased meal size and duration, is not observed in Mc3rTB/TB mice. Analysis of hypothalamic and neuroendocrine responses to HFS throughout the light-dark cycle suggests uncoupling of hypothalamic responses involving appetite-stimulating fasting-responsive hypothalamic neurons expressing agouti-related peptide (AgRP) and neuropeptide Y (Npy). Rescuing Mc3rs expression in Nkx2.1(+ve) neurons is sufficient to restore normal hypothalamic responses to negative energy balance. In addition, Mc3rs expressed in Nkx2.1(+ve) neurons are also sufficient to restore FAA and caloric loading of B6 mice subjected to HFS. In summary, MC3Rs expressed in Nkx2.1(+ve) neurons are sufficient to coordinate hypothalamic response and expression of compulsive behavioral responses involving meal anticipation and consumption of large meals during situations of prolonged negative energy balance.

Leukemia inhibitory factor (LIF) has been suggested to function as a potent inhibitor of feed intake in rodents. In sheep, intravenous injection of lipopolysaccharide (LPS) resulted in an increase in gene expression for LIF in the arcuate nucleus ( < 0.01). In the same experiment, agouti related protein (AgRP) expression was elevated ( < 0.05) but there were no effects on proopiomelanocortin expression. Another group of sheep were provided intracerebroventricular (ICV) injections of LIF at 250, 500, 1,000, and 2,500 ng per sheep. Cumulative feed intake was inhibited by the 1,000- and 2,500-ng doses at 8 and 10 h after ICV injection ( < 0.03). All doses of LIF elevated temperature above 40°C, indicating a fever. When AgRP was intracerebroventricularly injected before LIF, there was no effect of LIF to reduce feed intake, suggesting the LIF inhibition of feed intake is consistent with the concept that the effect is mediated by the melanocortin-4 receptor. In an experiment to determine whether endocrine and metabolic effects of LIF were similar to reported effects of LPS, sheep were intracerebroventricularly injected with 2,500 ng LIF, and blood samples were collected at 10-min intervals for 6 h for assay of LH, samples from the first 3 h were assayed for GH, and samples at 30-min intervals were assayed for glucose and free fatty acids. The effect of treatment and treatment × time interaction was significant, indicating elevated plasma free fatty acids ( < 0.03 and < 0.001, respectively) and glucose ( < 0.01 and < 0.0001, respectively). There was also a treatment × time interaction on circulating concentrations of LH such that LIF caused LH to decrease ( < 0.0001). Additionally, there was a tendency for LIF treatment to increase circulating concentrations of GH (P = 0.0874). The effects of LIF on feed intake and other parameters was similar to the effects of LPS and leads to a hypothesis that LIF expression in response to LPS may be a component of the mechanism for feed intake inhibition and perhaps for changes in selected hormone and metabolites in disease models.

Animals exhibit a rapid and sustained anorexia when fed a diet that is deficient in a single indispensable amino acid (IAA). The chemosensor for IAA deficiency resides within the anterior piriform cortex (APC). Although the cellular and molecular mechanisms by which the APC detects IAA deficiency are well established, the efferent neural pathways that reduce feeding in response to an IAA-deficient diet remain to be fully characterized. In the present work, we investigated whether 1) central melanocortin signaling is involved in IAA deficiency-induced anorexia (IAADA) and 2) IAADA engages other key appetite-regulating neuronal populations in the hypothalamus. Rats and mice that consumed a valine-deficient diet (VDD) for 2–3 wk exhibited marked reductions in food intake, body weight, fat and lean body mass, body temperature, and white adipose tissue leptin gene expression, as well as a paradoxical increase in brown adipose tissue uncoupling protein-1 mRNA. Animals consuming the VDD had altered hypothalamic gene expression, typical of starvation. Pharmacological and genetic blockade of central melanocortin signaling failed to increase long-term food intake in this model. Chronic IAA deficiency was associated with a marked upregulation of corticotropin-releasing hormone expression in the lateral hypothalamus, particularly in the parasubthalamic nucleus, an area heavily innervated by efferent projections from the APC. Our observations indicate that the hypothalamic melanocortin system plays a minor role in acute, but not chronic, IAADA and suggest that the restraint on feeding is analogous to that observed after chronic dehydration.

Adipose tissue macrophages are important mediators of inflammation and insulin resistance in obesity. IFN-γ is a central regulator of macrophage function. The role of IFN-γ in regulating systemic inflammation and insulin resistance in obesity is unknown. We studied obese IFN-γ knockout mice to identify the role of IFN-γ in regulating inflammation and insulin sensitivity in obesity. IFN-γ-knockout C57Bl/6 mice and wild-type control litter mates were maintained on normal chow or a high fat diet for 13 weeks and then underwent insulin sensitivity testing then sacrifice and tissue collection. Flow cytometry, intracellular cytokine staining, and QRTPCR were used to define tissue lymphocyte phenotype and cytokine expression profiles. Adipocyte size was determined from whole adipose tissue explants examined under immunofluorescence microscopy. Diet-induced obesity induced systemic inflammation and insulin resistance, along with a pan-leukocyte adipose tissue infiltrate that includes macrophages, T-cells, and NK cells. Obese IFN-γ-knockout animals, compared with obese wild-type control animals, demonstrate modest improvements in insulin sensitivity, decreased adipocyte size, and an M2-shift in ATM phenotype and cytokine expression. These data suggest a role for IFN-γ in the regulation of inflammation and glucose homeostasis in obesity though multiple potential mechanisms, including effects on adipogenesis, cytokine expression, and macrophage phenotype.

