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Obstructive sleep apnea (OSA) is recurrent obstruction of the upper airway leading to sleep fragmentation and intermittent hypoxia (IH) during sleep. There is growing evidence from animal models of OSA that IH is independently associated with metabolic dysfunction, including dyslipidemia and insulin resistance. The precise mechanisms by which IH induces metabolic disturbances are not fully understood. Over the last decade, several groups of investigators developed a rodent model of IH, which emulates the oxyhemoglobin profile in human OSA. In the mouse model, IH induces dyslipidemia, insulin resistance and pancreatic endocrine dysfunction, similar to those observed in human OSA. Recent reports provided new insights in possible mechanisms by which IH affects lipid and glucose metabolism. IH may induce dyslipidemia by up-regulating lipid biosynthesis in the liver, increasing adipose tissue lipolysis with subsequent free fatty acid flux to the liver, and inhibiting lipoprotein clearance. IH may affect glucose metabolism by inducing sympathetic activation, increasing systemic inflammation, increasing counter-regulatory hormones and fatty acids, and causing direct pancreatic beta cell injury. IH models of OSA have improved our understanding of the metabolic impact of OSA, but further studies are needed before we can translate recent basic research findings to clinical practice.
Obstructive sleep apnea (OSA) describes recurrent collapse of the upper airway during sleep.1 OSA is a common disorder affecting 4–24% of men and 2–9% of women in the US,2 but the prevalence of OSA in obese individuals exceeds 50%.3 OSA is particularly prevalent in individuals with central (visceral) obesity. Emerging evidence suggests that OSA leads to high cardiovascular mortality and morbidity.4–6 The cardiovascular risk imposed by OSA may be mediated through effects of OSA on glucose and lipid metabolism.
Several studies have shown that the impact of OSA on metabolic function is acutely or chronically reversible with continuous positive airway pressure (CPAP).7–8 The respiratory events associated with OSA lead to changes in intrathoracic pressure, hypercapnea, arousals from sleep, and intermittent hypoxia (IH). IH is the best studied aspect of OSA in terms of metabolic effects. A number of animal and human studies demonstrated that IH causes disturbances in lipid and glucose metabolism.9,10 In the present review, we will discuss experimental models of IH and effects of IH on metabolic function.
Animal models of sleep disordered breathing were extensively reviewed elsewhere.9–10 IH models are the most commonly used research models of OSA. IH has been predominantly employed in rodents. IH is administered by cyclic delivery of nitrogen, oxygen and air to a sealed chamber. There are two different types of rodent models of IH. The first type is sleep-dependent IH.11 In this model, IH is delivered exclusively during sleep. Sleep-dependent IH requires implantation of EEG and EMG electrodes in mice and sophisticated monitoring techniques resulting in low throughput. The second type of IH system delivers the stimulus, regardless of sleep/wake state. However, investigators usually attempt to deliver the stimulus during the light phase, when rodent sleep primarily occurs. In the subsequent narrative we will discuss the latter sleep-independent IH model. IH models vary in both frequency and severity of the hypoxic stimulus. Protocols deliver "apneas" varying in frequency from 9 to 60 times/hr and varying in severity with the delivered oxygen nadir ranging from 3 to 10%.12–16 The main advantage of sleep-independent IH is high throughput. Therefore, this approach has been exclusively employed to study metabolic outcomes of IH and OSA. In our experiments we exposed mice to IH with a FiO2 nadir of 5% during the 12 hr light phase (9am – 9pm). According to our data, this regimen resulted in the mean PaO2 of 51.7 mm Hg18 and oxyhemoglobin saturation fluctuating between 99% and ~ 70%,19 which is similar to the oxyhemoglobin profile in severe OSA. In the following discussion, we will refer to this regimen, unless specified otherwise.
Finally, IH has recently been used in human experiments to study glucose metabolism in healthy subjects.19 In this study, volunteers were exposed to hypoxic gas mixtures that induced decreases in arterial oxygen saturation (SaO2) to 85% around 25 times/hr.19
We have recently reviewed relationships between OSA and dyslipidemia.20 Several cross-sectional studies suggest that OSA is independently associated with increased levels of total cholesterol, LDL and triglycerides, whereas others report no such relationships.21–24 Several studies show that OSA treatment with CPAP may have a beneficial effect on lipid profile.8,25,26 However, the majority of the studies were not specifically designed to evaluate the lipid profile, ignoring important confounding factors such as diet, physical activity and body composition.20 There are no long-term studies with follow-up beyond 6 months of CPAP treatment. Therefore, the clinical data are unclear.
