Am J Physiol Regul Integr Comp Physiol 303: R700–R709, 2012.
First published August 15, 2012; doi:10.1152/ajpregu.00258.2012.
TRANSLATIONAL PHYSIOLOGY
Metabolic effects of intermittent hypoxia in mice: steady versus
high-frequency applied hypoxia daily during the rest period
Alba Carreras, Foaz Kayali, Jing Zhang, Camila Hirotsu, Yang Wang, and David Gozal
Department of Pediatrics, Comer Children’s Hospital, Biological Sciences Division, Pritzker School of Medicine,
The University of Chicago, Chicago, Illinois
Submitted 5 June 2012; accepted in final form 13 August 2012
high-frequency intermittent hypoxia; low-frequency intermittent hypoxia; insulin resistance
is the most common form of sleep disordered
breathing. It is highly prevalent (2–10% of the population
across the age spectrum), and it is associated with multiple and
important morbid consequences including metabolic disturbances (21, 50, 52, 78). One of the major perturbations of sleep
apnea is the occurrence of recurrent hypoxic events, i.e.,
high-frequency intermittent hypoxia (IHH), resulting from periodic collapse of the upper airway during sleep. These events
are often terminated by arousal, and therefore elicit fragmenSLEEP APNEA
Address for reprint requests and other correspondence: D. Gozal, Dept. of
Pediatrics, Univ. of Chicago, 5721 S. Maryland Ave., MC 8000, Suite K-160
Chicago, IL 60637 (e-mail: [email protected]).
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tation of normal sleep architecture. Epidemiological studies
have shown a disruption of metabolic pathways in sleep disordered breathing, with an association between indices of
insulin resistance and the degree of hypoxemia as a consequence of obstructive sleep apnea (OSA) being identified (27,
53, 55–56). However, a detailed analysis of the metabolic
adaptations to long-term IH has not been extensively pursued
to date.
It is now well established that low-frequency IH (IHL) as
seen in multiple cardiopulmonary disorders include alteration
of cellular and systemic physiology, polycythemia, pulmonary
hypertension, and weight loss (25, 48 – 49, 79). Previous studies have also shown effects of IHL on energy metabolism via
regulation of genes encoding for glucose transporters and
glycolytic enzymes (29, 61, 63), primarily through modulation
of hypoxia-inducible factor 1-␣ (HIF-1␣) subunit expression
and transcriptional activity. Indeed, HIF-1␣ was reciprocally
modulated by insulin and insulin-like growth factor pathways
(1, 32, 69, 72, 80).
Other studies exploring the effects of moderate IH on body
weight and blood sugar in mice showed lower body weight and
glycemic levels than those in normoxic mice after 40 days of
IH (40). In addition, previous studies conducted at altitude or
in patients with OSA have shown that glucose homeostasis is
influenced by hypoxia, both acutely, and also after more
prolonged exposures, suggesting that insulin resistance and
glucose intolerance are positively associated with the severity
of IH (27, 56, 68, 73). Liyori and colleagues (26) showed an
acute decrease in whole body insulin sensitivity in animals
exposed to IH mimicking the severe hypoxic stress of OSA
suggesting a causal relationship between IH exposures and the
development of insulin resistance in healthy animals (26).
Furthermore, 2–3 days of continuous hypoxia elicited insulin
resistance (7, 35), whereas more than 6 wk of continuous
hypoxia reduced fasting blood glucose levels but did not affect
insulin resistance or glucose tolerance (9, 15, 35).
One of the most widely physiological tests used to identify
impaired glucose tolerance is the glucose tolerance test (GTT).
In mouse models, GTT is carried out following a period of
fasting and glucose administration, both optimized in a relevant study performed by Andrikopoulos and colleagues (3).
Besides GTT, insulin tolerance test (ITT) is another widely
used assay in humans and animal models, and therefore these
two tests provide the opportunity to examine aspects of insulin
sensitivity. However, cellular glucose uptake is facilitated
through transport mediated by the hexose transporter or translocator family of membrane proteins (GLUT), specifically
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Carreras A, Kayali F, Zhang J, Hirotsu C, Wang Y, Gozal D.
Metabolic effects of intermittent hypoxia in mice: steady versus
high-frequency applied hypoxia daily during the rest period. Am J
Physiol Regul Integr Comp Physiol 303: R700 –R709, 2012. First
published August 15, 2012; doi:10.1152/ajpregu.00258.2012.—Intermittent hypoxia (IH) is a frequent occurrence in sleep and respiratory
disorders. Both human and murine studies show that IH may be
implicated in metabolic dysfunction. Although the effects of nocturnal
low-frequency intermittent hypoxia (IHL) have not been extensively
examined, it would appear that IHL and high-frequency intermittent
hypoxia (IHH) may elicit distinct metabolic adaptations. To this effect,
C57BL/6J mice were randomly assigned to IHH (cycles of 90 s 6.4%
O2 and 90 s 21% O2 during daylight), IHL (8% O2 during daylight
hours), or control (CTL) for 5 wk. At the end of exposures, some of
the mice were subjected to a glucose tolerance test (GTT; after
intraperitoneal injection of 2 mg glucose/g body wt), and others were
subjected to an insulin tolerance test (ITT; 0.25 units Humulin/kg
body wt), with plasma leptin and insulin levels being measured in
fasting conditions. Skeletal muscles were harvested for GLUT4 and
proliferator-activated receptor gamma coactivator 1-␣ (PGC1-␣) expression. Both IHH and IHL displayed reduced body weight increases
compared with CTL. CTL mice had higher basal glycemic levels, but
GTT kinetics revealed marked differences between IHL and IHH, with
IHL manifesting the lowest insulin sensitivity compared with either
IHH or CTL, and such findings were further confirmed by ITT. No
differences emerged in PGC1-␣ expression across the three experimental groups. However, while cytosolic GLUT4 protein expression
remained similar in IHL, IHH, and CTL, significant decreases in
GLUT4 membrane fraction occurred in hypoxia and were most
pronounced in IHL-exposed mice. Thus IHH and IHL elicit differential
glucose homeostatic responses despite similar cumulative hypoxic
profiles.
