Senolytic-facilitated Reversal of End-Organ Dysfunction in a Murine Model of Obstructive Sleep Apnea.

Rationale: Obstructive sleep apnea (OSA) is a highly prevalent condition that is associated with accelerated biological aging and multiple end-organ morbidities. Current treatments, such as continuous positive airway pressure (CPAP), have shown limited cognitive, metabolic, and cardiovascular beneficial outcomes despite adherence. Thus, adjunct therapies aiming to reduce OSA burden, such as senolytics, could improve OSA outcomes.Objectives: To assess if targeting senescence in addition to partial normoxia mimicking "good" CPAP adherence can improve physiological outcomes in mice exposed to chronic intermittent hypoxia.Methods: We compared the effects of 6 weeks of therapy with either partial normoxic recovery alone or combined with the senolytic navitoclax after 16 weeks of intermittent hypoxia exposures, a hallmark of OSA, on multiphenotypic cardiometabolic and neurocognitive parameters.Measurements and Main Results: Our findings indicate that only when combined with navitoclax, partial normoxic recovery significantly improved sleepiness (sleep in the dark phase: 34% ± 4% vs. 26% ± 3%; P < 0.01), cognition (preference score: 51% ± 19% vs. 70% ± 11%; P = 0.048), coronary artery function (response to acetylcholine [vasodilation]: 56% ± 13% vs. 72% ± 10%; P < 0.001), glucose, and lipid metabolism and reduced intestinal permeability and senescence in multiple organs.Conclusions: These findings indicate that the reversibility of end-organ morbidities induced by OSA is not only contingent on restoration of normal oxygenation patterns but can be further enhanced by targeting other OSA-mediated detrimental cellular processes, such as accelerated senescence.

Monocarboxylate Transporter-2 Expression Restricts Tumor Growth in a Murine Model of Lung Cancer: A Multi-Omic Analysis.

Monocarboxylate transporter 2 (MCT2) is a major high-affinity pyruvate transporter encoded by the SLC16A7 gene, and is associated with glucose metabolism and cancer. Changes in the gut microbiota and host immune system are associated with many diseases, including cancer. Using conditionally expressed MCT2 in mice and the TC1 lung carcinoma model, we examined the effects of MCT2 on lung cancer tumor growth and local invasion, while also evaluating potential effects on fecal microbiome, plasma metabolome, and bulk RNA-sequencing of tumor macrophages. Conditional MCT2 mice were generated in our laboratory using MCT2loxP mouse intercrossed with mCre-Tg mouse to generate MCT2loxP/loxP; Cre+ mouse (MCT2 KO). Male MCT2 KO mice (8 weeks old) were treated with tamoxifen (0.18 mg/g BW) KO or vehicle (CO), and then injected with mouse lung carcinoma TC1 cells (10 × 105/mouse) in the left flank. Body weight, tumor size and weight, and local tumor invasion were assessed. Fecal DNA samples were extracted using PowerFecal kits and bacterial 16S rRNA amplicons were also performed. Fecal and plasma samples were used for GC-MS Polar, as well as non-targeted UHPLC-MS/MS, and tumor-associated macrophages (TAMs) were subjected to bulk RNAseq. Tamoxifen-treated MCT2 KO mice showed significantly higher tumor weight and size, as well as evidence of local invasion beyond the capsule compared with the controls. PCoA and hierarchical clustering analyses of the fecal and plasma metabolomics, as well as microbiota, revealed a distinct separation between the two groups. KO TAMs showed distinct metabolic pathways including the Acetyl-coA metabolic process, activation of immune response, b-cell activation and differentiation, cAMP-mediated signaling, glucose and glutamate processes, and T-cell differentiation and response to oxidative stress. Multi-Omic approaches reveal a substantial role for MCT2 in the host response to TC1 lung carcinoma that may involve alterations in the gut and systemic metabolome, along with TAM-related metabolic pathway. These findings provide initial opportunities for potential delineation of oncometabolic immunomodulatory therapeutic approaches.

Cell-Selective Altered Cargo Properties of Extracellular Vesicles Following In Vitro Exposures to Intermittent Hypoxia.

Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with cardiovascular and metabolic dysfunction. However, the mechanisms underlying these morbidities remain poorly delineated. Extracellular vesicles (EVs) mediate intercellular communications, play pivotal roles in a multitude of physiological and pathological processes, and could mediate IH-induced cellular effects. Here, the effects of IH on human primary cells and the release of EVs were examined. Microvascular endothelial cells (HMVEC-d), THP1 monocytes, THP1 macrophages M0, THP1 macrophages M1, THP1 macrophages M2, pre-adipocytes, and differentiated adipocytes (HAd) were exposed to either room air (RA) or IH for 24 h. Secreted EVs were isolated and characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. The effects of each of the cell-derived EVs on endothelial cell (EC) monolayer barrier integrity, on naïve THP1 macrophage polarity, and on adipocyte insulin sensitivity were also evaluated. IH did not alter EVs cell quantal release, but IH-EVs derived from HMVEC-d (p < 0.01), THP1 M0 (p < 0.01) and HAd (p < 0.05) significantly disrupted HMVEC-d monolayer integrity, particularly after H2O2 pre-conditioning. IH-EVs from HMVEC-d and THP1 M0 elicited M2-polarity changes did not alter insulin sensitivity responses. IH induces cell-selective changes in EVs cargo, which primarily seem to target the emergence of endothelial dysfunction. Thus, changes in EVs cargo from selected cell sources in vivo may play causal roles in some of the adverse outcomes associated with OSA.

Circulating Exosomal miRNAs Signal Circadian Misalignment to Peripheral Metabolic Tissues.

Night shift work increases risk of metabolic disorders, particularly obesity and insulin resistance. While the underlying mechanisms are unknown, evidence points to misalignment of peripheral oscillators causing metabolic disturbances. A pathway conveying such misalignment may involve exosome-based intercellular communication. Fourteen volunteers were assigned to a simulated day shift (DS) or night shift (NS) condition. After 3 days on the simulated shift schedule, blood samples were collected during a 24-h constant routine protocol. Exosomes were isolated from the plasma samples from each of the blood draws. Exosomes were added to naïve differentiated adipocytes, and insulin-induced pAkt/Akt expression changes were assessed. ChIP-Seq analyses for BMAL1 protein, mRNA microarrays and exosomal miRNA arrays combined with bioinformatics and functional effects of agomirs and antagomirs targeting miRNAs in NS and DS exosomal cargo were examined. Human adipocytes treated with exosomes from the NS condition showed altered Akt phosphorylation responses to insulin in comparison to those treated with exosomes from the DS condition. BMAL1 ChIP-Seq of exosome-treated adipocytes showed 42,037 binding sites in the DS condition and 5538 sites in the NS condition, with a large proportion of BMAL1 targets including genes encoding for metabolic regulators. A significant and restricted miRNA exosomal signature emerged after exposure to the NS condition. Among the exosomal miRNAs regulated differentially after 3 days of simulated NS versus DS, proof-of-concept validation of circadian misalignment signaling was demonstrated with hsa-mir-3614-5p. Exosomes from the NS condition markedly altered expression of key genes related to circadian rhythm in several cultured cell types, including adipocytes, myocytes, and hepatocytes, along with significant changes in 29 genes and downstream gene network interactions. Our results indicate that a simulated NS schedule leads to changes in exosomal cargo in the circulation. These changes promote reduction of insulin sensitivity of adipocytes in vitro and alter the expression of core clock genes in peripheral tissues. Circulating exosomal miRNAs may play an important role in metabolic dysfunction in NS workers by serving as messengers of circadian misalignment to peripheral tissues.

Activation of the Integrated Stress Response and Metabolic Dysfunction in a Murine Model of Sleep Apnea.

