Obstructive sleep apnoea increases lipolysis and deteriorates glucose homeostasis in patients with type 2 diabetes mellitus

Obstructive sleep apnoea (OSA) is associated with type 2 diabetes mellitus (T2DM). However, mechanisms mediating association between these two conditions remain unclear. This study investigated, whether the OSA-associated changes in adipose tissue lipolysis might contribute to impaired glucose homeostasis in patient with T2DM. Thirty-five matched subjects were recruited into three groups: T2DM + severe OSA (T2DM + OSA, n = 11), T2DM with mild/no OSA (T2DM, n = 10) and healthy controls (n = 14). Subcutaneous abdominal adipose tissue microdialysis assessed spontaneous, epinephrine- and isoprenaline-stimulated lipolysis. Glucose metabolism was assessed by intravenous glucose tolerance test. Spontaneous lipolysis was higher in the T2DM + OSA compared with the T2DM (60.34 ± 23.40 vs. 42.53 ± 10.16 μmol/L, p = 0.013), as well as epinephrine-stimulated lipolysis (236.84 ± 103.90 vs. 167.39 ± 52.17 µmol/L, p < 0.001). Isoprenaline-stimulated lipolysis was unaffected by the presence of OSA (p = 0.750). The α2 anti-lipolytic effect was decreased in T2DM + OSA by 59% and 315% compared with T2DM and controls (p = 0.045 and p = 0.007, respectively). The severity of OSA (AHI) was positively associated with spontaneous (p = 0.037) and epinephrine-stimulated (p = 0.026) lipolysis. The α2-adrenergic anti-lipolytic effect (p = 0.043) decreased with increasing AHI. Spontaneous lipolysis was positively associated with Insulin resistance (r = 0.50, p = 0.002). Epinephrine-stimulated lipolysis was negatively associated with the Disposition index (r =  − 0.34, p = 0.048). AHI was positively associated with Insulin resistance (p = 0.017) and negatively with the Disposition index (p = 0.038). Severe OSA in patients with T2DM increased adipose tissue lipolysis, probably due to inhibition of the α2-adrenergic anti-lipolytic effect. We suggest that dysregulated lipolysis might contribute to OSA-associated impairments in insulin secretion and sensitivity.

glucose output and stimulated adipose tissue lipolysis in mice 5,[10][11][12][13][14] . Multiple intermittent-hypoxia induced metabolic derangements were only partially reversed after cessation of the exposure, whereas β-cell dysfunction gradually worsened 10 . Furthermore, similar effects were observed in humans, as acute exposure to intermittent hypoxia decreased insulin sensitivity and impaired β-cell function in healthy volunteers 15 . Among multiple suggested mechanisms (oxidative stress, increased sympathetic activity, elevated corticoid levels, elevated plasma endothelin-1 levels and activation of inflammatory pathways) increased levels of circulating free fatty acids (FFA) were recently proposed as the potential causal link between hypoxic exposure and impaired glucose metabolism 16,17 . Elevated circulating FFA levels impair glucose uptake and metabolism in muscle due to inhibition of key glycolytic enzymes 18,19 , induce Insulin resistance in liver, manifested as augmented hepatic glucose output, through effects on intracellular signalling pathways and gene expression [20][21][22] and decrease insulin secretion and stimulate apoptosis in β-cells 23,24 . Furthermore, increased FFA levels further worsen metabolic control and insulin secretion in patients with T2DM 25,26 .
Importantly, adipose tissue oxygen levels as low as 4% O 2 were observed during apnoeic episodes in a mouse model of OSA 27 , whereas in-vitro and rodent studies 5 showed that lipolysis in adipocytes (a major source of circulating FFA) was up-regulated by low pericellular oxygen levels 28 . It is thus plausible to hypothesize that OSAassociated hypoxic episodes might augment adipose tissue lipolysis and stimulate release of FFA into circulation with its adverse metabolic consequences.
