Obstructive sleep apnea (OSA) occurs during the whole pediatric age. Its actual prevalence (2%-4%) is difficult to measure because it is widely under-diagnosed among children.1 The morbidity of OSA in children is 33%, similar to asthma.2 The main causes of OSA in childhood are enlarged tonsils and/or adenoids (T&A), but many children present residual OSA symptoms after T&A surgery,3 and many of these OSA patients are obese children. There must be some other factors that cause these OSA symptoms.
Excessive daytime sleep (EDS) as one of symptoms in OSA that has been correlated with diabetes mellitus, after controlling for obesity, and age, severity of sleep apnea.4 Feinberg5 warned sleep experts in 1993 that diabetes mellitus patients who did not received systemic therapy for diabetes might present with serious hypersomnolence. Therefore, glycometabolism disturbance should be considered when EDS is present. Diabetes mellitus/insulin resistance (IR) are the predictor factors of EDS.
OSA can cause systemic diseases in adults and children, which is characterized by repeated upper airway collapse during sleep. OSA influences blood pressure, blood glucose and blood fat levels,6-9 which are all signs of metabolic disorder.10-12 Evidence is also emerging for a link between OSA and metabolic dysfunction that is independent of obesity in adults and children, although obesity is an independent risk factor for both OSA and the metabolic syndrome.13-15 It is an important finding that the severity of IR correlates with the severity of OSA independent of body mass index (BMI), despite the high prevalence of obesity among adults and children in the study groups.16,17
Mechanisms for IR have been widely studied. Visceral fat can influence the pathophysiology of lipid metabolic disorder and IR.18 Lipid tissue volume is an independent predictor of metabolic disorder, because this tissue secretes adipokines and promotes IR.19 IR may be also be related to changes in glucose transporter (GLUT) expression. The four GLUTs, numbered 1-4, have different expression patterns in different tissues.20 GLUT-1 and GLUT-3 are widely distributed, and govern basal glucose transport. GLUT-1 is sensitive to hypoxia, and GLUT-2 is prominent in hepatic and pancreatic tissue. Muscles and adipose tissue are target organs in glucose utilization, and GLUT-4 is the predominated GLUT in these tissues. GLUT-4 is the most important kind of GLUT which affects the periphery levels of glucose and insulin.21 Abnormal signaling in the insulin receptor substrates/phosphatidylinositol-3 kinase/GLUT pathway is widely accepted as an IR mechanism,22 and GLUTs play a major role. GLUTs sense glycometabolism changes in the internal environment, prophase changes of organ ischemia and anoxemia and are sensitive to glucose utilization under these conditions.20 We studied the changes of GLUTs in introabdominal adipose tissue of pedo-rats in our research, because visceral fat is the major problem in OSA patients, especially those with EDS symptoms.23-25
We tested the hypothesis that repeated hypoxiemia caused by intermittent hypoxia promote the development of IR in pedo-rats, and insulin resistance may be one of reasons that persistence of residual EDS symptoms after treatment of T&A. Therefore, because rats have no tonsil, we could preclude the impact of enlarged T&A and measure peripheral insulin sensitivity and GLUT expression in intraabdominal adipose tissues, after varying exposures to hypoxia.
All experiments were approved by the ethics committee, and performed in compliance with the Animal Management Rule of the People's Republic of China, and the Care and Use of the Laboratory Animals Guide of the Peking University People's Hospital. Newborn Sprague-Dawley (SD) male rats (Laboratory Animal Unit of the People's Hospital, Peking University) were housed in cages with their mothers under the standard laboratory conditions until weaning at 21 days. All pedo-rats used in experiments were 21 days old and approximately 55 g, and were randomly assigned to a control group, chronic continuous hypoxia (CCH) group and chronic intermittent hypoxia (CIH) group. Chronic exposure to intermittent hypoxia has been used as an animal model for studying intermittent hypoxia during sleep.26,27 CCH was used to exclude the effect of hypoxia.
