Omar Mesarwi

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.

Sleep Disorders and the Development of Insulin Resistance and Obesity

Normal sleep is characterized both by reduced glucose turnover by the brain and other metabolically active tissues, and by changes in glucose tolerance. Sleep duration has decreased over the last several decades; data suggest a link between short sleep duration and type 2 diabetes. Obstructive sleep apnea (OSA) results in intermittent hypoxia and sleep fragmentation, and also is associated with impaired glucose tolerance. Obesity is a major risk factor for OSA, but whether OSA leads to obesity is unclear. The quality and quantity of sleep may profoundly affect obesity and glucose tolerance, and should be routinely assessed by clinicians.

Intermittent hypoxia-induced glucose intolerance is abolished by α-adrenergic blockade or adrenal medullectomy

Obstructive sleep apnea causes intermittent hypoxia (IH) during sleep and is associated with dysregulation of glucose metabolism. We developed a novel model of clinically realistic IH in mice to test the hypothesis that IH causes hyperglycemia, glucose intolerance, and insulin resistance via activation of the sympathetic nervous system. Mice were exposed to acute hypoxia of graded severity (21, 14, 10, and 7% O2) or to IH of graded frequency [oxygen desaturation index (ODI) of 0, 15, 30, or 60, SpO2 nadir 80%] for 30 min to measure levels of glucose fatty acids, glycerol, insulin, and lactate. Glucose tolerance tests and insulin tolerance tests were then performed under each hypoxia condition. Next, we examined these outcomes in mice that were administered phentolamine (α-adrenergic blockade) or propranolol (β-adrenergic blockade) or that underwent adrenal medullectomy before IH exposure. In all experiments, mice were maintained in a thermoneutral environment. Sustained and IH induced hyperglycemia, glucose intolerance, and insulin resistance in a dose-dependent fashion. Only severe hypoxia (7% O2) increased lactate, and only frequent IH (ODI 60) increased plasma fatty acids. Phentolamine or adrenal medullectomy both prevented IH-induced hyperglycemia and glucose intolerance. IH inhibited glucose-stimulated insulin secretion, and phentolamine prevented the inhibition. Propranolol had no effect on glucose metabolism but abolished IH-induced lipolysis. IH-induced insulin resistance was not affected by any intervention. Acutely hypoxia causes hyperglycemia, glucose intolerance, and insulin resistance in a dose-dependent manner. During IH, circulating catecholamines act upon α-adrenoreceptors to cause hyperglycemia and glucose intolerance.

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.

Hepatocyte hypoxia inducible factor-1 mediates the development of liver fibrosis in a mouse model of nonalcoholic fatty liver disease

Background Obstructive sleep apnea (OSA) is associated with the progression of non-alcoholic fatty liver disease (NAFLD) to steatohepatitis and fibrosis. This progression correlates with the severity of OSA-associated hypoxia. In mice with diet induced obesity, hepatic steatosis leads to liver tissue hypoxia, which worsens with exposure to intermittent hypoxia. Emerging data has implicated hepatocyte cell signaling as an important factor in hepatic fibrogenesis. We hypothesized that hepatocyte specific knockout of the oxygen sensing α subunit of hypoxia inducible factor-1 (HIF-1), a master regulator of the global response to hypoxia, may be protective against the development of liver fibrosis. Methods Wild-type mice and mice with hepatocyte-specific HIF-1α knockout (Hif1a-/-hep) were fed a high trans-fat diet for six months, as a model of NAFLD. Hepatic fibrosis was evaluated by Sirius red stain and hydroxyproline assay. Liver enzymes, fasting insulin, and hepatic triglyceride content were also assessed. Hepatocytes were isolated from Hif1a-/-hep mice and wild-type controls and were exposed to sustained hypoxia (1% O2) or normoxia (16% O2) for 24 hours. The culture media was used to reconstitute type I collagen and the resulting matrices were examined for collagen cross-linking. Results Wild-type mice on a high trans-fat diet had 80% more hepatic collagen than Hif1a-/-hep mice (2.21 μg collagen/mg liver tissue, versus 1.23 μg collagen/mg liver tissue, p = 0.03), which was confirmed by Sirius red staining. Body weight, liver weight, mean hepatic triglyceride content, and fasting insulin were similar between groups. Culture media from wild-type mouse hepatocytes exposed to hypoxia allowed for avid collagen cross-linking, but very little cross-linking was seen when hepatocytes were exposed to normoxia, or when hepatocytes from Hif1a-/-hep mice were used in hypoxia or normoxia. Conclusions Hepatocyte HIF-1 mediates an increase in liver fibrosis in a mouse model of NAFLD, perhaps due to liver tissue hypoxia in hepatic steatosis. HIF-1 is necessary for collagen cross-linking in an in vitro model of fibrosis.

Sleep apnea, metabolic disease, and the cutting edge of therapy

Obstructive sleep apnea (OSA) is common, and many cross-sectional and longitudinal studies have established OSA as an independent risk factor for the development of a variety of adverse metabolic disease states, including hypertension, insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, dyslipidemia, and atherosclerosis. Nasal continuous positive airway pressure (CPAP) has long been the mainstay of therapy for OSA, but definitive studies demonstrating the efficacy of CPAP in improving metabolic outcomes, or in reducing incident disease burden, are lacking; moreover, CPAP has variable rates of adherence. Therefore, the future of OSA management, particularly with respect to limiting OSA-related metabolic dysfunction, likely lies in a coming wave of alternative approaches to endophenotyping OSA patients, personalized care, and defining and targeting mechanisms of OSA-induced adverse health outcomes.