Systemic and CNS-delimited inflammation triggers skeletal muscle catabolism in a manner dependent on glucocorticoid signaling., Skeletal muscle catabolism is a co-morbidity of many chronic diseases and is the result of systemic inflammation. Although direct inflammatory cytokine action on muscle promotes atrophy, nonmuscle sites of action for inflammatory mediators are less well described. We demonstrate that central nervous system (CNS)–delimited interleukin 1β (IL-1β) signaling alone can evoke a catabolic program in muscle, rapidly inducing atrophy. This effect is dependent on hypothalamic–pituitary–adrenal (HPA) axis activation, as CNS IL-1β–induced atrophy is abrogated by adrenalectomy. Furthermore, we identified a glucocorticoid-responsive gene expression pattern conserved in models of acute and chronic inflammatory muscle atrophy. In contrast with studies suggesting that the direct action of inflammatory cytokines on muscle is sufficient to induce catabolism, adrenalectomy also blocks the atrophy program in response to systemic inflammation, demonstrating that glucocorticoids are requisite for this process. Additionally, circulating levels of glucocorticoids equivalent to those produced under inflammatory conditions are sufficient to cause profound muscle wasting. Together, these data suggest that a significant component of inflammation-induced muscle catabolism occurs indirectly via a relay in the CNS.

The melanocortin-3 receptor (MC3R) gene is pleiotropic, influencing body composition, natriuresis, immune function, and entrainment of circadian rhythms to nutrient intake. MC3Rs are expressed in hypothalamic and limbic regions of the brain and in peripheral tissues. To investigate the roles of central MC3Rs, we inserted a "lox-stop-lox" (LoxTB) 5' of the translation initiation codon of the mouse Mc3r gene and reactivated transcription using neuron-specific Cre transgenic mice. As predicted based on earlier observations of Mc3r knock-out mice, Mc3r(TB/TB) mice displayed reduced lean mass, increased fat mass, and accelerated diet-induced obesity. Surprisingly, rescuing Mc3r expression in the nervous system using the Nestin-Cre transgene only partially rescued obesity in chow-fed conditions and had no impact on the accelerated diet-induced obesity phenotype. The ventromedial hypothalamus (VMH), a critical node in the neural networks regulating feeding-related behaviors and metabolic homeostasis, exhibits dense Mc3r expression relative to other brain regions. To target VMH MC3R expression, we used the steroidogenic factor-1 Cre transgenic mouse. Although restoring VMH MC3R signaling also had a modest impact on obesity, marked improvements in metabolic homeostasis were observed. VMH MC3R signaling was not sufficient to rescue the lean mass phenotype or the regulation of behaviors anticipating food anticipation. These results suggest that actions of MC3Rs impacting on energy homeostasis involve both central and peripheral sites of action. The impact of central MC3Rs on behavior and metabolism involves divergent pathways; VMH MC3R signaling improves metabolic homeostasis but does not significantly impact on the expression of behaviors anticipating nutrient availability.

BACKGROUND: Under physiological conditions, the melanocortin system is a crucial part of the complex network regulating food intake and energy expenditure. In pathological states, like cachexia, these two parameters are deregulated, i.e., food intake is decreased and energy expenditure is increased-a vicious combination leading to catabolism. Agouti-related protein (AgRP), the endogenous antagonist at the melanocortin-4 receptor (MC-4R), was found to increase food intake and to reduce energy expenditure. This qualifies MC-4R blockade as an attractive mode of action for the treatment of cachexia. Based on this rationale, a novel series of small-molecule MC-4R antagonists was designed, from which the orally active compound BL-6020/979 (formerly known as SNT207979) emerged as the first promising development candidate showing encouraging pre-clinical efficacy and safety properties which are presented here. METHODS AND RESULTS: BL-6020/979 is an orally available, selective and potent MC-4R antagonist with a drug-like profile. It increased food intake and decreased energy expenditure in healthy wild-type but not in MC-4R deficient mice. More importantly, it ameliorated cachexia-like symptoms in the murine C26 adenocarcinoma model; with an effect on body mass and body composition and on the expression of catabolic genes. Moreover, BL-6020/979 showed antidepressant-like properties in the chronic mild stress model in rats and exhibits a favorable safety profile. CONCLUSION: The properties of BL-6020/979 demonstrated in animal models and presented here make it a promising candidate suitable for further development towards a first-in-class treatment option for cachexia that potentially opens up the opportunity to treat two hallmarks of the disease, i.e., decreased food intake and increased energy expenditure, with one drug.

Exposure to chronic and acute oxidative stress is correlated with many human diseases, including, but not limited to, cancer, heart disease, diabetes, and obesity. In addition to cellular lipids and proteins, cellular oxidative stress can result in damage to DNA bases, especially in mitochondrial DNA. We previously described the development of spontaneous late-onset obesity, hepatic steatosis, hyperinsulinemia, and hyperleptinemia in mice that are deficient in the DNA glycosylase nei-like 1 (NEIL1), which initiates base excision repair of several oxidatively damaged bases. In the current study, we report that exposure to a chronic oxidative stress in the form of a high-fat diet greatly accelerates the development of obesity in neil1−/−mice. Following a 5-wk high-fat diet challenge, neil1−/−mice gained significantly more body weight than neil1+/+littermates and had increased body fat accumulation and moderate to severe hepatic steatosis. Analysis of oxygen consumption by indirect calorimetry indicated a modest reduction in total oxygen consumption in neil1−/−mice that was abolished upon correction for lean body mass. Additionally, hepatic expression of several inflammatory genes was significantly upregulated in neil1−/−mice following high-fat diet challenge compared with chow-fed or neil1+/+counterparts. A long-term high-fat diet also induced glucose intolerance as well as a significant reduction in mitochondrial DNA and protein content in neil1−/−mice. Collectively, these data indicate that NEIL1 deficiency results in an increased susceptibility to obesity and related complications potentially by lowering the threshold for tolerance of cellular oxidative stress in neil1−/−mice.

