Melanocortin 3/4 Receptor Research Articles

Evidence for a feeding related association between melanocortin in the NTS and Neuropeptide-Y in the PVN

Melanocortin and neuropeptide-Y (NPY) are both involved in feeding and energy regulation, and they have opposite effects in the paraventricular nucleus of the hypothalamus (PVN). The present study examined an interaction between melanocortin in the nucleus of the solitary tract (NTS) and NPY in the PVN. Male Sprague-Dawley rats were implanted with cannulae in the injection sites of interest. In Experiment 1, subjects received either the melanocortin 3/4-receptor (MC3/4) antagonist SHU9119 (0, 10, 50 and 100 pmol/0.5 μl) or the MC3/4 agonist MTII (0, 10, 50, 100 and 200 pmol/0.5 μl) into the NTS. Food intake was measured at 1, 2, 4, 6 and 24-h post-injection. Administration of SHU9119 into the NTS significantly and dose-dependently increased food intake at 1, 2, 4, 6 and 6-24-h, and administration of MTII into the NTS significantly and dose-dependently decreased 24-h free feeding. In Experiment 2, subjects received the MC3/4 agonist MTII (0, 10, 50, 100 and 200 pmol/0.5 μl) into the NTS just prior to NPY (0 and 1μg/0.5 μl) in the PVN. PVN injection of NPY stimulated feeding, and administration of MTII (50, 100 and 200 pmol) into the NTS significantly and dose-dependently decreased NPY-induced feeding at 2, 4, 6 and 6-24-h. These data suggest that there could be a neuronal association between melanocortin in the NTS and NPY in the PVN, and that the melanocortin system in the NTS has an antagonistic effect on NPY-induced feeding in the PVN.

Short-term Weight Trajectory of Severely Obese Individuals With and Without Pathogenic Satiety-Regulation Melanocortin 3/4 Receptor (MC3/4R) Mutations From a Multi-ethnic Asian Large Bariatric Surgery Program.

The melanocortin (3 or 4) receptor (MC3/4R) is involved in regulating satiety and body weight. Therefore, pathogenic mutation in MC3/4R is associated with severe obesity, for which bariatric surgery is one of the treatment options. However, there is limited data on whether individuals with MC3/4R mutation will have differential weight response to surgery, especially among the Asian populations-the epi-center of the evolving global obesity epidemic. From our large prospective Obesity-Metabolism & Intervention Cohort Study (OMICS; N = 654, recruited between 2007 and 2022), 5 individuals with pathogenic MC3/4R mutations ("case") were identified using candidate-genes panel next-generation sequencing (Illumina iSeq). These subjects were carefully propensity score-matched (baseline body mass index [BMI], age, sex, ethnicity, proportion with diabetes, type of bariatric surgery) in a 1:4 ratio to other controls. We performed linear mixed model analysis (for repeated measurements) to compare their longitudinal weight trajectories (percentage total weight loss, %TWL) over 12 months. The 5 cases with MC3/4R mutations were 48 ± 11 years, BMI 40.8 ± 11.2 kg/m2, 60% with diabetes, and all males. Their weight at baseline (pre-op), and 6 months and 12 months after surgery were 120 ± 38, 100 ± 31, and 101 ± 30 kg, respectively. Compared with propensity score-matched controls (N = 20), linear mixed model analysis suggested no difference in surgically induced %TWL (β coefficient = -5.8 ± 3.7, P = .13) over 12 months between the groups. Therefore, we conclude that rare pathogenic MC3/4R mutations do not significantly modify weight change (%TWL) in response to bariatric surgery.

Transient receptor potential cation channel 6 deficiency leads to increased body weight and metabolic dysfunction.

