We examined the role of melanocortin-4 receptors (MC4R) in proopiomelanocortin (Pomc) neurons in regulating metabolic and cardiovascular functions. Using Cre-loxP technology, we selectively rescued MC4R in Pomc neurons of mice with whole body MC4R deficiency (MC4R-Pomc-Cre mice). Body weight, food intake, and whole body oxygen consumption (V̇o2) were determined daily, and blood pressure (BP), heart rate (HR), and body temperature were measured 24 h/day by telemetry. An intracerebroventricular cannula was placed in the right lateral ventricle for intracerebroventricular infusions. Littermate MC4R-deficient (LoxTB-MC4R) mice were used as controls. After control measurements, the MC4R antagonist (SHU-9119; 1 nmol/h) was infused intracerebroventricularly for 7 days. Compared with LoxTB-MC4R mice, MC4R-Pomc-Cre mice were less obese (47 ± 2 vs. 52 ± 2 g) and had increased energy expenditure (2,174 ± 98 vs. 1,990 ± 68 ml·kg−1·min−1), but food intake (4.4 ± 0.2 vs. 4.3 ± 0.3 g/day), BP (112 ± 1 vs. 109 ± 3 mmHg), and HR [557 ± 9 vs. 551 ± 14 beats per minute (bpm)] were similar between groups. Chronic SHU-9119 infusion increased food intake (4.2 ± 0.2 to 6.1 ± 0.5 g/day) and body weight (47 ± 2 to 52 ± 2 g) in MC4R-Pomc-Cre mice, while no changes were observed in LoxTB-MC4R mice. Chronic SHU-9119 infusion also increased BP and HR by 5 ± 1 mmHg and 60 ± 8 bpm in MC4R-Pomc-Cre mice without altering BP or HR in LoxTB-MC4R mice. These results indicate that MC4Rs in Pomc neurons are important for regulation of energy balance. In contrast, while activation of MC4R in Pomc neurons facilitates the BP response to acute stress, our data do not support a major role of MC4R in Pomc neurons in regulating baseline BP and HR.
- blood pressure
- heart rate
- energy expenditure
- sympathetic nervous system
the central nervous system (CNS) melanocortin pathway plays an important role in regulating appetite, energy expenditure, sympathetic nervous system (SNS) activity, and blood pressure (BP). These effects are mediated mainly via activation of G protein-coupled melanocortin-4 receptors (MC4R) that are stimulated by α-melanocyte-stimulating hormone (α-MSH), a cleavage by-product of proopiomelanocortin (Pomc) peptide in Pomc-containing neurons (2, 3, 12, 13, 17, 20). Pomc neurons, which are mainly located in the arcuate nucleus (ARC) of the hypothalamus, send projections to different areas of the brain, including paraventricular nucleus (PVN), lateral hypothalamus (LH), and the dorsal-vagal complex, where these projections release α-MSH and activate MC4R (13, 14, 20, 23, 24). Other regions of the brain, including the nucleus of tractus solitarius (NTS) (10), also contain Pomc neurons, although the physiological role of Pomc neurons in the NTS is still largely unknown. A previous study by Balthasar et al. (1) showed that rescuing MC4R in the PVN attenuated 60% of weight gain found in mice with total body deletion of MC4R. These findings suggest that MC4R in other neuronal populations beside PVN are also important in body weight regulation by altering energy expenditure, as well as food intake. However, the locations of these additional neuronal populations in which MC4R activation controls metabolic and cardiovascular functions are still unclear.
The importance of the CNS melanocortin pathway in body weight homeostasis is demonstrated by the finding that humans and experimental animals with defective Pomc or MC4R signaling have early-onset severe obesity associated with hyperphagia and reduced energy expenditure. The Pomc-MC4R axis may also be important in linking obesity with increased SNS activation and hypertension. For instance, mice with MC4R deficiency do not develop hypertension despite obesity and many features of metabolic syndrome, including insulin resistance, hyperinsulinemia, hyperleptinemia, dyslipidemia, and increased visceral adiposity (22). We have also shown that selective deletion of leptin receptors in Pomc neurons leads to mild obesity, hyperglycemia, and hyperinsulinemia and completely abolishes leptin's ability to raise BP (7). These results in mice are relevant to humans and are supported by the finding that the prevalence of hypertension is markedly reduced in obese humans with MC4R deficiency compared with control obese subjects (10, 11).
