obstructive sleep apnea ( osa ) is a public - health problem as it affects at least 10% of the middle aged men and represents a main cause of cardiovascular morbidity and mortality .
an independent association between osa , insulin resistance , and type 2 diabetes has been consistently demonstrated by a number of cross - sectional , observational , and large population - based studies [ 2 , 3 ] .
moreover , osa patients have increased carotid intima - media thickness ( imt ) , an early sign of atherosclerosis , which correlates with nocturnal oxygen desaturation , independently of other cardiovascular risk factors [ 4 , 5 ] .
osa severity may also predict occult coronary atherosclerosis in healthy overweight or obese male subjects .
repetitive upper airway collapses during sleep result in intermittent hypoxia ( ih ) which is thought to be responsible for osa - associated cardiometabolic complications , including atherosclerosis and insulin resistance .
data obtained from animals exposed to ih , a validated experimental model of sleep apnea , showed that activation of the sympathetic nervous system and systemic inflammation underlie ih - induced metabolic and vascular consequences [ 79 ] .
we also recently demonstrated that ih - induced inflammatory changes of epididymal white adipose tissue ( ewat ) contributed to these outcomes . in obesity , it is increasingly recognized that chronic activation of inflammatory signaling pathways is causally linked to insulin resistance   and vascular alterations .
recent studies suggest that these deleterious effects could be mediated , at least in part , through the activation of toll - like receptors ( tlr ) , and in particular the tlr4 .
tlrs are a family of pattern - recognition receptors that play a critical role in the innate immune system by activating proinflammatory signaling pathways in response to microbial pathogens .
lipopolysaccharide ( lps ) binds to tlr4 , triggering a downstream cascade , which leads to the activation of the proinflammatory nuclear factor kappa - b ( nfb ) pathway and finally to the expression of numerous proinflammatory molecules , such as interleukin ( il)-6 and tumor necrosis factor ( tnf)- .
furthermore , studies on different strains of mice showed that expression and activation of tlr4 are involved in aortic inflammation   and atherogenesis . together
these experimental data confirm the major role of tlr4/nfb pathway in the crosstalk between inflammation , atherosclerosis , and metabolism dysfunctions . in c57bl/6 mice
, we previously found that ih - induced cardiovascular inflammation was characterized by an increased activity of nfb in aortic   and cardiac tissues ( unpublished data ) .
we also reported that , in lean animals , ewat exposed to ih became pathological , behaving like excess fat in obesity , as it exhibited increases in macrophage recruitment and secretion of il-6 and tnf- .
collectively , these data suggest that cardiometabolic complications due to ih and obesity may share some pathophysiological mechanisms . therefore , we assessed whether metabolic and vascular consequences induced by ih involved the proinflammatory tlr4/nfb pathway activation .
shizuo akira ( research institute for microbial diseases , osaka , japan ) and were obtained from the emma ( european mouse mutant archive ) network in orleans , france .
seventeen - week - old male tlr4 mice and their control groups ( c57bl/6 mice ) fed on a standard - chow diet were used .
the study was conducted in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ( council of europe , european treaties ets 123 , strasbourg , 18 march 1986 ) and with the guide for care and use of laboratory animals ( nih publication no .
tlr4 and their control c57bl/6 mice were divided into 2 subgroups , exposed to either intermittent hypoxia ( ih ) or normoxia ( n ) .
the four groups of animals were exposed to the ih stimulus during daytime ( n = 10 per cage , 8  h / day , cyclic 21 - 5% fio2 , 60  s cycle ( 60 events / h ) , lowest blood oxygen saturation up to 60% ) for 4 weeks . fio2 was measured with a gas analyzer ( ml206 , adinstruments ) throughout the experiment .
control animals ( normoxic mice , n ) were exposed to air in similar cages to reproduce similar noise and turbulences to those of the ih stimulus .
ambient temperature was maintained at 2022c . during the first week of ih exposure , intraperitoneal insulin tolerance test ( ipitt )
was performed to assess global insulin sensitivity and , on the day following the last exposure period , fasted animals were sacrificed under anesthesia with intraperitoneal injection of ketamine ( 100  mgkg ) and xylazine ( 10  mgkg ) for further analysis .
mice were fasted for 5 hours and then weighted before blood was collected from the tail tip for baseline glucose determination ( t = 0 ) .
insulin ( 0.5  iukg body weight , novo nordisk a / s , bagsvaerd , denmark ) was injected intraperitoneally , followed by further blood glucose measurements at 15 , 30 , 60 , and 90 minutes after the injection . the lowest blood glucose level ( nadir ) following insulin administration was calculated for each experimental group . at the time of sacrifice , blood was collected by cardiac puncture on edta tubes .
