The central melanocortin system directly controls peripheral lipid metabolism
2007

Abstract
Disruptions of the melanocortin signaling system have been linked to obesity. We investigated a possible role of the central nervous melanocortin system (CNS-Mcr) in the control of adiposity through effects on nutrient partitioning and cellular lipid metabolism independent of nutrient intake. We report that pharmacological inhibition of melanocortin receptors (Mcr) in rats and genetic disruption of Mc4r in mice directly and potently promoted lipid uptake, triglyceride synthesis, and fat accumulation in white adipose tissue (WAT), while increased CNS-Mcr signaling triggered lipid mobilization. These effects were independent of food intake and preceded changes in adiposity. In addition, decreased CNS-Mcr signaling promoted increased insulin sensitivity and glucose uptake in WAT while decreasing glucose utilization in muscle and brown adipose tissue. Such CNS control of peripheral nutrient partitioning depended on sympathetic nervous system function and was enhanced by synergistic effects on liver triglyceride synthesis. Our findings offer an explanation for enhanced adiposity resulting from decreased melanocortin signaling, even in the absence of hyperphagia, and are consistent with feeding-independent changes in substrate utilization as reflected by respiratory quotient, which is increased with chronic Mcr blockade in rodents and in humans with loss-of-function mutations in MC4R. We also reveal molecular underpinnings for direct control of the CNS-Mcr over lipid metabolism. These results suggest ways to design more efficient pharmacological methods for controlling adiposity.

Introduction
Energy homeostasis, the balance of caloric intake and energy expenditure, is regulated by closely interconnected neuroendocrine and autonomic pathways emanating from and controlled by the CNS. Specific neurocircuitry, which is mainly located in hypothalamic and brain stem areas, continuously monitors signals reflecting energy status and initiates appropriate behavioral and metabolic responses to fluctuations in nutrient availability (1–4). Melanocortin neurons expressing genes encoding the endogenous ligands for melanocortin receptors (Mcr) are essential components of the system within the CNS that controls nutrient intake and energy metabolism (5–10). The central nervous melanocortin system (CNS-Mcr) is also the direct central target of crucial afferent vagal and endocrine signals indicating acute and chronic energy availability, including leptin, insulin, cholecystokinin, and ghrelin (11–18).

Endogenous Mcr agonists result from post-translational processing of proopiomelanocortin (POMC) into melanocyte-stimulating hormones (?MSH, ?MSH, ?MSH) in the hypothalamic arcuate nucleus and elsewhere. The endogenous antagonist (inverse Mcr agonist), agouti-related protein (Agrp), is coexpressed with neuropeptide Y (Npy), both neuropeptides being potent orexigenic and anabolic factors, in a distinct subset of neurons in the mediobasal hypothalamus. Melanocortin ligands bind with varying affinity at 5 subtypes of G protein–coupled Mcr. In the CNS, Mc3rs and Mc4rs have been demonstrated to be most relevant to the regulation of energy homeostasis (19). In rodent models, activation of CNS-Mcr (20) or ablation of Agrp/Npy-coexpressing neurons (21–23) results in anorexia and weight loss, whereas blockade or targeted gene disruption of CNS-Mcr or removal of agonist-producing neurons leads to hyperphagia and obesity (24, 25). In humans, loss-of-function mutations in MC4R are associated with hyperphagia, severe early-onset obesity, hyperinsulinemia, and increased lean mass (26–30), a phenotype that closely mirrors that seen in the Mc4r-KO mouse (25), supporting an essential role for the melanocortin system in energy homeostasis across mammalian species.

It has recently been reported that the CNS-Mcr also directly controls hepatic glucose metabolism (31) and cellular thermogenesis in brown adipose tissue (BAT) (32). These findings confirmed the existence of a direct neuroendocrine control over peripheral cell metabolism as an important part of the central nervous control of body adiposity (7, 33, 34). Furthermore, a study by Rahmouni and colleagues suggested a possible role of the sympathetic nervous system (SNS) as a mediator of the Mcr-induced changes in peripheral metabolism (35).