Transient receptor potential cation channel 6 (TRPC6), a member of the TRPC family, is expressed in the hypothalamus and modulates cell Ca2+ influx. However, the role of TRPC6 in controlling metabolic and cardiovascular functions under normal conditions has not been previously determined. Thus the impacts of TRPC6 deletion on energy balance, metabolic, and cardiovascular regulation as well as the anorexic responses to leptin and melanocortin 3/4 receptor (MC3/4R) activation were investigated in this study. Extensive cardiometabolic phenotyping was conducted in male and female TRPC6 knockout (KO) and control mice from 6 to 24 wk of age to assess mechanisms by which TRPC6 influences regulation of energy balance and blood pressure (BP). We found that TRPC6 KO mice are heavier with greater adiposity, are hyperphagic, and have reduced energy expenditure, impaired glucose tolerance, hyperinsulinemia, and increased liver fat compared with controls. TRPC6 KO mice also have smaller brains, reduced proopiomelanocortin mRNA levels in the hypothalamus, and impaired anorexic response to leptin but not to MC3/4R activation. BP and heart rate, assessed by telemetry, were similar in TRPC6 KO and control mice, and BP responses to air-jet stress were attenuated in TRPC6 KO mice despite increased body weight and metabolic disorders that normally raise BP and increase BP responses to stress. Our results provide evidence for a novel and important role of TRPC6 in controlling energy balance, adiposity, and glucose homeostasis, which suggests that normal TRPC6 function may be necessary to link weight gain and hyperleptinemia with BP responses to acute stress.

Anti-Obesity Effect of Pine Needle Extract on High-Fat Diet-Induced Obese Mice.

Background: Obesity due to an excessive intake of nutrient disturbs the hypothalamus-mediated energy metabolism subsequently develops metabolic disorders. In this study, we investigated the effect of pine needle extract (PNE) on the hypothalamic proopiomelanocortin (POMC) neurons involved in the regulation of energy balance via melanocortin system and fat tissue metabolism. Methods: We performed electrophysiological and immunohistochemical analyses to determine the effect of PNE on POMC neurons. Mice were fed a normal or high-fat diet for 12 weeks, then received PNE for the last 2 weeks to measure the following physiological indices: Body weight, food intake, fat/lean mass, glucose metabolism, and plasma leptin levels. In addition, changes of thermogenic, lipolytic, and lipogenetic markers were evaluated in brown adipose tissue (BAT) and white adipose tissue (WAT) by western blotting, respectively. Results: PNE increased hypothalamic POMC neuronal activity, and the effect was abolished by blockade of melanocortin 3/4 receptors (MC3/4Rs). PNE decreased body weight, fat mass, plasma leptin levels, and improved glucose metabolism after high-fat-induced obesity. However, PNE did not change the expression of thermogenic markers of the BAT in HFD fed groups, but decreased only the lipogenetic markers of WAT. This study suggests that PNE has a potent anti-obesity effect, inhibiting lipogenesis in WAT, even though HFD-induced leptin resistance-mediated disruption of POMC neuronal activity.

Hindbrain melanocortin 3/4 receptors modulate the food intake and body weight suppressive effects of the GLP-1 receptor agonist, liraglutide

Simultaneously targeting multiple energy balance control systems is a promising direction for the development of obesity pharmacotherapies. Here, we explore the interaction between the GLP-1 and melanocortin system within the dorsal vagal complex (DVC) of the caudal brainstem. Using a pharmacological approach, we demonstrate that the full anorectic potential of liraglutide, an FDA-approved GLP-1 analog for the treatment of obesity, requires DVC melanocortin 3/4 receptor (MC3/4R) signaling. Specifically, the food intake and body weight suppressive effects of liraglutide were attenuated by DVC administration of the MC3/4R antagonist SHU9119. In contrast, the anorectic effects of liraglutide were enhanced by combined activation of DVC MC3/4Rs using the agonist MTII. Our findings highlight the modulation of liraglutide-induced anorexia by DVC MC3/4R signaling, thereby suggesting a site of action at which two important energy balance control systems interact.

Hypothalamic nesfatin-1 mediates feeding behavior via MC3/4R-ERK signaling pathway after weight loss in obese Sprague-Dawley rats

Nesfatin-1 is an anorexic peptide derived from nucleobindin 2 (NUCB2). An increase in hypothalamic nesfatin-1 inhibits feeding behavior and promotes weight loss. However, the effects of weight loss on hypothalamic nesfatin-1 levels are unclear. In this study, obese rats lost weight in three ways: Calorie Restriction diet (CRD), Sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB). We found an increase in nesfatin-1 serum and cerebrospinal fluid levels after weight loss in obese Sprague-Dawley (SD) rats. Moreover, weight loss also increased hypothalamic melanocortin 3/4 receptor (MC3/4R) and extracellular regulated kinase phosphorylation (p-ERK) signaling. Third ventricle administration of antisense morpholino oligonucleotide (MON) against the gene encoding NUCB2 inhibited hypothalamic nesfatin-1 and p-ERK signaling, increased food intake and reduced body weight loss in SG and RYGB obese rats. Third ventricle administration of SHU9119 (MC3/4R blocker) blocked hypothalamic MC3/4R, inhibited p-ERK signaling, increased food intake and reduced body weight loss in SG and RYGB obese rats. These findings indicate that weight loss leads to an increase in hypothalamic nesfatin-1. The increase in hypothalamic nesfatin-1 participates in regulating feeding behavior through the MC3/4R-ERK signaling especially after SG and RYGB.

