Effect of adrenal medullectomy on metabolic responses to chronic intermittent hypoxia in the frequently sampled intravenous glucose tolerance test.

Obstructive sleep apnea is associated with type 2 diabetes. We have previously developed a mouse model of intermittent hypoxia (IH) mimicking oxyhemoglobin desaturations in patients with sleep apnea and have shown that IH increases fasting glucose, hepatic glucose output, and plasma catecholamines. We hypothesize that adrenal medulla modulates glucose responses to IH and that such responses can be prevented by adrenal medullectomy. We performed adrenal medullectomy or sham surgery in lean C57BL/6J mice, which were exposed to IH or intermittent air (control) for 4 wk followed by the frequently sampled intravenous glucose tolerance test (FSIVGTT) in unanesthetized unrestrained animals. IH was administered during the 12-h light phase (9 AM to 9 PM) by decreasing inspired oxygen from 21 to 6.5% 60 cycles/h. Insulin sensitivity (SI), insulin independent glucose disposal [glucose effectiveness (SG)], and the insulin response to glucose (AIRG) were determined using the minimal model method. In contrast to our previous data obtained in restrained mice, IH did not affect fasting blood glucose and plasma insulin levels in sham-operated mice. IH significantly decreased SG but did not affect SI and AIRG Adrenal medullectomy decreased fasting blood glucose and plasma insulin levels and increased glycogen synthesis in the liver in hypoxic mice but did not have a significant effect on the FSIVGTT metrics. We conclude that, in the absence of restraints, IH has no effect on glucose metabolism in lean mice with exception of decreased SG, whereas adrenal medullectomy decreases fasting glucose and insulin levels in the IH environment.NEW & NOTEWORTHY To our knowledge, this is the first study examining the role of adrenal catecholamines in glucose metabolism during intermittent hypoxia (IH) in unanesthetized unrestrained C57BL/6J mice. We report that IH did not affect fasting glucose and insulin levels nor insulin sensitivity and insulin secretion during, whereas glucose effectiveness was decreased. Adrenal medullectomy decreased fasting blood glucose and insulin levels in mice exposed to IH but had no effect on glucose metabolism, insulin secretion, and insulin sensitivity.

Lysyl Oxidase as a Serum Biomarker of Liver Fibrosis in Patients with Severe Obesity and Obstructive Sleep Apnea.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) is associated with the progression of nonalcoholic fatty liver disease (NAFLD). We hypothesized that the hypoxia of OSA increases hepatic production of lysyl oxidase (LOX), an enzyme that cross-links collagen, and that LOX may serve as a biomarker of hepatic fibrosis. DESIGN: Thirty-five patients with severe obesity underwent liver biopsy, polysomnography, and serum LOX testing. A separate group with severe OSA had serum LOX measured before and after 3 mo of CPAP or no therapy, as did age-matched controls. LOX expression and secretion were measured in mouse hepatocytes following exposure to hypoxia. SETTING: The Johns Hopkins Bayview Sleep Disorders Center, and the Hypertension Unit of the Heart Institute at the University of São Paulo Medical School. MEASUREMENTS AND RESULTS: In the bariatric cohort, the apnea-hypopnea index was higher in patients with hepatic fibrosis than in those without fibrosis (42.7 ± 30.2 events/h, versus 16.2 ± 15.5 events/h; P = 0.002), as was serum LOX (84.64 ± 29.71 ng/mL, versus 45.46 ± 17.16 ng/mL; P < 0.001). In the sleep clinic sample, patients with severe OSA had higher baseline LOX than healthy controls (70.75 ng/mL versus 52.36 ng/mL, P = 0.046), and serum LOX decreased in patients with OSA on CPAP (mean decrease 20.49 ng/mL) but not in untreated patients (mean decrease 0.19 ng/mL). Hypoxic mouse hepatocytes demonstrated 5.9-fold increased LOX transcription (P = 0.046), and enhanced LOX protein secretion. CONCLUSIONS: The hypoxic stress of obstructive sleep apnea may increase circulating lysyl oxidase (LOX) levels. LOX may serve as a biomarker of liver fibrosis in patients with severe obesity and nonalcoholic fatty liver disease.

The effect of adrenal medullectomy on metabolic responses to chronic intermittent hypoxia.