Decreased appetite and involuntary weight loss are common occurrences in chronic disease and have a negative impact on both quality of life and eventual mortality. Weight loss in chronic disease comes from both fat and lean mass, and is known as cachexia. Both alterations in appetite and body weight loss occur in a wide variety of diseases, including cancer, heart failure, renal failure, chronic obstructive pulmonary disease and HIV. An increase in circulating inflammatory cytokines has been implicated as a uniting pathogenic mechanism of cachexia and associated anorexia. One of the targets of inflammatory mediators is the central nervous system, and in particular feeding centers in the hypothalamus located in the ventral diencephalon. Current research has begun to elucidate the mechanisms by which inflammation reaches the hypothalamus, and the neural substrates underlying inflammatory anorexia. Research into these neural mechanisms has suggested new therapeutic possibilities, which have produced promising results in preclinical and clinical trials. This review will discuss inflammatory signaling in the hypothalamus that mediates anorexia, and the opportunities for therapeutic intervention that these mechanisms present.

The central melanocortin system plays a key role in the regulation of food intake and energy homeostasis. We investigated whether genetic or pharmacologic blockade of central melanocortin signaling attenuates cardiac cachexia in mice and rats with heart failure. Permanent ligation of the left coronary artery (myocardial infarction (MI)) or sham operation was performed in wild-type (WT) or melanocortin-4 receptor (MC4R) knockout mice. Eight weeks after surgery, WT-Sham mice had significant increases in lean body mass (LBM; PPPPPPP

The hypothalamic melanocortin system, which controls appetite and energy expenditure, develops during the third trimester in primates. Thus, maternal nutrition and health may have a profound influence on the development of this system. To study the effects of chronic maternal high-fat diet (HFD) on the development of the melanocortin system in the fetal nonhuman primate, we placed adult female macaques on either a control (CTR) diet or a HFD for up to 4 yr. A subgroup of adult female HFD animals was also switched to CTR diet during the fifth year of the study (diet reversal). Third-trimester fetuses from mothers on HFD showed increases in proopiomelanocortin mRNA expression, whereas agouti-related protein mRNA and peptide levels were decreased in comparison with CTR fetuses. Proinflammatory cytokines, including IL-1β and IL-1 type 1 receptor, and markers of activated microglia were elevated in the hypothalamus, suggesting an activation of the local inflammatory response. Fetuses of diet-reversal mothers had normal melanocortin levels. These results raise the concern that chronic consumption of a HFD during pregnancy, independent of maternal obesity and diabetes, can lead to widespread activation of proinflammatory cytokines that may alter the development of the melanocortin system. The abnormalities in the fetal POMC system, if maintained into the postnatal period, could impact several systems, including body weight homeostasis, stress responses, and cardiovascular function. Indeed, the HFD offspring develop early-onset excess weight gain. These abnormalities may be prevented by healthful nutrient consumption during pregnancy even in obese and severely insulin-resistant individuals.

Background Pediatric acute lymphoblastic leukemia (ALL) therapies have been associated with many late effects, including obesity, hyperglycemia, and insulin resistance. Few data are available linking these abnormalities to specific risk factors present during ALL treatment. Methods Retrospective cohort study with prospective follow-up. Subjects had been diagnosed with ALL at ages 1–18 years and had been off chemotherapy for >9 months. Oral glucose tolerance testing (OGTT) was performed and these results compared to demographic, treatment, and anthropomorphic data from medical records. Results Twenty-seven subjects (11 female) were evaluated. Mean (±SD) diagnosis age 5.7 ± 3.5 years, mean study age 11.3 ± 3.7 years, mean time off therapy 2.8 ± 1.5 years. Six subjects had transient hyperglycemia during ALL treatment. At study time, one subject had prediabetes; eight (29.6%) had insulin resistance. Insulin resistance was not predicted by glucose levels during treatment, cumulative steroid or asparaginase dose, or type of steroid received. Body mass index (BMI) for age correlated significantly with several measures of insulin resistance, including fasting insulin, HOMA index, Matsuda index and insulin AUC (P = 0.001–0.009). Waist/hip ratio and BMI at ALL diagnosis also correlated with insulin resistance, but these factors' effects could not be separated from BMI at study time. Conclusions Variations in ALL therapy and presence of transient hyperglycemia do not appear to increase risk of glucose intolerance or insulin resistance in the first few years after completion of therapy. Elevated BMI strongly predicted insulin resistance in this study, as it does in the general population. Pediatr Blood Cancer 2009;53:58–63. © 2009 Wiley-Liss, Inc.

