Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1–RIP1 interaction is dispensable for necroptosis; RIP1–RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1–RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3–RIP3 interaction is required for necroptosis and RIP3–RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1–RIP3 heterodimer itself cannot induce necroptosis, the RIP1–RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1–RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis.Necroptosis is a type of programmed necrosis characterized by necrotic morphological changes, including cellular organelle swelling, cell membrane rupture,^{1, 2, 3} and dependence of receptor-interacting protein (RIP)1⁴ and RIP3.^{5, 6, 7} Physiological function of necroptosis has been illustrated in host defense,^{8, 9, 10, 11} inflammation,^{12, 13, 14, 15, 16} tissue injury,^{10, 17, 18} and development.^{19, 20, 21}Necroptosis can be induced by a number of different extracellular stimuli such as tumor necrosis factor (TNF). TNF stimulation leads to formation of TNF receptor 1 (TNFR1) signaling complex (named complex I), and complex II containing RIP1, TRADD, FAS-associated protein with a death domain (FADD), and caspase-8, of which the activation initiates apoptosis. If cells have high level of RIP3, RIP1 recruits RIP3 to form necrosome containing FADD,^{22, 23, 24} caspase-8, RIP1, and RIP3, and the cells undergo necroptosis.^{25, 26} Caspase-8 and FADD negatively regulates necroptosis,^{27, 28, 29, 30} because RIP1, RIP3, and CYLD are potential substrates of caspase-8.^{31, 32, 33, 34} Necrosome also suppresses apoptosis but the underlying mechanism has not been described yet. Mixed-lineage kinase domain-like (MLKL) is downstream of RIP3,^{35, 36} and phosphorylation of MLKL is required for necroptosis.^{37, 38, 39, 40, 41, 42}Apoptosis inducing complex (complex II) and necrosome are both supramolecular complexes.^{43, 44, 45} A recent study showed that RIP1 and RIP3 form amyloidal fibrils through their RIP homotypic interaction motif⁴⁶ (RHIM)-mediated polymerization, and suggested that amyloidal structure is essential for necroptosis signaling.⁴⁷ The RIP1–RIP3 heterodimeric amyloid complex is believed to function as a scaffold that brings signaling proteins into proximity to permit their activation. However, RIP1 and RIP3 also can each form fibrils on their own RHIM domains in vitro. It is unclear how the homo- and hetero-interactions are coordinated and organized on the amyloid scaffold to execute their functions in necroptosis. Here, we used inducible dimerization systems to study the roles of RIP1–RIP1, RIP1–RIP3, and RIP3–RIP3 interactions in necroptosis signaling. Our data suggested that it is the RIP1–RIP3 interaction in the RIP1–RIP3 heterodimeric amyloid complex that empowers to recruit other free RIP3; homodimerization of RIP3 triggers its autophosphorylation and only the phosphorylated RIP3 can recruit MLKL to execute necroptosis.     

Clinicopathologic Characteristics,Prevalence, and Risk Factors of Spontaneous Diabetes in Sooty Mangabeys (Cercocebus atys)

In 2008, clinical observations in our colony of sooty mangabeys (Cercocebus atys) suggested a high frequency of type 2 diabetes. Postmortem studies of diabetic animals revealed dense amyloid deposits in pancreatic islets. To investigate these findings, we screened our colony (97 male mangabeys; 99 female mangabeys) for the disease from 2008 to 2012. The overall prevalence of diabetes was 11% and of prediabetes was 7%, which is nearly double that reported for other primate species (less than 6%). Fructosamine and triglyceride levels were the best indicators of diabetes; total cholesterol and glycated hemoglobin were not associated with disease. Increasing age was a significant risk factor: prevalence increased from 0% in infants, juveniles, and young adults to 11% in adults and 19% in geriatric mangabeys. Sex, medroxyprogesterone acetate exposure, and SIV status were unrelated to disease. Weight was marginally higher in prediabetics, but body condition did not indicate obesity. Of the 49 mangabeys that were necropsied after clinical euthanasia or death from natural causes, 22 were