Johan Renes

The secretory function of adipocytes in the physiology of white adipose tissue.

White adipose tissue, previously regarded as a passive lipid storage site, is now viewed as a dynamic tissue. It has the capacity to actively communicate by sending and receiving different types of signals. An overview of these signals, the external modulators that affect adipose tissue and the secreted signaling molecules, the adipokines, is presented. The secretory function is highlighted in relation to energy metabolism, inflammation and the extracellular matrix and placed in the context of adipose tissue biology. We observe that the endocrine function of adipocytes receives much attention, while its paracrine and autocrine functions are underestimated. Also, we provide examples that species specificity should not be neglected. We conclude that adipose tissue primarily is an energy storage organ, well supported by its secretory function. J. Cell. Physiol. 216: 3–13, 2008.

ATP‐ and glutathione‐dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1

The present study was performed to investigate the ability of the multidrug resistance protein (MRP1) to transport different cationic substrates in comparison with MDR1‐P‐glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC4/Adr) and a MRP1‐transfected cell line (S1(MRP)). As substrates we used 3H‐labelled derivatives of the hydrophilic monoquaternary cation N‐(4′,4′‐azo‐n‐pentyl)‐21‐deoxy‐ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1‐substrates. MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP‐dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC4/Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1‐inhibitor MK571. ATP‐ and GSH‐dependent transport of daunorubicin and vincristine into GLC4/Adr membrane vesicles was inhibited by the MRP1‐specific monoclonal antibody QCRL‐3. MRP1‐mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations 10 mM. The apparent KM value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC50 of 0.4 μM. In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP‐ and GSH‐dependent manner. APDA is not a substrate for MRP1.

Profiling of the secreted proteins during 3T3-L1 adipocyte differentiation leads to the identification of novel adipokines

AbstractAdipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation was screened, and 41 proteins that responded to blocking of secretion by 20°C treatment and/or brefeldin A treatment were identified. Prohibitin, stress-70 protein, and adhesion-regulating molecule 1 are reported for the first time as secreted proteins. In addition, procollagen C-proteinase enhancer protein, galectin-1, cyclophilin A and C, and SF20/IL-25 are newly identified as adipocyte secreted factors. Secretion profiles indicated a dynamic environment including an actively remodeling extracellular matrix and several factors involved in growth regulation.

Proteomics Investigations of Drug-Induced Hepatotoxicity in HepG2 Cells.

Unexpected hepatotoxicity is one of the major reasons of drugs failing in clinical trials. This emphasizes the need for new screening methods that address toxicological hazards early in the drug discovery process. Here, proteomics techniques were used to gain further insight into the mechanistic processes of the hepatotoxic compounds. Drug-induced hepatotoxicity is mainly divided in hepatic steatosis, cholestasis, or necrosis. For each class, a compound was selected, respectively amiodarone, cyclosporin A, and acetaminophen. The changes in protein expressions in HepG2, after exposure to these test compounds, were studied using quantitative two-dimensional differential gel electrophoresis. Identification of differentially expressed proteins was performed by Maldi-TOF/TOF MS and liquid chromatography-tandem mass spectrometry. In this study, 254 differentially expressed protein spots were detected in a two-dimensional proteome map from which 86 were identified, showing that the proteome of HepG2 cells is responsive to hepatotoxic compounds. cyclosporin A treatment was responsible for most differentially expressed proteins and could be discriminated in the hierarchical clustering analysis. The identified differential proteins show that cyclosporin A may induce endoplasmic reticulum (ER) stress and disturbs the ER-Golgi transport, with an altered vesicle-mediated transport and protein secretion as result. Moreover, the differential protein pattern seen after cyclosporin A treatment can be related to cholestatic mechanisms. Therefore, our findings indicate that the HepG2 in vitro cell system has distinctive characteristics enabling the assessment of cholestatic properties of novel compounds at an early stage of drug discovery.

