Distinct metabolic responses to heme in inflammatory human and mouse macrophages - Role of nitric oxide.

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

An orally active carbon monoxide-releasing molecule enhances beneficial gut microbial species to combat obesity in mice.

Carbon monoxide (CO), a gaseous signaling molecule, has shown promise in preventing body weight gain and metabolic dysfunction induced by high fat diet (HFD), but the mechanisms underlying these effects are largely unknown. An essential component in response to HFD is the gut microbiome, which is significantly altered during obesity and represents a target for developing new therapeutic interventions to fight metabolic diseases. Here, we show that CO delivered to the gut by oral administration with a CO-releasing molecule (CORM-401) accumulates in faeces and enriches a variety of microbial species that were perturbed by a HFD regimen. Notably, Akkermansia muciniphila, which exerts salutary metabolic effects in mice and humans, was strongly depleted by HFD but was the most abundant gut species detected after CORM-401 treatment. Analysis of bacterial transcripts revealed a restoration of microbial functional activity, with partial or full recovery of the Krebs cycle, ß-oxidation, respiratory chain and glycolysis. Mice treated with CORM-401 exhibited normalization of several plasma and fecal metabolites that were disrupted by HFD and are dependent on Akkermansia muciniphila's metabolic activity, including indoles and tryptophan derivatives. Finally, CORM-401 treatment led to an improvement in gut morphology as well as reduction of inflammatory markers in colon and cecum and restoration of metabolic profiles in these tissues. Our findings provide therapeutic insights on the efficacy of CO as a potential prebiotic to combat obesity, identifying the gut microbiota as a crucial target for CO-mediated pharmacological activities against metabolic disorders.

Biomarkers of NRF2 signalling: Current status and future challenges.

The cytoprotective transcription factor NRF2 regulates the expression of several hundred genes in mammalian cells and is a promising therapeutic target in a number of diseases associated with oxidative stress and inflammation. Hence, an ability to monitor basal and inducible NRF2 signalling is vital for mechanistic understanding in translational studies. Due to some caveats related to the direct measurement of NRF2 levels, the modulation of NRF2 activity is typically determined by measuring changes in the expression of one or more of its target genes and/or the associated protein products. However, there is a lack of consensus regarding the most relevant set of these genes/proteins that best represents NRF2 activity across cell types and species. We present the findings of a comprehensive literature search that according to stringent criteria identifies GCLC, GCLM, HMOX1, NQO1, SRXN1 and TXNRD1 as a robust panel of markers that are directly regulated by NRF2 in multiple cell and tissue types. We assess the relevance of these markers in clinically accessible biofluids and highlight future challenges in the development and use of NRF2 biomarkers in humans.

An Oral Carbon Monoxide-Releasing Molecule Protects against Acute Hyper-hemolysis in Sickle Cell Disease.

Acute hyper-hemolysis is a severe life-threatening complication in patients with sickle cell disease (SCD) that may occur during delayed hemolytic transfusion reaction (DHTR), or vaso-occlusive crises associated with multi-organ failure. Here, we developed in vitro and in vivo animal models to mimic endothelial damage during the early phase of hyper-hemolysis in SCD. We then used the carbon monoxide (CO)-releasing molecule CORM-401 and examined its effects against endothelial activation, damage, and inflammation inflicted by hemolysates containing red blood cell membrane-derived particles. The in vitro results revealed that CORM-401: 1) prevented the up-regulation of relevant pro-inflammatory, and pro-adhesion controlled by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and 2) abolished the expression of the nuclear factor erythroid-2-related factor 2 (Nrf2) that regulates the inducible antioxidant cell machinery. We also show in SCD mice that CORM-401 protects against hemolysate-induced acute damage of target organs such as the lung, liver, and kidney through modulation of NF-kB pro-inflammatory and Nrf2 antioxidant pathways. Our data demonstrate the efficacy of CORM-401 as a novel therapeutic agent to counteract hemolysate-induced organ damage during hyper-hemolysis in SCD. This approach might be considered as possible preventive treatment in high-risk situations such as SCD patients with history of DHTR.