diabetic; all 22 animals demonstrated pancreatic amyloid, and most had more than 75% of islets replaced with amyloid. We conclude that type 2 diabetes is more common in mangabeys than in other primate species. Diabetes in mangabeys has some unusual pathologic characteristics, including the absence of altered cholesterol levels and glycated hemoglobin but a robust association of pancreatic insular amyloidosis with clinical diabetes. Future research will examine the genetic basis of mangabey diabetes and evaluate additional diagnostic tools using imaging and serum markers.Abbreviations: HbA1c, glycated hemoglobin; MPA, medroxyprogesterone acetate; YNPRC, Yerkes National Primate Research CenterSooty mangabeys (Cercocebus atys) are Old World NHP that are native to West Africa. Historically their use in research has been limited to infectious disease studies, leprosy studies, and behavioral research.^14,25 Over the past 20 to 30 y, they have been used in HIV–AIDS research. Mangabeys are natural hosts of SIV_smm, which is recognized as the origin of HIV2 infection in humans.^7,8,30,36,42 SIV typically is nonpathogenic in mangabeys despite high levels of virus replication, which makes this species a unique and invaluable model in AIDS research.^7,30,36,42 Our facility maintains a colony of approximately 200 sooty mangabeys. In 2008 clinical observations of relative hyperglycemia, glucosuria, and weight loss in our colony suggested that type 2 diabetes mellitus occurred at a relatively high frequency in this population. Spontaneous diabetes was found in 10% of the colony, and 5% of animals were prediabetic; this incidence is higher than that typically reported for other NHP species, such as cynomolgus macaques (less than 1% to 2%)²² and chimpanzees (less than 1%).³⁷ The prevalence of spontaneous diabetes in humans is typically 8.3%.^2,6,22,37 In addition, necropsies revealed that many affected animals had dense amyloid deposits in pancreatic islet cells. Insular amyloidosis was seen on histology, with a total replacement of islets by amyloid deposition in advanced diabetes. Advanced diabetes was determined by increased weight loss and severity of relative hyperglycemia. The increased clinical prevalence of diabetes in our mangabey colony prompted additional characterization of the clinicopathologic profile, risk factors, and prevalence of diabetes in our mangabey colony.The form of diabetes in this mangabey colony is characterized as type 2 diabetes mellitus, as they have hyperglycemia, hypertriglyceridemia, and islet amyloidosis. Type 2 diabetes mellitus is the most common of the 3 forms of diabetes, and has been documented in humans and NHP,^22,31,37,55 including rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), Celebes crested macaques (Macaca nigra), bonnet macaques (Macaca radiate), pigtailed macaques (Macaca nemestrina), vervet monkeys (Chlorocebus pygerythrus), squirrel monkeys (Saimiri sciureus), chimpanzees (Pan troglodytes), and woolly monkeys (Lagothrix spp.).^{1,24,31,52,55} Type 2 diabetes is a chronic metabolic disorder in which insulin resistance occurs in liver, muscle, and adipose tissue. As type 2 diabetes progresses, it also can be characterized as a relative insulin deficiency.^{1,6,15,22,29,31,37,55} The initial clinical presentation of diabetes in humans and NHP includes polydipsia, polyuria, polyphagia, weight loss, and lethargy.^{1,6,22,27,31,37,55} Similar presentation was observed in our colony of diabetic mangabeys.Diagnostic criteria of diabetes in NHP species is similar to that for humans and is based on clinical symptoms and routine lab tests, including serum chemistry panel to evaluate persistent fasting hyperglycemia, hypertriglyceridemia, and hypercholesterolemia.^{2,6,11,16-18,21,22,29,31,37,48-50,52,55} Hypertriglyceridemia and hypercholesterolemia frequently are elevated due to diabetes and therefore are used as supportive diagnostic markers. In addition, the disease is characterized by transient hyperinsulinemia followed by insulin deficiency subsequent to glucose challenge. Urinalysis is used to evaluate glucosuria and ketonuria. These tests are not exclusive for diagnosing diabetes and can be inconsistent between species, thus making conclusive diagnosis challenging. For example, hyperglycemia can be a transient finding associated with recent food intake or stress associated with restraint for blood sample collection or anesthetic access, whereas hypertriglyceridemia can be seen in obese animals and those with other metabolic diseases such as pancreatitis and hypothyroidism.