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. 2003 Jul 23;552(Pt 1):253–264. doi: 10.1113/jphysiol.2003.048173

Intermittent Hypoxia Increases Insulin Resistance in Genetically Obese Mice

Vsevolod Y Polotsky 1, Jianguo Li 1, Naresh M Punjabi 1, Arnon E Rubin 1, Philip L Smith 1, Alan R Schwartz 1, Christopher P O'Donnell 1
PMCID: PMC2343324  PMID: 12878760

Abstract

Obstructive sleep apnoea, a syndrome that leads to recurrent intermittent hypoxia, is associated with insulin resistance in obese individuals, but the mechanisms underlying this association remain unknown. We utilized a mouse model to examine the effects of intermittent hypoxia on insulin resistance in lean C57BL/6J mice and leptin-deficient obese (C57BL/6J−Lepob) mice. In lean mice, exposure to intermittent hypoxia for 5 days (short term) resulted in a decrease in fasting blood glucose levels (from 173 ± 11 mg dl−1 on day 0 to 138 ± 10 mg dl−1 on day 5, P < 0.01), improvement in glucose tolerance without a change in serum insulin levels and an increase in serum leptin levels in comparison with control (2.6 ± 0.3 vs. 1.7 ± 0.2 ng ml−1, P < 0.05). Microarray mRNA analysis of adipose tissue revealed that leptin was the only upregulated gene affecting glucose uptake. In obese mice, short-term intermittent hypoxia led to a decrease in blood glucose levels accompanied by a 607 ± 136 % (P < 0.01) increase in serum insulin levels. This increase in insulin secretion after 5 days of intermittent hypoxia was completely abolished by prior leptin infusion. Obese mice exposed to intermittent hypoxia for 12 weeks (long term) developed a time-dependent increase in fasting serum insulin levels (from 3.6 ± 1.1 ng ml−1 at baseline to 9.8 ± 1.8 ng ml−1 at week 12, P < 0.001) and worsening glucose tolerance, consistent with an increase in insulin resistance. We conclude that the increase in insulin resistance in response to intermittent hypoxia is dependent on the disruption of leptin pathways.

Obstructive sleep apnoea (OSA) is one of the most common complications of obesity. The prevalence of sleep-disordered breathing in individuals with a body mass index greater than 30 kg m−2 ranges from 40 % (apnoea-hypopnoea index (AHI) = 15 h−1) to 60 % (AHI = ≥5 h−1; Punjabi et al. 2002). The syndrome of OSA is characterized by recurrent collapse of the upper airway during sleep leading to periods of intermittent hypoxia (IH) and fragmentation of sleep (Gastaut et al. 1966). Several cardiovascular complications have been attributed to OSA, including increased risk of systemic hypertension (Nieto et al. 2000; Peppard et al. 2000), coronary artery disease (Mooe et al. 2001; Shahar et al. 2001) and stroke (Shahar et al. 2001). Recent clinical studies suggest that insulin resistance and glucose intolerance are positively associated with OSA, independent of the degree of obesity (Strohl et al. 1994; Vgontzas et al. 2000; Ip et al. 2002; Punjabi et al. 2002). Moreover, the extent of insulin resistance was related to the severity of the hypoxic stress of OSA (Ip et al. 2002; Punjabi et al. 2002).

Although little is known about the effects of IH on metabolic function, some studies have addressed the changes in glucose and insulin homeostasis that occur during short- and long-term exposure to continuous hypoxia (i.e. high altitude). In general, short-term continuous hypoxia (2-3 days) causes acute insulin resistance (Larsen et al. 1997; Braun et al. 2001), whereas long-term continuous hypoxia (more than 6 weeks) reduces fasting blood glucose levels, but does not affect insulin resistance or glucose tolerance (Calderon et al. 1966; Davidson & Aoki, 1970; Larsen et al. 1997). The corresponding short-term and long-term effects of IH on metabolic function have not been studied previously. It is conceivable that the IH stimulus of OSA impairs glucose homeostasis and exacerbates the insulin resistance associated with obesity.

Obesity and OSA are likely to disrupt several metabolic pathways. One common pathway could involve leptin, an adipocyte-derived hormone that regulates satiety and metabolic rate (Zhang et al. 1994; Maffei et al. 1995; Considine et al. 1996; Friedman, 1998; Breslow et al. 1999), which is present in the circulation at levels that are in proportion to the degree of obesity (Considine et al. 1996) and the severity of OSA (Chin et al. 1999; Ip et al. 2000; Vgontzas et al. 2000). In addition to a CNS, or central, role in regulating food intake (Halaas et al. 1995; Schwartz et al. 1996), leptin can act peripherally to inhibit insulin secretion (Kulkarni et al. 1997; Seufert et al. 1999) and increase glucose uptake in vivo (Sivitz et al. 1997; Barzilai et al. 1997). The presence of inappropriately low levels of leptin for a given degree of adiposity has been associated with a high level of insulin resistance (Montague et al. 1997; Ravussin et al. 199; Farooqi et al. 2001 7). The degree of insulin resistance in obese patients with concomitant OSA may therefore be dependent on the circulating level of leptin.

The purpose of the current study was to examine the combined impact of IH and obesity on glucose tolerance and insulin resistance in the presence and absence of leptin. We hypothesized that IH in the presence of leptin deficiency exacerbates the insulin resistance associated with obesity. Our approach was to utilize an established rodent model of IH (Fletcher et al. 1992) and determine the acute (5 days) and chronic (12 weeks) effects of IH on glucose tolerance and insulin resistance. Specifically, we examined the changes in blood glucose and serum insulin levels during (a) short-term IH in the presence and absence of obesity and leptin and (b) long-term IH in a mouse model of leptin-deficient obesity.