Inflammation-associated cachexia is associated with multiple chronic diseases and involves activation of appetite regulating centers in the arcuate nucleus of the hypothalamus (ARH). The nucleus of the solitary tract (NTS) in the brainstem has also been implicated as an important nucleus involved in appetite regulation. We set out to determine whether the NTS may be involved in inflammation-associated anorexia by injecting IL-1 beta into the 4th ventricle and assessing food intake and NTS neuronal activation. Injection of IL-1 beta produced a decrease in food intake at 3 and 12h after injection which was ameliorated at the 12h time point by a sub-threshold dose of agouti-related peptide (AgRP). Investigation into neuron types in the NTS revealed that IL-1 beta injection was associated with an increase in c-Fos activity in NTS neurons expressing tyrosine hydroxylase (TH). Additionally, injection of IL-1 beta into the 4th ventricle did not produce c-Fos activation of neurons expressing pro-opiomelanocortin (POMC) in the ARH, cells known to be involved in producing anorexia in response to systemic inflammation. Double-label in situ hybridization revealed that TH neurons did not express IL-1 receptor I (IL1-RI) transcript, demonstrating that c-Fos activation of TH neurons in this setting was not via direct stimulation of IL-1 beta on TH neurons themselves. We conclude that IL-1 beta injection into the 4th ventricle produces anorexia and is accompanied by an increase in activation in TH neurons in the NTS. This provides evidence that the brainstem may be an important mediator of anorexia in the setting of inflammation.

Melanocortin-4 receptors (MC4R) are key factors in the depression of appetite during disease. This study was designed to determine the role of agouti-related protein (AgRP) in the effect of endotoxin (lipopolysaccharide, LPS) on appetite. Sheep received an intracerebroventricular injection of either saline or AgRP (0.5 nmol/kg of BW) 1 h before intravenous injection of either saline or LPS (0.6 microg/kg of BW) at time 0 and again at 4 h. Agouti-related protein prevented the reduction in feed intake due to LPS (P < 0.05). In a second experiment, AgRP gene expression was unaffected at 3 h and increased (P < 0.01) at 6 h after LPS. Immunohistochemical evidence indicated that there was an increase in the percentage of AgRP neurons with c-Fos immunoreactive nuclei 6 h after sheep were injected with LPS (P < 0.04) and a corresponding decrease in a-melanocyte-stimulating hormone neurons coexpressing c-Fos (P < 0.001). In situ hybridization provided evidence for an increase in AgRP gene expression and a decrease in proopiomelanocortin gene expression 6 h after LPS (P < 0.05). In a final experiment, physiological elevation of orexigenic agents by short-term fasting kept feed intake at the same level as controls, in spite of the presence of LPS, similar to the effects of AgRP in Exp. 1. The AgRP inhibition of the MC4R prevents appetite inhibition in response to LPS and well after LPS inhibition of feed intake, both AgRP and a-melanocyte-stimulating hormone may change in a pattern that favors appetite increases. These studies support the notion of the MC4R as a critical component of the mechanism for appetite suppression due to endotoxin.

Agouti-related protein (AgRP) is an orexigenic neuropeptide produced by neurons in the hypothalamic arcuate nucleus (ARC) that is a key component of central neural circuits that control food intake and energy expenditure. Disorders in energy homeostasis, characterized by hypophagia and increased metabolic rate, frequently develop in animals with either acute or chronic diseases. Recently, studies have demonstrated that proopiomelanocortin-expressing neurons in the ARC are activated by the proinflammatory cytokine IL-1beta. In the current study, we sought to determine whether inflammatory processes regulate the expression of AgRP mRNA and to characterize the response of AgRP neurons to IL-1beta. Here, we show by real-time RT-PCR and in situ hybridization analysis that AgRP mRNA expression in rodents is increased in models of acute and chronic inflammation. AgRP neurons were found to express the type I IL-1 receptor, and the percentage of expression was significantly increased after peripheral administration of lipopolysaccharide. Furthermore, we demonstrate that IL-1beta inhibits the release of AgRP from hypothalamic explants. Collectively, these data indicate that proinflammatory signals decrease the secretion of AgRP while increasing the transcription of the AgRP gene. These observations suggest that AgRP neurons may participate with ARC proopiomelanocortin neurons in mediating the anorexic and metabolic responses to acute and chronic disease processes.

Background Steroid-induced sleep disturbance is a common and highly distressing morbidity for children receiving steroid chemotherapy for the treatment of pediatric acute lymphoblastic leukemia (ALL). Sleep disturbance can negatively impact overall quality of life, neurodevelopment, memory consolidation, and wound healing. Hypothalamic orexin neurons are influential wake-promoting neurons, and disturbances in orexin signaling leads to abnormal sleep behavior. A new class of drug, the orexin receptor antagonists, could be an intriguing option for sleep disorders caused by increased orexinergic output. Our aim was to examine the impact of ALL treatment doses of corticosteroids on the orexin system in rodents and in children undergoing treatment for childhood ALL. Methods We administered repeated injections of dexamethasone to rodents and measured responsive orexin neural activity compared to controls. In children with newly diagnosed standard risk B-cell ALL receiving dexamethasone therapy per Children’s Oncology Group (COG) induction therapy from 2014–2016, we collected pre- and during-steroids matched CSF samples and measured the impact of steroids on CSF orexin concentration. Results In both rodents, all markers orexin signaling, including orexin neural output and orexin receptor expression, were preserved in the setting of dexamethasone. Additionally, we did not detect a difference in pre- and during-dexamethasone CSF orexin concentrations in children receiving dexamethasone. Conclusions Our results demonstrate that rodent and human orexin physiology is largely preserved in the setting of high dose dexamethasone. The data obtained in our experimental model fail to demonstrate a causative role for disruption of the orexin pathway in steroid-induced sleep disturbance.