Intermittent hypoxia (IH) induces activation of the integrated stress response (ISR), but its role in IH-induced visceral white adipose tissue (vWAT) insulin resistance is unknown. CHOP is activated by chronic ISR, whereas GADD34 dephosphorylates the subunit of translation initiation factor 2 (eIF2α), leading to termination of the ISR. We hypothesized that CHOP/Gadd34 null mice would not manifest evidence of insulin resistance after IH exposures. Eight-week-old CHOP/GADD34-/- (double mutant [DM]) and wild-type (WT) littermates were randomly assigned to IH or room air (RA) exposures for 6 weeks. Glucose and insulin tolerance tests were performed, and regulatory T cells (Tregs) and macrophages in vWAT were assessed. Phosphorylated eIF2α:total eIF2α, ATF4, XBP1 expression, and insulin-induced pAKT/AKT expression changes were examined in vWATs. Single GADD34-/- and PERK+/- mice were also evaluated. Body weight and vWAT mass were reduced in DM and WT mice after IH. M1/M2 macrophages and inflammatory macrophages (Ly-6chigh) were significantly increased in WT vWAT but remained unchanged in DM mice. Tregs were significantly decreased in WT vWAT but not in DM mice. Systemic insulin and glucose tolerance tests revealed insulin resistance in IH-WT but not in IH-DM mice. Similarly, decreased pAKT/AKT responses to exogenous insulin emerged in IH-WT compared with RA-WT mice, whereas no significant differences emerged in IH-DM compared with DM-RA. Chronic ISR activation appears to contribute to the insulin resistance and vWAT inflammation that characteristically emerge after long-term IH exposures in a murine model of obstructive sleep apnea.

Visceral White Adipose Tissue after Chronic Intermittent and Sustained Hypoxia in Mice.

Angiogenesis, a process induced by hypoxia in visceral white adipose tissues (vWAT) in the context of obesity, mediates obesity-induced metabolic dysfunction and insulin resistance. Chronic intermittent hypoxia (IH) and sustained hypoxia (SH) induce body weight reductions and insulin resistance of different magnitudes, suggesting different hypoxia inducible factor (HIF)-1α-related activity. Eight-week-old male C57BL/6J mice (n = 10-12/group) were exposed to either IH, SH, or room air (RA). vWAT were analyzed for insulin sensitivity (phosphorylated (pAKT)/AKT), HIF-1α transcription using chromatin immunoprecipitation (ChIP)-sequencing, angiogenesis using immunohistochemistry, and gene expression of different fat cell markers and HIF-1α gene targets using quantitative polymerase chain reaction or microarrays. Body and vWAT weights were reduced in hypoxia (SH > IH > RA; P < 0.001), with vWAT in IH manifesting vascular rarefaction and increased proinflammatory macrophages. HIF-1α ChIP-sequencing showed markedly increased binding sites in SH-exposed vWAT both at 6 hours and at 6 weeks compared with IH, the latter also showing decreased vascular endothelial growth factor, endothelial nitric oxide synthase, P2RX5, and PAT2 expression, and insulin resistance (IH > > > SH = RA; P < 0.001). IH induces preferential whitening of vWAT, as opposed to prominent browning in SH. Unlike SH, IH elicits early HIF-1α activity that is unsustained over time and is accompanied by concurrent vascular rarefaction, inflammation, and insulin resistance. Thus, the dichotomous changes in HIF-1α transcriptional activity and brown/beige/white fat balance in IH and SH should enable exploration of mechanisms by which altered sympathetic outflow, such as that which occurs in apneic patients, results in whitening, rather than the anticipated browning of adipose tissues that occurs in SH.

Late gestational intermittent hypoxia induces metabolic and epigenetic changes in male adult offspring mice.

KEY POINTS: Late gestation during pregnancy has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia, a hallmark of OSA, could impose significant long-term effects on somatic growth, energy homeostasis and metabolic function in offspring. Here we show that late gestation intermittent hypoxia induces metabolic dysfunction as reflected by increased body weight and adiposity index in adult male offspring that is paralleled by epigenomic alterations and inflammation in visceral white adipose tissue. Fetal perturbations by OSA during pregnancy impose long-term detrimental effects manifesting as metabolic dysfunction in adult male offspring. ABSTRACT: Pregnancy, particularly late gestation (LG), has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia (IH), a hallmark of OSA, could impose significant long-term effects on somatic growth, energy homeostasis, and metabolic function in offspring. We hypothesized that IH during late pregnancy (LG-IH) may increase the propensity for metabolic dysregulation and obesity in adult offspring via epigenetic modifications. Time-pregnant female C57BL/6 mice were exposed to LG-IH or room air (LG-RA) during days 13-18 of gestation. At 24 weeks, blood samples were collected from offspring mice for lipid profiles and insulin resistance, indirect calorimetry was performed and visceral white adipose tissues (VWAT) were assessed for inflammatory cells as well as for differentially methylated gene regions (DMRs) using a methylated DNA immunoprecipitation on chip (MeDIP-chip). Body weight, food intake, adiposity index, fasting insulin, triglycerides and cholesterol levels were all significantly higher in LG-IH male but not female offspring. LG-IH also altered metabolic expenditure and locomotor activities in male offspring, and increased number of pro-inflammatory macrophages emerged in VWAT along with 1520 DMRs (P < 0.0001), associated with 693 genes. Pathway analyses showed that genes affected by LG-IH were mainly associated with molecular processes related to metabolic regulation and inflammation. LG-IH induces metabolic dysfunction as reflected by increased body weight and adiposity index in adult male offspring that is paralleled by epigenomic alterations and inflammation in VWAT. Thus, perturbations to fetal environment by OSA during pregnancy can have long-term detrimental effects on the fetus, and lead to persistent metabolic dysfunction in adulthood.

Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4ß6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1ß1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the ß2-AR*·Gαsß1γ2 complex crystal structure, the Gαi1ß1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1ß1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins.

Involvement of a non-CB1/CB2 cannabinoid receptor in the aqueous humor outflow-enhancing effects of abnormal-cannabidiol.

The purpose of this study was to investigate the effects of abnormal-cannabidiol (abn-cbd), a non-psychoactive cannabinoid agonist, on aqueous humor outflow via the trabecular meshwork (TM) of porcine eye, and to examine the involvement of a non-CB1/CB2 cannabinoid receptor and the p42/44 mitogen-activated protein kinase (p42/44 MAPK) pathway. The effects of abn-cbd on aqueous humor outflow were measured using a porcine anterior segment perfused organ culture model. The activation of p42/44 MAPK by abn-cbd was determined in cultured TM cells with western blot analysis using an anti-phospho-p42/44 MAPK antibody. Administration of abn-cbd caused a concentration-dependent enhancement of aqueous humor outflow facility with a maximum effect (155.0 ± 11.7% of basal outflow facility) after administration of 30 nM abn-cbd. Pretreatment with 1 µM of O-1918, a cannabidiol analog that acts as a selective antagonist at the non-CB1/CB2 receptor, produced a full antagonism of 30 nM abn-cbd induced increase of aqueous humor outflow facility. Pretreatment with 1 µM of CB1 antagonist SR141716A partially blocked, whereas pretreatment with either 1 µM of CB1 antagonist AM251 or 1 µM of CB2 antagonist SR144528 had no effect on abn-cbd induced enhancement of outflow facility. Treatment of TM cells with 30 nM of abn-cbd activated p42/44 MAPK, which was blocked completely by pretreatment with O-1918, and partially by pretreatment with SR141716A, but not by either AM251 or SR144528. In addition, PD98059, an inhibitor of p42/44 MAPK pathway, blocked completely the abn-cbd induced p42/44 MAPK activation and blocked partially the abn-cbd induced enhancement of outflow facility. In conclusion, the results from this study demonstrate that abn-cbd increases aqueous humor outflow through the TM pathway of the eye, and this effect is mediated by a non-CB1/CB2 cannabinoid receptor, with an involvement of p42/44 MAPK signaling pathway.

Explicit memory, anxiety and depressive like behavior in mice exposed to chronic intermittent hypoxia, sleep fragmentation, or both during the daylight period.

Obstructive sleep apnea (OSA) is a chronic and highly prevalent condition characterized by chronic intermittent hypoxia (IH) and sleep fragmentation (SF), and can lead to a vast array of end-organ morbidities, particularly affecting cardiovascular, metabolic and neurobehavioral functioning. OSA can induce cognitive and behavioral and mood deficits. Male C57Bl/6J 8-week-old mice were housed in custom-designed cages with a silent motorized mechanical sweeper traversing the cage floor at 2-min intervals (SF) during daylight for four weeks. Sleep control (SC) consisted of keeping sweeper immobile. IH consisted of cycling FiO2 21% 90 seconds-6.3% 90s or room air (RA; FiO2 21%) for sixteen weeks and combined SF-IH was conducted for nine weeks. Open field novel object recognition (NOR) testing, elevated-plus maze test (EPMT), and forced swimming test (FST) were performed. SF induced cognitive NOR performance impairments in mice along with reduced anxiety behaviors while IH induced deficits in NOR performance, but increased anxiety behaviors. SF-IH induced impaired performance in NOR test of similar magnitude to IH or SF alone. Combined SF-IH exposures did not affect anxiety behaviors. Thus, both SF an IH altered cognitive function while imposing opposite effects on anxiety behaviors. SF-IH did not magnify the detrimental effects of isolated SF or IH and canceled out the effects on anxiety. Based on these findings, the underlying pathophysiologic processes underlying IH and SF adverse effects on cognitive function appear to differ, while those affecting anxiety counteract each other.

