The role of CD1d in adipogenesis

Atherosclerosis-associated coronary heart disease is the number one cause of death in the western society, and often triggered by metabolic disorders, such as hyperlipidemia, hypertension, obesity, and diabetes. The CD1 molecules are a group of evolutionarily conservative transmembrane proteins that have recently emerged as novel lipid-binding and transporting molecules. The current study was aimed at illustrating the role of CD1d in regulation of lipid metabolism and adipogenesis. In stably transfected smooth muscle cells where CD1d is overexpressed via a pCMV promoter, high levels of binding of the oxysterol 7-ketocholesterol was found. Adipogenic treatment induces the cells to transdifferentiate into an adipocyte-like morphology. This adipocyte morphology of CD1d transfected SMCs strongly resembles that of the pre-adipocytes 3T3-L1 cells grown in the same adipogenic media. Adipogenic treatment of CD1d transfected 3T3-L1 cells led to an increased accumulation of lipids compared to mock transfected cells. Induction of adipogenic gene expression and activation, such as PPARγ and lipoprotein lipase (LPL), was achieved in adipogenically treated smooth muscle cells as well as 3T3-L1 cells with overexpression of CD1d. For determination of the role of CD1d in regulation of adipogenesis, a CD1d transgenic mouse strain was created using the CD1d-smooth muscle promoter construct. Compared to wild type control mice matched in age and sex, the transgenic mice show an age-dependent increase in abdominal and visceral fat tissue. Histopathological examination demonstrated marked enlargement of adipocytes in the transgenic fat tissue which otherwise remained a normal fat tissue structure. Immunohistochemical analysis of CD1d expression in the fat tissue revealed much stronger membrane CD1d immunostaining in the transgenic tissue than the wild type fat tissue. Under normal chow diets, CD1d-transgenic mice also developed fatty livers. In conclusion, CD1d serves as a regulator of lipid metabolism, which may transducer signals from oxysterols to induce expression of genes important in lipogenesis. These experimental results point to a novel mechanism by which CD1d mediates lipid metabolism in adipose tissue and contributes to the development of obesity. ^

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.

Development of an in vitro reconstitution assay for glucose transporter 4 translocation

In an attempt to define the mechanism of insulin-regulated glucose transporter 4 (Glut4) translocation, we have developed an in vitro reconstitution assay. Donor membranes from 3T3-L1 adipocytes transfected with mycGlut4 were incubated with plasma membrane (PM) from nontransfected 3T3-L1 cells, and the association was assessed by using two types of centrifugation assays. Association of mycGlut4 vesicles derived from donor membranes with the PM was concentration-, temperature-, time-, and Ca2+-dependent but ATP-independent. Addition of a syntaxin 4 fusion protein produced a biphasic response, increasing association at low concentration and inhibiting association at higher concentrations. PM from insulin-stimulated cells showed an enhanced association as compared with those from untreated cells. Use of donor membranes from insulin-stimulated cells further enhanced the association and also enhanced association to the PM from isolated rat adipocytes. Addition of cytosol, GTP, or guanosine 5′-[γ-thio]triphosphate decreased the association. In summary, insulin-induced Glut4 translocation can be reconstituted in vitro to a limited extent by using isolated membranes. This association appears to involve protein–protein interactions among the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. Finally, the ability of insulin to enhance association depends on insulin-induced changes in the PM and, to a lesser extent, in the donor membranes.

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.

Identification and characterization of an immunophilin expressed during the clonal expansion phase of adipocyte differentiation.

Mouse 3T3-L1 cells differentiate into fat-laden adipocytes in response to a cocktail of adipogenic hormones. This conversion process occurs in two discrete steps. During an early clonal expansion phase, confluent 3T3-L1 cells proliferate and express the products of the beta and delta members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. The cells subsequently arrest mitotic growth, induce the expression of the alpha form of C/EBP, and acquire the morphology of fully differentiated adipocytes. Many of the genes induced during the terminal phase of adipocyte conversion are directly activated by C/EBP alpha, and gratuitous expression of this transcription factor is capable of catalyzing adipose conversion in a number of different cultured cell lines. The genetic program undertaken during the clonal expansion phase of 3T3-L1 differentiation, controlled in part by C/EBP beta and C/EBP delta, is less clearly understood. To study the molecular events occurring during clonal expansion, we have identified mRNAs that selectively accumulate during this phase of adipocyte conversion. One such mRNA encodes an immunophilin hereby designated FKBP51. In this report we provide the initial molecular characterization of FKBP51.

Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.