METHODS

Animals

A total of 31 wild-type, male, lean C57BL/6J mice (lean) and 59 male obese C57BL/6J-Lepob (obese) mice from Jackson Laboratory (Bar Harbor, ME, USA) were used in the study. The study was approved by the Johns Hopkins University Animal Use and Care Committee and complied with the American Physiological Society Guidelines for Animal Studies. For all blood samples, injections and surgical procedures, anaesthesia was induced and maintained with 1-2 % isoflurane administered through a facemask.

Model of intermittent hypoxia

A gas control delivery system was designed to regulate the flow of room air, nitrogen and oxygen into customized cages housing the mice. A maximum of three lean mice or two obese mice were housed continuously in a single customized cage (dimensions 27 × 17 × 17 cm) with constant access to food and water. A series of programmable solenoids and flow regulators altered the inspired oxygen fraction (FI,O2) over a defined and repeatable profile that simulated the timing and magnitude of arterial oxygen desaturation changes seen in OSA patients (Tagaito et al. 2001). During each period of IH, the FI,O2 was reduced from 20.9 to 4.8-5.0 % over a 30 s period and then rapidly reoxygenated to room air levels in the subsequent 30 s period. The use of multiple inputs into the cage produced a uniform nadir FI,O2 level throughout the cage.

Leptin replacement

In 16 obese mice, anaesthesia was induced and maintained by 1-2 % isoflurane, and a small 0.25 cm skin incision was made in the midline, just caudal to the shoulder blades. A 100 μl Alzet (Mt View, CA, USA) mini-osmotic pump (0.5 μl h−1) was inserted subcutaneously and used to deliver leptin (Amgene, Thousand Oaks, CA, USA) at a dose of 200 μg kg−1 day−1. Mice were monitored for 6-8 h under room air conditions to assure the integrity of the surgical site and the return of normal behavioural activities. After the recovery period animals were placed in the IH chamber.

Experimental design

A. Short-term exposure to IH

Sixteen lean mice, 15 obese mice, and eight obese mice with leptin replacement were placed in the IH chamber for five consecutive days. Food intake and body weight were monitored daily for each animal. All animals were kept in a controlled environment (22-24 °C with a 12 h : 12 h light : dark cycle; lights on at 08.00) on a standard chow diet with free access to water. In a separate series of animals, 15 lean mice, 14 obese mice, and eight obese mice with leptin replacement were exposed to intermittent room air (IA, control groups) for 5 days in identical chambers and were weight-matched to the IH group daily during the experiment by varying food intake (Table 1). The IH and IA states were induced from 16.00 to 08.00 (i.e. for 16 h) each day of the experiment, alternating with 8 h of constant room air. The 16 h exposure period was chosen to represent the upper end of the physiological range for IH exposure and was based on our previous work in a mouse model of sleep-disordered breathing during polysomnographically determined sleep (Tagaito et al. 2001).

Table 1.

Body weight, daily food intake and fasting blood glucose of lean (C57BL/6J) and obese (C57BL/6J/Lepob) mice before and after exposure to IH or IA for 5 days

n Age (weeks) Body weight (g) Daily food intake (g) Fasting blood glucose (mg dl−1)
Day 0 Day 5 Day 0 Day 5 Day 0 Day 5
Lean mice
  IH 16 12 ± 1 28.4 ± 0.5 26.7 ± 0.7* 4.2 ± 0.2 3.0 ± 0.2* 173 ± 11 138 ± 10*†
  IA 15 12 ± 1 28.2 ± 0.5 26.8 ± 0.4* 4.1 ± 0.2 2.9 ± 0.2* 176 ± 9 169 ± 11
Obese mice
  IH 15 13 ± 1 55.7 ± 1.2 54.9 ± 1.4 6.1 ± 0.4 3.2 ± 0.1*† 250 ± 17 165 ± 13*†
  IA 14 13 ± 1 55.9 ± 1.3 53.7 ± 1.4 6.0 ± 0.3 4.1 ± 0.2 260 ± 21 228 ± 15
Obese mice with leptin replacement
  IH 8 11 ± 1 57.1 ± 10 49.2 ± 0.8* 6.0 ± 0.5 1.2 ± 0.3* 295 ± 18 131 ± 13*
  IA 8 11 ± 1 57.0 ± 1.0 50.4 ± 0.7 5.9 ± 0.4 1.0 ± 0.2* 322 ± 18 162 ± 16*
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The reslts are presented as means ± s.e.m. Statistical significance of the differnce between day 0 and day 5 within each group of animals exposed to hypxia and control conditions was derived with paired or unpaired t tests, respectively. IH, intermittent hypoxia; IA, intermittent air.

*

P < 0.01 for difference between day 0 day 5 in the same group

†

P < 0.01 for difference between IH and IA on the same day.

B. Long-term exposure to IH in obese mice

Obese mice were exposed to either IH (n = 7) or IA (n = 7) for 12 weeks. The experiment was performed as described for short-term IH exposure (above) with the exception that IH and IA states were induced for only 12 h each day of the experiment, alternating with 12 h of constant room air. The shorter period of IH exposure was chosen to lessen any potential adverse cardiovascular outcomes (Fletcher et al. 1992) associated with long-term IH in the obese mice.