Abstract P312: A Novel Selective Melanocortin-4 Receptor Agonist Attenuates Bradycardia and Hyperglycemia in Diabetic Rats

We previously demonstrated that functional brain melanocortin 3/4 receptors (MC3/4R) are required for leptin to exert its chronic cardiovascular and antidiabetic actions. To determine if chronic treatment with a selective MC4R agonist reduces blood glucose and prevents bradycardia in diabetic rats, we used a novel compound with high affinity to MC4R. Male 12-week-old Sprague-Dawley rats (n=5/group) were instrumented with telemetry probes for determination of mean arterial pressure (MAP) and heart rate (HR) 24-hrs/day and an intracerebroventricular (ICV) cannula was placed in the brain lateral ventricle for continuous infusion of the selective MC4R agonist PL6214 (2.5 μg/hr) or MTII (a non-selective MC3/4R agonist, 10 ng/hr) via osmotic minipump. Induction of diabetes caused hyperphagia (20±1 to 32±2 g), hyperglycemia (89±3 to 494±44 mg/dl) and bradycardia (-47 bpm). Chronic infusion of PL6214 for 11 days transiently reduced food intake which returned to diabetic values by day 6 after starting the infusions, whereas chronic infusion of MTII caused a reduction in food intake lasting only 3-4 days. PL6214 reduced blood glucose by 63% on day 2 and 16% by day 11 of infusion, and prevented further bradycardia induced by diabetes (-35±14 bpm on the last day of infusion). Chronic MTII infusion reduced blood glucose by 31% on day 2 and by day 6 glucose levels had already returned to values observed before treatment was started. MTII infusion did not attenuate the bradycardia (-99±13 bpm on the last day of infusion). Diabetes did not alter MAP, while the MC4R agonist increased MAP by 5±1 mmHg compared to control values. These results indicate that selective activation of MC4R agonist attenuates bradycardia and hyperglycemia in type 1 diabetes, and may provide a new strategy for treatment of diabetes. (P20GM104357, NHLBI-PO1HL51971, AHA-SDG5680016 and Palatin Technologies).

Regulation of Blood Pressure, Appetite, and Glucose by CNS Melanocortin System in Hyperandrogenemic Female SHR.

Hyperandrogenemia in females may be associated with sympathetic nervous system (SNS) activation and increased blood pressure (BP). However the importance of hyperandrogenemia in causing hypertension in females and the mechanisms involved are still unclear. We tested whether chronic hyperandrogenemia exacerbates hypertension in young female spontaneously hypertensive rats (SHR) and whether endogenous melanocortin-3/4 receptor (MC3/4R) activation contributes to the elevated BP. Cardiovascular and metabolic effects of chronic MC3/4R antagonism were assessed in female SHR treated with dihydrotestosterone (DHT, beginning at 5 weeks of age) and placebo-treated female SHR. BP and heart rate (HR) were measured by telemetry and an intracerebroventricular (ICV) cannula was placed in the lateral ventricle for infusions. After control measurements, the MC3/4R antagonist (SHU-9119) was infused for 10 days (1 nmol/hour, ICV, at 15 weeks of age) followed by a 5-day recovery period. MC3/4R antagonism increased food intake and body weight in DHT-treated SHR (14±1 to 35±1g/day and 244±3 to 298±8g) and controls (14±1 to 34±2g/day and 207±4 to 269±8g). Compared to untreated SHR, DHT-treated SHR had similar BP but lower HR (146±3 vs. 142±4mm Hg and 316±2 vs. 363±4 bpm). Chronic SHU-9119 infusion reduced BP and HR in DHT-treated SHR (-12±2mm Hg and -14±4 bpm) and control female SHR (-19±2mm Hg and -21±6 bpm). These results indicate that hyperandrogenemia does not exacerbate hypertension in female SHR. MC3/4R antagonism reduces BP and HR despite marked increases in food intake and body weight in hyperandrogenemic and control female SHR.

