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Obesity is associated with sleep-disordered breathing (SDB) and unrefreshing sleep. Residual daytime sleepiness and sleep impairments often persist after SDB treatment in patients with obesity, which suggests an independent effect of obesity on breathing and sleep. However, examining the relationship between sleep architecture and SDB in patients with obesity is complex and can be confounded by multiple factors. The main goal of this study was to examine the relationship between obesityrelated changes in sleep architecture and SDB. Sleep recordings were performed in 15 lean C57BL/6J and 17 diet-induced obesity (DIO) mice of the same genetic background. Arousals from sleep and apneas were manually scored. Respiratory arousals were classified as events associated with >30% drops in minute ventilation (VE) from baseline. We applied Poincaré analysis of VE during sleep to estimate breathing variability. Obesity augmented the frequency of arousals by 45% and this increase was independent of apneas. Respiratory arousals comprised only 15% of the arousals in both groups of mice. Breathing variability during non-rapid-eye-movment (NREM) sleep was significantly higher in DIO mice, but it was not associated with arousal frequency. Our results suggest that obesity induces sleep fragmentation independently of SDB severity.

Sleep-disordered breathing (SDB) affects over 50% of obese individuals. Exaggerated hypoxic chemoreflex is a cardinal trait of SDB in obesity. We have shown that leptin acts in the carotid bodies (CB) to augment chemoreflex and that leptin activates the transient receptor potential melastatin 7 (TRPM7) channel. However, the effect of leptin-TRPM7 signalling in CB on breathing and SDB has not been characterized in diet-induced obesity (DIO). We hypothesized that leptin acts via TRPM7 in the CB to increase chemoreflex leading to SDB in obesity. DIO mice were implanted with EEG/EMG electrodes and transfected with Lepr

