Restoring leptin signaling reduces hyperlipidemia and improves vascular
stiffness induced by chronic intermittent hypoxia
Ronghua Yang,*1Gautam Sikka,*2Jill Larson,1Vabren L. Watts,1Xiaolin Niu,1Carla L. Ellis,3
Karen L. Miller,1Andre Camara,2Christian Reinke,4Vladimir Savransky,4Vsevolod Y. Polotsky,4
Christopher P. O’Donnell,5Dan E. Berkowitz,2and Lili A. Barouch1
Departments of1Medicine, Division of Cardiology,2Biomedical Engineering,3Pathology, and4Division of Pulmonary
and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland;5Division of Pulmonary, Allergy,
and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Submitted 6 July 2009; accepted in final form 13 January 2011
Yang R, Sikka G, Larson J, Watts VL, Niu X, Ellis CL,
Miller KL, Camara A, Reinke C, Savransky V, Polotsky VY,
O’Donnell CP, Berkowitz DE, Barouch LA. Restoring leptin
signaling reduces hyperlipidemia and improves vascular stiffness
induced by chronic intermittent hypoxia. Am J Physiol Heart Circ
Physiol 300: H1467–H1476, 2011. First published January 28,
hypoxia (IH) during sleep can result from obstructive sleep apnea
(OSA), a disorder that is particularly prevalent in obesity. OSA is
associated with high levels of circulating leptin, cardiovascular dys-
function, and dyslipidemia. Relationships between leptin and cardio-
vascular function in OSA and chronic IH are poorly understood. We
exposed lean wild-type (WT) and obese leptin-deficient ob/ob mice to
IH for 4 wk, with and without leptin infusion, and measured cardio-
vascular indices including aortic vascular stiffness, endothelial func-
tion, cardiac myocyte morphology, and contractile properties. At
baseline, ob/ob mice had decreased vascular compliance and endo-
thelial function vs. WT mice. We found that 4 wk of IH decreased
vascular compliance and endothelial relaxation responses to acetyl-
choline in both WT and leptin-deficient ob/ob animals. Recombinant
leptin infusion in both strains restored IH-induced vascular abnormal-
ities toward normoxic WT levels. Cardiac myocyte morphology and
function were unaltered by IH. Serum cholesterol and triglyceride
levels were significantly decreased by leptin treatment in IH mice, as
was hepatic stearoyl-Coenzyme A desaturase 1 expression. Taken
together, these data suggest that restoring normal leptin signaling can
reduce vascular stiffness, increase endothelial relaxation, and correct
dyslipidemia associated with IH.
sleep apnea; heart; hypertrophy; vascular compliance; pulse-wave
velocity; endothelial function; nitric oxide
OBSTRUCTIVE SLEEP APNEA (OSA) is increasingly being recognized
as a risk factor in the obesity-related pathogenesis of cardiovas-
cular disease. OSA independently contributes to the development
of systemic and pulmonary hypertension (21, 39), right and left
ventricular dysfunction (9, 10), and coronary artery disease and
stroke (2), all of which are associated with failure of endothelium-
dependent vasodilation (8, 13). Chronic intermittent hypoxia (IH),
which has independently been shown to increase systemic arterial
hypertension in rodents (14, 24) and humans (44), is a primary
clinical feature of OSA. Our previous studies using a mouse
model of IH reproduced sleep-related hypoxemia and sleep frag-
mentation comparable with the levels found in OSA (22, 35) and
demonstrated that these animals develop moderate systemic and
pulmonary hypertension and left- and right-sided ventricular hy-
Chronic IH-induced cardiovascular remodeling is thought to
result from interactions between hemodynamic and inflamma-
tory changes. The adipocytokine leptin has been a focus of
these investigations as a result of the role of leptin in sympa-
thetic activation (29), nitric oxide (NO) production (23, 30),
inflammation, and oxidative stress, which can all affect cardio-
vascular function. Circulating leptin concentration is generally
proportional to body fat mass, although studies have suggested
that leptin levels are further elevated in OSA patients compared
with weight-matched controls (12, 18) with a proportional
increase with OSA severity (45). Whether hyperleptinemia in
obesity and OSA serves a protective role or causes detrimental
effects in the cardiovascular system is still under debate. While
some suggest that elevated leptin induces oxidative stress and
sympathetic overactivation (20, 42), others have found the
insulin-sensitizing and lipid lowering actions of leptin to be
cardioprotective (31, 46).