Role of hindbrain melanocortin-4 receptor activity in controlling cardiovascular and metabolic functions in spontaneously hypertensive rats

Although we previously demonstrated that activation of central nervous system (CNS) melanocortin3/4 receptors (MC3/4R) play a key role in blood pressure (BP) regulation, especially in spontaneously hypertensive rats (SHRs), the importance of hindbrain MC4R is still unclear. In the present study, we examined the cardiovascular and metabolic effects of chronic inhibition of MC3/4R in the hindbrain of SHRs and normotensive Wistar-Kyoto (WKY) rats. Male WKY rats (n = 6) and SHRs (n = 7) were implanted with telemetry probes to measure BP and heart rate (HR) 24 h/day, and an intracerebroventricular cannula was placed into the fourth ventricle. After 10 days of recovery and 5 days of control measurements, the MC3/4R antagonist (SHU-9119) was infused into the fourth ventricle (1 nmol/h) to antagonize hindbrain MC4R for 10 days, followed by a 5-day recovery period. Chronic hindbrain MC3/4R antagonism significantly increased food intake and body weight in WKY rats (17 ± 1 to 35 ± 2 g/day and 280 ± 8 to 353 ± 8 g) and SHRs (19 ± 2 to 35 ± 2 g/day and 323 ± 7 to 371 ± 11 g), and markedly increased fasting insulin and leptin levels while causing no changes in blood glucose levels (99 ± 4 to 87 ± 4 and 89 ± 5 to 89 ± 4 mg/dl, respectively, for WKY rats and SHRs). Chronic SHU-9119 infusion reduced mean arterial pressure and HR similarly in WKY rats (-8 ± 1 mmHg and -47 ± 3 b.p.m.) and SHRs (-11 ± 3 mmHg and -44 ± 3 b.p.m.). These results suggest that although hindbrain MC4R activity contributes to appetite and HR regulation, it does not play a major role in mediating the elevated BP in SHRs.

PVN Neuropeptide Y Contributes to the Sympathoexcitatory Effect of Leptin

Intracerebroventricular (ICV) leptin administration increases sympathetic nerve activity (SNA) via a neuronal pathway that includes the paraventricular nucleus of the hypothalamus (PVN). PVN Neuropeptide Y (NPY) tonically inhibits SNA via type 1 receptors (Y1R). Leptin inhibits NPY neurons; however, whether leptin increases SNA in part by decreasing tonic PVN NPY inhibition is unknown. To test this hypothesis, we determined if blockade of PVN Y1R (Y1Rx) elicits smaller increases in lumbar SNA (LSNA) after ICV leptin (3 μg) in α‐chloralose anesthetized male SD rats. LSNA was increased 90 min after ICV leptin (to 158±8% of control, n=5; P<0.05), but not after ICV artificial cerebrospinal fluid (aCSF; to 112±2%, n=5). In rats given ICV aCSF, subsequent PVN Y1Rx (bilateral BIBO3304, 60 pmol/60 nL) increased LSNA to 146±5 % of control (P<0.05). However, after ICV leptin, neither PVN Y1Rx (to 171±10 %) nor PVN aCSF (160±18 to 177±19%, n=5) significantly increased LSNA. These data support our hypothesis that leptin reduces tonic PVN NPY inhibition of LSNA. Leptin increases SNA in part via activation of PVN melanocortin 3/4 receptors (MC3/4R), and PVN presympathetic neurons express both Y1R and MC4R. Therefore, to test if leptin‐induced decreases in PVN NPY inhibition unmask MC3/4R excitation, we next determined if the MC3/4R agonist MTII increases LSNA more after Y1Rx. Bilateral PVN MTII (60 pmol/60 nL) did not alter LSNA (110±5% to 112±10 %, n=3); however, after Y1Rx, MTII increased LSNA from 124±3% to 164±6% (P<0.05, n=4). In conclusion, leptin decreases tonic PVN NPY inhibition of SNA, which may contribute to leptin‐induced increases in SNA by exposing enhanced MC3/4R excitatory drive of PVN presympathetic neurons. Supported by NIH HL088552 & AHA.