Metabolic syndrome, a complex of highly debilitating disorders that includes insulin resistance, hypertension, and dyslipidemia, is associated with the development of obesity in humans as well as rodent models. White adipose tissue (WAT) inflammation, caused in part by macrophage infiltration, and fat accumulation in the liver are both linked to development of the metabolic syndrome. Despite large increases in body fat, melanocortin 3-receptor (MC3-R)-deficient mice do not get fatty liver disease or severe insulin resistance. This is in contrast to obese melanocortin 4-receptor (MC4-R)-deficient mice and diet-induced obese (DIO) mice, which show increased adiposity, fatty liver disease, and insulin resistance. We hypothesized that defects in the inflammatory response to obesity may underlie the protection from metabolic syndrome seen in MC3-R null mice. MC4-R mice fed a chow diet show increased proinflammatory gene expression and macrophage infiltration in WAT, as do wild-type (WT) DIO mice. In contrast, MC3-R-deficient mice fed a normal chow diet show neither of these inflammatory changes, despite their elevated adiposity and a comparable degree of adipocyte hypertrophy to the MC4-R null and DIO mice. Furthermore, even when challenged with high-fat chow for 4 wk, a period of time shown to induce an inflammatory response in WAT of WT animals, MC3-R nulls showed an attenuated up-regulation in both monocyte chemoattractant protein-1 (MCP-1) and TNFα mRNA in WAT compared with WT high-fat-fed animals.

Chronic kidney disease (CKD) is associated with an increase in inflammatory cytokines and can result in cachexia with loss of muscle and fat stores. We previously demonstrated the efficacy of treating a model of cancer cachexia with ghrelin and a ghrelin receptor agonist. Currently, we examine a surgical model of CKD in rats, resulting in uremia and decreased accrual of lean body mass. Treatment with ghrelin and two ghrelin receptor agonists (BIM-28125 and BIM-28131) resulted in increased food intake and an improvement in lean body mass accrual that was related in part to a decrease in muscle protein degradation as assessed by muscle levels of the 14-kDa actin fragment resulting from cleaved actomyosin. Additionally, there was a decrease in circulating inflammatory cytokines in nephrectomized animals treated with ghrelin relative to saline treatment. Ghrelin-treated animals also had a decrease in the expression of IL-1 receptor in the brainstem and a decrease in expression of prohormone convertase-2, an enzyme involved in the processing of proopiomelanocortin to the anorexigenic peptide α-MSH. We conclude that ghrelin treatment in uremia results in improved lean mass accrual in part due to suppressed muscle proteolysis and possibly related to antiinflammatory effects.

Anorexia and involuntary weight loss are common and debilitating complications of a number of chronic diseases and inflammatory states. Proinflammatory cytokines, including IL-1 beta, are hypothesized to mediate these responses through direct actions on the central nervous system. However, the neural circuits through which proinflammatory cytokines regulate food intake and energy balance remain to be characterized. Here we report that IL-1 beta activates the central melanocortin system, a key neuronal circuit in the regulation of energy homeostasis. Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) were found to express the type I IL-1 receptor. Intracerebroventricular injection of IL-1 beta induced the expression of Fos protein in ARC POMC neurons but not in POMC neurons in the commissural nucleus of the tractus solitarius. We further show that IL-1 beta increases the frequency of action potentials of ARC POMC neurons and stimulates the release of alpha-MSH from hypothalamic explants in a dose-dependent fashion. Collectively, our data support a model in which IL-1 beta increases central melanocortin signaling by activating a subpopulation of hypothalamic POMC neurons and stimulating their release of alpha-MSH.

Some animals and humans fed a high-energy diet (HED) are diet-resistant (DR), remaining as lean as individuals who were naive to HED. Other individuals become obese during HED exposure and subsequently defend the obese weight (Diet-Induced Obesity- Defenders, DIO-D) even when subsequently maintained on a low-energy diet. We hypothesized that the body weight setpoint of the DIO-D phenotype resides in the hypothalamic paraventricular nucleus (PVN), where anorexigenic melanocortins, including melanotan II (MTII), increase presynaptic GABA release, and the orexigenic neuropeptide Y (NPY) inhibits it. After prolonged return to low-energy diet, GABA inputs to PVN neurons from DIO-D rats exhibited highly attenuated responses to MTII compared with those from DR and HED-naive rats. In DIO-D rats, melanocortin-4 receptor expression was significantly reduced in dorsomedial hypothalamus, a major source of GABA input to PVN. Unlike melanocortin responses, NPY actions in PVN of DIO-D rats were unchanged, but were reduced in neurons of the ventromedial hypothalamic nucleus; in PVN of DR rats, NPY responses were paradoxically increased. MTII-sensitivity was restored in DIO-D rats by several weeks’ refeeding with HED. The loss of melanocortin sensitivity restricted to PVN of DIO-D animals, and its restoration upon prolonged refeeding with HED suggest that their melanocortin systems retain the ability to up- and downregulate around their elevated body weight setpoint in response to longer-term changes in dietary energy density. These properties are consistent with a mechanism of body weight setpoint.

Many chronic diseases produce a condition called cachexia, which involves anorexia, decreased lean body mass and increased energy expenditure, worsening the prognosis for the underlying disease. This article describes a promising new approach to treating cachexia, targeting a key appetite-regulating center in the hypothalamus called the melanocortin system. Cachexia is a process that accompanies many chronic diseases, and consists of a combination of wasting of lean body mass, increased energy expenditure, and a paradoxical loss of appetite. Cachexia both worsens quality of life and negatively affects treatment of the underlying disease. Conditions as diverse as cancer, renal failure, and heart failure show a remarkable similarity in their associated cachexia, exhibiting changes in metabolism and endocrinology, including marked increases in levels of cytokines that accompany these diseases. So far, it has been difficult to treat disease-associated cachexia successfully. One treatment that has shown promise in animal trials, however, involves antagonism of the central melanocortin system, an anorexigenic pathway in the hypothalamus and brainstem. Humans who have genetic mutations involving pro-opiomelanocortin or the melanocortin 4 receptor in this pathway exhibit increased appetite and increased lean body mass. Recent research has shown that in rodent models of cancer and renal failure, administration of melanocortin 4 receptor antagonists results in an attenuation of symptoms of cachexia, including maintenance of appetite, lean body mass, and basal energy expenditure. Although this research needs to be substantiated in humans, it provides a promising direction for treating the wasting that is associated with a variety of disease states.