Obstructive sleep apnea causes intermittent hypoxia (IH) and is associated with insulin resistance and type 2 diabetes. IH increases plasma catecholamine levels, which may increase insulin resistance and suppress insulin secretion. The objective of this study was to determine if adrenal medullectomy (MED) prevents metabolic dysfunction in IH. MED or sham surgery was performed in 60 male C57BL/6J mice, which were then exposed to IH or control conditions (intermittent air) for 6 weeks. IH increased plasma epinephrine and norepinephrine levels, increased fasting blood glucose and lowered basal and glucose-stimulated insulin secretion. MED decreased baseline epinephrine and prevented the IH induced increase in epinephrine, whereas the norepinephrine response remained intact. MED improved glucose tolerance in mice exposed to IH, attenuated the impairment in basal and glucose-stimulated insulin secretion, but did not prevent IH-induced fasting hyperglycemia or insulin resistance. We conclude that the epinephrine release from the adrenal medulla during IH suppresses insulin secretion causing hyperglycemia.

Carotid body denervation prevents fasting hyperglycemia during chronic intermittent hypoxia.

Obstructive sleep apnea causes chronic intermittent hypoxia (IH) and is associated with impaired glucose metabolism, but mechanisms are unknown. Carotid bodies orchestrate physiological responses to hypoxemia by activating the sympathetic nervous system. Therefore, we hypothesized that carotid body denervation would abolish glucose intolerance and insulin resistance induced by chronic IH. Male C57BL/6J mice underwent carotid sinus nerve dissection (CSND) or sham surgery and then were exposed to IH or intermittent air (IA) for 4 or 6 wk. Hypoxia was administered by decreasing a fraction of inspired oxygen from 20.9% to 6.5% once per minute, during the 12-h light phase (9 a.m.-9 p.m.). As expected, denervated mice exhibited blunted hypoxic ventilatory responses. In sham-operated mice, IH increased fasting blood glucose, baseline hepatic glucose output (HGO), and expression of a rate-liming hepatic enzyme of gluconeogenesis phosphoenolpyruvate carboxykinase (PEPCK), whereas the whole body glucose flux during hyperinsulinemic euglycemic clamp was not changed. IH did not affect glucose tolerance after adjustment for fasting hyperglycemia in the intraperitoneal glucose tolerance test. CSND prevented IH-induced fasting hyperglycemia and increases in baseline HGO and liver PEPCK expression. CSND trended to augment the insulin-stimulated glucose flux and enhanced liver Akt phosphorylation at both hypoxic and normoxic conditions. IH increased serum epinephrine levels and liver sympathetic innervation, and both increases were abolished by CSND. We conclude that chronic IH induces fasting hyperglycemia increasing baseline HGO via the CSN sympathetic output from carotid body chemoreceptors, but does not significantly impair whole body insulin sensitivity.

Chronic intermittent hypoxia induces atherosclerosis via activation of adipose angiopoietin-like 4.

RATIONALE: Obstructive sleep apnea is a risk factor for dyslipidemia and atherosclerosis, which have been attributed to chronic intermittent hypoxia (CIH). Intermittent hypoxia inhibits a key enzyme of lipoprotein clearance, lipoprotein lipase, and up-regulates a lipoprotein lipase inhibitor, angiopoietin-like 4 (Angptl4), in adipose tissue. The effects and mechanisms of Angptl4 up-regulation in sleep apnea are unknown. OBJECTIVES: To examine whether CIH induces dyslipidemia and atherosclerosis by increasing adipose Angptl4 via hypoxia-inducible factor-1 (HIF-1). METHODS: ApoE(-/-) mice were exposed to intermittent hypoxia or air for 4 weeks while being treated with Angptl4-neutralizing antibody or vehicle. MEASUREMENTS AND MAIN RESULTS: In vehicle-treated mice, hypoxia increased adipose Angptl4 levels, inhibited adipose lipoprotein lipase, increased fasting levels of plasma triglycerides and very low density lipoprotein cholesterol, and increased the size of atherosclerotic plaques. The effects of CIH were abolished by the antibody. Hypoxia-induced increases in plasma fasting triglycerides and adipose Angptl4 were not observed in mice with germline heterozygosity for a HIF-1α knockout allele. Transgenic overexpression of HIF-1α in adipose tissue led to dyslipidemia and increased levels of adipose Angptl4. In cultured adipocytes, constitutive expression of HIF-1α increased Angptl4 levels, which was abolished by siRNA. Finally, in obese patients undergoing bariatric surgery, the severity of nocturnal hypoxemia predicted Angptl4 levels in subcutaneous adipose tissue. CONCLUSIONS: HIF-1-mediated increase in adipose Angptl4 and the ensuing lipoprotein lipase inactivation may contribute to atherosclerosis in patients with sleep apnea.