Because Pomc neurons send projections to areas outside the ARC, it has generally been assumed that MC4R in neurons downstream from Pomc neurons, especially in the PVN and LH, mediate most of the effects on metabolic and cardiovascular functions. However, MC4R may also be located on Pomc neurons, and an in vitro study suggests that specific agonists of MC4R may depolarize Pomc neurons (18). These observations are consistent with the possibility that MC4R located on Pomc neurons may act as an autoexcitatory and/or autopotentiation mechanism that enhances Pomc activation, leading to more efficient control of energy homeostasis and SNS activity. On the basis of our previous studies showing an important role for Pomc neurons in regulating BP and cardiovascular responses to acute stress (7) and the important role of MC4R in BP regulation (4–6, 8), we hypothesized that MC4R located on Pomc neurons may mediate, at least in part, the physiological effects of the brain melanocortin system on cardiovascular and metabolic function. Therefore, if our hypothesis is correct, mice with MC4R present only in Pomc neurons should exhibit decreased food intake or increased energy expenditure, as well as increases in basal blood pressure; moreover, chronic inhibition of MC4R should reverse these expected effects. Because no previous studies, to our knowledge, have examined the physiological role of MC4R on Pomc neurons in vivo, we generated mice in which MC4R were present only in Pomc neurons and nowhere else in the body and compared their cardiovascular and metabolic phenotypes, as well as their responses to chronic pharmacological antagonism of MC4R to those observed in mice with total body deletion of MC4R.
MATERIALS AND METHODS
The experimental procedures and protocols of this study conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.
Male wild-type (WT; n = 10), MC4R-green fluorescent protein (GFP; n = 3), LoxTB-MC4R (n = 35), and MC4R-Pomc-Cre mice (n = 34) were used in these studies. MC4R-Pomc-Cre mice were generated by crossing Pomc-Cre mice, which express Cre-recombinase specifically in POMC neurons (generously provided by Dr. Joel Elmquist, Southwestern Texas University, Dallas, TX), with whole body MC4R-deficient LoxTB-MC4R mice (generously provided by Dr. Bradford Lowell, Harvard University, Cambridge, MA). The MC4R deficiency in LoxTB-MC4R mice has been described elsewhere (1). In brief, a disrupted MC4R-null allele was generated by inserting a loxP-flanked transcriptional blocker (loxTB) between the transcription initiation (+1) and the ATG of the MC4R-coding sequence. Expression of Cre-recombinase removes the transcriptional blocker and allows “rescue” of normal MC4R transcription. Thus, by crossing LoxTB-MC4R with Pomc-cre mice, we generated mice in which MC4R were rescued only in Pomc neurons. Homozygous mice for the LoxTB-MC4R allele that also expressed Cre-recombinase were used as MC4R-Pomc-Cre mice, while littermate homozygous mice for the LoxTB-MC4R allele that did not express Cre-recombinase were used as controls (Fig. 1A). MC4R-GFP mice, in which GFP is driven by the MC4R promoter (kindly provided by Dr. Roger Cone, Vanderbilt University, Nashville, TN), were used for fluorescence immunohistochemistry (Fig. 1B).
Polymerase Chain Reaction
Genotyping was performed as previously described (1). Briefly, after weaning at 4 wk of age, mice were genotyped using tail snips to perform PCR across the loxP exon 6 and for Cre-recombinase using the following primers: 5′-GGAAGATGAACTCCACCCACC-3′, 5′-ACGATGGTTTCCGACCCATTC-3′, GAGCCCAGAAAGCGAAGGAAC-3′; and for Cre positive 5′-CTGCCACGACCAAGTGACAGC-3′, and Cre negative 5′-CTTCTCTACACCTGCGGTGCT-3′. Only animals tested positive for the loxP (homozygous) and Cre (heterozygous) were used as MC4R-Pomc-cre mice (Fig. 1A).
Blood Pressure Telemetry Probe Implantation
Male WT (n = 5), LoxTB-MC4R (n = 7), and MC4R-Pomc-Cre mice (n = 6) were anesthetized with isoflurane (1.5%), and atropine sulfate (0.37 mg/kg) was administered to prevent excessive airways secretion. A telemetric pressure transmitter device (model TA11PAC-10; Data Sciences International, Minneapolis, MN) was implanted in the left carotid artery under sterile conditions, as previously described (8, 22). Mean arterial pressure (MAP) and heart rate (HR) were continuously measured 24 h/day and were derived from average MAP and HR measured by bursts of 10 s every 10 min using the software provided by the manufacturer (Dataquest 4.0).
After telemetry probe implantation, a stainless-steel cannula (33 gauge, 5 mm long) was implanted into the right lateral cerebral ventricle using the coordinates as previously described (16). The guide cannula was anchored into place with two stainless-steel machine screws, a plastic cap, and dental acrylic, and a stylet was inserted to seal the cannula until use. During stereotaxic manipulation, anesthesia was maintained with 1.5–0.5% isoflurane. Several days after recovery from surgery, accuracy of the cannula placement was tested by measuring the dipsogenic response (immediate drinking of at least 1 ml of water in 5 min) to an intracerebroventricular injection of 100 ng of ANG II.
After the surgical procedures, mice were housed individually in cages for determination of daily food consumption. Mice were provided a normal sodium diet (0.5 mmol sodium/g food; Harlan Teklad, Madison, WI) ad libitum. Mice were allowed to recover for 10–12 days before control measurements of food intake, body weight, and plasma hormones were performed.
Body weight and body composition analyses.