Inhibition of the central melanocortin system decreases brown adipose tissue activity

The melanocortin system is an important regulator of energy balance, and melanocortin 4 receptor (MC4R) deficiency is the most common monogenic cause of obesity. We investigated whether the relationship between melanocortin system activity and energy expenditure (EE) is mediated by brown adipose tissue (BAT) activity. Therefore, female APOE*3-Leiden.CETP transgenic mice were fed a Western-type diet for 4 weeks and infused intracerebroventricularly with the melanocortin 3/4 receptor (MC3/4R) antagonist SHU9119 or vehicle for 2 weeks. SHU9119 increased food intake (+30%) and body fat (+50%) and decreased EE by reduction in fat oxidation (-42%). In addition, SHU9119 impaired the uptake of VLDL-TG by BAT. In line with this, SHU9119 decreased uncoupling protein-1 levels in BAT (-60%) and induced large intracellular lipid droplets, indicative of severely disturbed BAT activity. Finally, SHU9119-treated mice pair-fed to the vehicle-treated group still exhibited these effects, indicating that MC4R inhibition impairs BAT activity independent of food intake. These effects were not specific to the APOE*3-Leiden.CETP background as SHU9119 also inhibited BAT activity in wild-type mice. We conclude that inhibition of central MC3/4R signaling impairs BAT function, which is accompanied by reduced EE, thereby promoting adiposity. We anticipate that activation of MC4R is a promising strategy to combat obesity by increasing BAT activity.

Role of melanocortin signaling in neuroendocrine and metabolic actions of leptin in male rats with uncontrolled diabetes.

Although the antidiabetic effects of leptin require intact neuronal melanocortin signaling in rodents with uncontrolled diabetes (uDM), increased melanocortin signaling is not sufficient to mimic leptin's glucose-lowering effects. The current studies were undertaken to clarify the role of melanocortin signaling in leptin's ability to correct metabolic and neuroendocrine disturbances associated with uDM. To accomplish this, bilateral cannulae were implanted in the lateral ventricle of rats with streptozotocin-induced diabetes, and leptin was coinfused with varying doses of the melanocortin 3/4 receptor (MC3/4R) antagonist, SHU9119. An additional cohort of streptozotocin-induced diabetes rats received intracerebroventricular administration of either the MC3/4R agonist, melanotan-II, or its vehicle. Consistent with previous findings, leptin's glucose-lowering effects were blocked by intracerebroventricular SHU9119. In contrast, leptin-mediated suppression of hyperglucagonemia involves both melanocortin dependent and independent mechanisms, and the degree of glucagon inhibition was associated with reduced plasma ketone body levels. Increased central nervous system melanocortin signaling alone fails to mimic leptin's ability to correct any of the metabolic or neuroendocrine disturbances associated with uDM. Moreover, the inability of increased melanocortin signaling to lower diabetic hyperglycemia does not appear to be secondary to release of the endogenous MC3/4R inverse agonist, Agouti-related peptide (AgRP), because AgRP knockout mice did not show increased susceptibility to the antidiabetic effects of increased MC3/4R signaling. Overall, these data suggest that 1) AgRP is not a major driver of diabetic hyperglycemia, 2) mechanisms independent of melanocortin signaling contribute to leptin's antidiabetic effects, and 3) melanocortin receptor blockade dissociates leptin's glucose-lowering effect from its action on other features of uDM, including reversal of hyperglucagonemia and ketosis, suggesting that brain control of ketosis, but not blood glucose levels, is glucagon dependent.

Postmenopausal hypertension: role of the sympathetic nervous system in an animal model

In postmenopausal women the mechanisms responsible for hypertension have not been completely elucidated, and there are no gender-specific guidelines for women despite studies showing that blood pressure is not as well controlled to goal in women as in men. In the present study we tested the hypotheses that the sympathetic nervous system and the renal sympathetic nerves contribute to hypertension in aging female rats, that sympathetic activation may be mediated by the melanocortin 3/4 receptor (MC3/4R), and that MC3/4R activation may be due to increases in leptin. α-1, β-1,2-Adrenergic blockade reduced blood pressure in both young (3-4 mo) and old (18-19 mo) female spontaneously hypertensive rats (SHR). Renal denervation attenuated the hypertension more in old females than young females. MC3/4R antagonism with SHU-9119 given intracerebroventricularly had no effect on blood pressure in either young or old females but significantly reduced blood pressure in old males. Plasma leptin levels were similar in old male and female SHR and in old versus young females. These data suggest that the hypertension in old female SHR is in part due to activation of the sympathetic nervous system, that the renal nerves contribute to the hypertension, and that the mechanism responsible for sympathetic activation in old females is independent of the MC3/4R.