Glucose oxidation drives trunk neural crest cell development and fate.

Bioenergetic metabolism is a key regulator of cellular function and signaling, but how it can instruct the behavior of cells and their fate during embryonic development remains largely unknown. Here, we investigated the role of glucose metabolism in the development of avian trunk neural crest cells (NCCs), a migratory stem cell population of the vertebrate embryo. We uncovered that trunk NCCs display glucose oxidation as a prominent metabolic phenotype, in contrast to what is seen for cranial NCCs, which instead rely on aerobic glycolysis. In addition, only one pathway downstream of glucose uptake is not sufficient for trunk NCC development. Indeed, glycolysis, mitochondrial respiration and the pentose phosphate pathway are all mobilized and integrated for the coordinated execution of diverse cellular programs, epithelial-to-mesenchymal transition, adhesion, locomotion, proliferation and differentiation, through regulation of specific gene expression. In the absence of glucose, the OXPHOS pathway fueled by pyruvate failed to promote trunk NCC adaptation to environmental stiffness, stemness maintenance and fate-decision making. These findings highlight the need for trunk NCCs to make the most of the glucose pathway potential to meet the high metabolic demands appropriate for their development.

Development of carbon monoxide-releasing molecules conjugated to polysaccharides (glyco-CORMs) for delivering CO during obesity.

Metal carbonyls have been developed as carbon monoxide-releasing molecules (CO-RMs) to deliver CO for therapeutic purposes. The manganese-based CORM-401 has been recently reported to exert beneficial effects in obese animals by reducing body weight gain, improving glucose metabolism and reprogramming adipose tissue towards a healthy phenotype. Here, we report on the synthesis and characterization of glyco-CORMs, obtained by grafting manganese carbonyls on dextrans (70 and 40 kDa), based on the fact that polysaccharides facilitate the targeting of drugs to adipose tissue. We found that glyco-CORMs efficiently deliver CO to cells in vitro with higher CO accumulation in adipocytes compared to other cell types. Oral administration of two selected glyco-CORMs (5b and 6b) resulted in CO accumulation in various organs, including adipose tissue. In addition, glyco-CORM 6b administered for eight weeks elicited anti-obesity and positive metabolic effects in mice fed a high fat diet. Our study highlights the feasibility of creating carriers with multiple functionalized CO-RMs.

A synthetic porphyrin as an effective dual antidote against carbon monoxide and cyanide poisoning.

Simultaneous poisoning by carbon monoxide (CO) and hydrogen cyanide is the major cause of mortality in fire gas accidents. Here, we report on the invention of an injectable antidote against CO and cyanide (CN-) mixed poisoning. The solution contains four compounds: iron(III)porphyrin (FeIIITPPS, F), two methyl-ß-cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) and imidazole (Im3CD, I), and a reducing agent (Na2S2O4, S). When these compounds are dissolved in saline, the solution contains two synthetic heme models including a complex of F with P (hemoCD-P) and another one of F with I (hemoCD-I), both in their iron(II) state. hemoCD-P is stable in its iron(II) state and captures CO more strongly than native hemoproteins, while hemoCD-I is readily autoxidized to its iron(III) state to scavenge CN- once injected into blood circulation. The mixed solution (hemoCD-Twins) exhibited remarkable protective effects against acute CO and CN- mixed poisoning in mice (~85% survival vs. 0% controls). In a model using rats, exposure to CO and CN- resulted in a significant decrease in heart rate and blood pressure, which were restored by hemoCD-Twins in association with decreased CO and CN- levels in blood. Pharmacokinetic data revealed a fast urinary excretion of hemoCD-Twins with an elimination half-life of 47 min. Finally, to simulate a fire accident and translate our findings to a real-life scenario, we confirmed that combustion gas from acrylic cloth caused severe toxicity to mice and that injection of hemoCD-Twins significantly improved the survival rate, leading to a rapid recovery from the physical incapacitation.

CORM-401, an orally active carbon monoxide-releasing molecule, increases body temperature by activating non-shivering thermogenesis in rats.