Effects of different acute hypoxic regimens on tissue oxygen profiles and metabolic outcomes.

Obstructive sleep apnea (OSA) causes intermittent hypoxia (IH) during sleep. Both obesity and OSA are associated with insulin resistance and systemic inflammation, which may be attributable to tissue hypoxia. We hypothesized that a pattern of hypoxic exposure determines both oxygen profiles in peripheral tissues and systemic metabolic outcomes, and that obesity has a modifying effect. Lean and obese C57BL6 mice were exposed to 12 h of intermittent hypoxia 60 times/h (IH60) [inspired O2 fraction (Fi(O2)) 21-5%, 60/h], IH 12 times/h (Fi(O2) 5% for 15 s, 12/h), sustained hypoxia (SH; Fi(O2) 10%), or normoxia while fasting. Tissue oxygen partial pressure (Pti(O2)) in liver, skeletal muscle and epididymal fat, plasma leptin, adiponectin, insulin, blood glucose, and adipose tumor necrosis factor-α (TNF-α) were measured. In lean mice, IH60 caused oxygen swings in the liver, whereas fluctuations of Pti(O2) were attenuated in muscle and abolished in fat. In obese mice, baseline liver Pti(O2) was lower than in lean mice, whereas muscle and fat Pti(O2) did not differ. During IH, Pti(O2) was similar in obese and lean mice. All hypoxic regimens caused insulin resistance. In lean mice, hypoxia significantly increased leptin, especially during SH (44-fold); IH60, but not SH, induced a 2.5- to 3-fold increase in TNF-α secretion by fat. Obesity was associated with striking increases in leptin and TNF-α, which overwhelmed effects of hypoxia. In conclusion, IH60 led to oxygen fluctuations in liver and muscle and steady hypoxia in fat. IH and SH induced insulin resistance, but inflammation was increased only by IH60 in lean mice. Obesity caused severe inflammation, which was not augmented by acute hypoxic regimens.

Sleep apnea determines soluble TNF-α receptor 2 response to massive weight loss.

BACKGROUND: The effects of surgical weight loss (WL) on inflammatory biomarkers associated with sleep apnea remain unknown. We sought to determine if any biomarkers can predict amelioration of sleep apnea achieved by bariatric surgery. We hypothesized that surgical WL would substantially reduce severity of sleep apnea and levels of proinflammatory cytokines. METHODS: Twenty-three morbidly obese adults underwent anthropometric measurements, polysomnography, and serum biomarker profiling prior to and 1 year following bariatric surgery. We examined the effect of WL and amelioration of sleep apnea on metabolic and inflammatory markers. RESULTS: Surgical WL resulted in significant decreases in BMI (16.7 ± 5.97 kg/m(2)/median 365 days), apnea-hypopnea index (AHI), CRP, IL-6, sTNFαR1, sTNFαR2, and leptin levels, while ghrelin, adiponectin, and soluble leptin receptor concentrations increased significantly. Utilizing an AHI cutoff of 15 events/h, we found significantly elevated levels of baseline sTNFαR2 and greater post-WL sTNFαR2 decreases in subjects with baseline AHI ≥15 events/h compared to those with AHI <15 events/h despite no significant differences in baseline BMI, age, and ΔBMI. In a multivariable linear regression model adjusting for sex, age, impaired glucose metabolism, ΔBMI, and follow-up period, the post-WL decreases in AHI were an independent predictor of the decreases in sTNFαR2 and altogether accounted for 46% of the variance of ΔsTNFαR2 (P = 0.011) in the entire cohort. CONCLUSIONS: Of all the biomarkers, the decrease in sTNFαR2 was independently determined by the amelioration of sleep apnea achieved by bariatric surgery. The results suggest that sTNFαR2 may be a specific sleep apnea biomarker across a wide range of body weight.

Chronic intermittent hypoxia induces lung growth in adult mice.