Opioid-induced respiratory depression (OIRD) is the primary cause of death associated with opioids and individuals with obesity are particularly susceptible due to comorbid obstructive sleep apnea (OSA). Repeated exposure to opioids, as in the case of pain management, results in diminished therapeutic effect and/or the need for higher doses to maintain the same effect. With limited means to address the negative impact of repeated exposure it is critical to develop drugs that prevent deaths induced by opioids without reducing beneficial analgesia or causing withdrawal symptoms. We have previously shown that intranasal (IN) leptin can reverse apneas, hypoventilation, and upper airway obstruction while enhancing analgesia following acute morphine administration. Here we hypothesize that OIRD as a result of chronic opioid use can be attenuated by administration of IN leptin without acute withdrawal while also maintaining analgesia in both lean mice and mice with diet-induced obesity (DIO). To test this hypothesis, an opioid tolerance protocol was developed and a model of OIRD in mice chronically receiving morphine and tolerant to morphine analgesia was established. Subsequently, sleep and breathing were recorded by barometric plethysmography following acute or chronic administration of IN leptin. Operant behavioral assays and Pavlovian conditioning procedures were used to determine the impact of IN leptin on the analgesic efficacy of morphine and opioid withdrawal symptoms. Acute administration of IN leptin significantly attenuated OIRD in DIO male mice decreasing the apnea index by 58.9% and apnea time by 60.1%. In lean mice leptin was ineffective. Chronic IN leptin induced > 50% reduction in the OIRD in morphine tolerant DIO male mice suggesting the protective effect of leptin remained after the two-week treatment. Morphine caused a complete loss of temperature aversion which was not reduced by intranasal leptin indicating IN leptin does not decrease morphine analgesia. IN leptin did not modify naloxone conditional place aversion or naloxone withdrawal symptoms, suggesting that IN treatment does not induce opioid withdrawal. We conclude that IN leptin is ineffective in lean mice but prevents OIRD in obesity by increasing hypercapnic sensitivity when leptin resistance at the BBB is present without reducing analgesia or inducing withdrawal. NIH R61HL156240; NIH R01HL128970; NIH R41DA056239 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Individuals with obesity hypoventilation syndrome (OHS) are unable to increase ventilation to match increased CO2 production (VCO2). Obstructive sleep apnea (OSA), defined by the closure of the upper airway during sleep, is present in 90% of OHS patients. Leptin, an adipocyte-produced hormone, acts in leptin receptor (LEPRb) positive neurons and was implicated in the pathophysiology of OSA and OHS. Diet-induced obese (DIO) C57BL/6J mice shows features of OHS and OSA. Leptin-mediated neural pathways may link metabolism and breathing in obesity. We hypothesized that LEPRb-positive neurons in the dorsomedial hypothalamus (DMH) are necessary and sufficient to match control of breathing to VCO2 and that the stimulation of these neurons relieves OHS in DIO mice. We examined our hypothesis using in vivo and in vitro studies. We expressed designer receptors exclusively activated by designer drugs (DREADDs) in DMH of DIO Leprb-Cre mice and measured breathing during sleep, the hypercapnic ventilatory response (HCVR), VCO2, and O2 consumption (VO2). Activation of LEPRb-positive DMH neurons increased inspiratory flow and minute ventilation during REM sleep, without affecting sleep architecture. The effect was pronounced during inspiratory flow limitation, suggesting that upper airway obstruction during REM sleep was relieved. Activation of these neurons increased the HCVR and this response was blunted by a serotonin receptor antagonist, methysergide. Activation of these neurons augmented VCO2, VO2, and VE/VCO2, suggesting that breathing was stimulated out of proportion to increases in metabolism. In vitro experiments revealed that LEPRb-positive DMH neurons project to 5-HT neurons in the dorsal raphe (DR) and that optogenetic stimulation of these neurons evoked excitatory postsynaptic currents in 5-HT neurons of the DR. Administration of retrograde Cre-dependent AAV harboring caspase to the DR in Leprb-Cre mice deleted LEPRb-positive DMH neurons and abolished respiratory and metabolic effects of chemogenetic stimulation. Apoptosis of LEPRb-positive DMH neurons projecting to the DR also prevented the increase in ventilation during REM sleep induced by intranasal leptin. Leptin increases the HCVR, improves upper airway patency during sleep, and increases VCO2 acting on LEPRb-positive DMH neurons projecting to the DR. We conclude that LEPRb-positive DMH neurons projecting to the DR couple metabolism and breathing in obesity. NIH (R01 HL128970, R01 HL133100, and R01 HL138932), AHA (#915167). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diet-induced obesity (DIO) in male mice is a well-established model of sleep-disordered breathing (SDB). Male DIO mice manifest obesity hypoventilation, increased apnea severity, and sleep fragmentation. The effects of DIO on breathing during sleep and sleep architecture in females have been insufficiently investigated. In this study, we aimed to compare the breathing and sleep patterns of lean and obese female mice of similar genetic background. We hypothesized that female mice are less susceptible to the detrimental effects of DIO on sleep and SDB compared to males. Full-polysomnographies were performed in 15 lean female C57BL/6J and 14 female DIO-C57BL/6J mice using a whole-body plethysmography chamber. Arousals from sleep and apneas were manually scored. Ventilatory response to an arousal was calculated as ratio of the peak minute ventilation (VE) during each arousal relative to the baseline VE (from 2.5s to 5s preceding the arousal). Breathing stability was measured using Poincaré plots of VE. Mice were acutely exposed to 8% CO2 to measure the hypercapnic ventilatory response (HCVR). An additional 10 mice (5 each/group) underwent 24-h metabolic studies. We found that obesity was associated with hypometabolism, indicated by reduced oxygen consumption (VO2) and CO2 production in DIO mice. VE in 8% CO2 and HCVR were significantly suppressed suggesting a decreased CO2 sensitivity in obese animals. NREM sleep duration was reduced, but REM sleep was preserved. Arousal frequency was similar between groups (~78 events/h). DIO mice hypoventilated during NREM sleep. However, apnea severity was 4-fold augmented in lean females compared to DIO mice (14.7±3.9 vs 74.2±11.9 events/h; PE during arousal and at baseline. Our results suggest potential sex differences in the effects of DIO on breathing during sleep and sleep architecture. DIO in females appears to be less detrimental regarding SDB severity. The mechanisms underlying the augmented SDB severity in lean females are unknown, but an exacerbated CO2 responsiveness during sleep could play an important role. American Heart Association Postdoctoral Fellowship Award 828142 (to L.J.K.), NHLBI grant NIH R01 HL128970, R01 HL133100, and R01 HL13892 (to V.Y.P.), American Academy of Sleep Medicine Foundation 238-BS-20, and American Thoracic Society Unrestricted Award (to L.V.P.) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduction Diet-induced obesity (DIO) in male mice is a well-established model of sleep-disordered breathing (SDB). Male DIO mice manifest hypoventilation, increased apnea severity, and sleep fragmentation. The effects of DIO on breathing during sleep and sleep architecture in females have been insufficiently investigated. We hypothesized that female mice are less susceptible to the detrimental effects of DIO on sleep and SDB compared to males. Methods Full-polysomnographies were performed in 15 lean female C57BL/6J and 14 female DIO-C57BL/6J mice using a whole-body plethysmography chamber. Ventilatory response to an arousal was calculated as ratio of the average minute ventilation (VE) during each arousal (2.5s) relative to the baseline VE. Breathing stability was measured using Poincaré plots of VE. Mice were exposed to 8% CO2 to measure the hypercapnic ventilatory response (HCVR). Mice underwent 24-h metabolic studies. Results Obesity was associated with hypometabolism, indicated by reduced oxygen consumption (VO2) and CO2 production in DIO mice. VE in 8% CO2 and HCVR were significantly suppressed suggesting a decreased CO2 sensitivity in obese animals. NREM sleep duration was reduced, but REM sleep was preserved. Arousal frequency and VE during NREM and REM sleep were similar between groups. However, apnea severity was 4-fold augmented in lean females compared to DIO mice (14.7±3.9 vs 74.2±11.9 events/h; P< 0.001). In lean mice, the increased frequency of apneas was mainly attributed to sleep fragmentation since the events were predominantly post-arousals. Breathing stability during sleep was significantly increased in lean mice. Ventilatory response to an arousal was 30% increased in lean females compared to DIO group. Conclusion Our results suggest potential sex differences in the effects of DIO on breathing during sleep and sleep architecture. DIO in females appears to be less detrimental regarding SDB severity. The mechanisms underlying the augmented SDB severity in lean females are unknown, but an exacerbated CO2 responsiveness during sleep related to sleep fragmentation could play an important role. Support (if any) American Heart Association Postdoctoral Fellowship Award 828142, NHLBI grant NIH R01s HL128970, HL133100, and HL13892, American Academy of Sleep Medicine Foundation 238-BS-20, and American Thoracic Society Unrestricted Award.