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HYPOXIA AND GLUCOSE HOMOSTASIS
MATERIALS AND METHODS
GLUT1 and GLUT4. GLUT4 function is highly regulated
through not only changes in expression, but also by alterations
in the topographic cellular distribution of GLUT4, such that
increases in the GLUT4 within the plasma membrane (PM)
will facilitate the reduction of plasma glucose levels (8).
Overall, around 50% of intracellular GLUT4 is translocated to
the cell surface upon insulin activation (44). Apart from GLUT
isoforms, transcriptional coactivators can be also important
targets for physiological regulation of glucose. For instance,
peroxisome proliferator-activated receptor gamma coactivator 1-␣ (PGC-1␣) has been described as a transcriptional
coactivator that operates as a key regulator of the activation
a broad range of transcription factors for downstream regulation of genes encoding mitochondrial proteins and GLUT4 (10,
37, 39).
Another metabolic pathway potentially affected by hypoxia
could involve leptin, the adipocyte-derived adipokine product
of the ob gene. It has been shown that this hormone induces a
negative energy balance by reducing appetite and increasing
energy expenditure (20), which is found in circulation at
proportional levels to the OSA severity (13, 28, 73). Presence
of low levels of leptin or leptin resistance via its cognate
receptor have been associated with high levels of insulin
resistance (18, 47, 57).
Based on aforementioned considerations, we hypothesized
that high- and low-frequency intermittent hypoxia would elicit
differential metabolic effects on glycemic control in mice.
B
Adult male C57BL/6J mice (7 wk old, 20 –25 g) were 1) purchased
from Jackson Laboratories (Bar Harbor, ME), 2) allowed to acclimatize to their surroundings for at least 1 wk, and 3) always housed in
groups of five in standard clear polycarbonate cages. Mice were
maintained in a 12-h light/dark cycle (light on 7:00 AM to 7:00 PM)
at a constant temperature (26 ⫾ 1°C) and were allowed access to food
and water ad libitum. At 8 wk of age, mice were randomly assigned
into three hypoxic/normoxic group exposures (26 animals per group):
control, IHH, and IHL for a period of 5 wk (wk). At the end of the
experimental procedures, mice were euthanized by cervical dislocation, and all testing was conducted during the light phase. Animal
experiments were performed according to protocols approved by
IACUC of the University of Chicago and are in close agreement with
the National Institutes of Health “Guide for the Care and Use of
Laboratory Animals.” All efforts were made to minimize animal
suffering and to reduce the number of animals used.
Exposures To High- Or Low-Frequency Intermittent Hypoxia
Mice were placed in identical commercially designed chambers
(30 ⫻ 20 ⫻ 20 inches; Oxycycler A44XO, BioSpherix, Redfield, NY)
(Fig. 1A) operated under a 12:12 h light-dark cycle (7:00 AM to 7:00
PM) for a period of 5 wk before any assay (either GTT or ITT testing
or skeletal muscle harvesting). Programmed gas concentrations were
circulated into each chamber, and an internal O2 analyzer measured
the O2 concentration continuously. Deviations from the fixed concentrations were automatically corrected by a computerized system of
25.0
Setpoint
Process Variable
22.5
FiO2
20.0
17.5
15.0
12.5
Gas
Inlet
10.0
7.5
5.0
18:00
18:30
19:00
Time (min)
C
25.0
Setpoint
Process Variable
22.5
FiO2
20.0
17.5
15.0
12.5
10.0
7.5
5.0
18:00
18:30
19:00
Time (min)
Fig. 1. Schematics of the hypoxic chambers. Commercially available ventilated cages that mimic usual housing conditions are placed in computerized hypoxic
chambers to achieve intermittent hypoxia (IH) exposures in mice (A). Hypoxia results from targeted nitrogen enrichment of the air, followed by reoxygenation
using oxygen. The two different IH patterns are shown on right based on O2 sensor-acquired data within the experimental hypoxic chambers. IH cycles
correspond to the different hypoxic patterns where the fraction of inspired oxygen (FIO2) oscillates from 21% to 6.4% during the 12-h daylight in the
high-frequency IH (IHH) pattern (B) and 8% of inspired oxygen during the 12-h daylight in the low-frequency IH (IHL) pattern (C).
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A
Animals
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HYPOXIA AND GLUCOSE HOMOSTASIS
Body Weight
Body weight was checked weekly for a period of 5 wk always at
the same time of the day (middle of the light cycle period). Body
weight gain was determined by subtracting the body weight on first
day of hypoxia exposure from the body weight on subsequent days.
