Aging: silencing the PKA and AkT/PKB signaling pathways alters the antioxidant capacity of resveratrol.

One of the theories related to aging is the increase in oxidative stress. Given this, the objective of the study is to evaluate the cellular mechanisms responsible for the resveratrol antioxidant effect on leukocytes from donors aged between 20 and 80 years old. For this, leukocytes from donors of three age groups (20-39, 40-59 and 60-80) were isolated. Image-iT™LIVE Green Reactive Oxygen Species (ROS) Kit was used. Reactive Nitrogen Species (RNS) analysis was performed by measuring nitric oxide and peroxynitrite. The PKA, Akt/PKB and p38-MAPK were evaluated by chemiluminescence. The statistical analysis between age and treatments were performed by Pearson correlation (*p < 0.05). It was possible to observe the antioxidant effect of resveratrol in all age groups. The correlation results show loss of resveratrol effect in decreasing ROS in leukocytes from older donors. We observed an active antioxidant effect of p38-MAPK in all ages, with resveratrol acting on it. The PKA and Akt/PKB were active in leukocytes from donors aged 20-59. In cells from donors older than 60, these pathways are silenced, and an effect is also not observed in cells treated with resveratrol. Therefore, resveratrol showed antioxidant effect in all age, although it was more pronounced in leukocytes from younger. One of resveratrol's mechanisms is due to the activation of the PKA and Akt/PKB, which were activated in younger donor cells.

Albumin Expands Albumin Reabsorption Capacity in Proximal Tubule Epithelial Cells through a Positive Feedback Loop between AKT and Megalin.

Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as "albumin-induced albumin endocytosis". Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.

Lithium modulates multiple tau kinases with distinct effects in cortical and hippocampal neurons according to concentration ranges.

The hyperphosphorylation of tau is a central mechanism in the pathogenesis of Alzheimer's disease (AD). Lithium is a potent inhibitor of glycogen synthase kinase-3beta (GSK3ß), the most important tau kinase in neurons, and may also affect tau phosphorylation by modifying the expression and/or activity of other kinases, such as protein kinase A (PKA), Akt (PKB), and calcium calmodulin kinase-II (CaMKII). The aim of the present study is to determine the effect of chronic lithium treatment on the protein expression of tau and its major kinases in cortical and hippocampal neurons, at distinct working concentrations. Primary cultures of cortical and hippocampal neurons were treated with sub-therapeutic (0.02 mM and 0.2 mM) and therapeutic (2 mM) concentrations of lithium for 7 days. Protein expression of tau and tau-kinases was determined by immunoblotting. An indirect estimate of GSK3ß activity was determined by the GSK3ß ratio (rGSKß). Statistically significant increments in the protein expression of tau and CaMKII were observed both in cortical and hippocampal neurons treated with subtherapeutic doses of lithium. GSK3ß activity was increased in cortical, but decreased in hippocampal neurons. Distinct patterns of changes in the expression of the remaining tau tau-kinases were observed: in cortical neurons, lithium treatment was associated with consistent decrements in Akt and PKA, whereas hippocampal neurons displayed increased protein expression of Akt and decreased PKA. Our results suggest that chronic lithium treatment may yield distinct biological effects depending on the concentration range, with regional specificity. We further suggest that hippocampal neurons may be more sensitive to the effect of lithium, presenting with changes in the expression of tau-related proteins at subtherapeutic doses, which may not be mirrored by the effects observed in cortical neurons.

Antioxidant effect of Resveratrol: Change in MAPK cell signaling pathway during the aging process.

Aging is characterized by a progressive loss of physiological integrity. One common denominator is the increase of reactive oxygen species (ROS) caused by inhibition of important antioxidant pathways. Resveratrol is a polyphenol known for its potent antioxidant activity. However, antioxidant pathways activated by them change with aging. The objective of our study was to verify the antioxidant effect of resveratrol in an oxidative stress environment in Human Mononuclear Cells (PBMC) from donors with different ages. Resveratrol (5 µM), a stimulus with H2O2 (0,64 % v/v) in addition to inhibitors of PKA, AkT/PKB and MAPK signaling pathways were used in chemiluminescence assay. An incresed basal production of ROS was observed in the elderly than in the middle-aged group. Resveratrol was able to reduce ROS in both groups, but with greater efficiency in the middle-aged group. By inhibiting PKA, Akt/PKB and MAPK signaling pathways we observed that resveratrol presented an altered performance in the aging process, changing signaling pattern of MAPK pathway.

Angiotensin (1-7) Decreases Myostatin-Induced NF-κB Signaling and Skeletal Muscle Atrophy.

Myostatin is a myokine that regulates muscle function and mass, producing muscle atrophy. Myostatin induces the degradation of myofibrillar proteins, such as myosin heavy chain or troponin. The main pathway that mediates protein degradation during muscle atrophy is the ubiquitin proteasome system, by increasing the expression of atrogin-1 and MuRF-1. In addition, myostatin activates the NF-κB signaling pathway. Renin-angiotensin system (RAS) also regulates muscle mass. Angiotensin (1-7) (Ang-(1-7)) has anti-atrophic properties in skeletal muscle. In this paper, we evaluated the effect of Ang-(1-7) on muscle atrophy and signaling induced by myostatin. The results show that Ang-(1-7) prevented the decrease of the myotube diameter and myofibrillar protein levels induced by myostatin. Ang-(1-7) also abolished the increase of myostatin-induced reactive oxygen species production, atrogin-1, MuRF-1, and TNF-α gene expressions and NF-κB signaling activation. Ang-(1-7) inhibited the activity mediated by myostatin through Mas receptor, as is demonstrated by the loss of all Ang-(1-7)-induced effects when the Mas receptor antagonist A779 was used. Our results show that the effects of Ang-(1-7) on the myostatin-dependent muscle atrophy and signaling are blocked by MK-2206, an inhibitor of Akt/PKB. Together, these data indicate that Ang-(1-7) inhibited muscle atrophy and signaling induced by myostatin through a mechanism dependent on Mas receptor and Akt/PKB.

Involvement of PI3K/Akt and p38 MAPK in the induction of COX-2 expression by bacterial lipopolysaccharide in murine adrenocortical cells.

Previous studies from our laboratory demonstrated the involvement of COX-2 in the stimulation of steroid production by LPS in murine adrenocortical Y1 cells, as well as in the adrenal cortex of male Wistar rats. In this paper we analyzed signaling pathways involved in the induction of this key regulatory enzyme in adrenocortical cells and demonstrated that LPS triggers an increase in COX-2 mRNA levels by mechanisms involving the stimulation of reactive oxygen species (ROS) generation and the activation of p38 MAPK and Akt, in addition to the previously demonstrated increase in NFκB activity. In this sense we showed that: (1) inhibition of p38 MAPK or PI3K/Akt (pharmacological or molecular) prevented the increase in COX-2 protein levels by LPS, (2) LPS induced p38 MAPK and Akt phosphorylation, (3) antioxidant treatment blocked the effect of LPS on p38 MAPK phosphorylation and in COX-2 protein levels, (4) PI3K inhibition with LY294002 prevented p38 MAPK phosphorylation and, (5) the activity of an NFκB reporter was decreased by p38 MAPK or PI3K inhibition. These results suggest that activation of both p38 MAPK and PI3K/Akt pathways promote the stimulation of NFκB activity and that PI3K/Akt activity might regulate both p38 MAPK and NFκB signaling pathways. In summary, in this study we showed that in adrenal cells, LPS induces COX-2 expression by activating p38 MAPK and PI3K/Akt signaling pathways and that both pathways converge in the modulation of NFκB transcriptional activity.