Sample collection and intraperitoneal glucose tolerance test (IPGTT)

All blood samples and tissue collections were performed under 1-2 % isoflurane anaesthesia 6 h after the cessation of either IH or IA. A retro-orbital sinus puncture was performed for glucose and insulin measurements in peripheral blood (100 μl) before and after short-term IH exposure and during long-term exposure at weeks 0, 4, 8 and 12. After short-term exposure, an IPGTT was performed in 17 lean mice (n = 9 IH; n = 8 IA), 15 obese mice without leptin replacement (n = 8 IH; n = 7 IA) and in 16 obese mice with leptin replacement (n = 8 IH; n = 8 IA). After long-term exposure to IH and IA, an IPGTT was performed in all animals. A glucose load of 0.5 g kg−1 body weight was administered I.P. for the glucose tolerance test. During the IPGTT, glucose measurements were obtained by retro-orbital puncture (15 μl) at 15, 30, 45, 60, 75, 90 and 120 min after administration of the glucose load. Retro-orbital puncture was used due to large blood volume requirements associated with repetitive sampling. Haemostasis was assured by applying pressure and, in survival experiments, all animals were observed to assure that there were no signs of distress or abnormal behaviour.

At the termination of all short-term and long-term IH and IA experiments, arterial blood (1 ml) was obtained by direct cardiac puncture under 1-2 % isoflurane anaesthesia; animals were then killed with an overdose of pentobarbital (60 mg I.P). Subcutaneous white adipose tissue was collected, immediately frozen in liquid nitrogen and stored at −70 °C for future analysis.

Blood glucose and plasma leptin, insulin and free fatty acid (FFA) determination

Glucose was measured in blood from the retro-orbital plexus using an Accu-Chek Comfort Curve kit from Roche Diagnostics (Indianapolis, IN, USA). Serum leptin levels were measured with a mouse leptin radioimmunoassay kit from Linco Research (St Charles, MO, USA). Serum insulin levels were measured with a rat insulin ultrasensitive radioimmunoassay kit (cross-reactivity with mouse insulin 100 %) from Linco Research. Serum FFA were measured with a NEFA C test kit from Wako Diagnostics (Richmond, VA, USA).

To evaluate the degree of insulin resistance, the homeostasis model assessment (HOMA) was calculated using the following formula: HOMA = fasting serum insulin (μu ml−1) × fasting blood glucose (mmol l−1)/22.5 (Matthews et al. 1985).

RNA purification

The subcutaneous white adipose tissue was homogenized on ice using an Omni EZ Connect Homogenizer (Omni International, Warrington, VA, USA). Total RNA was isolated using Trizol Reagent (Life Technologies, Rockville, MD, USA) with additional RNA clean-up using the RNAeasy (Qiagen, Valencia, CA, USA) purification kit.

Gene expression microarray analyses

RNA samples of the subcutaneous white adipose tissue from mice exposed to short-term IH were pooled for animals of each strain for each experimental condition: lean mice exposed to IH (n = 5), lean mice exposed to IA (n = 5), obese mice exposed to IH (n = 5) and obese mice exposed to IA (n = 5). A gene expression microarray cRNA was produced from double-stranded cDNA and fragmented to a size of approximately 50 base pairs (bp). Biotinylated cRNA was hybridized to the Affymetrix Murine GeneChip U74A (Affymetrix, Santa Clara, CA, USA), which represents approximately 6000 sequences from the UniGene database that have been functionally characterized and approximately 6000 additional expressed sequence tag (EST) clusters. Absolute analysis of Affymetrix image data was carried out using the Affymetrix Microarray Suite 4.0, as described previously (Chen et al. 2000). Duplicate Genechips were used for quality control, and alterations in gene expression were determined on the basis of a twofold change in specific genes or ESTs.

RT-PCR

cDNA was produced from total RNA using the Advantage RT for PCR kit from Clontech (Palo Alto, CA, USA). PCR was performed with the SuperTaq Plus Polymerase kit from Ambion RNA Company (Austin, TX, USA) in an iCycler from Bio-Rad (Richmond, CA, USA) using primers for mouse leptin

(5′-3′: F, TGACACCAAAACCCTCATCA;

R, AGCATTCAGGGCTAACATCC)

and mouse 18 S rRNA

(5′-3′: F, CTGTTCCGCCTAGTCCTGTC;

R, GTTTCTCAGGCTCCCTCTCC)

derived from GeneBank sequences. PCR results were resolved on 1.2 % agarose gel stained with ethidium bromide. Densitometry was performed using a Kodak DC290 ZOOM digital camera with 2 150 000 pixels (2 megapixels) and an UN-SCAN-IT gel Automated Digitizing System, version 5.1 software from Silk Scientific Corporation (Orem, VT, USA).

Statistical analyses

All values are reported as means ± s.e.m. In short-term exposure, comparisons of body weights, food intake, fasting blood glucose, fasting serum insulin levels, and leptin and fatty acid levels within and between the IH and IA groups of obese and lean mice were performed using either a paired t test (between days 0 and 5 within a group) or an unpaired t test (between groups). In long-term exposure, statistical differences in body weight, food intake, fasting blood glucose, and fasting serum insulin levels were determined across time and between mice exposed to IH and IA, by two-way ANOVA. To test whether serum glucose levels during the IPGTT were different between IH and IA conditions, we used the technique of longitudinal data analysis (Zeger & Liang, 1986) to determine whether the response to glucose load was different at any time point. Because glucose levels during the IPGTT are inherently correlated, determination of whether significant differences exist between two groups at any time point requires that the autocorrelation between the repeated measurements be accounted for in these assessments. To account for the correlation between repeated measurements, the method of generalized estimating equations (Liang & Zeger, 1986) was used to obtain unbiased estimates (and associated standard errors) that quantify the differences between the two groups. This approach also allowed us to adjust for baseline differences in the glucose levels and estimate whether the change from the baseline level was different between two groups at any time point after the glucose load. Thus, all of the IPGTT results are presented as a change in the glucose value from baseline. The GENMOD procedure in the SAS statistical software package (version 9.0) was used to fit models using the method of generalized estimating equations. Statistical significance was based on whether the difference in glucose values from baseline was different under IH and IA conditions. A P value of less than 0.05 was considered significant.

