As plasma glucose level falls, protective mechanisms are activated to defend against hypoglycemia. Those mechanisms include counterregulatory hormone responses such as lowering of insulin secretion, increased secretion of glucagon and an increase in the adrenomedullary hormone, epinephrine [1]. In addition, the body activates behavioral defenses that make the individual aware of the hypoglycemia prompting food intake to ensure a continuous supply of glucose to the brain [2]. 

Patients with absolute deficiency of endogenous insulin, such as those with established type 1 diabetes or advanced type 2 diabetes who depend on exogenous insulin, have compromised defenses against hypoglycemia including failure to decrease insulin levels, failure of glucagon stimulation, and attenuated epinephrine secretion [1, 3-4]. Hypoglycemia is a significant mortality risk for type I diabetic patients and type II diabetic patients treated with advanced disease who are also treated with exogenous insulin [5]. Hypoglycemia is responsible for 6-10% of deaths in type 1 diabetics [6]. 

The concept of hypoglycemia-associated autonomic failure (HAAF) refers to an attenuated sympathoadrenal response to hypoglycemia. This response normally includes release of epinephrine, increased neural sympathetic tone, and onset of neurogenic symptoms such as pallor (adrenergic vasoconstriction), diaphoresis (cholinergic stimulation of sweat glands), anxiety, and tachycardia (adrenergic). The lack of such symptoms known as impaired hypoglycemia awareness blunts the behavioral defenses and predisposes the individual to repeated hypoglycemic events establishing a vicious cycle [1]. The risk of recurrent severe hypoglycemia is increased by at least 6 fold by impaired hypoglycemia awareness, and severe hypoglycemic events have been associated with fatal arrhythmias in animals, and death in humans [1, 7-8]. A key finding that led to the concept of HAAF in diabetics is that an event of hypoglycemia (as may occur during sleep or exercise), impairs the sympathetic response to a subsequent hypoglycemic event in nondiabetics and diabetics [9]. Thus, frequent hypoglycemic events can contribute to HAAF. HAAF is a functional disorder different from diabetic autonomic neuropathy, which is a structural disorder; however, HAAF prevalence is greater in patients with diabetic autonomic neuropathy [10]. 

Obstructive sleep apnea (OSA) is especially common in obese patients with type 2 diabetes. In the Look Ahead study, 86% of type 2 diabetic patients had undiagnosed OSA and 22% had severe OSA [11]. A recent meta-analysis indicates the prevalence of OSA is also significantly higher in non-obese type 1 diabetic patients (52%, mean BMI 22.9-25.8 kg/m2) compared to the general population . These data are consistent with a large clinical study that reported OSA in 46% of type 1 diabetic patients (mean BMI 24.4-26.4 kg/m2 ), suggesting the comorbidity of diabetes and OSA may not be due to obesity. Diabetic patients with severe OSA have higher prevalence rates of diabetic complications including diabetic peripheral neuropathy and diabetic retinopathy compared to diabetic patients with mild-moderate OSA [12]. Furthermore, the apnea hypopnea index was associated with PARP activation (a marker of oxidative stress), decreased intraepidermal nerve fiber density, and diabetic foot ulcers in patients with type 2 diabetes [13]. Importantly, controlled experiments in healthy human subjects have shown that key features of OSA including sleep restriction, sleep fragmentation, and intermittent hypoxia can lead to glucose dysregulation [14-18]. Further supporting comorbidity of OSA and type 2 diabetes, a recent meta-analysis by Reutrakul et al. reports OSA is independently associated with type 2 diabetes (unadjusted pooled relative risk: 1.62, 95% CI, 1.45-1.80) which is larger than the risk of type 2 diabetes by being physically inactive (adjusted relative risk: 1.2) [19]. In addition to the association between OSA and type 2 diabetes, OSA is associated with several other risk factors for atherosclerosis including endothelial dysfunction [20], vascular inflammation [21] hypertension [22], dyslipidemia [23], type 2 diabetes [11, 24], and the metabolic syndrome. 

Intermittent hypoxia that occurs during obstructive sleep apnea (OSA) also triggers a response that involves the activation of the sympathetic system, including the activation of the carotid body chemoreceptor which results in increased ventilatory drive and minute ventilation to restore normoxia [25-27]. Thus, in a similar mechanism of repeated sympathetic stimulation, OSA could potentially contribute to autonomic dysfunction, predisposing patients with OSA and diabetes to impaired hypoglycemia awareness.  