The aim of this study was to investigate whether adipose tissue lipolysis is modified by the presence of OSA in patients with T2DM to expand the knowledge on adipose-tissue pathophysiology in OSA, provide mechanistic links for epidemiological associations and identify possible treatment targets. To achieve this goal, we studied patients with T2DM with severe OSA compared to matched T2DM patients with mild/no OSA and control subjects without OSA. Subsequently, we assessed the impact of OSA on metabolic parameters such as Insulin resistance and β-cell function and we investigated the possible role of lipolysis-a proven drug target representing a novel treatment option for OSA-associated metabolic impairments 5 .
There were no differences among groups in age and body composition (BMI, adiposity); however, waist circumference was higher in the T2DM + OSA group (111.91 ± 6.83 cm) compared with both the T2DM and control groups (104.50 ± 7.15 cm and 102.07 ± 8.39 cm, respectively, p = 0.033 and p = 0.003, respectively) as summarized in Table 1. Similarly, no differences were observed in plasma lipid, FFA and glycerol levels; nevertheless, subjects with severe OSA presented elevated plasmatic cortisol (by 20%), alanine transaminase (by 52%), and gammaglutamyl transferase (by 224%) levels compared with T2DM group and the healthy controls, as shown in Table 2. Additionally, subjects diagnosed with T2DM showed 47% higher Insulin resistance (assessed by HOMA-IR) as well as 73% higher fasting plasma glucose and 69% higher HbA1c levels, independently of the presence/severity Table 1. Anthropometric and sleep characteristics of recruited subjects. Data are mean (SD), n (%). BMI, body mass index; AHI = apnoea hypopnea index; ODI, oxygen desaturation index. T90 = percentage of total sleep time with oxygen saturation less than 90%. T85 = percentage of total sleep time with oxygen saturation less than 85%. *Significant difference (p < 0.05) compared with control group. † Significant difference (p < 0.05) compared with T2DM group. www.nature.com/scientificreports/ of OSA, as presented in Table 3. However, after adjustment the analysis for BMI, plasma insulin levels were higher in T2DM and T2DM + OSA group and similarly, after adjustment for gender, plasma insulin levels were higher in T2DM + OSA group compared to control group, as summarized in Supplementary material Tables A and B. Subjects with T2DM showed decreased Disposition index (reflecting the ability of pancreas to secrete insulin accordingly to prevalent Insulin resistance) by 23% and 201% increased Insulin resistance compared with healthy control subjects. Glucose-induced insulin secretion (represented by AIR g ) was reduced by 35% in T2DM (not significantly) and further reduced by 79% (p < 0.05) in T2DM + OSA compared to a control group. After adjustment for waist circumference, reduction in AIR g of T2DM and T2DM + OSA reached 49% and 95%, respectively (both p < 0.05). Controlling for gender, BMI or gender + BMI + waist circumference had no impact on AIR g differences between groups, as summarized in Supplementary material Table C. Spontaneous, epinephrine, isoprenaline-stimulated lipolysis and α 2 -adrenergic anti-lipolytic effect. The investigation of the spontaneous lipolysis showed that the presence of severe OSA increased the lipolytic rate by 42% in T2DM + OSA compared with T2DM (60.34 ± 23.40 μmol/L vs. 42.53 ± 10.16 μmol/L, p = 0.013). Furthermore, the epinephrine-stimulated lipolysis was increased in T2DM + OSA by 41% and 82% compared with T2DM and healthy control subjects, respectively (236.84 ± 103.90 µmol/L vs. 167.39 ± 52.17 µmol/L and 130.33 ± 40.31 μmol/L, respectively, both p < 0.001). In contrast, no differences were observed after in-situ administration of isoprenaline (173.12 ± 43.40 μmol/L vs. 179.65 ± 49.19 μmol/L, for T2DM + OSA and T2DM, respectively, p = 0.750). Moreover, we investigated the α 2 -adrenergic anti-lipolytic effect in subcutaneous abdominal adipose tissue by evaluation of the difference between epinephrine-stimulated lipolysis (α-and β-adrenergic receptor agonist) and isoprenaline-stimulated lipolysis (selective β-adrenergic receptor agonist). The obtained data showed that the presence of severe OSA led to a decrease in the α 2 -adrenergic anti-lipolytic effect by 59% Table 2. Biochemical characteristics of recruited subjects. Data are mean (SD). HDL, high density lipoprotein; LDL, low density lipoprotein; FFA, free fatty acids; ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transferase. *Significant difference (p < 0.05) compared with control group. † Significant difference (p < 0.05) compared with T2DM group.  www.nature.com/scientificreports/ and 315% compared with the T2DM and control groups, respectively (p = 0.045 and p = 0.007, respectively). The data are summarized in Fig. 1.

