High copper levels induce premature senescence in 3T3-L1 preadipocytes.

Copper (Cu) dyshomeostasis has been linked to obesity and related morbidities and also to aging. Cu levels are higher in older or obese individuals, and adipose tissue (AT) Cu levels correlate with body mass index. Aging and obesity induce similar AT functional and structural changes, including an accumulation of senescent cells. To study the effect of Cu-mediated stress-induced premature senescent (Cu-SIPS) on preadipocytes, 3T3-L1 cell line was exposed to a subcytotoxic concentration of copper sulfate. After Cu treatment, preadipocytes acquired typical senescence characteristics including diminished cell proliferation, cell and nuclei enlargement and increased lysosomal mass (higher Lamp2 expression and a slight increased number of cells positive for ß-galactosidase associated with senescence (SA-ß-Gal)). Cell cycle arrest was due to upregulation of p16Ink4aInk4a and p21Waf1/Cip1. Accordingly, protein levels of the proliferation marker KI67 were reduced. Cu-SIPS relates with oxidative stress and, in this context, an increase of SOD1 and HO-1 expression was detected in Cu-treated cells. The mRNA expression of senescence-associated secretory phenotype factors, such as Mmp3, Il-6 and Tnf-α, increased in Cu-SIPS 3T3-L1 cells but no effect was observed on the expression of heterochromatin-associated protein 1(HP1). Although the downregulation of Lamin B1 expression is considered a hallmark of senescence, Cu-SIPS cells presented higher levels of Lamin B1. The dysregulation of nuclear lamina was accompanied by an increase of nuclear blebbing, but not of micronuclei number. To conclude, a Cu-SIPS model in 3T3-L1 preadipocytes is here described, which may be an asset to the study of AT dysregulation observed in obesity and aging.

A hint for the obesity paradox and the link between obesity, perirenal adipose tissue and Renal Cell Carcinoma progression.

Increasing evidence supports a role for local fat depots in cancer outcomes. Despite the robust positive association of obesity with renal cell carcinoma (RCCa) diagnosis, increased adiposity is inversely related to RCCa oncological outcomes. Here, we sought to ascertain whether imagiologically assessed local fat depots associate with RCCa progression and survival and account for this apparent paradox. A retrospective cohort of renal carcinoma patients elective for nephrectomy (n = 137) were included. Beyond baseline clinicopathological characteristics, computed tomography (CT)-scans at the level of renal hilum evaluated areas and densities of different adipose tissue depots (perirenal, subcutaneous, visceral) and skeletal muscle (erector spinae, psoas and quadratus lumborum muscles) were analyzed. Univariate and multivariable Cox proportional hazards models were estimated following empirical analysis using stepwise Cox regression. Age, visceral adipose tissue (VAT) area and body mass index (BMI) predicted tumour-sided perirenal fat area (R2 = 0.584), which presented upregulated UCP1 expression by 27-fold (P = 0.026) and smaller adipocyte areas, compared with subcutaneous depot. Multivariate analyses revealed that increased area of perirenal adipose tissue (PRAT) on the contralateral and tumour side associate with improved progression-free survival (HR = 0.3, 95CI = 0.1-0.8, P = 0.019) and overall survival (HR = 0.3, 95CI = 0.1-0.7, P = 0.009). PRAT measurements using CT, might become a possible tool, well correlated with other measures of obesity such as VAT and BMI, that will improve determination of obesity and contribute to assess the risk for disease progression and mortality in renal cancer patients. Present data supports the obesity paradox in RCCa, assumed that larger PRAT areas seem to protect from disease progression and death.

Anti-oxidant effect of metformin through AMPK/SIRT1/PGC-1α/SIRT3- independent GPx1 expression in the heart of mice with endometriosis.