The CIH group was exposed to intermittent hypoxia each day, from 8 a.m. to 6 p.m., for 40 days. Specifically, rat cages were placed inside plexiglas chambers (28.5 cm × 30.0 cm × 51.5 cm), where the oxygen level was controlled by alternating flows of nitrogen and oxygen. Intermittent hypoxia was created by alternating from 21% oxygen for 120 seconds to 8% oxygen for 60 seconds every 3 minutes during the light period. Oxygen concentration in these chambers was continuously measured by an oxygen analyzer, and feedback-controlled by a computerized system connected to a gas valve outlet.
Any deviation from the determined settings was corrected by the addition of pure nitrogen or oxygen through solenoid valves. Ambient CO2 in the chamber was periodically monitored and maintained at 0.03%, via adjustment of the basal chamber ventilation. Humidity was measured and maintained between 40%-50% and temperature was maintained at 22°C-24°C.
Rats in CCH group were exposed to CCH. The rats were also placed in plexiglas chambers, where the oxygen level was maintained at 10.5%. When the oxygen level reached higher than 10.5%, nitrogen was added to the chamber. Soda lime was placed inside the chambers to absorb CO2, and the CO2 level was maintained at less than 2%.
Rats in the control group were housed in plexiglas chambers adjacent to the other groups. However, only compressed room air was delivered to the chambers.
Measurement of blood levels
On the first and last day of the experiment, blood samples were taken from each animal. After 12 hours of fasting, these blood samples were obtained by puncturing the opthalmic venous plexus. Blood glucose was measured by glucose oxidase-peroxidase (GOD-POD) reagents (Co-health Beijing Laboratories Co. Ltd., China). Serum insulin level was determined by ELISA (Beijing North Institute of Biological Technology, China).
To maintain a hyperinsulemic and euglycemic state, blood levels were exogenously controlled with an artificial clamp apparatus that included delivery of insulin and glucose by a pump. After 40 experimental days, all animals were fasted for 12-14 hours overnight, then anesthetized by an intraperitoneal injection of amobarbital sodium (25 mg/kg), and laid on a heating table to maintain normal body temperature. Cannula were inserted into the right jugular vein for infusion of glucose and insulin via separate lines. Another cannula was placed in the left carotid artery to retrieve blood samples.
Glucose and insulin solutions were stored in two digital syringe pumps and joined by a "Y" connector to the jugular catheter. Insulin (Novolin R, Novo Nordisk Pharmaceuticals, Denmark) was infused at a rate of 4 mU·kg-1·min-1 through the jugular vein catheter. Blood glucose (BG) levels were serially monitored with a Glucometer (Surestep, Johnson, USA), and glucose infusion rates (GIR) were adjusted every 5 minutes to maintain BG at euglycemic levels ((5.0±0.5) mmol/L).
Clamping was achieved by 90 minutes and maintained for 30 minutes. The GIR during the last 30 minutes of insulin infusion was considered to be an estimate of whole body glucose utilization. At the end of the clamp study rats were euthanized by decapitation. White adipose tissue surrounding the ovaries (para-ovary) was rapidly removed and immediately frozen in liquid nitrogen.
Quantitative real-time reverse transcription PCR
Quantitative real-time polymerase chain reaction (rt-PCR) was performed to determine GLUT mRNA levels in all experimental groups. Total RNA from each sample of intraabdominal WAT was extracted with an Rneasy Mini kit (QIAGEN, Valencia, CA, USA). Two μg of RNA from each sample were reverse-transcribed to cDNA with standard commercial kits (Promega UK Ltd., Southamptom, UK). For rt-PCR, relative gene expression was determined with an SYB Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA) and an Applied Biosystems 7300 rt-PCR system, per the manufacturer's protocol. PCR was performed in a thermal cycler as follows: 50°C for 2 minutes, initial denaturation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds, and 60°C for 1 minute. A sample containing no cDNA was used as a negative control. Each quantitative PCR was performed in triplicate. Target cDNA was amplified by primer sets for GLUT-1, 2, 3, and 4. GenBank accession number and gene primer sequences are summarized in Table 1. β-actin was used as the housekeeping gene.