Introduction Individuals with obesity hypoventilation syndrome (OHS) are unable to increase ventilation to match increased CO2 production (VCO2). Obstructive sleep apnea (OSA), defined by the closure of the upper airway during sleep, is present in 90% of OHS patients. Leptin, an adipocyte-produced hormone, acts in leptin receptor (LEPRb) positive neurons and was implicated in the pathophysiology of OSA and OHS. Diet-induced obese (DIO) C57BL/6J mice shows features of OHS and OSA. Leptin-mediated neural pathways may link metabolism and breathing in obesity. We hypothesized that LEPRb-positive neurons in the dorsomedial hypothalamus (DMH) are necessary and sufficient to match control of breathing to VCO2 and that the stimulation of these neurons relieves OHS in DIO mice. Methods We expressed designer receptors exclusively activated by designer drugs (DREADDs) in DMH of DIO Leprb-Cre mice and measured breathing during sleep, the hypercapnic ventilatory response (HCVR), VCO2, and O2 consumption (VO2). Results Activation of LEPRb-positive DMH neurons increased inspiratory flow and minute ventilation during REM sleep, without affecting sleep architecture. The effect was pronounced during inspiratory flow limitation, suggeting that upper airway obstruction during REM sleep was relieved. Activation of these neurons increased the HCVR and this response was blunted by a serotonin receptor antagonist, methysergide. Activation of these neurons augmented VCO2, VO2, and VE/VCO2, suggesting that breathing was stimulated out of proportion to increases in metabolism. In vitro experiments revealed that LEPRb-positive DMH neurons project to 5-HT neurons in the dorsal raphe (DR) and that optogenetic stimulation of these neurons evoked excitatory postsynaptic currents in 5-HT neurons of the DR. Administration of retrograde Cre-dependent AAV harboring caspase to the DR in Leprb-Cre mice deleted LEPRb-positive DMH neurons and abolished respiratory and metabolic effects of chemogenetic stimulation. Apoptosis of LEPRb-positive DMH neurons projecting to the DR also prevented the increase in ventilation during REM sleep induced by intranasal leptin. Conclusion Leptin increases the HCVR, improves upper airway patency during sleep, and increases VCO2 acting on LEPRb-positive DMH neurons projecting to the DR. We conclude that LEPRb-positive DMH neurons projecting to the DR couple metabolism and breathing in obesity. Support (if any) NIH (R01 HL128970, R01 HL133100, and R01 HL138932), AHA (#915167).

Serotonin is an important mediator modulating behavior, metabolism, sleep, control of breathing, and upper airway function, but the role of aging in serotonin-mediated effects has not been previously defined. Our study aimed to examine the effect of brain serotonin deficiency on breathing during sleep and metabolism in younger and older mice. We measured breathing during sleep, hypercapnic ventilatory response (HCVR), CO2 production (VCO2), and O2 consumption (VO2) in 16–18- week old and 40–44-week old mice with deficiency of tryptophan hydroxylase 2 (Tph2), which regulates serotonin synthesis specifically in neurons, compared to Tph2+/+ mice. As expected, aging decreased VCO2 and VO2. Tph2 knockout resulted in an increase in both metabolic indexes and no interaction between age and the genotype was observed. During wakefulness, neither age nor genotype had an effect on minute ventilation. The genotype did not affect hypercapnic sensitivity in younger mice. During sleep, Tph2−/− mice showed significant decreases in maximal inspiratory flow in NREM sleep, respiratory rate, and oxyhemoglobin saturation in REM sleep, compared to wildtype, regardless of age. Neither serotonin deficiency nor aging affected the frequency of flow limited breaths (a marker of upper airway closure) or apneas. Serotonin deficiency increased the amount and efficiency of sleep only in older animals. In conclusion, younger Tph2−/− mice were able to defend their ventilation and phenotypically did not differ from wildtype during wakefulness. In contrast, both young and old Tph2−/− mice showed sleep-related hypoventilation, which was manifested by hypoxemia during REM sleep.

BackgroundObesity can cause hypertension and exacerbates sleep-disordered breathing (SDB). Leptin is an adipocyte-produced hormone, which increases metabolic rate, suppresses appetite, modulates control of breathing, and increases blood pressure. Obese individuals with high circulating levels of leptin are resistant to metabolic and respiratory effects of leptin, but they appear to be sensitive to hypertensive effects of this hormone. Obesity-induced hypertension has been associated with hyperleptinemia. New Zealand obese (NZO) mice, a model of polygenic obesity, have high levels of circulating leptin and hypertension, and are prone to develop SDB, similarly to human obesity. We hypothesize that systemic leptin receptor blocker Allo-aca will treat hypertension in NZO mice without any effect on body weight, food intake, or breathing.MethodsMale NZO mice, 12–13 weeks of age, were treated with Allo-aca (n = 6) or a control peptide Gly11 (n = 12) for 8 consecutive days. Doses of 0.2 mg/kg were administered subcutaneously 2×/day, at 10 AM and 6 PM. Blood pressure was measured by telemetry for 48 h before and during peptide infusion. Ventilation was assessed by whole-body barometric plethysmography, control of breathing was examined by assessing the hypoxic ventilatory response (HVR), and polysomnography was performed during light-phase at baseline and during treatment. Heart rate variability analyses were performed to estimate the cardiac autonomic balance.ResultsSystemic leptin receptor blockade with Allo-aca did not affect body weight, body temperature, and food intake in NZO mice. Plasma levels of leptin did not change after the treatment with either Allo-aca or the control peptide Gy11. NZO mice were hypertensive at baseline and leptin receptor blocker Allo-aca significantly reduced the mean arterial pressure from 134.9 ± 3.1 to 124.9 ± 5.7 mmHg during the light phase (P < 0.05), whereas the control peptide had no effect. Leptin receptor blockade did not change the heart rate or cardiac autonomic balance. Allo-aca did not affect minute ventilation under normoxic or hypoxic conditions and HVR. Ventilation, apnea index, and oxygen desaturation during NREM and REM sleep did not change with leptin receptor blockade.ConclusionSystemic leptin receptor blockade attenuates hypertension in NZO mice, but does not exacerbate obesity and SDB. Thus, leptin receptor blockade represents a potential pharmacotherapy for obesity-associated hypertension.

