|
|
| |||||||||||||||||||||||||||||||||||||||||
Background Weight gain and weight loss are associated with changes in blood pressure through unknown mechanisms. Central melanocortinergic signaling is implicated in the control of energy balance and blood pressure in rodents, but there is no information regarding such an association with blood pressure in humans.
Methods We assessed blood pressure, heart rate, and urinary catecholamines in overweight or obese subjects with a loss-of-function mutation in MC4R, the gene encoding the melanocortin 4 receptor, and in equally overweight control subjects. We also examined the effects of an MC4R agonist administered for 7 days in 28 overweight or obese volunteers.
Results The prevalence of hypertension was markedly lower in the MC4R-deficient subjects than in the control subjects (24% vs. 53%, P=0.009). After the exclusion of subjects taking antihypertensive medications, blood-pressure levels were significantly lower in MC4R-deficient subjects than in control subjects, with mean (±SE) systolic blood pressures of 123±14 mm Hg and 131±12 mm Hg, respectively (P=0.02), and mean diastolic blood pressures of 73±10 mm Hg and 79±7 mm Hg, respectively (P=0.03). As compared with control subjects, MC4R-deficient subjects had a lower increase in heart rate on waking (P=0.007), a lower heart rate during euglycemic hyperinsulinemia (P<0.001), and lower 24-hour urinary norepinephrine excretion (P=0.04). The maximum tolerated daily dose of 1.0 mg of the MC4R agonist led to significant increases of 9.3±1.9 mm Hg in systolic blood pressure and of 6.6±1.1 mm Hg in diastolic blood pressure (P<0.001 for both comparisons) at 24 hours, as compared with placebo. Differences in blood pressure were not explained by changes in insulin levels; there were no significant adverse events.
Conclusions Results of our genetic and pharmacologic studies implicate melanocortinergic signaling in the control of human blood pressure through an insulin-independent mechanism.
The hypothalamic leptin–melanocortin pathway is critically involved in the control of energy balance, and genetic disruption of molecules in this pathway leads to severe obesity in rodents and humans.6 A key component of this pathway is the melanocortin system, which includes leptin-responsive neurons expressing neuropeptide Y and agouti-related-protein and those expressing proopiomelanocortin, which is cleaved to produce the melanocyte-stimulating hormones (MSH) (alpha-, beta-, and gamma-MSH).7 Downstream targets of these neurons express the melanocortin 3 receptor (MC3R) and the melanocortin 4 receptor (MC4R). Melanocortins are agonists of MC3R and MC4R, whereas agouti-related-protein is a high-affinity antagonist.8
Studies in rodents suggest that the melanocortin system is important in cardiovascular regulation.9 Acute central administration of alpha-MSH increases mean arterial pressure and heart rate.10 Pharmacologic studies indicate that this effect is attributable to activation of the sympathetic nervous system.11 Chronic pharmacologic blockade of MC3R and MC4R causes weight gain and a reduction in heart rate but no increase in arterial pressure.12 These pressor and depressor effects are mediated predominantly by signaling through MC4R-expressing neuronal pathways, because Mc4r-null mice maintain a normal blood pressure despite marked obesity and are unresponsive to the pressor effects of central alpha-MSH administration.10
MC4R deficiency is the most common form of inherited human obesity, with a mutation prevalence of approximately 6% in subjects with severe early-onset obesity,13 2.5% in unselected obese adults,14,15 and 0.1% in a population-based study of unselected subjects.16 We have previously described the clinical characteristics of human MC4R deficiency.13 Many of these phenotypes are closely replicated in the Mc4r-deficient mouse, suggesting that it is an excellent model for the human disease.17
To examine the effects of loss of function of the melanocortin pathway on blood pressure in humans, we studied adults who were heterozygous for complete loss-of-function mutations in MC4R (i.e., with haploinsufficiency) and equally overweight or obese control subjects who did not have MC4R mutations. To examine the effects of increased signaling through melanocortin receptors, we studied the short-term effects of a melanocortin receptor agonist in overweight or obese volunteers.