GLUT proteins were measured from a total cellular membrane fraction, as previously described.28 Briefly, each intraabdominal white adipose tissue (WAT) sample was homogenized in buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, 250 mmol/L sucrose, pH 7.4), with a polytron homogenizer, then centrifuged at 3000 ×g for 15 minutes. Fat cakes were discarded, and the infranatant, a fat-free extract, was centrifuged at 12 000 ×g for 15 minutes. The pellet was resuspended as a plasma membrane fraction in 1 ml of buffer. The supernatant was centrifuged at 28 000 ×g for 15 minutes, the pellet was discarded and the supernatant was centrifuged at 146 000 ×g for 75 minutes. The final pellet was resuspended as a microsomal fraction in 1 ml of buffer. The tissues were homogenized in the same buffer, and centrifuged at 1000 ×g for 10 minutes. The supernatant was saved and the pellet was resuspended in 1/3 of the initial volume, and centrifuged again at 1000 ×g for 10 minutes. Both supernatants were mixed and submitted to a 150 000 ×g centrifugation for 75 minutes. The final pellet was resuspended as a total membrane fraction in 1 ml of buffer. The resulting supernatant was then used to measure proteins. All procedures were performed at 4°C.
Total protein content for each sample was evaluated by Lowry's method.29 Aliquots of each sample (20 μg of protein) were added to sample buffer. This mixture was boiled for 5 minutes, and then loaded onto a 12% sodium dodecyl sulfate polyacrylamide gel. Following gel electrophoresis, the separated proteins were transferred onto a nitrocellulose membrane (Amersham Biosciences, UK) via a wet transfer method. After immersion in blocking solution (5% non-fat milk, 0.05% Tween 20 in Tris-buffered saline), the membranes were incubated with multiple anti-GLUT antibodies (rabbit polyclonal anti-GLUT-1, or rabbit polyclonal anti-GLUT-2, or rabbit polyclonal anti-GLUT-3, or mouse monoclonal anti-GLUT-4; Abcam, Cambridge, UK) at 4°C overnight. To verify equal protein loading, blots were also incubated with rabbit polyclonal anti-actin antibody (1:2000, Sigma, USA). After rinsing with TTBS, membranes were incubated in a 1:10 000 dilution of horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz, USA) in TTBS at room temperature for 1 hour, with agitation. Protein bands were detected with ECL Western blotting detection reagents (Zhongshan Goldenbridge Biotechnology, China) on X-ray film (Kodak, USA).
Immunohistochemical staining was performed on 10 μm slide-mounted paraffin sections after they were deparaffinized in xylene, then hydrated through a series of graded alcohol solutions. Each experimental group's intraabdominal WAT samples included ten different animals. After blocking and embedding and cutting the adipose tissues, endogenous peroxidase was inactivated by incubating the slide-mounted sections in 0.3% H2O2 for 30 minutes. And then blocking in 5% normal goat serum, slides were incubated in 1:250 rabbit polyclonal anti-GLUT-1, rabbit polyclonal anti-GLUT-2, rabbit polyclonal anti-GLUT-3, or mouse monoclonal anti-GLUT-4 overnight at 4°C. After rinsing, slides were then incubated with rabbit anti-goat secondary antibody conjugated to a peroxidase-linked dextran polymer in 1:4000 (Rabbit Polymer Kit or Mouse Polymer Kit, Zhongshan Goldenbridge Biotechnology, China) at 37°C for 30 minutes. Signals were visualized by 3,3-diaminobenzidine tetrahydrochloride, which formed a brown precipitate at the site of the immunolabeled transporter. Slides were air-dried, then cleared in xylene, and coverslipped.
All data were expressed as mean ± standard deviation (SD). Statistical differences were evaluated by one-way analysis of variance (ANOVA), followed by post hoc comparisons (LSD test) among the three groups. Significant differences between two groups were evaluated by Student's t test. A value with an associated P <0.05 was considered statistically significant. All analyses were performed with SPSS 13.0 software (SPSS, USA).
Insulin and glucose level in plasma
Animal characteristics are presented in Table 2. Initial body weight was approximately 55 g, and no significant differences were found among final body weights in any group. The fasting plasma glucose (PG) in the CIH group was higher than normal, but there was no statistical significance compared with the control group. The fasting plasma insulin level in CIH group was much higher than in the control group (P=0.038, ANOVA). While both fasting PG and insulin levels in CCH showed no difference compared with the control group.
