Depot-specific modulation of adipokine levels in rat adipose tissue by diet-induced obesity: The effect of aerobic training and energy restriction

The present study examined the effects of aerobic training and energy restriction on adipokines levels in mesenteric (MEAT) and retroperitoneal (RPAT) white adipose tissue from obese rats. Male Wistar rats were fed with standard laboratory diet (Control group) or high fat diet (HFD). After 15 weeks, HFD rats were randomly assigned to the following groups: rats submitted to HFD, which were sedentary (sedentary HFD, n = 8) or trained (trained HFD, n = 8); or submitted to energy-restriction (ER), which were sedentary (sedentary ER, n = 8) or trained (trained ER, n = 8). Trained rats ran on a treadmill at 55% VO(2max) for 60 min/day, 5 days/week, for 10 weeks. ER rats were submitted to a reduction of 20% daily caloric ingestion compared to the Control group. ER and aerobic training decreased body weight, MEAT and RPAT absolute weight, and fat mass. IL-6, IL-10 and TNF-alpha levels were decreased and adiponectin did not change in RPAT in response to ER protocol. On the other hand, ER and the aerobic training protocol decreased IL-6, TNF-alpha and adiponectin levels in MEAT. Absolute MEAT weight showed a positive correlation with IL-6 (r = 0.464), INF-alpha (r = 0.508); and adiponectin (r = 0.342). These results suggest a tissue-specific heterogeneous response in adipokines level. The combination of the protocols (aerobic training and energy restriction) did not induce an enhanced effect. Published by Elsevier Ltd.

In vitro basal cytotoxicity assay applied to estimate acute oral systemic toxicity of grandisin and its major metabolite

Preclinical investigations can start with preliminary in vitro studies before using animal models. Following this approach, the number of animals used in preclinical acute toxicity testing can be reduced. In this study, we employed an in-house validated in vitro cytotoxicity test based on the Spielmann approach for toxicity evaluation of the lignan grandisin, a candidate anticancer agent, and its major metabolite. the 4-O-demethylgrandisin, by neutral red uptake (NRU) assay, on mouse fibroblasts Balb/c 3T3 cell line. Using different concentrations of grandisin and its major metabolite (2.31; 1.16; 0.58; 0.29; 0.14; 0.07; 0.04; 0.002 mu M) in Balb/c 3T3-A31 NRU cytotoxicity assay, after incubation for 48 h, we obtained IC(50) values for grandisin and its metabolite of 0.078 and 0.043 mu M, respectively. The computed LD(50) of grandisin and 4-O-demethylgrandisin were 617.72 and 429.95 mg/kg, respectively. Both were classified under the Globally Harmonized System as category 4. Since pharmacological and toxicological data are crucial in the developmental stages of drug discovery, using an in vitro assay we demonstrated that grandisin and its metabolite exhibit distinct toxicity profiles. Furthermore, results presented in this work can contribute to reduce the number of animals required in subsequent pharmacological/toxicological studies. (C) 2010 Elsevier GmbH. All rights reserved.

Produção e caracterização de quitooligossacarídeos produzidos pelo fungo Metarhizium anisopliae e avaliação da citotoxicidade em células tumorais

Chitosan is a natural polymer, biodegradable, nontoxic, high molecular weight derived from marine animals, insects and microorganisms. Oligomers of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) have interesting biological activities, including antitumor effects, antimicrobial activity, antioxidant and others. The alternative proposed by this work was to study the viability of producing chitooligosaccharides using a crude enzymes extract produced by the fungus Metarhizium anisopliae. Hydrolysis of chitosan was carried out at different times, from 10 to 60 minutes to produce chitooligosaccharides with detection and quantification performed by High Performace Liquid Chromatography (HPLC). The evaluation of cytotoxicity of chitosan oligomers was carried out in tumor cells (HepG2 and HeLa) and non-tumor (3T3). The cells were treated for 72 hours with the oligomers and cell viability investigated using the method of MTT. The production of chitosan oligomers was higher for 10 minutes of hydrolysis, with pentamers concentration of 0.15 mg/mL, but the hexamers, the molecules showing greater interest in biological properties, were observed only with 30 minutes of hydrolysis with a concentration of 0.004 mg/mL. A study to evaluate the biological activities of COS including cytotoxicity in tumor and normal cells and various tests in vitro antioxidant activity of pure chitosan oligomers and the mixture of oligomers produced by the crude enzyme was performed. Moreover, the compound with the highest cytotoxicity among the oligomers was pure glucosamine, with IC50 values of 0.30; 0.49; 0.44 mg/mL for HepG2 cells, HeLa and 3T3, respectively. Superoxide anion scavenging was the mainly antioxidant activity showed by the COS and oligomers. This activity was also depending on the oligomer composition in the chitosan hydrolysates. The oligomers produced by hydrolysis for 20 minutes was analyzed for the ability to inhibit tumor cells showing inhibition of proliferation only in HeLa cells, did not show any effect in HepG2 cells and fibroblast cells (3T3)