Methods
Physiologic Studies in MC4R-Deficient Subjects
We analyzed blood pressure and metabolic data for 46 MC4R-deficient adults from 32 nuclear families identified as part of the Genetics of Obesity Study.13 All subjects were heterozygous for complete loss-of-function mutations in MC4R (for details, see Table 1 in the Supplementary Appendix, available with the full text of this article at NEJM.org). We recruited 30 adult volunteers whom we determined to be overweight, which was defined as having a body-mass index (BMI, the weight in kilograms divided by the square of the height in meters) of more than 25, or obese (BMI, >30) and who were shown to have a normal MC4R genetic sequence. All subjects provided written informed consent.
Studies were conducted at the Wellcome Trust Clinical Research Facility at Addenbrooke's Hospital in accordance with the principles of the Declaration of Helsinki after approval by the ethics committee at Addenbrooke's Hospital. Eight MC4R-deficient adults of European descent and eight control subjects who were matched in age, ancestral background, and BMI agreed to undergo more detailed studies (Table 2 in the Supplementary Appendix). European descent was determined by the reported country of birth of at least three generations of family members. Details of measurement of blood pressure, autonomic nervous system activation, body composition, measurement of abdominal and liver fat by magnetic resonance imaging, hyperinsulinemic–euglycemic and hyperglycemic clamp studies, along with analytical methods, definitions, and calculations, are provided in the Methods section in the Supplementary Appendix.
Pharmacologic Study in Overweight or Obese Volunteers
We conducted a double-blind, dose-escalating, crossover trial of an MC4R agonist, LY2112688 (Eli Lilly), as compared with placebo, in 28 healthy men and women who were overweight or obese and whose weight had been stable during the previous 6 months (Table 3 in the Supplementary Appendix). The MC4R agonist was administered in two doses (0.45 mg and 1.0 mg) as a subcutaneous infusion over a 24-hour period. After each 24-hour infusion of either the MC4R agonist or placebo, there was a washout interval of 3 to 6 days before the next round of infusions was started in the same subjects. Seven-day infusions of the MC4R agonist at one dose or infusions of placebo were also administered with a washout interval of 7 days in a crossover design. Medications that were known to alter appetite, blood pressure, and autonomic function were prohibited.
Studies were conducted in the Clinical Research Unit at Biotrial in Rennes, France, under Good Clinical Practice guidelines as part of a program for the development of drugs targeting obesity. The study protocol was approved by the ethics review board (Comité de Protection des Personnes de Brest, France). Details regarding the study design, methods for measurement of blood pressure and biochemical values, statistical analysis, and a synopsis of the protocol are provided in the Methods section in the Supplementary Appendix.
Results
Blood Pressure and MC4R Deficiency
We studied the blood pressure and metabolic phenotype in 46 adults with MC4R haploinsufficiency and in 30 control subjects with a normal MC4R genotype (Table 1). The prevalence of hypertension was significantly lower in the MC4R-deficient group than in the control group (Figure 1A). After the exclusion of subjects receiving antihypertensive medications, the mean systolic and diastolic blood-pressure levels were lower in MC4R-deficient adults than in control subjects (Figure 1B and 1C). The prevalence of diabetes was similar in the MC4R-deficient group and the control group (18% and 20%, respectively). After the exclusion of subjects with diabetes, fasting plasma glucose levels were similar in the two groups (Table 1). Consistent with our previous observation that the relative hyperinsulinemia associated with childhood MC4R deficiency becomes less marked with age,13 there was no significant difference in fasting plasma insulin levels between the two groups (Table 1).
|
|
To examine autonomic nervous system function, we measured variability in heart rate in MC4R-deficient subjects and in control subjects (Table 2 in the Supplementary Appendix). There was no difference in sleeping heart rate between the two study groups (Figure 2A). On waking, the heart rate increased in the two groups; however, the magnitude of this increase was significantly attenuated in MC4R-deficient subjects (P=0.007) (Figure 2A). Infusion of insulin increased the heart rate in the two groups. Although the magnitude of the increase was similar, MC4R-deficient subjects maintained significantly lower heart rates throughout the clamp procedure, with a difference of 7 bpm between the two groups during the steady-state period (P<0.001) (Figure 2A).
|
The root mean square of successive differences (RMSSD) between adjacent normal RR intervals, an index of parasympathetic activity, was similar in MC4R-deficient subjects and control subjects during sleep (Figure 2C). The RMSSD decreased in response to waking in control subjects, but there was no change in MC4R-deficient subjects. Infusion of insulin led to a reduction in the RMSSD values in both groups, although the values were higher in MC4R-deficient subjects throughout the hyperinsulinemic–euglycemic clamp procedure (P=0.002) (Figure 2C).