OBJECTIVES: Endometriosis is a gynecological disease associated with an imbalance between oxidative species production and anti-oxidative defenses. In women, endometriosis has been reported to associate with increased incidence of cardiovascular events. As such, this study aimed to analyze the oxidation-responsive AMPK/SIRT1/PGC-1α/SIRT3 pathway in the heart of a mouse model of endometriosis. The effect of metformin, an insulin-sensitizing and anti-oxidative drug with already shown positive results in endometriotic tissue was studied. METHODS: Thirty-six female B6CBA/F1 mice were divided into 4 groups (Control-C, Surgery-induced Endometriosis and Metformin-EM (50 mg/kg/day orally administrated for 3 months), Endometriosis-E and Metformin-M). Immunofluorescent labelling of SIRT1 and SIRT3 was performed in the heart tissue. Assessment of expression of AMPKα, SIRT1, PGC-1α, SIRT3, SOD2, and GPx1 was performed by Western Blotting. The quantification of microRNA(miR)-34a, miR-195, miR-217, miR-155 and miR-421, involved in the regulation of expression of SIRT1 and SIRT3, was performed by Real-Time PCR. RESULTS: Data showed an increase in phospho-AMPKα and in GPx1 expression in the EM group when compared to the C group, but not in the total AMPK, SIRT1, PGC-1α, SIRT3 and SOD2, suggesting a GPx1 expression increase independently of the AMPK/SIRT1/PGC-1α/SIRT3 pathway. MicroRNAs, excepting miR-217, showed a consistent trend of increase in the M group. CONCLUSIONS: Our study showed that endometriosis does not significantly affect the expression of the components of the AMPK/SIRT1/PGC-1α/SIRT3 pathway in the heart. However, it indicates that an oxidative condition underlying endometriosis is required for metformin to evidence an increment in the expression of the anti-oxidative enzyme GPx1.

Characterization of α-MSH browning effect in diverse mice white adipose tissue depots.

White adipose tissue (WAT) browning is a potent mechanism to dissipate energy as heat and, thus, its activation constitutes a promise therapeutic approach to obesity. We previously reported the melanocortin α-melanocyte stimulating hormone (α-MSH) ability to increase the number of beige cells in subcutaneous inguinal WAT (ingWAT) in high fat diet (HFD)-fed mice. The current study examined the browning effect of intraperitoneally administered α-MSH on diverse fat depots from mice fed with HFD or standard rodent diet (SD). For this, mRNA expression of browning hallmark genes was quantified concomitantly with histological examination of the adipose tissue samples (epidydimal (eWAT), mesenteric (mWAT), retroperitoneal (rpWAT) or ingWAT). As well, α-MSH impact on body weight, serum profile, WAT mass and lipolytic rates were evaluated. In the visceral depots mWAT, eWAT and rpWAT from HFD-fed mice, α-MSH was not able to induce a browning mechanism. Surprisingly, in SD-fed mice, α-MSH decreased the expression of several beige-specific genes in rpWAT and promoted an increase of the size of lipid droplets. No browning effect was observed in ingWAT from SD-fed mice. We also verified that HFD ingestion per se stimulated the browning mechanisms in rpWAT, but not in mWAT and eWAT. In conclusion, the fat depots from diverse anatomical locations respond differently to α-MSH treatment when exposed to different diets.

Cumulative Effect of Cardiovascular Risk Factors on Regulation of AMPK/SIRT1-PGC-1α-SIRT3 Pathway in the Human Erectile Tissue.

