Differential disruption on glucose and insulin metabolism in two rat models of diet-induced obesity, based on carbohydrates or lipids.

Obesity is a relevant health public issue and is the main factor for glucose metabolism dysregulation and diabetes progression; however, the differential role of a high-fat diet or high sugar diet consumption on glucose metabolism and insulin processing is not well understood and has been scarcely described. Our research aimed to analyze the effects of chronic consumption of both high sucrose and high-fat diets on glucose and insulin metabolism regulation. Wistar rats were fed with high-sugar or high-fat diets for 12 months; after that, fasting glucose and insulin levels were measured along with a glucose tolerance test (GTT). Proteins related to insulin synthesis and secretion were quantified in pancreas homogenates, whereas islets were isolated to analyze ROS generation and size measurement. Our results show that both diets induce metabolic syndrome, linked with central obesity, hyperglycemia, and insulin resistance. We observed alterations in the expression of proteins related with insulin synthesis and secretion, along with diminution of Langerhans islets size. Interestingly, the severity and number of alterations were more evident in the high-sugar diet than in the high-fat diet group. In conclusion, obesity and glucose metabolism dysregulation induced by carbohydrate consumption, led to worst outcomes than high-fat diet.

Chronic exposure to ozone induces cardiac antioxidant response and overexpression of either mitochondrial fision protein DRP1 and hipertrophyc-related proteins.

Pollution is considered a risk factor for cardiovascular disease; however, the mechanisms to explain this relationship are not well understood; ozone is one of the most abundant and studied air contaminants. Our study aimed to evaluate the effect of chronic exposition of rats to controlled low doses of ozone on oxidative stress, apoptosis, mitochondrial dynamics, and cardiac hypertrophy. Male Wistar rats were daily exposed to low ozone doses during 7, 15, 30, and 60 days, 4 h/day. Hearts were dissected, and homogenates were prepared. Oxidative stress was evaluated by TBARS and protein nitrosylation in addition to Superoxide dismutase 1 (SOD1) and Catalase levels; the apoptosis related-proteins caspase 3, caspase 9, Bax, Bcl-2, and the mitochondrial dynamic-associated proteins Fis1, Drp1, OPA1, and Mfn1 were quantified by western blot among the cardiac hypertrophy indicator alpha-actin (cardiac actin). There were no changes in the oxidative stress markers, however SOD1 expression increases. Caspase 3 expression decreased, whereas caspase 9 increased without changes in Bax or Bcl-2. Mitochondrial fission may be favored according to the increased expression of Drp1 but not changes in fusion-related proteins OPA1 and Mfn1. Finally, the molecular marker for cardiac hypertrophy was overexpressed after 30 and 60 days of ozone exposition. The chronic exposition to ozone induces a deleterious effect on cardiac mitochondria. Antioxidant defenses also show changes in relation to exposure time, as well as an apparent pro-hypertrophic effect associated with altered mitochondrial dynamics.

Identification of functional leptin receptors expressed in ventricular mitochondria.

Leptin is a 16 kDa pro-satiety peptide produced primarily not only by white adipocytes but also by numerous other tissues including the heart. Circulating leptin exerts its effect through specific receptors, although its principle actions are dependent on the activation of the long form of the leptin receptor, termed OBRb. As leptin is also produced within the cardiomyocyte, we hypothesized that the peptide can also exert effects by targeting intracellular sites. Accordingly, we determined whether cardiac mitochondria express functional leptin receptors. The presence of mitochondrial OBRb was identified through Western blotting of isolated mitochondria, immunofluorescence as well as immunogold labeling with electron microscopy. Although leptin had no direct effect on mitochondrial integrity, it profoundly enhanced the ability of calcium to induce mitochondrial swelling, an effect partially reversed by an OBR antagonist. 24 h exposure to leptin (50 ng/ml) was without effect on mitochondria in cultured neonatal rat ventricular myocytes in contrast to leptin tagged with a 10 amino acid membrane translocation sequence which significantly induced mitochondrial permeability transition pore opening, whereas both leptins produced a hypertrophic response. Our results therefore show that mitochondria express functional OBR which may be of importance toward understanding the role of intracellularly derived leptin in cardiac physiology and pathology.

Chronic consumption of imbalance diets high in sucrose or fat induces abdominal obesity with different pattern of metabolic disturbances and lost in Langerhans cells population.

