Eduard Peris

Lipopolysaccharide-Binding Protein Plasma Levels in Children: Effects of Obstructive Sleep Apnea and Obesity

BACKGROUND Obstructive sleep apnea (OSA) has been linked to obesity, inflammation, and metabolic syndrome. The gut microbiota, which serves as reservoir for bacterial lipopolysaccharides (LPS), could be altered by OSA and trigger inflammation. LPS-binding protein (LBP) serves as a surrogate marker of underlying low-grade endotoxemia by LPS from the gut. We hypothesized that systemic LBP levels would be higher in obese children and in those with OSA. METHODS Consecutive snoring and nonsnoring children (mean age 6.8 ± 1.3 y) were included after overnight polysomnography, and fasting levels of lipids, insulin glucose, and high-sensitivity C-reactive protein were obtained. Children were subdivided into four subgroups based on the presence of obesity or OSA. Plasma LBP levels were assayed using ELISA. RESULTS Of 219 participants, nonobese controls had the lowest levels of LBP, and the presence of obesity without OSA was associated with significant LBP increases. Nonobese children with OSA exhibited increased LBP levels, with obese children with OSA demonstrating the highest LBP levels of all four groups. Furthermore, LBP was independently associated with body mass index and with measures of OSA severity as well as with metabolic dysfunction, particularly insulin resistance as indicated by the homeostasis model assessment of insulin resistance. CONCLUSIONS Systemic low-level endotoxemia and resultant systemic inflammation is present in children who are either obese or suffer from OSA and is particularly prominent when both conditions are present. We postulate that disrupted sleep and other factors facilitating obesity such as a high-fat diet may disrupt the gut microbiome and lead to increased systemic LPS levels with resultant inflammation, promoting downstream metabolic dysfunction.

Chronic sleep fragmentation induces endothelial dysfunction and structural vascular changes in mice.

STUDY OBJECTIVES Sleep fragmentation (SF) is a common occurrence and constitutes a major characteristic of obstructive sleep apnea (OSA). SF has been implicated in multiple OSA-related morbidities, but it is unclear whether SF underlies any of the cardiovascular morbidities of OSA. We hypothesized that long-term SF exposures may lead to endothelial dysfunction and altered vessel wall structure. METHODS AND RESULTS Adult male C57BL/6J mice were fed normal chow and exposed to daylight SF or control sleep (CTL) for 20 weeks. Telemetric blood pressure and endothelial function were assessed weekly using a modified laser-Doppler hyperemic test. Atherosclerotic plaques, elastic fiber disruption, lumen area, wall thickness, foam cells, and macrophage recruitment, as well as expression of senescence-associated markers were examined in excised aortas. Increased latencies to reach baseline perfusion levels during the post-occlusive period emerged in SF mice with increased systemic BP values starting at 8 weeks of SF and persisting thereafter. No obvious atherosclerotic plaques emerged, but marked elastic fiber disruption and fiber disorganization were apparent in SF-exposed mice, along with increases in the number of foam cells and macrophages in the aorta wall. Senescence markers showed reduced TERT and cyclin A and increased p16INK4a expression, with higher IL-6 plasma levels in SF-exposed mice. CONCLUSIONS Long-term sleep fragmentation induces vascular endothelial dysfunction and mild blood pressure increases. Sleep fragmentation also leads to morphologic vessel changes characterized by elastic fiber disruption and disorganization, increased recruitment of inflammatory cells, and altered expression of senescence markers, thereby supporting a role for sleep fragmentation in the cardiovascular morbidity of OSA.

Chronic sleep disruption alters gut microbiota, induces systemic and adipose tissue inflammation and insulin resistance in mice.