Male WT (n = 5), LoxTB-MC4R (n = 7), and MC4R-Pomc-Cre (n = 6) mice were individually housed and fed standard chow (Harlan Teklad) starting at 6 wk through 18 wk of age. To examine the role of MC4R in Pomc neurons in regulating food consumption and body weight, food intake was measured daily and body weight was measured twice a week. We also examined weekly changes in body composition using magnetic resonance imaging (EchoMRI-900TM, Echo Medical System, Houston, TX). MRI measurements were performed in conscious mice placed in a thin-walled plastic cylinder with a cylindrical plastic insert added to limit movement of the mice. Mice were briefly submitted to a low-intensity electromagnetic field and fat mass, lean mass, free water, and total water were measured. At death, the livers of LoxTB-MC4R and MC4R-Pomc-Cre mice were removed, and liver lean and fat mass content were measured using the tissue biopsy probe component of the EchoMRI scanner.
Oil Red-O staining.
To confirm the results obtained by tissue biopsy measurements of liver lean and fat mass content, we also performed Oil Red-O staining in frozen liver sections (10 μm thick) of WT, LoxTB-MC4R, and MC4R-Pomc-Cre mice. Sections were fixed in 10% buffered formalin for 5 min and stained for 10 min with 0.5% Oil Red-O in 60% isopropyl alcohol. The slides were washed several times in water and counterstained in Mayer's hematoxylin for 30 s and mounted in aqueous mounting media.
MC4R-GFP and Pomc immunohistochemistry.
Frozen brain coronal sections of MC4R-GFP mice were cut (30 μm thick) and processed for double-labeling of Pomc (Phoenix Pharmaceutics, Burlingame, CA) protein and GFP. Sections were placed free-floating in 0.01 M PBS and then incubated in blocking solution (PBS, 0.3% Triton X-100, and 5% normal donkey serum) for 1 h at room temperature. The expression of GFP was detected by incubating with primary antibody 1:250 goat anti-GFP biotin (Abcam, Cambridge, MA) diluted in blocking solution for 24 h at 4°C. After rinses with PBS (5 times for 5 min), sections were incubated with rabbit anti-Pomc at a dilution of 1:100 for 1 h at room temperature. Sections were rinsed 10 times for 5 min and then incubated with secondary antibody 1:500 Alexa Fluor 488 (Invitrogen, Molecular Probes, Carlsbad, CA) and donkey anti-rabbit Alexa Fluor 594 (Molecular Probes) diluted in 0.01 M PBS plus 0.3% Triton X-100 overnight at 4°C. The sections were washed 10 times for 5 min in 0.01 M PBS and mounted on gelatin-coated glass slides. Slides were examined in a fluorescence microscope at appropriate wavelengths.
Glucose tolerance test.
d-glucose (1.5 mg/kg body wt) was administered intraperitonealy after a 6-h fast in 22-wk-old male LoxTB-MC4R (n = 5) and MC4R-Pomc-Cre (n = 5) mice. Blood samples were collected by tail snip, and blood glucose was measured at 0, 15, 30, 60, 90, and 120 min after glucose injection using glucose strips (Reli On).
Western blot for uncoupling protein-1.
Interscapular brown adipose tissues (BAT) from LoxTB-MC4R (n = 5) and MC4R-Pomc-Cre (n = 5) mice at 20 wk of age were homogenized in lysis buffer (KPO4, pH 7.4) and cleared by centrifugation (1,000 g, 5 min at 4°C). After protein concentration of supernatant was determined by the Bradford method (Bio-Rad, Hercules, CA), 50 μg of protein was separated in a 4–15% precast linear gradient polyacrylamide gel (Bio-Rad). After being transferred to nitrocellulose membrane, blots were rinsed in PBS and blocked in Odyssey blocking buffer (LI-COR, Lincoln, NE) for 1 h at room temperature, and incubated with mouse monoclonal antiuncoupling protein-1 (UCP-1) antibody (1:1,000; Sigma, Saint Louis, MO) overnight at 4°C. The membrane was probed for rabbit anti-β-actin (1:5,000; Abcam) as a loading control. The membrane was then incubated with IR700-conjugated donkey anti-mouse IgG and IR800-conjugated donkey anti-rabbit (1:2,000; Rockland Immunologicals, Gilbertsville, PA). Antibody labeling was visualized using the Odyssey infrared scanner (LI-COR) for simultaneous detection of two fluorprobes. Fluorescence intensity analyses were performed using Odyssey software (LI-COR). Measurement of UCP-1 was normalized to β-actin.
Oxygen consumption (V̇o2) and body temperature measurements.
