Erika Cortez

Mitochondrial localization of non-histone protein HMGB1 during human endothelial cell–Toxoplasma gondii infection

Toxoplasma gondii is an obligate intracellular pathogen, replicating only within a specialized membrane‐bounded cytoplasmic vacuole, the parasitophorous vacuole (PV), which interacts with host cell mitochondria. High mobility group box 1 (HMGB1), a known nuclear transcription factor, also may be involved in pathological conditions, whose function is to signal tissue damage. Using confocal microscopy, we have investigated the localization of HMGB1 and the mitochondria performance during interaction between human umbilical vein endothelial cells (HUVEC) and Toxoplasma. Immunofluorescence showed HMGB1 localization in HUVEC tubular mitochondria stained with Mito Tracker (MT). At 2 h post‐infection, MT labeled spherical structures scattered throughout the cytoplasm and HMGB1 were still present. After 24 h of infection, long and tubular structures were localized around PVs and were double labeled by MT and HMGB1, suggesting a structural reorganization of the mitochondria over a long period of infection. For the first time, these results show there is HMGB1 in HUVEC mitochondria and that this protein could be playing a part in mitochondrial DNA events which are important for fission and fusion processes reported here during HUVEC‐T. gondii infection.

Heart energy metabolism impairment in Western-diet induced obese mice

Nutritional transition has contributed to growing obesity, mainly by changing eating habits of the population. The mechanisms by which diet-induced obesity leads to cardiac injury are not completely understood, but it is known that obesity is associated to impaired cardiac function and energy metabolism, increasing morbidity and mortality. Therefore, our study aimed to investigate the mechanisms underlying cardiac metabolism impairment related to Western diet-induced obesity. After weaning, male Swiss mice were fed a Western diet for 16 weeks in order to induce obesity. After this period, the content of proteins involved in heart energy metabolism GLUT1, cytosolic lysate and plasma membrane GLUT4, AMPK, pAMPK, IRβ, IRS-1, PGC-1α, CPT1 and UCP2 was evaluated. Also, the oxidative phosphorylation of myocardial fibers was measured by high-resolution respirometry. Mice in the Western diet group (WG) presented altered biometric parameters compared to those in control group, including higher body weight, increased myocardial lipid deposition and glucose intolerance, which demonstrate the obesogenic role of Western diet. WG presented increased CPT1 and UCP2 contents and decreased IRS-1, plasma membrane GLUT4 and PGC-1α contents. In addition, WG presented cardiac mitochondrial dysfunction and reduced biogenesis, demonstrating a lower capacity of carbohydrates and fatty acid oxidation and also decreased coupling between oxidative phosphorylation and adenosine triphosphate synthesis. Cardiac metabolism impairment related to Western diet-induced obesity is probably due to damaged myocardial oxidative capacity, reduced mitochondrial biogenesis and mitochondria uncoupling, which compromise the bioenergetic metabolism of heart.

Ghrelin signaling in heart remodeling of adult obese mice

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), has been suggested to be associated to obesity, insulin secretion, cardiovascular growth and homeostasis. GHS-R has been found in most of the tissues, and among the hormone action it is included the regulation of heart energy metabolism. Therefore, hypernutrition during early life leads to obesity, induces cardiac hypertrophy, compromises myocardial function, inducing heart failure in adulthood. We examined ghrelin signaling process in cardiac remodeling in these obese adult mice. The cardiomyocytes (cmy) of left ventricle were analyzed by light microscopy and stereology, content and phosphorilation of cardiac proteins: ghrelin receptor (growth hormone secretagogue receptor 1a, GHSR-1a), protein kinase B (AKT and pAKT), phosphatidil inositol 3 kinase (PI3K), AMP-activated protein kinase (AMPK and pAMPK) and actin were achieved by Western blotting. GHSR-1a gene expression was analyzed by Real Time-PCR. We observed hyperglycemia and higher liver and visceral fat weight in obese when compared to control group. Obese mice presented a marked increase in heart weight/tibia length, indicating an enlarged heart size or a remodeling process. Obese mice had increased GHSR-1a content and expression in the heart associated to PI3K content and increased AKT content and phosphorylation. In contrast, AMPK content and phosphorylation in heart was not different between experimental groups. Ghrelin plasma levels in obese group were decreased when compared to control group. Our data suggest that remodeled myocardial in adult obese mice overnourished in early life are associated with higher phosphorylation of GHSR-1a, PI3K and AKT but not with AMPK.

Progenitor cells and TNF-alpha involvement during morphological changes in pancreatic islets of obese mice

Overnutrition during pregnancy and lactation lend increasing support to the development of obesity and several chronic diseases in adulthood such as type 2 diabetes mellitus, which leads to beta-cell dysfunction and insulin resistance. In this work, we aimed to study the effects of early life overnutrition on the development of obesity, analyzing the morphological changes, expression of TNF-α, and also the stem cell marker CD133 in the pancreatic islets of young and adult mice. Overnutrition during lactation phase was used as an experimental model to induce obesity. The animals were analyzed at 28 and 150 days of age, when pancreata were collected for histological, ultrastructural and western blotting analysis. The results showed that islet hypertrophy is established in obese groups at day 28 and remained until adulthood. CD133+ cells were observed as small cells within pancreatic islets in both control and obese young mice. However, at day 150, these cells were observed only in the islet peripheries and near ducts of the obese group. Furthermore, TNF-α expression in pancreatic islets was increased in both young and adult obese groups when compared to control groups. This work shows interesting data about CD133 receptor and TNF-α roles in the pancreas during obesity development.