Leptin is an adipocyte-derived hormone that acts as a major regulator of food intake and energy homeostasis. It circulates both as a free and as a protein-bound entity. Leptin is released into the blood in proportion to the amount of body fat and exerts sustained inhibitory effects on food intake while increasing energy expenditure. The leptin receptor belongs to the class I cytokine receptor superfamily and possesses strong homology to the signal-transducing subunits of the IL-6 receptor. The hypothalamic melanocortin system, and specifically the melanocortin-4 receptor (MC-4R), is critical in mediating leptin's effect on appetite and metabolism. Serum leptin concentrations are elevated in patients with chronic kidney disease (CKD) and correlate with C-reactive protein levels suggesting that inflammation is an important factor that contributes to hyperleptinemia in CKD. Hyperleptinemia may be important in the pathogenesis of inflammation-associated cachexia in CKD. We showed that experimental uremic cachexia was attenuated in db/db mice, a model of leptin receptor deficiency. Nephrectomy in these animals did not result in any change in weight gain, body composition, resting metabolic rate, and efficiency of food consumption. Furthermore, experimental uremic cachexia could be ameliorated by blocking leptin signaling through the hypothalamic MC-4R. MC-4R knockout mice or mice administered the MC-4R and MC-3R antagonist, agouti-related peptide, resisted uremia-induced loss of lean body mass and maintained normal basal metabolic rates. Thus, melanocortin receptor antagonism may provide a novel therapeutic strategy for inflammation-associated cachexia in CKD.

High levels of binding sites for melanocortin peptides exist within the arcuate nucleus, and a functional response to melanocortin peptides has been demonstrated in arcuate POMC neurons. Because the MC3R is thought to function as an inhibitory autoreceptor on POMC neurons, we reasoned that peripheral injections of MC3R-specific agonists would act within the arcuate nucleus to inhibit POMC neurons and thereby stimulate feeding. We demonstrate that the peptidergic MC3R agonist, d-Trp8-γ-MSH, stimulates feeding via the MC3R when injected peripherally. These data provide the first evidence that feeding can be stimulated by peripheral injection of MC3R-specific agonists.

The pathogenesis of cachexia in patients with uremia is unknown. We tested the hypothesis that uremia-associated cachexia is caused by leptin signaling through the hypothalamic melanocortin receptor 4 (MC4-R). We performed either subtotal nephrectomy (N) or sham operations in WT, leptin receptor-deficient (db/db), and MC4-R knockout (MC4-RKO) mice. The animals were on 17% protein diets, and none of the uremic animals were acidotic. WT-N mice produced a classic syndrome of cachexia characterized by decreased food intake, increased metabolic rate, and loss of lean body mass. Corrected leptin levels were elevated. db/db mice and MC4-RKO mice resisted the cachexic effects of uremia on weight gain, body composition, and metabolic rate. Likewise, treatment of WT mice with intracranial agouti-related peptide reversed the cachexic effects of uremia on appetite, weight gain, body composition, and metabolic rate. Gene expression of ubiquitin C and proteasome subunits C2, C3, and C9 was not changed in the uremic animals, suggesting that other pathways are involved in this model of nonacidotic uremic cachexia. The results of this study suggest that elevated circulating levels of cytokines such as leptin may be an important cause of uremia-associated cachexia via signaling through the central melanocortin system.

Cancer anorexia-cachexia syndrome is observed in 80% of patients in the advanced stages of cancer and is a strong independent risk factor for mortality. Numerous cytokines produced by tumor and immune cells, interacting with the neuropeptidergic system, mediate the cachectic effect of cancer. Since there is currently no effective pharmacological treatment and the anorexia-cachexia syndrome continues to be defined biochemically, we review the role of cytokines and neuropeptides in this process.Currently data suggest that cancer anorexia-cachexia syndrome results from a multifactorial process involving many mediators, including hormones (e.g. leptin), neuropeptides (e.g. neuropeptide Y, melanocortin, melanin-concentrating hormone and orexin) and cytokines (e.g. interleukin 1, interleukin 6, tumor necrosis factor alpha and interferon gamma). It is likely that close interrelation among these mediators exists in the hypothalamus, decreasing food intake and leading to cachexia.In the pathogenesis of cancer anorexia, cytokines play a pivotal role influencing the imbalance of orexigenic and anorexigenic circuits that regulate the homeostatic loop of body-weight regulation, leading to cachexia. Interfering pharmacologically with cytokine expression or neural transduction of cytokine signals can be an effective therapeutic strategy in anorectic patients before they develop cancer anorexia-cachexia syndrome.