It is known that diabetic autonomic neuropathy is associated with impaired hypoglycemia awareness and that diabetic autonomic neuropathy can be associated with more severe obstructive sleep apnea [28]. Thus, it is plausible that other conditions associated with diabetic autonomic neuropathy, such as impaired hypoglycemia awareness and HAAF, may be affected by obstructive sleep apnea, particularly as they trigger similar pathophysiologic responses at the sympathetic level. Interestingly, adrenergic activation mediates the reduced sympathoadrenal response to subsequent hypoglycemia (following a preceding hypoglycemic episode), which is a key feature of HAAF and the use of adrenergic blockage prevents antecedent hypoglycemia from impairing adrenergic activation to subsequent hypoglycemia [29], thus beta-adrenergic antagonists may prevent the development of HAAF. Furthermore, patients with untreated OSA have increased sympathetic tone [30], perhaps providing a chronically activated sympathetic environment, thus predisposing the patient to the development of HAAF. It has been established that sleep and exercise can increase the sympathetic tone and also predispose to HAAF, but the relationship between OSA and HAAF has not been investigated in the current literature.

Our overall hypothesis is that OSA in part influences the severity of impaired hypoglycemia awareness. The purpose of the study is to evaluate whether or not OSA is associated with attenuation of the sympathoadrenal response to hypoglycemia (HAAF), presenting clinically as impaired hypoglycemia awareness.

Subjects 

Type 2 diabetic patients undergoing overnight diagnostic polysomnography for the evaluation of sleep problems at the Sleep Center at Alton Memorial Hospital (Alton, IL) were invited to participate and sign an institutionally-approved consent form. Diabetic patients were considered eligible if they were using medication for diabetes, either insulin or noninsulin therapies. Exclusion criteria were: 1) unable to provide informed consent; 2) unable to read or understand hypoglycemia questions; 3) children or pregnant women; 4) alcohol intake in the last 24h; and 5) patients on stimulants (i.e. methylphenidate, mixed amphetamine salts). 

Questionnaires 

Upon reporting to the sleep center and following completion of informed consent, patients self-administered the Hypoglycemia Awareness Questionnaire [31] (Appendix 1). This survey has been previously validated for assessment of hypoglycemia awareness [31] and compared to other surveys, it is easier to administer. The Hypoglycemia Awareness Questionnaire validation study [31] meets requirements for a positive rating for the following quality criteria for measurement properties of health status questionnaires: content validity, criterion validity, construct validity, and reproducibility, as described in [32] and included in the Consensus-Based Standards for the Selection of Health Measurement Instruments (COSMIN) checklist. Thus, we are confident the Hypoglycemia Awareness Questionnaire is an appropriate instrument to assess hypoglycemia awareness. 

Specifically, the question “Do you recognize symptoms when you have a hypo?” was used to classify the respondents. The categories for answers were always, usually, occasionally, never, or don’t know. All subjects who selected ‘always or usually’ were classified as having no impaired hypoglycemia awareness (NIHA). Subjects who selected ‘occasionally or never’ were classified as having impaired hypoglycemia awareness (IHA). However, subjects who selected ‘never’ and were not using insulin or oral antihyperglycemics associated with hypoglycemia were classified as NIHA, as the ‘never’ response indicates that they do not experience hypoglycemia versus IHA. In addition, the included patients completed a sleep history and medical history questionnaire, as part of usual care prior to undergoing polysomnography. 

Polysomnography 

Diagnostic polysomnography was performed using the Natus Sleepworks 7.1.1 (Pleasanton, CA). This methodology records brain activity, eye movements, heart rate, blood pressure, oxygen, carbon dioxide, air flow, chest movement, and oxygen saturation among other variables. The American Academy of Sleep Medicine (AASM) “recommended” guidelines were used for scoring apnea/hypopnea index [33]. Apnea hypopnea index was scored using positional data (body position: supine, lateral, or prone) for each patient. An apnea/hypopnea index greater than or equal to 5 events per hour is consistent with a diagnosis of OSA according to current AASM guidelines [34]. In the current study, severity of sleep apnea was classified according to  the apnea/hypopnea index with 5-15 events per hour categorized as mild OSA, 16-30 events per hour categorized as moderate OSA, and > 30 events per hour categorized as severe OSA. 