Heterogeneity of Melanoma Cell Responses to Sleep Apnea-Derived Plasma Exosomes and to Intermittent Hypoxia.

Obstructive sleep apnea (OSA) is associated with increased cutaneous melanoma incidence and adverse outcomes. Exosomes are secreted by most cells, and play a role in OSA-associated tumor progression and metastasis. We aimed to study the effects of plasma exosomes from OSA patients before and after adherent treatment with continuous positive airway pressure (CPAP) on melanoma cells lines, and also to identify exosomal miRNAs from melanoma cells exposed to intermittent hypoxia (IH) or normoxia. Plasma-derived exosomes were isolated from moderate-to-severe OSA patients before (V1) and after (V2) adherent CPAP treatment for one year. Exosomes were co-incubated with three3 different melanoma cell lines (CRL 1424; CRL 1619; CRL 1675) that are characterized by genotypes involving different mutations in BRAF, STK11, CDKN2A, and PTEN genes to assess the effect of exosomes on cell proliferation and migration, as well as on pAMK activity in the presence or absence of a chemical activator. Subsequently, CRL-1424 and CRL-1675 cells were exposed to intermittent hypoxia (IH) and normoxia, and exosomal miRNAs were identified followed by GO and KEG pathways and gene networks. The exosomes from these IH-exposed melanoma cells were also administered to THP1 macrophages to examine changes in M1 and M2 polarity markers. Plasma exosomes from V1 increased CRL-1424 melanoma cell proliferation and migration compared to V2, but not the other two cell lines. Exposure to CRL-1424 exosomes reduced pAMPK/tAMPK in V1 compared to V2, and treatment with AMPK activator reversed the effects. Unique exosomal miRNAs profiles were identified for CRL-1424 and CRL-1675 in IH compared to normoxia, with six miRNAs being regulated and several KEGG pathways were identified. Two M1 markers (CXCL10 and IL6) were significantly increased in monocytes when treated with exosomes from IH-exposed CRL-1424 and CRL-1625 cells. Our findings suggest that exosomes from untreated OSA patients increase CRL-1424 melanoma malignant properties, an effect that is not observed in two other melanoma cell lines. Exosomal cargo from CRL-1424 cells showed a unique miRNA signature compared to CRL-1675 cells after IH exposures, suggesting that melanoma cells are differentially susceptible to IH, even if they retain similar effects on immune cell polarity. It is postulated that mutations in STK-11 gene encoding for the serine/threonine kinase family that acts as a tumor suppressor may underlie susceptibility to IH-induced metabolic dysfunction, as illustrated by CRL-1424 cells.

Plasma exosomes in OSA patients promote endothelial senescence: effect of long-term adherent continuous positive airway pressure.

Obstructive sleep apnea (OSA) is associated with increased risk for end-organ morbidities, which can collectively be viewed as accelerated aging. Vascular senescence is an important contributor to end-organ dysfunction. Exosomes are released ubiquitously into the circulation, and transfer their cargo to target cells facilitating physiological and pathological processes. Plasma exosomes from 15 patients with polysomnographically diagnosed OSA at baseline (OSA-T1) after 12 months of adherent continuous positive airway pressure (CPAP) treatment (OSA-T2), 13 untreated OSA patients at 12-month intervals (OSA-NT1, OSA-NT2), and 12 controls (CO1 and CO2) were applied on naïve human microvascular endothelialcells-dermal (HMVEC-d). Expression of several senescence gene markers including p16 (CDKN2A), SIRT1, and SIRT6 and immunostaining for ß-galactosidase activity (x-gal) were performed. Endothelial cells were also exposed to intermittent hypoxia (IH) or normoxia (RA) or treated with hydrogen peroxide (H2O2), stained with x-gal and subjected to qRT-PCR. Exosomes from OSA-T1, OSA-NT1, and OSA-NT2 induced significant increases in x-gal staining compared to OSA-T2, CO1, and CO2 (p-value < 0.01). p16 expression was significantly increased (p < 0.01), while SIRT1 and SIRT6 expression levels were decreased (p < 0.02 and p < 0.009). Endothelial cells exposed to IH or to H2O2 showed significant increases in x-gal staining (p < 0.001) and in senescence gene expression. Circulating exosomes in untreated OSA induce marked and significant increases in senescence of naïve endothelial cells, which are only partially reversible upon long-term adherent CPAP treatment. Furthermore, endothelial cells exposed to IH or H2O2 also elicit similar responses. Thus, OSA either directly or indirectly via exosomes may initiate and exacerbate cellular aging, possibly via oxidative stress-related pathways.