WT (n = 5), LoxTB-MC4R (n = 5), and MC4R-Pomc-Cre (n = 5) mice at 22–24 wk of age were placed individually in metabolic cages (CLAMS, Columbus OH) equipped with oxygen sensors to measure oxygen consumption (V̇o2) and receivers to measure body temperature. Body temperature was measured using a telemetry probe (Respironics Mini Emitter 4000, Bend, OR) implanted intraperitonealy 8 to 10 days prior to the beginning of the experiment. V̇o2 was measured using a Zirconia oxygen sensor. V̇o2 and body temperature were measured for 2 min at every 10-min interval continuously 24 h/day. After the mice were acclimatized to the new environment for 3 days, V̇o2 and body temperature were measured for five consecutive days, and then they were lightly anesthetized with isoflurane, and a minipump was implanted in the scapular region and attached to the intracerebroventricular cannula to deliver SHU-9119 (1 nmol/h at 0.5 μl/h) for 7 days. At the end of day 7 of SHU-9119 infusion, the tubing connecting the minipump to the intracerebroventricular cannula was severed, and measurements were continued for an additional 5-day recovery period.
Acute melanotan II injection.
Food intake and V̇o2 were measured 12 and 24 h after an intraperitoneal injection of melanotan II (MTII; 400 μg ip) or saline vehicle given between 5:15 PM and 5:30 PM in LoxTB-MC4R (n = 5) and MC4R-Pomc-Cre mice (n = 5) at 22 wk of age.
Acute air-jet stress test.
To determine whether rescuing MC4R in Pomc neurons alters MAP and HR responses to an acute pressor stress, LoxTB-MC4R (n = 6) and MC4R-Pomc-Cre (n = 7) mice were placed in a special cage used for air jet stress testing, as previously described (7). After the 5-min air-jet stress, MAP and HR were measured for an additional 30-min recovery period. BP and HR responses during air-jet stress and the recovery period were calculated as the change in MAP and HR from the average baseline values obtained 10 min before the 5-min after the air-jet stress was initiated. The area under the MAP curve was calculated during the air-jet stress and recovery periods using the following parameters: average change in MAP for each minute during the 5-min air-jet stress test and for each 5 min during the 30-min recovery period.
Chronic MC4R antagonism.
After 10 days to recover from surgery, daily MAP, HR, and food intake were recorded for 5 consecutive days in mice implanted with telemetry probes and intracerebroventricular cannulas; then SHU-9119 (1 nmol/h at 0.5 μl/h; Polypeptide Laboratories, Torrance, CA) was infused intracerebroventricularly for 7 days via osmotic minipump (model 1007D, Durect Corp., Cupertino, CA). The minipump was placed subcutaneously in the scapular region and connected intracerebroventricularly using RenaPulse tubing (RenaPulse; Braintree Scientific, Braintree, MA). The rate of SHU-9119 infusion was based on previous studies in rodents showing that this dose effectively blocks MC4R (4–6) and our preliminary results showing increased appetite in WT mice. On the last day of SHU-9119 infusion, the tubing connecting the minipump to the intracerebroventricular cannula was severed, and measurements were continued for an additional 5-day recovery period.
Plasma hormones and glucose measurements.
Blood samples (100 μl) were collected via a tail snip after 6 h of fasting (8:00 AM to 2:00 PM) during the control period (day 5), on the last day of SHU 9119 infusion (day 7), and at the end of the posttreatment period for measurements of plasma glucose, leptin, and insulin concentrations. Plasma leptin and insulin concentrations were measured using ELISA [R&D Systems (Minneapolis, MN) and Crystal Chem (Downers Grove, IL), respectively], and plasma glucose concentrations were determined using the glucose oxidation method (Beckman glucose analyzer 2; Beckman Coulter, Brea, CA). The quantitative insulin-sensitivity check index (QUICKI) was also calculated from fasting insulin and glucose values. QUICKI = 1/[log (insulin in μU/ml) + log (fasting glucose in mg/dl)].
The results are expressed as means ± SE. The data were analyzed by paired t-test or one-way ANOVA with repeated measures followed by Dunnett's post hoc test for comparisons between control and experimental values within each group when appropriate. Comparisons between different groups were made by two-way ANOVA followed by Bonferroni's post hoc test when appropriate. Statistical significance was accepted at a level of P < 0.05.
Selective Rescue of MC4R in Pomc Neurons Reduces Body Weight, Visceral Adiposity, and Liver Fat Content, and Improves Glucose Tolerance
Homozygous mice for the LoxTB-MC4R allele that also expressed Cre-recombinase were used as MC4R-Pomc-Cre mice, while littermate homozygous mice for the LoxTB-MC4R allele that did not express Cre-recombinase were used as controls (Fig. 1A). To demonstrate colocalization between MC4R and Pomc neurons, we used double-labeling immunofluorescence for GFP and Pomc protein immunoreactivity in MC4R-GFP mice. As expected, we observed positive MC4R staining in Pomc neurons (Fig. 1B). Despite no differences in body length (nasal to anal distance, 10.6 ± 0.1 vs. 10.7 ± 0.1 cm at 24 wk of age) or average daily food intake from 8 to 18 wk of age (Fig. 1C), MC4R-Pomc-Cre mice were less obese compared with LoxTB-MC4R mice (Fig. 1D). Food intake was significantly higher in LoxTB-MC4R compared with MC4R-Pomc-Cre mice only between 6 and 7 wk of age. MC4R-Pomc-Cre mice exhibited higher lean and less fat body mass than LoxTB-MC4R mice as early as 6 wk of age (Fig. 1, E and F), smaller livers with less fat infiltration measured by magnetic resonance imaging (EchoMRI) and oil red-O staining (Fig. 1, G–J), and reduced mesenteric and retroperitoneal fat content (Fig. 1, K and L). Compared with WT mice, however, MC4R-Pomc-Cre mice were still 67% heavier and consumed 22% more food (Fig. 1, C–I).