A role for melanocortin signaling in the regulation of body weight in humans has been clearly established. Haploinsufficiency of the type 4 melanocortin receptor is associated with early-onset obesity, implying that this receptor provides an important tonic inhibition of weight gain. Agouti-related peptide (AGRP) is an endogenous antagonist of melanocortin signaling. Therefore, loss of AGRP function could lead to the expression of a lean phenotype. We investigated the potential role of AGRP in human weight regulation by examining the association between the Ala67Thr AGRP polymorphism and indices of body composition. Significant associations were found between homozygosity for this mutation (n = 8) and body composition phenotype in 874 subjects of the Quebec family study (QFS). By PCR-RFLP analysis, we have identified eight individuals who are homozygous for the 67Thr variant allele within the QFS population, where none were observed in SAFHS. The eight QFS homozygote individuals have lower weight (−16%; P = 0.02), body mass index (−17%; P = 0.01), fat free mass (−9%; P = 0.002), fat mass (FM) (−20%; P = 0.04), and leptin (−20%; P = 0.02) when compared to those carrying at least one 67Ala allele. Individuals homozygous for the 67Thr allele had a BMI that was either at or slightly below an ideal range for their age. Thus, the Ala67Thr AGRP polymorphism is associated with lower body weight in humans, with the largest effect being observed on body FM. We did not observe any difference in the stability or cellular distribution of the mutant protein in a heterologous expression system, thus the mechanism of this effect requires further investigation. © 2004 Wiley-Liss, Inc.

Knowledge of specific neurotransmitters as well as the pathways and mechanisms regulating appetite in ruminants that continually graze, such as sheep, is incomplete. Although fundamentally agouti-related protein (AGRP) has a similar function across species to increase food intake, the regulation of AGRP may vary across grazing and intermittent feeders. To investigate the role of orexigenic peptides in the regulation of feed intake, we first extracted messenger RNA from sheep that were fasted for 3 days, which was then used for PCR followed by cloning and sequencing to demonstrate the presence of hypothalamic AGRP expression. Ovine AGRP was closely related to the bovine, but contained sequence differences with human and mouse AGRP. Analysis of genomic DNA also revealed a similar gene structure to other published species. Secondly, using dual-labeled immunohistochemistry, we determined that there was both increased AGRP immunoreactivity and increased abundance of c-Fos immunoreactivity in AGRP neurons in the arcuate nucleus of fasted sheep. Because AGRP neurons are activated by fasting, we hypothesized that AGRP would stimulate feeding in this ruminant species. Sheep fed ad libitum were injected intracerebroventricularly with concentrations of AGRP at 0.2 and 2.0 nmol/kg. AGRP at 2.0 nmol/kg significantly increased food intake at 4, 6 and 12 h (p < 0.05). A 4th study was done to investigate the interactions of AGRP and neuropeptide Y (NPY) on food intake over a 24-hour period. Intracerebroventricular injections of either AGRP or NPY significantly increased cumulative food intake over saline controls. When AGRP and NPY were injected in combination, food intake was increased over saline controls; however, AGRP did not potentiate the effects of NPY. These results demonstrate that AGRP stimulates food intake in sheep and highlights the important differences between this species and rodent models.

Anorexia is a common symptom in chronic illness. It contributes to malnutrition and strongly affects survival and quality of life. A common denominator of many chronic diseases is an elevated inflammatory status, which is considered to play a pivotal role in the failure of food-intake regulating systems in the hypothalamus. In this review, we summarize findings on the role of hypothalamic inflammation on food intake regulation involving hypothalamic neuropeptide Y (NPY) and pro-opiomelanocortin (POMC). Furthermore, we outline the role of serotonin in the inability of these peptide based food-intake regulating systems to respond and adapt to changes in energy metabolism during chronic disease.

Glucocorticoids are highly conserved fundamental regulators of energy homeostasis. In response to stress in the form of perceived danger or acute inflammation, glucocorticoids are released from the adrenal gland, rapidly mobilizing energy from carbohydrate, fat and protein stores. In the case of inflammation, mobilized protein is critical for the rapid synthesis of acute phase reactants and an efficient immune response to infection. While adaptive in response to infection, chronic mobilization can lead to a p rofound depletion of energy stores. Skeletal muscle represents the major body store of protein, and can become substantially atrophied under conditions of chronic inflammation. Glucocorticoids elicit the atrophy of muscle by increasing the rate of protein degradation by the ubiquitin-proteasome system and autophagy lysosome system. Protein synthesis is also suppressed at the level of translational initiation, preventing the production of new myofibrillar protein. Glucocorticoids also antagonize the action of anabolic regulators such as insulin further exacerbating the loss of protein and muscle mass. The loss of muscle mass in the context of chronic disease is a key feature of cachexia and contributes substantially to morbidity and mortality. A growing body of evidence demonstrates that glucocorticoid signaling is a common mediator of wasting, irrespective of the underlying initiator or disease state. This review will highlight fundamental mechanisms of glucocorticoid signaling and detail the mechanisms of glucocorticoid-induced muscle atrophy. Additionally, the evidence for glucocorticoids as a driver of muscle wasting in numerous disease states will be discussed. Given the burden of wasting diseases and the nodal nature of glucocorticoid signaling, effective anti-glucocorticoid therapy would be a valuable clinical tool. Therefore, the progress and potential pitfalls in the development of glucocorticoid antagonists for muscle wasting will be discussed.

Cancer cachexia is a syndrome of weight loss that results from the selective depletion of skeletal muscle mass and contributes significantly to cancer morbidity and mortality. The driver of skeletal muscle atrophy in cancer cachexia is systemic inflammation arising from both the cancer and cancer treatment. While the importance of tumor derived inflammation is well described, the mechanism by which cytotoxic chemotherapy contributes to cancer cachexia is relatively unexplored. We found that the administration of chemotherapy to mice produces a rapid inflammatory response. This drives activation of the hypothalamic-pituitary-adrenal axis, which increases the circulating level of corticosterone, the predominant endogenous glucocorticoid in rodents. Additionally, chemotherapy administration results in a significant loss of skeletal muscle mass 18 hours after administration with a concurrent induction of genes involved with the ubiquitin proteasome and autophagy lysosome systems. However, in mice lacking glucocorticoid receptor expression in skeletal muscle, chemotherapy-induced muscle atrophy is completely blocked. This demonstrates that cytotoxic chemotherapy elicits significant muscle atrophy driven by the production of endogenous glucocorticoids. Further, it argues that pharmacotherapy targeting the glucocorticoid receptor, given in concert with chemotherapy, is a viable therapeutic strategy in the treatment of cancer cachexia.