We hypothesized that exposure to hypoxia will destabilize breathing during sleep in our mouse model of sleep-disordered breathing (SDB), and the isocapnic administration of CO2 during severe hypoxia will improve SDB.

Rationale: Obstructive sleep apnea is recurrent upper airway obstruction caused by a loss of upper airway muscle tone during sleep. The main goal of our study was to determine if designer receptors exclusively activated by designer drugs (DREADD) could be used to activate the genioglossus muscle as a potential novel treatment strategy for sleep apnea. We have previously shown that the prototypical DREADD ligand clozapine-N-oxide increased pharyngeal diameter in mice expressing DREADD in the hypoglossal nucleus. However, the need for direct brainstem viral injections and clozapine-N-oxide toxicity diminished translational potential of this approach, and breathing during sleep was not examined.Objectives: Here, we took advantage of our model of sleep-disordered breathing in diet-induced obese mice, retrograde properties of the adeno-associated virus serotype 9 (AAV9) viral vector, and the novel DREADD ligand J60.Methods: We administered AAV9-hSyn-hM3(Gq)-mCherry or control AAV9 into the genioglossus muscle of diet-induced obese mice and examined the effect of J60 on genioglossus activity, pharyngeal patency, and breathing during sleep.Measurements and Main Results: Compared with control, J60 increased genioglossus tonic activity by greater than sixfold and tongue uptake of 2-deoxy-2-[18F]fluoro-d-glucose by 1.5-fold. J60 increased pharyngeal patency and relieved upper airway obstruction during non-REM sleep.Conclusions: We conclude that following intralingual administration of AAV9-DREADD, J60 can activate the genioglossus muscle and improve pharyngeal patency and breathing during sleep.

Introduction Obesity is a major cause of sleep-disordered breathing (SDB). Conventional metrics of SDB can be confounded by the effects of obesity on oxygenation and lack of standard definitions. Sleep fragmentation is frequently observed in obese individuals, but whether it occurs independently of SDB remains unknown. Quantitative analysis of ventilation may delineate the effects of obesity on breathing patterns and sleep fragmentation. We aimed to examine the effects of obesity on respiratory patterns during sleep and the relationship between obesity-related respiratory variability and sleep fragmentation. Methods Sleep recordings were performed in 15 lean C57BL/6J and 17 diet-induced obese (DIO) mice on the same genetic background. We applied Poincaré analysis of minute ventilation (VE) during sleep to estimate the breathing variability. Arousals were classified as respiratory when associated with ≥30% drops in VE from baseline. Results Breathing variability was significantly higher in DIO mice during NREM sleep, but not during REM sleep. Obesity-induced breathing variability could not be entirely attributed to apneas or arousals. Sleep fragmentation was 45% greater in DIO mice. Respiratory arousals comprised 15% of the arousals in both strains. Breathing variability was inversely associated with sleep fragmentation regardless of obesity. Conclusion Obesity increased respiratory variability during NREM sleep, which was not fully attributed to apneas and macro-sleep architecture. Obesity caused sleep fragmentation that was not entirely explained by SDB severity. Our quantitative analysis of VE identified differences in breathing variability in obesity that were not captured by traditional SDB metrics, which may be applicable for human SDB. Support (If Any) NHLBI NIH R01 HL135483, 2R01 HL133100-05, R01 HL128970, and R01 HL13892; NINDS NIH R01 NS112266; American Academy of Sleep Medicine Foundation 238-BS-20; American Thoracic Society Unrestricted Award; Johns Hopkins Blaustein Pain Research Grant; American Heart Association Postdoctoral Fellowship Award 828142.

Rationale: Leptin treats upper airway obstruction and alveolar hypoventilation in leptin-deficient ob/ob mice. However, obese humans and mice with diet-induced obesity (DIO) are resistant to leptin because of poor permeability of the blood–brain barrier. We propose that intranasal leptin will bypass leptin resistance and treat sleep-disordered breathing in obesity. Objectives: To assess if intranasal leptin can treat obesity hypoventilation and upper airway obstruction during sleep in mice with DIO. Methods: Male C57BL/6J mice were fed with a high-fat diet for 16 weeks. A single dose of leptin (0.4 mg/kg) or BSA (vehicle) were administered intranasally or intraperitoneally, followed by either sleep studies (n = 10) or energy expenditure measurements (n = 10). A subset of mice was treated with leptin daily for 14 days for metabolic outcomes (n = 20). In a separate experiment, retrograde viral tracers were used to examine connections between leptin receptors and respiratory motoneurons. Measurements and Main Results: Acute intranasal, but not intraperitoneal, leptin decreased the number of oxygen desaturation events in REM sleep, and increased ventilation in non-REM and REM sleep, independently of metabolic effects. Chronic intranasal leptin decreased food intake and body weight, whereas intraperitoneal leptin had no effect. Intranasal leptin induced signal transducer and activator of transcription 3 phosphorylation in hypothalamic and medullary centers, whereas intraperitoneal leptin had no effect. Leptin receptor–positive cells were synaptically connected to respiratory motoneurons. Conclusions: In mice with DIO, intranasal leptin bypassed leptin resistance and significantly attenuated sleep-disordered breathing independently of body weight.