Comparative proteomic analysis of cell lines and scrapings of the human intestinal epithelium

BackgroundIn vitro models are indispensable study objects in the fields of cell and molecular biology, with advantages such as accessibility, homogeneity of the cell population, reproducibility, and growth rate. The Caco-2 cell line, originating from a colon carcinoma, is a widely used in vitro model for small intestinal epithelium. Cancer cells have an altered metabolism, making it difficult to infer their representativity for the tissue from which they are derived. This study was designed to compare the protein expression pattern of Caco-2 cells with the patterns of intestinal epithelial cells from human small and large intestine. HT-29 intestinal cells, Hep G2 liver cells and TE 671 muscle cells were included too, the latter two as negative controls.ResultsTwo-dimensional gel electrophoresis was performed on each tissue and cell line protein sample. Principal component and cluster analysis revealed that global expression of intestinal epithelial scrapings differed from that of intestinal epithelial cell lines. Since all cultured cell lines clustered together, this finding was ascribed to an adaptation of cells to culture conditions and their tumor origin, and responsible proteins were identified by mass spectrometry. When investigating the profiles of Caco-2 cells and small intestinal cells in detail, a considerable overlap was observed.ConclusionNumerous proteins showed a similar expression in Caco-2 cells, HT-29 cells, and both the intestinal scrapings, of which some appear to be characteristic to human intestinal epithelium in vivo. In addition, several biologically significant proteins are expressed at comparable levels in Caco-2 cells and small intestinal scrapings, indicating the usability of this in vitro model. Caco-2 cells, however, appear to over-express as well as under-express certain proteins, which needs to be considered by scientists using this cell line. Hence, care should be taken to prevent misinterpretation of in vitro obtained findings when translating them to the in vivo situation.

Transport of glutathione conjugates into secretory vesicles is mediated by the multidrug‐resistance protein 1

Intracellular glutathione‐conjugate transport was evaluated in the human small cell lung carcinoma cell line GLC4 with low multidrug resistance protein (MRP1) expression and its 300× doxorubicin‐resistant, MRP1‐over‐expressing, GLC4‐Adr subline. Transport of non‐toxic concentrations of monochlorobimane and 5‐chloro‐methyl fluorescein diacetate was evaluated using fluorescence microscopy. After exposure to these compounds, fluorescence was observed especially in intracellular vesicles in GLC4‐Adr. Immunotransmission electron microscopy showed that MRP1 was present in the vesicle membranes and plasma membrane, while inside the vesicles the glutathione conjugate of 1‐chloro‐2,4‐dinitrobenzene could be detected. Experiments with brefeldin A, which induces arrest in vesicle release from the Golgi complex, indicated that these vesicles may originate from the trans‐Golgi network. In GLC4‐Adr cells, doxorubicin also was transported in vesicles, with an arrest in vesicle release from the Golgi complex. Our results indicate that MRP1 functions as a glutathione‐conjugate transporter not only at the plasma membrane but also in intracellular secretory vesicles. Int. J. Cancer 76:55–62, 1998.© 1998 Wiley‐Liss, Inc.

Absence of an adipogenic effect of rosiglitazone on mature 3T3-L1 adipocytes: increase of lipid catabolism and reduction of adipokine expression

Aims/hypothesisThe thiazolidinedione (TZD) rosiglitazone is a peroxisome proliferator-activated receptor-γ agonist that induces adipocyte differentiation and, hence, lipid accumulation. This is in apparent contrast to the long-term glucose-lowering, insulin-sensitising effect of rosiglitazone. We tested whether the action of rosiglitazone involves specific effects on mature adipocytes, which are different from those on preadipocytes.Materials and methodsDifferentiated mature 3T3-L1 adipocytes were used as an in vitro model. Transcriptomics, proteomics and assays of metabolism were applied to assess the effect of rosiglitazone in different insulin and glucose conditions.ResultsRosiglitazone does not induce an increase, but rather a decrease in the lipid content of mature adipocytes. Analysis of transcriptome data, confirmed by quantitative RT-PCR and measurements of lipolysis, indicates that an altered energy metabolism may underlie this change. The pathway analysis shows a consistent picture dominated by lipid catabolism. In addition, we confirmed at both mRNA level and protein level that rosiglitazone represses adipokine expression and production, except for genes encoding adiponectin and apolipoprotein E. Moreover, transcriptome changes indicate that a general repression of genes encoding secreted proteins occurs.Conclusions/interpretationOur findings suggest that the change of adiposity as seen in vivo reflects a shift in balance between the different effects of TZDs on preadipocytes and on mature adipocytes, while the changes in circulating adipokine levels primarily result from an effect on mature adipocytes.

