5-HT Obesity Medication Efficacy via POMC Activation is Maintained During Aging

Department of Pharmacology (L.K.B., B.D., G.D., A.S.G., G.F., D.B., B.B., L.K.H.) and Wellcome Trust/ Medical Research Council Institute of Metabolic Science (M.L.E.), University of Cambridge, Cambridge, CB2 0QQ, United Kingdom; Rowett Institute of Nutrition and Health (G.D., L.K.H.), University of Aberdeen, Aberdeen, AB21 9SB, United Kingdom; Department of Molecular and Integrative Physiology (M.J.L., M.R.), University of Michigan Medical School, Ann Arbor, Michigan 48105; and Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (M.R.), Consejo Nacional de Investigaciones Científicas y Técnicas, 1428 Buenos Aires, Argentina

D-fenfluramine, which stimulates 5-HT release/blocks 5-HT reuptake, and sibutramine, which blocks 5-HT/noradrenaline (NA) reuptake.However, both compounds have been withdrawn from clinical use due to off-target effects (3,4).Transgenic technology has aided in the identification of 5-HT receptors (5-HTRs) responsible for the therapeutic effect of 5-HT obesity medications; an effect principally mediated via G q -coupled 5-HT 2C Rs (3).The therapeutic potential of selective activation of 5-HT 2C Rs for obesity treatment was realized in the summer of 2012 with the U.S. Food and Drug Administration approval of 5-HT 2C R agonist lorcaserin (Arena Pharmaceuticals) and its release in 2013.
Over the last decade, a primary mechanism through which 5-HT action, via 5-HT 2C R activation, achieves effects on appetite and body weight has been revealed.Specifically, D-fenfluramine and 5-HT 2C R agonists chiefly elicit appetitive effects through activation of pro-opiomelanocortin (POMC) neurons (5)(6)(7)(8)(9)(10).Brain POMC, a critical mediator of energy balance, is abundantly expressed in the arcuate nucleus of the hypothalamus (ARC), and a smaller popula-tion is localized in the nucleus of the solitary tract (11,12).Although the 5-HT 2C R-POMC axis represents a critical target in the clinical treatment of obesity, recent reports demonstrate that endogenous ARC POMC regulation of energy balance is impaired with aging.In middle-aged mice (12 months old, comparable to a human 40 years old), ARC POMC neurons display an age-dependent upregulation of mammalian target of rapamycin (mTOR) signaling, which functions to reduce POMC neuronal activity, an effect reversed by mTOR inhibitor rapamycin (13).Furthermore, genetic ablation of endogenous inhibitors of mTOR promoted aged characteristics in ARC POMC neurons of young mice (14,15).Middle-aged mice also display increased inhibitory agouti-related protein drive onto ARC POMC neurons, which functionally reduced POMC firing rate and decreased resting membrane potential compared with younger mice (16).
Given the importance of POMC in mediating 5-HT's appetitive effect, we investigated whether age-associated perturbed ARC POMC activity thereby reduces 5-HT compound anorectic potency.

Animals
Mice on a C57BL/6 background expressing enhanced green fluorescent protein (EGFP) under the control of POMC regulatory elements (POMC-EGFP mice) (8,17), ARC-specific POMC-knockout mice (POMC fneo/fneo ) (12), and wild-type littermates were maintained on a 12-hour light, 12-hour dark cycle (lights on at 7:00 AM) with ad libitum access to chow diet and water unless otherwise stated.All experiments were in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986.