GIR levels measured by hyperinsulinemic-euglycemic clamping
During the hyperinsulinemic-euglycemic clamp, the coefficient of variation in PG level was <10% between 60-120 minutes in all groups. Glucose was clamped at about 5.0 mmol/L in all three groups. We found that the mean GIR (GIR60-120 min) levels were significantly decreased in the CIH group compared with the control group, (7.25±1.29) mg·kg-1·min-1 vs. (13.34±1.54) mg· kg-1 ·min-1, (P <0.001). GIR60-120 min levels in the CIH, CCH and control groups were (7.25±1.29) mg·kg-1 ·min-1, (12.58±0.66) mg·kg-1 ·min-1 and (13.34±1.54) mg·kg-1·min-1 respectively, and there were no significant differences between the CCH and control groups (Figure 1).
CIH hypoxia exacerbates GLUT mRNA expression
The relative GLUT mRNA levels varied widely among the experimental groups (Figure 2). Compared with the control group, GLUT-1 mRNA increased in the CIH group, 0.033±0.006 vs. 0.023±0.003 (P <0.001). There were no similar changes in the CCH group. Although GLUT-2 mRNA is not abundant in adipose tissue, significant increases (1.2 fold) were observed in the CIH group, compared with the control group, 0.84±0.04 vs 1.00±0.07 (P <0.001). In contrast, the relative levels of GLUT-3 and GLUT-4 mRNA were significantly lower in the CIH group, 0.004±0.002 vs. 0.019±0.006 (P <0.001) and 0.002±0.002 vs. 0.039±0.009 (P <0.001), respectively. Except for GLUT-3, the relative levels of GLUTs showed no difference in the CCH group compared with the control group.
GLUTs proteins in WAT
All GLUT protein measurements were normalized to levels of β-actin protein and are expressed as ratios. GLUT-1 protein expression was much higher in the CCH and CIH groups compared with the control (Figure 3), especially the CIH group, 1.71±0.14 vs. 1.00±0.10, P <0.001. The relative GLUT-1 expression level among groups was as follows: CIH >CCH >control. GLUT-2 expression was increased significantly in both the CCH and CIH groups. The relative expression of the GLUT-3 protein was slightly decreased in all groups compared with control. GLUT-4 protein expression in the CIH group was significantly lower than control as well as changes in the level of GLUT-4 mRNA (Figure 2). No significant differences in GLUT-4 protein expression were observed between the CCH and control group, 0.90±0.093 vs. 1.00±0.10, P=0.12. All trends in GLUTs protein expression were similar to those observed for GLUT-1/2/3 mRNA levels (Figure 2).
While the GLUT-4 immunolabel was intense in the control adipose tissue, it was remarkably less abundant in CIH animals compared with controls (Figure 4). A similar pattern was seen with GLUT-3 immunolabel, but was less dramatic than what was seen for GLUT-4. There were no obvious differences in GLUT-1 and GLUT-2 immunolabels among the groups.
Any child, from the neonate to the adolescent, may experience OSA syndrome, although it is most common in preschool children, especially those with enlarged T&A. But Tasker et al30 considered other factors may partly account for the occurrence symptoms of OSA after T&A surgery. A recent study confirmed that removal of T&A was not the perfect treatment for eliminating the risk of residual or recurrent sleep disordered breathing (SDB). There are must be some other factors that engaged in the persistence of OSA in childhood. de la Eva et al had 62 obese children undergo polysomnography and metabolic studies, to examine links between OSA, insulin resistance, and dyslipidemia. They found that the severity of OSA (log10(respiratory disturbance index)) correlated with fasting insulin levels, independent of BMI.31 Insulin levels may be further elevated as a consequence of OSA in obese children. However, the interrelationship between insulin levels and OSA is complex. The effect of adiposity on many clinical cohort samples is hard to be isolated, even when sophisticated statistical analyses are used.
Our results support the hypothesis that CIH exacerbates IR from pedo-period when weights were matched among different groups. Compared with the control animals, CIH animals showed a higher peripheral blood fasting insulin and lower GIR levels, which demonstrates that CIH may be engaged in the development of IR. However, GIR levels in CCH group were not significantly different from control, which indicates that CCH had no impact on whole-body insulin sensitivity.