Fibroblast growth factor 1: A key regulator of human adipogenesis

Obesity, with its related problems, is recognized as the fastest growing disease epidemic facing the world, yet we still have limited insight into the regulation of adipose tissue mass in humans. We have previously shown that adipose-derived microvascular endothelial cells (MVECs) secrete a factor(s) that increases proliferation of human preadipocytes. We now demonstrate that coculture of human preadipocytes with MVECs significantly increases preadipocyte differentiation, evidenced by dramatically increased triacylglycerol accumulation and glycerol-3-phosphate dehydrogenase activity compared with controls. Subsequent analysis identified fibroblast growth factor (FGF)-1 as an adipogenic factor produced by MVECs. Expression of FGF-1 was demonstrated in MVECs but not in preadipocytes, while preadipocytes were shown to express FGF receptors 1-4. The proliferative effect of MVECs on human preadipocytes was blocked using a neutralizing antibody specific for FGF-1. Pharmacological inhibition of FGF-1 signaling at multiple steps inhibits preadipocyte replication and differentiation, supporting the key adipogenic role of FGF-1. We also show that 3T3-L1 cells, a highly efficient murine model of adipogenesis, express FGF-1 and, unlike human preadipocytes, display no increased differentiation potential in response to exogenous FGF-1. Conversely, FGF-1-treated human preadipocytes proliferate rapidly and differentiate with high efficiency in a manner characteristic of 3T3-L1 cells. We therefore suggest that FGF-1 is a key human adipogenic factor, and these data expand our understanding of human fat tissue growth and have significant potential for development of novel therapeutic strategies in the prevention and management of human obesity.

The suitability of biodegradable poly(dl-lactide-co-glycolide)75:25 microspheres for the sustained release of antibiotics

Post-operative infections resulting from total hip arthroplasty are caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa entering the wound perioperatively or by haemetogenous spread from distant loci of infection. They can endanger patient health and require expensive surgical revision procedures. Gentamicin impregnated poly (methyl methacrylate) bone cement is traditionally used for treatment but is often removed due to harbouring bacterial growth, while bacterial resistance to gentamicin is increasing. The aim of this work was to encapsulate the antibiotics vancomycin, ciprofloxacin and rifampicin within sustained release microspheres composed of the biodegradable polymer poly (dl-lactide-co-glycolide) [PLCG] 75:25. Topical administration to the wound in hydroxypropylmethylcellulose gel should achieve high local antibiotic concentrations while the two week in vivo half life of PLCG 75:25 removes the need for expensive surgical retrieval operations. Unloaded and 20% w/w antibiotic loaded PLCG 75:25 microspheres were fabricated using a Water in Oil emulsification with solvent evaporation technique. Microspheres were spherical in shape with a honeycomb-like internal matrix and showed reproducible physical properties. The kinetics of in vitro antibiotic release into newborn calf serum (NCS) and Hank's balanced salt solution (HBSS) at 37°C were measured using a radial diffusion assay. Generally, the day to day concentration of each antibiotic released into NCS over a 30 day period was in excess of that required to kill St. aureus and Ps. auruginosa. Only limited microsphere biodegradation had occurred after 30 days of in vitro incubation in NCS and HBSS at 37°C. The moderate in vitro cytotoxicity of 20% w/w antibiotic loaded microspheres to cultured 3T3-L1 cells was antibiotic induced. In conclusion, generated data indicate the potential for 20% w/w antibiotic loaded microspheres to improve the present treatment regimens for infections occurring after total hip arthroplasty such that future work should focus on gaining industrial collaboration for commercial exploitation.

Zinc-α2-glycoprotein, a lipid mobilizing factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia

Zinc-α2-glycoprotein (ZAG), a 43-kDa protein, is overexpressed in certain human malignant tumors and acts as a lipid-mobilizing factor to stimulate lipolysis in adipocytes leading to cachexia in mice implanted with ZAG-producing tumors. Because white adipose tissue (WAT) is an endocrine organ secreting a wide range of protein factors, including those involved in lipid metabolism, we have investigated whether ZAG is produced locally by adipocytes. ZAG mRNA was detected by RT-PCR in the mouse WAT depots examined (epididymal, perirenal, s.c., and mammary gland) and in interscapular brown fat. In WAT, ZAG gene expression was evident in mature adipocytes and in stromal-vascular cells. Using a ZAG Ab, ZAG protein was located in WAT by Western blotting and immunohistochemistry. Mice bearing the MAC16-tumor displayed substantial losses of body weight and fat mass, which was accompanied by major increases in ZAG mRNA and protein levels in WAT and brown fat. ZAG mRNA was detected in 3T3-L1 cells, before and after the induction of differentiation, with the level increasing progressively after differentiation with a peak at days 8-10. Both dexamethasone and a β 3 agonist, BRL 37344, increased ZAG mRNA levels in 3T3-L1 adipocytes. ZAG gene expression and protein were also detected in human adipose tissue (visceral and s.c.). It is suggested that ZAG is a new adipose tissue protein factor, which may be involved in the modulation of lipolysis in adipocytes. Overexpression in WAT of tumor-bearing mice suggests a local role for adipocyte-derived ZAG in the substantial reduction of adiposity of cancer cachexia.

