Functional imaging and neurochemistry identify in vivo neuroprotection mechanisms counteracting excitotoxicity and neurovascular changes in the hippocampus and visual cortex of obese and type 2 diabetic animal models.

Functional MRI (fMRI) with 1 H-MRS was combined on the hippocampus and visual cortex of animal models of obesity (high-fat diet, HFD) and type 2 diabetes (T2D) to identify the involved mechanisms and temporal evolution of neurometabolic changes in these disorders that could serve as potentially reliable clinical biomarkers. HFD rats presented elevated levels of N-acetylaspartylglutamate (NAAG) (p = 0.0365 vs. standard diet, SD) and glutathione (GSH) (p = 0.0494 vs. SD) in the hippocampus. NAAG and GSH levels in this structure proved to be correlated (r = 0.4652, p = 0.0336). This mechanism was not observed in diabetic rats. Combining MRS and fMRI-evaluated blood-oxygen-level-dependent (BOLD) response, elevated taurine (p = 0.0326 vs. HFD) and GABA type A receptor (GABAA R) (p = 0.0211 vs. SD and p = 0.0153 vs. HFD) were observed in the visual cortex of only diabetic rats, counteracting the elevated BOLD response and suggesting an adaptative mechanism against hyperexcitability observed in the primary visual cortex (V1) (p = 0.0226 vs. SD). BOLD amplitude was correlated with the glutamate levels (r = 0.4491; p = 0.0316). Therefore, here we found evidence for several biological dichotomies regarding excitotoxicity and neuroprotection in different brain regions, identifying putative markers of their different susceptibility and response to the metabolic and vascular insults of obesity and diabetes.

Biodistribution and intestinal inflammatory response following voluntary oral intake of silver nanoparticles by C57BL/6J mice.

Silver nanoparticles (AgNP) are among the most widely commercialized nanomaterials globally, with applications in medicine and the food industry. Consequently, the increased use of AgNP in the food industry has led to an unavoidable rise  in human exposure to these nanoparticles. Their widespread use raises concerns about potential hazards to human health, specifically their intestinal pro-inflammatory effects. Thus, the main objective of this study was to evaluate the biological effects of two subacute doses of 5 nm polyvinylpyrrolidone (PVP)-AgNP in C57BL/6J mice. One mg/kg body weight or 10 mg/kg bw was provided once a day for 14 days, using a new technology (HaPILLness) that allows voluntary, stress-free, and accurate oral dosing. It was observed that after oral ingestion, while AgNP is biodistributed throughout the entire organism, most of the ingested dose is excreted in the feces. The passage and accumulation of AgNP throughout the intestine instigated a prominent inflammatory response, marked by significant histological, vascular, and cellular transformations. This response was driven by the activation of the nuclear factor-кB (NF-кB) inflammatory pathway, ultimately leading to the generation of multiple cytokines and chemokines.

New Therapeutic Strategies for Obesity and Its Metabolic Sequelae: Brazilian Cerrado as a Unique Biome.

Brazil has several important biomes holding impressive fauna and flora biodiversity. Cerrado being one of the richest ones and a significant area in the search for new plant-based products, such as foods, cosmetics, and medicines. The therapeutic potential of Cerrado plants has been described by several studies associating ethnopharmacological knowledge with phytochemical compounds and therapeutic effects. Based on this wide range of options, the Brazilian population has been using these medicinal plants (MP) for centuries for the treatment of various health conditions. Among these, we highlight metabolic diseases, namely obesity and its metabolic alterations from metabolic syndrome to later stages such as type 2 diabetes (T2D). Several studies have shown that adipose tissue (AT) dysfunction leads to proinflammatory cytokine secretion and impaired free fatty acid (FFA) oxidation and oxidative status, creating the basis for insulin resistance and glucose dysmetabolism. In this scenario, the great Brazilian biodiversity and a wide variety of phytochemical compounds make it an important candidate for the identification of pharmacological strategies for the treatment of these conditions. This review aimed to analyze and summarize the current literature on plants from the Brazilian Cerrado that have therapeutic activity against obesity and its metabolic conditions, reducing inflammation and oxidative stress.

Circulating Dopamine Is Regulated by Dietary Glucose and Controls Glucagon-like 1 Peptide Action in White Adipose Tissue.