Modulation of thyroid hormone receptors, TR alpha and TR beta, by using different doses of triiodothyronine (T3) at different times

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Modulation of thyroid hormone receptors, TRα and TRβ, by using different doses of triiodothyronine (T3) at different times

Objective: To examine the effect of different doses of triiodothyronine (T3) on mRNA levels of thyroid hormone receptors, TRα and TRβ, at different times. Materials and methods: 3T3-L1 adipocytes were incubated with T3 (physiological dose: F; supraphysiological doses: SI or SII), or without T3 (control, C) for 0.5, 1, 6, or 24h. TRα and TRβ mRNA was detected using real-time polymerase chain reaction. Results: F increased TRβ mRNA levels at 0.5h. After 1h, TRα levels increased with F and SI and TRβ levels decreased with SII compared with C, F, and SI. After 6h, both genes were suppressed at all concentrations. In 24h, TRα and TRβ levels were similar to those of C group. Conclusions: T3 action with F began at 1h for TRα and at 0.5h for TRβ. These results suggest the importance of knowing the times and doses that activate T3 receptors in adipocytes.

Endostatin gene therapy stimulates upregulation of ICAM-1 and VCAM-1 in a metastatic renal cell carcinoma model

One of the greatest challenges in urological oncology is renal cell carcinoma (RCC), which is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and respond positively to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of ES-based antiangiogenic therapy to activate tumor-associated endothelial cells in metastatic RCC (mRCC). Balb/c-bearing Renca cells were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. The T-cell subsets and lymphocyte populations of tumors, mediastinal lymph nodes and the spleen were assessed by flow cytometry. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed by real-time PCR, flow cytometry and immunohistochemistry analysis. ES gene therapy led to an increase in the percentage of infiltrating CD4-interferon (IFN)-gamma cells (P<0.05), CD8-IFN-gamma cells (P<0.01) and CD49b-tumor necrosis factor-alpha cells (P<0.01). In addition, ES therapy caused an increase at the mRNA level of ICAM-1 (1.4-fold; P<0.01) and VCAM-1 (1.5-fold) (control vs treated group; P<0.001). Through flow cytometry, we found a significant increase in the CD34/ICAM-1 cells (8.1-fold; P<0.001) and CD34/VCAM-1 cells (1.6-fold; P<0.05). ES gene therapy induced a significant increase in both T CD4 and CD8 cells in the lymph nodes and the spleen, suggesting that ES therapy may facilitate cell survival or clonal expansion. CD49b cells were also present in increased quantities in all of these organs. In this study, we demonstrate an antitumor inflammatory effect of ES in an mRCC model, and this effect is mediated by an increase in ICAM-1 and VCAM-1 expression in tumor-associated endothelial cells.

DEPTOR Cell-Autonomously Promotes Adipogenesis, and Its Expression Is Associated with Obesity

DEP domain-containing mTOR-interacting protein (DEPTOR) inhibits the mechanistic target of rapamycin (mTOR), but its in vivo functions are unknown. Previous work indicates that Deptor is part of the Fob3a quantitative trait locus (QTL) linked to obesity/leanness in mice, with Deptor expression being elevated in white adipose tissue (WAT) of obese animals. This relation is unexpected, considering the positive role of mTOR in adipogenesis. Here, we dissected the Fob3a QTL and show that Deptor is the highest-priority candidate promoting WAT expansion in this model. Consistently, transgenic mice overexpressing DEPTOR accumulate more WAT. Furthermore, in humans, DEPTOR expression in WAT correlates with the degree of obesity. We show that DEPTOR is induced by glucocorticoids during adipogenesis and that its overexpression promotes, while its suppression blocks, adipogenesis. DEPTOR activates the proadipogenic Akt/PKB-PPAR-gamma axis by dampening mTORC1-mediated feedback inhibition of insulin signaling. These results establish DEPTOR as a new regulator of adipogenesis.