To test whether the reduced body weight and higher lean/fat mass ratio in MC4R-Pomc-Cre mice translated into better glucose handling, we performed glucose tolerance tests (GTT) in both groups of mice at 20–22 wk of age. MC4R-Pomc-Cre mice exhibited greater tolerance to a glucose load as reflected by a 40% smaller area under the blood glucose curve during the GTT (Fig. 1, M and N).
Selective Rescue of MC4R in Pomc Neurons Is Associated With Increases in V̇o2, Body Temperature, and BAT UCP-1 Expression
Since restoration of MC4R in Pomc neurons attenuated the obesity observed in LoxTB-MC4R mice by ∼9% without altering food intake, we investigated whether the reduced body weight of MC4R-Pomc-Cre mice was accompanied by increased energy expenditure. We measured V̇o2 and body temperature 24 h/day for 5 consecutive days in 22-wk-old mice and found that MC4R-Pomc-Cre mice had 10% greater V̇o2 and 0.5°C higher body temperature than control LoxTB-MC4R mice (Fig. 2, A and B). Compared with WT controls, MC4R-Pomc-Cre mice had similar body temperature but a 25% lower V̇o2 (Fig. 2, A and B). Interscapular BAT UCP-1 protein content was also significantly higher in MC4R-Pomc-Cre compared with LoxTB-MC4R mice (Fig. 2C). These observations suggest that reduced body weight in MC4R-Pomc-Cre mice is likely due to increased energy expenditure. Thus, MC4R in Pomc neurons appear to play an important role in regulating basal energy expenditure, while exerting little effect on basal control of appetite.
MC4R Activation With Melanotan II Reduced Food Intake and Increased V̇o2 in MC4R-Pomc-Cre Mice but not in LoxTB-MC4R Mice
We examined whether exogenous activation of MC4R in Pomc neurons alters appetite and energy expenditure by administering 400 μg of MTII (single intraperitoneal injection) to both groups just before lights out (6:00 PM) and measured food intake and V̇o2 for the following 24 h. The dose of MTII was chosen on the basis of previous studies showing that it was effective in reducing food intake and increasing oxygen consumption in control mice (1). To account for the potential effect of stress caused by the intraperitoneal injection on appetite, the mice were previously injected with saline vehicle (250 μl) 3 days before MTII was administered. Saline injections did not alter V̇o2 (data not shown) or food intake in MC4R-Pomc-Cre (3.4 ± 1.0 vs. 3.8 ± 0.8 g/day) or LoxTB-MC4R mice (3.8 ± 0.3 vs. 4.2 ± 1.0 g/day). The overall effects of MTII to suppress food intake and to increase V̇o2 were calculated relative to the effects of saline injection. MTII caused a 20% reduction in food intake (12-h: 2.3 ± 0.2 to 1.8 ± 0.1 g and 24-h: 3.4 ± 0.2 to 2.7 ± 0.2 g, P < 0.05) and an 8% increase in V̇o2 (12-h: 1,866 ± 130 to 1,958 ± 133 and 24-h: 2,199 ± 148 to 2,417 ± 163 ml·kg−1·min−1, P < 0.05) in MC4R-Pomc-Cre mice, whereas no significant changes were observed in LoxTB-MC4R mice at 12-h (2.6 ± 0.2 to 2.7 ± 0.2 g and 1,968 ± 57 to 1,902 ± 71 ml·kg−1·min−1) or 24-h postinjection (3.8 ± 0.3 to 3.7 ± 0.2 g and 2,264 ± 92 to 2,235 ± 40 ml·kg−1·min−1) (Fig. 2, D and E). These results suggest that although baseline endogenous activation of MC4R in Pomc neurons plays a more important role in regulating basal energy expenditure than appetite, acute pharmacological activation of MC4R in these neurons can also suppress food intake in addition to increasing V̇o2.