Beneficial effects of nicotine, cotinine and its metabolites as potential agents for Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder, which is characterized by neuroinflammation, dopaminergic neuronal cell death and motor dysfunction, and for which there are no proven effective treatments. The negative correlation between tobacco consumption and PD suggests that tobacco-derived compounds can be beneficial against PD. Nicotine, the more studied alkaloid derived from tobacco, is considered to be responsible for the beneficial behavioral and neurological effects of tobacco use in PD. However, several metabolites of nicotine, such as cotinine, also increase in the brain after nicotine administration. The effect of nicotine and some of its derivatives on dopaminergic neurons viability, neuroinflammation, and motor and memory functions, have been investigated using cellular and rodent models of PD. Current evidence shows that nicotine, and some of its derivatives diminish oxidative stress and neuroinflammation in the brain and improve synaptic plasticity and neuronal survival of dopaminergic neurons. In vivo these effects resulted in improvements in mood, motor skills and memory in subjects suffering from PD pathology. In this review, we discuss the potential benefits of nicotine and its derivatives for treating PD.

Diet-induced obesity induces endoplasmic reticulum stress and insulin resistance in the amygdala of rats.

Insulin acts in the hypothalamus, decreasing food intake (FI) by the IR/PI3K/Akt pathway. This pathway is impaired in obese animals and endoplasmic reticulum (ER) stress and low-grade inflammation are possible mechanisms involved in this impairment. Here, we highlighted the amygdala as an important brain region for FI regulation in response to insulin. This regulation was dependent on PI3K/AKT pathway similar to the hypothalamus. Insulin was able to decrease neuropeptide Y (NPY) and increase oxytocin mRNA levels in the amygdala via PI3K, which may contribute to hypophagia. Additionally, obese rats did not reduce FI in response to insulin and AKT phosphorylation was decreased in the amygdala, suggesting insulin resistance. Insulin resistance was associated with ER stress and low-grade inflammation in this brain region. The inhibition of ER stress with PBA reverses insulin action/signaling, decreases NPY and increases oxytocin mRNA levels in the amygdala from obese rats, suggesting that ER stress is probably one of the mechanisms that induce insulin resistance in the amygdala.

Modification of Akt by SUMO conjugation regulates alternative splicing and cell cycle.

Akt/PKB is a key signaling molecule in higher eukaryotes and a crucial protein kinase in human health and disease. Phosphorylation, acetylation, and ubiquitylation have been reported as important regulatory post-translational modifications of this kinase. We describe here that Akt is modified by SUMO conjugation, and show that lysine residues 276 and 301 are the major SUMO attachment sites within this protein. We found that phosphorylation and SUMOylation of Akt appear as independent events. However, decreasing Akt SUMOylation levels severely affects the role of this kinase as a regulator of fibronectin and Bcl-x alternative splicing. Moreover, we observed that the Akt mutant (Akt E17K) found in several human tumors displays increased levels of SUMOylation and also an enhanced capacity to regulate fibronectin splicing patterns. This splicing regulatory activity is completely abolished by decreasing Akt E17K SUMO conjugation levels. Additionally, we found that SUMOylation controls Akt regulatory function at G1/S transition during cell cycle progression. These findings reveal SUMO conjugation as a novel level of regulation for Akt activity, opening new areas of exploration related to the molecular mechanisms involved in the diverse cellular functions of this kinase.

Calix[6]arene bypasses human pancreatic cancer aggressiveness: downregulation of receptor tyrosine kinases and induction of cell death by reticulum stress and autophagy.

Pancreatic cancer ranks fourth among cancer-related causes of death in North America. Minimal progress has been made in the diagnosis and treatment of patients with late-stage tumors. Moreover, pancreatic cancer aggressiveness is closely related to high levels of pro-survival mediators, which can ultimately lead to rapid disease progression, resistance and metastasis. The main goal of this study was to define the mechanisms by which calix[6]arene, but not other calixarenes, efficiently decreases the aggressiveness of a drug resistant human pancreas carcinoma cell line (Panc-1). Calix[6]arene was more potent in reducing Panc-1 cell viability than gemcitabine and 5-fluorouracil. In relation to the underlying mechanisms of cytotoxic effects, it led to cell cycle arrest in the G0/G1 phase through downregulation of PIM1, CDK2, CDK4 and retinoblastoma proteins. Importantly, calix[6]arene abolished signal transduction of Mer and AXL tyrosine kinase receptors, both of which are usually overexpressed in pancreatic cancer. Accordingly, inhibition of PI3K and mTOR was also observed, and these proteins are positively modulated by Mer and AXL. Despite decreasing the phosphorylation of AKT at Thr308, calix[6]arene caused an increase in phosphorylation at Ser473. These findings in conjunction with increased BiP and IRE1-α provide a molecular basis explaining the capacity of calix[6]arene to trigger endoplasmic reticulum stress and autophagic cell death. Our findings highlight calix[6]arene as a potential candidate for overcoming pancreatic cancer aggressiveness. Importantly, we provide evidence that calix[6]arene affects a broad array of key targets that are usually dysfunctional in pancreatic cancer, a highly desirable characteristic for chemotherapeutics.