Identification of intracellular peptides in rat adipose tissue: Insights into insulin resistance

Intracellular peptides generated by the proteasome and oligopeptidases have been suggested to function in signal transduction and to improve insulin resistance in mice fed a high-caloric diet. The aim of this study was to identify specific intracellular peptides in the adipose tissue of Wistar rats that could be associated with the physiological and therapeutic control of glucose uptake. Using semiquantitative mass spectrometry and LC/MS/MS analyses, we identified ten peptides in the epididymal adipose tissue of the Wistar rats; three of these peptides were present at increased levels in rats that were fed a high-caloric Western diet (WD) compared with rats fed a control diet (CD). The results of affinity chromatography suggested that in the cytoplasm of epididymal adipose tissue from either WD or CD rats, distinctive proteins bind to these peptides. However, despite the observed increase in the WD animals, the evaluated peptides increased insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with palmitate. Thus, intracellular peptides from the adipose tissue of Wistar rats can bind to specific proteins and facilitate insulin-induced glucose uptake in 3T3-L1 adipocytes.

A Low-Protein, High-Carbohydrate Diet Increases Fatty Acid Uptake and Reduces Norepinephrine-Induced Lipolysis in Rat Retroperitoneal White Adipose Tissue

A low-protein, high-carbohydrate (LPHC) diet for 15 days increased the lipid content in the carcass and adipose tissues of rats. The aim of this work was to investigate the mechanisms of this lipid increase in the retroperitoneal white adipose tissue (RWAT) of these animals. The LPHC diet induced an approximately two- and tenfold increase in serum corticosterone and TNF-alpha, respectively. The rate of de novo fatty acid (FA) synthesis in vivo was reduced (50%) in LPHC rats, and the lipoprotein lipase activity increased (100%). In addition, glycerokinase activity increased (60%), and the phosphoenolpyruvate carboxykinase content decreased (27%). Basal [U-C-14]-glucose incorporation into glycerol-triacylglycerol did not differ between the groups; however, in the presence of insulin, [U-C-14]-glucose incorporation increased by 124% in adipocytes from only control rats. The reductions in IRS1 and AKT content as well as AKT phosphorylation in the RWAT from LPHC rats and the absence of an insulin response suggest that these adipocytes have reduced insulin sensitivity. The increase in NE turnover by 45% and the lack of a lipolytic response to NE in adipocytes from LPHC rats imply catecholamine resistance. The data reveal that the increase in fat storage in the RWAT of LPHC rats results from an increase in FA uptake from circulating lipoproteins and glycerol phosphorylation, which is accompanied by an impaired lipolysis that is activated by NE.

Fibronectin expression is decreased in metastatic renal cell carcinoma following endostatin gene therapy

Tumor cells induce the disruption of homeostasis between cellular and extracellular compartments to favor tumor progression. The expression of fibronectin (FN), a matrix glycoprotein, is increased in several carcinoma cell types, including renal cell carcinoma (RCC). RCC are highly vascularized tumors and are often amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the modulation of FN gene expression by ES gene therapy in a murine metastatic renal cell carcinoma (mRCC) model. Balb/C mice bearing Renca cells were treated with NIH/3T3-LXSN cells or NIH/3T3-LendSN cells. At the end of the experiment, the ES serum levels were measured, and the FN gene expression was assessed using real-time PCR. The tissue FN was evaluated by western blotting and by immunofluorescence analysis. The ES serum levels in treated mice were higher than those in the control group (P < 0.05). ES treatment led to significant decreases at the FN mRNA (P < 0.001) and protein levels (P < 0.01). Here, we demonstrate the ES antitumor effect that is mediated by down-regulation of FN expression in mRCC. (C) 2012 Elsevier Masson SAS. All rights reserved.