Cardiovascular disease risk factors (CVDRF), especially diabetes mellitus (DM), disrupt oxidative stress response. This condition underlies endothelial dysfunction, early manifested in men as erectile dysfunction. The current study is aimed at elucidating the impact of CVDRF in the oxidation responsive AMPK/SIRT1-PGC-1α-SIRT3 pathway and related miRNAs in the human corpus cavernosum. Human penile tissue fragments from individuals submitted to programmed urological surgeries (n = 27), aged 43-63 years, were clustered depending on the presence of CVDRF; the control group included samples from patients without CVDRF, and groups A and B included samples from patients with DM and additional CVDRF, totalizing ≤2 CVDRF (group A) and ≥3 CVDRF (group B). Dual-immunolabelling of SIRT3, SOD2, or GPX1 with α-actin in tissue sections was carried out. The assessment of expression levels of NOX1, phospho-AMPKα, total AMPKα, SIRT1, PGC-1α, SIRT3, SOD2, and GPX1 was performed by western blotting and of miR-200a, miR-34a, miR-421, and miR-206 by real-time PCR. Phospho-AMPKα and SIRT3 expression was found significantly increased in group B relative to other groups, suggesting a marked influence of CVDRF, additional to DM, in the regulation of these enzymes. NOX1 was also increased in group B relative to controls. Only an increasing tendency was observed in the phospho-AMPKα/total AMPKα ratio, SIRT1, and PGC-1α expression in groups A and B when compared with controls. Concerning antioxidant enzymes, GPX1 expression was found incremented in group A, but SOD2 expression was decreased in groups A and B, comparative with controls. Group B presented significantly diminished levels of miR-421 and miR-200a, but only a decreasing trend on miR-34 and miR-206 expression was observed. Taken together, our findings demonstrated that besides DM, additional CVDRF presented a cumulative effect in the cellular response to oxidative unbalance, contributing to AMPK/SIRT1-PGC-1α-SIRT3 pathway activation. SOD2, a major mitochondrial antioxidant defence, did not follow the same variation.

Peripherally administered melanocortins induce mice fat browning and prevent obesity.

BACKGROUND/OBJECTIVES: The browning of white adipose tissue (WAT) has been in the spotlight during the last years, becoming an attractive approach to combat obesity. Melanocortin neuropeptides, such as α-melanocyte-stimulating hormone (α-MSH), are well-known regulators of appetite at the central nervous system, but its role in adipocyte metabolism is poorly elucidated. This study sought to verify if α-MSH can induce transdifferentiation of white to brown/beige adipocytes and to determine whether it can ameliorate the obesity phenotype. METHODS: The browning effect of α-MSH was determined in isolated adipocytes using the 3T3-L1 cell line and in inguinal subcutaneous adipose tissue (ingWAT) of diet-induced obese (DIO) mice by quantifying the expression of browning hallmark genes, oxygen consumption, and mitochondrial biogenesis. α-MSH protection from diet-induced obesity was evaluated by analyzing mice body weight, fat mass, and lipid and glucose serum profiles. RESULTS: Here, we report that α-MSH activates a thermogenic gene program and increases the mitochondrial respiratory rate in 3T3-L1 adipocytes and ingWAT of DIO mice. Without affecting food intake, peripheral administration of α-MSH decreases body weight and ingWAT mass, promoting a significant rise in the number of smaller adipocytes, whereas it lowered the larger ones. Additionally, there was an increase in the mass of brown adipose tissue. Browning activation occurs concomitantly with improvement on serum lipid profile, insulin resistance, and glucose homeostasis. CONCLUSIONS: This study highlights the anti-obesity properties of melanocortins by promoting ingWAT browning and provides new perspectives for future designing of more effective therapeutic strategies.

Characterization of TGF-ß expression and signaling profile in the adipose tissue of rats fed with high-fat and energy-restricted diets.

Transforming growth factor beta (TGF-ß) plays an important role in the pathogenesis of obesity, influencing the release of inflammation mediators and promoting remodeling and collagen deposition in the adipose tissue (AT). In this context, this work aims to elucidate whether TGF-ß and Smad-dependent or Smad-independent signaling pathways contribute to regional differentiation of AT in high-fat diet (HFD) and energy-restricted (ER) rat models. For this, TGF-ß, TGF-ß receptors I and II, PAI-1 and GLUT4 mRNA levels were quantified by real-time PCR, and western blotting assays allowed the semiquantification of TGF-ß and proteins from Smad3, ERK1/2 and Akt signaling pathways in subcutaneous and visceral (epididymal, retroperitoneal and mesenteric) fat depots from control, HFD and ER-treated rats. HFD was associated to increased levels of TGF-ß and PAI-1 mRNA in epididymal and retroperitoneal visceral fat depots, while ER diet induced a reduction of TGF-ß mRNA levels in mesenteric, but surprisingly an increase in retroperitoneal fat. Regarding the different signaling pathways, contrarily to what was found for Smad3, activation of ERK1/2 and Akt in response to HFD was detected in all the visceral but not in subcutaneous fat depots. ER-treated rats presented a more heterogeneous signaling response, as well as decreased TGF-ß receptors expression, in the different visceral fat depots. In conclusion, subcutaneous and visceral AT respond differently to distinct diet patterns regarding TGF-ß expression and activated signaling pathways. Furthermore, the present study points that visceral AT should not be understood as a homogeneous entity since that response also varied in the different fat depots.