In situ hybridization histochemistry
Young adult (males: 3.2 months Ϯ 9 days, n ϭ 7; females: 3.2 months Ϯ 3 days, n ϭ 7) and middle-aged adult (males: 13.1 month Ϯ 11 days, n ϭ 5; females: 13.3 months Ϯ 5 days, n ϭ 7) C57BL/6 mice were anesthetized with pentobarbitone (50 mg/kg ip) and transcardially perfused with diethylpyrocarbonatetreated PBS followed by 10% neutral buffered formalin (Sigma).Brains were extracted, immersed in the same fixative overnight, cryoprotected in 20% sucrose for 24 to 48 hours and then sectioned coronally on a freezing sliding microtome at 25 m.Tissue was collected in 5 equal series.In situ hybridization histochemistry was conducted using antisense radiolabeled riboprobes specific to the mRNA sequences of ht2cr (5-HT 2C R) and Pomc (7,9,18,19).Riboprobes were generated from cDNA templates by in vitro transcription with a T3 (ht2cr) or SP6 (Pomc) polymerase, according to the manufacturer's protocol (Promega and Ambion Inc) in the presence of 35 S-labeled UTP.cRNA riboprobes were diluted to 2 ϫ 10 7 cpm/mL in hybridization solution.Sections were mounted onto microscope slides, air-dried, and exposed to Biomax MR film (Kodak).Density of ht2cr mRNA and Pomc mRNA was examined in adjacent sections of brain tissue using densitometry analysis by computing the integrated density (the sum of the gray values minus background) using ImageJ (National Institutes of Health).This was performed at 3 ARC levels (Ϫ1.46,Ϫ1.82, and Ϫ2.30 mm from bregma) and the average integrated density was calculated.

Data analysis
Data were analyzed with paired or independent t test, 2 , or repeated-mea- sures ANOVA followed by Tukey's post hoc tests.For all analyses, significance was assigned at P Ͻ .05.Data are presented as mean Ϯ SEM.

5-HT obesity medication appetite suppression requires ARC POMC signaling
We first sought to evaluate the contribution of ARC POMC to the appetitive effects of 5-HT obesity medications.Previous reports indicate that D-fenfluramine and 5-HT 2C R agonists require melanocortin 4 receptors to produce effects on appetite (6, 7, 9); however, the role of the melanocortin system in sibutramine's appetitive effects has not been established.Although selective genetic manipulation of 5-HT 2C R expression on POMC neurons illustrates the importance of the subpopulation of 5-HT 2C R expressed with POMC in communicating 5-HT obesity drug effects (5,10), these studies manipulate 5-HT 2C R receptor expression, not POMC.Here we probe specifically whether ARC POMC neurons are required for the therapeutic effect of 5-HT obesity drugs D-fenfluramine and sibutramine.D-Fenfluramine produced a significant reduction in food intake in male and female wild-type mice (Figure 1,  A and B), but this effect was prevented in discrete ARC-deficient POMC fneo/fneo littermates (Figure 1, C and D).Surprisingly, sibutramine, which blocks the reuptake of both 5-HT and NA, also required ARC POMC neurons to achieve its appetitive effects in male and female mice (Figure 1, A-D).Our results reveal a necessary mechanism through which obesity medications D-fenfluramine and sibutramine achieve their therapeutic appetitive effect, via activation of ARC POMC neurons.

Preserved ARC POMC and 5-HT 2C R appetitive machinery with aging
Given that 5-HT anorectic efficacy is maintained with aging, we surmised that it is POMC basal tone that is compromised with aging, not the 5-HT 2C R and POMC neuroanatomical appetitive machinery.We therefore compared Pomc mRNA and ht2cr mRNA abundance in young adult (3-5 months old) and middle-aged (12-15 months old) male and female mice.Despite observing ageassociated obesity, ARC Pomc mRNA (Figure 4, A and B) and ht2cr mRNA (Figure 4, C and D) did not vary with age in male and female mice.These data suggest that critical neural components for 5-HT appetite regulation at the level of gene expression are not reduced with age in parallel with weight gain.
To investigate the significance of this finding at the functional level, we determined whether pharmacological stimulation of the G q -coupled 5-HT 2C Rs was equally effective in increasing ARC POMC cellular activity in brain slices from young adult and middle-aged POMC-EGFP mice.Using 2-photon fluorescence imaging to monitor changes in intracellular calcium, we observed that 5-HT 2C R agonist WAY-161503 (20M) activated a similar proportion of ARC POMC neurons in both young adult and middle-aged mice (Figure 4, E-G).Together, these data reveal that the density of POMC and 5-HT 2C Rs expressed on an anatomical level is not altered with aging and, furthermore, that 5-HT 2C R signaling within POMC neurons is not affected by age.This provides a mechanism through which 5-HT and 5-HT 2C R agonist anorectic potency is maintained with age.