Previous observations in other laboratories (36, 37) indicate that pharmacological or genetic blockade of CNS-Mcr increases fat mass in the absence of hyperphagia, whereas stimulation of CNS-Mcr reduces adiposity (38). Our current study supports those findings. We therefore hypothesized that the CNS-Mcr may directly regulate peripheral lipid metabolism and nutrient partitioning, as suggested in previous studies (39–42). In the present work, we demonstrate for the first time to our knowledge that the level of CNS-Mcr activity potently and rapidly determines the balance among cellular glucose uptake, triglyceride (TAG) synthesis, lipid deposition, and lipid mobilization in liver, muscle, and adipose tissue. These effects precede changes in adiposity and are independent of changes in food intake, consistent with the hypothesis that the CNS-Mcr controls adiposity levels rapidly and perhaps more efficiently than other known CNS pathways.

Results
CNS-Mcr activity controls adiposity independent of food intake.
Blockade of CNS-Mcr via chronic intracerebroventricular (i.c.v.) infusion of the Agrp-like Mcr antagonist (inverse-agonist) SHU9119 (24 nmol/d for 7 days) increased food intake in ad libitum–fed rats (SHU9119-ad lib) compared with i.c.v. saline–infused controls (Figure ?(Figure1A).1A). A second control group of i.c.v. SHU9119–infused animals was pair-fed to match the intake of saline-infused controls (SHU9119-pf). Activation of CNS-Mcr via chronic i.c.v. infusion of the Mcr agonist Melanotan 2 (MTII) (1 nmol/d for 7 days) only transiently modified feeding behavior and did not change total food intake over the 72 hours before euthanasia. In addition, a pair-fed saline-infused control group was also included that was limited to the daily amount of calories ingested by the i.c.v. MTII-treated animals. Weight gain of i.c.v. SHU9119-ad lib rats was significantly higher than that of controls or SHU9119-pf rats, whereas the i.c.v. MTII–treated group exhibited an expected net loss of body weight, the body weight of these animals remained comparable to that of the saline pair-fed group (Figure ?(Figure1B).1B). SHU9119 treatment potently increased metabolic efficiency, calculated as the ratio of body weight gain per amount of food consumed during the 7-day experimental period (Figure ?(Figure1C).1C). In rats with ad libitum access to food, body weight increase following CNS-Mcr blockade correlated with a net gain of fat mass as determined by nuclear magnetic resonance (Figure ?(Figure1D).1D). Interestingly, rats with CNS-Mcr blockade that were pair-fed with saline-infused controls also had a substantial increase in fat mass that was consistent with their increased metabolic efficiency, even in the absence of hyperphagia (Figure ?(Figure1D),1D), whereas no differences in lean mass were observed between rats with CNS-Mcr blockade and saline pair-fed (Figure ?(Figure1E).1E). On the other hand and as expected, activation of the CNS-Mcr significantly decreased fat mass.

Discussion
Understanding how specific CNS circuits control fat storage and metabolism is essential in order to achieve a breakthrough in this important area of research.* Our current data demonstrate that the most potent brain circuit known to control food intake, the CNS-Mcr, also directly regulates peripheral lipid metabolism. Our central finding is that melanocortin neurons and Mcr in the CNS directly and potently affect cellular glucose utilization, lipid uptake, and TAG synthesis in the periphery. This most likely occurs through effects on autonomic outflow, thereby efficiently shifting substrate metabolism to modulate energy storage and adiposity. More specifically, we can conclude from our results that largely independent of changes in food intake, the CNS-Mcr directly and rapidly controls TAG synthesis, lipid deposition, and lipid mobilization in WAT. These findings provide a compelling explanation for how fat mass increases upon blockade of CNS-Mcr, even in the absence of hyperphagia or detectable changes in energy expenditure, and are consistent with earlier observations by Raposinho et al. (38). Our results also suggest a molecular basis for a CNS-Mcr–induced switch in nutrient partitioning and substrate utilization, which is indirectly reflected by changes in RQ (Figure ?(Figure1,1, F–H). We also observed an increase in RQ in MC4R-deficient subjects, implicating melanocortin signaling in the regulation of substrate utilization in humans. In view of our findings in rodents, it is plausible that an impaired central control of nutrient partitioning, lipid deposition, and lipid mobilization may contribute to the obesity phenotype in human MC4R deficiency.