Statistical analysis 

Clinical and demographic characteristics (Table 1), comorbid conditions (Table 2), medication use (Table 3), and apnea-related symptoms (Table 4) were categorized by NIHA or IHA status. Data were examined for normality using a histogram. Continuous data that were normally distributed were compared using a two-sided t-test with unequal variances. Non-normally distributed continuous data were analyzed usingthe Wilcoxon Rank Sum test. Categorical variables were analyzed using Pearson Chi-Square or Fisher’s Exact Tests. 

Self-reported hypoglycemia symptoms from the Hypoglycemia Awareness Questionnaire (Appendix 1) were stratified by mechanism (i.e., autonomic, neuroglycopenic, or nonspecific, Table 5). Univariate and multivariate logistic regression models were constructed to assess the relation of autonomic (Table 6), neuroglycopenic (Table 7), or nonspecific symptoms of hypoglycemia (Table 8) to the presence other disease states and medication use. All variables were included in the multivariate models at the same time. Explanatory variables were selected based on a theoretical or hypothesized relation to the outcomes being modeled. The baseline set of covariate values was for a person who was 59.98 years of age, male, obese, with no comorbid conditions or use of medications from the drug classes studied. Statistical significance was assumed when p < .05. All analyses were performed using Stata MP 14.2 (StataCorp. 2015. Stata Statistical Software: Release 14. College Station, TX: StataCorp LP.). 

For this study, we included all diabetic patients undergoing diagnostic sleep studies in our center in one calendar year. We calculated approximately 250-300 diabetic patients would have a  diagnostic polysomnography, and assumed 30-40% of them would agree to participate in our study, aiming for approximately 90-100 patients.

Table 1: Clinical and demographic characteristics (n = 78)

Table 2 : Comorbid conditions (n = 78, %)

Table 3: Medication use (n = 78, %)

Table 4: Apnea-related symptoms (n = 78, column %)

Table 5: Categorization of common hypoglycemia symptoms

Table 6: Logistic regression of autonomic symptoms (n = 76)

Table 7: Logistic regression of neuroneuroglycopenic symptoms (n = 76)

Despite the failure to identify a relationship between OSA and impaired hypoglycemia awareness, this study identified important relationships between specific medications and comorbid conditions associated neurodegeneration and dysfunction. Future studies with more stringent methods to stratify patients into NIHA vs. IHA, are warranted to determine a relationship between OSA and impaired hypoglycemia awareness. Hypoglycemia is a life-threatening complication in diabetic patients, thus there is urgent need to further understand and reduce the prevalence of impaired hypoglycemia awareness in effort to reduce hypoglycemia-induced mortality. This study lays the groundwork or future studies on the relationship between OSA and impaired hypoglycemia awareness in diabetic patients.

HAAF: hypoglycemia-associated autonomic failure 
IHA: impaired hypoglycemia awareness 
NIHA: no impaired hypoglycemia awareness 
OSA: obstructive sleep apnea 
SNRIs: selective serotonin and norepinephrine reuptake inhibitors 

Ethics Approval and Consent to Participate This study was approved by the Christian Hospital Institutional Review Board (no reference number assigned). All participants signed an informed consent before participating in this study. 

Availability of Data and Material 

The datasets used and/or analyzed during this study are not publicly available (due to no funding received for this study), but are available from the corresponding author upon reasonable request. 

Competing Interests 

The authors declare that they have no competing interests.

Funding 

Data collection occurred during a diagnostic sleep study as part of usual care for patients seeking treatment for sleep disorders, thus no funding was required or obtained in support of this study. 

Authors’ Contributions 

BLG assisted with study design, data interpretation, and was a major contributor in writing the manuscript. SAS performed statistical analysis. JAL was responsible for study design, data collection management, data interpretation, and assisted with manuscript writing. AR recruited patients, assisted with study design and methodology, and assisted with polysomnography data interpretation. CMH performed statistical analysis, data interpretation, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript. 

Acknowledgements 

The authors would like to thank the staff at the Alton Memorial Hospital Sleep Center for their assistance with data collection: Julie Hodge, secretary, and technicians Vanessa Harvey, Sandra Rongey, Lynne Kloemken, Rachel Hill, and Karon Signorino.

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