Effects of Aging and Cardiovascular Disease Risk Factors on the Expression of Sirtuins in the Human Corpus Cavernosum.

INTRODUCTION: Sirtuin (SIRT)1 was recently identified in human corpus cavernosum (CC). We hypothesized that other sirtuins could also be expressed in the CC. Expression of these enzymes in tissues is affected by aging, the main independent risk factor for erectile dysfunction besides other cardiovascular disease risk factors (CVDRF), such as diabetes or obesity. AIM: The aim of this study was to characterize the expression of SIRT1-3 and SIRT5-7 in human CC relatively to age and CVDRF. METHODS: Samples of CC collected from patients submitted to programmed surgeries or organ donors were divided in three groups according to age and presence of CVDRF. Expression of SIRT1-3 and SIRT5-7 mRNAs was assessed by real-time polymerase chain reaction. Cellular localization and semi-quantification of sirtuins proteins were performed by immunofluorescence and Western blotting (WB), respectively. Nuclear factor kappa B (NFkB)-p65, inducible (iNOS) and endothelial nitric oxide synthase (eNOS) levels were also assayed by WB. MAIN OUTCOME MEASURES: The main outcome measure was to characterize the expression of SIRT1-3 and SIRT5-7 in human CC. RESULTS: SIRT1-3 and SIRT5-7 mRNAs were detected in all individuals, without statistical differences among groups, excepting SIRT7 that decreased four times in aged groups relatively to young (P = 0.013). WB analysis demonstrated that aged individuals with CVDRF presented higher levels of SIRT7 protein relatively to young (P = 0.0495) and lower levels of SIRT3 protein relatively to healthy aged (P = 0.0077). Expression of NFkB-p65 and iNOS were higher in aged than in young individuals (P = 0.0185; P = 0.004, respectively). No differences in other sirtuins or total eNOS were seen among groups although phospho eNOS Ser(1177) levels decreased in groups of aged men relatively to young (P = 0.0043; P = 0.0099). CONCLUSIONS: Our results demonstrate for the first time expression of SIRT2-3 and SIRT5-7 in the human CC. Aged individuals with CVDRF presented an increase in SIRT7 protein levels and a decrease in mitochondrial SIRT3. This finding suggests that CVDRF induces the loss of antioxidant defense mechanisms leading to endothelial injury.

Intracellular signaling mechanisms of the melanocortin receptors: current state of the art.

The melanocortin system is composed by the agonists adrenocorticotropic hormone and α, ß and γ-melanocyte-stimulating hormone, and two naturally occurring antagonists, agouti and agouti-related protein. These ligands act by interaction with a family of five melanocortin receptors (MCRs), assisted by MCRs accessory proteins (MRAPs). MCRs stimulation activates different signaling pathways that mediate a diverse array of physiological processes, including pigmentation, energy metabolism, inflammation and exocrine secretion. This review focuses on the regulatory mechanisms of MCRs signaling, highlighting the differences among the five receptors. MCRs signal through G-dependent and independent mechanisms and their functional coupling to agonists at the cell surface is regulated by interacting proteins, namely MRAPs and ß-arrestins. The knowledge of the distinct modulation pattern of MCRs signaling and function may be helpful for the future design of novel drugs able to combine specificity, safety and effectiveness in the course of their therapeutic use.

Effect of aging and cardiovascular risk factors on receptor Tie1 expression in human erectile tissue.