GTT and ITT
Both tests were performed following 5 wk of either control IHH or
IHL conditions. In both tests, animals were fasted for 3 h with water
available ad libitum. An intraperitoneal injection (26 gauge 3/8⬙
needle) of sterile glucose (2 mg/g of body wt for GTT) or an
intraperitoneal injection of sterile humulin (0.25 U/kg of body wt for
ITT) was administered. At the beginning of both tests, the tip of the
tail was nicked using a sterile surgical blade. Blood recovered from
the tip of the tail at different time points (for GTT: 0, 4, 15, 30, 60, 90,
120 min after injection; ITT: 0, 4, 15, 30, 60, 75, 90, 105, 120 min
after injection) was tested using an OneTouch Ultra2 glucometer (Life
Scan; Milpitas, CA). At the indicated time points, venous blood
samples were collected in heparin-coated capillary tubes from the tail
vein. Insulin and leptin assays were carried out on selected time points
using enzyme-linked immunosorbent assay (ELISA) kits (Millipore;
St. Charles, MO) according to the manufacturer’s protocol. The linear
range of the insulin assay was 0.2–10 ng/ml, with the limit of
sensitivity at 0.2 ng/ml (35 pM), and intra- and interindividual
coefficients of variation up to 8.37% and 17.9% respectively at lower
concentrations (0.32 ng/ml) of this analyte. Similarly for the leptin
assay, the linear range was 0.2 up to 30 ng/ml with the sensitivity
threshold at 0.05 ng/ml (⬃3.13 pM). The intra-assay variation coefficient was up to 1.76% at high concentrations of leptin (17.60 ng/ml),
and 4.59% of interindividual coefficient of variation at low concentrations (1.66 ng/ml) of this analyte.
PGC1-␣ and GLUT4 Protein Expression
Total cellular protein or cytosolic and membrane protein fractions were extracted from frozen skeletal muscle tissue (quadriceps) following a slightly modified protocol, as previously described by Wang and colleagues (74). Frozen tissues were homogenized using a BBX24 Bullet Blender homogenizer (Next
Advance, Averill Park, NY) such that 100 mg of tissue and a
volume of beads (0.5 mm zirconium oxide beads-ZSB05) equal to
the mass of tissue were added to the sample. Two volumes of
buffer containing 50 mM Tris·HCl (pH 7.5), 5 mM EDTA, 10 mM
EGTA, 10 mM benzamidine, 50 ␮g/ml phenylmethylsulfonyl fluoride, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 10 ␮g/ml pepstatin,
and 10% glycerol (vol/vol) were also added before placing the
sample into the homogenizer. At the end of the homogenization
process, the homogenate was incubated on a rocking platform in
the presence of 20 mM 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate (CHAPS) at 4°C for 2 h. After centrifugation at
14,000 g for 30 min at 4°C, the supernatant was collected as total
cellular protein. In another set of skeletal muscle samples, the
homogenate was centrifuged without CHAPS incubation at 14,000
g for 30 min at 4°C, and the supernatant was collected as the
cytosolic fraction. Next, the pellet was washed once with the
previous buffer, resuspended in the sample buffer with 20 mM
CHAPS, sonicated using a F60 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA), and incubated on a rocking platform at 4°C
for 2 h. Finally, after a centrifugation at 14,000 g for 30 min at 4°C,
the supernatant was collected as the membrane fraction. For all
samples, protein concentration was determined using the modified
Bradford method-DC Protein Assay (Bio-Rad Laboratories, Hercules, CA) using Protein Assay Standard II (bovine serum albumin) (Bio-Rad Laboratories) as standard.
Protein samples were electrophoresed under reducing denaturing conditions in 8% polyacrylamide-SDS gels and transferred by
electroblotting onto a nitrocellulose membrane. Equal loading and
transfer efficiency of total cellular protein for PGC1-␣ test or either
cytosolic or membranous protein for GLUT4 test were carefully
documented using membrane reversible Ponceau staining and
anti-␤-tubulin antibody (Upstate-Millipore; St. Charles, MO). After being blocked in 5% albumin solution from bovine serum
(Sigma; St. Louis, MO) for 1 h, membranes were incubated with
either an anti-PGC1-␣ monoclonal antibody (Calbiochem-EMD
Millipore, St. Charles, MO) or an anti-GLUT4 polyclonal antibody
(Santa Cruz; Santa Cruz, CA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz).
Signal detection was facilitated with Immun-Star WesternC
Chemiluminescence Kit (Bio-Rad Laboratories). PGC1-␣,
GLUT4, and ␤-tubulin signals were quantitated using a Molecular
Imager ChemiDoc XRS⫹ Imaging System (Bio-Rad Laboratories). PGC1-␣ and GLUT4 expression were normalized to the
␤-tubulin.