Dopamine directly acts in the liver and white adipose tissue (WAT) to regulate insulin signaling, glucose uptake, and catabolic activity. Given that dopamine is secreted by the gut and regulates insulin secretion in the pancreas, we aimed to determine its regulation by nutritional cues and its role in regulating glucagon-like peptide 1 (GLP-1) action in WAT. Solutions with different nutrients were administered to Wistar rats and postprandial dopamine levels showed elevations following a mixed meal and glucose intake. In high-fat diet-fed diabetic Goto-Kakizaki rats, sleeve gastrectomy upregulated dopaminergic machinery, showing the role of the gut in dopamine signaling in WAT. Bromocriptine treatment in the same model increased GLP-1R in WAT, showing the role of dopamine in regulating GLP-1R. By contrast, treatment with the GLP-1 receptor agonist Liraglutide had no impact on dopamine receptors. GLP-1 and dopamine crosstalk was shown in rat WAT explants, since dopamine upregulated GLP-1-induced AMPK activity in mesenteric WAT in the presence of the D2R and D3R inhibitor Domperidone. In human WAT, dopamine receptor 1 (D1DR) and GLP-1R expression were correlated. Our results point out a dietary and gut regulation of plasma dopamine, acting in the WAT to regulate GLP-1 action. Together with the known dopamine action in the pancreas, such results may identify new therapeutic opportunities to improve metabolic control in metabolic disorders.

Improvement of Glycaemia and Endothelial Function by a New Low-Dose Curcuminoid in an Animal Model of Type 2 Diabetes.

Curcumin has been suggested as a promising treatment for metabolic diseases, but the high doses required limit its therapeutic use. In this study, a new curcuminoid is synthesised to increase curcumin anti-inflammatory and antioxidant potential and to achieve hypoglycaemic and protective vascular effects in type 2 diabetic rats in a lower dose. In vitro, the anti-inflammatory effect was determined through the Griess reaction, and the antioxidant activity through ABTS and TBARS assays. In vivo, Goto-Kakizaki rats were treated for 2 weeks with the equimolar dose of curcumin (40 mg/kg/day) or curcuminoid (52.4 mg/kg/day). Fasting glycaemia, insulin tolerance, plasma insulin, insulin signalling, serum FFA, endothelial function and several markers of oxidative stress were evaluated. Both compounds presented a significant anti-inflammatory effect. Moreover, the curcuminoid had a marked hypoglycaemic effect, accompanied by higher GLUT4 levels in adipose tissue. Both compounds increased NO-dependent vasorelaxation, but only the curcuminoid exacerbated the response to ascorbic acid, consistent with a higher decrease in vascular oxidative and nitrosative stress. SOD1 and GLO1 levels were increased in EAT and heart, respectively. Altogether, these data suggest that the curcuminoid developed here has more pronounced effects than curcumin in low doses, improving the oxidative stress, endothelial function and glycaemic profile in type 2 diabetes.

Lactation as a programming window for metabolic syndrome.

The concept of developmental origins of health and disease (DOHaD) was initially supported by the low birth weight and higher risk of developing cardiovascular disease in adult life, caused by nutrition restriction during foetal development. However, other programming windows have been recognized in the last years, namely lactation, infancy, adolescence and even preconception. Although the concept has been developed in order to study the impact of foetal calorie restriction in adult life, it is now recognized that maternal overweight during programming windows is also harmful to the offspring. This article explores and summarizes the current knowledge about the impact of maternal obesity and obesogenic diets during lactation in the metabolic programming towards the development of metabolic syndrome in the adult life. The impact of maternal obesity and obesogenic diets in milk quality is discussed, including the alterations in specific micro and macronutrients, as well as the impact of such alterations in the development of metabolic syndrome-associated features in the newborn, such as insulin resistance and adiposity. Moreover, the impact of milk quality and formula feeding in infants' gut microbiota, immune system maturation and in the nutrient-sensing mechanisms, namely those related to gut hormones and leptin, are also discussed under the current knowledge.

Sex-specific changes in peripheral metabolism in a model of chronic anxiety induced by prenatal stress.