Pharmacologically (or genetically) decreased CNS-Mcr tone promotes WAT TAG uptake, synthesis, and deposition as well as WAT glucose uptake for energy storage. With increased CNS-Mcr tone, these processes are reversed and lipid mobilization is increased in WAT. These effects occur rapidly and are not a consequence of massive changes in food intake or adiposity. One recent study indicates that the hypothalamic melanocortin system may undergo activity changes shortly before expected meals (47), suggesting the possibility that in addition to regulating chronic energy balance control, one of the functional roles of the CNS-Mcr is to prepare glucose and lipid metabolism pathways in peripheral organs to be able to more efficiently store incoming nutrients, as discussed elsewhere (48).

We have confirmed that the CNS-Mcr system can function globally by regulating hepatic fat metabolism in addition to the control of TAG metabolism in WAT. Thus, modulation of CNS melanocortin tone also controls TAG synthesis and content in the liver, although these effects on hepatic metabolism were less independent of nutrient intake, perhaps because during blockade of CNS-Mcr, pair-fed rats may lack the necessary substrate abundance for increased metabolic TAG production, while pair-feeding is sufficient for the metabolically less “costly” WAT TAG synthesis. CNS-Mcr blockade increased hepatic levels of SCD1, and this also occurred in pair-fed rats, indicating partial independence from changes in food intake, but TAG content or changes in FAS mRNA levels did not reach significant levels in the absence of ad libitum access to dietary nutrients (Supplemental Figure 2).

Interestingly, rats with blocked CNS-Mcr displayed an increase in hepatic MTP gene expression, which appears to reflect increased hepatic lipoprotein assembly and secretion (49). In parallel, these rats had increased hepatic TAG storage along with upregulation of the respective hepatic TAG metabolism enzymes. These observations, together with the unchanged plasma levels of TAG and unchanged fecal TAG levels, suggest that blockade of CNS-Mcr stimulates synthesis of hepatic TAGs, which are rapidly transported to WAT, resulting in unchanged plasma TAG concentrations. Based on a series of elegant studies by Rossetti and colleagues, the vagus nerve appears to be the crucial communication link mediating such CNS control of hepatic metabolism (1, 50).

The SNS connects WAT cells directly with homeostatic control areas in the CNS, including Mcr-positive neurons in the hypothalamus as well as in other nonhypothalamic forebrain areas, midbrain, and brainstem areas (5, 34). We therefore tested the hypothesis that the SNS may be responsible for informing WAT cells about changes in CNS-Mcr tone by measuring SNA in the WAT after stimulation and blockade of the CNS-Mcr and using mice lacking ?1,2,3-adrenergic receptors. Our results indicate that CNS-Mcr modulates SNA in WAT and that SNS signaling via functional ?-adrenergic receptors is required for CNS-Mcr–induced changes in adipocyte metabolism, changes that failed to occur in TKO mice. Nevertheless, we acknowledge that complete disruption of SNS signaling in the TKO mice may create undesired tangential phenotypes. Further studies will be necessary to completely elucidate the likely participation of additional pathways in the observed CNS-WAT crosstalk. In vitro studies indicate that insulin upregulates LPL expression and activity in human WAT (51, 52) and increases LPL and FAS gene expression in brown adipocytes (53). In our experiments, insulin was increased upon blockade of CNS-Mcr. Therefore, it appeared to be possible that changes in plasma insulin levels could contribute to the CNS-WAT communication necessary for rapid and potent switching of metabolic substrate choice. However, we observed similar effects on lipogenic enzymes in chronically i.c.v. SHU9119–treated rats studied in the basal state or during hyperinsulinemia at the end of euglycemic-hyperinsulinemic clamps, suggesting that CNS-Mcr blockade does not need increased insulin to exert its effects (Supplemental Figure 3).