Thermoregulation is critical in health and disease and is tightly controlled to maintain body temperature homeostasis. Carbon monoxide (CO), an endogenous gasotransmitter produced during heme degradation by heme oxygenases, has been suggested to play a role in body core temperature (Tb) regulation. However, a direct involvement of CO in thermoregulation has not been confirmed and its mechanism(s) of action remain largely unknown. In the present study we characterized the effects of systemic delivery of CO by administration of an orally active CO-releasing molecule (CORM-401) on Tb regulation in conscious freely moving rats. Specifically, we evaluated the main thermo effectors in rats treated with CORM-401 by assessing: (i) non-shivering thermogenesis, i.e. the increased metabolism of brown fat measured through oxygen consumption and (ii) the rate of heat loss from the tail through calculations of heat loss index. We found that oral administration of CORM-401 (30 mg/kg) resulted in augmented CO delivery into the blood circulation as evidenced a by significant increase in carbon monoxy hemoglobin levels(COHb). In addition, treatment with CORM-401 increased Tb, which was caused by an elevated non-shivering thermogenesis indicated by increased oxygen consumption without significant changes in the tail heat loss. On the other hand, CORM-401 did not affect blood pressure, but significantly decreased heart rate. In summary, the findings of the present study reveal that increased circulating CO levels lead to a rise in Tb, which could have important implications in the emerging role of CO in the modulation of energetic metabolism.

Inhibition of Adipose Tissue Beiging by HIV Integrase Inhibitors, Dolutegravir and Bictegravir, Is Associated with Adipocyte Hypertrophy, Hypoxia, Elevated Fibrosis, and Insulin Resistance in Simian Adipose Tissue and Human Adipocytes.

For people living with HIV, treatment with integrase-strand-transfer-inhibitors (INSTIs) can promote adipose tissue (AT) gain. We previously demonstrated that INSTIs can induce hypertrophy and fibrosis in AT of macaques and humans. By promoting energy expenditure, the emergence of beige adipocytes in white AT (beiging) could play an important role by limiting excess lipid storage and associated adipocyte dysfunction. We hypothesized that INSTIs could alter AT via beiging inhibition. Fibrosis and gene expression were measured in subcutaneous (SCAT) and visceral AT (VAT) from SIV-infected, dolutegravir-treated (SIVART) macaques. Beiging capacity was assessed in human adipose stromal cells (ASCs) undergoing differentiation and being exposed to dolutegravir, bictegravir, or raltegravir. Expression of beige markers, such as positive-regulatory-domain-containing-16 (PRDM16), were lower in AT of SIVART as compared to control macaques, whereas fibrosis-related genes were higher. Dolutegravir and bictegravir inhibited beige differentiation in ASCs, as shown by lower expression of beige markers and lower cell respiration. INSTIs also induced a hypertrophic insulin-resistant state associated with a pro-fibrotic phenotype. Our results indicate that adipocyte hypertrophy induced by INSTIs is involved via hypoxia (revealed by a greater hypoxia-inducible-factor-1-alpha gene expression) in fat fibrosis, beiging inhibition, and thus (via positive feedback), probably, further hypertrophy and associated insulin resistance.

TLR4 Signaling and Heme Oxygenase-1/Carbon Monoxide Pathway Crosstalk Induces Resiliency of Myeloma Plasma Cells to Bortezomib Treatment.

Relapse in multiple myeloma (MM) decreases therapy efficiency through unclear mechanisms of chemoresistance. Since our group previously demonstrated that heme oxygenase-1 (HO-1) and Toll-like receptor 4 (TLR4) are two signaling pathways protecting MM cells from the proteasome inhibitor bortezomib (BTZ), we here evaluated their cross-regulation by a pharmacological approach. We found that cell toxicity and mitochondrial depolarization by BTZ were increased upon inhibition of HO-1 and TLR4 by using tin protoporphyrin IX (SnPP) and TAK-242, respectively. Furthermore, the combination of TAK-242 and BTZ activated mitophagy and decreased the unfolded protein response (UPR) survival pathway in association with a downregulation in HO-1 expression. Notably, BTZ in combination with SnPP induced effects mirroring the treatment with TAK-242/BTZ, resulting in a blockade of TLR4 upregulation. Interestingly, treatment of cells with either hemin, an HO-1 inducer, or supplementation with carbon monoxide (CO), a by-product of HO-1 enzymatic activity, increased TLR4 expression. In conclusion, we showed that treatment of MM cells with BTZ triggers the TLR4/HO-1/CO axis, serving as a stress-responsive signal that leads to increased cell survival while protecting mitochondria against BTZ and ultimately promoting drug resistance.