RESULTS

A. Short-term exposure to intermittent hypoxia

a.Lean mice: fasting parameters on day 0 and day 5

Exposure to IH for 5 days decreased fasting blood glucose levels by 35 ± 14 mg dl−1 (P < 0.01, Table 1), whereas insulin (Fig. 1) and FFA (1.1 ± 0.2 mmol l−1 on day 0 and 1.4 ± 0.2 mmol l−1 on day 5) remained unchanged. In control mice exposed to 5 days of IA, there were no changes in glucose (Table 1), insulin (Fig. 1 left panel) or FFA levels (0.9 ± 0.1 mmol l−1 on day 0 and 1.1 ± 0.3 mmol l−1 on day 5). During exposure to IH, the HOMA index of insulin resistance decreased from 9.1 ± 1.0 on day 0 to 2.8 ± 0.5 on day 5 (P < 0.05), whereas in control animals exposed to IA, it did not change (5.6 ± 2.1 and 4.4 ± 0.6, respectively). The decrease in insulin resistance induced by IH in lean mice was independent of changes in food intake and body weight, which were matched between the IH and the IA groups (Table 1).

Figure 1. Fasting serum insulin levels in C57BL/6J (lean) and C57Bl/6J-Lepob (obese) mice were determined before (day 0) and after (day 5) exposure to either IH or IA for 5 days.

Figure 1

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These results are presented as the means ± s.e.m. The statistical significance of the difference between day 0 and day 5 within each group of animals or between animals exposed to intermittent hypoxia or intermittent air was derived with paired or unpaired t tests, respectively. * P < 0.01 for difference between day 0 and day 5; †P < 0.01 between mice exposed to IH and IA on day 5.

b. Lean mice: intraperitoneal glucose tolerance on day 5

In lean mice, IH resulted in significant improvement of glucose tolerance, as assessed by IPGTT on day 5 (Fig. 2A). After adjustment for the difference in fasting blood glucose (see Table 1), the glucose levels throughout the IPGTT were on average 70 ± 25 mg dl−1 lower (P < 0.01) in mice exposed to IH compared with control animals exposed to IA (Fig. 2A). In lean mice, the 2 h IPGTT insulin levels were unchanged from the day 5 baseline levels in both the IH and IA groups (Fig. 3, left panel).

Figure 2. Blood glucose levels as a result of an IPGTT after a 5 day exposure to either IH or IA in lean mice, leptin-deficient obese mice and leptin-deficient obese mice receiving supplemental leptin.

Figure 2

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The change in the blood glucose from fasting levels was determined over a 2 h period after I.P. injection of glucose at 0.5 g kg−1 body weight in lean C57BL/6J mice (A), leptin-deficient obese C57Bl/6J-Lepob mice (B) and in C57Bl/6J-Lepob (obese) mice receiving leptin subcutaneously at 200 μg kg−1 day−1 (C). The IPGTT was performed after exposure to IH or IA for 5 days. The results show the mean of the change in blood glucose level (blood glucose level –fasting blood glucose level) ± s.e.m. The statistical significance of the difference between groups was derived with the method of generalized estimating equations (see Methods for the details). * P < 0.05 for the difference between groups.

Figure 3. Serum insulin levels as a result of an IPGTT after exposure to either IH or IA in lean mice, leptin-deficient obese mice and leptin-deficient obese mice receiving supplemental leptin.

Figure 3

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Serum insulin levels in C57BL/6J (lean) and C57Bl/6J-Lepob (obese) mice were determined before (fasting level) and 2 h after I.P. glucose injection (0.5 g kg−1 body weight) in the IPGTT. The IPGTT was performed after exposure to either IH or IA for 5 days. Results are presented as the means ± s.e.m. The statistical significance of the difference between fasting and 2 h IPGTT levels within each group of animals or between animals exposed to intermittent hypoxia or intermittent air was derived with paired or unpaired t tests, respectively. * P < 0.001 for difference between fasting and 2 h IPGTT levels; †P < 0.01 between mice exposed to IH and IA.

c. Obese mice: fasting parameters on day 0 and day 5

Exposure to IH for 5 days led to a decrease (P < 0.001) in fasting blood glucose levels of 85 ± 5 mg dl−1 (Table 1) and unchanged levels of FFA (1.2 ± 0.1 mmol l−1 on day 0 and 1.2 ± 0.2 mmol l−1 on day 5). In contrast to lean mice, fasting insulin levels rose markedly (P < 0.01) in response to 5 days of IH in obese mice (Fig. 1). In obese control mice exposed to 5 days of IA, there were no changes in glucose (Table 1), insulin (Fig. 1) or FFA levels (1.4 ± 0.2 mmol l−1 on day 0 and 1.4 ± 0.1 mmol l−1 on day 5). During exposure to IH, the HOMA index increased more than fourfold (from 44.9 ± 11.0 on day 0 to 204.7 ± 39.3 on day 5; P = 0.01), whereas there was no change in control animals exposed to IA (65.9 ± 8.1 and 58.9 ± 18.1, respectively). The increase in insulin resistance induced by IH in obese mice was independent of changes in food intake and body weight (Table 1).