Rescuing MC4R in Pomc Neurons Augments the Cardiovascular Response to Acute Stress Without Altering Baseline BP or HR
We have previously shown that brain MC4R play an important role in regulating BP and HR and that they are required for obesity to be associated with increased sympathetic activation and hypertension (8, 22). However, the brain regions where MC4R contribute to the regulation of cardiovascular function are still largely unknown. To test the hypothesis that MC4R on Pomc neurons are important for the regulation of cardiovascular function, we measured BP and HR 24 h/day using telemetry under normal conditions and during an acute air-jet stress test. We observed that although baseline BP (109 ± 3 vs. 112 ± 1 mmHg) and HR (551 ± 14 vs. 557 ± 9 bpm) were not different between LoxTB-MC4R and MC4R-Pomc-Cre mice (Fig. 3, A and B), the response to acute air-jet stress in MC4R-Pomc-Cre mice was more pronounced than observed in LoxTB-MC4R control mice. For instance, we noticed a 55% and 40% greater rise in BP and HR in MC4R-Pomc-Cre mice during the 5-min air-jet stress period (Fig. 3, C–F). Moreover, BP and HR quickly returned to baseline values in control LoxTB-MC4R mice during the 30-min recovery period post air-jet stress test, whereas in MC4R-Pomc-Cre mice, BP and HR remained elevated (Fig. 3, C–F). These results indicate that MC4R in Pomc neurons may also modulate cardiovascular responses to acute stress.
Chronic Central MC4R Antagonism Increased Food Intake, Body Weight, and Fasting Plasma Glucose Levels in MC4R-Pomc-Cre Mice But not in LoxTB-MC4R Mice
To examine whether chronic blockade of MC4R in mice with selective rescue of MC4R in Pomc neurons would result in hyperphagia and weight gain, as we have observed in rats and lean wild-type animals (4, 5, 6), we continuously infused SHU-9,119 (1 nmol/h) directly into the brain lateral ventricle for 7 days using osmotic minipumps connected to the intracerebroventricular cannula. Chronic antagonism of MC4R increased food intake in MC4R-Pomc-Cre mice by ∼60% over the 7-day treatment period (Fig. 4B), which resulted in a cumulative net food intake of almost 11 g (the sum of the daily difference in food intake during treatment compared with the average daily food intake during baseline period, Fig. 4C). This increase in food intake caused significant weight gain in MC4R-Pomc-Cre mice, which eliminated the initial difference in body weight between MC4R-Pomc-Cre and LoxTB-MC4R mice (Fig. 4A). Chronic SHU-9119 infusion in LoxTB-MC4R mice produced no effect on food intake or body weight (Fig. 4, A–C), indicating that the chronic effects of SHU-9119 on appetite are due mainly to antagonism of MC4R and not to a nonspecific effect of SHU-9119 or blockade of MC3R. In WT mice, chronic MC4R antagonism increased food intake by ∼27%, leading to a cumulative net food intake of almost 7 g (Fig. 4, A–C) and significant weight gain (8.5%, Fig. 4A). However, baseline fasting plasma glucose and insulin levels were significantly lower in MC4R-Pomc-Cre mice compared with LoxTB-MC4R control mice (Fig. 4, D and E), suggesting that rescue of MC4R in Pomc neurons improved insulin sensitivity, as evidenced by an almost 50% reduction in plasma insulin levels, while blood glucose levels were also reduced by ∼20%. Moreover, chronic MC4R antagonism raised fasting glucose levels in MC4R-Pomc-Cre mice, so that they were no longer different compared with LoxTB-MC4R mice, while insulin levels remained unchanged, indicating that MC4R blockade impaired insulin sensitivity in MC4R-Pomc-Cre mice (Fig. 4, D and E). We further examined insulin sensitivity by calculating the QUICKI index and found that MC4R-Pomc-Cre had ∼10% higher insulin sensitivity (0.253 ± 0.006 vs. 0.233 ± 0.007) compared with LoxTB-MC4R mice. These observations suggest that MC4R in Pomc neurons also contribute to the CNS control of glucose homeostasis. Baseline leptin levels were not different between groups and did not change during SHU-9119 infusion (Fig. 4F).
We also tested whether SHU-9119 would reduce energy expenditure and body temperature in MC4R-Pomc-Cre mice. However, SHU-9119 ICV infusion did not alter V̇o2 or body temperature in both groups (Fig. 4, G and H). In fact, SHU-9119 tended to increase V̇o2 in MC4R-Pomc-Cre mice. This small increase in V̇o2 may have been caused, in part, by increased food intake and rapid weight gain during SHU-9119 treatment. At the end of the experiment, the animals were killed, and the brains were removed and sectioned to confirm the placement of the cannula into the right lateral ventricle (Fig. 4I).
Chronic Intracerebroventricular SHU-9119 Infusion Raised BP and HR in MC4R-Pomc-Cre Mice
We previously demonstrated that MC4Rs provide a key link between obesity, increased SNS activity, and hypertension (8, 22). Since we observed augmented BP and HR responses to stress in MC4R-Pomc-Cre mice, we further hypothesized that SHU-9119 would reduce BP and HR in these mice. Surprisingly, blockade of MC4R raised BP and HR in MC4R-Pomc-Cre mice, whereas no changes were observed in LoxTB-MC4R control mice (Fig. 5, A–D). In WT mice, SHU-9119 infusion reduced MAP and HR by −5 mmHg and −38 bpm, respectively. These data are consistent with our previous studies showing that chronic CNS MC4R inhibition reduces BP and HR, despite increasing food intake and promoting weight gain (4, 5).