BACKGROUND: Prenatal stress is associated with increased susceptibility to psychiatric and metabolic disorders later in life. Prenatal exposure to stress mediators may have sex-dependent effects on offspring brain and metabolic function, promoting a sex-specific vulnerability to psychopathology and metabolic alterations at adulthood. In this work, the impact of prenatal stress on glucose homeostasis and peripheral metabolism of male and female offspring was investigated in a chronic anxiety animal model. METHODS: Pregnant Wistar rats were injected with saline or glucocorticoid (dexamethasone: 1 mg/kg, subcutaneous) at gestational days 18 and 19. Male and female offspring weight was monitored, and anxious-like behaviour and peripheral insulin-sensitive tissues were analysed at adulthood. RESULTS: At birth, females and males prenatally exposed to stress presented decreased body weight which remained low in females. At adulthood, a morphological disorganization of the Langerhans islets was observed in both sexes prenatally exposed to stress, yet not changes in insulin levels were detected. Also, prenatal stress increased glucose transporter 4 (GLUT-4) levels in female and male adipose tissues and decreased insulin receptor levels in the liver and skeleton muscle but only in females. CONCLUSIONS: Exposure to stress mediators in critical periods of development negatively affects behaviour and metabolism. Prenatal stress programmes offspring peripheral metabolism in a sex-specific manner, emphasizing that the response to stress in critical periods of development may be sex-specific having each sex different vulnerabilities to psychiatric and metabolic disorders. Considering sex-specificities may provide critical clues for the design of preventive strategies and for early therapeutic intervention.

Metabolic Disease Programming: From Mitochondria to Epigenetics, Glucocorticoid Signalling and Beyond.

Embryonic and foetal development are critical periods of development in which several environmental cues determine health and disease in adulthood. Maternal conditions and an unfavourable intrauterine environment impact foetal development and may programme the offspring for increased predisposition to metabolic diseases and other chronic pathologic conditions throughout adult life. Previously, non-communicable chronic diseases were only associated with genetics and lifestyle. Now the origins of non-communicable chronic diseases are associated with early-life adaptations that produce long-term dysfunction. Early-life environment sets the long-term health and disease risk and can span through multiple generations. Recent research in developmental programming aims at identifying the molecular mechanisms responsible for developmental programming outcomes that impact cellular physiology and trigger adulthood disease. The identification of new therapeutic targets can improve offspring's health management and prevent or overcome adverse consequences of foetal programming. This review summarizes recent biomedical discoveries in the Developmental Origins of Health and Disease (DOHaD) hypothesis and highlight possible developmental programming mechanisms, including prenatal structural defects, metabolic (mitochondrial dysfunction, oxidative stress, protein modification), epigenetic and glucocorticoid signalling-related mechanisms suggesting molecular clues for the causes and consequences of programming of increased susceptibility of offspring to metabolic disease after birth. Identifying mechanisms involved in DOHaD can contribute to early interventions in pregnancy or early childhood, to re-set the metabolic homeostasis and break the chain of subsequent events that could lead to the development of disease.

Impairment of the angiogenic process may contribute to lower success rate of root canal treatments in diabetes mellitus.

AIM: To investigate the association between root canal treatment outcome, diabetes mellitus, and alterations of the angiogenic process. METHODOLOGY: A retrospective observational study was conducted in healthy (control group, CG) and diabetic (type II diabetes mellitus group, DG) patients after root canal treatment. The follow-up appointments were performed to clinically and radiographically observe symptoms, the healing of periapical lesions and the quality of root fillings. In the animal model study, diabetic Goto-Kakizaki (GK) rats and control Wistar rats were used. After 21 days of pulp exposure and the development of apical periodontitis (AP), the mandibles were removed for scintigraphic, radiographic, histopathological and molecular analyses. Chi-square tests were performed to examine the variables related to endodontic outcome and differences between animal groups were assessed using the Student's t-test. RESULTS: The group of patients with diabetes had a significantly lower rate of success following root canal treatment than the CG (p < .001). Logistic regression suggested that diabetes is a risk factor for success of root canal treatment. In the animal study, GK rats had significantly higher fasting glycaemia at t0 and t21 (p < .001) and triglycerides levels (p < .05) and area under the curve (AUC) during the insulin tolerance test at t21 (p < .001). AP area was significantly greater in GK rats (p < .05). Histologically, diabetic rats had increased signs of periodontal ligament inflammation 21 days after the induction of apical periodontitis, with fibro-hyaline matrix filling and vessel with undefined walls. Wistar rats had significantly increased vascular endothelial growth factor (VEGF) levels and VEGF/Ang-2 ratio 21 days after AP induction (p < .08; p < .07). GK rats had intrinsically lower levels of VEGF than control rats (p < .05), which did not change after AP. CONCLUSION: Diabetes mellitus should be considered as an important factor in the prognosis of root canal treatment and its outcomes over time. Future strategies to improve angiogenesis and tissue repair should be pursued to achieve better root canal treatment outcomes in diabetic patients.