AIM: Obesity is a worldwide health issue, associated with development of type 2 Diabetes Mellitus. The aim of this study is to analyze the effect of consumption of two hypercaloric diets on metabolic disturbance and beta cells damage. MAIN METHODS: Male Wistar rats were subjected to twelve months consumption of three diets: a Control balanced diet (CTD, carbohydrates 58 %, proteins 29 %, lipids 13 %) and two hypercaloric diets, high in sucrose (HSD, carbohydrates 68 %, proteins 22 %, lipids 10 %) or high in fat (HFD, carbohydrates 31 %, proteins 14 %, lipids 55 %). Serum levels of glucose, triglycerides and free fatty acids were measured after zoometric parameters determination. Antioxidant enzymes activity and oxidative stress-marker were measured in pancreas tissue among histological analysis of Langerhans islets. KEY FINDINGS: Although diets were hypercaloric, the amount of food consumed by rats decreased, resulting in an equal caloric consumption. The HSD induced hypertriglyceridemia and hyperglycemia with higher levels in free fatty acids (FFA, lipotoxicity); whereas HFD did not increased neither the triglycerides nor FFA, nevertheless the loss of islets' cell was larger. Both diets induced obesity with hyperglycemia and significant reduction in Langerhans islets size. SIGNIFICANCE: Our results demonstrate that consumption of HSD induces more significant metabolic disturbances that HFD, although both generated pancreas damage; as well hypercaloric diet consumption is not indispensable to becoming obese; the chronic consumption of unbalanced diets (rich in carbohydrates or lipids) may lead to abdominal obesity with metabolic and functional disturbances, although the total amount of calories are similar.

Bax induces cytochrome c release by multiple mechanisms in mitochondria from MCF7 cells.

Bax, a pro-apoptotic member of the Bcl-2 family of proteins has the ability to form transmembrane pores large enough to allow cytochrome c (Cyt c) release, as well as to activate the mitochondrial permeability transition pore (mPTP); however, no differential study has been conducted to clarify which one of these mechanisms predominates over the other in the same system. In the present study, we treated isolated mitochondria from MCF7 cells with recombinant protein Bax and tested the efficacy of the mPTP inhibitor cyclosporin A (CsA) and of the Bax channel blocker (Bcb) to inhibit cytochrome c release. We also, induced apoptosis in MCF7 cell cultures with TNF-α plus cycloheximide to determine the effect of such compounds in apoptosis induction via mPTP or Bax oligomerization. Cytochrome c release was totally prevented by CsA and partially by Bcb when apoptosis was induced with recombinant Bax in isolated mitochondria from MCF7 cells. CsA increased the number of living cells in cell culture, as compared with the effect of Bax channel blocker. These results indicate that mPTP activation is the predominant pathway for Bax-induced cytochrome c release from MCF7 mitochondria and for apoptosis induction in the whole cell.

Postconditioning protects against reperfusion injury in hypertensive dilated cardiomyopathy by activating MEK/ERK1/2 signaling.

BACKGROUND: Postconditioning (PostC) cardioprotection has been related to up-regulation of survival kinases; however, the efficacy of PostC and the role of ERK1/2 (extracellular signal-regulated kinase 1/2) remain to be substantiated in hypertension states that may produce "pathologic remodeling." Therefore, in this work we compared PostC effect and assessed the role of ERK1/2 activation in a model of hypertensive dilated cardiomyopathy (DCM), versus normal (Sham) and compensated hypertrophy (CH) models. METHODS AND RESULTS: Rats were subjected to angiotensin II administration until development of cardiovascular diseases. Then, isolated hearts underwent ischemia followed by PostC and reperfusion. PostC maintained the double product in all groups. PostC reduced infarct size from 36.16 ± 3% to 9.8% ± 2.2 in Sham, from 37.5 ± 2.4% to 12 ± 3% in CH, and from 40 ± 2.4% to 11.55 ± 3% in DCM. Inhibition of the mitogen-activated protein kinase kinase (MEK)/ERK1/2 pathway had different effects on PostC-conferred cardioprotection in the evaluated groups. Interestingly, although phosphatidylinositol-3-kinase activation was negligible in PostC DCM hearts, we observed Akt activation. CONCLUSIONS: PostC confers cardioprotection through alternative survival pathways in normal and CH hearts, and cardiac function recovery in DCM relies mainly on MEK/ERK1/2 cascade. Down-regulation of phosphatidylinositide 3-kinase does not affect the cardioprotective response in DCM, because MEK/ERK1/2 cascade may convey direct Akt activation, strengthening downstream signaling.