Our studies indicate that MC4R on Pomc neurons contribute to the regulation of body weight, body composition, and energy expenditure. In addition, our observations indicate that MC4R on Pomc neurons modulate the cardiovascular responses to acute stress but do not support a major role of MC4R in Pomc neurons in regulating baseline BP and HR. We also found other important effects of restoring MC4R function in Pomc neurons, including improved glucose handling, as evidenced by reductions in baseline circulating levels of insulin and glucose, and greater tolerance to a glucose load compared with control MC4R-deficient LoxTB-MC4R mice.
Control of Metabolic Functions by MC4R on POMC Neurons
It has generally been assumed that MC4R on second-order neurons, especially those located in the PVN and other brain regions downstream of Pomc neurons, mediate most of the effects of Pomc neuron activation. However, Balthasar et al. (1) showed that rescue of MC4R in the PVN attenuated weight gain by only about 60%, compared with that observed in mice with total body deletion of MC4R, suggesting that MC4R in other neuronal populations are also important in body weight regulation. Moreover, they found that restoration of MC4R in PVN decreased food intake but did not alter energy expenditure compared with LoxTB-MC4R mice. These findings are consistent with the possibility that MC4R in other brain regions besides the PVN may play an important role in regulating body weight by altering energy expenditure as well as food intake. However, the locations of neuronal populations in which MC4R activation controls metabolic and cardiovascular functions are unclear.
Our current study suggests that Pomc neurons may be another site where MC4R activation plays an important role in regulation of energy balance and cardiovascular function. Reactivation of MC4R only in Pomc neurons significantly attenuated hyperphagia and obesity as early 6–7 wk of age in LoxTB-MC4R mice with total body deficiency of MC4R, an effect that could have important effects on body weight later in life. After 9–10 wk of age, we did not observe significant differences in food intake in LoxTB-MC4R and MC4R-Pomc-Cre mice. However, reduced body weight, decreased total fat mass, improved glucose tolerance, and decreased liver fat were observed in 18–20-wk-old mice with MC4R reactivated only in Pomc neurons, compared with LoxTB-MC4R mice, despite no differences in food intake. At 18 wk of age, mice with reactivation of MC4R also had increases in V̇o2, body temperature, and UCP 1, compared with mice with total body MC4R deficiency, suggesting that their reduced body weight was maintained mainly by increased metabolic rate. However, we cannot rule out the possibility that the small difference in body weight between groups may have contributed to the improved metabolic phenotype of MC4R-Pomc-Cre mice.
The potential importance of MC4R on Pomc neurons for controlling metabolic and cardiovascular function had not, to our knowledge, been previously reported. In vitro studies indicate that specific agonists of MC4R depolarize Pomc neurons, whereas MC4R antagonists hyperpolarize these neurons (18), consistent with the possibility that MC4R located on Pomc neurons may act as an autoexcitatory and/or as an autopotentiation mechanism that enhances Pomc neuron activation (Fig. 6). To our knowledge, the results of the current study provide the first in vivo evidence that MC4Rs on Pomc neurons contribute importantly to several metabolic functions, including regulation of body weight and body composition, as well as BP and HR responses to acute stress.
Previous studies have shown that activation of MC4R in the CNS increases sympathetic activation to BAT and promotes thermogenesis (9, 19). Here, we showed that baseline V̇o2 and MTII-stimulated increases in V̇o2 were higher in MC4R-Pomc-Cre than in LoxTB-MC4R mice, which suggests that MC4R on Pomc neurons may be important in regulating SNS activity to BAT and thermogenesis. However, additional studies are needed to directly test this hypothesis.
Increased tolerance to an acute glucose load and reduced fasting plasma insulin and glucose levels observed in MC4R-Pomc-Cre mice may be explained, at least in part, by the combination of greater lean mass and reduced liver lipid infiltration in these mice. Therefore, our observations provide evidence for an important role of MC4R in Pomc neurons in glucose homeostasis and in preventing lipid deposition in nonadipose tissue. Although rescue of MC4R in Pomc neurons did not substantially alter baseline food intake after 8–10 wk of age, chronic central blockade of MC4R with SHU-9119 led to a 60–70% increase in food intake in MC4R-Pomc-Cre mice, while not changing appetite in LoxTB-MC4R mice. In addition, blockade of MC4R receptors in lean WT mice significantly increased food intake and body weight, although less than in MC4R-Pomc-Cre mice. A likely explanation for the enhanced effects of MC4R blockade on food intake and body weight in MC4R-Pomc-Cre mice is that they exhibit increased sensitivity to MC4R antagonism or an attenuated response by compensatory factors that restrain hyperphagia caused by MC4R antagonism. These possibilities, however, are only speculative and whether MC4R-Pomc-Cre mice exhibit impaired modulation of orexigenic/anorexigenic factors in response to chronic MC4R blockade is still unknown.