We have previously shown that vascular and myocardial
abnormalities in leptin-deficient ob/ob animals are attributable
to the disruption of intact leptin signaling independent of body
weight (4, 28, 43). Because IH exposure leads to exacerbation
of well-established cardiovascular risk factors of hyperlipid-
emia and insulin resistance (17, 26), and ob/ob animals lack
leptin-mediated protection against these developments, we hy-
pothesized that IH may disrupt normal leptin signaling in WT
and result in worsening of lipid-induced injury in ob/ob,
leading to further exacerbation of cardiovascular function. We
predicted that leptin repletion could rescue such defects in both
WT and ob/ob. Moreover, the inhibitory actions of leptin on
fatty acid synthesis may also offer cardiovascular benefits in
lean WT mice exposed to IH. To test these hypotheses, we
treated C57BL6J wild-type (WT) and ob/ob animals with IH
vs. room air control (RA), with or without leptin, resulting in
eight groups of animals. We examined vascular compliance
with noninvasive Doppler detection of pulse-wave velocity
(PWV) and endothelium-dependent vasodilation response to
acetylcholine (ACh). We also measured myocyte contractility
and calcium transients in response to isoproterenol (ISO)
stimulation and evaluated myocyte histology and morphome-
MATERIALS AND METHODS
Animals and study design. Male 10-wk-old, WT C57BL/6 and
leptin-deficient ob/ob mice were purchased from The Jackson Labo-
* R. Yang and G. Sikka contributed equally to this work.
Address for reprint requests and other correspondence: L. Barouch, Johns
Hopkins Univ. School of Medicine, Div. of Cardiology, Ross 1050, 720
Rutland Ave., Baltimore, MD, 21205 (e-mail: Barouch@jhmi.edu).
Am J Physiol Heart Circ Physiol 300: H1467–H1476, 2011.
First published January 28, 2011; doi:10.1152/ajpheart.00604.2009.
0363-6135/11 Copyright © 2011 the American Physiological Society http://www.ajpheart.orgH1467
ratory (n ? 15–20 per group; Bar Harbor, ME) and housed in a
university animal facility with a 12-h:12-h light/dark cycle and al-
lowed water and food ad libitum. Subsets of both WT and ob/ob
strains were exposed to chronic IH, leptin treatment, or a combination
of the two. This gives a total of eight experimental groups: 1) WT-RA,
2) WT-IH, 3) WT-RA ? Leptin, 4) WT-IH ? Leptin, 5) OB-RA,
6) OB-IH, 7) OB-RA ? Leptin, and 8) OB-IH ? Leptin.
IH exposure followed a previously described protocol (7). Briefly,
animals exposed to IH were housed in custom cages that delivered an
intermittent hypoxic stimulus [5–21% Fi(O2), 30-s cycles during the
12-h light cycle]. A constant flow of RA was delivered to the cages
during the dark cycle. The average arterial partial pressure of O2over
each hypoxic cycle under this protocol was previously determined to
be 51.7 ? 4.2 mmHg, whereas air delivered at the same rate as
hypoxic gases produced an average arterial partial pressure of O2of
99.7 ? 2.1 mmHg (41). IH or RA exposure lasted for 4 wk. Mouse
leptin (Amgen, Thousand Oaks, CA) was delivered at 0.3 mg/kg per
day via osmotic minipumps (Alzet, Cupertino, CA) that were surgi-
cally implanted 1 day before commencing hypoxia exposure as
previously described (4). At the end of the protocol, animals under-
went noninvasive studies before being euthanized. They were hepa-
rinized (0.1 ml heparin sodium sc) for 10 min and then anesthetized
with isoflurane before the hearts and aortas were excised for tissue
collection or cardiac myocyte isolation/aortic ring sectioning. Animal
treatment and care was provided in accordance with institutional
guidelines. The Institutional Animal Care and Use Committee of The
Johns Hopkins University School of Medicine approved all protocols
and experimental procedures.