Results from IH animal studies unambiguously show that IH is a direct cause of hyperlipidemia. IH causes increases in total cholesterol, HDL-C and triglycerides after 5 days, while an increase in LDL cholesterol is evident after 4 weeks.27,28 The severity of increases in lipids was proportional to the severity of the hypoxic stimulus.28 In C57BL/6J mice on a high cholesterol diet, exposure to IH for 12 wks predominantly increases VLDL and LDL.29 Similar changes in response to IH occurred in atherosclerosis-prone apolipoprotein E deficient mice.18
Early in our observations of IH-induced hyperlipidemia, we reported up-regulation of a key hepatic transcription factor of lipid biosynthesis, sterol regulatory element binding protein 1c (SREBP-1c) and a SREBP-1c -regulated enzyme, stearoyl coenzyme A desaturase 1 (SCD-1).27–30 These findings are in agreement with other studies of acute hypoxia.31 SCD-1 converts saturated fatty acids into monounsaturated fatty acids. Abundance of monounsaturated fatty acids increases the biosynthesis of cholesterol esters and triglycerides, which are incorporated into secreted VLDL particles.32,33 Indeed, we have shown that IH up-regulates lipoprotein secretion.28 Interruption of SREBP-1 signaling in transgenic mice and depletion of SCD-1 with anti-sense oligonucleotides in C57BL/6J mice prevented hyperlipidemia during IH.30,34 The increases in SCD-1 may be mediated through hypoxia inducible factor 1 (HIF-1), a master-regulator of metabolic responses to hypoxia.35 Mice with partial deficiency of functional HIF-1 are partially protected against hypertriglyceridemia and hepatic lipid accumulation during IH36 and show attenuated increases in the active nuclear isoform of SREBP-1 and SCD-1. Thus, dyslipidemia of IH may be a consequence of IH-induced up-regulation of lipid biosynthetic pathways in the liver.
Under most circumstances, in the post-absorptive state, triglycerides are derived from the re-esterification of hepatic free fatty acids (FFA). Most FFA are derived from peripheral lipolysis, but in the absorptive state, can also be synthesized from carbohydrate "de novo" in the liver.37 Simultaneously, a tightly controlled cholesterol synthesis pathway manufactures lipoproteins that are used by the liver to export triglycerides in the form of VLDL. As previously discussed, SCD-1 is involved in the conversion of saturated fatty acids to monounsaturated fatty acids for triglyceride synthesis and VLDL export. VLDL particles enter the bloodstream and undergo hydrolysis by tissue enzymes including lipoprotein lipase (LpL) to liberate FFA (see lipoprotein clearance section). We have previously shown that IH increases hepatic triglyceride and hepatic VLDL secretion, but does not cause an increase in de novo fatty acid synthesis.27 These data suggest that peripheral lipolysis is supplying hepatic fatty acids for the synthesis of triglycerides and VLDL. Our experiments in apolipoprotein E-deficient mice have shown increases in serum FFA levels suggesting increased adipose tissue lipolysis during IH.18 IH activates the sympathetic nervous system,9–10 which could potentially induce lipolysis.38–40 In addition, more FFA may be available for triglycerides and cholesterol ester synthesis if FFA do not undergo beta-oxidation, the oxygen-dependent mitochondrial combustion of fatty acids.38–40 The confluence of increased FFA delivery and impaired beta oxidation may also underlie the association between OSA and accumulation of fat in the liver, liver injury, oxidative stress, and non-alcoholic steatohepatitis.41–48 Chronic IH resulted in liver injury and mild elevation of liver enzymes in lean mice.49 In mice fed a high cholesterol high fat diet, IH converted hepatic steatosis to steatohepatitis, increasing pro- inflammatory cytokines TNF-α, IL-1β, IL-6 and MIP-2 in liver tissue, lobular inflammation and fibrosis.17 Thus, IH may lead to dyslipidemia and steatohepatitis due to increased FFA flux from adipose tissue to the liver but additional studies are needed to explore this mechanism and its relevance for human disease.
Another putative mechanism of dyslipidemia during IH is inhibited VLDL clearance. Mice exposed to IH show higher peak levels and prolonged decay of plasma chylomicrons (CM) after a fatty meal (Drager, Polotsky, Submitted). Triglyceride-rich lipoproteins, CM and VLDL, disproportionally elevated in IH are cleared from the circulation in a multi-step process, which begins with hydrolysis of triglycerides by LpL. LpL is a key enzyme in plasma lipoprotein metabolism that is preferentially expressed in adipose tissue, skeletal muscle and the heart.50,51 LpL is synthesized in adipocytes, myocytes and macrophages, secreted and transported to the luminal surface of blood vessels, where it is anchored by heparan sulfate proteoglycans and glycosylphosphatidylinositol-anchored HDL- binding protein 1.52 Our unpublished data show that IH selectively inhibits LpL in adipose tissue, but the mechanisms of this phenomenon are unknown.
In summary, IH may disrupt lipid metabolism by increasing adipose tissue lipolysis and FFA flux to the liver, up-regulating hepatic triglyceride biosynthesis and lipoprotein secretion, and suppressing lipoprotein clearance (Figure 1).