d. Obese mice: intraperitoneal glucose tolerance on day 5

A 5 day exposure to IH improved glucose tolerance in obese mice in a manner comparable to lean mice, but, after correction for fasting blood glucose levels, the difference with control mice reached statistical significance only at two time points, 30 and 45 min (P < 0.05; Fig. 2B). Unlike lean mice, IH in obese mice induced a twofold increase in post-IPGTT serum insulin from an already elevated fasting level (28.6 ± 3.0 ng ml−1vs. 13.6 ± 2.3 ng ml−1; P < 0.001), whereas there was no change in the IA group (Fig. 3, right panel).

e. Leptin replacement in obese mice: fasting parameters on day 0 and day 5

The leptin infusion rate of 200 μg kg−1 day−1 produced serum leptin levels of 11.2 ± 2.4 ng ml−1 in the IA group and 10.2 ± 2.1 ng ml−1 in the IH group, and resulted in reduced food intake and weight loss in both groups (Table 1). Fasting blood glucose levels were significantly decreased (P < 0.001) after 5 days in both the IH and IA groups. Fasting serum insulin levels on day 5 were 1.0 ± 0.3 ng ml−1 and 1.4 ± 0.5 ng ml−1 in mice subjected to IH and IA, respectively, which was a substantial decrease compared to leptin-deficient mice (Fig. 1, right panel). The HOMA levels of 9.0 ± 2.7 (IH group) and 10.4 ± 3.1 (IA group) were similar to that of lean mice (see sections Aa and Ab), and 15-20 times lower than those in leptin-deficient obese mice exposed to IH (see sections Ac and Ad). Thus, the presence of elevated circulating levels of leptin reversed the marked increase in insulin resistance that occurred in obese leptin-deficient mice exposed to IH.

f. Leptin replacement in obese mice: intraperitoneal glucose tolerance on day 5

In the presence of leptin, glucose tolerance in obese mice was improved comparably in both the IH and IA groups (compare Fig. 2C and 2B) to a level similar to lean mice exposed to IH (compare Fig. 2C and 2A). Serum insulin levels at 2 h averaged 4.1 ± 0.9 ng ml−1 in the IH group and 2.8 ± 0.7 ng ml−1 in the IA group, both of which were considerably lower than the values of 28.6 ± 3.0 ng ml−1 and 9.1 ± 1.9 ng ml−1 seen in the leptin-deficient obese mice during IH (P < 0.001) and IA (P < 0.05), respectively. Thus, leptin administration led to an improvement in glucose tolerance and serum insulin levels 2 h after the glucose challenge in obese mice, and reversed the metabolic dysfunction evident in leptin-deficient obese mice exposed to IH.

g. Gene expression in adipose tissue of lean and obese mice

In lean mice exposed to IH, six genes were upregulated greater than twofold and 47 genes were downregulated greater than twofold compared with mice exposed to IA. In obese mice subjected to IH, 12 genes were upregulated greater than twofold and three genes were downregulated greater than twofold compared with mice exposed to IA. Among the genes involved in glucose metabolism and insulin signalling (Misbin et al. 1981; Arnalich et al. 2000; Kim et al. 2000; Li et al. 2000; James et al. 2001; Saltiel & Kahn, 2001; Ceddia et al. 2002) only six genes were differentially expressed in mice subjected to IH (Table 2). In lean mice, leptin was significantly upregulated, phosphotidylinositol-3-kinase (PI(3)K), interleukin 6 (IL-6) receptor, insulin-degrading enzyme and caveolin 3 were downregulated, and uncoupling protein was unchanged. In obese mice, leptin was unchanged, caveolin 3 was upregulated and uncoupling protein was downregulated. Notably, there were no significant changes in the expression of other genes associated with insulin signalling (i.e. insulin receptor, insulin receptor substrates (IRS), glucose transporters), glucose utilization (i.e. glycolysis enzymes) or insulin resistance (i.e. tumour necrosis factor (TNF)α, peroxisome proliferator activated receptor (PPAR)γ, adiponectin and resistin) (Tamemoto et al. 1994; Bruning et al. 1998; Withers et al. 1998; Zisman et al. 2000; Steppan et al. 2001; Yamauchi et al. 2001).

Table 2.

Genes involved in glucose metabolism and insulin signalling in the subcutaneous white adipose tissue of lean (C57BL/6J) and obese (C57BL/6J-Lepob) mice exhibiting a twofold or greater change after exposure to IH for 5 days

n GenBank accession number Name Up (+) or down (−) regulation (fold difference)
Lean mice Obese mice
1 AI882416 Leptin +3.3 NC
2 M21247 Uncoupling protein 1 NC −5.6
3 U50413 Phosphatidylinositol-3-kinase, p85 alpha subunit −2.2 NC
4 X51975 Interleukin 6 receptor alpha −6.4 NC
5 AI574278 Insulin degrading enzyme −7 NC
6 AV023068 Caveolin 3 −15.7 +4.2
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NC, no change or less than twofold difference was found in comparison with intermittent air control. IH, intermittent hypoxia.