Urinary Catecholamine Excretion
We measured 24-hour urinary catecholamine excretion in 10 adults with haploinsufficiency for MC4R with a mean (±SD) age of 33.8±13.6 years and a mean BMI of 43±7.5 and in 19 control subjects with a mean age of 39.8±8.5 years and a mean BMI of 37.2±3.8. MC4R-deficient subjects had lower 24-hour norepinephrine excretion than did the control subjects (199±23 nmol and 291±28 nmol, respectively; P<0.05) (Figure 3A). Although 24-hour urinary epinephrine excretion also tended to be lower in MC4R-deficient subjects, the difference was not significant (Figure 3B). There was no significant difference in 24-hour dopamine excretion between the two groups (Figure 3C). Nonparametric tests gave the same results for these comparisons.
|
We measured body composition, fat distribution, insulin secretion, and insulin sensitivity to ascertain whether there might be other phenotypic signatures associated with MC4R deficiency. MC4R-deficient subjects and control subjects had similar percentages of total body fat and fat-free mass, intraabdominal adipose tissue area, subcutaneous abdominal adipose tissue area, and liver fat (Table 4 in the Supplementary Appendix).
During the hyperinsulinemic–euglycemic clamp procedure in MC4R-deficient subjects and control subjects, there were similar mean (±SE) steady-state levels of blood glucose (5.0±0 mmol per liter in the two groups) and geometric mean steady-state levels of plasma insulin (1308 pmol per liter [1-SE range, 1231 to 1390] and 1299 pmol per liter [1-SE range, 1217 to 1386], respectively). During steady state, there was no significant difference between the two groups in insulin-mediated glucose uptake, with similar rates of insulin-stimulated oxidative and nonoxidative glucose disposal (Figure 4A). Posthepatic insulin clearance rates were also similar (data not shown). Reduced activity in the sympathetic nervous system would be expected to increase insulin secretion in the MC4R-deficient subjects. Although there was a trend in this direction, there was no significant difference between MC4R-deficient subjects and control subjects in geometric mean levels of first-phase insulin secretion (614 pmol per liter [1-SE range, 471 to 801] and 474 pmol per liter [1-SE range, 368 to 612], respectively) or second-phase insulin secretion (405 pmol per liter [1-SE range, 332 to 494] and 332 pmol per liter [1-SE range, 272 to 405], respectively) during the intravenous glucose tolerance test (Figure 4B). Results were similar when insulin responses were compared with the use of area-under-the-curve analysis and repeated-measures analysis of variance (data not shown). Geometric mean levels of second-phase insulin secretion that was measured during the hyperglycemic clamp procedure were also similar in the two groups (944 pmol per liter [1-SE range, 760 to 1172] and 729 pmol per liter [1-SE range, 575 to 924], respectively). There was no significant difference in the disposition index (the product of insulin sensitivity and first-phase insulin secretion) between the two groups. Nonparametric tests provided the same results for these comparisons.
|
Of the 28 healthy overweight or obese subjects who were enrolled, 26 completed the study (Table 3 in the Supplementary Appendix). Steady-state plasma levels of drugs were achieved within 24 hours and were proportional to the dose. Infusion of the MC4R agonist LY2112688 resulted in symptoms known to be mediated by melanocortin pathways, such as yawning, a sensation of muscular stiffness, and increased penile erection. The need to stretch or yawn was significantly increased at all doses during the 7-day infusion. A significant increase in male erectile function was observed at the 0.45-mg and 1.0-mg daily doses only. Adverse effects, including headache, asthenia, nausea, vomiting, and diarrhea, are presented in Table 5 in the Supplementary Appendix.