Obstructive sleep apnea (OSA) is recurrent obstruction of the upper airway due to the loss of upper airway muscle tone during sleep. OSA is highly prevalent, especially in obesity. There is no pharmacotherapy for OSA. Previous studies have demonstrated the role of leptin, an adipose-tissue-produced hormone, as a potent respiratory stimulant. Leptin signaling via a long functional isoform of leptin receptor, LEPRb, in the nucleus of the solitary tract (NTS), has been implicated in control of breathing. We hypothesized that leptin acts on LEPRb positive neurons in the NTS to increase ventilation and maintain upper airway patency during sleep in obese mice. We expressed designer receptors exclusively activated by designer drugs (DREADD) selectively in the LEPRb positive neurons of the NTS of Leprb-Cre-GFP mice with diet-induced obesity (DIO) and examined the effect of DREADD ligand, J60, on tongue muscle activity and breathing during sleep. J60 was a potent activator of LEPRb positive NTS neurons, but did not stimulate breathing or upper airway muscles during NREM and REM sleep. We conclude that, in DIO mice, the stimulating effects of leptin on breathing during sleep are independent of LEPRb signaling in the NTS.

Rationale: North America is currently facing an opioid epidemics. Over 130 Americans die every day from an opioid overdose and the COVID-19 pandemic has increased opioid usage and opioid-related deaths. Respiratory depression is the main cause of morbidity and mortality from opioids. We have previously shown that leptin acts as respiratory stimulant and prevents opioid-induced respiratory depression and upper airway obstruction without reducing analgesia. In this study, we aimed to examine if intranasal (IN) administration of leptin effectively delivers leptin to the brain and prevents opioid-related deaths. Methods: To determine leptin delivery to the brain male C57BL/6J mice, were treated with IN leptin at 1.2 mg/kg in 1% BSA (n=5) or 1% BSA (n=5) and sacrificed 20 min later. Brains were harvested, the olfactory bulbs, medullas and hypotalami were isolated, quick frozen and stored at-80°C. For leptin level measurements, brain tissue was homogenized, protease inhibitors and protein concentrations were determined and ELISA was performed. To determine the survival probability, male C57BL/6J mice, 12 weeks of age, received IN leptin (n=26) or vehicle (n=25) at 1.2 mg/kg and 30 minutes later received a bolus of intraperitoneal (IP) morphine at 400mg/kg. Mice were video monitored for 24 hours and survival time after morphine injection was recorded Results: IN leptin significantly increased leptin levels in the olfactory bulb (from 0 ng/mL to 7.53 ng/mL, p = 0.004) and medulla (from 0.008 ng/mL to 0.107 ng/mL) and presented a trend in the hypothalamus (from 0.018 ng/mL to 0.291 ng/mL, p = 0.1). Mice that received IN leptin had a higher survival rate when compared to mice that received vehicle (69% vs 92%, p = 0.044, Figure). Conclusion: We demonstrated that the intranasal route is effective for the delivery of leptin to the brain and that leptin reduced opioid-induced mortality. (Table Presented).

Study Objectives Obesity leads to obstructive sleep apnea (OSA), which is recurrent upper airway obstruction during sleep, and obesity hypoventilation syndrome (OHS), hypoventilation during sleep resulting in daytime hypercapnia. Impaired leptin signaling in the brain was implicated in both conditions, but mechanisms are unknown. We have previously shown that leptin stimulates breathing and treats OSA and OHS in leptin-deficient ob/ob mice and leptin-resistant diet-induced obese mice and that leptin’s respiratory effects may occur in the dorsomedial hypothalamus (DMH). We hypothesized that leptin receptor LepRb-deficient db/db mice have obesity hypoventilation and that restoration of leptin signaling in the DMH will increase ventilation during sleep in these animals. Methods We measured arterial blood gas in unanesthetized awake db/db mice. We subsequently infected these animals with Ad-LepRb or control Ad-mCherry virus into the DMH and measured ventilation during sleep as well as CO2 production after intracerebroventricular (ICV) infusions of phosphate-buffered saline or leptin. Results Awake db/db mice had elevated CO2 levels in the arterial blood. Ad-LepRb infection resulted in LepRb expression in the DMH neurons in a similar fashion to wildtype mice. In LepRb-DMH db/db mice, ICV leptin shortened REM sleep and increased inspiratory flow, tidal volume, and minute ventilation during NREM sleep without any effect on the quality of NREM sleep or CO2 production. Leptin had no effect on upper airway obstruction in these animals. Conclusion Leptin stimulates breathing and treats obesity hypoventilation acting on LepRb-positive neurons in the DMH.

Obesity can lead to recurrent upper airway obstruction (obstructive sleep apnea, OSA) during sleep as well as alveolar hypoventilation. We have previously shown that leptin stimulates breathing and treats OSA in leptin-deficientob/obmice and leptin-resistant diet-induced obese mice. Our previous data also suggest that leptin’s respiratory effects may occur in the dorsomedial hypothalamus (DMH). We selectively expressed leptin receptorLepRbin the DMH neurons of obeseLepRb-deficientdb/dbmice (LepRb-DMH mice), which hypoventilate at baseline, and showed that intracerebroventricular injection of leptin in these animals increased inspiratory flow, tidal volume and minute ventilation during NREM sleep without any effect on the quality of NREM sleep or CO2production. Leptin had no effect on upper airway obstruction inLepRb-DMHanimals. We conclude that leptin stimulates breathing and treats obesity related hypoventilation acting on LepRb-positive neurons in the DMH.