OSA is associated with increased prevalence of type 2 diabetes53 and has recently been shown to be a risk factor for incident diabetes.54 In non-diabetics, OSA is associated with insulin resistance in proportion to the degree of nocturnal hypoxemia.47,55–57 CPAP can reverse the insulin resistance of OSA both acutely (within 2 days) and chronically (after 4 months).7,58 In patients with type 2 diabetes, OSA may worsen glycemic control,59 which improves after CPAP.60,61 We will focus on the mechanisms that might explain these observations, drawing from IH experiments.
IH induces acute insulin resistance. In lean C57BL/6J mice, IH (60 cycles/hr with an FiO2 nadir 5–6%, 9 hr) resulted in a significant increase in insulin resistance measured by hyperinsulinemic euglycemic clamp.62 The insulin resistance rapidly normalized after cessation of each diurnal IH exposure.63 Chronic IH greatly exacerbates insulin resistance and glucose intolerance in obese leptin deficient ob/ob mice64 and in C57BL/6J mice with diet induced obesity (Polotsky, unpublished data). Recently, healthy human volunteers were exposed to IH simulating moderate OSA (at an average rate of 24.3 events/h).19 Alveolar hypoxia was established by inspiring hypoxic N2 –O2 gas mixture until the oxyhemoglobin saturation dropped to 85%.19 After 5 hours, an intravenous glucose tolerance test demonstrated a decrease in both insulin sensitivity and glucose effectiveness by minimal modeling methods. Moreover, IH did not induce an appropriate increase in insulin secretion, demonstrating inadequate pancreatic responses to insulin resistance.
There are several potential mechanisms of insulin resistance and impaired insulin secretion during IH. First, activation of hepatic lipid biosynthesis discussed in the previous section may lead to hepatic insulin resistance. Second, IH activates the sympathetic nervous system, which is a potent stimulator of lipolysis. FFAs reduce insulin-mediated whole body glucose uptake within hours due to interruption of insulin signaling in skeletal muscle.65,66 In addition, catecholamines directly stimulate the mobilization of glycogen and inhibit glucose uptake from muscle, stimulate secretion of glucagon, inhibit secretion of insulin, and increase gluconeogenesis in the liver.9 Up-regulation of pro-inflammatory pathways by cellular hypoxia may also lead to insulin resistance.67,68 IH also activates the hypothalamic-pituitary-adrenal axis.63 The resulting release of corticosteroids have well-defined effects leading to insulin resistance,69 including an increase in lipolysis, inhibition of insulin-dependent translocation of glucose transporter type 4 (Glut4) to the cell surface in the muscle, suppression of glycogen synthesis and an increase in gluconeogenesis. Steroid hormones also inhibit insulin secretion.70 IH may also lead to insulin resistance altering production of adipokines, hormones produced in adipose tissue. IH increases leptin gene expression and circulating protein levels levels. Leptin acts both centrally and peripherally, to inhibits insulin secretion while increasing glucose uptake.71–77
The development of insulin resistance during IH despite high leptin levels observed in OSA patients78–80 suggests a currently inexplicable phenomenon of leptin resistance.81 Finally, IH may suppress secretion of adiponectin, an insulin-sensitizing hormone.82
Diabetes is the final manifestation of insulin resistance and a failure of compensatory pancreatic beta cell insulin secretion. Experimental evidence on the effect of IH on pancreatic β-cells is scant and presented only in two publications. Immunohistochemistry of pancreatic islets showed that short-term IH increases both apoptosis and proliferation of β-cells.63,83 Xu et al. have shown that overexpression of antioxidant enzyme superoxide dismutase in pancreatic islets protected them against IH-induced apoptosis, whereas IH-induced β-cell proliferation was not affected.83 The net effect of IH exposure was increased turnover83 or increased replication of pancreatic β-cells. In contrast, human data in healthy volunteers suggest that IH may impair pancreatic endocrine function.19 Thus, IH induces insulin resistance and may have a detrimental effect on pancreatic endocrine function.
OSA is associated with dysregulation of lipid and glucose metabolism, but studying of these phenomena in patients with OSA has been challenging due to confounding effects of obesity. Animal and human work has determined that IH has an impact on metabolism that parallel findings in patients with OSA. However, mechanisms of metabolic effects of IH are still poorly understood. Potential mechanisms include tissue hypoxia/reoxygenation, systemic catecholamine-mediated lipolysis and lipotoxicity, hepatic transcriptional upregulation of lipid synthesis, impaired lipid clearance, systemic inflammation and disruption of hypothalamic-pituitary-adrenal and adipokine hormonal balance. Future clinical, basic and translational studies are warranted to determine effects of IH and OSA on metabolic function and identify the mechanisms.
Sources and Funding
Luciano F. Drager and Jonathan Jun are Post-Doctoral Fellow at Johns Hopkins University. Dr. Drager is supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq # 200032/2009-7) and Fundação Zerbini, Brazil. Dr.Jun is supported by the National Sleep Foundation/American Lung Association Pickwick Grant (SF-78568 N) and NIH T32 training grant (HL07534).
Vsevolod Y. Polotsky is supported by NIH (R01 HL80105, 5P50HL084945) and the United States Israel Binational Science Foundation (grant BSF No. 2005265). .
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