Residues accessible in the binding-site crevice of transmembrane helix 6 of the CB2 cannabinoid receptor.

We have used the substituted-cysteine accessibility method (SCAM) to map the residues in the sixth membrane-spanning segment of the CB2 cannabinoid receptor that contribute to the surface of the water-accessible binding-site crevice. Using a background of the mutant C2.59S which is relatively insensitive to the methanethiosulfonate (MTS) reagents, we mutated to cysteine, one at a time, 34 consecutive residues in TMH6 of the CB2 receptor. These mutant receptors were then expressed in HEK293 cells. By incubating HEK293 cells stably transfected with CB2 receptors with the small, charged, hydrophilic, thiol-specific reagent methanethiosulfonate ethylammonium (MTSEA), [(3)H]CP55940 binding was significantly inhibited for six mutant receptors. All six of the mutants that reacted with MTSEA were protected from the reaction when pretreated with the cannabinoid agonist WIN55212-2, suggesting that MTSEA modification occurred within the binding crevice. Therefore, the side chains of the residues at these reactive loci (V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62) are on the water-accessible surface of the binding-site crevice. These residues are extracellular to the TMH6 CWXP hinge motif. The pattern of accessibility is consistent with a alpha-helical conformation for this segment of TMH6. Molecular modeling studies performed in the context of the CB2 model show that V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62 face into the CB2 binding pocket, further confirming our SCAM results. These results are similar to the accessibility patterns determined by SCAM studies of TMH6 in the opioid and dopamine D2 receptors.

Aqueous humor outflow effects of 2-arachidonylglycerol.

This study was conducted to test the effects of 2-arachidonylglycerol (2-AG), an endocannabinoid, on aqueous humor outflow facility, to study the cellular mechanisms of 2-AG, and to investigate the possible existence and activity of monoacylgylcerol lipase (MGL), a 2-AG metabolic enzyme, in the trabecular meshwork (TM). The effects of 2-AG on aqueous humor outflow facility were measured using an anterior segment perfused organ culture model. The expression and activity of MGL in TM tissues were assessed using Western blot analysis and an enzyme activity assay respectively. 2-AG induced activation of p42/44 mitogen-activated protein (MAP) kinase was determined by Western blot analysis using an anti-phospho p42/44 MAP kinase antibody. AlexaFluor 488-labeled phalloidin staining was used to examine actin filament in cultured TM cells. Administration of 10nM of 2-AG caused a transient enhancement of aqueous humor outflow. In the presence of 100nM of LY2183240, an inhibitor of MGL, the effect of 10nM of 2-AG on outflow was prolonged by at least 4h. The 2-AG-induced enhancement of outflow was blocked by SR141716A, a CB1 antagonist, and SR144528, a CB2 antagonist. In Western blot studies, a 35kDa band representing MGL was detected on TM tissues with an anti-MGL antibody. The 2-AG enzymatic hydrolysis activity was detected in TM tissues and this activity was reduced by 70.1+/-5.3% with the addition of 100 nM of LY2183240. Treatment of trabecular meshwork cells with 10nM of 2-AG plus 100 nM LY2183240 for 5h evoked phosphorylation of p42/44 MAP kinase. The 2-AG-induced enhancement of p42/44 MAP kinase phosphorylation was blocked by pretreatment with SR141716A, SR144528, as well as PD98059, an inhibitor of the p42/44 MAP kinase pathway. In addition, the outflow-enhancing effect of 2-AG was blocked by pretreatment with PD98059. Furthermore, treatment with 2-AG plus LY2183240 caused rounding of TM cells and a reduction of actin stress fibers in TM cells. Pretreatment with SR141716A, SR144528, and PD98059 blocked these 2-AG-induced morphology and cytoskeleton changes in TM cells. In conclusion, the results from this study demonstrate that administration of 2-AG increases aqueous humor outflow facility and this effect of 2-AG is mediated through both the CB1 and CB2 cannabinoid receptors. In addition, this study reveals the existence and the activity of MGL, a 2-AG metabolizing enzyme, in the TM tissues. Furthermore, this study suggests that 2-AG-induced enhancement of outflow facility involves the p42/44 MAP kinase signaling pathway and changes in actin cytoskeletons in TM cells.