Obstructive sleep apnea (OSA) increases cardiovascular morbidity and mortality, which have been attributed to intermittent hypoxia (IH). The effects of IH on lung structure and function are unknown. We used a mouse model of chronic IH, which mimics the O(2) profile in patients with OSA. We exposed adult C57BL/6J mice to 3 mo of IH with a fraction of inspired oxygen (F(I)(O(2))) nadir of 5% 60 times/h during the 12-h light phase. Control mice were exposed to room air. Lung volumes were measured by quasistatic pressure-volume (PV) curves under anesthesia and by water displacement postmortem. Lungs were processed for morphometry, and the mean airspace chord length (Lm) and alveolar surface area were determined. Lung tissue was stained for markers of proliferation (proliferating cell nuclear antigen), apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling), and type II alveolar epithelial cells (surfactant protein C). Gene microarrays were performed, and results were validated by real-time PCR. IH increased lung volumes by both PV curves (air vs. IH, 1.16 vs. 1.44 ml, P < 0.0001) and water displacement (P < 0.01) without changes in Lm, suggesting that IH increased the alveolar surface area. IH induced a 60% increase in cellular proliferation, but the number of proliferating type II alveolocytes tripled. There was no increase in apoptosis. IH upregulated pathways of cellular movement and cellular growth and development, including key developmental genes vascular endothelial growth factor A and platelet-derived growth factor B. We conclude that IH increases alveolar surface area by stimulating lung growth in adult mice.

Intermittent and sustained hypoxia induce a similar gene expression profile in human aortic endothelial cells.

Obstructive sleep apnea may cause vascular inflammation and atherosclerosis, which has been attributed to intermittent hypoxia (IH). Recent data suggest that IH, but not sustained hypoxia (SH), activates proinflammatory genes in HeLa cells. Effects of IH and SH on the gene expression profile in human aortic endothelial cells (HAEC) have not been compared. We perfused media with alternating flow of 16% and 0% O2 (IH) or constant flow of 4% O2 (SH-4%), 8% O2 (SH-8%), or 16% O2 (control) for 8 h. Illumina gene microarrays were performed, with subsequent verification by real-time PCR. Proinflammatory cytokines in the media were measured by ELISA. Both IH and SH-4% upregulated proinflammatory genes, including heat shock protein 90-kDa B1, tumor necrosis factor superfamily member 4, and thrombospondin 1. Among all proinflammatory genes, only IL-8 mRNA showed significantly higher levels of expression (1.78-fold) during IH, compared with SH-4%, but both types of hypoxic exposure elicited striking three- to eightfold increases in IL-8 and IL-6 protein levels in the media. IH and SH-4% also upregulated antioxidant genes, including heme oxygenase-1 and nuclear factor (erythroid-derived 2)-like 2 (NRF2), whereas classical genes regulated by hypoxia-inducible factor 1 (HIF-1), such as endothelin and glucose transporter GLUT1, were not induced. SH-8% induced changes in gene expression and cytokine secretion that were similar to those of IH and SH-4%. In conclusion, short exposures to IH and SH upregulate proinflammatory and antioxidant genes in HAEC and increase secretion of proinflammatory cytokines IL-8 and IL-6 into media in similar fashions.

Obstructive sleep apnea, insulin resistance, and steatohepatitis in severe obesity.

RATIONALE: Obstructive sleep apnea is associated with insulin resistance and liver injury. It is unknown whether apnea contributes to insulin resistance and steatohepatitis in severe obesity. OBJECTIVES: To examine whether sleep apnea and nocturnal hypoxemia predict the severity of insulin resistance, systemic inflammation, and steatohepatitis in severely obese individuals presenting for bariatric surgery. METHODS: We performed sleep studies and measured fasting blood glucose, serum insulin, C-reactive protein, and liver enzymes in 90 consecutive severely obese individuals, 75 women and 15 men, without concomitant diabetes mellitus or preexistent diagnosis of sleep apnea or liver disease. Liver biopsies (n = 20) were obtained during bariatric surgery. MEASUREMENTS AND MAIN RESULTS: Obstructive sleep apnea with a respiratory disturbance index greater than 5 events/hour was diagnosed in 81.1% of patients. The median respiratory disturbance index was 15 +/- 29 events/hour and the median oxygen desaturation during apneic events was 4.6 +/- 1.8%. All patients exhibited high serum levels of C-reactive protein, regardless of the severity of apnea, whereas liver enzymes were normal. Oxygen desaturation greater than 4.6% was associated with a 1.5-fold increase in insulin resistance, according to the homeostasis model assessment index. Histopathology data suggested that significant nocturnal desaturation might predispose to hepatic inflammation, hepatocyte ballooning, and liver fibrosis. Fasting blood glucose levels and steatosis scores were not affected by nocturnal hypoxia. There was no relationship between the respiratory disturbance index and insulin resistance or liver histopathology. CONCLUSIONS: Hypoxic stress of sleep apnea may be implicated in the development of insulin resistance and steatohepatitis in severe obesity.