Overnourishment during lactation induces metabolic and haemodynamic heart impairment during adulthood

AIM In this study, the effects of postnatal overfeeding on heart energy homoeostasis and cardiac haemodynamics in adult male Swiss mice were examined. METHODS AND RESULTS During the suckling period, the mice were divided into four groups of control or overfed pups in combination with baseline or ischaemia/reperfusion treatments (control group baseline, CGBL; overfed group baseline, OGBL; control group ischaemia/reperfusion, CGIR; and overfed group ischaemia/reperfusion, OGIR). End diastolic pressure (EDP), heart contraction speed (Max dP/dt), relaxation speed (Min dP/dt), isovolumetric relaxation time (Tau) and frequency by beats per minute (BPM) were measured. During baseline and ischaemia/reperfusion, key proteins such as AKT1, AKT2, AKT3, pAKT, adenosine monophosphate-activated protein kinase (AMPK), pAMPK, insulin receptor beta (IRβ), protein tyrosine phosphatase 1B (PTP1B), insulin receptor substrate 1 (IRS1), fatty acid binding protein (FABP), CD36, phosphoinositide 3-kinase (PI3K) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were studied. The expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), carnitine palmitoyltransferase 1 (CPT1) and uncoupling protein 3 (UCP3) was studied as a marker of cardiac hypertrophy and energetic metabolism. Cardiac fibrosis was analyzed by quantifying collagen deposition, which is increased in the OGBL and OGIR groups compared with the control groups. CONCLUSIONS The OGBL group showed reduced EDP compared with the CGBL group and high Max dP/dt compared with the OGBL group. Ischaemia/reperfusion increased EDP and Min dP/dt in the intragroup comparison. By contrast, Tau and frequency were not significantly different among groups. The OGIR mice showed significant alterations in heart metabolism proteins, including AKT2, pAKT/AKT1, pAKT/AKT2, AMPK, pAMPK/AMPK, PTP1B, IRS1, FABP and CD36. Furthermore, alterations in ANP, BNP, CPT1 and UCP3 messenger RNA (mRNA) expression indicated hypertrophy and reduction in their efficiency, such that exclusive overnutrition in childhood induces a long-term effect on haemodynamics, metabolism and heart remodelling.

Lymphocytes Mitochondrial Physiology as Biomarker of Energy Metabolism during Fasted and Fed Conditions

Mitochondria are central coordinators of energy metabolism, and changes of their physiology have long been associated with metabolic disorders. Thus, observations of energy dynamics in different cell types are of utmost importance. Therefore, tools with quick and easy handling are needed for consistent evaluations of such interventions. In this paper, our main hypothesis is that during different nutritional situations lymphocytes mitochondrial physiology could be associated with the metabolism of other cell types, such as cardiomyocytes, and consequently be used as metabolic biomarker. Blood lymphocytes and heart muscle fibers were obtained from both fed and 24 h-fasted mice, and mitochondrial analysis was assessed by high-resolution respirometry and western blotting. Carbohydrate-linked oxidation and fatty acid oxidation were significantly higher after fasting. Carnitine palmitoil transferase 1 and uncouple protein 2 contents were increased in the fasted group, while the glucose transporters 1 and 4 and the ratio phosphorylated AMP-activated protein kinase/AMPK did not change between groups. In summary, under a nutritional status modification, mitochondria demonstrated earlier adaptive capacity than other metabolic sensors such as glucose transporters and AMPK, suggesting the accuracy of mitochondria physiology of lymphocytes as biomarker for metabolic changes.

Overnutrition during lactation leads to impairment in insulin signaling, up-regulation of GLUT1 and increased mitochondrial carbohydrate oxidation in heart of weaned mice.

Several studies have demonstrated that overnutrition during early postnatal period can increase the long-term risk of developing obesity and cardiac disorders, yet the short-term effects of postnatal overfeeding in cardiac metabolism remains unknown. The aim of our study was to investigate the cardiac metabolism of weaned mice submitted to overnutrition during lactation, particularly as to mitochondrial function, substrate preference and insulin signaling. Postnatal overfeeding was induced by litter size reduction in mice at postnatal day 3. At 21 days of age (weaning), mice in the overfed group (OG) presented biometric and biochemical parameters of obesity, including increased body weight, visceral fat, liver weight and increased left ventricle weight/tibia length ratio; indicating cardiac hypertrophy, hyperglycemia, hyperinsulinemia and increased liver glycogen content compared to control group. In the heart, we detected impaired insulin signaling, mainly due to decreased IRβ, pTyr-IRS1, PI3K, GLUT4 and pAkt/Akt and increased PTP1B, GLUT1 and pAMPKα/AMPKα content. Activities of lactate dehydrogenase and citrate synthase were increased, accompanied by enhanced carbohydrate oxidation, as observed by high-resolution respirometry. Moreover, OG hearts had lower CPT1, PPARα and increased UCP2 mRNA expression, associated with increased oxidative stress (4-HNE content), BAX/BCL2 ratio and cardiac fibrosis. Ultrastructural analysis of OG hearts demonstrated mild mitochondrial damage without alterations in OXPHOS complexes. In conclusion, overnutrition during early life induces short-term metabolic disturbances, impairment in heart insulin signaling, up-regulates GLUT-1 and switch cardiac fuel preference in juvenile mice.