Respiratory depression is the main cause of morbidity and mortality associated with opioids. Obesity increases opioid-related mortality, which is mostly related to comorbid obstructive sleep apnea. Naloxone, a μ-opioid receptor blocker, is an effective antidote, but it reverses analgesia. Like humans with obesity, mice with diet-induced obesity hypoventilate during sleep and develop obstructive sleep apnea, which can be treated with intranasal leptin. We hypothesized that intranasal leptin reverses opioid-induced sleep-disordered breathing in obese mice without decreasing analgesia. To test this hypothesis, mice with diet-induced obesity were treated with morphine at 10 mg/kg subcutaneously and with leptin or placebo intranasally. Sleep and breathing were recorded by barometric plethysmography, and pain sensitivity was measured by the tail-flick test. Excitatory postsynaptic currents were recorded in vitro from hypoglossal motor neurons after the application of the μ-opioid receptor agonist [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin and leptin. Morphine dramatically increased the frequency of apneas and greatly increased the severity of hypoventilation and obstructive sleep apnea. Leptin decreased the frequency of apneas, improved obstructive sleep apnea, and completely reversed hypoventilation, whereas morphine analgesia was enhanced. Our in vitro studies demonstrated that [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin reduced the frequency of excitatory postsynaptic currents in hypoglossal motoneurons and that application of leptin restored excitatory synaptic neurotransmission. Our findings suggest that intranasal leptin may prevent opioid respiratory depression during sleep in patients with obesity receiving opioids without reducing analgesia.

Introduction Obstructive sleep apnea (OSA) is characterized by recurrent periods of upper airway obstruction. The prevalence of OSA exceeds 50% in obese individuals and in 10–20% of obese patients OSA coexists with obesity hypoventilation syndrome (OHS) defined as daytime hypercapnia and hypoventilation during sleep attributed to the depressed control of breathing. There is no effective pharmacotherapy for OSA and OHS. Leptin is a potent respiratory stimulant and a potential therapeutic candidate. However, diet-induced obesity (DIO) results in reduced permeability of the blood-brain barrier (BBB) for leptin. Previous studies have shown that the BBB can be penetrated by exosomes, natural nanoparticles that can be used as drug delivery vehicles. In this study, we aimed to determine if exosomes overcome the BBB and treat OSA and OHS in DIO mice. Methods o examine the ability of exosomes to cross the BBB, male, lean (n=5) and DIO (n=5) C57BL/6J mice were injected with fluorescent exosomes or saline into the lateral tail vein. After 4h fluorescent exosomes biodistribution was evaluated by an in vitro imaging system (IVIS). Saline injected mice images were used for background adjustment. A separate subgroup of male, DIO (n=10) and lean (n=10) mice were headmounted with EEG and nuchal EMG leads. Sleep studies were performed in a plethysmography chamber and mice received saline, empty exosomes, free leptin, or leptin-loaded exosomes in a crossover manner. Results Exosomes were successfully delivered to the brain and the transport across the BBB was more efficient in DIO mice with 2-times greater relative fluorescence units measured in DIO when compared to lean mice (p Conclusion We demonstrated that exosomes overcome the BBB and that leptin-loaded exosomes treat OSA and OHS in DIO mice. Support (if any) R01HL 128970, R01HL 138932, R61 HL156240, U18 DA052301, FAPESP 2018/08758-3

To assess effects of low-level continuous positive airway pressure (CPAP) on snoring in habitual snorers without obstructive sleep apnea (OSA).A multicenter prospective in-laboratory reversal crossover intervention trial was conducted between September 2013 and August 2014. Habitual snorers were included if they snored (inspiratory sound pressure level ≥ 40 dBA) for ≥ 30% all sleep breaths on a baseline sleep study (Night 1), and if significant OSA and daytime somnolence were absent. Included participants then underwent a CPAP titration study at 2, 4, or 6 cm HOn baseline sleep studies, participants demonstrated snoring at ≥ 40 dBA on 53 ± 3% and ≥ 45 dBA on 35 ± 4% of breaths. Snoring frequency decreased progressively as nasal pressure increased from 0 to 4 cm HLow-level CPAP below the range required to treat OSA diminished nocturnal snoring, and produced uniform reduction in nightly noise production below the World Health Organization's limit of 45 dBA.ClinicalTrials.gov, identifier: NCT01949584.

