nature publishing group
Hemorphins are small peptides generated by enzymatic cleav-
age from β-globin chains during catabolism of human hemo-
globin. These peptides exhibit biological activities that interfere
with the endorphin system, the inflammatory response, blood-
pressure control, and cognitive function (1).
VV-hemorphin-7 and LVV-hemorphin-7 globin fragments
peptides have been described to exert a hypotensive effect by
specifically inhibiting the angiotensin-converting enzyme (2,3)
and mimicking angiotensin IV cellular actions (4). We have
earlier shown decreased serum VV-hemorphin-7 like immu-
noreactivity (VVH7-i.r.) levels in different type 1 and type 2
diabetic populations (5). Furthermore, a negative correlation
between serum VVH7-i.r. level and diastolic blood pressure
(DBP) has been found in non-albuminuric patients with type
1 diabetes, suggesting that decreased VVH7-i.r. may influence
vascular and renal complications in diabetes (5).
Unfortunately, the mechanisms involved in the reduction
of VVH7-i.r. concentration in diabetes remain unexplained.
Some hypotheses, such as interference of the glycation phe-
nomenon at the cleavage site of the hemoglobin β chain (6),
or dysfunction of enzymes involved in hemorphin production
(cathepsin D) or catabolism (dipeptidyl peptidase IV and angi-
otensin-converting enzyme) have been ruled out (5).
Because obesity and type 2 diabetes share insulin resistance,
high glomerular-filtration rate (GFR), microinflammation,
and high cardiovascular risk, we investigated whether obesity
could also exhibit such hemorphin abnormalities.
SubjectS and MethodS
The obese group was composed of 54 adult obese subjects (BMI ≥30 kg/
m2) attending our outpatient clinic Nutrition department. Obese sub-
jects were selected with an absence of diabetes (diagnostic criteria for
type 2 diabetes as defined by the American Diabetes Association) and
were not being treated for hypertension. The control group was com-
posed of 33 healthy adult normal-weight subjects (18.5 ≤ BMI <25 kg/
m2) issued from the local Blood Donor Center Bank. Subjects with
chronic hemolysis, attested by low haptoglobin levels, were excluded.
Blood samples were collected after an overnight fast. Sera were imme-
diately separated by centrifugation (3,500 rpm, 15 min, 4 °C) and stored
at −80 °C. Systolic blood pressure and DBP were measured in the sitting
position after 15 min of rest using a Dynamap monitor (Dinamap pro
1000; GE Healthcare, Freiburg, Germany). Mean blood pressure was
calculated as the result of the following equation: DBP + 1/3 (systolic
The first two authors contributed equally to the work.
1APHM, La Timone Hospital, Department of Nutrition, Metabolic Diseases and Endocrinology, Marseille, France; 2Aix-Marseille Univ, UMR 1062 INSERM/1260 INRA
(NORT), Marseille, France; 3University of la Rochelle, UMR 7266 CNRS-URL (LIENSS), AMES team, Pole of Science and Technology, La Rochelle, France; 4APHM,
La Timone Hospital, Biostatistics Research Unit, Marseille, France; 5Aix-Marseille Univ, UMR 912 (SESSTIM), Marseille, France.. Correspondence: Bernard Vialettes
Received 3 November 2011; accepted 31 May 2012; advance online publication 00 Month 2012. doi:10.1038/oby.2012.186
Serum Hemorphin-7 Levels Are Decreased
Marie Maraninchi1,2, Delphine Feron3, Ingrid Fruitier-Arnaudin3, Audrey Bégu-Le Corroller1,2,
Juan P. Nogueira1,2, Julien Mancini4,5, René Valéro1,2, Jean M. Piot3 and Bernard Vialettes1,2
Hemorphin peptides exhibit biological activities that interfere with the endorphin system, the inflammatory response,
and blood-pressure control. VV-hemorphin-7 and LVV-hemorphin-7 peptides exert a hypotensive effect, in particular,
by inhibiting the renin–angiotensin system. Furthermore, levels of circulating hemorphin-7 peptides have been found
to be decreased in diseases such as type 1 and type 2 diabetes. Because type 2 diabetes and obesity share common
features, such as insulin resistance, microinflammation, high glomerular-filtration rate (GFR), and cardiovascular risk,
we evaluated serum VV-hemorphin-7 like immunoreactivity (VVH7-i.r.) levels, using an enzyme-linked immunosorbent
assay method, on a group of 54 obese subjects without diabetes or hypertension, compared with a group of
33 healthy normal-weight subjects. Circulating VVH7-i.r. levels were significantly decreased in the obese group
compared with the control group (1.98 ± 0.19 vs. 4.86 ± 0.54 μmol/l, respectively, P < 0.01), and a significant negative
correlation between VVH7-i.r. and diastolic blood pressure (DBP) was found in obese patients (r = −0.35, P = 0.011).
There was no significant correlation between VVH7-i.r. level and insulin resistance, metabolic syndrome, or GFR.
The decreased serum hemorphin-7 found in obese subjects, as in diabetes, may contribute to the development of
hypertension and to the cardiovascular risk associated with these metabolic diseases.
Obesity (2012) doi:10.1038/oby.2012.186
Insulin resistance was estimated using the quantitative insulin-
sensitivity check index (7). Metabolic syndrome was defined by the
International Diabetes Federation criteria. GFR was estimated using the
modification of diet in renal disease equation (8).
This study was conducted in accordance with the Declaration of
Helsinki and approved by the Ethics Committee of Marseille. Written
informed consent was obtained from all patients.
VVh7-i.r. serum level measurement by competitive antigen
enzyme-linked immunosorbent assay
Serum VVH7-i.r. was measured by an enzyme-linked immunosorbent
assay method as described earlier (5). A 96-well enzyme-linked immuno-
sorbent assay plate was coated overnight at 4 °C with the antigen VVH7
(Altergen, Strasbourg, France) at a final concentration of 2.6 × 10−9
mol/l. Then, the wells were blocked by a solution of PBS-BSA 1% for 2 h.
Rabbit polyclonal antibodies, raised against the C-part of VVH7 (diluted
1:10,000), and antirabbit immunoglobulin G peroxidase conjugate sec-
ondary antibody (diluted 1:20,000) were used sequentially. Thereafter,
tetramethyl-benzidine liquid substrate was added and the reaction was
stopped with H2SO4 0.5 mol/l. The absorbance was read at 450 nm.
To determine serum VVH7-i.r. concentrations, a standard range of
synthetic VVH7 was made, which included concentrations from 10−13 to
10−5 mol/l. The ratio between the absorbance at 450 nm in the presence
(B) and absence (Bmax) of VVH7, against a logarithmic plot concentra-
tion of VVH7, displayed a typical calibration graph. The IC50 (i.e., the
concentration of peptides able to inhibit 50% of coated antigen–antibody
binding) was 6.87 × 10−7 mol/l.
Plasma glucose was assayed using the hexokinase oxidase method
(Beckman Coulter, Galway, Ireland) and plasma insulin levels
were assessed using an electrochemiluminescence method (Roche
Diagnostic, Mannheim, Germany). Total cholesterol, high-density
lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and trig-
lycerides were determined using enzymatic methods (CHOD-PAP,
high-density lipoprotein-c plus, and GPOPAP, respectively, Roche,
Grenoble, France). Plasma creatinine was assessed by the Jaffé method
(Beckman Coulter, Galway, Ireland). Haptoglobin and lactic-dehy-
drogenase activity were measured by the nephelometric method and
the ultraviolet test, respectively (Beckman Coulter, Galway, Ireland).
Results were expressed as means ± s.e.m. Nonparametric statistical tests
were used (Mann–Whitney test and Spearman’s rank correlation test).
A P value <0.05 was considered significant for all the analyses.
The main characteristics of the obese population are described
in Supplementary Table S1 online.
Serum VVH7-i.r. was significantly decreased in the obese
group compared with the control group (1.98 ± 0.19 vs. 4.86
± 0.54 μmol/l, respectively, P < 0.01) (Figure 1a). Age (35.85 ±
1.60 vs. 45.3 ± 2.07 years, respectively) and gender (44 women
and 10 men vs. 18 women and 15 men, respectively) did not
influence VVH7-i.r. levels in either group.
In the obese group, we found a significant negative correla-
tion between VVH7-i.r. level and DBP (r = −0.35, P = 0.011)
(Figure 1b) but not with systolic blood pressure (P = 0.29) or
mean blood pressure (P = 0.07). We found no significant cor-
relation between VVH7-i.r. levels and BMI (P = 0.10) or adi-
pose tissue distribution (waist circumference (W), P = 0.34; hip
circumference (H), P = 0.06; W/H ratio, P = 0.89; or waist-to-
height ratio, P = 0.28). VVH7-i.r. levels showed no significant
difference between the obese subgroup that exhibited meta-
bolic syndrome (40% prevalence of the total obese population)
and the obese subgroup without this phenotype (1.83 ± 0.32
vs. 2.03 ± 0.26 μmol/l, respectively, P = 0.43). Insulin sensitiv-
ity, assessed by fasting insulin levels (P = 0.45) and quantitative
insulin-sensitivity check index (P = 0.33), GFR (P = 0.28) or cir-
culating lipid parameters, such as triglycerides (P = 0.45), total
cholesterol (P = 0.52), high-density lipoprotein (P = 0.10), and
low-density lipoprotein-cholesterol (P = 0.73), were not related
to VVH7-i.r. serum concentrations in our obese population.