Genetic BACH1 deficiency alters mitochondrial function and increases NLRP3 inflammasome activation in mouse macrophages.

BTB-and-CNC homologue 1 (BACH1), a heme-regulated transcription factor, mediates innate immune responses via its functional role in macrophages. BACH1 has recently been shown to modulate mitochondrial metabolism in cancer cells. In the current study, we utilized a proteomics approach and demonstrate that genetic deletion of BACH1 in mouse macrophages is associated with decreased levels of various mitochondrial proteins, particularly mitochondrial complex I. Bioenergetic studies revealed alterations of mitochondrial energy metabolism in BACH1-/- macrophages with a shift towards increased glycolysis and decreased oxidative phosphorylation. Moreover, these cells exhibited enhanced mitochondrial membrane potential and generation of mitochondrial reactive oxygen species (mtROS) along with lower levels of mitophagy. Notably, a higher inducibility of NLRP3 inflammasome activation in response to ATP and nigericin following challenge with lipopolysaccharide (LPS) was observed in BACH1-deficient macrophages compared to wild-type cells. Mechanistically, pharmacological inhibition of mtROS markedly attenuated inflammasome activation. In addition, it is shown that inducible nitric oxide synthase and cyclooxygenase-2, both of which are markedly induced by LPS in macrophages, are directly implicated in BACH1-dependent regulation of NLRP3 inflammasome activation. Taken together, the current findings indicate that BACH1 is critical for immunomodulation of macrophages and may serve as a target for therapeutic approaches in inflammatory disorders.

Luminal Administration of a Water-soluble Carbon Monoxide-releasing Molecule (CORM-3) Mitigates Ischemia/Reperfusion Injury in Rats Following Intestinal Transplantation.

BACKGROUND: The protective effects of carbon monoxide (CO) against ischemia/reperfusion (IR) injury during organ transplantation have been extensively investigated. Likewise, CO-releasing molecules (CORMs) are known to exert a variety of pharmacological activities via liberation of controlled amounts of CO in organs. Therefore, we hypothesized that intraluminal administration of water-soluble CORM-3 during cold storage of intestinal grafts would provide protective effects against IR injury. METHODS: Orthotopic syngeneic intestinal transplantation was performed in Lewis rats following 6 h of cold preservation in Ringer solution or University of Wisconsin solution. Saline containing CORM-3 (100 µmol/L) or its inactive counterpart (iCORM-3) was intraluminally introduced in the intestinal graft before cold preservation. RESULTS: Histopathological analysis of untreated and iCORM-3-treated grafts revealed a similar erosion and blunting of the intestinal villi. These changes in the mucosa structure were significantly attenuated by intraluminal administration of CORM-3. Intestinal mucosa damage caused by IR injury led to considerable deterioration of gut barrier function 3 h postreperfusion. CORM-3 significantly inhibited upregulation of proinflammatory mRNA levels, ameliorated intestinal morphological changes, and improved graft blood flow and mucosal barrier function. Additionally, CORM-3-treated grafts increased recipient survival rates. Pharmacological blockade of soluble guanylyl cyclase activity significantly reversed the protective effects conferred by CORM-3, indicating that CO partially mediates its therapeutic actions via soluble guanylyl cyclase activation. CONCLUSIONS: Our study demonstrates that luminally delivered CORM-3 provides beneficial effects in cold-stored rat small intestinal grafts and could be an attractive therapeutic application of CO in the clinical setting of organ preservation and transplantation.