Chronic sleep fragmentation (SF) commonly occurs in human populations, and although it does not involve circadian shifts or sleep deprivation, it markedly alters feeding behaviors ultimately promoting obesity and insulin resistance. These symptoms are known to be related to the host gut microbiota. Mice were exposed to SF for 4 weeks and then allowed to recover for 2 weeks. Taxonomic profiles of fecal microbiota were obtained prospectively, and conventionalization experiments were performed in germ-free mice. Adipose tissue insulin sensitivity and inflammation, as well as circulating measures of inflammation, were assayed. Effect of fecal water on colonic epithelial permeability was also examined. Chronic SF-induced increased food intake and reversible gut microbiota changes characterized by the preferential growth of highly fermentative members of Lachnospiraceae and Ruminococcaceae and a decrease of Lactobacillaceae families. These lead to systemic and visceral white adipose tissue inflammation in addition to altered insulin sensitivity in mice, most likely via enhanced colonic epithelium barrier disruption. Conventionalization of germ-free mice with SF-derived microbiota confirmed these findings. Thus, SF-induced metabolic alterations may be mediated, in part, by concurrent changes in gut microbiota, thereby opening the way for gut microbiome-targeted therapeutics aimed at reducing the major end-organ morbidities of chronic SF.

Resveratrol attenuates intermittent hypoxia-induced macrophage migration to visceral white adipose tissue and insulin resistance in male mice.

Chronic intermittent hypoxia during sleep (IH), as occurs in sleep apnea, promotes systemic insulin resistance. Resveratrol (Resv) has been reported to ameliorate high-fat diet-induced obesity, inflammation, and insulin resistance. To examine the effect of Resv on IH-induced metabolic dysfunction, male mice were subjected to IH or room air conditions for 8 weeks and treated with either Resv or vehicle (Veh). Fasting plasma levels of glucose, insulin, and leptin were obtained, homeostatic model assessment of insulin resistance index levels were calculated, and insulin sensitivity tests (phosphorylated AKT [also known as protein kinase B]/total AKT) were performed in 2 visceral white adipose tissue (VWAT) depots (epididymal [Epi] and mesenteric [Mes]) along with flow cytometry assessments for VWAT macrophages and phenotypes (M1 and M2). IH-Veh and IH-Resv mice showed initial reductions in food intake with later recovery, with resultant lower body weights after 8 weeks but with IH-Resv showing better increases in body weight vs IH-Veh. IH-Veh and IH-Resv mice exhibited lower fasting glucose levels, but only IH-Veh had increased homeostatic model assessment of insulin resistance index vs all 3 other groups. Leptin levels were preserved in IH-Veh but were significantly lower in IH-Resv. Reduced VWAT phosphorylated-AKT/AKT responses to insulin emerged in both Mes and Epi in IH-Veh but normalized in IH-Resv. Increases total macrophage counts and in M1 to M2 ratios occurred in IH-Veh Mes and Epi compared all other 3 groups. Thus, Resv ameliorates food intake and weight gain during IH exposures and markedly attenuates VWAT inflammation and insulin resistance, thereby providing a potentially useful adjunctive therapy for metabolic morbidity in the context of sleep apnea.

Inflammatory Markers and Obstructive Sleep Apnea in Obese Children: The NANOS Study

Introduction. Obesity and obstructive sleep apnea syndrome (OSA) are common coexisting conditions associated with a chronic low-grade inflammatory state underlying some of the cognitive, metabolic, and cardiovascular morbidities. Aim. To examine the levels of inflammatory markers in obese community-dwelling children with OSA, as compared to no-OSA, and their association with clinical and polysomnographic (PSG) variables. Methods. In this cross-sectional, prospective multicenter study, healthy obese Spanish children (ages 4–15 years) were randomly selected and underwent nocturnal PSG followed by a morning fasting blood draw. Plasma samples were assayed for multiple inflammatory markers. Results. 204 children were enrolled in the study; 75 had OSA, defined by an obstructive respiratory disturbance index (RDI) of 3 events/hour total sleep time (TST). BMI, gender, and age were similar in OSA and no-OSA children. Monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in OSA children, with interleukin-6 concentrations being higher in moderate-severe OSA (i.e., AHI > 5/hrTST; P < 0.01), while MCP-1 levels were associated with more prolonged nocturnal hypercapnia (P < 0.001). Conclusion. IL-6, MCP-1, and PAI-1 are altered in the context of OSA among community-based obese children further reinforcing the proinflammatory effects of sleep disorders such as OSA. This trial is registered with ClinicalTrials.gov NCT01322763.