Discussion
It is a clinical imperative to gain a greater understanding of the mechanisms underlying age-dependent obesity and, in turn, how these mechanisms impact the efficacy of obesity treatments.Like adult humans, adult mice accumulate adiposity with age, in the face of constant environmental conditions (ie, ad libitum access to the same diet and existing at a constant temperature).Although the biological factors driving increased fat accumulation with age are not well understood, recent reports reveal age-related melanocortin system remodeling and link these functional changes with age-associated obesity (13,16).This agerelated remodeled circuitry could thereby impact the efficacy of obesity medications requiring melanocortin system function to exert therapeutic effects.Here we distill a critical and necessary mechanism responsible for communicating the anorectic effect of obesity medications D-fenfluramine and sibutramine to ARC POMC neurons.The mechanistic specificity of the hypophagic effect achieved via this small population of POMC neurons is remarkable given the wholesale effect on 5-HT and 5-HT/NA bioavailability produced by D-fenfluramine and sibutramine, respectively.Our findings are consistent with earlier reports demonstrating that 5-HT depolarizes and stimulates expression of ARC POMC neurons and that functional melanocortin 4 receptors (the primary appetitive receptor target of POMC) are required for D-fenfluramine and 5-HT 2C R agonists to suppress food intake (7,9,20,21).Subsequent work genetically manipulating 5-HT 2C Rs expressed in global POMC neurons has emphasized the importance of this population of 5-HT 2C Rs in 5-HT hypophagia.Specifically, D-fenfluramine and 5-HT 2C R appetite suppression is attenuated in 5-HT 2C Rnull mice but fully restored in mice with 5-HT 2C Rs exclusively re-expressed in POMC cells (5).These data are complemented by recent findings that selective inactivation of 5-HT 2C Rs in POMC neurons abolishes D-fenfluramine and 5-HT 2C R agonist hypophagia (10).Our results reveal a new level of regional specificity and furthermore indicate that independent of their expression of 5-HT 2C Rs, POMC neurons specifically within the ARC are a critical component of the neural circuit through which obesity medications D-fenfluramine and sibutramine influence appetite.
Given that functional POMC is mechanistically required for 5-HT pharmacotherapy appetite suppression, recent reports revealing perturbed POMC function and melanocortin system remodeling in middle-age presented the possibility that 5-HT compound anorectic efficacy may be diminished with age.Specifically, middle-age is associated with an upregulation in mTOR signaling in ARC POMC cells, coupled with an age-dependent increase in inhibitory agouti-related protein inputs, which promotes lower resting membrane potential and reduction in POMC neuronal activity (13,16).
Nevertheless, in the present study, we demonstrate that 5-HT compounds suppress feeding with equal potency in young adult and middle-aged mice.Furthermore, we report that the 5-HT 2C R-POMC appetitive machinery is not compromised with middle-age; both ARC ht2cr and Pomc abundance is preserved, as is 5-HT 2C R signaling in POMC cells.Specifically, plasma membrane G q -coupled 5-HT 2C Rs activate phospholipase C, which hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate into the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, promoting calcium mobilization (3).Using 2-photon calcium imaging in the ARC of POMC-EGFP mice, we report that a 5-HT 2C R agonist was equally ef-fective in stimulating intracellular calcium mobilization in young and middle-aged mice, which suggests that there is no inherent defect in components of the intracellular signaling cascade lying downstream of the 5-HT 2C R. Thereby, these findings provide a mechanism through which 5-HT medication anorectic potency is maintained with age and suggest that pharmacological activation of the 5-HT 2C R-POMC system is sufficient to overcome any age-related reduction in endogenous POMC neuronal activity.
In summary, we report that 5-HT compound anorectic potency is maintained with aging in association with equivalent 5-HT medication activation of POMC neurons at comparable 5-HT 2C R densities in the young adult and middle-aged brain.These data indicate that 5-HT compounds are capable of overcoming components of agerelated changes in physiology to activate POMC neurons and suppress feeding.These data reveal that although former and current 5-HT obesity medications require ARC POMC to achieve appetitive effects, a pathway that is remodeled with aging, the anatomical 5-HT 2C R-POMC machinery is preserved and the anorectic potency is maintained, findings of clinical significance to the global obese population.