A Rapid and Sensitive High-Throughput Screening Method to Identify Compounds Targeting Protein–Nucleic Acids Interactions

DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein–nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian highmobility- group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2–DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2–DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2–DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

Proteomic analysis of the influence of the adipocyte secretome on Glioma Gl261 cells

Glioma is the most frequent form of malignant brain tumor in the adults and childhood. There is a global tendency toward a higher incidence of gliomas in highly developed and industrialized countries. Simultaneously obesity is reaching epidemic proportions in such developed countries. It has been highly accepted that obesity may play an important role in the biology of several types of cancer. We have developed an in vitro method for the understanding of the influence of obesity on glioma mouse cells (Gl261). 3T3-L1 mouse pre-adipocytes were induced to the maturity. The conditioned medium was harvested and used into the Gl261 cultures. Using two-dimension electrophoresis it was analyzed the proteome content of Gl261 in the presence of conditioned medium (CGl) and in its absence (NCGl). The differently expressed spots were collected and analyzed by means of mass spectroscopy (MALDI-TOF-MS). Significantly expression pattern changes were observed in eleven proteins and enzymes. RFC1, KIF5C, ANXA2, N-RAP, RACK1 and citrate synthase were overexpressed or only present in the CGl. Contrariwise, STI1, hnRNPs and phosphoglycerate kinase 1 were significantly underexpressed in CGl. Aldose reductase and carbonic anhydrase were expressed only in NCGl. Our results show that obesity remodels the physiological and metabolic behavior of glioma cancer cells. Also, proteins found differently expressed are implicated in several signaling pathways that control matrix remodeling, proliferation, progression, migration and invasion. In general our results support the idea that obesity may increase glioma malignancy, however, some interesting paradox finding were also reported and discussed.

A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis

NFAT (nuclear factor of activated T cells) is a family of transcription factors implicated in the control of cytokine and early immune response gene expression. Recent studies have pointed to a role for NFAT proteins in gene regulation outside of the immune system. Herein we demonstrate that NFAT proteins are present in 3T3-L1 adipocytes and, upon fat cell differentiation, bind to and transactivate the promoter of the adipocyte-specific gene aP2. Further, fat cell differentiation is inhibited by cyclosporin A, a drug shown to prevent NFAT nuclear localization and hence function. Thus, these data suggest a role for NFAT transcription factors in the regulation of the aP2 gene and in the process of adipocyte differentiation.

The effect of the senescence-associated secretory phenotype (SASP) on glucose homeostasis in metabolic cells

Aim: Dysregulated glucose homeostasis is a hallmark of Type 2diabetes. A distinctive feature of ageing is the accumulation ofsenescent cells, defined as cells that have undergone irreversible lossof proliferative capacity. Characteristic of senescent cells is thesenescence-associated secretory phenotype (SASP) involving theproduction of factors which reinforce senescence arrest in neigh-bouring tissue environments. We hypothesise that SASP inducesmetabolic dysfunction in non-senescent cells, impairing glucosemetabolism and propagating insulin resistance. We sought todetermine the effect of SASP on glucose homeostasis in hepatic,adipose and skeletal muscle cell lines. Methods: Human dermal fibroblasts were subjected to a geno-toxic dose of doxorubicin to induce senescence, confirmed using ab-galactosidase assay. Conditioned media containing SASP werecollected post 24h and 48h of inducing senescence and used at20% and 40% concentrations to treat AML-12 hepatocytes, 3T3-L1 adipocytes and C2C12 myocytes for 24h and 48h. Cells andmedia were collected and glucose and lipid concentrations weremeasured before and after the respective incubation periods. Results: Cell media obtained from C2C12 myocytes exposed to40% SASP for 24h and 48h and AML-12 hepatocytes after 48hexhibited significantly higher concentrations of glucose in com-parison to control media (p < 0.0001, p < 0.05) suggesting areduced glucose uptake. Glucose utilisation remained unchanged in3T3-L1 cells. Conclusion: Our data suggest an important role for SASP inaltering glucose homeostasis and identify SASP as a potentialmediator between ageing and the increase in age-related insulinresistance.

A role for protein kinase B beta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKB beta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKB alpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKB beta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKB beta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKB beta in insulin-stimulated glucose transport in adipocytes.

GLUT4 - At the cross roads between membrane trafficking and signal transduction

GLUT4 is a mammalian facilitative glucose transporter that is highly expressed in adipose tissue and striated muscle. In response to insulin, GLUT4 moves from intracellular storage areas to the plasma membrane, thus increasing cellular glucose uptake. While the verification of this 'translocation hypothesis' (Cushman SW. Wardzala LJ. J Biol Chem 1980;255: 4758-4762 and Suzuki K, Kono T. Proc Natl Acad Sci 1980;77: 2542-2545) has increased our understanding of insulin-regulated glucose transport, a number of fundamental questions remain unanswered. Where is GLUT4 stored within the basal cell? How does GLUT4 move to the cell surface and what mechanism does insulin employ to accelerate this process) Ultimately we require a convergence of trafficking studies with research in signal transduction. However, despite more than 30 years of intensive research we have still not reached this point. The problem is complex, involving at least two separate signal transduction pathways which feed into what appears to be a very dynamic sorting process. Below we discuss some of these complexities and highlight new data that are bringing us closer to the resolution of these questions.