Melanocortin 3/4 receptors in paraventricular nucleus modulate sympathetic outflow and blood pressure

Central melanocortin 3/4 receptors (MC3/4Rs) are known to regulate energy balance. Activation of MC3/4Rs causes a greater increase in the firing activity of the PVN neurons in obese Zucker rats than in lean Zucker rats. The present study was undertaken to determine the roles of MC3/4Rs in the hypothalamic paraventricular nucleus (PVN) in modulating the sympathetic activity and blood pressure and its downstream pathway. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in anaesthetized rats. Microinjection of the MC3/4R agonist melanotan II (MTII) into the PVN increased the RSNA and MAP. The MC3/4R antagonist agouti-related peptide (AgRP) or SHU9119 decreased the RSNA and MAP, but the MC4R antagonist HS024 had no significant effect on the RSNA and MAP. The effects of MTII were abolished by pretreatment of the PVN with AgRP, SHU9119, the adenylate cyclase inhibitor SQ22536 or the protein kinase A inhibitor Rp-cAMP, and substantially attenuated by HS024. Microinjection of SQ22536 alone into the PVN had no significant effect on the RSNA and MAP, but Rp-cAMP caused significant decreases in the RSNA and MAP. Furthermore, MTII increased the cAMP level in the PVN. These results indicate that activation of MC3/4Rs in the PVN increases the sympathetic outflow and blood pressure via the cAMP-protein kinase A pathway. Melanocortin 3 receptors in the PVN may exert a tonic excitatory effect on sympathetic activity.

Abstract 159: Role of Melanocortin 3/4 Receptor (MC3/4R) in a Model of Postmenopausal Hypertension.

The mechanisms responsible for hypertension in postmenopausal women have not been completely elucidated. We have shown previously that MAP in old female SHR is mediated in part by sympathetic activation. In male SHR leptin causes sympathetic activation via the melanocortin 3 / 4 receptor (MC3/4R) activation, and MC3/4R blockade reduces their blood pressure. The hypothesis of the present study was that MC3/4R also contributes to hypertension in old female SHR. Female SHR (aged 18 mos, n=6) received MC3/4 receptor antagonist SHU-9119 (1 nmol/h via minipump) or saline vehicle (n=5 controls). After two weeks recovery, MAP and heart rate (HR) were measured for 5 days during baseline period, and then rats received SHU 9119 or saline icv, and MAP and HR were recorded for 7 days. MC3/4R antagonism produced an increase in food intake (17.1±2 gr, n=5, vs 26.6±1 gr, n=6, p<0.001) and body weight (217±11 gr,n=5 vs 256±6 gr, n=6; p<0.001), compared to saline controls. However, MC3/4R antagonism had no effect on MAP (baseline period: 174±10 mmHg ,n=4 vs 181±5 mmHg, n=6; p:NS; treatment period, 175±13 mmHg, n=4 vs 172±10 mmHg, n=6; p:NS) or HR (Baseline period: 344±4 bpm, n=4 vs 357±12 bpm, n=6; Treatment period, V-R:345±9 bpm, n=4 vs 332±9 bpm, n=6; p:NS). These results show that MC3/4R plays no role in sympathetic activation and regulation of BP in old female SHR. These data suggest that aging women may need different antihypertensive medications than do men. Supported by NIH HL69192, HL55901; HL66072.

Central mechanisms activated by leptin to modify hypercapnia‐induced ventilatory responses