Impaired mitochondrial function and reduced viability in bone marrow cells of obese mice

Bone marrow cells (BMCs) are the main type of cells used for transplantation therapies. Obesity, a major world health problem, has been demonstrated to affect various tissues, including bone marrow. This could compromise the success of such therapies. One of the main mechanisms underlying the pathogenesis of obesity is mitochondrial dysfunction, and recent data have suggested an important role for mitochondrial metabolism in the regulation of stem cell proliferation and differentiation. Since the potential use of BMCs for clinical therapies depends on their viability and capacity to proliferate and/or differentiate properly, the analysis of mitochondrial function and cell viability could be important approaches for evaluating BMC quality in the context of obesity. We therefore compared BMCs from a control group (CG) and an obese group (OG) of mice and evaluated their mitochondrial function, proliferation capacity, apoptosis, and levels of proteins involved in energy metabolism. BMCs from OG had increased apoptosis and decreased proliferation rates compared with CG. Mitochondrial respiratory capacity, biogenesis, and the coupling between oxidative phosphorylation and ATP synthesis were significantly decreased in OG compared with CG, in correlation with increased levels of uncoupling protein 2 and reduced peroxisome proliferator-activated receptor-coactivator 1α content. OG also had decreased amounts of the glucose transporter GLUT-1 and insulin receptor (IRβ). Thus, Western-diet-induced obesity leads to mitochondrial dysfunction and reduced proliferative capacity in BMCs, changes that, in turn, might compromise the success of therapies utilizing these cells.

Laminin expression during bone marrow mononuclear cell transplantation in hepatectomized rats.

The adult bone marrow retains two populations of stem cells with emerging importance for the treatment of diverse liver diseases: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the mechanisms that control liver regeneration after bone marrow cell transplantation are still controversial. Liver regeneration after partial hepatectomy is a complex process that requires the proliferation of all hepatic cells. Growth factors, cytokines and extracellular matrix molecules are key elements in this process. Laminins are a family of extracellular matrix proteins with adhesive and chemotactic functions, expressed in the portal and centrolobular veins of the normal liver. The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy followed by bone marrow mononuclear cell (BMMNC) transplantation. Rat BMMNCs were isolated by Ficoll‐gradient centrifugation, stained with DAPI and injected into recently hepatectomyzed rats via the portal vein. Liver sections obtained 15 min, 1 day and 3 days after the surgery were immunolabeled with anti‐rat CD34 and/or laminin primary antibodies and observed under a laser scanning confocal microscope. Results showed that 15 min after partial hepatectomy, a transplanted CD34+ HSC was found in contact with laminin, which was localized in the portal and centrolobular veins of rat livers. Furthermore, 1 and 3 days after hepatectomy, transplanted BMMNCs were found in the hepatic sinusoids expressing laminin. These results strongly suggest that laminin might be an important extracellular matrix component for bone marrow cell attachment and migration in the injured liver.

Bone marrow mononuclear cell transplantation improves mitochondrial bioenergetics in the liver of cholestatic rats

Mitochondrial dysfunction has been associated with liver cholestatis. Toxic bile salt accumulation leads to chronic injury with mitochondrial damage, ROS increase and apoptosis, resulting in liver dysfunction. This study aimed to analyze mitochondrial bioenergetics in rats with hepatic fibrosis induced by bile duct ligation (BDL) after BMMNC transplantation. Livers were collected from normal rats, fibrotic rats after 14 and 21 days of BDL (F14d and F21d) and rats that received BMMNC at 14 days of BDL, analyzed after 7 days. F21d demonstrated increased collagen I content and consequently decrease after BMMNC transplantation. Both F14d and F21d had significantly reduced mitochondrial oxidation capacity and increased mitochondrial uncoupling, which were restored to levels similar to those of normal group after BMMNC transplantation. In addition, F21d had a significantly increase of UCP2, and reduced PGC-1α content. However, after BMMNC transplantation both proteins returned to levels similar to normal group. Moreover, F14d had a significantly increase in 4-HNE content compared to normal group, but after BMMNC transplantation 4-HNE content significantly reduced, suggesting oxidative stress reduction. Therefore, BMMNC transplantation has a positive effect on hepatic mitochondrial bioenergetics of cholestatic rats, increasing oxidative capacity and reducing oxidative stress, which, in turn, contribute to liver function recover.