Serum lipid biomarker determinations. Blood samples were ob-
tained by cardiac puncture at the time of euthanasia and centrifuged at
12,000 g for 10 min to separate plasma for measurements. Leptin was
assayed in triplicate by a sandwich ELISA kit specific for the detec-
tion of mouse leptin (Millipore, Billerica, MA) per the manufacturer’s
instructions. Fasting serum total cholesterol (TC) and triglyceride
(TG) levels were determined with kits from Wako Diagnostics and
serum free fatty acid (FFA) levels with a FFA Quantification Kit from
Biovision. Malondialdehyde (MDA) levels in plasma were deter-
mined using a colorimetric assay kit (Oxis International, Beverly
Hills, CA) on the basis of reaction with N-methyl-2-phenylindole.
Data were normalized to WT-RA as percentage of increase of MDA
Assessment of vascular compliance using PWV. Animals were
anesthetized with isoflurane (5% for induction and 1.5% for mainte-
nance in 100% oxygen) and positioned supine on a temperature-
controlled printed circuit board (Indus Instruments, Houston, TX)
with legs strapped to electrocardiogram electrodes. Body temperature
was monitored with a rectal probe (Physitemp, Clifton, NJ) and
maintained at 37°C. Doppler spectrograms of aortic outflow were
acquired with a 2 mm, 10-MHz pulsed Doppler probe (Indus Instru-
ments). Thoracic aortic outflow and abdominal aortic flow profiles
were captured. PWV was calculated as the quotient of the separation
distance between the thoracic and abdominal probe sites and the time
difference between pulse arrivals; time was recorded with respect to
the R-wave of the ECG.
Assessment of endothelium-dependent vascular relaxation. Excised
aortas were cleaned in ice-cold Krebs-Ringer-bicarbonate solution
containing the following (in mM): 118.3 NaCl, 4.7 KCl, 1.6 CaCl2,
1.2 KH2PO4, 25 NaHCO3, 1.2 MgSO4, and 11.1 dextrose. Equal-size
(2 mm) aortic rings were carefully cut. Vascular tension changes were
determined as previously described (50). Briefly, one end of the aortic
rings was connected to a transducer, and the other to a micromanip-
ulator, with the entire structure immersed in a bath chamber filled with
constantly oxygenated Krebs buffer at 37°C. All vessels were
mounted using a microscope, ensuring no damage to the smooth
muscle or endothelium. The aortas were brought to an optimal resting
tension using the micromanipulator, after which a dose of 60 mM KCl
was administered, and repeated after a wash with Krebs buffer. After
these washes, the vessels were allowed to equilibrate for 20–30 min
in indomethacine (3 ?M), and then phenylephrine (1 ?M) was
administered to cause vasoconstriction. A dose-dependent response
with the muscarinic agonist ACh was then performed to check
endothelial function, with increasing doses (10?9–10?5M; half logs)
administered when the vasorelaxation in response to the prior dose
reached steady state. Relaxation response is calculated as a percentage
of tension following preconstriction. Sigmoidal dose-response curves
were fitted to data with the minimum constrained to 0.