Discussion
In this study, we aimed to determine, whether the presence of severe OSA might contribute to impaired glucose homeostasis through augmented adipose tissue lipolysis in patients with already developed T2DM. Based on the fact that subcutaneous adipose tissue is the major contributor of FFA into the systemic pool 29 , an in-vivo in-situ microdialysis technique was employed together with metabolic phenotyping by IVGTT. We showed that the presence of severe OSA in subjects with T2DM increased spontaneous and epinephrine-stimulated lipolysis compared with T2DM subjects with mild/no OSA, possibly through modifications in the α 2 -adrenergic control of lipolysis. Furthermore, augmented adipose tissue lipolysis was associated with Insulin resistance and impaired insulin secretion, two clinical features determining the development and progression of T2DM.
The role of adipose tissue as a potential factor mediating connection between OSA and development of T2DM gained interest due to its proved impact on whole-body glucose metabolism 30,31 as well as due to reports that adipocyte lipolysis is regulated by pericellular oxygen levels 5,16,28,32 . Previously, we demonstrated that adipocytes exposed to intermittent hypoxia in vitro (modelling oxygen desaturation in the context of OSA) increase spontaneous lipolytic rate 33 which is in line with a current paper demonstrating higher FFA levels during IVGTT in subjects with OSA 34 pointing to adipose tissue Insulin resistance. However, it remained unclear, whether such impairments persist in T2DM subjects with OSA as insulin resistance is present in these subjects. It could also be assumed that contribution of low O 2 levels to lipolysis regulation is diminished in T2DM + OSA. We hypothesized that an OSA-associated increase in the release of FFA might not only induce an adipose tissue proinflammatory state 35 and modify its endocrine function 36 , but also might impair insulin signalling through ectopic accumulation of lipids in muscle, liver and pancreas [37][38][39] . We observed that epinephrine-stimulated lipolysis, representing a model for nocturnal catecholamine bursts during apnoeic episodes, was elevated in T2DM patients with severe OSA, demonstrating that adipose tissue remained sensitive to β-adrenergic lipolytic drive even after prolonged exposure to periods of haemoglobin desaturations. Our study is in line with previous reports demonstrating that nocturnal hypoxemia elevated plasma FFA levels in heart failure subjects as well as after withdrawal from CPAP therapy 17,40 and provides a mechanistic explanation of elevated plasma FFA levels.
Using microdialysis technique, our study documented a link between adipose tissue hypoxia and spontaneous www.nature.com/scientificreports/ as well as epinephrine-stimulated lipolysis. Furthermore, results of our lipolysis experiments performed during awake hours suggest that increased adipose tissue lipolysis is not only the result of acute tissue hypoxemia during hypoxic episodes 16,40 , but it persists into wakefulness, probably due to prolonged effects on lipolysis regulation or due to persistently increased sympathetic nerve activity [41][42][43][44] . Indeed, exposure to hypoxia induces molecular changes in key lipolytic regulators 45 and upregulation of its activators 46 , as well as by increased Insulin resistance to its anti-lipolytic effect 47 . On the other hand, exposure to hypoxia was reported to decrease FFA uptake and oxidation in muscle 48 , which would also result in FFA accumulation in the circulation. The IVGTT, assessing simultaneously insulin sensitivity and glucose-induced insulin secretion, showed that AHI was associated with insulin secretion and Insulin resistance, supporting the previously reported association between OSA and T2DM 4 . Furthermore, epinephrine-stimulated lipolysis was negatively associated with the Disposition index-a variable describing the ability of β-cells to secrete insulin taking into account individual insulin sensitivity. Additionally, a link between enhanced adipose tissue lipolysis and development of Insulin resistance was reported previously 47,49 and replicated in this study in subjects with T2DM and OSA, as spontaneous lipolysis was related to the severity of OSA and associated with multiple parameters of Insulin resistance. Augmented lipolysis during apnoeic episodes thus not only contributes to acute elevation of glucose levels 17 , but it also impairs insulin secretion and thus might participate in glycaemic control worsening in T2DM subjects or participate in the progressive pancreatic endocrine dysfunction. Although elevated FFA might represent an important mechanism inducing Insulin resistance and β-cell dysfunction, it should be noted that impaired insulin secretion, β-cell apoptosis, stimulated lipolysis and impaired muscle FFA metabolism were also observed in vitro as a direct consequence of hypoxia 28,50,51 . This study suggests a direct link between deoxygenation and metabolic impairments, as the level of hypoxic exposure (T90) was adversely associated with insulin sensitivity and insulin secretion.
Augmented sympathetic activity, as repeatedly reported in OSA 41,52,53 , was per se associated with metabolic impairments 54 ; however, catecholamines also play a key role in the regulation of lipolysis through their binding to β-and α 2 -adrenergic receptors 55,56 . Studies showed that activation of β-adrenergic receptors stimulates lipolysis, while α 2 -adrenergic receptor activation has anti-lipolytic effect 57,58 . Administering epinephrine and isoprenaline into adipose tissue enabled assessment of individual contributions of α 2 and β receptors to increased lipolysis. No differences in isoprenaline-stimulated lipolysis across groups suggest that hypoxia did not alter β-adrenergic receptor activation/signalling; however, lipolysis was significantly upregulated by epinephrine in OSA. A plausible explanation is that increased lipolysis observed in T2DM patients with OSA is mediated through diminished α 2 -adrenergic signalling. Indeed, the observation that pure β-adrenergic lipolysis is unaffected by hypoxia was reported by other authors as well. For example, no differences in isoprenaline-stimulated lipolysis were observed in normoxic vs intermittently hypoxic human isolated primary adipocytes 59 . Furthermore, modelling hypoxic responses by targeting HIF-signalling showed even reduced isoprenaline-stimulated lipolysis in human adipocytes ex vivo 60 and exposing humans to hypoxic environment decreased isoprenaline-stimulated lipolysis 61 .
Interestingly, the severity of OSA was associated with the loss of α 2 anti-lipolytic effect. Although verification of these observations is warranted, the fact that α 2 -adrenergic receptors outnumber β-adrenergic receptors in human adipose tissue 55 makes α 2 -receptors not only a possible explanation for increased lipolysis in OSA, but also a potential pharmacological target for OSA-associated metabolic abnormalities, as suggested in a recent rodent study 5 .
Limitations of the study should be noted. First, experiments were performed in awake individuals, whereas apnoeic episodes occur during sleep, which could affect metabolite levels (e.g. plasma FFA), which remained unchanged in our study in contrast to studies using blood collections during sleep 17 . On the other hand, we were still able to detect changes in adipose tissue lipolysis persisting throughout the day, suggesting prolonged modifications in lipolysis regulation. Second, IVGTT does not assess the incretin-mediated part of insulin secretion (representing almost 65% of postprandial insulin secretion) 62 . Third, the limited sample size could affect statistical power of the present study, reflected in higher probability of type II error. For example, the power of isoprenaline-stimulated lipolysis comparison was 0.17, while for AIR g and S I the power was 0.38 and 0.37 respectively. Furthermore, unequal representation of the sexes warrants caution when generalizing results reported in the study as gender differences were reported in lipolysis regulation, response to weight reduction as well as other features associated with OSA 63,64 . The origin of referral/selection bias might be attributed to multiple sources, e.g. higher prevalence of diabetic women than men in the Czech Republic by 11% (according to the Institute of Health Information and Statistics of the Czech Republic), higher willingness of women to participate in a research study and recruitment through advertisement in local media with uneven gender distribution of readers. Importantly, there were no differences in basal or catecholamine-induced lipolysis between men and women in neither of the investigated groups, as summarized in Supplementary material Table D. Finally, adipose tissue gene expression was not investigated in this study; however, previous studies reported no changes in α-or β-adrenergic receptor gene expression in visceral adipose tissue of OSA subjects compared with controls 65 .