the plasma fraction was collected after blood centrifugation during 10 minutes at 11000  rpm ( 4c ) .
total cholesterol was measured in plasma by a colorimetric enzymatic reaction using the infinity kit ( thermoelectron corporation , massachusetts , usa ) according to the manufacturer 's guidelines .
bilateral epididymal fat pads were collected , weighted , and either fixed in 90% ethanol for adipocyte morphology study or incubated for cytokine determinations .
ethanol - fixed , paraffin - embedded ewat were sectioned ( 3.5  m ) , deparaffinized in toluene and rehydrated in descending ethanol series , and then stained with hematoxylin - eosin to assess tissue morphology .
adipocyte size was measured from photographs ( 10  40 magnification ) using the nis - elements microscope imaging software ( nikon ) .
each ewat pad was divided into two equal pieces and incubated at 37c with mild shaking in rpmi medium .
after 120 minutes of incubation , il-6 and tnf- were measured in the supernatants using an elisa test according to manufacturer 's instructions ( r&d system europe , lille , france ) .
cytokine concentrations were expressed as ng / ml for 1  g of adipose tissue .
ih and n aortas were embedded in optimum cutting temperature ( oct ) compounds ( tissue - tek , sakura finetek europe bv , alphen aan den rijn , the netherlands ) , sectioned ( 10  m ) , and stained .
hematoxylin - eosin staining was used to assess the intima - media thickness ( imt ) .
morphometric analysis ( up to 15 measurements on 10 noncontiguous midthoracic descending aorta sections per animal ) was performed with a light microscope ( nikon eclipse 80i , nikon ) and the nis - elements microscope imaging software ( nikon instruments europe bv ) .
we investigated whether ih could activate nfb by assessing the expression of its activated subunit nfb - p50 which has translocated into the nucleus .
nuclear nfb - p50 was determined in thoracic aorta of mice exposed to n or ih .
tissue homogenization and proteins extraction were performed according to manufacturer 's instructions using a nuclear extract kit ( active motif europe , rixensart , belgium ) .
proteins concentration was evaluated using the bca assay ( thermoscientific , massachusetts , usa ) .
nuclear proteins were assayed for the presence of the activated p50 by elisa using the transam nfb - p65/p50/p52 kit ( active motif europe ) .
results were expressed as mean  standard errors of the means ( sem ) and analyzed using 2-way anova and subsequent bonferroni 's multiple post hoc comparisons or mann - whitney u test .
they had smaller fat pads with smaller adipocytes ( figures 1(a ) , 1(c ) , and 1(d ) ) , and ewat released more tnf- and il-6 compared to normoxic controls ( figures 1(e ) and 1(f ) ) .
regarding body weight alterations , normoxic and hypoxic tlr4 mice were not different from their respective control animals ( figure 1(b ) ) .
after one - week exposure , the 4 experimental groups were assessed for insulin tolerance test ( figure 2(a ) ) .
c57bl/6 mice exposed to ih exhibited a decreased response to insulin as shown by a lower glucose decrement ( figure 2(b ) ) and a trend for a smaller glucose nadir compared to their normoxic controls ( figure 2(d ) ) .
the insulin response was not affected in hypoxic tlr4 mice : the response curve was almost superposable with those of the normoxic tlr4 animals ( figures 2(c ) and 2(d ) ) and not significantly different from the curve of normoxic c57bl6 animals ( figure 2(a ) ) .
hypoxic c57bl/6 mice had morphological and functional alterations of their aorta , as they exhibited larger intima - media thickness ( figures 3(a ) and 3(b ) ) and higher nfb - p50 activity ( figure 3(c ) ) . these alterations were not observed in hypoxic tlr4 mice ( figures 3(a ) , 3(b ) , and 3(c ) ) .
plasma levels of total cholesterol were not different between the 4 experimental groups ( figure 3(d ) ) .
there are some similarities with cardiometabolic complications due to obesity , the latter resulting from inflammation involving tlr4 signaling . here
, we showed in nonobese c57bl/6 mice that ih induced morphological and inflammatory remodeling of vascular and white adipose tissues as well as insulin resistance .
these alterations were prevented in hypoxic tlr4-deficient mice suggesting that ih - induced cardiometabolic consequences involve tlr4 signaling - mediated inflammation .