Data Analysis
Body weight matching was not pursued among both IH conditions and CTL group, as will be discussed below. Slope A obtained
from the GTT (Fig. 3B) was calculated using the glucose levels
measured at times 0 –15 min after glucose injection. Slope B from
the same test (Fig. 3C) was computed between the peak serum
glucose levels (15 min) and 120 min after glucose injection. In
contrast, only slope A was calculated for the ITT (Fig. 3H), and
included glucose levels measured at time 4 min till nadir of glucose
levels (60 min) after humulin injection. In addition, area under the
curve (AUCg) analyses were calculated for each of the three
conditions during GTT. Furthermore, the homeostatic model of
insulin resistance (HOMA) was calculated using baseline glucose
and insulin concentrations using the following equation: fasting
glucose (mg/dl) ⫻ fasting insulin (␮U/ml)/405. All data are reported as means ⫾ SE. Comparison within glucose, hormones
(insulin and leptin), and protein expression levels of GLUT4 and
PGC1-␣ between hypoxic and normoxic conditions were assessed
by one-way ANOVA test followed by unpaired Student’s t-test
with Bonferroni posttests. A P value of ⬍0.05 was defined as
significant.
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solenoid valves controlling gas outlets adding either N2 or O2.
Ambient CO2 in the chamber was maintained at less than 0.01%, and
humidity was also maintained at 40 –50% by circulating the gas
through a freezer and silica gel. Ambient temperature was kept at
26°C as described previously by Gozal and colleagues (22). Two
episodic hypoxia profiles were used in the study, IHH and IHL, as
shown in Fig. 1, B and C, respectively. Low-frequency intermittent
hypoxia-exposed mice were subjected for 12 h during daylight to a
continuous FIO2 of 8% (Fig. 1C), a level that is associated with
reproducible nadir of oxyhemoglobin saturations in the 75– 80%
range. For the rest of the 12-h lights-off period, normoxic (FIO2, 21%)
conditions were applied. IHH-exposed mice were subjected for 12 h
during daylight to intermittent hypoxia/normoxia cycles of 3-min
duration [Hypoxia, nadir of FIO2: 6.4% for 90 s alternating with
normoxia (FIO2, 21%) for 90 s; Fig. 1B]. This IH profile is associated
with reproducible nadir of oxyhemoglobin saturations in the 65%72% range. Normoxic (FIO2, 21%) conditions were used during the
12-h lights-off period. Control animals were exposed to circulating
room air (FIO2, 21%) during daylight and lights-off period. The nadir
of FIO2 and the cycle duration were designed to obtain similar
cumulative hypoxic profiles between IHH and IHL during the 12-h
daylight time period. Immediately after the 5-wk exposures, 30
animals (10 per condition) were used for GTT, 30 animals (10 per
condition) were used for ITT, and 18 mice (6 per condition) were
euthanized, and skeletal muscles (quadriceps) were quickly removed,
frozen in liquid nitrogen, and kept at ⫺80°C until analysis.
HYPOXIA AND GLUCOSE HOMOSTASIS
RESULTS
Effect of Chronic IHH and IHL Exposures on Body
Weight Gain
Mice exposed either to IHH or IHL gained less weight over
5 wk (2.82 ⫾ 0.22 g and 3.67 ⫾ 0.24 g on week 5 after
exposure, respectively, n ⫽ 26 per group) compared with the
normoxic controls (n ⫽ 26; 5.45 ⫾ 0.22 g at the end of the
exposure; P ⬍ 0.05) with IHH being lower than IHL. This
difference reached statistical significance after the first week of
exposure (Fig. 2; P ⬍ 0.05).
Effect of IHH and IHL on Glucose and Insulin Sensitivity
Gain body weight (g)
6
CTL
5
IHH
4
IHL
3
2
1
0
-1
1
2
3
4
5
Time (weeks)
Fig. 2. Changes in body weight during hypoxia. Body weight evolution during
IHH, IHL, and normoxia (CTL) throughout 5 wk-exposures. Body weight
alteration in mice exposed to 5 wk of intermittent hypoxia, n ⫽ 26 per group.
This difference reached statistical significance after the first week of exposure
(P ⬍ 0.05).
(⫺2.63 ⫾ 0.13 means ⫾ SE, CTL; ⫺1.73 ⫾ 0.16 means ⫾ SE,
IHH; ⫺0.62 ⫾ 0.08 means ⫾ SE, IHL; P ⬍ 0.05).
Insulin tolerance test. As in the preceding experiments, low
baseline fasting glycemic levels emerged in IHH and IHL
exposed mice (133.2 and 65.1 mg/dl, respectively; n ⫽ 10 per
group) compared with CTL group (169 mg/dl; n ⫽ 10). After
IP humulin injection, the slope corresponding to the decreasing
levels of plasma glucose over time (slope A) was considered as
the indicator for insulin sensitivity. Calculations showed significantly attenuated slopes in IHH and IHL exposed mice
(⫺1.32 ⫾ 0.11 mg·dl⫺1·min⫺1, means ⫾ SE, ⫺1.16 ⫾ 0.28
mg·dl⫺1·min⫺1, means ⫾ SE, respectively) compared with
CTL mice (⫺1.87 ⫾ 0.12 mg·dl⫺1·min⫺1, means ⫾ SE; P ⬍
0.05).