Metformin alleviates stress-induced cellular senescence of aging human adipose stromal cells and the ensuing adipocyte dysfunction.

Aging is associated with central fat redistribution and insulin resistance. To identify age-related adipose features, we evaluated the senescence and adipogenic potential of adipose-derived stromal cells (ASCs) from abdominal subcutaneous fat obtained from healthy normal-weight young (<25 years) or older women (>60 years). Increased cell passages of young-donor ASCs (in vitro aging) resulted in senescence but not oxidative stress. ASC-derived adipocytes presented impaired adipogenesis but no early mitochondrial dysfunction. Conversely, aged-donor ASCs at early passages displayed oxidative stress and mild senescence. ASC-derived adipocytes exhibited oxidative stress, and early mitochondrial dysfunction but adipogenesis was preserved. In vitro aging of aged-donor ASCs resulted in further increased senescence, mitochondrial dysfunction, oxidative stress, and severe adipocyte dysfunction. When in vitro aged young-donor ASCs were treated with metformin, no alteration was alleviated. Conversely, metformin treatment of aged-donor ASCs decreased oxidative stress and mitochondrial dysfunction resulting in decreased senescence. Metformin's prevention of oxidative stress and of the resulting senescence improved the cells' adipogenic capacity and insulin sensitivity. This effect was mediated by the activation of AMP-activated protein kinase as revealed by its specific inhibition and activation. Overall, aging ASC-derived adipocytes presented impaired adipogenesis and insulin sensitivity. Targeting stress-induced senescence of ASCs with metformin may improve age-related adipose tissue dysfunction.

Carbon Monoxide Releasing Molecule A1 Reduces Myocardial Damage After Acute Myocardial Infarction in a Porcine Model.

ABSTRACT: Infarct size is a major determinant of outcomes after acute myocardial infarction (AMI). Carbon monoxide-releasing molecules (CORMs), which deliver nanomolar concentrations of carbon monoxide to tissues, have been shown to reduce infarct size in rodents. We evaluated efficacy and safety of CORM-A1 to reduce infarct size in a clinically relevant porcine model of AMI. We induced AMI in Yorkshire White pigs by inflating a coronary angioplasty balloon to completely occlude the left anterior descending artery for 60 minutes, followed by deflation of the balloon to mimic reperfusion. Fifteen minutes after balloon occlusion, animals were given an infusion of 4.27 mM CORM-A1 (n = 7) or sodium borate control (n = 6) over 60 minutes. Infarct size, cardiac biomarkers, ejection fraction, and hepatic and renal function were compared amongst the groups. Immunohistochemical analyses were performed to compare inflammation, cell proliferation, and apoptosis between the groups. CORM-A1-treated animals had significant reduction in absolute infarct area (158 ± 16 vs. 510 ± 91 mm2, P < 0.001) and infarct area corrected for area at risk (24.8% ± 2.6% vs. 45.2% ± 4.0%, P < 0.0001). Biochemical markers of myocardial injury also tended to be lower and left ventricular function tended to recover better in the CORM-A1 treated group. There was no evidence of hepatic or renal toxicity with the doses used. The cardioprotective effects of CORM-A1 were associated with a significant reduction in cell proliferation and inflammation. CORM-A1 reduces infarct size and improves left ventricular remodeling and function in a porcine model of reperfused MI by a reduction in inflammation. These potential cardioprotective effects of CORMs warrant further translational investigations.

Adipose tissue senescence is mediated by increased ATP content after a short-term high-fat diet exposure.

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high-fat diet (HFD, 1-10 weeks) in 5-month-old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence-associated ß-galactosidase activity and cyclin-dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD-derived preadipocytes, as compared with chow diet-derived preadipocytes. One-month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD-induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.

Sensitive quantification of carbon monoxide in vivo reveals a protective role of circulating hemoglobin in CO intoxication.