ATP and UTP stimulate bone morphogenetic protein-2,-4 and -5 gene expression and mineralization by rat primary osteoblasts involving PI3K/AKT pathway.

The modulation of purinergic receptors plays an important role in the regulation of bone formation by the osteoblast. On the other hand, bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß superfamily, regulate the differentiation of osteoprogenitor bone cells and stimulate bone formation. In this study, we investigate the effects of several nucleotides on osteoblast differentiation and function, and their relation with the gene expression of osteogenic proteins BMP-2, BMP-4 and BMP-5 as well as of differentiation markers alkaline phosphatase (ALP) and bone sialoprotein (BSP). Our results indicate that 100µM ATP, ATPγS and UTP, but not ADP, ADPßS or UDP, promote ALP activity in rat primary osteoblasts, showing a peak about day 7 of the treatment. ATP, ATPγS and UTP also increase the mRNA levels of ALP, BMP-2, BMP-4, BMP-5 and BSP. Both the ALP activity and ALP and BMP-4 mRNA increments induced by ATP and UTP are inhibited by Ly294002, a PI3K inhibitor, suggesting the involvement of PI3K/AKT signaling pathway in purinergic modulation of osteoblast differentiation. Furthermore, bone mineralization enhance 1 and 1.5 fold after culturing osteoblasts in the presence of 100µM ATP or UTP, respectively, but not of ADP or UDP for 22 days. This information suggests that P2Y2 receptors (responsive to ATP, ATPγS and UTP) enhance osteoblast differentiation involving PI3K/AKT signaling pathway activation and gene expression induction of ALP, BMP-2, BMP-4, BMP-5 and BSP. Our findings state a novel molecular mechanism that involves specific gene expression activation of osteoblast function by the purinoreceptors, which would be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.

Role of AKT / mTORC1 pathway in pancreatic β - cell proliferation / Participación de la vía de señalización AKT/mTORC1 en la proliferación de las células β del páncreas

Growth factors, insulin signaling and nutrients are important regulators of β-cell mass and function. The events linking these signals to regulation of β-cell mass are not completely understood. Recent findings indicate that mTOR pathway integrates signals from growth factors and nutrients with transcription, translation, cell size, cytoskeleton remodeling and mitochondrial metabolism. mTOR is a part of two distinct complexes; mTORC1 and mTORC2. The mammalian TORC1 is sensitive to rapamycin and contains Raptor, deptor, PRAS40 and the G protein β-subunit-like protein (GβL). mTORC1 activates key regulators of protein translation; ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1. This review summarizes current findings about the role of AKT/mTORC1 signaling in regulation of pancreatic β cell mass and proliferation. mTORC1 is a major regulator of β-cell cycle progression by modulation of cyclins D2, D3 and cdk4/cyclin D activity. These studies uncovered key novel pathways controlling cell cycle progression in β-cells in vivo. This information can be used to develop alternative approaches to expand β-cell mass in vivo and in vitro without the risk of oncogenic transformation. The acquisition of such knowledge is critical for the design of improved therapeutic strategies for the treatment and cure of diabetes as well as to understand the effects of mTOR inhibitors in β-cell function.