It has been known for decades that renal failure, and particularly end-stage renal disease, is a catabolic state often associated with anorexia and wasting ([1][1]). The problem of identifying the substance(s) responsible for anorexia, wasting, and cachexia of uremic patients with and without

On the day of proestrus in the rat, rising plasma levels of estradiol (E) act in concert with progesterone (P) to trigger a preovulatoryrelease of gonadotropins. Cellular levels of galanin mRNA in GnRH neurons are increased in association with the proestrous surge ofgonadotropin secretion; however, the relative contribution made by E and P to the induction of galanin mRNA expression in GnRH neuronsis unknown. We investigated the role of E and P in the induction of galanin gene expression in GnRH neurons by examining the effectsof different combinations of E (estradiol benzoate; 50 mg and P (5 mg)) on the LH surge and the concomitant induction of galanin mRNAin GnRH neurons. We sacrificed ovariectomized adult rats after 1 of 4 treatments: Group 1: vehicle control (n=6); Group 2: P alone (n=7) Group 3: E alone (n=7); Group 4: combined E/P (n=6); the animals were killed at 18.00 h at the time of the LH surge. The brains fromthese animals were processed by double-label in situ hybridization to allow measurement of galanin mRNA levels in GnRH neurons. GnRHneurons were identified with a digoxigenin-labeled cRNA probe for GnRH mRNA, and galanin mRNA was detected and measuredsimultaneously with an 35S-labeled cRNA probe coupled with computerized grain counting. Estimation of cellular levels of GnRH mRNAwas accomplished with single-label in situ hybridization, an 35S-labeled GnRH cRNA probe and computerized grain counting. We observeda 3-fold induction of galanin mRNA in the GnRH neurons of animals treated with E alone compared with those treated with the vehiclealone (vehicle: 13±2 vs E: 42±4 grains/cell (g/c); P

Galanin mRNA levels in GnRH neurons increase in association with a steroid-induced LH surge in female rats. Both the steroid-induced LH surge and the concomitant increase of galanin mRNA in GnRH neurons are blocked by non-specific inhibition of central nervous system activity imposed by pentobarbital and specific central alpha-adrenergic receptor blockade. Based on these observations, we hypothesized that galanin gene expression in GnRH neurons is induced whenever GnRH neurons become activated to generate an LH surge. If this were the case, then any neurotransmitter receptor blocking agent that inhibits the LH surge by central mechanisms would likewise block the associated increase in galanin mRNA in GnRH neurons. We tested this hypothesis by examining the effects of an N-methyl-D-aspartate (NMDA) receptor antagonist on the steroid-induced LH surge and on levels of galanin mRNA in GnRH neurons. Three groups of ovariectomized rats were used: Group 1 -treated with estradiol and progesterone (E/P) and sacrificed at the peak of the LH surge; Group 2-treated the same as Group 1 except that dizocilpine (MK801, an NMDA receptor antagonist) was used to block the LH surge; and Group 3-treated the same as Group 1 except they received vehicle instead of E/P. Double-and single-label in situ hybridization followed by computerized image analysis were used to measure levels of galanin mRNA and GnRH mRNA in GnRH neurons [as grains/cell (g/c)]. E/P treatment induced a 3-fold increase in LH levels and a 5-fold increase in the galanin mRNA signal content of GnRH neurons. Treatment with MK801 completely prevented the LH surge in all animals and also blocked the steroid-induced increase in galanin mRNA in GnRH neurons. As assessed by 2 independent GnRH single-labeled assays, neither GnRH message content nor the number of identifiable GnRH neurons differed among the experimental groups. We conclude that the increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a steroid-evoked LH surge, and we infer that induction of galanin gene expression in GnRH neurons is induced as a consequence of synaptic activation of GnRH neurons.

Introduction Hypoxia regulates adipocyte metabolism. Hexosamine biosynthesis is implicated in murine 3T3L1 adipocyte differentiation and is a possible underlying mechanism for hypoxia’s effects on adipocyte metabolism. Methods Lipid metabolism was studied in human visceral and subcutaneous adipocytes in in vitro hypoxic culture with adipophilic staining, glycerol release, and palmitate oxidation assays. Gene expression and hexosamine biosynthesis activation was studied with QRTPCR, immunofluorescence microscopy, and Western blotting. Results Hypoxia inhibits lipogenesis and induces basal lipolysis in visceral and subcutaneous human adipocytes. Hypoxia induces fatty acid oxidation in visceral adipocytes but had no effect on fatty acid oxidation in subcutaneous adipocytes. Hypoxia inhibits hexosamine biosynthesis in adipocytes. Inhibition of hexosamine biosynthesis with azaserine attenuates lipogenesis and induces lipolysis in adipocytes in normoxic conditions, while promotion of hexosamine biosynthesis with glucosamine in hypoxic conditions slightly increases lipogenesis. Conclusions Hypoxia’s net effect on human adipocyte lipid metabolism would be expected to impair adipocyte buffering capacity and contribute to systemic lipotoxicity. Our data suggest that hypoxia may mediate its effects on lipogenesis and lipolysis through inhibition of hexosamine biosynthesis. Hexosamine biosynthesis represents a target for manipulation of adipocyte metabolism.