Chronic sleep fragmentation during the sleep period induces hypothalamic endoplasmic reticulum stress and PTP1b-mediated leptin resistance in male mice.

BACKGROUND Sleep fragmentation (SF) is highly prevalent and may constitute an important contributing factor to excessive weight gain and the metabolic syndrome. Increased endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) leading to the attenuation of leptin receptor signaling in the hypothalamus leads to obesity and metabolic dysfunction. METHODS Mice were exposed to SF and sleep control (SC) for varying periods of time during which ingestive behaviors were monitored. UPR pathways and leptin receptor signaling were assessed in hypothalami. To further examine the mechanistic role of ER stress, changes in leptin receptor (ObR) signaling were also examined in wild-type mice treated with the ER chaperone tauroursodeoxycholic acid (TUDCA), as well as in CHOP-/+ transgenic mice. RESULTS Fragmented sleep in male mice induced increased food intake starting day 3 and thereafter, which was preceded by increases in ER stress and activation of all three UPR pathways in the hypothalamus. Although ObR expression was unchanged, signal transducer and activator of transcription 3 (STAT3) phosphorylation was decreased, suggesting reduced ObR signaling. Unchanged suppressor of cytokine signaling-3 (SOCS3) expression and increases in protein-tyrosine phosphatase 1B (PTP1B) expression and activity emerged with SF, along with reduced p-STAT3 responses to exogenous leptin. SF-induced effects were reversed following TUDCA treatment and were absent in CHOP -/+ mice. CONCLUSIONS SF induces hyperphagic behaviors and reduced leptin signaling in hypothalamus that are mediated by activation of ER stress, and ultimately lead to increased PTP1B activity. ER stress pathways are therefore potentially implicated in SF-induced weight gain and metabolic dysfunction, and may represent a viable therapeutic target.

Effects of adenotonsillectomy on plasma inflammatory biomarkers in obese children with obstructive sleep apnea: A community-based study

Background:Obesity and obstructive sleep apnea syndrome (OSA) are highly prevalent and frequently overlapping conditions in children that lead to systemic inflammation, the latter being implicated in the various end-organ morbidities associated with these conditions.Aim:To examine the effects of adenotonsillectomy (T&A) on plasma levels of inflammatory markers in obese children with polysomnographically diagnosed OSA who were prospectively recruited from the community.Methods:Obese children prospectively diagnosed with OSA, underwent T&A and a second overnight polysomnogram (PSG) after surgery. Plasma fasting morning samples obtained after each of the two PSGs were assayed for multiple inflammatory and metabolic markers including interleukin (IL)-6, IL-18, plasminogen activator inhibitor-1 (PAI-1), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9), adiponectin, apelin C, leptin and osteocrin.Results:Out of 122 potential candidates, 100 obese children with OSA completed the study with only one-third exhibiting normalization of their PSG after T&A (that is, apnea-hypopnea index (AHI) ≤1/hour total sleep time). However, overall significant decreases in MCP-1, PAI-1, MMP-9, IL-18 and IL-6, and increases in adropin and osteocrin plasma concentrations occurred after T&A. Several of the T&A-responsive biomarkers exhibited excellent sensitivity and moderate specificity to predict residual OSA (that is, AHI⩾5/hTST).Conclusions:A defined subset of systemic inflammatory and metabolic biomarkers is reversibly altered in the context of OSA among community-based obese children, further reinforcing the concept on the interactive pro-inflammatory effects of sleep disorders such as OSA and obesity contributing to downstream end-organ morbidities.