In this study, we have shown, for the first time, a significant
decrease of serum hemorphin-7 in obese subjects compared
with healthy control subjects, which is similar to the reduction
already described in subjects with type 1 and 2 diabetes. Only
a few data are available on hemorphin-7 peptides in human
pathological situations. Levels of circulating hemorphin-7
peptides have also been found to be decreased in breast cancer
(9), but increased in patients with an abdominal aortic aneu-
rysm (10). Our study shows a significant negative correlation
between VVH7-i.r. and DBP in the obese population, which is
similar to that described previously in non-albuminuric type
1 diabetic patients (5), and reinforces the hypothesis that an
abnormal decrease in VVH7-i.r indicates susceptibility to the
development of hypertension and cardiovascular disease.
Hemorphins are known to interfere with the renin–
angiotensin system. In vitro, VV- and LVV-hemorphin-7 pep-
tides exert competitive inhibition on angiotensin-converting
enzyme activity (3). Hemorphins are also a ligand of the
insulin-regulated amino peptidase/angiotensin-4 receptor and
reproduce angiotensin IV cellular actions that induce vasodila-
tation (2,4). Moreover, LVV-hemorphin-7 injections in hyper-
tensive rats reduce blood pressure and heart rate (11). These
converging observations suggest that hemorphin-7 peptides
could represent natural hypotensive substances. One could
postulate that the decrease of circulating VVH7-i.r. in obese
and diabetic patients contributes to increased DBP by interfer-
ing with the renin–angiotensin system.
The mechanisms of hypertension in obesity are complex.
Sympathetic nervous activation, impaired pressure-regulated
natriuresis, hormonal and adipocytokine anomalies, and
endothelial dysfunction have been implicated. Activation
of renin–angiotensin system seems to play a central role.
Renin–angiotensin system is activated in obesity despite
plasma volume expansion and sodium retention. In addition
to renal overproduction of renin, adipose tissue produces angi-
otensinogen, which could also increase blood pressure.
The decrease of hemorphin-7 serum levels in obese and
diabetic patients is a new mechanism and may be involved in
some of the pathways leading to hypertension.
The hypothesis that insulin resistance is shared by both diseases
and could be the common denominator has failed. Whatever the
marker is for insulin resistance, it is not related to the VVH7-i.r.
values. Another hypothesis is that high GFR, observed in both
diseases (12), could produce renal escape of hemorphins into the
Diastolic blood pressure (mm Hg)
Control subjects Obese subjects
P < 0.001
Figure 1 Decrease of circulating VV-hemorphin-7 like immunoreactivity (VVH7-i.r.) levels and negative correlation between VVH7-i.r. and diastolic
blood pressure (DBP) in obese subjects. (a) Distribution of VVH7-i.r. in the serum of control and obese subjects, using a box-plot representation.
The median is indicated by a thick black line inside the box. The top and bottom of the box are the upper and lower quartiles and indicate the
interquartile range (IQR). The whiskers indicate the lowest datum still within 1.5 IQR of the lower quartile, and the highest datum still within 1.5 IQR
of the upper quartile. Outliers (between 1.5 and 3 times the IQR) are marked with a circle and extreme values (more than three times the IQR) with
an asterisk. (b) Correlation between VVH7-i.r. serum levels and DBP in the obese group. Spearman correlation coefficient was determined to assess
the significant association between parameters (r = −0.351, P = 0.011).
urine, thus leading to its decrease in serum. Again, the absence
of a correlation between VVH7-i.r. level and creatinine clearance
has eliminated this hypothesis. Low-grade inflammation sta-
tus, which occurs in both diabetes and obesity, could represent
another hypothesis that remains to be explored, but requires a
larger population study. The association of reduced hemorphin
status with very disparate diseases such as diabetes, obesity and
breast cancer, could suggest that mechanisms shared by these
diseases as endothelial dysfunction and angiogenesis could lead
to the hemorphin anomaly. Indeed, Perrot-Applanat et al. has
shown that the nuclear factor κB gene signature is highly simi-
lar between tumor necrosis factor α stimulated endothelial cells
and breast tumor biopsies (13). It is possible that tumor necrosis
factor α activation is the common denominator governing these
processes and the decrease of hemorphin in these apparently
unrelated diseases. We have also hypothetized in the past that
hemorphin decrease in blood could be related to lysosomial and/
or extralysosomial protease hyperactivities. Indeed, such anoma-
lies of either cathepsin S or 20S proteasome have been described
in obesity (14) and breast cancer (15), respectively.
In conclusion, decreased serum hemorphin-7 is a com-
mon feature in metabolic diseases such as diabetes and obes-
ity, and could contribute to the risk of future development
of hypertension and, potentially, of cardiovascular and renal
Supplementary material is linked to the online version of the paper at http://
This study was supported by a grant from the French Health Ministry (PHRC-
2005-R24-07). The authors wish to thank the medical and paramedical
teams of the Nutrition, Metabolic Diseases and Endocrinology Department
of University Hospital “La Timone” (AP-HM) for their technical assistance.
The authors declared no conflict of interest.
See the online ICMJE Conflict of Interest Forms for this article.
© 2012 The Obesity Society
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