Adiponectin and sporadic Alzheimer's disease: Clinical and molecular links.

Obesity has been consistently associated with Alzheimer's disease (AD) though the exact mechanisms by which it influences cognition are still elusive and subject of current research. Adiponectin, the most abundant adipokine in circulation, is inversely correlated with adipose tissue dysfunction and seems to be a central player in this association. In fact, different signalling pathways are shared by adiponectin and proteins involved in AD pathophysiology and considerable amount of evidence supports its direct and indirect influence on ß-amyloid and tau aggregates formation. In this paper we present a critical review of cellular, animal and clinical studies which have contributed to a more thorough understanding of the extent to which adiponectin influences the risk of developing AD as well as its progression. Finally, the effect of acetylcholinesterase inhibitors on circulating adiponectin levels, possible therapeutic applications and future research strategies are also discussed.

GLP-1 improves adipose tissue glyoxalase activity and capillarization improving insulin sensitivity in type 2 diabetes.

Methylglyoxal was shown to impair adipose tissue capillarization and insulin sensitivity in obese models. We hypothesized that glyoxalase-1 (GLO-1) activity could be diminished in the adipose tissue of type 2 diabetic obese patients. Moreover, we assessed whether such activity could be increased by GLP-1-based therapies in order to improve adipose tissue capillarization and insulin sensitivity. GLO-1 activity was assessed in visceral adipose tissue of a cohort of obese patients. The role of GLP-1 in modulating GLO-1 was assessed in type 2 diabetic GK rats submitted to sleeve gastrectomy or Liraglutide treatment, in the adipose tissue angiogenesis assay and in the HUVEC cell line. Glyoxalase-1 activity was decreased in visceral adipose tissue of pre-diabetic and diabetic obese patients, together with other markers of adipose tissue dysfunction and correlated with increased HbA1c levels. Decreased adipose tissue GLO-1 levels in GK rats were increased by sleeve gastrectomy and Liraglutide, being associated with overexpression of angiogenic and vasoactive factors, as well as insulin receptor phosphorylation (Tyr1161). Moreover, GLP-1 increased adipose tissue capillarization and HUVEC proliferation in a glyoxalase-dependent manner. Lower adipose tissue GLO-1 activity was observed in dysmetabolic patients, being a target for GLP-1 in improving adipose tissue capillarization and insulin sensitivity.

Using Resistin, glucose, age and BMI to predict the presence of breast cancer.

BACKGROUND: The goal of this exploratory study was to develop and assess a prediction model which can potentially be used as a biomarker of breast cancer, based on anthropometric data and parameters which can be gathered in routine blood analysis. METHODS: For each of the 166 participants several clinical features were observed or measured, including age, BMI, Glucose, Insulin, HOMA, Leptin, Adiponectin, Resistin and MCP-1. Machine learning algorithms (logistic regression, random forests, support vector machines) were implemented taking in as predictors different numbers of variables. The resulting models were assessed with a Monte Carlo Cross-Validation approach to determine 95% confidence intervals for the sensitivity, specificity and AUC of the models. RESULTS: Support vector machines models using Glucose, Resistin, Age and BMI as predictors allowed predicting the presence of breast cancer in women with sensitivity ranging between 82 and 88% and specificity ranging between 85 and 90%. The 95% confidence interval for the AUC was [0.87, 0.91]. CONCLUSIONS: These findings provide promising evidence that models combining age, BMI and metabolic parameters may be a powerful tool for a cheap and effective biomarker of breast cancer.

High-fat diet induces a neurometabolic state characterized by changes in glutamate and N-acetylaspartate pools associated with early glucose intolerance: An in vivo multimodal MRI study.