High sugar but not high fat diet consumption induces hepatic metabolic disruption and up-regulation of mitochondrial fission-associated protein Drp1 in a model of moderate obesity.

Identification of new modifications and the association with diet patterns are essential for the prevention of non-alcoholic fatty liver disease (NAFLD). To address this problem, we feed rats with high caloric diets based on high sucrose (HSD) and high fat (HFD) and analysed metabolic and mitochondrial alterations. Both diets induce moderated obesity and fat accumulation in the liver after 8, 10 and 12 months of diet. The HSD induces both hyperleptinemia and hyperinsulinemia, as well as up-regulation of transcription factors SRBEP1 and PPARγ along slight increase nitrosylation of proteins and increased mitochondrial fission. In contrast, HFD induced hyperleptinemia without changes in neither insulin levels nor oxidative stress, SREBP1, PPARγ, or mitochondrial dynamics. In conclusion, chronic consumption of high sucrose content diets induces more pathological and metabolic alteration in liver in comparison with consumption of high-fat content diets, although both induces obesity and liver steatosis in these animal models.

A CRAC-like motif in BAX sequence: relationship with protein insertion and pore activity in liposomes.

Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, -L/V-(X)(1-5)-Y-(X)(1-5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein.

Reduced capacity of Ca²+ retention in liver as compared to kidney mitochondria. ADP requirement.

Ca²+ loading in mitochondria promotes the opening of a non-selective transmembrane pathway. Permeability transition is also associated with the interaction of cyclophilin D at the internal surface of the non-specific transmembrane pore. This interaction is circumvented by cyclosporin A and ADP. Our results show that, in the absence of ADP, liver mitochondria were unable to retain Ca²+, they underwent a fast and large amplitude swelling, as well as a rapid collapse of the transmembrane potential. In contrast, in the absence of ADP, kidney mitochondria retained Ca²+, swelling did not occur, and the collapse of the membrane potential was delayed. Ca²+ efflux was reversed by the addition of ADP and cyclosporin A. Our findings indicate that the differences between liver and kidney mitochondria are due to the low association of cyclophilin D to the ADP/ATP carrier found in kidney mitochondria as compared to liver mitochondria.

The high-risk HPV E6 proteins modify the activity of the eIF4E protein via the MEK/ERK and AKT/PKB pathways.

Previous studies have proposed that the human papillomavirus (HPV) E6 oncoproteins modify the transcriptional activity of eIF4E through mechanisms dependent on p53 degradation. However, the effect of these oncoproteins on pathways regulating the activity of the eIF4E protein remains poorly understood. Hence, we investigated the mechanisms whereby E6 proteins regulate the activity of the eIF4E protein and its effect on target genes. Overexpression of E6 constructs (HPV-6, HPV-16, HPV-18, and HPV52) showed that E6 oncoproteins increased phosphorylation of the eIF4E protein (Serine-209). This result was mainly mediated by phosphorylation of the 4EBP1 protein via the PI3K/AKT pathway. Additionally, the pharmacological inhibition of eIF4E phosphorylation in cervical cancer cell lines substantially reduced the protein levels of CCND1 and ODC1, indicating that E6 of the high-risk genotypes may modify protein synthesis of the eIF4E target genes by increasing the activity of the AKT and ERK pathways.

Molecular epidemiology of bacterial vaginosis and its association with genital micro-organisms in asymptomatic women.

Introduction. Bacterial vaginosis (BV) is dysbiosis associated with an increased risk of several sexually transmitted infections. It is primarily diagnosed via Gram staining, although molecular analyses have presented higher diagnostic accuracy.Aim. This study aimed to evaluate the molecular epidemiology of BV in asymptomatic women to determine its association with several commensal and pathogenic micro-organisms of the genitalia.Methodology. The prevalence of BV was investigated through semiquantitative assessment of 201 women recruited during their routine gynaecological inspection at an outpatient clinic in Tabasco, Mexico.Results. Women with BV showed an increased prevalence of Chlamydia trachomatis (P=0.021) and Mycoplasma hominis (P=0.001). Of the BV-associated micro-organisms, Gardnerella vaginalis was significantly associated with C. trachomatis (P=0.005) and/or Ureaplasma parvum (P=0.003), whereas Atopobium vaginae and Megasphaera type 1 correlated significantly with Mycoplasma hominis (P=0.001). No significant association was observed between human papillomavirus (HPV) infection and BV, although there was increased prevalence of HPV59, HPV73, HPV52 and HPV58 in women displaying cervical cytological abnormalities.Conclusion. Identification of BV-associated micro-organisms via molecular analysis may help to distinguish recurrent cases from new infections and identify micro-organisms potentially associated with pharmacological resistance.