Melanocortin 3/4 receptors (MC3/4R) play a pivotal role in mediated metabolic and cardiovascular actions of leptin. In this study, we evaluated the changes in pulmonary ventilation (VE) to hypercapnia (7% CO2) in rats treated with daily microinjections of leptin and SHU9119 (MC3/4R antagonist) alone or combined into the lateral ventricle (LV) for 7 days. Male Holtzman rats (n=6/group) with stainless steel cannula implanted into the LV were used. Ventilation was recorded by plethysmography. Leptin (5 μg/5μl/day) into the LV reduced body weight by ~17% and increased (P<0.05) the tidal volume (11.0±0.7 vs. saline: 7.8±0.7 ml.kg−1) and the ventilatory response to hypercapnia (1796±140 vs. saline: 1213±126 ml.min−1.kg−1), without changing respiratory frequency. SHU (1 nmol/5μl/day) increased body weight by ~ 13%, without changing the increase in ventilation (1185±53 ml.min−1.kg−1)and in the tidal volume (6.8±0.7 ml.kg−1) produced by hypercapnia. Combined treatment with SHU+leptin also increased body weight (~9%), however, decreased tidal volume (5.2±0.3 ml.kg−1) and ventilatory response to hypercapnia (795±34 ml.min− 1.kg−1, P<0.05). The results suggest that leptin‐induced increase in ventilation depends on the activation of central MC3/4 receptors. In addition, it seems that leptin produces also a parallel secondary inhibitory action on ventilation independent on MC3/4R activation.

Leptin increases baroreflex gain: role of melanocortin 3/4 receptors in the hypothalamic paraventricular nucleus

Intracerebroventricular (ICV) leptin increases lumbar sympathetic nerve activity (LSNA) and LSNA baroreflex gain (BRG), but the sites and neuronal pathways are unknown. To test the role of the paraventricular nucleus (PVN), baroreflex function was assessed in α‐chloralose anesthetized male SD rats at baseline and 1 hr after ICV leptin (3 μg), using a 4 parameter sigmoidal fit of LSNA responses to slow ramp (3–5 min) changes in arterial pressure, induced by iv infusion of nitroprusside and phenylephrine. Then, 90 min after ICV leptin, 60 nL of muscimol (mus; 1 mM/L; n=3), the melanocortin 3/4 receptor (MC3/4R) inhibitor SHU9119 (0.5 mM/L; n=6), or the aCSF vehicle (n=5) was microinjected bilaterally into the PVN. Leptin increased (P<0.05) LSNA (158±9%) and LSNA BRG (from 3.2±0.4 to 5.7±0.2 %/mmHg). In control experiments (after PVN aCSF), further increases (P<0.05) in LSNA (186±21%) and LSNA BRG (7.2±0.6 %/mmHg) were observed. In contrast, PVN inhibition with mus completely reversed (P<0.05) the effects of leptin to increase LSNA [to 160±9% (leptin) then 125±2% (mus)] and LSNA BRG [in %/mmHg: from 3.4±0.6 to 4.8±0.6 (leptin) then 3.6±0.4 (mus)]. PVN SHU9119 decreased (P<0.05) LSNA [to 169±9% (leptin) then 139±10% (SHU9119)] and LSNA BRG [in %/mmHg: from 3.1±0.4 to 4.8±0.7 (leptin) then 4.4±0.6 (SHU9119)] compared to aCSF; however, both LSNA and LSNA BRG remained elevated (P<0.05) versus baseline. In conclusion, the PVN contributes to the increases in LSNA and LSNA BRG induced by ICV leptin, in part via MC3/4R. Support: HL088552.

Activation of the central melanocortin system contributes to the increased arterial pressure in obese Zucker rats

We have previously demonstrated that leptin-mediated activation of the central nervous system (CNS) melanocortin system reduces appetite and increases sympathetic activity and blood pressure (BP). In the present study we examined whether endogenous melanocortin system activation, independent of leptin's actions, contributes to the regulation of BP and metabolic functions in obese Zucker rats, which have mutated leptin receptors. The long-term cardiovascular and metabolic effects of central melanocortin-3/4 receptor (MC3/4R) antagonism with SHU-9119 were assessed in lean (n = 6) and obese (n = 8) Zucker rats. BP and heart rate (HR) were measured 24-h/day by telemetry and an intracerebroventricular cannula was placed in the brain lateral ventricle. After stable control measurements, SHU-9119 was infused intracerebroventricularlly (1 nmol/h) for 10 days followed by a 10-day recovery period. Chronic CNS MC3/4R antagonism significantly increased food intake and body weight in lean (20 ± 1 to 45 ± 2 g and 373 ± 11 to 432 ± 14 g) and obese (25 ± 2 to 35 ± 2 g and 547 ± 10 to 604 ± 11 g) rats. No significant changes were observed in plasma glucose levels in lean or obese rats, whereas plasma leptin and insulin levels markedly increased in lean Zucker rats during CNS MC3/4R antagonism. Chronic SHU-9119 infusion in obese Zucker rats reduced mean arterial pressure (MAP) and HR by 6 ± 1 mmHg and 24 ± 5 beats/min, whereas in lean rats SHU-9119 infusion reduced HR by 31 ± 9 beats/min while causing only a transient decrease in MAP. These results suggest that in obese Zucker rats the CNS melanocortin system contributes to elevated BP independent of leptin receptor activation.