BACKGROUND: Type-2 diabetes mellitus (T2DM) is a metabolic disorder with a broad range of complications in the brain that depend on the conditions that precede its onset, such as obesity and metabolic syndromes. It has been suggested that neurotransmitter and metabolic perturbations may emerge even before the early stages of T2DM and that high-caloric intake could adversely influence the brain in such states. Notwithstanding, evidence for neurochemical and structural alterations in these conditions are still sparse and controversial. PURPOSE: To evaluate the influence of high-fat diet in the neurochemical profile and structural integrity of the rodent brain. STUDY TYPE: Prospective. SUBJECTS: Wistar rats (n = 12/group). FIELD STRENGTH/SEQUENCE: A PRESS, ISIS, RARE, and EPI sequences were performed at 9.4T. ASSESSMENT: Neurochemical and structural parameters were assessed by magnetic resonance spectroscopy, voxel-based morphometry, volumetry, and diffusion tensor imaging. STATISTICAL TESTS: Measurements were compared through Student and Mann-Whitney tests. Pearson correlation was used to assess relationships between parameters. RESULTS: Animals submitted to high-caloric intake gained weight (P = 0.003) and developed glucose intolerance (P < 0.001) but not hyperglycemia. In the hippocampus, the diet induced perturbations in glutamatergic metabolites reflected by increased levels of glutamine (P = 0.016) and glutamatergic pool (Glx) (P = 0.036), which were negatively correlated with glucose intolerance (glutamine, r = -0.804, P = 0.029), suggesting a link with neurometabolic dysregulation. At caudate-putamen, high-fat diet led to a surprising increase in the pool of N-acetylaspartate (P = 0.028). A relation with metabolic changes was again suggested by the negative correlation between glucose intolerance and levels of glutamatergic metabolites in this region (glutamate, r = -0.845, P = 0.014; Glx, r = -0.834, P = 0.020). Neither changes in phosphate compounds nor major structural alterations were observed for both regions. DATA CONCLUSION: We found evidence that high-fat diet-induced obesity leads to distinct early and region-specific metabolic/neurochemical imbalances in the presence of early glucose intolerance even when structural alterations or T2DM are absent. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 3 J. Magn. Reson. Imaging 2018.

Methylglyoxal in Metabolic Disorders: Facts, Myths, and Promises.

Glucose and fructose metabolism originates the highly reactive byproduct methylglyoxal (MG), which is a strong precursor of advanced glycation end products (AGE). The MG has been implicated in classical diabetic complications such as retinopathy, nephropathy, and neuropathy, but has also been recently associated with cardiovascular diseases and central nervous system disorders such as cerebrovascular diseases and dementia. Recent studies even suggested its involvement in insulin resistance and beta-cell dysfunction, contributing to the early development of type 2 diabetes and creating a vicious circle between glycation and hyperglycemia. Despite several drugs and natural compounds have been identified in the last years in order to scavenge MG and inhibit AGE formation, we are still far from having an effective strategy to prevent MG-induced mechanisms. This review summarizes the endogenous and exogenous sources of MG, also addressing the current controversy about the importance of exogenous MG sources. The mechanisms by which MG changes cell behavior and its involvement in type 2 diabetes development and complications and the pathophysiological implication are also summarized. Particular emphasis will be given to pathophysiological relevance of studies using higher MG doses, which may have produced biased results. Finally, we also overview the current knowledge about detoxification strategies, including modulation of endogenous enzymatic systems and exogenous compounds able to inhibit MG effects on biological systems.

Functional abolition of carotid body activity restores insulin action and glucose homeostasis in rats: key roles for visceral adipose tissue and the liver.

AIMS/HYPOTHESIS: We recently described that carotid body (CB) over-activation is involved in the aetiology of insulin resistance and arterial hypertension in animal models of the metabolic syndrome. Additionally, we have demonstrated that CB activity is increased in animal models of insulin resistance, and that carotid sinus nerve (CSN) resection prevents the development of insulin resistance and arterial hypertension induced by high-energy diets. Here, we tested whether the functional abolition of CB by CSN transection would reverse pre-established insulin resistance, dyslipidaemia, obesity, autonomic dysfunction and hypertension in animal models of the metabolic syndrome. The effect of CSN resection on insulin signalling pathways and tissue-specific glucose uptake was evaluated in skeletal muscle, adipose tissue and liver. METHODS: Experiments were performed in male Wistar rats submitted to two high-energy diets: a high-fat diet, representing a model of insulin resistance, hypertension and obesity, and a high-sucrose diet, representing a lean model of insulin resistance and hypertension. Half of each group was submitted to chronic bilateral resection of the CSN. Age-matched control rats were also used. RESULTS: CSN resection normalised systemic sympathetic nervous system activity and reversed weight gain induced by high-energy diets. It also normalised plasma glucose and insulin levels, insulin sensitivity lipid profile, arterial pressure and endothelial function by improving glucose uptake by the liver and perienteric adipose tissue. CONCLUSIONS/INTERPRETATION: We concluded that functional abolition of CB activity restores insulin sensitivity and glucose homeostasis by positively affecting insulin signalling pathways in visceral adipose tissue and liver.