In conclusion, the present study showed that the presence of OSA increased spontaneous lipolysis in T2DM patients, which was associated with the severity of OSA, as well as with Insulin resistance and impaired insulin secretion. The study identified reduced α 2 anti-lipolytic effect as a possible factor responsible for increased lipolysis in severe OSA. Based on these observations, we suggest that pharmacological inhibitors of lipolysis might represent a novel treatment modality for metabolic impairments associated with OSA in T2DM, particularly for subjects not tolerating CPAP therapy. Moreover, despite the strong evidence of relationship between OSA and T2DM, studies investigating the effect of CPAP treatment on glycaemic control in T2DM provide inconclusive results (with multiple differences in study designs). For example, no effect of CPAP treatment was observed in GlycOSA study 66 , while another study 67

Methods
Subjects. The subjects were recruited through referral from physicians and local media advertisement. Subjects were recruited into three groups: patients with T2DM with mild/no OSA (T2DM, n = 10, 7 postmenopausal females), patients with T2DM with severe OSA (T2DM + OSA, n = 11, 5 postmenopausal females) and healthy controls without OSA or T2DM (n = 14, 11 postmenopausal females). The inclusion criteria were set as 18 to 85 years of age and body mass index (BMI) of 22 to 40 kg/m 2 . T2DM was diagnosed based on the European Association for the Study of Diabetes criteria 69 . Importantly, all patients with acute illness, decompensated chronic disease, cardiac or renal insufficiency, treated with drugs interacting with lipolysis (i.e., beta blockers, corticoids, insulin treatment, sulfonylurea treatment, GLP-1 receptor agonists and gliflozins) and a body weight change > 5 kg in last 3 months were excluded. All subjects gave written informed consent before participation in the study. Clinical investigations. Within 2 weeks of the microdialysis experiment, subjects visited the research centre for metabolic and anthropometric assessments. The measurements included blood drawn for biochemical analysis, blood count, coagulation and urinalysis, multifrequency bioimpedance measurements (Body Impedance analyser NUTRIGUARD-M, Data Input GmbH, Frankfurt, Germany) for body composition data and recording of individual body weight, height and waist circumference. Subsequently, a frequent-sampling intravenous glucose tolerance test (IVGTT) was performed. as follows: two intravenous catheters were inserted in the antecubital vein in the dominant (for blood sampling) and non-dominant (for glucose and insulin administration) arm. Basal sampling at times − 15, − 10, − 5, and − 1 min were performed, followed by intravenous administration of 0.3 g/kg glucose at time 0 min. Twenty minutes after the glucose dose, 0.03 U/kg of insulin (Humulin R, Lilly France S.A.S, Fegersheim, France) was administered intravenously. Blood samples were collected at times 2, 3, 4, 5, 6,8,10,12,14,16,19,22,24,25,27,30,40,50,60,70,80, 90, 100, 120, 140, 160, and 180 min. The glucose and insulin levels in collected samples were determined. The values were subjected to the minimal model analysis 70 for insulin sensitivity and insulin secretion indices, including acute insulin response to glucose (AIR G ), Disposition index (DI), Insulin sensitivity (S I ), Glucose effectiveness (S G ), β-cell function, and Insulin resistance.
Statistical analysis. The differences in outcome variables between groups (control, T2DM and T2DM + OSA) were analysed using one-way analysis of variance (ANOVA) with least significant difference posthoc tests. Repeated measures ANOVA was employed to analyse differences between groups in epinephrine and isoprenaline-stimulated lipolysis. Correlations between continuous variables were analysed using the Pearson correlation coefficient. For the investigation of associations between outcome variables and severity of OSA, all www.nature.com/scientificreports/ subjects were first stratified into groups based on terciles of AHI and subsequently subjected to analysis using the general linear model adjusted for sex and age. For statistical tests, SPSS 23.0 for Windows (SPSS Inc., Chicago, IL, USA) was used, whereas GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA) was used for AUC insulin calculation and figure construction. The statistical significance was set to, p ≤ 0.05 in all tests. Data are presented as mean ± standard deviation.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.