Integrated stress response activation by sleep fragmentation during late gestation in mice leads to emergence of adverse metabolic phenotype in offspring.

BACKGROUND: Late gestational sleep fragmentation (SF) is highly prevalent particularly in obese women, and induces metabolic dysfunction in adult offspring mice. SF induces activation of the integrated stress response (ISR), which might be involved in metabolic disorders. We hypothesized that adult offspring of double mutant mice (DM) involving the critical ISR genes CHOP and GADD34 would be protected from developing obesity and insulin resistance following SF. METHODS: Time-pregnant CHOP/GADD34 DM and wild type (WT) mice were randomly assigned to sleep control (SC) or SF conditions during the last 5days of gestation. At 24-weeks of age, body weight, fat mass, and HOMA-IR were assessed in the offspring. Tregs lymphocytes, Lyc6chigh, M1 and M2 macrophages were examined in visceral white adipose tissues (vWAT) using flow cytometry. The effects of plasma exosomes on adipocyte cell line proliferation, differentiation and insulin sensitivity were also evaluated. RESULTS: SF-WT male showed significant increases in body weight, vWAT mass and HOMA-IR compared to SC-WT mice, while SF had no effect in SF-DM mice. Inflammatory macrophages (Ly-6chigh) and the ratio of M1/M2 macrophages were increased while FoxP3+ Tregs counts were decreased in SF-WT but not in SF-DM mice. Exosomes from SF-WT, but not from the SF-DM offspring increased pre-adipocyte proliferation and differentiation, and decreased in vitro adipocyte insulin sensitivity. CONCLUSION: Activation of the ISR during late gestation, as induced by late gestational SF, appears to underlie some of the transgenerational modifications in metabolic genes ultimately contributing to a metabolic syndrome phenotype in adult offspring.

Prolonged Exposures to Intermittent Hypoxia Promote Visceral White Adipose Tissue Inflammation in a Murine Model of Severe Sleep Apnea: Effect of Normoxic Recovery.

Study Objective: Increased visceral white adipose tissue (vWAT) mass results in infiltration of inflammatory macrophages that drive inflammation and insulin resistance. Patients with obstructive sleep apnea (OSA) suffer from increased prevalence of obesity, insulin resistance, and metabolic syndrome. Murine models of intermittent hypoxia (IH) mimicking moderate-severe OSA manifest insulin resistance following short-term IH. We examined in mice the effect of long-term IH on the inflammatory cellular changes within vWAT and the potential effect of normoxic recovery (IH-R). Methods: Male C57BL/6J mice were subjected to IH for 20 weeks, and a subset was allowed to recover in room air (RA) for 6 or 12 weeks (IH-R). Stromal vascular fraction was isolated from epididymal vWAT and mesenteric vWAT depots, and single-cell suspensions were prepared for flow cytometry analyses, reactive oxygen species (ROS), and metabolic assays. Results: IH reduced body weight and vWAT mass and IH-R resulted in catch-up weight and vWAT mass. IH-exposed vWAT exhibited increased macrophage counts (ATMs) that were only partially improved in IH-R. IH also caused a proinflammatory shift in ATMs (increased Ly6c(hi)(+) and CD36(+) ATMs). These changes were accompanied by increased vWAT insulin resistance with only partial improvements in IH-R. In addition, ATMs exhibited increased ROS production, altered metabolism, and changes in electron transport chain, which were only partially improved in IH-R. Conclusion: Prolonged exposures to IH during the sleep period induce pronounced vWAT inflammation and insulin resistance despite concomitant vWAT mass reductions. These changes are only partially reversible after 3 months of normoxic recovery. Thus, long-lasting OSA may preclude complete reversibility of metabolic changes.

Exosomes and Metabolic Function in Mice Exposed to Alternating Dark-Light Cycles Mimicking Night Shift Work Schedules.

Sleep is an important modulator of metabolic function. Disruptions of sleep in circadian rhythm are common in modern societies and are associated with increased risk of developing cardiometabolic disorders. Exosomes are ubiquitous extracellular vesicles that may play a mechanistic role in metabolic derangements. We hypothesized that alternating dark-light cycles mimicking shift work in mice would alter fecal microbiota and colonic epithelium permeability and alter plasma exosome cargo and metabolic function. C57BL/6 mice were randomly assigned to (i) control day light (CL), or (ii) inverted dark-light every 2 weeks for 8 weeks (IN). Body weight, fat mass and HOMA-IR were measured, along with Tregs, metabolic, and resident macrophages in visceral white adipose tissue (vWAT). Fecal water samples were incubated with confluent colonic epithelium cell cultures in electric cell-substrate impedance sensing (ECIS) arrays, and plasma exosomes were added to differentiated adipocytes and insulin-induced pAKT/AKT expression changes were assessed by western blots. Mice exposed to IN showed elevated HOMA-IR, and their fecal samples showed altered microbiota which promote increased permeability of the colonic epithelial cell barrier. Plasma exosomes decreased pAKT/AKT responses to exogenous insulin compared to CL, and altered expression of circadian clock genes. Inflammatory macrophages (Ly-6chigh) were increased in IN-exposed vWAT, while Tregs were decreased. Thus, gut microbiota and the cargo of plasma exosomes are altered by periodic shifts in environmental lighting, and effectively alter metabolic function, possibly via induction of systemic inflammation and altered clock expression in target tissues. Further exploration of exosomal miRNA signatures in shift workers and their putative metabolic organ cell targets appears warranted.

Noladin ether acts on trabecular meshwork cannabinoid (CB1) receptors to enhance aqueous humor outflow facility.

PURPOSE: To study the effects of 2-arachidonyl glyceryl ether (noladin ether), an endocannabinoid ligand selective for cannabinoid (CB)1 receptor, on aqueous humor outflow facility, to investigate the involvement of trabecular meshwork CB1 receptors and the p42/44 MAP kinase signaling pathway and to explore the cellular mechanisms of noladin ether-induced changes of outflow facility. METHODS: The effects of noladin ether on aqueous humor outflow facility were measured in a porcine anterior-segment-perfused organ culture model. The expression of CB1 receptors on cultured porcine trabecular meshwork cells and the coupling of these receptors to p42/44 MAP kinase was determined by immunofluorescence microscopy and Western blot analysis. Both Western blot and zymography were used to monitor the effects of noladin ether on matrix metalloproteinase (MMP)-2. In morphologic studies, AlexaFluor 488-labeled phalloidin staining was used to examine actin filament, and immunohistochemistry with anti-paxillin antibodies was used to detect focal adhesions. RESULTS: Within 1 hour after adding 3, 30, or 300 nM of noladin ether, the aqueous humor outflow facility increased concentration dependently. The effect of 30 nM of noladin ether was completely blocked by SR141716A, a selective CB1 antagonist. Positive signals were detected on cultured porcine trabecular meshwork cells with an anti-CB1 antibody in immunofluorescence microscopy and Western blot studies. Treatment of trabecular meshwork cells with 30 nM of noladin ether activated p42/44 MAP kinase, whereas pretreatment with SR141716A blocked the p42/44 MAP kinase-activating effects of noladin ether. In addition, the enhancement of outflow facility induced by noladin ether was blocked by pretreatment of porcine anterior segments with PD98059, an inhibitor of p42/44 MAP kinase pathway. Furthermore, noladin ether treatment caused rounding of trabecular meshwork cells, and there was a decrease of actin stress fibers, as well as a decrease in focal adhesions. These noladin ether-induced morphologic changes were also blocked by SR141716A and PD98059. CONCLUSIONS: The results demonstrate for the first time that administration of noladin ether, an endocannabinoid agonist selective for the CB1 receptor, increases aqueous humor outflow facility. The data also show that noladin ether-induced enhancement of outflow facility is mediated through the trabecular meshwork CB1 receptor, with an involvement of p42/44 MAP kinase signaling pathway and changes in actin cytoskeletons.

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.