Although one previous report suggested that high concentrations of Mcr ligands may directly affect lipolysis in cultured adipocytes (54), we conclude that changes in the endogenous CNS-Mcr are sufficient to control TAG metabolism, since chronic subcutaneous infusion of the same doses of Mcr ligands had no effect on TAG metabolism.
Energy balance in Mcr-KO mice has been studied in detail. In addition to hyperphagia, these mice exhibit decreased locomotor activity and energy expenditure (24, 25, 37, 39, 42), factors that could indirectly contribute to, or be a consequence of, their increased adiposity. The central regulation of lipid metabolism appears to be a physiological function of the endogenous Mcr system, since TAG metabolism profiles of Mcr-KO mice are comparable to those of rats during pharmacological blockade of CNS-Mcr. For example, we detected an increase in WAT LPL mRNA levels in Mc4r-KO mice, reflecting increased lipid uptake in WAT. However, enzymes responsible for WAT lipogenesis were not significantly different from those of WT controls, likely indicating that the morbidly obese MC4r-KO mice were close to reaching their maximum WAT storage capacity and therefore prevented from further TAG synthesis. Those differences in the extent to which pharmacological and genetic manipulation of the CNS melanocortin tone affect WAT TAG metabolism might also be due to diverse counterregulatory processes in the mutated mice triggered by the massive early-onset obesity.

Specific populations of Mc4r-expressing neurons within distinct regions of the brain may differentially regulate food intake and energy expenditure (55). It remains unknown, however, which exact Mcr-expressing neurons in the CNS are responsible for the regulation of peripheral TAG and glucose metabolism. For example, the brain regions relevant for Mc4r-induced lipid mobilization activity are unclear. Sympathetic preganglionic neurons in the intermediolateral nucleus of the spinal cord, which mediate the SNS, abundantly express MC4rs and thereby represent an important candidate population (56). The possibility that MTII and SHU9119 reach the intermediolateral nucleus of the spinal cord after i.c.v. injection cannot be excluded, and therefore the effects seen in the present study may in part have resulted from changes in Mcr signaling in the intermediolateral nucleus.

Unexpectedly, overall insulin sensitivity and hepatic glucose production were not changed upon chronic blockade of CNS-Mcr. However, glucose utilization was specifically decreased in muscle and BAT, while being increased in WAT. Consistent with a WAT-specific enhancement of glucose uptake, we found enhanced basal and stimulated insulin signaling in WAT of Mc4r-KO mice. These results are also consistent with a switch in nutrient partitioning from substrate utilization toward substrate storage, which may in part be promoted by CNS-Mcr–induced tissue-specific changes in insulin sensitivity. We were unable to replicate results of earlier reports (31) indicating that chronic central infusion of SHU9119 impairs insulin sensitivity and decreases hepatic glucose production. Those differences may be explained by variations in methodologies, animal models, and ligand doses used. However, our findings on the divergent effects on glucose utilization by adipose tissue and skeletal muscle are consistent with effects following central infusion of ghrelin (44) or Npy (57, 58). In one report, Npy produced increased insulin-stimulated glucose uptake by adipose tissue and decreased glucose uptake by several muscle types. The increased fat mass of rats with blocked CNS-Mcr may result from increased TAG uptake and synthesis as well as higher glucose utilization by WAT. Energy storage commands from the brain may directly suppress muscle and BAT glucose utilization or simply create a WAT glucose sink, whereby glucose is rapidly cleared from plasma and stored as fat.

In summary, we provide a combination of pharmacological and genetic evidence to demonstrate that the CNS-Mcr system promotes fat storage via direct and rapid modulation of liver, muscle, and fat cell metabolism. Such synergistic CNS control of peripheral lipid and glucose metabolism appears to be mainly mediated by the SNS and is largely independent of parallel changes in food intake and adiposity. Specifically, when CNS melanocortin activity is reduced, a coordinated response is elicited that promotes fat deposition by]