The HPA axis modulates the CNS melanocortin control of liver triacylglyceride metabolism

The central melanocortin system regulates lipid metabolism in peripheral tissues such as white adipose tissue. Alterations in the activity of sympathetic nerves connecting hypothalamic cells expressing melanocortin 3/4 receptors (MC3/4R) with white adipocytes have been shown to partly mediate these effects. Interestingly, hypothalamic neurons producing corticotropin-releasing hormone and thyrotropin-releasing hormone co-express MC4R. Therefore we hypothesized that regulation of hypothalamo-pituitary adrenal (HPA) and hypothalamo-pituitary thyroid (HPT) axes activity by the central melanocortin system could contribute to its control of peripheral lipid metabolism. To test this hypothesis, we chronically infused rats intracerebroventricularly (i.c.v.) either with an MC3/4R antagonist (SHU9119), an MC3/4R agonist (MTII) or saline. Rats had been previously adrenalectomized (ADX) and supplemented daily with 1mg/kg corticosterone (s.c.), thyroidectomized (TDX) and supplemented daily with 10μg/kgL-thyroxin (s.c.), or sham operated (SO). Blockade of MC3/4R signaling with SHU9119 increased food intake and body mass, irrespective of gland surgery. The increase in body mass was accompanied by higher epididymal white adipose tissue (eWAT) weight and higher mRNA content of lipogenic enzymes in eWAT. SHU9119 infusion increased triglyceride content in the liver of SO and TDX rats, but not in those of ADX rats. Concomitantly, mRNA expression of lipogenic enzymes in liver was increased in SO and TDX, but not in ADX rats. We conclude that the HPA and HPT axes do not play an essential role in mediating central melanocortinergic effects on white adipose tissue and liver lipid metabolism. However, while basal hepatic lipid metabolism does not depend on a functional HPA axis, the induction of hepatic lipogenesis due to central melanocortin system blockade does require a functional HPA axis.

Anatomical dissociation of melanocortin receptor agonist effects on taste- and gut-sensitive feeding processes

Injections of the melanocortin 3/4 receptor (MCR) agonist melanotan II (MTII) to a variety of brain structures produces anorexia, suggesting distributed brain MCR control of food intake. We performed a detailed analysis of feeding behavior (licking microstructure analysis) after a range of MTII doses (0.005 nM to 1 nM) was targeted to the forebrain (third ventricle, 3V) or hindbrain (fourth ventricle, 4V) regions. MTII (0.1 nM and 1 nM) delivered to the 3V or 4V significantly reduced 0.8 M sucrose intake. The anorexia was mediated by reductions in the number of licking bursts in the meal, intrameal ingestion rate, and meal duration; these measures have been associated with postingestive feedback inhibition of feeding. Anorexia after 3V but not 4V MTII injection was also associated with a reduced rate of licking in the first minute (initial lick rate) and reduced mean duration of licking bursts; these measures have been associated with taste evaluation. MTII effects on taste evaluation were further explored: In experiment 2, 3V MTII (1 nM) significantly reduced intake of noncaloric 4 mM saccharin and 0.1 M and 1 M sucrose solutions, but not water. The anorexia was again associated with reduced number of licking bursts, ingestion rate, meal duration, initial lick rate, and mean burst duration. In experiments 3 and 4, brief access (20 s) licking responses for sweet sucrose (0.015 M to 0.25 M) and bitter quinine hydrochloride (0.01 mM to 1 mM) solutions were evaluated. Licking responses for sucrose were suppressed, whereas those for quinine solutions were increased after 3V MTII, but not after 4V MTII injections (0.1 nM and 1 nM). The results suggest that multiple brain MCR sites influence sensitivity to visceral feedback, whereas forebrain MCR stimulation is necessary to influence taste responsiveness, possibly through attenuation of the perceived intensity of taste stimuli.