The increase in leptin expression was verified by semi-quantitative RT-PCR (Fig. 4A). Leptin expression in relationship to 18S rRNA (a house-keeping gene) was 44 % higher in lean mice exposed to IH compared to IA controls (Fig 4B). In lean mice exposed to IH, serum leptin levels were significantly elevated compared to the IA group (Fig. 4C), demonstrating that exposure to IH resulted in both an increase in leptin gene expression as well as increased circulating leptin levels in the peripheral blood.

Figure 4. Expression of leptin mRNA and 18S rRNA in subcutaneous adipose tissue, and serum leptin levels in lean mice after exposure to either IH or IA for 5 days.

Figure 4

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In lean C57BL/6J mice, leptin mRNA and 18S rRNA expression in subcutaneous adipose tissue was assessed by RT-PCR (A and B), and fasting serum leptin levels (C) were measured by radioimmunoassay after exposure to IH (n = 9) or IA (n = 9) for 5 days. Ethidium bromide staining of an agarose gel shows representative RT-PCR yielded products of predicted size, 416 bp for leptin and 500 bp for 18S (A). Semi-quantitative determination of leptin mRNA expression is presented as a percentage of 18S rRNA RT-PCR product expression (B). Results are presented as the means ± s.e.m. The statistical significance of the difference between animals exposed to IH and IA was derived with an unpaired t test.

B. Long-term exposure to IH

a. Fasting parameters

During the 12 week exposure to IH, two out of seven of the IH mice died (one mouse died during exposure and the other died during assessment of fasting glucose and insulin levels); data from these animals were excluded from all analyses. Throughout the 12 week exposure period, the mice exposed to IH and the control mice exposed to IA had similar food intake and body weight (Table 3) and maintained their normal weight gain trajectory (Tankersley et al. 1996). Fasting glucose levels also followed the expected course (Menahan, 1983), declining over time from the nadir at 9 weeks of age. In contrast to the 5 day exposure described above, fasting glucose levels during IH remained comparable to those of the IA control group throughout the 12 week experiment. However, similar to the hyperinsulinaemia seen during short-term IH exposure, the fasting insulin levels in the IH group also increased significantly (P < 0.01) over time, indicating that IH led to sustained insulin resistance (Fig. 5). Indeed, at 12 weeks, the HOMA index was more than double (P < 0.05) in the IH group (134.5 ± 23.1) compared to the IA group (57.3 ± 9.0).

Table 3.

Body weight, daily food intake and fasting blood glucose of obese (C57BL/6J-Lepob) mice during exposure to IH or IA for 12 weeks

IH IA
Body weight (g) Baseline 44.4 ± 1.8 45.0 ± 1.4
Week 4 51.5 ± 1.4* 52.4 ± 0.6*
Week 8 55.9 ± 2.0* 55.5 ± 1.3*
Week 12 60.1 ± 2.3* 60.3 ± 1.3*
Daily food intake (g) Baseline 5.5 ± 0.2 5.5 ± 0.2
Week 4 5.1 ± 0.4 5.1 ± 0.2
Week 8 5.3 ± 0.2 5.3 ± 0.4
Week 12 4.9 ± 0.1 4.6 ± 0.3
Fasting blood glucose (mg dl−1) Baseline 373 ± 41 349 ± 24
Week 4 245 ± 23* 242 ± 33*
Week 8 233 ± 39* 205 ± 32*
Week 12 200 ± 13* 185 ± 34*
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IH, intermittent hypoxia; IA, intermittent air. The age at of the animals at the onset of the experiment was 9 ± 1 week for both the IH (n = 5) and IA (n = 7) groups. Statistical differences were determined by two-way ANOVA across the intervention (IH vs. IA) and time (0, 4, 8 and 12 weeks).

*

P < 0.02 for difference between baseline and weeks 4, 8 or 12.

Figure 5. Fasting serum insulin levels in obese mice after exposure to either IH or IA for 12 weeks.

Figure 5

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Fasting serum insulin levels in C57Bl/6J-Lepob (obese) mice were determined before (baseline), during (weeks 4 and 8) and after exposure to IH or IA conditions for 12 weeks. Results are presented as the means ± s.e.m. Statistical significance of the difference between groups and over time was determined by two-way ANOVA. * P < 0.01 for difference between baseline, week 8 and week 12 levels for the IH group.

b. Intraperitoneal glucose tolerance at week 12

The degree of glucose intolerance present in the IA group at 12 weeks (Fig. 6; continuous line, squares) was identical to that described above at 5 days in the IA group (Fig. 2B; continuous line, squares). In contrast, the glucose tolerance was markedly different between the 5 day (Fig. 2B; dotted line, diamonds) and 12 week exposures (Fig. 6; dotted line, diamonds) in the IH group. After 12 weeks of exposure, all five animals exposed to IH reached blood glucose levels above 600 mg dl−1 during the IPGTT, whereas only two out of seven of the IA group exceeded 600 mg dl−1. Consequently, the blood glucose levels were significantly higher in the IH compared to the IA group at 90 min (a difference of 80 ± 26 mg dl−1; P = 0.002) and 120 min (a difference of 116 ± 24 mg dl−1; P < 0.001) after glucose administration (Fig. 6), demonstrating that glucose intolerance in obese mice is exacerbated by exposure to IH.

Figure 6. Blood glucose levels as a result of an IPGTT after a 12 week exposure to either IH or IA in obese mice.