Teduglutide effects on gene regulation of fibrogenesis on an animal model of intestinal anastomosis.

BACKGROUND: Teduglutide is an enterotrophic analog of glucagon-like peptide 2 approved for the rehabilitation of short-bowel syndrome. This study aims to analyze the effects of teduglutide administration on the gene regulation of fibrogenesis during the intestinal anastomotic healing on an animal model. METHODS: Wistar rats (n = 62) were assigned into four groups: "Ileal Resection and Anastomosis" or "Laparotomy," each one subdivided into "Postoperative Teduglutide Administration" or "No Treatment," and sacrificed at the third or at the seventh days, with ileal sample harvesting. Gene expression of matrix components and remodeling factors (matrix metalloproteinases [Mmp] and tissue inhibitors of metalloproteinases [Timp]) and growth factors was studied by real-time polymerase chain reaction. Net collagen deposition was assessed through the Collagen-to-Mmp-to-Timp ratio of fold change of relative gene expression. RESULTS: Gene expression profiles revealed a balance toward net degradation of collagen at the third day of the intestinal anastomotic healing. Teduglutide appeared to be associated with an overall accumulation of collagen at the third day of the anastomotic repair, attributable to the upregulation of Collagen type 1 alpha 1, Collagen type 3 alpha 1, and Collagen type 4 alpha 1, Timp1, and Timp2 and downregulation of Mmp13 and to a net degradation of collagen at the seventh day, derived from repression of Collagen type 3 alpha 1, Collagen type 5 alpha 1 and Timp1 expression. CONCLUSIONS: Teduglutide appeared to be associated with a favorable influence on fibrogenesis at the third day of the intestinal anastomotic repair and to a trend to fibrolysis at the seventh day.

The Force at the Tip--Modelling Tension and Proliferation in Sprouting Angiogenesis.

Sprouting angiogenesis, where new blood vessels grow from pre-existing ones, is a complex process where biochemical and mechanical signals regulate endothelial cell proliferation and movement. Therefore, a mathematical description of sprouting angiogenesis has to take into consideration biological signals as well as relevant physical processes, in particular the mechanical interplay between adjacent endothelial cells and the extracellular microenvironment. In this work, we introduce the first phase-field continuous model of sprouting angiogenesis capable of predicting sprout morphology as a function of the elastic properties of the tissues and the traction forces exerted by the cells. The model is very compact, only consisting of three coupled partial differential equations, and has the clear advantage of a reduced number of parameters. This model allows us to describe sprout growth as a function of the cell-cell adhesion forces and the traction force exerted by the sprout tip cell. In the absence of proliferation, we observe that the sprout either achieves a maximum length or, when the traction and adhesion are very large, it breaks. Endothelial cell proliferation alters significantly sprout morphology, and we explore how different types of endothelial cell proliferation regulation are able to determine the shape of the growing sprout. The largest region in parameter space with well formed long and straight sprouts is obtained always when the proliferation is triggered by endothelial cell strain and its rate grows with angiogenic factor concentration. We conclude that in this scenario the tip cell has the role of creating a tension in the cells that follow its lead. On those first stalk cells, this tension produces strain and/or empty spaces, inevitably triggering cell proliferation. The new cells occupy the space behind the tip, the tension decreases, and the process restarts. Our results highlight the ability of mathematical models to suggest relevant hypotheses with respect to the role of forces in sprouting, hence underlining the necessary collaboration between modelling and molecular biology techniques to improve the current state-of-the-art.

Ischaemia-induced autophagy leads to degradation of gap junction protein connexin43 in cardiomyocytes.