as previously published by our group and others , we used a deep intermittent hypoxia which mimics severe sleep apnea whereas patients mainly suffer from mild to moderate sleep apnea [ 7 , 17 , 18 ] . indeed , in the absence of additional factors such as obesity , high fat diet , and genetic vulnerability , ih needs to be severe enough to induce measurable and reproducible vascular alterations , especially in c57bl/6 mice which are atheroresistant animals . in lean c57bl/6 mice
, we showed that ih induced ewat alterations characterized by fat pad wasting and shrunken adipocytes .
these results are in agreement with previous reports in lean c57bl/6   and apoe   mice after 4 and 6 weeks of ih , respectively .
shrunken adipocytes are suggestive of lipolysis , as elevated circulating free fatty acids ( ffas ) have been reported by us and others in ih - exposed animals [ 10 , 20 , 21 ] and in patients suffering from sleep apnea . in the latter ,
activation of beta - adrenergic receptors is a well - known mechanism of lipolysis , and both ih and osa are commonly associated with sympathoadrenergic activation [ 10 , 24 ] .
adipose inflammation may have also contributed to lipolysis   as we found an increased release of the inflammatory cytokines tnf- and il6 from fat pads of hypoxic c57bl/6 mice .
this is again consistent with previous reports in lean apoe mice after 6 weeks of ih , and more recently in 3t3-l1 adipocytes exposed to fluctuating oxygen concentration .
these inflammatory changes may be due to local hypoxia , which is known to be a leading cause of ewat dysregulation , as well as to systemic effects such as elevated circulating ffas   and activation of the sympathoadrenergic system .
we found that both ih - induced morphological and inflammatory alterations of ewat were prevented in tlr4-deficient mice .
there is growing evidence from studies using murine models of obesity that activation of the proinflammatory tlr4/nfb pathway constitutes one mechanism that links inflammation and metabolic disorders [ 3032 ] .
although tlr4 activation is known to enhance lipolysis [ 33 , 34 ] , we were surprised to find that ewat wasting and adipocyte hypotrophy were completely prevented in tlr4-deficient mice suggesting that tlr4 signaling could be the main mechanism of these consequences .
as observed in mice models of diet - induced obesity [ 30 , 32 ] , tlr4 deficiency prevented the enhanced release of tnf- and il6 in our hypoxic mice .
this strengthens the role of tlr4 in ih - induced inflammation , as well as the pathophysiological similarities with obesity , that is , a normal amount of fat under hypoxia behaving like excess fat in obesity .
this is in agreement with previous findings obtained in various mouse strains ( genetically obese , lean c57bl/6 , and apoe mice ) and duration of ih exposure ( acute or chronic ih ) , using different methods to assess insulin sensitivity ( homa - ir , itt , hyperinsulinemic euglycemic clamp ) [ 10 , 19 , 35 , 36 ] .
this confirms the role of ih in the alterations of glucose homeostasis observed in sleep apnea , the insulin resistance worsening with osa severity , independently of obesity [ 3739 ] .
both chronic inflammation   and elevated ffa levels [ 41 , 42 ] are established factors causing insulin resistance in obesity .
indeed proinflammatory cytokines secreted from adipocytes are considered as a key step in obesity - induced insulin resistance , as the sole tnf- neutralization in obese rats is sufficient to improve insulin sensitivity . inversely
, upstream activation of the inflammatory cytokine cascade using agonists of toll - like receptors leads to insulin resistance [ 33 , 34 ] . in the present study , we observed increased release of tnf- and il6 from ewat and decreased insulin sensitivity in hypoxic c57bl6 mice .
both parameters were not impaired in tlr4-deficient mice suggesting that tlr4 mediated inflammation was involved in this metabolic consequence .
body weight alterations did not contribute to these improvements , as hypoxic c57bl/6 and tlr4-deficient mice had similar body weight .
we found that ih induced vascular remodeling , including morphological ( larger intima - media thickness ) and inflammatory changes ( higher nfb - p50 activity ) .
this confirms our previous results and others regarding the detrimental remodeling effects of ih on preatherosclerotic [ 17 , 18 ] and atherosclerotic [ 21 , 44 , 45 ] processes .
we confirmed in the present study that ih is indeed a powerful vascular stress , as only 28 days of exposure , which is very short compared to the duration of the human disease , induce early vascular alterations in the atheroresistant c57bl/6 mouse strain .
besides the well - known role of the sympathoadrenergic system and the related hemodynamic alterations , accumulating evidence suggests that inflammation is involved early in the pathophysiology of ih - related atherosclerosis .
regarding the aggravation of atherosclerosis by ih in apoe - deficient mice , we recently demonstrated that this deleterious effect involved inflammatory alterations of ewat , as ewat lipectomy prevented the proatherogenic effect of ih .