INTRODUCTION: Erectile dysfunction is highly prevalent in patients with advanced age or cardiovascular disease risk factors (CVDRFs). These conditions interfere on expression of vascular growth factors and respective receptors causing disturbance in endothelial function. AIM: This study aims to assess the effect of aging and CVDRF on the expression of tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie) 1 in human corpus cavernosum (CC). METHODS: CC fragments obtained from programmed surgeries or organ donors were divided into three groups: young, healthy aged, and aged with CVDRF. Angiopoietin (Ang) 1, Ang2, Tie1, and Tie2 mRNA and protein levels were assessed by real-time polymerase chain reaction and Western blotting, respectively. Dual-immunolabeling of Tie1 with specific markers of endothelium and smooth muscle and Ang1 and Ang2 was performed. MAIN OUTCOME MEASURES: To characterize the expression of Tie1 in human CC and elucidate its potential inhibitory effect in Ang-Tie2 system. RESULTS: Analysis of mRNAs demonstrated a decrease in Tie1 expression in CVDRF individuals compared with aged or young healthy individuals. No variation for Tie2, Ang1, or Ang2 expression was observed among the studied groups. In all analyzed CC fragments, a 125 kDa band, Tie1, was detected. This protein presented a significant age-related decrease, specially in individuals with CVDRF. Immunofluorescence study revealed Tie1 expression in the endothelium of samples of all experimental groups. CONCLUSIONS: Employing different methodological approaches, we show for the first time that Tie1 is expressed in human CC endothelium, and its level of expression diminishes in aged individuals, particularly those with CVDRF. This finding reinforces the view that delivery of Ang1 to the CC of erectile dysfunction affected CVDRF patients is able to activate a beneficial Tie2 response.

Alpha-MSH signalling via melanocortin 5 receptor promotes lipolysis and impairs re-esterification in adipocytes.

The melanocortin system has a clear effect on the mobilisation of stored lipids in adipocytes. The aim of the current study was to investigate the role of melanocortin 5 receptor (MC5R) on alpha-melanocyte-stimulating hormone (alpha-MSH)-induced lipolysis in 3T3-L1 adipocytes. To this end, MC5R expression was decreased by small interfering RNA (siRNA), which significantly impaired the alpha-MSH stimulation of lipolysis, as determined by glycerol and nonesterified fatty-acid (NEFA) quantification. The functional role of alpha-MSH/MC5R on triglyceride (TG) hydrolysis was mediated by hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), perilipin 1 (PLIN1) and acetyl-CoA carboxylase (ACC). Immunofluorescence microscopy revealed that phosphorylated HSL clearly surrounded lipid droplets in alpha-MSH-stimulated adipocytes, whereas PLIN1 left the immediate periphery of lipids. These observations were lost when the expression of MC5R was suppressed. In 3T3-L1 adipocytes, alpha-MSH-activated MC5R signals through the cAMP/PKA and MAPK/ERK1/2 pathways. PKA was fundamental for HSL and PLIN1 activation and lipolysis regulation. ERK1/2 inhibition strongly interfered with the release of NEFAs but not glycerol. In addition, the intracellular TG levels, which were decreased after MC5R activation, were restored after ERK1/2 inhibition, indicating that these kinases are involved in NEFA re-esterification rather than lipolysis regulation. This notion is also supported by the observation that the alpha-MSH-mediated activation of phosphoenolpyruvate carboxykinase (PEPCK) was abolished in the presence of ERK1/2 inhibitors. Altogether, these results indicate that alpha-MSH-activated MC5R regulates two tightly coupled pathways in adipocytes: lipolysis and re-esterification. The global effect is a decrease in adipocyte fat mass, which is important for strategies to ameliorate obesity.

Melanocortin 5 receptor signaling and internalization: role of MAPK/ERK pathway and ß-arrestins 1/2.

The Melanocortin 5 receptor (MC5R) is a G-protein coupled receptor (GPCR) that exhibits high affinity for α-MSH. Here we present evidence for MC5R-GFP internalization and subsequent recycling to cell surface, in α-MSH-stimulated HeLa cells. This melanocortin induces a biphasic activation of ERK1/2 with an early peak at 15min, a G(i)-protein driven, ß-arrestins 1/2 independent process, and a late sustained activation that is regulated by ß-arrestins 1/2. ERK1/2 lead to downstream phosphorylation of 90-kDa ribosomal S6 kinases (p90RSK) and mitogen- and stress-activated protein kinase 1 (MSK1). Only a small fraction (10%) of phosphorylated p90RSK and ERK1/2 translocates to the nucleus inducing c-Fos expression. α-MSH also activates CREB through cAMP/PKA pathway. In 3T3-L1 adipocytes, where MC5R is endogenously expressed, α-MSH also induces phosphorylation and cytosolic retention of the same signaling molecules. These findings provide new evidence on the signaling mechanisms underlying MC5R biological response to α-MSH.