There was a dose-dependent increase in blood pressure after infusion of LY2112688 in overweight or obese adults during a 24-hour period (Figure 5). Three hours after the start of infusion, the average systolic blood pressure was 4.0 to 8.5 mm Hg higher than placebo-controlled values (Figure 5A). Maximum increases from the placebo values were generally observed at 24 hours. The average difference in systolic blood pressure at 24 hours was 9.3±1.9 mm Hg for the maximum tolerated dose of 1.0 mg per day (P<0.001). Diastolic blood pressure 1 hour after the start of infusion was 2.5 to 8.2 mm Hg higher than placebo-controlled values for the dose range of 0.15 to 2.0 mg (Figure 5B). The average difference in diastolic blood pressure at 24 hours was 6.6±1.1 mm Hg for the maximum tolerated dose of 1.0 mg per day (P<0.001). Blood-pressure changes were sustained for the 7 days of LY2112688 infusion (Table 6 in the Supplementary Appendix). A decrease in mean heart rate of approximately 5 to 6 bpm was observed 1 hour after infusion in all dose groups, with the exception of the 0.45-mg dose. However, the average heart rate increased during the 7-day infusions, with an average difference of 3 bpm, as compared with placebo (Table 6 in the Supplementary Appendix). There were no significant differences in levels of plasma insulin and glucose and urinary norepinephrine after LY2112688 administration (Table 7 in the Supplementary Appendix).
|
Obesity is a major risk factor for hypertension,1 and weight loss reduces blood pressure.18 Studies in animals have indicated that induced weight gain results in increased blood pressure.19 Numerous mechanisms have been suggested,20 many of which involve the assumption that insulin resistance or hyperinsulinemia that accompanies weight gain is involved through effects on renal sodium retention, vascular structure and function, ion transport, or activity of the sympathetic nervous system.21
Melanocortinergic signaling is critical to the control of weight in humans6 and in animals.8 Studies in rodents have clearly implicated centrally expressed MC4R in the control of blood pressure.9,10 In this study, we combined observations from subjects with reduced MC4R signaling and pharmacologic studies using a centrally active melanocortinergic agonist to examine the relationship between melanocortin signaling and blood pressure in humans. We conclude that both increases and decreases in central melanocortin signaling influence blood pressure in humans and that the effects are not explained by changes in circulating insulin levels or insulin sensitivity. In the case of long-standing decreases in melanocortinergic tone, these changes are associated with reduced activity in the sympathetic nervous system.
We studied a clearly defined group of subjects with MC4R haploinsufficiency. Because some of these subjects were related to each other, they may share genetic factors other than mutated MC4R that contribute to blood-pressure regulation. Careful matching of control subjects to MC4R-deficient subjects with respect to BMI, age, and ancestry revealed striking differences both in the prevalence of hypertension and in mean levels of systolic and diastolic blood pressure. The prevalence of hypertension and mean blood pressures seen in the control group were very similar to those of unselected overweight or obese subjects,2 a finding that supported our assertion that the MC4R-deficient subjects were relatively protected from the expected relationship between overweight and raised blood pressure.
We studied a synthetic peptide agonist that is highly selective for MC4R. The drug, MC4R agonist LY2112688, had an increased affinity for MC4R over MC1R of more than a factor of 30 and over MC3R of more than a factor of 100. Administration of the MC4R agonist to overweight or obese volunteers produced the classic symptoms of yawning, stretching, and penile erection associated with activation of central MC4R and a marked short-term increase in blood pressure that was sustained for the 7 days of the drug's administration. The average increase in systolic and diastolic blood pressure in these studies was quantitatively similar to the difference in blood pressure between MC4R-deficient subjects and control subjects.
A deficiency in MC4R (in both children13 and mice22) appears to be associated with hyperinsulinemia at a rate that is disproportionate to the degree of obesity, and several studies have implicated hyperinsulinemia as a link between obesity and hypertension.21 Our finding that MC4R deficiency is associated with reduced, and not increased, blood pressure does not support such a link. In addition, we could find no difference in measures of insulin sensitivity and intraabdominal fat between MC4R-deficient subjects and control subjects. Finally, the administration of the MC4R agonist for 7 days did not significantly alter fasting insulin levels, despite having a marked effect on blood pressure.