Factores de crecimiento y nutrientes son reguladores muy importantes de la masa y función de las células β, pero las vías de señalización que unen estas señales a estos procesos no han sido completamente elucidadas. Estudios recientes han demostrado que la proteína mTOR integra señales provenientes de factores de crecimiento y disponibilidad de nutrientes con procesos celulares como transcripción, traducción, organización del citoesqueleto y metabolismo mitocondrial. mTOR puede hacer parte de dos complejos diferentes, mTORC1 y mTORC2. En el complejo mTORC1, la proteina mTOR la cual es sensible a rapamicina y se encuentra asociada a las proteínas Raptor, G β L, deptor y PRAS40, activa reguladores claves en la síntesis de proteínas, tales como la proteína cinasa ribosomal S6 (S6K) y la proteína de unión al factor eucariótico de iniciación 4E. El presente trabajo recopila información reciente sobre la participación de la vía de señalización AKT/mTORC1 en la regulación de la proliferación y masa de las células β del páncreas. mTORC1 regula la progresión del ciclo celular en células β, mediante la modulación de los niveles de las ciclinas D2 y D3 y la actividad del complejo Cdk4/ ciclina D. Estos estudios que revelan nuevos puntos de control del ciclo celular en células β, pueden ser utilizados en el desarrollo de nuevos enfoques para expandir la masa de células β, sin el riesgo de inducir una transformación oncogénica. Los resultados relacionados en el presente trabajo aportan información muy valiosa para el desarrollo de nuevas estrategias terapéuticas para el tratamiento la diabetes tipo 2.

Role of AKT/mTORC1 pathway in pancreatic ß-cell proliferation.

Growth factors, insulin signaling and nutrients are important regulators of ß-cell mass and function. The events linking these signals to regulation of ß-cell mass are not completely understood. Recent findings indicate that mTOR pathway integrates signals from growth factors and nutrients with transcription, translation, cell size, cytoskeleton remodeling and mitochondrial metabolism. mTOR is a part of two distinct complexes; mTORC1 and mTORC2. The mammalian TORC1 is sensitive to rapamycin and contains Raptor, deptor, PRAS40 and the G protein ß-subunit-like protein (GßL). mTORC1 activates key regulators of protein translation; ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1. This review summarizes current findings about the role of AKT/mTORC1 signaling in regulation of pancreatic ß cell mass and proliferation. mTORC1 is a major regulator of ß-cell cycle progression by modulation of cyclins D2, D3 and cdk4/cyclin D activity. These studies uncovered key novel pathways controlling cell cycle progression in ß-cells in vivo. This information can be used to develop alternative approaches to expand ß-cell mass in vivo and in vitro without the risk of oncogenic transformation. The acquisition of such knowledge is critical for the design of improved therapeutic strategies for the treatment and cure of diabetes as well as to understand the effects of mTOR inhibitors in ß-cell function.

Factores de crecimiento y nutrientes son reguladores muy importantes de la masa y función de las células ß, pero las vías de señalización que unen estas señales a estos procesos no han sido completamente elucidadas. Estudios recientes han demostrado que la proteína mTOR integra señales provenientes de factores de crecimiento y disponibilidad de nutrientes con procesos celulares como transcripción, traducción, organización del citoesqueleto y metabolismo mitocondrial. mTOR puede hacer parte de dos complejos diferentes, mTORC1 y mTORC2. En el complejo mTORC1, la proteina mTOR la cual es sensible a rapamicina y se encuentra asociada a las proteínas Raptor, G ß L, deptor y PRAS40, activa reguladores claves en la síntesis de proteínas, tales como la proteína cinasa ribosomal S6 (S6K) y la proteína de unión al factor eucariótico de iniciación 4E. El presente trabajo recopila información reciente sobre la participación de la vía de señalización AKT/mTORC1 en la regulación de la proliferación y masa de las células ß del páncreas. mTORC1 regula la progresión del ciclo celular en células ß, mediante la modulación de los niveles de las ciclinas D2 y D3 y la actividad del complejo Cdk4/ ciclina D. Estos estudios que revelan nuevos puntos de control del ciclo celular en células ß, pueden ser utilizados en el desarrollo de nuevos enfoques para expandir la masa de células ß, sin el riesgo de inducir una transformación oncogénica. Los resultados relacionados en el presente trabajo aportan información muy valiosa para el desarrollo de nuevas estrategias terapéuticas para el tratamiento la diabetes tipo 2.