Fausto Chiazza

The NLRP3 Inflammasome as a novel player of the intercellular crosstalk in metabolic disorders.

The combination of obesity and type 2 diabetes is a serious health problem, which is projected to afflict 300 million people worldwide by 2020. Both clinical and translational laboratory studies have demonstrated that chronic inflammation is associated with obesity and obesity-related conditions such as insulin resistance. However, the precise etiopathogenetic mechanisms linking obesity to diabetes remain to be elucidated, and the pathways that mediate this phenomenon are not fully characterized. One of the most recently identified signaling pathways, whose activation seems to affect many metabolic disorders, is the “inflammasome,” a multiprotein complex composed of NLRP3 (nucleotide-binding domain and leucine-rich repeat protein 3), ASC (apoptosis-associated speck-like protein containing a CARD), and procaspase-1. NLRP3 inflammasome activation leads to the processing and secretion of the proinflammatory cytokines interleukin- (IL-) 1β and IL-18. The goal of this paper is to review new insights on the effects of the NLRP3 inflammasome activation in the complex mechanisms of crosstalk between different organs, for a better understanding of the role of chronic inflammation in metabolic disease pathogenesis. We will provide here a perspective on the current research on NLRP3 inflammasome, which may represent an innovative therapeutic target to reverse the detrimental metabolic consequences of the metabolic inflammation.

Acute treatment with relaxin protects the kidney against ischaemia/reperfusion injury

Although recent preclinical and clinical studies have demonstrated that recombinant human relaxin (rhRLX) may have important therapeutic potential in acute heart failure and chronic kidney diseases, the effects of acute rhRLX administration against renal ischaemia/reperfusion (I/R) injury have never been investigated. Using a rat model of 1‐hr bilateral renal artery occlusion followed by 6‐hr reperfusion, we investigated the effects of rhRLX (5 μg/Kg i.v.) given both at the beginning and after 3 hrs of reperfusion. Acute rhRLX administration attenuated the functional renal injury (increase in serum urea and creatinine), glomerular dysfunction (decrease in creatinine clearance) and tubular dysfunction (increase in urinary excretion of N‐acetyl‐β‐glucosaminidase) evoked by renal I/R. These beneficial effects were accompanied by a significant reduction in local lipid peroxidation, free radical‐induced DNA damage and increase in the expression/activity of the endogenous antioxidant enzymes Mn‐ and CuZn‐superoxide dismutases (SOD). Furthermore, rhRLX administration attenuated the increase in leucocyte activation, as suggested by inhibition of myeloperoxidase activity, intercellular‐adhesion‐molecule‐1 expression, interleukin (IL)‐1β, IL‐18 and tumour necrosis factor‐α production as well as increase in IL‐10 production. Interestingly, the reduced oxidative stress status and neutrophil activation here reported were associated with rhRLX‐induced activation of endothelial nitric oxide synthase and up‐regulation of inducible nitric oxide synthase, possibly secondary to activation of Akt and the extracellular signal‐regulated protein kinase (ERK) 1/2, respectively. Thus, we report herein that rhRLX protects the kidney against I/R injury by a mechanism that involves changes in nitric oxide signalling pathway.

Pharmacological Inhibition of NLRP3 Inflammasome Attenuates Myocardial Ischemia/Reperfusion Injury by Activation of RISK and Mitochondrial Pathways

Although the nucleotide-binding oligomerization domain- (NOD-) like receptor pyrin domain containing 3 (NLRP3) inflammasome has been recently detected in the heart, its role in cardiac ischemia/reperfusion (IR) is still controversial. Here, we investigate whether a pharmacological modulation of NLRP3 inflammasome exerted protective effects in an ex vivo model of IR injury. Isolated hearts from male Wistar rats (5-6 months old) underwent ischemia (30 min) followed by reperfusion (20 or 60 min) with and without pretreatment with the recently synthetized NLRP3 inflammasome inhibitor INF4E (50 μM, 20 min before ischemia). INF4E exerted protection against myocardial IR, shown by a significant reduction in infarct size and lactate dehydrogenase release and improvement in postischemic left ventricular pressure. The formation of the NLRP3 inflammasome complex was induced by myocardial IR and attenuated by INF4E in a time-dependent way. Interestingly, the hearts of the INF4E-pretreated animals displayed a marked improvement of the protective RISK pathway and this effect was associated increase in expression of markers of mitochondrial oxidative phosphorylation. Our results demonstrate for the first time that INF4E protected against the IR-induced myocardial injury and dysfunction, by a mechanism that involves inhibition of the NLRP3 inflammasome, resulting in the activation of the prosurvival RISK pathway and improvement in mitochondrial function.