Relationship between oxidative stress and mitochondrial function in the post-conditioned heart.

The pathways activated by post-conditioning may converge on the mitochondria, in particular on the mitochondrial permeability transition pore. We sought to characterize the inhibition status of the mitochondrial permeability transition early after the post-conditioning maneuver and before long reperfusion was established. We observed that post-conditioning maneuvers applied to isolated rat hearts, after a prolonged ischemia and before reperfusion, promoted cardiac mechanical function recovery and maintained mitochondrial integrity. These effects were evaluated by mitochondrial swelling, calcium transport, and NAD(+) content measurements; the improvements were established before restoring a long lasting reperfusion period. Mitochondrial integrity was associated with a diminution in oxidative stress, since carbonylation of proteins was prevented and aconitase activity was preserved in the post-conditioned hearts, implying that ROS might mediate mitochondrial dysfunction and mPTP opening. In addition, we found that cytochrome release was significantly abolished in the post-conditioned heart, in contrast with conventionally reperfused hearts.

Leptin Modifies the Rat Heart Performance Associated with Mitochondrial Dysfunction Independently of Its Prohypertrophic Effects.

BACKGROUND: Functional receptors for leptin were described on the surface of cardiomyocytes, and there was a prohypertrophic effect with high concentrations of the cytokine. Therefore, leptin could be a link between obesity and the prevalence of cardiovascular diseases. On the other hand, a deleterious effect of leptin on mitochondrial performance was described, which was also associated with the evolution of cardiac hypertrophy to heart failure. The goal of our study was to analyze the effect of the exposure of rat hearts to a high concentration of leptin on cardiac and mitochondrial function. METHODS: Rat hearts were perfused continuously with or without 3.1 nM leptin for 1, 2, 3, or 4 hours. Homogenates and mitochondria were prepared by centrifugation and analyzed for cardiac actin, STAT3, and pSTAT3 by Western blotting, as well as for mitochondrial oxidative phosphorylation, membrane potential, swelling, calcium transport, and content of oxidized lipids. RESULTS: In our results, leptin induced an increased rate-pressure product as a result of increased heart rate and contraction force, as well oxidative stress. In addition, mitochondrial dysfunction expressed as a loss of membrane potential, decreased ability for calcium transport and retention, faster swelling, and less respiratory control was observed. CONCLUSIONS: Our results support the role of leptin as a deleterious factor for cardiac function and indicates that mitochondrial dysfunction could be a trigger for cardiac hypertrophy and failure.

On the opening of an insensitive cyclosporin A non-specific pore by phenylarsine plus mersalyl.

The purpose of this work was addressed to provide new information on the effect of thiol reagents on mitochondrial non-specific pore opening, and its response to cyclosporin A (CSA). To meet this proposal phenylarsine oxide (PHA) and mersalyl were employed as tools to induce permeability transition and CSA to inhibit it. PHA-induced mitochondrial dysfunction, characterized by Ca2+ efflux, swelling, and membrane de-energization, was inhibited by N-ethylmaleimide and CSA. Conversely, mersalyl failed to inhibit the inducing effect of phenylarsine oxide, it rather strengthened it. In addition, the effect of mersalyl was associated with cross-linking of membrane proteins. The content of membrane thiol groups accessible to react with PHA, mersalyl, and PHA plus mersalyl was determined. In all situations, permeability transition was accompanied by a significant decrease in the whole free membrane thiol content. Interestingly, it is also shown that mersalyl hinders the protective effect of cyclosporin A on PHA-induced matrix Ca2+ efflux.

Hypothyroidism provides resistance to kidney mitochondria against the injury induced by renal ischemia-reperfusion.