Figure 6

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The change in the blood glucose from fasting levels was determined over a 2 h period after I.P. injection of glucose at 0.5 g kg−1 body weight in C57Bl/6J-Lepob (obese) mice following 12 weeks of exposure to IH and IA. Results are presented as the means of the change in blood glucose level (blood glucose level –fasting blood glucose level) ± s.e.m. The statistical significance of the difference between groups was derived with the method of generalized estimating equations (see Methods for the details). * P = 0.002 for the difference between groups, †P < 0.001 for the difference between groups.

DISCUSSION

The purpose of our study was to assess the impact of IH, a key clinical manifestation of OSA, on glucose and insulin regulation in the presence and absence of obesity and leptin. Several new findings resulted from the study. Firstly, short-term exposure to IH led to a decrease in fasting blood glucose levels and improved glucose tolerance in both lean and obese, leptin-deficient mice. In lean mice, fasting and post-IPGTT insulin levels did not change and the HOMA estimate of insulin resistance decreased, consistent with IH increasing the sensitivity of peripheral tissues to insulin. In contrast to lean mice, a decrease in blood glucose levels in obese mice was accompanied by a marked rise in serum insulin levels and an increase in insulin resistance. Secondly, the increase in insulin resistance that occurred in obese, leptin-deficient mice in response to short-term IH was abolished by acute leptin replacement, suggesting that leptin deficiency, rather than obesity per se, may be responsible for the metabolic dysregulation that occurs during IH. Thirdly, short-term exposure to IH in lean mice led to an upregulation of leptin gene expression in adipose tissue and an increase in circulating leptin levels, indicating that elevations in leptin may represent a compensatory response to the presence of IH. Fourthly, chronic IH exposure led to a sustained increase in serum insulin levels and insulin resistance in obese mice. Moreover, in contrast to short-term IH exposure, long-term IH worsened glucose tolerance, suggesting that IH accelerated the progression of glucose intolerance and insulin resistance in a leptin-deficient murine model of obesity. In the discussion that follows we explore the relationship and putative pathways linking IH to insulin resistance.

IH and insulin resistance

In obese, leptin-deficient mice, short-term IH led to an increase in both fasting and post-IPGTT serum insulin levels in the presence of decreased fasting glucose and improved glucose tolerance, suggesting that IH can impact on pancreatic endocrine function and increase insulin secretion. However, the relatively small decrease in blood glucose levels (Table 1) was out of proportion to the marked increase in insulin secretion (Fig. 1), consistent with a progression of insulin resistance. The insulin resistance observed in obese leptin-deficient mice during short-term IH was also present after long-term IH, and was associated with a worsening of glucose tolerance. In contrast, in previous in vivo studies, long-term exposure to continuous hypoxia did not cause insulin resistance (Larsen et al. 1997) and resulted in a decrease in fasting blood glucose levels and unchanged glucose tolerance (Calderon et al. 1966; Davidson & Aoki, 1970). Thus, a key finding in our study is that the intermittent nature of the hypoxic stimulus is critical in the development of insulin resistance and impaired glucose tolerance.

Leptin deficiency probably plays an important role in the insulin and glucose responses to IH. Leptin can act at the level of the pancreas to downregulate insulin gene transcription and insulin secretion (Kulkarni et al. 1997; Seufert et al. 1999) and at the peripheral tissues to increase glucose uptake (Barzilai et al. 1997; Sivitz et al. 1997). Although it is unclear through what pathways leptin deficiency interacts with IH to cause insulin resistance in our model, the increase in gene expression of caveolin and decrease in that of uncoupling protein 1 observed in the adipose tissue of obese, but not lean mice, represent two putative mechanisms (Li et al. 2000; James et al. 2001). Moreover, our data in obese mice did not reveal any other mechanism potentially implicated in increased insulin resistance in response to IH. Specifically, serum FFA (Kim et al. 2001) and adipose tissue expression of insulin receptor, IRS, TNFα, PPARγ, glucose transporters, adiponectin and resistin were unchanged (Tamemoto et al. 1994; Bruning et al. 1998; Withers et al. 1998; Zisman et al. 2000; Saltiel & Kahn, 2001; Steppan et al. 2001; Yamauchi et al. 2001). Thus, leptin deficiency plays a key role in the acceleration of insulin resistance in mice exposed to IH, but it remains to be determined whether other models of obesity and insulin resistance with intact leptin pathways are also susceptible to metabolic dysfunction after exposure to IH.

Alternatively, the compensatory increases in leptin that occur in lean mice exposed to IH could increase glucose uptake in the periphery through various mechanisms. Potential mediators of increased glucose uptake include IRS (Tamemoto et al. 1994; Withers et al. 1998), PI(3)K (Kim et al. 2000; Saltiel & Kahn, 2001), Akt/protein kinase B kinase (Kim et al. 2000; Saltiel & Kahn, 2001) and uncoupling protein (Li et al. 2000), although none of these genes showed an increase in expression level in response to IH in adipose tissue of lean mice. Nevertheless, an increase in kinase activity or the amount of these proteins may account for the actions of leptin on glucose utilization in the peripheral tissues. Other potential pathways for improving glucose uptake in response to IH that were identified by gene expression analysis in lean mice include downregulation of several genes associated with increased insulin resistance, such as IL-6 receptor (Arnalich et al. 2000), insulin degrading enzyme (Misbin et al. 1981) and caveolin (James et al. 2001; Table 2). Thus, a number of pathways, in addition to elevated leptin levels, may contribute to the improved glucose tolerance that occurs in lean mice exposed to IH.