Melanocortin 5 receptor activates ERK1/2 through a PI3K-regulated signaling mechanism.

Melanocortin 5 receptor (MC5R) is a G protein coupled receptor (GPCR) with high affinity for alpha-melanocyte-stimulating hormone (alpha-MSH). To unravel some of the downstream cell-signaling pathways activated by this receptor, HEK293 cells were transiently and stably transfected with a vector encoding green fluorescent protein (GFP)-tagged MC5R. In these cells the receptor was correctly addressed to the cell surface and was functional, as shown by the MC5R-induced formation of intracellular cAMP. In fact, the MC5R agonist alpha-MSH induced a 10- or 16-fold increase (transient or stable cells, respectively) above the cAMP levels found in unstimulated cells. Moreover, in cells stably expressing MC5R-GFP, alpha-MSH promoted ERK1/2 phosphorylation in a dose-dependent manner (EC50=7.3 nM) with the maximal effect occurring after 5 min of agonist incubation. The signaling pathway conveyed through ERK1/2 is not linked to cAMP, since the phosphorylation of these kinases is unchanged by the inhibition of adenylyl cyclase. Also, ERK1/2 activation is not significantly affected by protein kinase A (PKA), protein kinase C (PKC) and protein kinase B or Akt (Akt/PKB) specific inhibitors. However, alpha-MSH-induced ERK1/2 activation is abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Altogether, these findings demonstrate that MC5R signals through a PI3K-regulated Akt-independent pathway leading to downstream activation of ERK1/2. The involvement of these MAPK suggests that MC5R could be implicated in cellular proliferation or differentiation mechanisms.

Linfoma não-Hodgkin em crianças com imunodeficiência: relato de cinco casos / Non-Hodgkin's lymphoma in children with immunodeficiency: report of five cases

Neste estudo é relatado o quadro clínico de cinco crianças com linfoma não-Hodgkin secundário a imunodeficiência ou imunossupressão: três portadoras do vírus da imunodeficiência humana, uma com imunodeficiência primária e uma após transplante hepático. De acordo com a classificação atual, os tipos histológicos foram: linfoma linfoblástico de células B precursoras (2), linfoma cutâneo de grandes células anaplásico (1), linfoma de células B periféricas, sugestivo de Burkitt (1), e linfoma linfoblástico de células T precursoras (1). Todos os pacientes foram submetidos a quimioterapia, sendo que dois estão em remissão clínica, dois morreram e um continua em tratamento. Após a introdução da terapia anti-retroviral combinada e o aumento dos transplantes de órgãos sólidos ocorre maior risco de neoplasia nesses pacientes. Desse modo, é importante o seguimento desses pacientes para determinar os fatores de risco para o desenvolvimento de neoplasias e definir adequada estratégia de tratamento.

The outcomes of five children with non-Hodgkin's lymphomas associated with immunodeficiency or immunosuppression is reported: three children with HIV, one with primary immunodeficiency and one after liver transplantation. According to the REAL classification, two patients had precursor B-lymphoblastic lymphomas, one had an anaplastic large cell lymphoma, one had a peripheral B-cell neoplasm suggestive of Burkitt's lymphoma, and one had precursor T-lymphoblastic lymphoma. All patients received chemotherapy. Two are in complete remission, two died and one remains under treatment. There has been an increasing awareness of the risk of non-Hodgkin's lymphoma and lymphoproliferative disorders after active antiretroviral therapy and with the expansion of solid organ transplant programs in the pediatric setting. Thus, for these patients it is important to establish risk factors for hematological disorders and determine the optimal and safest treatment.