Circulating Leptin and Insulin Levels
Insulin levels at time 0 min (T0) before glucose injection and
insulin levels at time 120 min (T120) after exogenous glucose
administration are shown in Fig. 4A. At T0, both IHH- and
IHL-exposed mice were associated with increased insulin
plasma concentrations, particularly among IHH-exposed animals (0.27 ⫾ 0.03 ng/ml, means ⫾ SE) compared with CTL
group (0.10 ⫾ 0.04 ng/ml, means ⫾ SE; P ⬍ 0.05). In contrast,
insulin levels at T120 were lower in both IH groups (0.78 ⫾
0.04 ng/ml, means ⫾ SE, CTL; 0.41 ⫾ 0.005 ng/ml, means ⫾
SE, IHH; 0.40 ⫾ 0.05 ng/ml, means ⫾ SE, IHL; n ⫽ 10 per
group; P ⬍ 0.05).
Leptin levels were significantly lower in animals exposed to
IH (0.55 ⫾ 0.07 ng/ml, means ⫾ SE, IHH; 0.49 ⫾ 0.005 ng/ml,
means ⫾ SE, IHL; P NS) compared with CTL (0.95 ⫾ 0.19
ng/ml, means ⫾ SE; n ⫽ 20 per group; P ⫽ 0.241; Fig. 4B).
Effect of IHH and IHL on PGC1-␣ and GLUT4 Protein
Expression in Skeletal Muscle
PGC1-␣ expression. Compared with CTL mice, PGC1-␣
protein expression was unaltered by either IHH or IHL exposures (Fig. 5A).
GLUT4 expression. Different trends in GLUT4 protein expression emerged in the cytosolic and membrane fractions
(Fig. 5, B and C). GLUT4 cytosolic protein expression was
increased in skeletal muscle of IHL exposed mice compared
with either CTL or IHH conditions [n ⫽ 6 per group; 1.71 ⫾
0.54 intensity units (IU), means ⫾ SE, IHL; 1.68 ⫾ 0.5IU,
means ⫾ SE, IHH; 1.39 ⫾ 0.45IU, means ⫾ SE, CTL; P NS]
(Fig. 5B). In contrast, expression of GLUT4 in the membrane
fraction was significantly lower in IHH (0.62 ⫾ 0.03 IU,
means ⫾ SE) and IHL (0.39 ⫾ 0.06 IU, means ⫾ SE) groups
compared with CTL mice (0.74 ⫾ 0.14 IU, means ⫾ SE; P ⬍
0.05), with IHL group being the lowest (Fig. 5C). Accordingly,
GLUT4 membrane-to-cytosolic protein ratios, which are indicative of translocated GLUT4 protein (i.e., biologically active),
were markedly lower in hypoxic mice compared with controls
(Fig. 5D), and a significant correlation between GLUT4
(Membr.Prot./Cyto.Prot) and slope A from ITT was apparent
(Fig. 5E).
DISCUSSION
In the present study, we aimed to assess the impact of
different cycling frequencies in the application of the IH
stimulus, a key clinical manifestation of OSA, on glucose
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Glucose tolerance test. As shown in Fig. 3A, IHH and IHL
had lower fasting glucose levels (138.3 and 65.6 mg/dl, respectively; n ⫽ 10 per group) compared with the CTL group
(198.3 mg/dl; n ⫽ 10). HOMA values were highest in IHH and
were higher in IHL compared with CTL (Fig. 3E; IHH vs. IHL
P ⬍ 0.05; IHH and IHL vs. CTL: P ⬍ 0.05). The complex
relationships between basal levels of plasma glucose and insulin are further illustrated in Fig. 3F. Indeed, glucose levels
were highest and insulin levels lowest in CTL under standard
fasting conditions. AUCg analysis showed that IHH and IHL
were smaller than CTL (Fig. 3D), and these findings were
replicated by slope analyses. Indeed, as shown in Fig. 3, B and
C, two distinct phases of blood glucose trajectory are apparent
and were represented by an initial exponential increase to peak
glycemic levels (mathematically represented by slope A) followed by an exponential decay (represented by the slope B).
Significant differences were observed in slope A between IHL
(slope A: 5.57 ⫾ 0.52 mg·dl⫺1·min⫺1, means ⫾ SE) and CTL
(12.82 ⫾ 1.11 mg·dl⫺1·min⫺1, means ⫾ SE; P ⬍ 0.05) and
between IHH (11.22 ⫾ 0.94 mg·dl⫺1·min⫺1, means ⫾ SE; n ⫽
10) and IHL (P ⬍ 0.05), with IHL being worse than IHH and the
latter worse than CTL.
Slope B represents how fast glucose levels in bloodstream
decreased from minute 15 to minute 120 after glucose IP
injection. The CTL group showed higher slope B values
compared with both IHH and IHL, with the latter condition
being the less efficient at decreasing plasma glucose levels
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B
Glucose (mg/dl)
CTL
IHH
IHL
Slope B
500
450
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Slope A
Slope A ((mg/dl)/min)
A
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HOMA units
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Slope A ((mg/dl)/min)
Glucose (mg/dl)
250
-0.5
-1.0
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*
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IHL
-2.5
CTL
Time (min)
Fig. 3. Changes in metabolic parameters during intermittent hypoxia in mice. Plasma glucose concentrations over 2 h during the intraperitoneal glucose tolerance
test (GTT, 2 mg glucose/g body wt) (A) and intraperitoneal insulin tolerance test (ITT, 0.25 U/kg body wt) (G) following fasting for 3 h in C57BL/6J mice
exposed to IHH, IHL, and CTL conditions. Slope A (B) was calculated using the glucose levels measured at times 0 –15 min after glucose injection; slope B (C)
was computed between times 15–120 min after glucose injection and slope A (H) was measured between times 4 – 60 min after humulin injection. The area under
the curve (AUC) was calculated for glycemic levels during GTT experiments (D), and the homeostatic model for insulin resistance (HOMA) was calculated
during baseline fasting conditions (E). Furthermore in F, a scatterplot of glycemic levels and corresponding plasma insulin concentrations at fasting baseline is
shown for CTL (solid circles), IHH (solid squares), and IHL (solidk triangles). Slopes results are means ⫾ SE (n ⫽ 10 per group). *Significance in both GTT
and ITT have been defined as P ⬍ 0.05 (significant differences between two groups; **significant differences between all three groups).
regulation. Despite similar cumulative hypoxic profiles, presentation of IH using different cycling frequencies was associated with distinct glycemic homeostatic responses. From the
present study, it becomes apparent that IH induces reductions
in body weight gain, lowers fasting glucose levels, and also
reduces the uptake of glucose after in vivo glucose administration, as well as insulin receptor sensitivity. Furthermore,
lower levels of translocated GLUT4 to the plasma membrane
in skeletal muscle are apparent. All of these phenomena are
exacerbated in the context of IHL compared with high-frequency hypoxic oscillations, the latter differing from control
conditions. Of note, although the concept that sustained and
intermittent chronic hypoxic exposures imposes divergent effects on metabolic regulation is not novel and has been explored in several studies in both human and animals using
exercise procedures (14, 66) or hypobaric hypoxia (46), we are
unaware of any specific studies addressing the issue of hypoxia
cycle duration on metabolic outcomes. In the discussion that
follows, we will further explore the relationships and putative
pathways linking IH frequency to insulin resistance. However,
before we address these issues, several technical points appear
worthy of mention. Indeed, the IH profile used herein is
markedly similar to those reported by previous investigators,
and as such our current findings can be potentially compared
with such earlier studies (53, 58 –59). Second, body weight
matching was not pursued among both IH exposed mice and
CTL group. Particularly, underfeeding CTL mice or overfeeding IH-exposed mice would be necessary to achieve weight
matching (30, 58) between groups. Such interventions may
however alter glycemic homeostasis per se and therefore mask
the effects of IH explored in the present study. We reasoned
that if an association between insulin resistance and IH
emerged, the present findings would be all the more compelling considering the reductions in weight anticipated in the
context of IH exposures. Third, GTT is routinely performed in
mice following an overnight fast, with the food being removed
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AUC (mg dl -1 min-1)
D
HYPOXIA AND GLUCOSE HOMOSTASIS
1.0
IHH
0.8
Insulin (ng/ml)
B
CTL
IHL
0.6
0.4
* *
*
1.4
1.2
Leptin (ng/ml)
A
R705
1.0
0.8
0.6
0.4
0.2
0.2
0.0
0.0
Time (min)
around 4 PM, and the glucose load being administered via IP
injection the following morning. However, it is well known
that mice are nocturnal feeders, with ⬃70% of their daily
caloric intake occurring during the dark cycle (6), and that their
metabolic rate is much higher than humans. Therefore, an
overnight fast represents a comparatively longer fasting time
compared with humans and is more akin to starvation. In
addition, there is no consistency in the amount of glucose
administered with studies using 1 g/kg (4, 51, 54) or 2 g/kg (5,
24, 31, 43, 81) or both doses to determine glucose tolerance
(23, 62). In 2008, GTT was reevaluated in mice to determine
the most appropriate fasting duration, route of glucose administration, and the ideal amount of glucose (3). Based on such
considerations, we fasted mice for 3 h in both GTT and ITT
and administered IP 2 mg glucose/g body wt during GTT.
Low- and High-Frequency IH and Body Weight Gain
Factors contributing to hypoxia-induced weight loss are
complex and incompletely understood. Several investigators
have demonstrated that body weight will continue to increase
in mice submitted to chronic IH (58), whereas other laboratories have reported decreases in body weight accrual velocity in
mice subjected to IH compared with controls (12, 17, 71, 76).
Row et al. in 2002, measured body weights in postnatal rats
and reported significantly lower body weight gain in rat pups
exposed to IH than age-match controls housed in room air (60).
In a recent study carried out in male lean mice exposed to
different hypoxic patterns (normoxia, IH 12 times/h, IH 60
times/h or sustained hypoxia), body weights were similar
among all hypoxic mice independent of the hypoxic regimen
used but gained less weight than control animals (58). Consistent with such findings, we also found lower body weight gain
in both hypoxic groups compared with room air-exposed adult
mice independent from the IH frequency pattern for the first 7
days of exposure. However, lower body weight increases
emerged in IHH compared with IHL, starting as of 14 days of
hypoxic exposures. Consequently, body weight gain appears to
be influenced by the IH frequency.
Low- and High-Frequency IH and Insulin Resistance
The selected IH profiles aimed to reproduce the overall
cumulative hourly oxygen desaturations patterns routinely observed in moderate to severe OSA patients with oxyhemoglobin saturation levels ranging from 65 to 80% (11, 19, 22, 67).
A large body of evidence derived from epidemiological cohorts
and clinical populations indicates that OSA may also contribute to the development of metabolic disorders including glycemic regulation impairments (27, 55–56, 65). Furthermore, in
healthy humans, acute IH induced decreased insulin sensitivity
and inadequate increases in pancreatic insulin secretion (42).
Iiyori and colleagues (26) used a euglycemic hyperinsulinemic
clamp in mice and showed that acute IH (60 times/h over 9 h)
induced insulin resistance. We now show that chronic IH will
lead to lower fasting glycemic levels and relatively higher
levels of insulin concentrations, specifically in IHL-exposed
mice and not as much in IHH-exposed mice, yet suggesting the
presence of insulin resistance state after both types of IH
exposures. There were however discrepant findings between
HOMA and either AUCg or slope analyses. Indeed, HOMA
results suggested that IHH would be worse affected. This is not
the case for the other two analytical approaches, which suggest
that IHL is most severely affected. An important caveat for
HOMA validity is that it imputes a dynamic ␤-cell function
(i.e., glucose-stimulated insulin secretion) from fasting steadystate data. Therefore, based on the concordance between the
two dynamic assessments of glucose and insulin relationships
(i.e., AUCg and slope analyses), we would postulate that the
IHL⬎IHH⬎CTL effect is most likely reflective of the true
changes in glucose homeostasis. In addition, as can be seen
from Fig. 3F, there were complex changes in both the baseline
glycemic and insulin levels with each type of exposure, potentially reflecting underlying changes in the ”homeostat“ level of
plasma glucose after prolonged hypoxic exposures, as well as
changes in either basal secretion of insulin or in insulin
sensitivity under those conditions. Reinke and collaborators
(58) showed that even infrequent episodic hypoxic events (12
times/h) were sufficient to disturb insulin and glucose regulation in lean mice. We should note that in the present study we
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Fig. 4. Changes in insulin and leptin plasma levels during IH. Insulin and leptin plasma levels in C57BL/6J mice exposed to IHH, IHL, and CTL conditions for
a period of 5 wk. A: plasma insulin levels at fasting glucose baseline levels and after 120 min of glucose administration in the GTT (n ⫽ 10; *significant
differences compared with control group; P ⬍ 0.05). B: leptin plasma levels measured at baseline after 3 h fasting period (n ⫽ 20; P NS). Results are
means ⫾ SE.
R706
HYPOXIA AND GLUCOSE HOMOSTASIS
CTL
IHH
IHL
CTL
IHH
IHL
PGC1-α
A
150
100
50
0
CTL
IHH
CTL
IHL
β-Tubulin
β-Tubulin
C
150
100
50
0
CTL
IHH
IHL
IHL
150
100
50
0
CTL
IHH
IHL
Glut4 (Memb Prot/Cyto Prot)
0.0
E
150
*
100
*
50
0
CTL
IHH
ITT-Slope A ((mg/dl)/min)
D
Glut4 (Membr Prot/Cyto Prot) (% of Controls)
B
Membrane Glut4 expression (% of Controls)
Glut4 (Membr)
Cytosolic Glut4 expression (% of Controls)
Glut4 (Cyto)
IHH
0.2
0.4
0.6
-1.2
0.8
1.0
CTL
IHH
IHL
-1.4
-1.6
-1.8
-2.0
IHL
Fig. 5. Effect of IH on glucose tranporter 4 (GLUT4) and proliferator-activated receptor gamma coactivator-1␣ (PGC-1␣) expression in skeletal muscle. Effect
of 5-wk IHH, IHL, and CTL on PGC-1␣ and GLUT4 protein content in skeletal muscle. Representative Western blots of PGC-1␣ protein expression (A), GLUT4
cytosolic protein expression (B), and GLUT4 membrane protein expression (C) are shown. Samples on the autoradiographs (Western blot images panels) are
expressed as percentage of the CTL group. Ratio between GLUT4 membrane protein expression and GLUT4 cytosolic protein expression were represented in
D. Intensity units results are means ⫾ SE and *P ⬍ 0.05 significantly different from the CTL group (n ⫽ 6 per group). Correlation between GLUT4
(Membr.Prot./Cyto.Prot) and slope A from ITT is shown (E).
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PGC1-alpha expression (% of Controls)
β-Tubulin
HYPOXIA AND GLUCOSE HOMOSTASIS
different IH profile, improved glucose tolerance in the absence
of increased GLUT4 protein expression was previously reported (14). The unbalanced distribution of GLUT4, as clearly
shown in Fig. 5D, may account for the IHL-animals inability to
facilitate the transport of glucose to the intracellular space, as
described in our experiments using both GTT and ITT (Fig.
5E). Thus the frequency of IH is critical for the development of
insulin resistance and impaired glucose tolerance, thereby
confirming Polotsky et al. previous findings (53).
Low- and High-Frequency IH and Leptin
Low fasting leptin levels probably play an important role in
the alterations reported on insulin and glucose responses to IH.
It has been shown that leptin can act at the level of pancreas,
by downregulating insulin gene transcription and insulin secretion (34, 64). Conversely, leptin deficiency plays a key role
in the acceleration of insulin resistance in mice exposed to IH,
yet it remains unclear whether mice with intact leptin pathways
are also susceptible to metabolic dysfunction after exposure to
IH (53). We found lower baseline levels of leptin expression in
both hypoxic groups with no significant differences between
high- and low-frequency hypoxia. Therefore, hypoxia per se,
rather than the frequency of IH, appears to be the determinant
factor underlying the emergence of lower leptin levels.
We should emphasize that a significant limitation of our
study included the absence of any assessments of differences in
the levels of stress hormones among the three experimental
groups. Indeed, a recent work by O’Donnell and colleagues
(77) has postulated that the initial stress response, i.e., corticosterone levels, was a major determinant to the acute metabolic changes observed after short-lasting period of IH. This
group of investigators has just published a study that emphasizes the differences between acute and chronic metabolic
adaptations to IH, whereby longer durations of IH exposures
appear to alleviate the magnitude of metabolic dysfunction
induced by IH (36).
Perspectives and Significance
The results of the present study demonstrate that intermittent
hypoxia, independently of the frequency, leads to impairments
in glucose metabolism in mice. Indeed, IH is associated with
reduced body weight, increase in insulin resistance, and low
leptin levels. Furthermore, the alterations in GLUT4 cellular
distribution in skeletal muscle are further supportive of a
putative mechanism for the effects of IH on insulin sensitivity.
These findings may have substantial implications for the understanding of metabolic regulation and deregulation in diseases associated with hypoxia, such a sleep apnea and chronic
obstructive lung disease.
GRANTS
This work was supported by the National Heart, Lung, and Blood Institute
Grants HL-065270 and HL-086662, and P50 HL-107160. F. Kayali was the
recipient of a T-32 Training Grant HL-094282 (N. R. Prabhakar, PI).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: A.C., F.K., J.Z., and C.H. performed experiments;
A.C., J.Z., and D.G. analyzed data; A.C. and D.G. interpreted results of
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did not explore the presence of hypoxic injury to pancreatic
␤-cells during IH, and therefore the contribution of such
previously reported effects of IH may have contributed to the
kinetics of glycemia and insulin levels following IP glucose
injection (75, 77). Another important comment pertains to the
marked differences in peak glycemic levels that occurred after
IP glucose injection. Notwithstanding the differences in body
weight, this glucose dosage has been previously shown to
reliably reflect existing differences in insulin sensitivity (3).
However, it will be important in future studies to compare
whether the oral glucose administration route will provide
further information on the deregulation of overall glycemic
homeostatic processes. Furthermore, the recent development of
frequent sampling approaches in the mouse may provide improved resolution on the regulation of various glucose disposition compartments (2).
In the present study, we also show that after glucose injection, the resultant circulating glucose levels require longer
times to be cleared from the circulation and to reach preinjection levels in both hypoxic groups, with the kinetics of glycemic decay in IHL-exposed mice being the most affected.
Although the oral route of exogenous glucose administration is
more physiological, we used an intraperitoneal administration
to reduce the variability associated with inconsistent rates of
gastric emptying, which can complicate data interpretation. On
the other hand, the ITT was developed as a simple way to
evaluate insulin action in vivo in humans. In mice, this test was
modified, where a larger bolus of insulin is given after a fast of
variable duration and the glucose concentration is monitored
for a longer duration (⬃90 –120 min). For this test, the fall in
blood glucose is used as a reflection of insulin action, such as
a fall by ⬃50% of glucose concentrations in wild-type mice on
a chow diet and in normoxia (45). For both GTT and ITT, we
opted to display the data as the kinetic slope from the baseline
point to the highest or lowest glucose levels, respectively,
thereby normalizing across potential confounders. Thus both
GTT and ITT findings coincide in their findings and clearly
establish a consistent perturbation of insulin sensitivity in the
context of IH that is further modulated by the IH stimulus
presentation frequency.
The changes in GLUT4 and PGC1-␣ were also examined in
skeletal muscle. As mentioned, PGC1-␣ serves as a transcriptional coactivator that plays a key role in coordinating cellular
metabolic flux in skeletal muscle, in which ⬃90% of insulinstimulated glucose uptake occurs (33). GLUT4 delivery to the
cell surface is coordinated by insulin signaling and requires
GLUT4 mobilization from intracellular membrane compartments, recognition of GLUT4-containing vesicles at the PM,
and finally fusion of these two membranes (38). Whereas total
GLUT 4 (data not shown) and PGC1-␣ remained unaltered by
hypoxic exposures, we found that expression of GLUT4 in the
membrane was significantly lower in IHL than IHH or controls,
with no changes in GLUT4 expression in the cytosol. A
potential explanation for such findings resides in the known
activity of insulin. Indeed, insulin stimulates constitutive exocytosis (8) and particularly that of GLUT4 (38). Although the
molecular mechanisms underlying the specificity of insulin
action on GLUT4 vesicles remain to be established, insulin
promotes tethering and fusion of GLUT4 vesicles to the plasma
membrane (41), within the characteristic time frames of appearance of GLUT4 in the PM (16, 70). Interestingly, using a
R707
R708
HYPOXIA AND GLUCOSE HOMOSTASIS
experiments; A.C. prepared figures; A.C. and D.G. drafted manuscript; A.C.,
F.K., J.Z., C.H., Y.W., and D.G. approved final version of manuscript; Y.W.
and D.G. conception and design of research; D.G. edited and revised manuscript.
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