Analyse der Mechanismen zur Regulation der Zellzyklus-abhängigen Stabilität der MARCKS-mRNA

Die Expression des PKC-Hauptsubstrates MARCKS (myristoylated alanine-rich C kinase substrate) wird in Swiss 3T3-Fibroblasten in Abhängigkeit des Zellzyklus durch Variation der mRNA-Stabilität reguliert. In der vorliegenden Arbeit wurde die Beteiligung der 3' nichttranslatierten Region (3'UTR) der MARCKS-mRNA an der Stabilitätskontrolle analysiert. Durch Einsatz der RNase/EMSA-Technik konnten zwei cis-Elemente der MARCKS 3'UTR identifiziert und lokalisiert werden, die mit RNA-bindenden Swiss 3T3-Proteinen (trans-Faktoren) interagieren. Diese neu identifizierten cis-Elemente sind AU-reiche Elemente (ARE) der Klasse III, da sie sehr große Sequenzhomologie zu ARE dieser Klasse aufweisen und der MARCKS 3'UTR, wie für ARE typisch, Instabilität vermitteln.Durch UV-crosslinking wurden vier Proteine mit Molekülmassen von 55, 40, 36 und 30 kDa nachgewiesen, die spezifisch an das 52nt lange Haupt-ARE (MARCKS 52nt) mit unterschiedlicher Affinität binden konnten. Mit Hilfe von rekombinant hergestellten ELAV/Hu-Proteinen und einem ELAV/Hu-spezifischen, affinitätsgereinigten Antiserum konnte eines der vier Proteine (p36) als das ELAV/Hu-Protein HuR identifiziert werden. Die Funktion der ELAV/Hu-Proteine für die Stabilitätskontrolle der MARCKS-mRNA ließ sich durch transiente und stabile Transfektion von HuR und neuronenspezifischem HuD mit dem Tetracyclin induzierbaren Expressionssystem (Tetoff) in Swiss 3T3- bzw. MEF/3T3-Tetoff-Zellen verdeutlichen: Durch Überexpression von HuR und HuD wurde die wachstumsinduzierte Destabilisierung der MARCKS-mRNA bei Wiedereintritt der Zellen in den Zellzyklus unterbunden.

Protein Interactions with the Glucose Transporter Binding Protein GLUT1CBP That Provide a Link between GLUT1 and the Cytoskeleton

Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin–Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, α-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.

The RD114/simian type D retrovirus receptor is a neutral amino acid transporter

The RD114/simian type D retroviruses, which include the feline endogenous retrovirus RD114, all strains of simian immunosuppressive type D retroviruses, the avian reticuloendotheliosis group including spleen necrosis virus, and baboon endogenous virus, use a common cell-surface receptor for cell entry. We have used a retroviral cDNA library approach, involving transfer and expression of cDNAs from highly infectable HeLa cells to nonpermissive NIH 3T3 mouse cells, to clone and identify this receptor. The cloned cDNA, denoted RDR, is an allele of the previously cloned neutral amino acid transporter ATB0 (SLC1A5). Both RDR and ATB0 serve as retrovirus receptors and both show specific transport of neutral amino acids. We have localized the receptor by radiation hybrid mapping to a region of about 500-kb pairs on the long arm of human chromosome 19 at q13.3. Infection of cells with RD114/type D retroviruses results in impaired amino acid transport, suggesting a mechanism for virus toxicity and immunosuppression. The identification and functional characterization of this retrovirus receptor provide insight into the retrovirus life cycle and pathogenesis and will be an important tool for optimization of gene therapy using vectors derived from RD114/type D retroviruses.

Repression of transcription mediated by dual elements in the CCAAT/enhancer binding protein α gene

During adipocyte differentiation, the expression of C/EBPα is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPα gene led to the identification of a potential repressive element within the proximal 5′ flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPα undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPα promoter-luciferase constructs containing both the proximal 5′ flanking and the entire 5′ untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPα gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5′ untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5′ untranslated region. Although mutation of either CUP element in constructs containing both the 5′ flanking and 5′ untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPα gene prior to induction of the adipocyte differentiation program.

Cell locomotion and focal adhesions are regulated by substrate flexibility

Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism.