Studies on the antiobesity effect of zinc-α2-glycoprotein in the ob/ob mouse

OBJECTIVE: To investigate the mechanism of the lipid depletion by zinc-a(2)-glycoprotein (ZAG). DESIGN: Studies were conducted in the ob/ob mouse, or on isolated adipocytes from these animals or their lean counterparts. RESULTS: Treatment of these animals for 15 days with ZAG (100? µg, intravenously, daily) resulted in a reduction of body weight of 6.55? g compared with phosphate-buffered saline-treated controls, without a change in food or water intake, but with a 0.4?°C rise in rectal temperature. ZAG-treated mice had a 30% reduction in carcass fat mass and a twofold increase in weight of brown adipose tissue. Epididymal adipocytes from ZAG-treated mice showed an increased expression of ZAG and hormone-sensitive lipase (HSL), and this was maintained for a further 3 days in the absence of ZAG. There was an increased lipolytic response to isoproterenol, which was retained for 3 days in vitro in the absence of ZAG. Expression of HSL was also increased in subcutaneous and visceral adipose tissue, as was also adipose triglyceride lipase (ATGL). There was a rapid loss of labelled lipid from epididymal adipose tissue of ZAG-treated mice, but not from the other depots, reflecting the difference in sensitivity to lipolytic stimuli. The increased expression of HSL and ATGL may involve the extracellular signal-regulated kinase (ERK) pathway, as the active (phospho) form was upregulated in all adipose depots after ZAG administration, whereas in vitro studies showed induction of HSL and ATGL by ZAG to be attenuated by PD98059, an inhibitor of the ERK pathway. CONCLUSION: These results suggest that ZAG not only induces direct lipolysis, but also sensitizes adipose tissue to other lipolytic stimuli.

Studies on the anti-obesity activity of zinc-α2-glycoprotein in the rat

OBJECTIVE: To investigate the anti-obesity effect of the adipokine zinc-a(2)-glycoprotein (ZAG) in rats and the mechanism of this effect. SUBJECTS: Mature male Wistar rats (540 ± 83 g) were administered human recombinant ZAG (50 µg per 100 g body weight given intravenously daily) for 10 days, while control animals received an equal volume of phosphate-buffered saline (PBS). RESULTS: Animals treated with ZAG showed a progressive decrease in body weight, without a decrease in food and water intake, but with a 0.4 °C rise in body temperature. Body composition analysis showed loss of adipose tissue, but an increase in lean body mass. The loss of fat was due to an increase in lipolysis as shown by a 50% elevation of plasma glycerol, accompanied by increased utilization of non-esterified fatty acids, as evidenced by the 55% decrease in plasma levels. Plasma levels of glucose and triglycerides were also reduced by 36-37% and there was increased expression of the glucose transporter 4 in both skeletal muscle and adipose tissue. Expression of the lipolytic enzymes adipose triglyceride lipase and hormone-sensitive lipase in the white adipose tissue (WAT) were increased twofold after ZAG administration. There was almost a twofold increased expression of uncoupling proteins 1 and 3 in brown adipose tissue and WAT, which would contribute to increased substrate utilization. Administration of ZAG increased ZAG expression twofold in the gastrocnemius muscle, BAT and WAT, which was probably necessary for its biological effect. CONCLUSION: These results show that ZAG produces increased lipid mobilization and utilization in the rat.

Targeting the glycoprotein 130 receptor subunit to control pain and inflammation

The glycoprotein 130 (gp130) is a shared signal-transducing-membrane-associated receptor for several hematopoietic cytokines. Its activation is implicated in pain and in a variety of diseases via signaling of proinflammatory cytokines. These include interleukin-6 (IL-6) subfamily cytokines, many of which play important roles in the pathogenesis of diseases such as rheumatoid arthritis, Castleman's disease, and Kaposi's sarcoma. Several strategies have been developed to block gp130-receptor-mediated signaling. These include the application of monoclonal antibodies, the creation of mutant form(s) of the gp130 with increased binding affinity for such ligands as IL-6/sIL-6R complex, and the generation of antagonists by selective mutagenesis of the specific cytokine/gp130 receptor binding site(s). Other strategies include targeting gp130-mediated signaling pathways such as that involving signal transducer and activator of transcription-3. This review provides a summary of the latest research pertaining to the role of gp130 in the pathogenesis of inflammatory and other diseases in which the gp130 receptor is implicated. An overview of antagonists targeting the gp130 receptor is included with particular emphasis on their mechanism of action and their limitations and potential for therapeutic application.

Common variance in amplitude envelope perception tasks and their impact on phoneme duration perception and reading and spelling in Finnish children with reading disabilities

Our goal was to investigate auditory and speech perception abilities of children with and without reading disability (RD) and associations between auditory, speech perception, reading, and spelling skills. Participants were 9-year-old, Finnish-speaking children with RD (N = 30) and typically reading children (N = 30). Results showed significant group differences between the groups in phoneme duration discrimination but not in perception of amplitude modulation and rise time. Correlations among rise time discrimination, phoneme duration, and spelling accuracy were found for children with RD. Those children with poor rise time discrimination were also poor in phoneme duration discrimination and in spelling. Results suggest that auditory processing abilities could, at least in some children, affect speech perception skills, which in turn would lead to phonological processing deficits and dyslexia.

The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at -80 °C. For example, at 20 °C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.

Methodology for development of a physiological model incorporating CYP3A and P-glycoprotein for the prediction of intestinal drug absorption

The small intestine poses a major barrier to the efficient absorption of orally administered therapeutics. Intestinal epithelial cells are an extremely important site for extrahepatic clearance, primarily due to prominent P-glycoprotein-mediated active efflux and the presence of cytochrome P450s. We describe a physiologically based pharmacokinetic model which incorporates geometric variations, pH alterations and descriptions of the abundance and distribution of cytochrome 3A and P-glycoprotein along the length of the small intestine. Simulations using preclinical in vitro data for model drugs were performed to establish the influence of P-glycoprotein efflux, cytochrome 3A metabolism and passive permeability on drug available for absorption within the enterocytes. The fraction of drug escaping the enterocyte (F(G)) for 10 cytochrome 3A substrates with a range of intrinsic metabolic clearances were simulated. Following incorporation of P-glycoprotein in vitro efflux ratios all predicted F(G) values were within 20% of observed in vivo F(G). The presence of P-glycoprotein increased the level of cytochrome 3A drug metabolism by up to 12-fold in the distal intestine. F(G) was highly sensitive to changes in intrinsic metabolic clearance but less sensitive to changes in intestinal drug permeability. The model will be valuable for quantifying aspects of intestinal drug absorption and distribution.

Role of β-adrenergic receptors in the oral activity of zinc-α2-glycoprotein (ZAG)

Zinc-a2-glycoprotein (ZAG) is an adipokine with the potential as a therapeutic agent in the treatment of obesity and type 2 diabetes. In this study we show that human ZAG, which is a 41-kDa protein, when administered to ob/ob mice at 50 µg/d-1 orally in the drinking water produced a progressive loss of body weight (5 g after 8 d treatment), together with a 0.5 C increase in rectal temperature and a 40% reduction in urinary excretion of glucose. There was also a 33% reduction in the area under the curve during an oral glucose tolerance test and an increased sensitivity to insulin. These results were similar to those after iv administration of ZAG. However, tryptic digestion was shown to inactivate ZAG. There was no evidence of human ZAG in the serum but a 2-fold elevation of murine ZAG, which was also observed in target tissues such as white adipose tissue. To determine whether the effect was due to interaction of the human ZAG with the ß-adrenergic (ß-AR) in the gastrointestinal tract before digestion, ZAG was coadministered to ob/ob mice together with propanolol (40 mg/kg-1), a nonspecific ß-AR antagonist. The effect of ZAG on body weight, rectal temperature, urinary glucose excretion, improvement in glucose disposal, and increased insulin sensitivity were attenuated by propanolol, as was the increase in murine ZAG in the serum. These results suggest that oral administration of ZAG increases serum levels through interaction with a ß-AR in the upper gastrointestinal tract, and gene expression studies showed this to be in the esophagus.