Effect of resveratrol on visceral white adipose tissue inflammation and insulin sensitivity in a mouse model of sleep apnea

Background:Sleep fragmentation (SF) increases food intake and the risk of obesity, and recruits macrophages to visceral white adipose tissue (VWAT) promoting tissue inflammation and insulin resistance. Administration of resveratrol (Resv) has been associated with significant improvements in high-fat diet-induced obesity, inflammation and insulin resistance.Methods:Male mice were subjected to SF or sleep control conditions for 8 weeks, and treated with either Resv or vehicle (Veh). Fasting plasma levels of glucose, insulin and leptin were obtained and VWAT insulin sensitivity tests were performed (phosphorylated AKT/total AKT), along with flow-cytometric assessments for VWAT macrophages (M1 and M2) and T-cell lymphocytes (CD4+, CD8+ and T regulatory cell (Treg)).Results:SF-Veh and SF-Resv mice showed increased food consumption and weight gain. However, although SF-Veh mice exhibited increased fasting insulin and leptin levels, and reduced VWAT p-AKT/AKT responses to insulin, such alterations were abrogated in SF-Resv-treated mice. Increases in M1, reduced M2 counts and increased tumor necrosis factor-α release emerged in SF-Veh macrophages compared with all other three groups. Similarly, increased CD8+ and reduced Treg lymphocyte counts were apparent in SF-Veh.Conclusions:Resveratrol does not reverse the SF-induced increases in food intake and weight gain, but markedly attenuates VWAT inflammation and insulin resistance, thereby providing a potentially useful adjunctive therapy in the context of sleep disorders manifesting metabolic morbidity.

Contextualised urinary biomarker analysis facilitates diagnosis of paediatric obstructive sleep apnoea

BACKGROUND Intrinsic variance of the urine proteome limits the discriminative power of proteomic analysis and complicates potential biomarker detection in the context of paediatric sleep disorders. METHODS AND RESULTS Using a rigorous workflow for proteomic analysis of urine, we demonstrate that gender and diurnal effects constitute two important sources of variability in healthy children. In the context of disease, complex pathophysiological perturbations magnify these proteomic differences and therefore require contextualised biomarker analysis. Indeed, by performing biomarker discovery in a gender- and diurnal-dependent manner, we identified ∼30-fold more candidate biomarkers of paediatric obstructive sleep apnoea (OSA), a highly prevalent condition in children characterised by repetitive episodes of intermittent hypoxia and hypercapnia, and sleep fragmentation in the context of recurrent upper airway obstructive events during sleep. Remarkably, biomarkers were highly specific for gender and sampling time as poor overlap (∼3%) was observed in the proteins identified in boys and girls across morning and bedtime samples. CONCLUSIONS As no clinical basis to explain gender-specific effects in OSA or healthy children is apparent, we propose that implementation of contextualised biomarker strategies will be applicable to a broad range of human diseases, and may be specifically applicable to paediatric OSA.

Vitamin D levels and obstructive sleep apnoea in children

AIMS/HYPOTHESIS Obstructive sleep apnoea (OSA) is a common health problem in children. African American (AA) and obese children have higher prevalence of OSA, and are also at a higher risk of reduced vitamin D levels. We hypothesised that OSA would be associated with lower levels of plasma 25-hydroxyvitamin D (25(OH)D) and increase in the risk of metabolic dysfunction and systemic inflammation. METHODS In this observational cross-sectional study, 176 prospectively recruited children (mean age: 6.8±0.8 years) underwent overnight polysomnographic evaluation and a fasting blood draw the morning after the sleep study. In addition to lipid profile, homeostatic model of insulin resistance (HOMA-IR) and high-sensitivity C-reactive protein (hsCRP) assays and plasma 25(OH)D levels were assessed using ELISA kits. RESULTS AA children, obese children and children with OSA had significantly lower 25(OH)D levels. Linear associations emerged between 25(OH)D plasma levels and body mass index (BMI) z-score, hsCRP and HOMA-IR, as well as with apnoea-hypopnoea index (AHI) and oxygen saturation (SpO2) nadir, the latter two associations remaining statistically significant even when controlling for all other potential confounders, and independently accounting for 17.7% of the variance in 25(OH)D (p<0.01). CONCLUSIONS 25(OH)D levels are reduced in paediatric OSA, in AA children and in obese children, particularly when all are present, and may play a role in modulating the degree of insulin resistance and systemic inflammation. The short-term and long-term significance of reduced 25(OH)D in paediatric OSA remains undefined.