Carbon monoxide (CO) is a gaseous molecule known as the silent killer. It is widely believed that an increase in blood carboxyhemoglobin (CO-Hb) is the best biomarker to define CO intoxication, while the fact that CO accumulation in tissues is the most likely direct cause of mortality is less investigated. There is no reliable method other than gas chromatography to accurately determine CO content in tissues. Here we report the properties and usage of hemoCD1, a synthetic supramolecular compound composed of an iron(II)porphyrin and a cyclodextrin dimer, as an accessible reagent for a simple colorimetric assay to quantify CO in biological samples. The assay was validated in various organ tissues collected from rats under normal conditions and after exposure to CO. The kinetic profile of CO in blood and tissues after CO treatment suggested that CO accumulation in tissues is prevented by circulating Hb, revealing a protective role of Hb in CO intoxication. Furthermore, hemoCD1 was used in vivo as a CO removal agent, showing that it acts as an effective adjuvant to O2 ventilation to eliminate residual CO accumulated in organs, including the brain. These findings open new therapeutic perspectives to counteract the toxicity associated with CO poisoning.

LIPE-related lipodystrophic syndrome: clinical features and disease modeling using adipose stem cells.

OBJECTIVE: The term Multiple Symmetric Lipomatosis (MSL) describes a heterogeneous group of rare monogenic disorders and multifactorial conditions, characterized by upper-body adipose masses. Biallelic variants in LIPE encoding hormone-sensitive lipase (HSL), a key lipolytic enzyme, were implicated in three families worldwide. We aimed to further delineate LIPE-related clinical features and pathophysiological determinants. METHODS: A gene panel was used to identify pathogenic variants. The disease features were reviewed at the French lipodystrophy reference center. The immunohistological, ultrastructural, and protein expression characteristics of lipomatous tissue were determined in surgical samples from one patient. The functional impact of variants was investigated by developing a model of adipose stem cells (ASCs) isolated from lipomatous tissue. RESULTS: We identified new biallelic LIPE null variants in three unrelated patients referred for MSL and/or partial lipodystrophy. The hallmarks of the disease, appearing in adulthood, included lower-limb lipoatrophy, upper-body and abdominal pseudo-lipomatous masses, diabetes and/or insulin resistance, hypertriglyceridemia, liver steatosis, high blood pressure, and neuromuscular manifestations. Ophthalmological investigations revealed numerous auto-fluorescent drusen-like retinal deposits in all patients. Lipomatous tissue and patient ASCs showed loss of HSL and decreased expression of adipogenic and mature adipocyte markers. LIPE-mutated ASCs displayed impaired adipocyte differentiation, decreased insulin response, defective lipolysis, and mitochondrial dysfunction. CONSLUSIONS: Biallelic LIPE null variants result in a multisystemic disease requiring multidisciplinary care. Loss of HSL expression impairs adipocyte differentiation, consistent with the lipodystrophy/MSL phenotype and associated metabolic complications. Detailed ophthalmological examination could reveal retinal damage, further pointing to the nervous tissue as an important disease target.

Increased Sirt1 secreted from visceral white adipose tissue is associated with improved glucose tolerance in obese Nrf2-deficient mice.

Obesity is associated with metabolic dysregulation characterized by insulin resistance and glucose intolerance. Nuclear factor E2-related factor (Nrf2) is a critical regulator of the stress response and Nrf2-deficient mice (Nrf2-/-) are protected against high fat diet (HFD)-induced metabolic derangement. We searched for factors that could underline this favorable phenotype and found that Nrf2-/- mice exhibit higher circulating levels of sirtuin 1 (Sirt1), a key player in cellular homeostasis and energy metabolism, compared to wild-type mice. Increased Sirt1 levels in Nrf2-/- mice were found not only in animals under standard diet but also following HFD. Interestingly, we report here that the visceral adipose tissue (eWAT) is the sole source of increased Sirt1 protein in plasma. eWAT and other fat depots displayed enhanced adipocytes lipolysis, increased fatty acid oxidation and glycolysis, suggesting autocrine and endocrine actions of Sirt1 in this model. We further demonstrate that removal of eWAT completely abolishes the increase in circulating Sirt1 and that this procedure suppresses the beneficial effect of Nrf2 deficiency on glucose tolerance, but not insulin sensitivity, following a HFD regime. Thus, in contrast to many other stressful conditions where Nrf2 deficiency exacerbates damage, our study indicates that up-regulation of Sirt1 levels specifically in the visceral adipose tissue of Nrf2-/- mice is a key adaptive mechanism that mitigates glucose intolerance induced by nutritional stress.