GJIC (gap junction intercellular communication) between cardiomyocytes is essential for synchronous heart contraction and relies on Cx (connexin)-containing channels. Increased breakdown of Cx43 has been often associated with various cardiac diseases. However, the mechanisms whereby Cx43 is degraded in ischaemic heart remain unknown. The results obtained in the present study, using both HL-1 cells and organotypic heart cultures, show that simulated ischaemia induces degradation of Cx43 that can be prevented by chemical or genetic inhibitors of autophagy. Additionally, ischaemia-induced degradation of Cx43 results in GJIC impairment in HL-1 cells, which can be restored by autophagy inhibition. In cardiomyocytes, ubiquitin signals Cx43 for autophagic degradation, through the recruitment of the ubiquitin-binding proteins Eps15 (epidermal growth factor receptor substrate 15) and p62, that assist in Cx43 internalization and targeting to autophagic vesicles, via LC3 (light chain 3). Moreover, we establish that degradation of Cx43 in ischaemia or I/R (ischaemia/reperfusion) relies upon different molecular players. Indeed, degradation of Cx43 during early periods of ischaemia depends on AMPK (AMP-activated protein kinase), whereas in late periods of ischaemia and I/R Beclin 1 is required. In the Langendorff-perfused heart, Cx43 is dephosphorylated in ischaemia and degraded during I/R, where Cx43 degradation correlates with autophagy activation. In summary, the results of the present study provide new evidence regarding the molecular mechanisms whereby Cx43 is degraded in ischaemia, which may contribute to the development of new strategies that aim to preserve GJIC and cardiac function in ischaemic heart.

Effects of methylglyoxal and pyridoxamine in rat brain mitochondria bioenergetics and oxidative status.

Advanced glycation end products (AGEs) and methylglyoxal (MG), an important intermediate in AGEs synthesis, are thought to contribute to protein aging and to the pathogenesis of age-and diabetes-associated complications. This study was intended to investigate brain mitochondria bioenergetics and oxidative status of rats previously exposed to chronic treatment with MG and/or with pyridoxamine (PM), a glycation inhibitor. Brain mitochondrial fractions were obtained and several parameters were analyzed: respiratory chain [states 3 and 4 of respiration, respiratory control ratio (RCR), and ADP/O index] and phosphorylation system [transmembrane potential (ΔΨm), ADP-induced depolarization, repolarization lag phase, and ATP levels]; hydrogen peroxide (H2O2) production levels, mitochondrial aconitase activity, and malondialdehyde levels as well as non-enzymatic antioxidant defenses (vitamin E and glutathione levels) and enzymatic antioxidant defenses (glutathione disulfide reductase (GR), glutathione peroxidase (GPx), and manganese superoxide dismutase (MnSOD) activities). MG treatment induced a statistical significant decrease in RCR, aconitase and GR activities, and an increase in H2O2 production levels. The administration of PM did not counteract MG-induced effects and caused a significant decrease in ΔΨm. In mitochondria from control animals, PM caused an adaptive mechanism characterized by a decrease in aconitase and GR activities as well as an increase in both α-tocopherol levels and GPx and MnSOD activities. Altogether our results show that high levels of MG promote brain mitochondrial impairment and PM is not able to reverse MG-induced effects.

Atorvastatin-mediated protection of the retina in a model of diabetes with hyperlipidemia.

Insulin resistance, a key feature of obesity, metabolic syndrome, and type 2 diabetes mellitus (T2DM), results in a variety of metabolic and vascular abnormalities. Metabolic disturbances associated with diabetes could contribute to disrupting the structural and (or) functional integrity of the retina. The effects of atorvastatin on retinal cells in hyperlipidemic T2DM rats have not yet been investigated. We used Goto-Kakizaki (GK) rats fed with an atherogenic diet (AD) for 4 months to investigate whether atorvastatin (administered for 1 month) would slow-down or reverse the progression of lesions in the diabetic retina. Fluorogenic substrates were used to measure the proteasome activities in retinal cells. The production of reactive oxygen species was determined by immunofluorescence in frozen retina sections, using dihydroethydium. Nitrotyrosine levels were assessed using immunohistochemistry. Protein levels of ubiquitin conjugates, free ubiquitin, and ubiquitin activating enzyme E1 were determined with Western blotting. Atorvastatin significantly reduced the levels of oxidative stress that were induced by the AD and restored the proteasome activities in the diabetic GK rats. Atorvastatin therapy significantly improved local oxidative stress levels in GK rats fed with AD. Atorvastatin can, at least in part, restore the ubiquitin proteasome system, and may represent a pharmacological approach to prevent some of the complications associated with diabetic retinopathy.