interestingly , this beneficial effect occurred while insulin resistance ( a well - known risk factor for atherosclerosis ) was not improved by ewat lipectomy , suggesting that ewat inflammation could be the main determinant of ih - induced atherogenicity . here , we showed that ewat from hypoxic c57bl/6 mice released higher amounts of inflammatory cytokines and that this effect , as well as the morphological and inflammatory changes of aorta , was prevented in tlr4-deficient animals .
given the role of tlr4 in adipose tissue inflammation and insulin resistance , and the relationship between adipose tissue inflammation , insulin resistance , and vascular dysfunction [ 12 , 47 ] , the beneficial effect of tlr4 deficiency on arterial remodeling could be explained by the prevention of adipose inflammation and insulin resistance . here , the role of ewat inflammation seems to be predominant compared to the light improvement of insulin response and the absence of cholesterol alterations in these animals .
ih - induced dyslipidemic alterations are indeed inconstant in mice [ 45 , 4850 ] , suggesting that dyslipidemia contributes only in part to the first steps of vascular remodeling in this model . reduced ih - driven hemodynamic alterations could be a further explanation , as tlr4-deficient mice are less susceptible to hypertension [ 51 , 52 ] .
finally , a direct effect on the vascular wall is also possible , as tlr4 has been evidenced in human   and murine   atherosclerotic plaques , and inhibition of tlr4 signaling pathway attenuated diet - induced atherosclerosis in apoe mice [ 16 , 55 ] .
a recent study also identified tlr4 signaling pathway as a direct key mediator of vascular inflammation and impairment of endothelial insulin signaling in the setting of obesity .
the direct effect on vasculature could even be predominant , as tlr4 deficiency prevented atherosclerosis in ldlr mice , with no effect on adipose tissue inflammation and whole - body insulin sensitivity .
we showed in nonobese c57bl/6 mice that ih induced morphological and inflammatory remodeling of aorta and epididymal white adipose tissue , as well as insulin resistance .
these alterations were prevented in tlr4-deficient mice suggesting that ih - induced cardiometabolic consequences involved inflammation mediated by tlr4 signaling .
the precise mechanisms and the specific role of one type of tissue or cell ( e.g. , adipose tissue ) remain to be determined as tlr4 knockout used in the study was not cell specific .
tlr4 is indeed expressed on many cell types , predominantly those of the immune system , but also on nonhematopoietic cell types ( e.g. , endothelial , epithelial cells , etc . ) . despite these limitations , as a practical point of view
, the whole body is exposed to hypoxia during sleep apnea , and tlr4 in various cells is likely to be involved in the numerous complications of sleep apnea .
moreover the available treatment that blocks tlr4 activation ( eritoran ) is not cell specific .
however , one clinical study has recently investigated the activation of tlr4 signaling pathway in osa .
the authors found increases in tlr4 expression , nfb nuclear binding , and release of ifn , tnf- , and il-6 in circulating monocytes .
there are therefore similarities between these clinical findings and our experimental results suggesting that targeting tlr4/nfb pathway could provide further therapeutic options for sleep apnea patients .
 
 objective . intermittent hypoxia ( ih ) is a major component of sleep apnea syndrome as its cardiometabolic complications have been mainly attributed to ih . the pathophysiology is still poorly understood but there are some similarities with the obesity - associated cardiometabolic complications . as the latter results from inflammation involving toll - like receptor-4 ( tlr4 ) signaling , we assessed this pathway in the cardiometabolic consequences of ih . methods . 
 lean adult male tlr4-deficient ( tlr4/ ) mice and their controls ( c57bl/6 mice ) were exposed to either ih ( fio2 21 - 5% , 1 min cycle , 8  h / day ) or air ( normoxic mice ) for 4 weeks . 
 animals were assessed at 1-week exposure for insulin tolerance test and after 4-week exposure for morphological and inflammatory changes of the epididymal fat and thoracic aorta . 
 results . 
 ih induced insulin resistance , morphological and inflammatory changes of the epididymal fat ( smaller pads and adipocytes , higher release of tnf- and il-6 ) and aorta ( larger intima - media thickness and higher nfb - p50 activity ) . 
 all these alterations were prevented by tlr4 deletion . conclusion . 
 ih induces metabolic and vascular alterations that involve tlr4 mediated inflammation . 
 these results confirm the important role of inflammation in the cardiometabolic consequences of ih and suggest that targeting tlr4/nfb pathway could represent a further therapeutic option for sleep apnea patients . 
1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion