Obstructive sleep apnea is associated with insulin resistance, lipid dysregulation, and hepatic steatosis and fibrosis in nonalcoholic fatty liver disease (NAFLD). We have previously shown that hepatocyte HIF-1 (hypoxia-inducible factor-1) mediates the development of liver fibrosis in a mouse model of NAFLD. We hypothesized that intermittent hypoxia (IH) modeling obstructive sleep apnea would worsen hepatic steatosis and fibrosis in murine NAFLD, via HIF-1. Mice with hepatocyte-specific deletion of Hif1a (Hif1a 2/2 hep) and wild-type (Hif1a F/F ) controls were fed a high trans-fat diet to induce NAFLD with steatohepatitis. Half from each group were exposed to IH, and the other half were exposed to intermittent air. A glucose tolerance test was performed just prior to the end of the experiment. Mitochondrial efficiency was assessed in fresh liver tissue at the time of death. The hepatic malondialdehyde concentration and proinflammatory cytokine levels were assessed, and genes of collagen and fatty acid metabolism were examined. Hif1a 2/2 hep mice gained less weight than wild-type Hif1a mice (22.3 g, P = 0.029). There was also a genotype-independent effect of IH on body weight, with less weight gain in mice exposed to IH (P = 0.003). Fasting glucose, homeostatic model assessment for insulin resistance, and glucose tolerance test results were all improved in Hif1a 2/2 hep mice. Liver collagen was increased in mice exposed to IH (P = 0.033) and was reduced in Hif1a 2/2 hep mice (P , 0.001), without any significant exposure/genotype interaction being demonstrated. Liver TNF-a and IL-1b were significantly increased in mice exposed to IH and were decreased in Hif1a 2/2 hep mice. We conclude that HIF-1 signaling worsens the metabolic profile and hastens NAFLD progression and that IH may worsen liver fibrosis. These effects are plausibly mediated by hepatic inflammatory stress.

Study objectives Chronic obstructive pulmonary disease and obstructive sleep apnea overlap syndrome is associated with excess mortality, and outcomes are related to the degree of hypoxemia. People at high altitude are susceptible to periodic breathing, and hypoxia at altitude is associated with cardio-metabolic dysfunction. Hypoxemia in these scenarios may be described as superimposed sustained plus intermittent hypoxia, or overlap hypoxia (OH), the effects of which have not been investigated. We aimed to characterize the cardio-metabolic consequences of OH in mice. Methods C57BL/6J mice were subjected to either sustained hypoxia (SH, FiO2=0.10), intermittent hypoxia (IH, FiO2=0.21 for 12 hours, and FiO2 oscillating between 0.21 and 0.06, 60 times/hour, for 12 hours), OH (FiO2=0.13 for 12 hours, and FiO2 oscillating between 0.13 and 0.06, 60 times/hour, for 12 hours), or room air (RA), n=8/group. Blood pressure and intraperitoneal glucose tolerance test were measured serially, and right ventricular systolic pressure (RVSP) was assessed. Results Systolic blood pressure transiently increased in IH and OH relative to SH and RA. RVSP did not increase in IH, but increased in SH and OH by 52% (p<0.001) and 20% (p=0.001). Glucose disposal worsened in IH and improved in SH, with no change in OH. Serum LDL and VLDL increased in OH and SH, but not in IH. Hepatic oxidative stress increased in all hypoxic groups, with the highest increase in OH. Conclusions Overlap hypoxia may represent a unique and deleterious cardio-metabolic stimulus, causing systemic and pulmonary hypertension, and without protective metabolic effects characteristic of sustained hypoxia.

Rationale: The overlap syndrome of chronic obstructive pulmonary disease (COPD) and sleep disordered breathing (SDB) is associated with higher morbidity and mortality relative to either disease alone, and poorer outcomes are associated with worsened hypoxia. People at high altitude are susceptible to SDB, and hypoxic burden in these individuals is associated with metabolic dysfunction and cardiovascular disease. We aimed to characterize the cardio‐metabolic consequences of superimposed sustained plus intermittent hypoxia, as a model of COPD/SDB overlap syndrome, or high‐altitude hypoxia with periodic breathing. Methods:C57BL/6J mice (n=8/group) were subjected to one of four oxygen profiles for six weeks. In room air (RA), mice were kept at FiO2=0.21; in sustained hypoxia (SH), they were kept at FiO2=0.10. In intermittent hypoxia (IH), mice were kept at FiO2=0.21 for 12 hours, and FiO2 fluctuated between 0.21 and 0.06, 60 times/hour, for 12 hours. In overlap hypoxia (OH), mice were kept at FiO2=0.13 for 12 hours, and FiO2 fluctuated between 0.13 and 0.06, 60 times/hour, for 12 hours. Blood pressure and heart rate variability were measured weekly, and right ventricular systolic pressure (RVSP) was measured prior to sacrifice. Intraperitoneal glucose tolerance test (GTT) was performed at baseline and before sacrifice. Results: Mice in RA gained weight (+1.3g), whereas mice in SH lost weight (‐0.5g, p=0.010 vs RA), and there were no changes in IH or OH (IH: ‐0.1g, p=0.056; OH: +0.9g, p=0.876). Blood pressure was highly variable and did not significantly differ by group. With respect to heart rate variability, the LF/HF ratio at 4 weeks increased in SH and IH, without any significant change in RA or OH mice. IH did not increase RVSP relative to RA (p=0.977), whereas SH and OH increased RVSP by 52% (p<0.001) and 20% (p=0.002), respectively. Fasting blood glucose did not change in RA (‐3 mg/dL, p=0.808), but decreased in both SH and OH (‐57 mg/dL, p=0.001, and ‐41 mg/dL, p=0.004), and increased in IH (+25 mg/dL, p=0.001). Hepatic glycogen content reflected fasting glucose levels. The difference in area under the GTT curve (final‐initial) was unchanged in OH versus RA (p=0.666), but increased in IH (p=0.015) and decreased in SH (p=0.004). Hepatic IL‐1β was increased 22% in IH (p=0.019) but not in other groups, and TNF‐α and IL‐6 were similar between groups. Hepatic malondialdehyde, an oxidative stress marker, was increased in each group relative to RA (SH: +14%, p=0.003, IH: +23%, p<0.001, OH: +35%, p<0.001). Conclusions:Overlap hypoxia may represent a unique and deleterious cardiometabolic phenotype, characterized by more severe pulmonary hypertension than chronic intermittent hypoxia, without the protective metabolic effects of sustained hypoxia. Hepatic oxidative stress was worse in OH than in other tested hypoxia profiles.

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