Massive Ca(2+) accumulation in mitochondria, plus the stimulating effect of an inducing agent, i.e., oxidative stress, induces the so-called permeability transition, which is characterized by the opening of a nonspecific pore. This work was aimed at studying the influence of thyroid hormone on the opening of such a nonspecific pore in kidney mitochondria, as induced by an oxidative stress. To meet this objective, membrane permeability transition was examined in mitochondria isolated from kidney of euthyroid and hypothyroid rats, after a period of ischemia/reperfusion. It was found that mitochondria from hypothyroid rats were able to retain accumulated Ca(2+) to sustain a transmembrane potential after Ca(2+) addition, as well as to maintain matrix NAD(+) and membrane cytochrome c content. The protective effect of hypothyroidism was clearly opposed to that occurring in ischemic reperfused mitochondria from euthyroid rats. Our findings demonstrate that these mitochondria were unable to preserve selective membrane permeability, except when cyclosporin A was added. It is proposed that the protection is conferred by the low content of cardiolipin found in the inner membrane. This phospholipid is required to switch adenine nucleotide translocase from specific carrier to a non-specific pore.

In female rat heart mitochondria, oophorectomy results in loss of oxidative phosphorylation.

Oophorectomy in adult rats affected cardiac mitochondrial function. Progression of mitochondrial alterations was assessed at one, two and three months after surgery: at one month, very slight changes were observed, which increased at two and three months. Gradual effects included decrease in the rates of oxygen consumption and in respiratory uncoupling in the presence of complex I substrates, as well as compromised Ca2+ buffering ability. Malondialdehyde concentration increased, whereas the ROS-detoxifying enzyme Mn2+ superoxide dismutase (MnSOD) and aconitase lost activity. In the mitochondrial respiratory chain, the concentration and activity of complex I and complex IV decreased. Among other mitochondrial enzymes and transporters, adenine nucleotide carrier and glutaminase decreased. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase also decreased. Data strongly suggest that in the female rat heart, estrogen depletion leads to progressive, severe mitochondrial dysfunction.

Sodium inhibits permeability transition by decreasing potassium matrix content in rat kidney mitochondria.

Inner membrane mitochondria undergo a permeability increase elicited after the opening of a nonspecific pore due to supraphysiological matrix Ca2+ load, and the presence of an inducer. Multiple inducers have been used to promote the transition in permeability; among them are carboxyatractyloside (CAT) and reactive oxygen-derived species. In contrast, inhibitors such as ADP and cyclosporin A have been commonly used. In this work, we show that the opening or closure of the nonspecific pore depends on the cationic composition of the incubation medium. It was found that when mitochondria were incubated in either 125 mM KCl or 125 mM LiCl, ADP was essential to maintain selective membrane permeability. Interestingly, the nucleotide was not required when the medium contained 125 mM NaCl. Furthermore, it was established that CAT promotes membrane leakage in K(+)- or Li(+)-incubated mitochondria, while it failed to do so in Na(+)-incubated mitochondria. Evidence is also presented on the ability of Na+ to induce resistance in mitochondria against membrane damage by oxidative stress. Mitochondrial Ca2+ discharge, swelling, and transmembrane electric gradient were analyzed to establish permeability transition. It is concluded that the protection provided by Na+ was accomplished by inducing matrix K+ depletion, which, in turn, diminished the free fraction of matrix Ca2+.

Lactation reduces stress-caused dopaminergic activity and enhances GABAergic activity in the rat medial prefrontal cortex.

We investigated the effect of restraint on the release of dopamine, GABA and glutamate in the medial prefrontal cortex (mPFC) of lactating compared with virgin Wistar female rats; besides the expression of D1, neuropeptide Y Y2, GABA receptors and corticotropin-releasing factor (CRF). Results from microdialysis experiments showed that basal dopamine and GABA, but not glutamate, concentrations were higher in lactating rats. In virgin animals, immobilization caused significant increase in dopamine, whereas GABA was unchanged and glutamate reduced. In lactating animals, restrain significantly decreased dopamine concentrations and, in contrast to virgin animals, GABA and glutamate concentrations increased. We found a higher expression of CRF, as well as the D1 and neuropeptide Y Y2 receptors in the left mPFC of virgin stressed rats; also, only stressed lactating animals showed a significant increase in immunopositive cells to GABA in the left cingulate cortex; meanwhile, a significant decrease was measured in virgin rats after stress in the left prelimbic region. The increased inhibition of the mPFC dopamine cells during stress and the down-regulated expression of the neuropeptide Y Y2 receptor may explain the lower CRF and hyporesponse to stress measured in lactating animals. Interestingly, participation of mPFC in stress regulation seems to be lateralized.