CIH animals also showed lower GLUT-4 expression at both the mRNA and protein levels. Of all GLUTs, GLUT-4 is the most important transporter in lipid tissue, and IR is related to decrease of GLUT-4 expression on plasma membrane of adipocytes in non-insulin-dependent diabetes mellitus patients, and insulin stimulation does not increase the level of GLUT-4.28 The reduced GLUT-4 mRNA and protein expression in CIH groups also suggests that the existence of peripheral IR.
The pronounced GLUT-4 immunolabel paucity in adipocyte membrane, particularly the membrane, may influence the uptake and utilization of glucose in intraabdominal adipocytes. This change may also simultaneously induce IR in liver and muscle and even throughout the body. Hyperlipidosis, especially in the abdominal region, could cause IR, which may have a positive feedback on accumulation of adipose tissue. Therefore, our observation of GLUT-4 expression, the most important GLUT in adipose tissue, shows that such reduced expression may be a direct reflection of severe IR.
Changes in GLUTs protein expression were parallel with changes in GLUTs mRNA expression except for GLUT-3. The expression of hypoxia-sensitive GLUT-1 increased more in the CIH group than that in CCH group, which may be a result of basic energy demand modulation in introabdominal adipose tissue.
Our results strongly suggest that there is peripheral IR in CIH animals from pedo-period. After CCH, the fasting blood insulin level, GIR level and relative GLUT-4 mRNA and protein levels in adipocyte membrane showed little change compared with the control group. Apparently it is CIH that triggered IR from pedo-animals, not CCH. The changes in glycometabolism are caused by reputats hypoxia-reoxygen, not continuous hypoxia.
How can CIH cause IR? Some studies suggest an autonomic neural mechanism. Autonomic neuropathy is a dysfunction of the central respiratory motor control of the diaphragm, and decreased ability of the upper airway to maintain patency during sleep. Sleep disturbances negatively affect carbohydrate metabolism and endocrine function.32 Proposed mechanisms for this metabolic dysfunction are stimulation of the sympathetic nervous system, hypothalamic-pituitary-adrenal axis, and release of adipocyte-derived inflammatory factors33-35 that increase expression of interleukin-6, tumor necrosis factor α, and leptin.36 Results from animal model studies and human studies of sleep disorders both suggested that chronic intermittent hypoxia could cause a degree of sympathetic activity37 and IR increase.38-44 The possibility that OSA contributes to IR is strengthened by the efficacy of continuous positive airway pressure (CPAP) in decreasing the degree of IR in OSA patients, particularly in less obese subjects.45
Several studies demonstrated the persistence of EDS and fatigue in children and teenagers who underwent T&A surgery. Guilleminault et al46 showed that recurrentapnea was seen in children at puberty despite T&A surgery before eight year. This information lead us consider other causes of airway obstruction in patients who underwent T&A surgery for SDB. Metabolic disorders from early age may be part of the reason for persistent SDB after T&A surgery. In addition, OSA influences metabolic dysfunction or IR in obese subjects, and IR and recurrent OSA trigger weight gain. The feedback between of OSA and metabolic dysfunction can have a negative influence the therapeutic effect of T&A surgery. Waters and colleagues47 found that over time the resolution of OSA could influence on changes in metabolic markers (cholesterol) in children. So maintaining the glycometabolism balance and weight from an early age may alleviate some of these symptoms, such as EDS in children.
We found that CIH decreased GLUT expression in the adipocyte membrane, and caused insulin insensitivity. These changes may be related to high export of sympathesis, fragments of sleep, loss of sleep, imbalance of the hypothalamic-pituitary axis, dysfunction of endothelium and changes in cytokine or adipokine levels.12,48 Some researchers have reported the efficacy of CPAP in treating OSA patients,49 although there were no specific mechanisms as to how CPAP promoted GLUT production and rhagiocrine cell glucose utilization. CPAP decrease of IR might terminate the state of hormone disturbance.
Only long-term effects of CIH were investigated, not short-term effects of hypoxia-reoxygenation. Moreover, the increased oxygen consumption caused by movement or eating under CIH is hard to control, and we ignored these effects in statistical analysis. In the future, we should test the effects of metabolic therapy in clinical patients who underwent T&A surgery. It does not matter which comes first: obese or metabolic disturbance, the mutually destructive effect of their co-morbidity on OSA is related to the decreased expression of GLUT-4 in intraabdominal adipose tissues.
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