Background Obesity causes multiorgan dysfunction, specifically metabolic abnormalities in the liver. Obese patients are opioid-sensitive and have high rates of respiratory complications after surgery. Obesity also has been shown to cause resistance to leptin, an adipose-derived hormone that is key in regulating hunger, metabolism, and respiratory stimulation. We hypothesized that obesity and leptin deficiency impair opioid pharmacokinetics (PK) independently of one another. Methods Morphine PK were characterized in C57BL/6J wild-type (WT), diet-induced obese (DIO), and leptin-deficient (ob/ob) mice, and in ob/ob mice given leptin-replacement (LR) therapy. WT mice received several dosing regimens of morphine. Obese mice (30 g) received one 80 mg/kg bolus of morphine. Blood was collected at fixed times after morphine injection for quantification of plasma morphine and morphine 3-glucuronide (M3G) levels. PK parameters used to evaluate morphine metabolism included area-under the curve (AUC150), maximal morphine concentration (CMAX), and M3G-to-morphine ratio, and drug elimination was determined by clearance (Cl/F), volume of distribution, and half-life (T1/2). PK parameters were compared between mouse groups by the use of 1-way analysis of variance, with P values less than .05 considered significant. Results DIO compared with WT mice had significantly decreased morphine metabolism with lower M3G-to-morphine ratio (mean difference [MD]: -4.9; 95% confidence interval [CI]: -8.8 to -0.9) as well as a decreased Cl/F (MD: -4.0; 95% CI: -8.9 to -0.03) Ob/ob compared with WT mice had a large increase in morphine exposure with a greater AUC150 (MD: 980.4; 95% CI: 630.1-1330.6), CMAX (MD: 6.8; 95% CI: 2.7-10.9), and longer T1/2 (MD: 23.1; 95% CI: 10.5-35.6), as well as a decreased Cl/F (MD: -7.0; 95% CI: -11.6 to -2.7). Several PK parameters were significantly greater in ob/ob compared with DIO mice, including AUC150 (MD: 636.4; 95% CI: 207.4-1065.4), CMAX (MD: 5.3; 95% CI: 3.2-10.3), and T1/2 (MD: 18.3; 95% CI: 2.8-33.7). When leptin was replaced in ob/ob mice, PK parameters began to approach DIO and WT levels. LR compared with ob/ob mice had significant decreases in AUC150 (MD: -779.9; 95% CI: -1229.8 to -330), CMAX (MD: -6.1; 95% CI: -11.4 to -0.9), and T1/2 (MD: -19; 95% CI: -35.1 to -2.8). Metabolism increased with LR, with LR mice having a greater M3G-to-morphine ratio compared with DIO (MD: 5.3; 95% CI: 0.3-10.4). Conclusions Systemic effects associated with obesity decrease morphine metabolism and excretion. A previous study from our laboratory demonstrated that obesity and leptin deficiency decrease the sensitivity of central respiratory control centers to carbon dioxide. Obesity and leptin deficiency substantially decreased morphine metabolism and clearance, and replacing leptin attenuated the PK changes associated with leptin deficiency, suggesting leptin has a direct role in morphine metabolism.

Study objectives Obesity hypoventilation and obstructive sleep apnea are common complications of obesity linked to defects in respiratory pump and upper airway neural control. Leptin-deficient ob/ob mice have impaired ventilatory control and inspiratory flow limitation during sleep, which are both reversed with leptin. We aimed to localize central nervous system (CNS) site(s) of leptin action on respiratory and upper airway neuroventilatory control. Methods We localized the effect of leptin to medulla versus hypothalamus by administering intracerbroventricular leptin (10 μg/2 μL) versus vehicle to the lateral (n = 14) versus fourth ventricle (n = 11) of ob/ob mice followed by polysomnographic recording. Analyses were stratified for effects on respiratory (nonflow-limited breaths) and upper airway (inspiratory flow limitation) functions. CNS loci were identified by (1) leptin-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and (2) projections of respiratory and upper airway motoneurons with a retrograde transsynaptic tracer (pseudorabies virus). Results Both routes of leptin administration increased minute ventilation during nonflow-limited breathing in sleep. Phrenic motoneurons were synaptically coupled to the nucleus of the solitary tract, which also showed STAT3 phosphorylation, but not to the hypothalamus. Inspiratory flow limitation and obstructive hypopneas were attenuated by leptin administration to the lateral but not to the fourth cerebral ventricle. Upper airway motoneurons were synaptically coupled with the dorsomedial hypothalamus, which exhibited STAT3 phosphorylation. Conclusions Leptin relieves upper airway obstruction in sleep apnea by activating the forebrain, possibly in the dorsomedial hypothalamus. In contrast, leptin upregulates ventilatory control through hindbrain sites of action, possibly in the nucleus of the solitary tract.

The present disclosure generally relates to compositions and methods of treating opioid-induced respiratory depression in a subject in need thereof, the method comprising administering leptin.

Leptin is a potent respiratory stimulant. The carotid bodies (CB) express the long functional isoform of leptin receptor, LepRb, but the role of leptin in CB has not been fully elucidated. The objectives of the current study were (1) to examine the effect of subcutaneous leptin infusion on minute ventilation (VE) and the hypoxic ventilatory response to 10% O2 (HVR) in C57BL/6J mice before and after CB denervation; (2) to express LepRb in CB of LepRb‐deficient obese db/db mice and examine its effects on breathing during sleep and wakefulness and on HVR. We found that leptin enhanced carotid sinus nerve activity at baseline and in response to 10% O2 in vivo. In C57BL/6J mice, leptin increased VE from 1.1 to 1.5 mL/min/g during normoxia (P < 0.01) and from 3.6 to 4.7 mL/min/g during hypoxia (P < 0.001), augmenting HVR from 0.23 to 0.31 mL/min/g/Δurn:x-wiley:00223751:media:tjp13266:tjp13266-math-0001 (P < 0.001). The effects of leptin on VE and HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB increased VE from 1.1 to 1.3 mL/min/g during normoxia (P < 0.05) and from 2.8 to 3.2 mL/min/g during hypoxia (P < 0.02), increasing HVR. Compared to control db/db mice, LepRb transfected mice showed significantly higher VE throughout non‐rapid eye movement (20.1 vs. −27.7 mL/min respectively, P < 0.05) and rapid eye movement sleep (16.5 vs 23.4 mL/min, P < 0.05). We conclude that leptin acts in CB to augment VE and HVR, which may protect against sleep disordered breathing in obesity., This work was funded by the NIH R01s HL133100 to V.Y.P. and J.S.K.S., R01 HL128970 to V.Y.P. and A.R.S., R01 HL138932 to V.Y.P. V.Y.P. is also supported by NIEHS grant P50 ES018176 and EPA Agreements 83615201 & 83451001. C.C.E. was supported in part by ‘Beca de estancias formativas de la Consejería de Salud de Andalucía 2017. C. Caballero Eraso. Número de expediente: EF‐0128‐2016’.