Chronic intermittent hypoxia and acetaminophen induce synergistic liver injury in mice.

Obstructive sleep apnoea (OSA) leads to chronic intermittent hypoxia (CIH) during sleep. Obstructive sleep apnoea has been associated with liver injury. Acetaminophen (APAP; known as paracetamol outside the USA) is one of the most commonly used drugs which has known hepatotoxicity. The goal of the present study was to examine whether CIH increases liver injury, hepatic oxidative stress and inflammation induced by chronic APAP treatment. Adult C57BL/6J mice were exposed to CIH or intermittent air (IA) for 4 weeks. Mice in both groups were treated with intraperitoneal injections of either APAP (200 mg kg(-1)) or normal saline daily. A combination of CIH and APAP caused liver injury, with marked increases in serum alanine aminotransferase, aspartate aminotransferase (AST), gamma-glutamyl transferase and total bilirubin levels, whereas CIH alone induced only elevation in serum AST levels. Acetaminophen alone did not affect serum levels of liver enzymes. Histopathology revealed hepatic necrosis and increased apoptosis in mice exposed to CIH and APAP, whereas the liver remained intact in all other groups. Mice exposed to CIH and APAP exhibited decreased hepatic glutathione in conjunction with a fivefold increase in nitrotyrosine levels, suggesting formation of toxic peroxynitrite in hepatocytes. Acetaminophen or CIH alone had no effect on either glutathione or nitrotyrosine. A combination of CIH and APAP caused marked increases in pro-inflammatory chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory protein-2, which were not observed in mice exposed to CIH or APAP alone. We conclude that CIH and chronic APAP treatment lead to synergistic liver injury, which may have clinical implications for patients with OSA.

Dyslipidemia and atherosclerosis induced by chronic intermittent hypoxia are attenuated by deficiency of stearoyl coenzyme A desaturase.

Obstructive sleep apnea leads to chronic intermittent hypoxia (CIH) and is associated with atherosclerosis. We have previously shown that C57BL/6J mice exposed to CIH and a high-cholesterol diet develop dyslipidemia, atherosclerosis of the aorta, and upregulation of a hepatic enzyme of lipoprotein secretion, stearoyl coenzyme A desaturase 1 (SCD-1). We hypothesized that (1) SCD-1 deficiency will prevent dyslipidemia and atherosclerosis during CIH; and (2) human OSA is associated with dyslipidemia and upregulation of hepatic SCD. C57BL/6J mice were exposed to CIH or normoxia for 10 weeks while being treated with either SCD-1 or control antisense oligonucleotides. Obese human subjects underwent sleep study and bariatric surgery with intraoperative liver biopsy. In mice, hypoxia increased hepatic SCD-1 and plasma very-low-density lipoprotein cholesterol levels and induced atherosclerosis lesions in the ascending aorta (the cross-section area of 156514+/-57408 microm(2)), and descending aorta (7.0+/-1.2% of the total aortic surface). In mice exposed to CIH and treated with SCD-1 antisense oligonucleotides, dyslipidemia and atherosclerosis in the ascending aorta were abolished, whereas lesions in the descending aorta showed 56% reduction. None of the mice exposed to normoxia developed atherosclerosis. In human subjects, hepatic SCD mRNA levels correlated with the degree of nocturnal hypoxemia (r=0.68, P=0.001). Patients exhibiting oxyhemoglobin desaturations at night showed higher plasma triglyceride and low-density lipoprotein cholesterol levels, compared to subjects without hypoxemia. In conclusion, CIH is associated with dyslipidemia and overexpression of hepatic SCD in both humans and mice alike; SCD-1 deficiency attenuates CIH-induced dyslipidemia and atherosclerosis in mice.