^1,22,37,55The typical clinical approach to the diagnosis of diabetes in NHP and other veterinary patients includes evaluation of fructosamine and glycated hemoglobin (HbA1c) levels and glucose tolerance testing. These tests are indices of glycemic control and are used in clinical settings primarily to assess prognosis and response to treatment; they are also useful for the initial diagnosis of diabetes when used in parallel with serum chemistry markers. Fructosamine and HbA1c can both provide information on long-term glycemic control, because fructosamine reflects average blood glucose levels over 2 to 3 wk whereas HbA1c reflects average blood glucose over 2 to 3 mo preceding blood collection. HbA1c is the primary test for diabetes in human medicine,^6,31,35,37 whereas fructosamine is commonly used in veterinary medicine. Glucose tolerance testing provides an indirect measure of insulin sensitivity, but it is not frequently used clinically in NHP because of the requirement for prolonged physical restraint or sedation.^{1,21,22,26,27,34,37,55}Prevention and management of diabetes in NHP and humans can be achieved by identifying potential risk factors, including age, weight, sex, genetics, hormone drug exposure, and viral status.^{1,6,15,22,29,31,37,42,55} Advanced age, obesity, sex, and genetics are associated with diabetes in some species of NHP and humans.^{1,6,15,22,29,31,37,55} In addition, exposure to drugs such as medroxyprogesterone acetate (MPA) is suspected to be linked to diabetes due to the hormonal effects of progesterone impacting glucoregulatory function.^{1,6,10,22,23,31,34,55} MPA exposure is of interest, because it is used regularly in our mangabey colony as both a contraceptive and as therapy for endometriosis. In addition, SIV status is being evaluated as a risk factor, because a portion of our colony is SIV positive. Although HIV is not thought to be associated with diabetes in people, SIV pathogenesis in mangabeys differs; therefore it was of interest to explore the possible association of SIV and diabetes in mangabeys.^7,30,36,42 Pancreatic insular amyloidosis has been documented to be associated with type 2 diabetes in several species. Amyloidosis is a group of disorders that are caused by extracellular deposition of misfolded proteins that can result in impaired function of any organ.^{15,20,23,28,32,43,45,48,49} Because a high incidence of pancreatic insular amyloid was noted at necropsy, we sought to document the relationship with clinical diabetes in mangabeys.Spontaneous type 2 diabetes mellitus has been well documented in several species of NHP. Because the literature contains little information regarding the clinicopathologic features (the ‘profile’), risk factors, and prevalence of spontaneous diabetes mellitus in sooty mangabeys, the primary aims of the current study were 1) to determine whether elevated levels of fasting blood glucose, fructosamine, HbA1c, triglycerides, and total cholesterol levels are reliable diagnostic markers of type 2 diabetes mellitus in this NHP species; 2) to determine whether age, sex, MPA exposure, and SIV status influence the risk of diabetes; 3) to determine whether body weight influences diabetic status; 4) to evaluate the relationship between pancreatic amyloidosis and diabetes mellitus; and 5) to characterize the prevalence of diabetes mellitus in the mangabey population at our institution. To our knowledge, this report is the first to describe the natural occurrence of type 2 diabetes mellitus within a captive colony of sooty mangabeys. We hypothesized that blood glucose, fructosamine, HbA1c, triglyceride, and total cholesterol would be reliable diagnostic markers and that age, sex, and MPA exposure would influence the risk of diabetes in this species.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.     

Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF''s neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF''s effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.Glial cell line-derived neurotrophic factor (GDNF) is the founding member of the four ligands in the GDNF family, which belong to the transforming growth factor-β superfamily.¹ GDNF was characterized as a potent survival factor for many neurons in culture such as dopaminergic, motor, sympathetic, parasympathetic, sensory and enteric neurons.^{1, 2} In addition, in dopaminergic neuron cultures GDNF stimulates neuronal differentiation, neurite outgrowth, synapse formation and dopamine release.^{1, 2}As degeneration of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNpc) represents a major hallmark of Parkinson disease (PD), the most common neurodegenerative movement disorder, GDNF has raised considerable interest as a therapeutic molecule for the treatment of PD.^{3, 4, 5} PD affects >2% of individuals over the age of 60 years, but no curative treatment is available to date, mainly due to a lack of understanding disease etiology.^{6, 7, 8} Preclinical studies in the established 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) rodent and primate models of PD demonstrated a substantial neuroprotection and regeneration effect by striatal provided GDNF or its close relative neurturin.^{3, 4, 9} However, clinical phase II trials on PD patients using GDNF or neurturin did so far not convincingly recapitulate their beneficial effects on the dopaminergic system in humans most likely due to technical problems and the selection of advanced PD patients.^{10, 11, 12, 13}GDNF signaling is highly complex as this neurotrophic factor can bind to a variety of receptors, thus being able to induce pleiotropic effects. GDNF efficiently binds to the GPI-linked GDNF family receptor α1 (GFRα1).^{1, 2} It has been shown that the GDNF/GFRα1 complex can activate not only the canonical GDNF receptor Ret, a receptor tyrosine kinase which signals through the sarcoma protein (Src)/rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt, NF-κB (nuclear factor ''kappa-light-chain-enhancer'' of activated B cells), JNK (c-Jun N-terminal kinases) and PLCγ (phospholipase γ) pathway, but also with other signaling inducing receptors.^{1, 2, 4, 5, 13} So far, at least four alternative GDNF receptors have been described which are all expressed in midbrain dopaminergic neurons, NCAM,^{14, 15} the integrins αV and βI,^{14, 16} syndecan 3¹⁷ and N-cadherin.¹⁸ Interestingly, Ret is not essential during pre- and postnatal development of the mouse dopaminergic system,^{19, 20, 21, 22, 23} but specifically required for the maintenance of SNpc dopaminergic neurons and their striatal innervation in aged mice.^{23, 24, 25} In contrast, GDNF seems most likely under physiological conditions to be dispensable during development and maintenance of midbrain dopaminergic neurons in mice, although conflicting results exist.^{26, 27, 28} Thus, Ret might be activated by a GDNF-independent mechanism to stimulate SNpc dopaminergic neuron survival. In addition, the in vivo function of the alternative GDNF receptors in the dopaminergic system under physiological and pathophysiological conditions, like PD, and their dependence on GDNF has not yet been addressed in detail. This raised the important question which GDNF receptor might be required to mediate GDNF''s reported neuroprotective and regenerative effect in the dopaminergic system in PD animal models and potentially in PD patients.^{5, 29}Previously, we showed in dopaminergic neuron-specific Ret knockout mice that Ret receptor loss does not result in a higher vulnerability of midbrain dopaminergic neurons against MPTP but to less resprouting of left over dopaminergic neuron axons in the striatum after MPTP intoxication.³⁰ In adult mice endogenous GDNF levels are rather low.^{26, 31} Therefore, we could not rule out in that study the possibility, that higher levels of GDNF—as also used in the clinical GDNF trials in PD patients—might have neuroprotective and regenerating effects even in the absence of the Ret receptor. Here we addressed now this question by viral overexpression of GDNF in MPTP-treated mice lacking expression of Ret again specifically in dopaminergic neurons.^{23, 30} We found that in the absence of Ret in dopaminergic neurons even a substantial overexpression of GDNF in the striatum does not have a neuroprotective and regenerative effect. Thus, despite the expression of alternative GDNF receptors on midbrain dopaminergic neurons, the presence of the canonical GDNF receptor Ret seems to be mandatory for mediating GDNF''s beneficial survival and axonal resprouting effect in these neurons.     

Omentin protects against LPS-induced ARDS through suppressing pulmonary inflammation and promoting endothelial barrier via an Akt/eNOS-dependent mechanism

