each poster 3' presentation + 2' discussion
SFRR-E PP 4.1 [YIA]:
Restructuring of the Redox Proteome in Insulin Resistance
Jonathan Scavuzzo | The University of Sydney | Australia
Recent years have seen the field of redox biology evolve its understanding of free radicals from the toxic by-products of cellular metabolism to hormetic metabolites with a potent second messenger capacity. An emerging area of interest regarding free radical hormesis is that of growth factor stimulus, particularly insulin signalling in relation to both physiology and pathophysiology. This interest is driven by numerous growth factors driving a cellular ‘oxidative shift’ upon signal transduction and suggests a dynamic role for thiol-based free radical signalling in signalling pathways and their dysregulated, diseased states. Insulin Resistance, a pathophysiological state defined as a loss of insulin sensitivity in several tissues, is defined on the molecular level as the perturbation of glucose transporter GLUT4 trafficking to the plasma membrane by the insulin signal cascade. Indeed, we predict a complex network of cysteine-based thiol groups exists that comes under oxidation and reduction upon insulin stimulus, fine-tuning the activity of target proteins and eliciting the canonical insulin response. Our study utilises a protocol that labels all oxidised cysteines in the proteome with Cysteine-reactive Phosphate Tags (CPTs), phospho-tagging thiols of interest. From here, we utilise the EasyPhos phosphoproteomic workflow developed in house by Dr Sean Humphrey to enrich for never-before-seen coverage of the redox proteome. We apply this technical leap forward to newly optimised models of insulin resistance, designed around chronic exposure of adipocytes to subtle concentrations of perturbations to minimise off-target effects, revealing the hypothesised network of protein thiols responsive to acute insulin stimulus and characterising a reorganisation of this network under insulin resistant conditions. In future, our findings will serve as a launching pad for describing the underlying crossplay between canonical insulin signalling and redox signalling, hopefully contributing a better understanding of insulin resistance that will one day progress to therapeutic applications within a clinical setting.
SFRR-E PP 4.3:
Fatty acid amide hydrolase deficiency shows harmful effects on ischemic cardiomyopathy
Dr. Sanela Kalinovic | University of medicine Mainz | Germany
Introduction: Ischemic cardiomyopathy leads to inflammation and left ventricular (LV) dysfunction. Animal studies provided evidence for cardioprotective effects of the endocannabinoid system, including cardiomyocyte adaptation, inflammation and remodeling. Since endocannabinoid receptor CB2-deficiency led to increased apoptosis and infarct size with worsened LV-function, we investigated the impact of elevated level of the endocannabinoid anandamide in fatty acid amide hydrolase (FAAH)-/--mice undergoing repetitive I/R.
Methods: Repetitive daily 15 min. left anterior descending artery occlusion over 1, 3 and 7d in C57/Bl6 (WT)- and FAAH-/--mice (n≥8). Possible PPAR-α mediated effects of anandamide in FAAH-/--mice were eliminated with selective PPAR-α antagonist GW6471 i.v. LV-function was assessed using M-mode echocardiography. Immunohistochemical analysis revealed collagen deposition, macrophage accumulation and remodeling. Hypertrophy was determined by cardiomyocyte area and heart weight/tibia length. Molecular analyses involved Taqman® RT-qPCR and ELISA.
Results: FAAH-/--mice showed cardiomyocyte loss after 7dI/R, accompanied by scar formation with persistent LV-dysfunction 60d after discontinuation of I/R, while WT-mice recovered after 60d. Collagen deposition was reduced to WT-levels when FAAH-/--mice were treated with GW6471. CCL2-expression was significantly higher in FAAH-/--mice, followed by higher macrophage infiltration in infarcted areas, which was reversed by GW6471. Further, abolished HMOX-1 induction in FAAH-/--mice, as well as enhanced hypertrophy and adverse remodeling, were normalized by PPAR-α antagonism. Finally, FAAH-/- -mice showed stronger downregulation of PPAR-α when compared to WT, suggesting a compensatory mechanism as endocannabinoids are also ligands for PPAR-α, and its activation causes lipotoxicity leading to cardiomyocyte apoptosis.
Discussion: Our study gives novel insights into the role of endocannabinoids acting via PPAR-α. We hypothesize that increase in endocannabinoids may have partially detrimental effects on cardiomyocyte survival due to PPAR-α activation.
SFRR-E PP 4.4:
UCP1 - a lever of the redox-metabolic seesaw in the regulation of lipid-buffering function of white adipose tissue
Dr. Aleksandra Jankovic | University of Belgrade, Institute for Biological Research "Siniša Stanković" National Institute of the Republic of Serbia | Serbia
Uncoupling protein 1 (UCP1) is a molecular hallmark of thermogenic adipocytes. It can also reside in the unilocular adipocytes of white adipose tissue (WAT) that do not possess almost any oxidation and thermogenesis capacity. Therefore, the significance of UCP1 in WAT is vague. To clarify the physiological role of UCP1 in white adipocytes we aimed to investigate the relation of UCP1 expression with the components of redox-adaptive homeostasis and metabolic function in WAT. Toward this, we investigated the expression pattern of UCP1 with Nrf2 and its downstream targets, glutathione (GSH), and lipid peroxidation levels during extensive lipolysis induced by long-term cold exposure and during reversal, when lipid deposits in adipocytes recover, upon re-acclimation from cold to room temperature (RT). To this end, stated molecular targets were investigated at different time points during 45 days of cold acclimation and re-acclimation in the rat retroperitoneal WAT (rpWAT) and compared to respective RT and cold-acclimated controls. The results have shown that in response to cold-induced lipid mobilization transient induction of UCP1 precedes Nrf2 expression and upregulation of downstream antioxidant enzymes (such as MnSOD and GST). The reverse sequence of molecular events was observed during the early (1-12. days) and late (12-45. days) periods of re-acclimation from cold to RT. Namely, in the initial days of re-acclimation high lipogenesis and redox threshold (GSH, and expression of CuZnSOD and MnSOD) correspond to lower UCP1 levels. From the moment of restitution of lipid reserves (revealed by rpWAT mass) and on, UCP1, GSH, and most antioxidant enzymes return to their RT control values. The results emphasize that UCP1 and Nrf2 represent levers of redox-metabolic seesaw fine-tuning of redox homeostasis for optimal regulation of lipid mobilization and deposition in white adipocytes.
Research is supported by the Science Fund of the Republic of Serbia, PROMIS, #6066747-WARMED.
SFRR-E PP 4.5 [YIA]:
Mechanistic insights into inorganic nitrite-mediated vasodilation of isolated aortic rings under oxidative/hypertensive conditions and S-nitros(yl)ation of proteins in germ-free mice
Dr. Paul Stamm | University Medical Center Mainz | Germany
The prevalence and clinical importance of arterial hypertension is still growing. Inorganic nitrite (NO2-) represents an attractive dietary antihypertensive agent but its metabolism and mode of action are not completely understood, which we aimed to investigate with the present study.
Isolated aortic rings from rats were treated ex vivo with oxidants or rats were infused in vivo with angiotensin-II. Vascular responses to acetylcholine (ACh) and nitrite were assessed by isometric tension recording. The loss of vasodilatory potency in response to oxidants (but not in vivo angiotensin-II) was much more pronounced for ACh as compared to nitrite. This effect may be caused by the redox regulation of conversion to xanthine oxidase (XO). Conventionally-raised and germ-free mice were treated with nitrite by gavage, which did not improve ACh-mediated vasodilation but increased plasma levels of S-nitros(yl)ated proteins in the conventionally-raised but not in the germ-free mice.
In conclusion, inorganic nitrite represents a dietary drug option to treat arterial hypertension in addition to already established pharmacological treatment. Short-term oxidative stress did not impair the vasodilatory properties of nitrite, which may be beneficial in cardiovascular disease patients. The gastrointestinal microbiome appears to play a key role in nitrite metabolism and bioactivation.
SFRR-E PP 4.6:
Peri/epicellular (pec) protein disulphide isomerase A1 (PDI) regulates platelet-endothelium interaction in cells exposed to high glucose levels
Dr. Renato Simoes Gaspar | School of Medicine - University of Sao Paulo | Brazil
Background: Diabetes leads to endothelial dysfunction and thrombus formation in a context of oxidative stress. Our group has shown that peri/epicellular (pec) protein disulphide isomerase A1 (pecPDI), a thiol isomerase protein, influences vascular cell adhesion and that platelet PDI is positively correlated with glycaemia. Therefore, we investigated the impact of pecPDI in platelet-endothelium interactions upon the exposure to high glucose levels.
Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in a normo- (5.5 mM) or high-glucose (25 mM) medium for 48 hours in the presence or absence of PDI inhibitors while platelets were isolated from healthy donors. Hydrogen peroxide was measured through amplex red and cell adhesion was assessed through immunofluorescence.
Results: Platelets adhered more onto HUVECs exposed to high glucose levels, which also presented thicker actin fibres when compared to those of normo-glucose HUVECs. High-glucose HUVECs produced more hydrogen peroxide and were more adhesive onto a collagen matrix. High-glucose cells also showed an augmented fractal dimension, suggestive of increased spreading. Interestingly, there was an increased co-localization between PDIA1 and collagen receptor integrin beta 1 in high-glucose cells when compared to normal HUVECs. Rac1 and RhoA, which are a key GTPases downstream of integrin activation, were markedly translocated to the cell membrane when HUVECs were exposed to 25 mM glucose. PecPDI inhibitors impaired all of the abovementioned processes.
Conclusion: Platelets adhere more onto HUVECs exposed to high glucose levels, in a process largely mediated by pecPDI. Mechanistically, pecPDI was shown to regulate oxidant production, cell adhesion, cytoskeleton organization and activation of RhoGTPases. We propose that pecPDI is a master regulator of platelet-endothelium interactions in high-glucose conditions, thus fostering the development of improved targets to treat cardiovascular diseases of diabetic individuals.
SFRR-E PP 4.7:
Fibroblast growth factor FGF21, a regulator of oxidative stress cell responses, in early metabolic disturbances of health-to-disease transition in non-communicable diseases (NCD)
Christina Morgenstern | University of Graz | Austria
Recently, fibroblast growth factor 21 (FGF21) has been recognized as a stress-responsive hormone that plays a key role in glucose and lipid metabolism and in the control of energy balance. It is also considered as a key regulator of the oxidative stress cell responses, due to relations of the FGF21 gene with NRF2, UCP3, SOD2, ERK and others. Its expression is induced by oxidative stress, and it also exerts protective effects, e.g. by inhibiting inflammation in response to oxidative stress.
We hypothesized that circulating levels of FGF21 are affected in health-to-disease transition and focused on early stages of metabolic disturbances in NCDs in comparison to full health.
Apparently healthy subjects with mildly impaired renal function (eGFR 30-60 ml/min/1.73 m2) (group A), mildly impaired glucose tolerance (HOMA index >2.5 and HbA1c 38.8-44 mmol/mol) (group B) or early stages of arteriosclerotic lesions (carotid intima-media thickness left and right >75th percentile) (group C) were studied and compared to subjects with clinically and biochemically proven full health. Overlaps between groups were excluded to allow for identifying FGF21 behavior in different types of disturbances. Along with FGF21, a comprehensive panel of metabolic and oxidative stress biomarkers was assessed.
Both groups A and B showed higher FGF21 concentrations compared to subjects with full health and group C; they also showed higher levels of branched-chain amino acid-derived C5-acylcarnitine (suggesting incomplete fatty acid oxidation), leptin, triglycerides and fatty liver index, BMI and waist circumference, and lower HDL-cholesterol, vitamin C and β-cryptoxanthin; human mercaptalbumin (reduced:oxidized form) was impaired in group A (all P < 0.001). Glutathione peroxidase, myeloperoxidase and malondialdehyde did not differ.
These results demonstrate that circulating FGF21 levels are elevated in early stages of metabolic disturbances in insulin resistance and impaired kidney function, but not in early vascular changes in the absence of such disturbances.
SFRR-E PP 4.8:
Spatial and temporal H2O2 production in hepatocytes during non-alcoholic fatty liver disease
Tzu-Keng Shen | Signal Transduction and Metabolism Laboratory, Laboratoire de Gastroentérologie Expérimental et Endotools, Université Libre de Bruxelles, Brussels, Belgium | Belgium
The incidence and prevalence of non-alcoholic fatty liver disease (NAFLD) are rapidly rising worldwide due to the global obesity epidemic. Obesity-induced oxidative stress plays an important role during the progression of NAFLD through different mechanisms, including oxidative modifications of protein thiols, stimulation of transcription pathways, and promoting the recruitment of inflammatory cells. Indeed, it is known that obesogenic environments in NAFLD, characterized by high levels of glucose, fatty acids, insulin, and pro-inflammatory cytokines (i.e., IFN-γ, TNF-α and IL-6), increase the generation of reactive oxygen species like hydrogen peroxide (H2O2). Chronic hepatic oxidative stress in obesity is mainly attributed to palmitate-induced mitochondrial and peroxisomal H2O2 generation, UPR/ER stress, and an increased flux of the DAG-PKC-NOX signaling pathway. However, the contribution of H2O2 originating from different cellular compartments during NAFLD development remains unclear. Likewise, it remains to be elucidated which environmental compounds contribute to H2O2 generation from each of the contributing compartments. To address this, we express the latest ultrasensitive pH-independent H2O2 probe HyPer7 in different cellular compartments of hepatocytes including the cytosol, mitochondrial matrix, peroxisomes, and nucleus. This allows us to monitor H2O2 levels and dynamics live in different cellular compartments with spatial and temporal resolution. By exposing the HyPer7-expressing hepatocytes to obesogenic environments, we attempt to identify the subcellular source(s) of H2O2 responsible for the progression of NAFLD. In the long term, the results of this study can be used to design targeted therapeutic options to curb NAFLD progression.