Therapeutic effects of CO-releaser/Nrf2 activator hybrids (HYCOs) in the treatment of skin wound, psoriasis and multiple sclerosis.

Carbon monoxide (CO) produced by heme oxygenase-1 (HO-1) or delivered by CO-releasing molecules (CO-RMs) exerts anti-inflammatory action, a feature also exhibited by the nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of the stress response. We have recently developed new hybrid molecules (HYCOs) consisting of CO-RMs conjugated to fumaric esters known to activate Nrf2/HO-1. Here we evaluated the biological activities of manganese (Mn) and ruthenium (Ru)-based HYCOs in human monocytes and keratinocytes in vitro as well as in vivo models of inflammation. The effects of HYCOs were compared to: a) dimethyl fumarate (DMF), a known fumaric ester used in the clinic; b) a CO-RM alone; or c) the combination of the two compounds. Mn-HYCOs donated CO and up-regulated Nrf2/HO-1 in vitro more efficiently than Ru-HYCOs. However, irrespective of the metal, a strong reduction in anti-inflammatory markers in monocytes stimulated by LPS was observed with specific HYCOs. This effect was not observed with DMF, CO-RM alone or the combination of the two, indicating the enhanced potency of HYCOs compared to the separate entities. Selected HYCOs given orally to mice accelerated skin wound closure, reduced psoriasis-mediated inflammation and disease symptoms equalling or surpassing the effect of DMF, and ameliorated motor dysfunction in a mouse model of multiple sclerosis. Thus, HYCOs have potent anti-inflammatory activities that are recapitulated in disease models in which inflammation is a prominent component. Prolonged daily administration of HYCOs (up to 40 days) is well tolerated in animals. Our results clearly confirm that HYCOs possess a dual mode of action highlighting the notion that simultaneous Nrf2 targeting and CO delivery could be a clinically relevant application to combat inflammation.

The CO-releasing molecule CORM-3 protects adult cardiomyocytes against hypoxia-reoxygenation by modulating pH restoration.

Several studies have reported that CORM-3, a water-soluble carbon monoxide releasing molecule, elicits cardioprotection against myocardial infarction but the mechanism remains to be investigated. Numerous reports indicate that inhibition of pH regulators, the Na+/H+ exchanger (NHE) and Na+/HCO3- symporter (NBC), protect cardiomyocytes from hypoxia/reoxygenation injury by delaying the intracellular pH (pHi) recovery at reperfusion. Our goal was to explore whether CORM-3-mediated cytoprotection involves the modulation of pH regulation. When added at reoxygenation, CORM-3 (50 µM) reduced the mortality of cardiomyocytes exposed to 3 h of hypoxia and 2 h of reoxygenation in HCO3--buffered solution. This effect was lost when using inactive iCORM-3, which is depleted of CO and used as control, thus implicating CO as the mediator of this cardioprotection. Interestingly, the cardioprotective effect of CORM-3 was abolished by switching to a bicarbonate-free medium. This effect of CORM-3 was also inhibited by 5-hydroxydecanoate, a mitochondrial ATP-dependent K+ (mKATP) channel inhibitor (500 µM) or PD098059, a MEK1/2 inhibitor (10 µM). In additional experiments and in the absence of hypoxia-reoxygenation, intracellular pH was monitored in cardiomyocytes exposed to cariporide to block NHE activity. CORM-3 inhibited alkalinisation and this effect was blocked by PD098059 and 5-HD. In conclusion, CORM-3 protects the cardiomyocyte against hypoxia-reoxygenation injury by inhibiting a bicarbonate transporter at reoxygenation, probably the Na+/HCO3- symporter. This cardioprotective effect of CORM-3 requires the activation of mKATP channels and the activation of MEK1/2.