Exosomes and metabolic functionin 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

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

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

Aorta macrophage inflammatory and epigenetic changes in a murine model of obstructive sleep apnea: Potential role of CD36

Obstructive sleep apnea (OSA) affects 8-10% of the population, is characterized by chronic intermittent hypoxia (CIH), and causally associates with cardiovascular morbidities. In CIH-exposed mice, closely mimicking the chronicity of human OSA, increased accumulation and proliferation of pro-inflammatory metabolic M1-like macrophages highly expressing CD36, emerged in aorta. Transcriptomic and MeDIP-seq approaches identified activation of pro-atherogenic pathways involving a complex interplay of histone modifications in functionally-relevant biological pathways, such as inflammation and oxidative stress in aorta macrophages. Discontinuation of CIH did not elicit significant improvements in aorta wall macrophage phenotype. However, CIH-induced aorta changes were absent in CD36 knockout mice, Our results provide mechanistic insights showing that CIH exposures during sleep in absence of concurrent pro-atherogenic settings (i.e., genetic propensity or dietary manipulation) lead to the recruitment of CD36(+)high macrophages to the aortic wall and trigger atherogenesis. Furthermore, long-term CIH-induced changes may not be reversible with usual OSA treatment

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

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-tosevere 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 CRL1424 cells

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

International audienc

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

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

Exosomes and metabolic functionin 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

Aorta macrophage inflammatory and epigenetic changes in a murine model of obstructive sleep apnea: Potential role of CD36

Obstructive sleep apnea (OSA) affects 8-10% of the population, is characterized by chronic intermittent hypoxia (CIH), and causally associates with cardiovascular morbidities. In CIH-exposed mice, closely mimicking the chronicity of human OSA, increased accumulation and proliferation of pro-inflammatory metabolic M1-like macrophages highly expressing CD36, emerged in aorta. Transcriptomic and MeDIP-seq approaches identified activation of pro-atherogenic pathways involving a complex interplay of histone modifications in functionally-relevant biological pathways, such as inflammation and oxidative stress in aorta macrophages. Discontinuation of CIH did not elicit significant improvements in aorta wall macrophage phenotype. However, CIH-induced aorta changes were absent in CD36 knockout mice, Our results provide mechanistic insights showing that CIH exposures during sleep in absence of concurrent pro-atherogenic settings (i.e., genetic propensity or dietary manipulation) lead to the recruitment of CD36(+)high macrophages to the aortic wall and trigger atherogenesis. Furthermore, long-term CIH-induced changes may not be reversible with usual OSA treatment