Assessment of NO production in aortic tissue. Excised aortas were
cleaned and carefully dissected in ice-cold Krebs-HEPES buffer
containing the following (in mM): 110 NaCl, 4.7 KCl, 25 NaHCO3,
1.2 MgSO4, 1.03 KH2PO4, 11.1 D-(?)-glucose, 20.0 HEPES, and
1.87 CaCl2, pH 7.4 at 24°C. Aortic rings (2–4 mm) were cut open and
pinned, endothelial side up, in a Silastic-coated Petri dish. Tissues
were incubated with fresh Krebs-HEPES buffer containing 1 nM of
the NO-sensitive fluorescent dye 4-amino-5methylamino-2=,7=-di-
aminofluoroscein (DAF-FM) diacetate (Molecular Probes, Eugene,
OR) for 30 min at 37°C, followed by washout of DAF-FM and a
20-min equilibration period. Fluorescence intensity data were col-
lected using the Nikon NIS-Element suite, with excitation and emis-
sion wavelengths set to 485 and 510 nm, respectively. Fluorescence
intensity was recorded every 30 s for a period of 15 min, and the slope
of intensity change was calculated to indicate rate of NO production.
Data is then normalized to WT-RA ? 100%.
Cardiac myocyte isolation. Cardiac myocytes were prepared from
each treatment group as previously described (28, 51), with slight
modifications. Briefly, excised hearts were perfused retrograde with
Ca2?-free HEPES buffer containing (in mM): 134 NaCl, 4.0 KCl, 1.2
MgSO4, 1.2 NaH2PO4, 11 glucose, 1.2 HEPES, 1.0 2,3-butanedione
monoxime (Sigma, St. Louis, MO) and digested with collagenase type
2 (1.2 mg/ml; Worthington Biochemicals, Lakewood, NJ) and pro-
tease type XIV (0.05 mg/ml; Sigma). Myocytes were then separated
by mechanically mincing digested hearts, followed by filtration,
centrifugation, and suspension in 0.125 mM1Ca2?Tyrode solution
containing (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, 5.5
glucose, and 1.2 NaH2PO4 adjusted to a pH of 7.4 with NaOH.
Myocytes were resuspended first in 0.250 mM Ca2?Tyrode solution,
then in 0.5 mM Ca2?Tyrode solution, and finally stored in Tyrode
solution containing 1.0 mM Ca2?. Functional studies were completed
within 6 h after isolation.
Cell shortening and Ca2?transient measurements. Isolated myo-
cytes were incubated with 5 ?M Fura-2 AM (Molecular Probes) for
10 min then transferred to a Lucite chamber on the stage of an
inverted microscope (Nikon TE 200). The stage was kept at 37°C and
continuously perfused at ?2 ml/min with Tyrode solution containing
2.4 mM Ca2?. Rod-shaped myocytes with clear striations and no
spontaneous contraction were selected for recording. Myocytes were
stimulated with suprathreshold voltage at 1 Hz, and their sarcomere
lengths were recorded with an IonOptix iCCD camera (IonOptix,
Milton, MA). Intracellular Ca2?concentration ([Ca2?]i) indicated by
the Fura-2 AM fluorescence intensity (FFI) was simultaneously de-
termined by calculating the 360-nm/380-nm dual-excitation fluores-
cence ratio of the probe. Isoproterenol (Tocris, 10?9-10?7M), a
nonselective ?-adrenergic receptor agonist, was administered in 10-
fold increasing doses. Each administration took place when myocyte
response to the previous dose reached a steady state. Peak shortening
and peak calcium rise were calculated as the absolute difference in
sarcomere length and FFI, respectively. ?-Adrenergic responses to
isoproterenol are calculated as percent increases from baseline short-
ening and calcium transient and fitted with a sigmoidal dose-response
curve with the bottom constrained to 0.
Echocardiographic evaluation. In vivo cardiac geometry and func-
tion were assessed by transthoracic echocardiography (Sonos 5500, 15
MHz linear transducer; Agilent, Palo Alto, CA) in mice anesthetized
with 1–2% isoflurane. LV end-systolic and -diastolic cross-sectional
diameter and the mean of septal and posterior wall thicknesses were
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