Putting It Together: Sleep Apnea, the Integrated Stress Response, and Metabolic Dysfunction

Obstructive sleep apnea (OSA) is a very common illness, with prevalence estimates of at least 10% in the general population (1, 2), and considerably higher in certain subgroups, such as those with refractory hypertension or diabetes (3, 4). For years, researchers have recognized the apparent link between OSA and metabolic dysfunction (5). OSA has been independently associated with a variety of metabolic disorders, including elevated fasting glucose, dyslipidemia, hypertension, atherosclerosis, and nonalcoholic fatty liver disease. Although trials of continuous positive airway pressure to treat OSA have led to conflicting results regarding the causal role of OSA in these adverse metabolic outcomes, experimental data from animals and humans suggest that sympathetic overactivation, oxidative stress, and inflammation in OSA may all contribute to the development of metabolic dysfunction (6). In this issue of the Journal, Khalyfa and colleagues (pp. 477– 486) suggest that the integrated stress response (ISR) may account for some of the aspects of dysregulated glucose handling observed in OSA (7). The ISR is a eukaryotic signaling pathway that is activated in response to intrinsic and extrinsic stresses such as hypoxia, oncogene activation, viral infection, and endoplasmic reticulum stress, or depletion of necessary ligands such as amino acids and glucose (8). In response to one or more of these factors, various kinases may phosphorylate the a subunit of eukaryotic translation initiation factor 2 (eIF2a), thereby reducing global protein synthesis and aiding in cell survival, mainly via enhanced activating transcription factor 4 (ATF4) expression. There is increasing evidence for the role of the ISR in various lung diseases (9), but there are relatively few data regarding the role it may play in sleep-disordered breathing. OSA may be modeled in animals by a variety of methods. Two commonly used techniques are (1) chronic sleep fragmentation, usually using a mechanical apparatus to force repeated arousals from sleep; and (2) chronic intermittent hypoxia (IH), which involves the repeated lowering of ambient FIO2 to induce oxyhemoglobin desaturation mimicking that seen in OSA (10). Previous work in the field has demonstrated that each of these animal models of OSA may lead to ISR activation and eIF2a phosphorylation (11, 12), and IH was linked to myocardial infarction via ISR activation in a rodent model (12). However, the role of the ISR in OSA-mediated metabolic dysfunction has not yet been explored. The authors therefore sought to determine whether disruption of the ISR would protect against IH-induced glucose dysregulation. To that end, they developed a mouse strain with knockout of both GADD34 and CHOP, mediators of the ISR via eIF2a (double-mutant [DM] mice), and additional strains with GADD34 complete knockout and PERK partial knockout (PERK is a kinase that phosphorylates eIF2a to initiate the ISR). These strains were exposed to severe IH (nadir FIO2 6.4%, 20 cycles per hour, for 12 hours per day for 6 weeks) or room air. Glucose regulation was evaluated by means of the intraperitoneal glucose tolerance test and insulin tolerance test, and visceral white adipose tissue (vWAT) insulin response and immune response were also assessed. The authors found that IH induced minor but statistically significant weight loss in wild-type animals, but this effect was not seen in any other genotype. Moreover, IH induced dysglycemia during the intraperitoneal glucose tolerance test and insulin tolerance test relative to room-air–exposed mice, but this difference was abrogated in DM mice, suggesting that the ISR may account for these specific IH-induced metabolic changes. Homeostatic model assessment of insulin resistance (HOMA-IR), a product of fasting glucose and insulin levels that is sometimes used to gauge the severity of insulin resistance, was elevated in IH-exposed wild-type mice, but this effect of IH was not observed in DM mice, GADD34 mice, or PERK mice. Similarly, vWAT insulin sensitivity, assessed by changes in AKT phosphorylation, was worsened in IH, but again this effect of IH was not observed in DM mice. To help explain these changes, the authors investigated T regulatory lymphocyte and macrophage populations in vWAT, and noted an IH-induced reduction in macrophage number and shift toward M1 phenotype, as well as a reduction in T regulatory cells. No knockout strains showed any of these IH-induced effects. Finally, they evaluated ISR activation at the time the animals were killed by examining eIF2a and ATF4 phosphorylation, and as expected, IH appeared to induce the ISR in wild-type animals but not in any knockout strain. Taken together, these results suggest that IH may induce metabolic dysfunction via prolonged activation of the ISR. Although the independent effects of OSA on glucose dysregulation have been noted for several years, mechanisms to explain these findings have been relatively less well elucidated. Because OSA is a heterogeneous disease with several potential mechanistic factors (anatomic occlusion, elevated loop gain, reduced arousal threshold, and blunted neuromuscular response to airway occlusion) and with myriad effects (IH and hypercapnia, sleep fragmentation, intrathoracic pressure swings, and sympathetic overactivation), teasing out the specific targets of investigation can be daunting. In this series of experiments, Khalyfa and colleagues advance the field by validating ISR activation in IH as another potential means by which OSA may induce metabolic dysfunction. As with many novel findings, the new data raise interesting questions and have some important limitations. First and most significantly, in evaluating the metabolic responses to IH, we note that DM mice seem to have abnormal glucose handling at baseline, and that there is a genotype effect independent of IH. Thus, it becomes unclear whether between-group differences are due to the baseline characteristics, potential ceiling effects, or complex interactions of the genetic background and IH exposure. Similarly, although IH did not activate the ISR in DM mice as gauged by eIF2a phosphorylation per se, DM mice did seem to have increased baseline eIF2a phosphorylation relative to unexposed wild-type mice. It is unclear whether this may be due in part to the GADD34 knockout specifically, as GADD34 serves to terminate the early phase of the ISR. GADD34 mice also