Central control of the sympathetic nervous system can clearly influence blood pressure,23 and the administration of an MC4R agonist in mice is associated with increased activity of the sympathetic nervous system.24,25 The rarity of finding subjects with MC4R deficiency without other coexisting illnesses and the complexity of our protocol made it impossible to incorporate direct electrophysiological studies of activity in the sympathetic nervous system. However, surrogate measures support such an effect. Although the sleeping heart rate (under strong vagal influence) was similar in MC4R-deficient subjects and control subjects, the increases in heart rate on waking and during a hyperinsulinemic clamp procedure were significantly lower in the MC4R-deficient subjects than in the control subjects. Also, urinary norepinephrine levels were significantly reduced in the MC4R-deficient subjects. The urinary norepinephrine level is usually thought to be a relatively crude measure of activity in the sympathetic nervous system and is useful only in large populations, but we believe that it is all the more striking that we observed significant differences in relatively small numbers of subjects, presumably because the effect of MC4R haploinsufficiency in this carefully defined group was both substantial and consistent. This pattern has been observed in persons with recent weight loss, in whom activation of the autonomic nervous system has been more directly quantified.26 Finally, direct measures of sympathetic nerve activity are unlikely to have provided additional information, because studies involving animals have shown that sympathetic nerve output to the renal area may be most relevant to the control of blood pressure.27,28
Despite a significant increase in heart rate, we did not observe a significant increase in levels of urinary norepinephrine in the pharmacologic study, although we saw a nonsignificant trend for an increase in this measure. The explanation for this lack of symmetry with the genetic studies is unclear but may imply that discernible effects on this measure require alterations in signaling through MC4R of a longer duration or that the effect of LY2112688 on other melanocortin receptors might modify the effects that we observed.
In conclusion, we have demonstrated that central melanocortinergic tone significantly influences blood pressure in humans. The therapeutic use of a melanocortin agonist in common human obesity may be complicated by the increase in blood pressure, as seen in our short-term studies. However, the effects of administration of a melanocortin agonist to patients with MC4R haploinsufficiency would be of great interest. Given the effects of MC4R tone on blood pressure, we conclude that the well-established effects of weight loss and obesity on human blood pressure may involve alterations in central signaling at MC4R, mediated through changes in sympathetic neural activity.
Supported by grants from the National Health and Medical Research Council of Australia, the Royal Australasian College of Physicians, and St. Vincent's Clinic Foundation, Sydney (to Dr. Greenfield); from the Wellcome Trust and the Cambridge Biomedical Research Centre of the National Institute for Health Research (to Drs. O'Rahilly and Farooqi); and from the Medical Research Council (to Drs. O'Rahilly, Brage, and Farooqi); and by Eli Lilly for the pharmacologic studies.
Drs. Miller, Satterwhite, Cameron, and Mayer report being employed by Eli Lilly and having an equity interest in the company; and Dr. O'Rahilly, receiving consulting fees from Eli Lilly. No other potential conflict of interest relevant to this article was reported.
We thank Adrian Dixon, Sri Aitken, and the radiography staff at Addenbrooke's Hospital for their assistance with imaging studies; Fiona Tulloch and Keith Burling, who performed the biochemical assays, and David Moller for helpful discussions; the patients and volunteers for their participation; and the physicians involved in the Genetics of Obesity Study.
Source Information
From the University of Cambridge Metabolic Research Laboratories (J.R.G., J.M.K., E.H., S.O., I.S.F.) and Medical Research Council Epidemiology Unit (S.B.), the Institute of Metabolic Science and the Department of Radiology (T.C.S., D.J.L.), Addenbrooke's Hospital, Cambridge, United Kingdom; Eli Lilly, Lilly Corporate Center, Indianapolis (J.W.M., J.H.S., G.S.C., J.P.M.); and Biotrial, Rennes, France (B.A.).
This article (10.1056/NEJMoa0803085) was published at NEJM.org on December 17, 2008.
Address reprint requests to Dr. Farooqi at the Institute of Metabolic Science, Metabolic Research Laboratories, Addenbrooke's Hospital, Hills Rd., Box 289, Cambridge CB2 0QQ, United Kingdom, or at isf20{at}cam.ac.uk.
References
| |||||||||||||||||||||||||||||||||||||||||
This article has been cited by other articles:
HOME | SUBSCRIBE | SEARCH | CURRENT ISSUE | PAST ISSUES | COLLECTIONS | PRIVACY | TERMS OF USE | HELP | beta.nejm.org Comments and questions? Please contact us. The New England Journal of Medicine is owned, published, and copyrighted © 2010 Massachusetts Medical Society. All rights reserved. |
Previous