These observations lead us to speculate that despite not playing a major role in normal regulation of appetite in adult mice, MC4R on Pomc neurons may be important for tonic CNS melanocortin system regulation by other neurons that control food intake. Therefore, when MC4R signaling in Pomc neurons is removed, this may facilitate activation of orexigenic neurons and/or inhibition of anorexigenic neurons leading to marked increases in food intake. Further studies are necessary to address this hypothesis.
Control of Blood Pressure by MC4R on POMC Neurons
Clinical (10, 11) and experimental (4, 5, 6, 8, 22) evidence suggests that the brain melanocortin system is an important regulator of cardiovascular function. For instance, MC4R-deficient mice are not hypertensive despite severe early-onset obesity and metabolic syndrome (8, 22). Also, humans with MC4R mutations exhibit lower prevalence of hypertension and reduced 24-h norepinephrine excretion despite severe obesity (11). To test the hypothesis that MC4Rs on Pomc neurons are important for the regulation of cardiovascular function by the melanocortin system, we measured BP and HR, and their responses to acute stress in mice with MC4R rescued in Pomc neurons, as well as in mice with whole body deletion of MC4R. Although we found no differences in baseline BP and HR between groups, we noted a greater response to acute stress in MC4R-Pomc-Cre mice. This suggests that despite not playing a major role in baseline regulation of BP and HR, MC4Rs on Pomc neurons may modulate the cardiovascular responses to acute stress. Surprisingly, we also observed an increase in BP and HR in MC4R-Pomc-Cre but not in LoxTB-MC4R mice during chronic blockade of MC4R. It is possible that when MC4R signaling in Pomc neurons is removed, this may facilitate activation of excitatory and/or inhibition of inhibitory neurons, leading to an increase in SNA activity and BP. However, further studies are necessary to address this hypothesis. In addition, blockade of MC4R receptors in Pomc neurons located in the NTS of the brain stem may exert an opposite effect on BP and HR compared with the response to MC4R blockade in other brain regions, especially in the hypothalamus. Previous studies have shown that acute injection of α-MSH or MTII into the NTS produced a rapid decrease in BP and HR in anesthetized normotensive and hypertensive rats, and these responses were attenuated in a dose-dependent manner by MC4R blockade (15, 21), whereas acute or chronic lateral ventricle infusions of MC4R agonists invariably increase BP and HR in rodents (4, 5). Validation analysis of the MC4R in the brain stem revealed a high degree of colocalization (∼43%) with Pomc neurons in the NTS of GFP-MC4R mice, which supports the notion that blockade of MC4R in these Pomc neurons may have contributed to the BP and HR responses to SHU-9119 infusion. Nevertheless, our results indicate that MC4R in other neuronal populations besides Pomc neurons play a more important role in the regulation of baseline BP and HR.
Perspectives and Significance
The CNS Pomc-melanocortin system is a powerful regulator of cardiovascular and metabolic functions. Understanding the complexity of the brain melanocortin system and how cardiovascular and metabolic functions are differentially regulated by this system may lead to novel therapies for modulating appetite and energy expenditure without adverse effects on the cardiovascular system, thus providing safer ways to reduce the burden of the obesity epidemic. We have demonstrated that rescuing MC4R in Pomc neurons leads to differential control of appetite, energy expenditure, and cardiovascular function. Our results suggest that MC4R in Pomc neurons are important for regulation of appetite and energy balance and indicate that rescuing MC4R only in Pomc neurons is sufficient to augment BP and HR responses to acute stress but do not support a major role of MC4R in Pomc neurons in regulating baseline BP and HR. MC4Rs in other neurons appear to be more important for BP regulation since previous studies have shown that MC4R play a key role in mediating obesity-induced hypertension and that chronic blockade of total CNS MC4R lowers BP in models of hypertension with high SNS activity (5). The unexpected results of the present study showing that MC4R blockade actually raised BP in MC4R-Pomc-Cre suggest that the Pomc-MC4R system may play a more complex role in BP regulation than previously recognized. We speculate that activation MC4R in some neurons (e.g., in the hypothalamus) may raise BP, whereas in other neurons (e.g., NTS), MC4R activation may tend to lower BP.
This research was supported by the National Heart, Lung and Blood Institute Grant PO1-HL-51971 and by a Scientist Development Grant from the American Heart Association to Jussara M. do Carmo.
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: J.M.d.C. and A.A.d.S. conception and design of research; J.M.d.C., J.S.R., and B.R.P. performed experiments; J.M.d.C. and A.A.d.S. analyzed data; J.M.d.C., A.A.d.S., and J.E.H. interpreted results of experiments; J.M.d.C. and A.A.d.S. prepared figures; J.M.d.C. drafted manuscript; J.M.d.C., A.A.d.S., and J.E.H. edited and revised manuscript; J.M.d.C., A.A.d.S., J.S.R., B.R.P., and J.E.H. approved final version of manuscript.
We thank Haiyan Zhang for performing the insulin and leptin assays.
- Copyright © 2013 the American Physiological Society