Identification of Novel Human Adipocyte Secreted Proteins by Using SGBS Cells

Adipose tissue is an endocrine organ secreting different types of proteins, known as adipokines. These adipokines play important roles in homeostasis and metabolism. Adipocyte differentiation leads to a change in adipokine secretion profile which is probably involved in disruption of homeostasis. Many adipokines have been identified but species differences and limitations of human adipose tissue material urged the need for better model systems. Here we used a human cell strain derived from a Simpson Golabi Behmel syndrome (SGBS) patient. SGBS cells have already been used in functional studies on adipocytes but not in a proteomic search for adipokines. In this study, 2D-MS/MS and nLC-MALDI-MS/MS were applied to investigate secretion profiles of SGBS adipokines. A total of 80 secreted proteins were identified; 6 proteins are novel adipocyte secreted proteins, 20 proteins have not been detected before in human adipose material and 23 additional proteins previously detected in visceral adipose tissue have been found here secreted by SGBS-cells of subcutaneous origin. It can be concluded that SGBS cells are both a valid human cell model for adipocyte secretion profiling and for searching for novel human (pre)adipocytes secreted proteins.

Proteomics in the search for mechanisms and biomarkers of drug-induced hepatotoxicity

The safety assessment for pharmaceuticals includes in vivo repeated dose toxicity tests in laboratory animals. These in vivo studies often generate false negative results and unexpected toxicity. The appearance of this unexpected toxicity is one of the major reasons for the drawback of a drug from the market. The liver is often a target organ in toxicology since it is responsible for the metabolism and elimination of chemical compounds. Therefore, there is need for new screening methods which classify hepatotoxic compounds earlier in development. This will lead to safer drugs and a more efficient drug discovery process. Furthermore, these new screening methods are preferably in vitro test systems, aiming at reducing the use of laboratory animals. In this review the possibilities of proteomics and its promising results for improving current predictive and mechanistic toxicological studies are described. Biomarkers or protein panels for hepatotoxic mechanisms, which reflect the in vivo situation, need to be identified to allow a better toxicity screening. Therefore, in vivo studies and in vitro cell models are discussed and evaluated with regard to the protein expression of their metabolic enzymes, their similarities with liver, their use for analyzing toxicological mechanisms and hepatotoxicity screening. Studies in which proteomics are combined with other omics-technologies are also presented. The results from these integrated data analyses can be used for the development of improved panels of biomarkers for toxicity screening.

Resveratrol-Induced Changes of the Human Adipocyte Secretion Profile

Enlarged white adipose tissue (WAT) is a feature of obesity and leads to changes in its paracrine and endocrine function. Dysfunction of WAT cells is associated with obesity-associated disorders like type 2 diabetes and cardiovascular diseases. Resveratrol (RSV), a natural polyphenolic compound, mimics beneficial effects of calorie restriction. As such, RSV seems a promising therapeutic target for obesity-associated disorders. The effect of RSV on the human adipokine profile is still elusive. Therefore, a proteomic study together with bioinformatical analysis was performed to investigate the effect of RSV on the secretion profile of mature human SGBS adipocytes. RSV incubation resulted in elevated basal glycerol release and reduced intracellular TG content. This increased intracellular lipolysis was accompanied by profound changes in the adipocyte secretion profile. Extracellular matrix proteins were down-regulated while processing proteins were mostly up-regulated after RSV treatment. Interestingly, RSV induced secretion of proteins protective against cellular stress and proteins involved in the regulation of apoptosis. Furthermore, we found a RSV-induced up-regulation of adiponectin and ApoE accompanied by a down-regulation of PAI-1 and PEDF secretion which may improve anti-inflammatory processes and increased insulin sensitivity. These effects may contribute to alleviate obesity-induced metabolic complications. In addition, two novel RSV-regulated adipocyte-secreted proteins were identified.