High Sugar Intake and Development of Skeletal Muscle Insulin Resistance and Inflammation in Mice: A Protective Role for PPAR-δ Agonism

Peroxisome Proliferator Activated Receptor (PPAR)-δ agonists may serve for treating metabolic diseases. However, the effects of PPAR-δ agonism within the skeletal muscle, which plays a key role in whole-body glucose metabolism, remain unclear. This study aimed to investigate the signaling pathways activated in the gastrocnemius muscle by chronic administration of the selective PPAR-δ agonist, GW0742 (1 mg/kg/day for 16 weeks), in male C57Bl6/J mice treated for 30 weeks with high-fructose corn syrup (HFCS), the major sweetener in foods and soft-drinks (15% wt/vol in drinking water). Mice fed with the HFCS diet exhibited hyperlipidemia, hyperinsulinemia, hyperleptinemia, and hypoadiponectinemia. In the gastrocnemius muscle, HFCS impaired insulin and AMP-activated protein kinase signaling pathways and reduced GLUT-4 and GLUT-5 expression and membrane translocation. GW0742 administration induced PPAR-δ upregulation and improvement in glucose and lipid metabolism. Diet-induced activation of nuclear factor-κB and expression of inducible-nitric-oxide-synthase and intercellular-adhesion-molecule-1 were attenuated by drug treatment. These effects were accompanied by reduction in the serum concentration of interleukin-6 and increase in muscular expression of fibroblast growth factor-21. Overall, here we show that PPAR-δ activation protects the skeletal muscle against the metabolic abnormalities caused by chronic HFCS exposure by affecting multiple levels of the insulin and inflammatory cascades.

Urinary metabolic fingerprinting of mice with diet-induced metabolic derangements by parallel dual secondary column-dual detection two-dimensional comprehensive gas chromatography

This study investigates the potential of a parallel dual secondary column-dual detection two-dimensional comprehensive GC platform (GC×2GC-MS/FID) for metabolic profiling and fingerprinting of mouse urine. Samples were obtained from a murine model that mimics a typical unhealthy Western diet featuring both high fat and sugar (HFHS) intake, which induces obesity, dyslipidemia, and insulin resistance. Urines collected at different steps of the study were used to obtain pivotal and comparative data on the presence and relative distributions of early markers of metabolic disease. The data elaboration and interpretation work-flow includes an advanced untargeted fingerprinting approach, with peak-region features to locate relevant features to be quantified by external standard calibration. The reliability of untargeted fingerprinting is confirmed by quantitative results on selected relevant features that showed percentages of variations consistent with those observed by comparing raw data quantitative descriptors (2D peak-region volumes and percent of response). Analytes that were up-regulated with % of variation ranging from 30 to 1000, included pyruvic acid, glycerol, fructose, galactose, glucose, lactic acid, mannitol and valine. Down-regulation was evidenced for malonic acid, succinic acid, alanine, glycine, and creatinine. Advanced fingerprinting also is demonstrated for effectively evaluating individual variations during experiments, thus representing a promising tool for personalized intervention studies. In this context, it is interesting to observe that informative features that were not discriminant for the entire population may be relevant for individuals.

Fructose-derived advanced glycation end-products drive lipogenesis and skeletal muscle reprogramming via SREBP-1c dysregulation in mice.

Advanced Glycation End-Products (AGEs) have been recently related to the onset of metabolic diseases and related complications. Moreover, recent findings indicate that AGEs can endogenously be formed by high dietary sugars, in particular by fructose which is widely used as added sweetener in foods and drinks. The aim of the present study was to investigate the impact of a high-fructose diet and the causal role of fructose-derived AGEs in mice skeletal muscle morphology and metabolism. C57Bl/6J mice were fed a standard diet (SD) or a 60% fructose diet (HFRT) for 12 weeks. Two subgroups of SD and HFRT mice received the anti-glycative compound pyridoxamine (150 mg/kg/day) in the drinking water. At the end of protocol high levels of AGEs were detected in both plasma and gastrocnemius muscle of HFRT mice associated to impaired expression of AGE-detoxifying AGE-receptor 1. In gastrocnemius, AGEs upregulated the lipogenesis by multiple interference on SREBP-1c through downregulation of the SREBP-inhibiting enzyme SIRT-1 and increased glycation of the SREBP-activating protein SCAP. The AGEs-induced SREBP-1c activation affected the expression of myogenic regulatory factors leading to alterations in fiber type composition, associated with reduced mitochondrial efficiency and muscular strength. Interestingly, pyridoxamine inhibited AGEs generation, thus counteracting all the fructose-induced alterations. The unsuspected involvement of diet-derived AGEs in muscle metabolic derangements and proteins reprogramming opens new perspectives in pathogenic mechanisms of metabolic diseases.

Accumulation of Advanced Glycation End-Products and Activation of the SCAP/SREBP Lipogenetic Pathway Occur in Diet-Induced Obese Mouse Skeletal Muscle.

Aim of this study was to investigate whether advanced glycation end-products (AGEs) accumulate in skeletal myofibers of two different animal models of diabesity and whether this accumulation could be associated to myosteatosis. Male C57Bl/6j mice and leptin-deficient ob/ob mice were divided into three groups and underwent 15 weeks of dietary manipulation: standard diet-fed C57 group (C57, n = 10), high-fat high-sugar diet-fed C57 group (HFHS, n = 10), and standard diet-fed ob/ob group (OB/OB, n = 8). HFHS mice and OB/OB mice developed glycometabolic abnormalities in association with decreased mass of the gastrocnemius muscle, fast-to-slow transition of muscle fibers, and lipid accumulation (that occurred preferentially in slow compared to fast fibers). Moreover, we found in muscle fibers of HFHS and OB/OB mice accumulation of AGEs that was preferential for the lipid-accumulating cells, increased expression of the lipogenic pathway SCAP/SREBP, and co-localisation between AGEs and SCAP-(hyper)expressing cells (suggestive for SCAP glycosylation). The increased expression of the SCAP/SREBP lipogenic pathway in muscle fibers is a possible mechanism underlying lipid accumulation and linking myosteatosis to muscle fiber atrophy and fast-to-slow transition that occur in response to diabesity.

Targeting the NLRP3 Inflammasome to Reduce Diet-Induced Metabolic Abnormalities in Mice

Although the molecular links underlying the causative relationship between chronic low-grade inflammation and insulin resistance are not completely understood, compelling evidence suggests a pivotal role of the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome. Here we tested the hypothesis that either a selective pharmacological inhibition or a genetic downregulation of the NLRP3 inflammasome results in reduction of the diet-induced metabolic alterations. Male C57/BL6 wild-type mice and NLRP3−/− littermates were fed control diet or high-fat, high-fructose diet (HD). A subgroup of HD-fed wild-type mice was treated with the NLRP3 inflammasome inhibitor BAY 11-7082 (3 mg/kg intraperitoneally [IP]). HD feeding increased plasma and hepatic lipids and impaired glucose homeostasis and renal function. Renal and hepatic injury was associated with robust increases in profibrogenic markers, while only minimal fibrosis was recorded. None of these metabolic abnormalities were detected in HD-fed NLRP3−/− mice, and they were dramatically reduced in HD-mice treated with the NLRP3 inflammasome inhibitor. BAY 11-7082 also attenuated the diet-induced increase in NLRP3 inflammasome expression, resulting in inhibition of caspase-1 activation and interleukin (IL)-1 β and IL-18 production (in liver and kidney). Interestingly, BAY 11-7082, but not gene silencing, inhibited nuclear factor (NF)-κB nuclear translocation. Overall, these results demonstrate that the selective pharmacological modulation of the NLRP3 inflammasome attenuates the metabolic abnormalities and the related organ injury/dysfunction caused by chronic exposure to HD, with effects similar to those obtained by NLRP3 gene silencing.

A non-erythropoietic peptide derivative of erythropoietin decreases susceptibility to diet-induced insulin resistance in mice.

The haematopoietic activity of erythropoietin (EPO) is mediated by the classic EPO receptor (EpoR) homodimer, whereas tissue‐protective effects are mediated by a heterocomplex between EpoR and the β‐common receptor (βcR). Here, we investigated the effects of a novel, selective ligand of this heterocomplex – pyroglutamate helix B surface peptide (pHBSP) – in mice fed a diet enriched in sugars and saturated fats.

High-fructose intake as risk factor for neurodegeneration: Key role for carboxy methyllysine accumulation in mice hippocampal neurons

Several studies indicate the involvement of advanced glycation end-products (AGEs) in neurodegenerative diseases. Moreover, the rising consumption of fructose in industrialized countries has been related to cognitive impairment, but the impact of fructose-derived AGEs on hippocampus has never been investigated. The present study aimed to evaluate in the hippocampus of C57Bl/6 mice fed a standard (SD) or a 60% fructose (HFRT) diet for 12 weeks the production of the most studied AGEs, carboxy methyllysine (CML), focusing on the role of the glutathione-dependent enzyme glyoxalase (Glo-1), the main AGEs-detoxifying system, in relation to early signs of neuronal impairment. HFRT diet evoked CML accumulation in the cell body of pyramidal neurons, followed by RAGE/NFkB signaling activation. A widespread reactive gliosis and altered mitochondrial respiratory complexes activity have been evidenced in HFRT hippocampi, paralleled by oxidative stress increase due to impaired activity of Nrf2 signaling. In addition, a translocation of Glo-1 from axons toward cell body of pyramidal neurons has been observed in HFRT mice, in relation to CML accumulation. Despite increased expression of dimeric Glo-1, its enzymatic activity was not upregulated in HFRT hippocampi, due to reduced glutathione availability, thus failing to prevent CML accumulation. The prevention of CML production by administration of the specific inhibitor pyridoxamine was able to prevent all the fructose-induced hippocampal alterations. In conclusion, a high-fructose consumption, through CML accumulation and Glo-1 impairment, induces in the hippocampus the same molecular and metabolic alterations observed in early phases of neurodegenerative diseases, and can thus represent a risk factor for their onset.