Obesity leads to sleep-disordered breathing (SDB) manifested by recurrent upper airway obstructions termed obstructive sleep apnea (OSA) and carbon dioxide retention due to hypoventilation. The objective of this work was to characterize breathing during sleep in C57BL6/J mice with diet-induced obesity (DIO). Arterial blood gas was measured in nine obese and nine lean mice during wakefulness. Nine male mice with DIO and six lean male C57BL/6J mice were head mounted with electroencephalogram (EEG) and electromyogram (EMG) electrodes. Sleep recordings were performed in the whole body plethysmography chamber; upper airway obstruction was characterized by the presence of inspiratory flow limitation in which airflow plateaus with increases in inspiratory effort. Obese mice showed significantly lower pH and higher partial pressure of arterial CO(2) (PaCO(2)) in arterial blood gas compared to lean mice, 7.35 ± 0.04 versus 7.46 ± 0.06 (p < 0.001) and 38 ± 8 mm Hg versus 30 ± 5 mm Hg (p < 0.001). Obese mice had similar levels of minute ventilation to lean mice during sleep and wakefulness, despite higher body weight and temperature, indicating an increase in the metabolic rate and hypoventilation. Obese mice also showed baseline hypoxemia with decreased mean oxyhemoglobin saturation across sleep/wake states. Obese mice had a higher prevalence of flow-limited breathing compared to lean mice during sleep. However, the oxygen desaturation index in lean and obese mice did not differ. We conclude that DIO in mice leads to hypoventilation. Obesity also increases the frequency of inspiratory limited breaths, but it does not translate into progression of OSA.

Obese leptin-deficient ( ob/ob) mice demonstrate defects in upper airway structural and neuromuscular control. We hypothesized that these defects predispose to upper airway obstruction during sleep, and improve with leptin administration. High-fidelity polysomnographic recordings were conducted to characterize sleep and breathing patterns in conscious, unrestrained ob/ob mice (23 wk, 67.2 ± 4.1 g, n = 13). In a parallel-arm crossover study, we compared responses to subcutaneous leptin (1 μg/h) vs. vehicle on respiratory parameters during NREM and REM sleep. Upper airway obstruction was defined by the presence of inspiratory airflow limitation (IFL), as characterized by an early inspiratory plateau in airflow at a maximum level (V̇imax) with increasing effort. The severity of upper airway obstruction (V̇imax) was assessed along with minute ventilation (V̇e), tidal volume (VT), respiratory rate (RR), inspiratory duty cycle, and mean inspiratory flow at each time point. IFL occurred more frequently in REM sleep (37.6 ± 0.2% vs. 1.1 ± 0.0% in NREM sleep, P < 0.001), and leptin did not alter its frequency. V̇imax (3.7 ± 1.1 vs. 2.7 ± 0.8 ml/s, P < 0.001) and V̇e increased (55.4 ± 22.0 vs. 39.8 ± 16.4 ml/min, P < 0.001) with leptin vs. vehicle administration. The increase in V̇e was due to a significant increase in VT(0.20 ± 0.06 vs. 0.16 ± 0.05 ml, P < 0.01) rather than RR. Increases in V̇e were attributable to increases in mean inspiratory flow (2.5 ± 0.8 vs. 1.8 ± 0.6 ml/s, P < 0.001) rather than inspiratory duty cycle. Similar increases in V̇e and its components were observed in non-flow-limited breaths during NREM and REM sleep. These responses suggest that leptin stabilized pharyngeal patency and increased drive to both the upper airway and diaphragm during sleep.

Obstructive sleep apnea (OSA) is characterized by recurrent upper airway obstruction during sleep. OSA leads to high cardiovascular morbidity and mortality. The pathogenesis of OSA has been linked to a defect in neuromuscular control of the pharynx. There is no effective pharmacotherapy for OSA. The objective of this study was to determine whether upper airway patency can be improved using chemogenetic approach by deploying designer receptors exclusively activated by designer drug (DREADD) in the hypoglossal motorneurons. DREADD (rAAV5-hSyn-hM3(Gq)-mCherry) and control virus (rAAV5-hSyn-EGFP) were stereotactically administered to the hypoglossal nucleus of C57BL/6J mice. In 6–8 weeks genioglossus EMG and dynamic MRI of the upper airway were performed before and after administration of the DREADD ligand clozapine-N-oxide (CNO) or vehicle (saline). In DREADD-treated mice, CNO activated the genioglossus muscle and markedly dilated the pharynx, whereas saline had no effect. Control virus treated mice showed no effect of CNO. Our results suggest that chemogenetic approach can be considered as a treatment option for OSA and other motorneuron disorders.