Acute respiratory distress syndrome (ARDS) is characterized by increased pulmonary inflammation and endothelial barrier permeability. Omentin has been shown to benefit obesity-related systemic vascular diseases; however, its effects on ARDS are unknown. In the present study, the level of circulating omentin in patients with ARDS was assessed to appraise its clinical significance in ARDS. Mice were subjected to systemic administration of adenoviral vector expressing omentin (Ad-omentin) and one-shot treatment of recombinant human omentin (rh-omentin) to examine omentin''s effects on lipopolysaccharide (LPS)-induced ARDS. Pulmonary endothelial cells (ECs) were treated with rh-omentin to further investigate its underlying mechanism. We found that a decreased level of circulating omentin negatively correlated with white blood cells and procalcitonin in patients with ARDS. Ad-omentin protected against LPS-induced ARDS by alleviating the pulmonary inflammatory response and endothelial barrier injury in mice, accompanied by Akt/eNOS pathway activation. Treatment of pulmonary ECs with rh-omentin attenuated inflammatory response and restored adherens junctions (AJs), and cytoskeleton organization promoted endothelial barrier after LPS insult. Moreover, the omentin-mediated enhancement of EC survival and differentiation was blocked by the Akt/eNOS pathway inactivation. Therapeutic rh-omentin treatment also effectively protected against LPS-induced ARDS via the Akt/eNOS pathway. Collectively, these data indicated that omentin protects against LPS-induced ARDS by suppressing inflammation and promoting the pulmonary endothelial barrier, at least partially, through an Akt/eNOS-dependent mechanism. Therapeutic strategies aiming to restore omentin levels may be valuable for the prevention or treatment of ARDS.Acute respiratory distress syndrome (ARDS) is a devastating condition with a 30–60% mortality rate.^{1, 2} Although the pathogenesis of ARDS is complex, the inflammatory response and endothelial barrier disruption play important roles in the development of ARDS.^{3, 4, 5} Therefore, in addition to conventional anti-inflammatory treatments, therapeutic strategies aim to restore pulmonary endothelial barrier integrity and function through regulating inter-endothelial AJs and the endothelial cytoskeleton to minimize protein leakage and leukocyte infiltration under ARDS conditions.^{6, 7}Obesity, especially visceral obesity, has clearly been shown to impair systemic vasculature and to lead to the initiation and progression of vascular disorders.^{8, 9, 10} Although different from the well-documented impacts of obesity on cardiovascular disease, the relationships between obesity and ARDS have not been well elucidated. Clinical and experimental data focused on pertinent physiological changes in obesity indicate that the obesity may alter ARDS pathogenesis by ‘priming'' the pulmonary endothelial barrier for insult and amplifying the early inflammatory response, thus lowering the threshold to initiate ARDS.^{11, 12} Contrary to conventional dogma, adipose tissue is now appreciated as an important endocrine tissue that secretes various bioactive molecules called adipokines, which contribute to the progression of diverse vascular diseases, including hypertension, cardiovascular disease and atherosclerosis.^{13, 14, 15, 16} Although ARDS is not a classified pulmonary vascular disease, it is a severe inflammatory lung condition with widespread pulmonary endothelial breakdown. Clinical evidence has indicated that the obesity might be an emerging risk factor for ARDS and that circulating adipokines levels are associated with the initiation and progression of ARDS.^{11, 12, 17, 18} Moreover, experimental studies have suggested that some anti-inflammatory adipokines, such as adiponectin and apelin, exert beneficial actions on ARDS.^{19, 20, 21}Omentin is an anti-inflammatory adipokine that is abundant in human visceral fat tissue.^{22, 23} Paradoxically, higher circulating omentin-1 levels are present in lean and healthy individuals compared with the obese and diabetic patients. Moreover, as a novel biomarker of endothelial dysfunction, reduced circulating omentin levels are related to the pathological mechanism of obesity-linked vascular disorders, including type 2 diabetes, atherosclerosis, hypertension and cardiovascular disease.^{24, 25, 26, 27, 28} Furthermore, experimental studies have found that omentin stimulates vasodilation in isolated blood vessels and suppresses cytokine-stimulated inflammation in endothelial cells (ECs).^{29, 30, 31} Thus, these data suggest that omentin may protect against obesity-related vascular complications through its anti-inflammatory and vascular-protective properties; however, little is known regarding its role in lung tissue. It was reported that decreased circulating omentin-1 levels could be regarded as an independent predictive marker for the obstructive sleep apnea syndrome and that omentin protects against pulmonary arterial hypertension through inhibiting vascular structure remodeling and abnormal contractile reactivity.^{32, 33, 34} However, to our knowledge, no study has assessed the impact of omentin on ARDS.Akt-related signaling pathways function as an endogenous negative feedback mechanism in response to the injurious stimulus. Our prior studies have demonstrated that Akt-related signaling contributes to protection against ARDS.^{35, 36} Moreover, omentin has been reported to exert anti-inflammatory, pro-survival and pro-angiogenic functions in various cells via an Akt-dependent mechanism.^{30, 31, 37, 38, 39, 40, 41, 42}Collectively, given that ARDS is ultimately an obesity-related disorder of vascular function and that omentin is a favorable pleiotropic adipokine capable of anti-inflammatory, pro-angiogenic and anti-apoptotic abilities; omentin may exert beneficial effects on ARDS. In the present study, we first aimed to appraise the clinical significance of omentin in ARDS and then specifically evaluated its impact on inflammation and the endothelial barrier. Furthermore, we mechanistically investigated the role of Akt-related signaling pathways in these effects induced by omentin in vivo and in vitro.