IH and leptin

Several clinical studies have shown that patients with OSA have significantly higher leptin levels than weight-matched subjects without OSA (Chin et al. 1999; Ip et al. 2000; Vgontzas et al. 2000). Moreover, treatment with continuous positive airway pressure can result in significant reductions in leptin levels in OSA patients (Chin et al. 1999). The mediating mechanisms and the biological and clinical significance of the increased leptin levels associated with OSA are unknown. Experiments on cell cultures of adipocytes (Grosfeld et al. 2002) and fibroblasts (Ambrosini et al. 2002) have demonstrated that continuous hypoxia increases leptin gene expression via hypoxia-inducible factor (HIF)-1α transcription factor (Ambrosini et al. 2002). Our data show that IH causes an elevation in leptin expression and protein level (Fig. 4), suggesting that the hypoxic stress caused by OSA may be the mechanism by which leptin levels are increased. In addition to providing insight into the mechanism by which OSA increases leptin levels, our data provide evidence that leptin plays an important role in mitigating the metabolic disturbances that accompany IH. Both upregulation of leptin and leptin replacement in leptin-deficient mice protected the animals against the development of glucose intolerance and insulin resistance during IH (Fig. 1 and Fig. 2A and C). Thus, the elevation of leptin levels caused by the hypoxic stress induced by OSA may represent an important compensatory response that acts to minimize metabolic dysfunction.

Caveats

Several caveats associated with our study need to be acknowledged. First, fasting blood glucose levels declined over the course of the 12 week (long-term) exposure to IH in obese, leptin-deficient mice, reflecting the natural aging process in C57Bl/6J-Lepob mice (Menahan, 1983). This time-related decline in fasting glucose levels, however, does not impact on our results and conclusions since (1) the decline in fasting blood glucose was identical in both the IH and the IA control groups and (2) the glucose tolerance curve was similar in control animals experiencing either long-term IA (Fig. 6) or short-term IA (Fig. 2B), whereas animals exposed to long-term IH (Fig. 6) showed a marked deterioration in glucose tolerance compared to the short-term IH exposure (Fig. 2B). A second caveat is that mild food restriction and weight loss could potentially contribute to the decreases in blood glucose and HOMA index observed in lean mice during short-term IH exposure. However, control mice that were identically weight-matched to mice exposed to 5 days of IH did not exhibit any change in either blood glucose, serum insulin levels or HOMA index in response to IA, indicating that the mild food restriction did not have an impact on baseline metabolic function. Third, the pooling of mRNA for microarray analysis, despite the animals being genetically identical, is potentially less sensitive than assessing gene expression responses from individual animals. We acknowledge that many of the changes in gene expression discussed above require subsequent verification, although we did show an increase in gene expression, elevated serum levels and physiological relevance for an increase in leptin in response to IH. Fourth, it may be expected that several other metabolic genes that can increase glucose uptake, including glucose transporters, glycolysis enzymes and leptin, would be upregulated by an increase in HIF-1α transcription factor, which is a major regulator of the cellular responses to hypoxia (Iyer et al. 1998; Ambrosini et al. 2002). However, since HIF-1α is almost instantly degraded with the cessation of a hypoxic stimulus (Hon et al. 2002), the absence of any upregulation of HIF-1α-controlled genes, with the exception of leptin, is probably due to our experimental approach assessing responses 6 h after removal of IH.

Clinical implications

Our approach of assessing metabolic function in obese mice exposed to IH was designed to simulate the relationship between obesity and OSA in the clinical environment. The data are relevant for daytime metabolic assessment in patients with severe OSA, since the IH stimulus represented significant and repeated decreases in arterial oxygen levels (Tagaito et al. 2001) and all measurements were performed 6 h after cessation of the IH stimulus. The results from our animal model have several clinical implications. We have shown that IH may be an underlying mechanism contributing to the previously described clinical association between OSA and insulin resistance (Ip et al. 2002; Punjabi et al. 2002). Based on our data, we propose that IH may account for the insulin resistance observed in OSA if leptin pathways are disrupted. Although the leptin deficiency of the obese mouse is rare in humans (Montague et al. 1997), partial leptin deficiency (Ravussin et al. 1997; Farooqi et al. 2001), and, in particular, leptin resistance (Considine et al. 1996; Schwartz et al. 1996; Ceddia et al. 2002) are common. It is conceivable that administration of high doses of either leptin or other factors that stimulate leptin pathways (e.g. ciliary neurotrophic factor; Lambert et al. 2001) could attenuate the insulin resistance and glucose intolerance that occurs in obese patients with OSA.

Conclusion

In conclusion, we have shown that in the absence of leptin, IH can accelerate the progression of insulin resistance and glucose intolerance associated with obesity. Leptin administration and elevations in endogenous leptin levels may have a protective effect against the progression of glucose intolerance and insulin resistance in the presence of IH. Finally, our data suggest that the previously observed association between OSA and insulin resistance are due to the detrimental effects of IH, and that OSA exacerbates the insulin resistance and glucose intolerance associated with obesity when leptin deficiency is present.

Acknowledgments

This work was supported by National Heart, Lung and Blood Institute Grants K08 HL68715 to V. Y. Polotsky, K23 HL04065 to N. M. Punjabi, F32 HL71469 to A. E. Rubin, R01 HL71506 to A. R. Schwartz, R01 HL37379 to P. L. Smith and R01 HL63767 and R01 HL66324 to C. P. O'Donnell. The authors gratefully acknowledge Mrs Raisa Gelman for invaluable technical assistance with radioimmune assays, Dr Eric P. Hoffman for performing gene expression analysis, and Drs Brock A. Beamer and Josephine M. Egan for critical reading of the manuscript.

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