The expression of P-glycoprotein does influence the distribution of novel fluorescent compounds in solid tumour models

Solid tumours display a complex drug resistance phenotype that involves inherent and acquired mechanisms. Multicellular resistance is an inherent feature of solid tumours and is known to present significant barriers to drug permeation in tumours. Given this barrier, do acquired resistance mechanisms such as P-glycoprotein (P-gp) contribute significantly to resistance? To address this question, the multicellular tumour spheroid (MCTS) model was used to examine the influence of P-gp on drug distribution in solid tissue. Tumour spheroids (TS) were generated from either drug-sensitive MCF7WT cells or a drug-resistant, P-gp-expressing derivative MCF7Adr. Confocal microscopy was used to measure time courses and distribution patterns of three fluorescent compounds; calcein-AM, rhodamine123 and BODIPY-taxol. These compounds were chosen because they are all substrates for P-gp-mediated transport, exhibit high fluorescence and are chemically dissimilar. For example, BODIPY-taxol and rhodamine 123 showed high accumulation and distributed extensively throughout the TSWT, whereas calcein-AM accumulation was restricted to the outermost layers. The presence of P-gp in TSAdr resulted in negligible accumulation, regardless of the compound. Moreover, the inhibition of P-gp by nicardipine restored intracellular accumulation and distribution patterns to levels observed in TSWT. The results demonstrate the effectiveness of P-gp in modulating drug distribution in solid tumour models. However, the penetration of agents throughout the tissue is strongly determined by the physico-chemical properties of the individual compounds.

Induction of lipolysis in vitro and loss of body fat in vivo by zinc-alpha(2)-glycoprotein

Loss of adipose tissue in cancer cachexia has been associated with tumour production of a lipid-mobilizing factor (LMF) which has been shown to be homologous with the plasma protein zinc-a2-glycoprotein (ZAG). The aim of this study was to compare the ability of human ZAG with LMF to stimulate lipolysis in vitro and induce loss of body fat in vivo, and to determine the mechanisms involved. ZAG was purified from human plasma using a combination of Q Sepharose and Superdex 75 chromatography, and was shown to stimulate glycerol release from isolated murine epididymal adipocytes in a dose-dependent manner. The effect was enhanced by the cyclic AMP phosphodiesterase inhibitor Ro20-1724, and attenuated by freeze/thawing and the specific ß3-adrenoreceptor antagonist SR59230A. In vivo ZAG caused highly significant, time-dependent, decreases in body weight without a reduction in food and water intake. Body composition analysis showed that loss of body weight could be attributed entirely to the loss of body fat. Loss of adipose tissue may have been due to the lipolytic effect of ZAG coupled with an increase in energy expenditure, since there was a dose-dependent increase in expression of uncoupling protein-1 (UCP-1) in brown adipose tissue. These results suggest that ZAG may be effective in the treatment of obesity.

Effect of zinc-alpha 2-glycoprotein (ZAG) on expression of uncoupling proteins in skeletal muscle and adipose tissue

The plasma protein zinc-α2-glycoprotein (ZAG) has been shown to be identical with a lipid mobilizing factor capable of inducing loss of adipose tissue in cancer cachexia through an increased lipid mobilization and utilization. The ability of ZAG to induce uncoupling protein (UCP) expression has been determined using in vitro models of adipose tissue and skeletal muscle. ZAG induced a concentration-dependent increase in the expression of UCP-1 in primary cultures of brown, but not white, adipose tissue, and this effect was attenuated by the β3-adrenergic receptor (β3-AR) antagonist SR59230A. A 6.5-fold increase in UCP-1 expression was found in brown adipose tissue after incubation with 0.58 μM ZAG. ZAG also increased UCP-2 expression 3.5-fold in C2C12 murine myotubes, and this effect was also attenuated by SR59230A and potentiated by isobutylmethylxanthine, suggesting a cyclic AMP-mediated process through interaction with a β3-AR. ZAG also produced a dose-dependent increase in UCP-3 in murine myotubes with a 2.5-fold increase at 0.58 μM ZAG. This effect was not mediated through the β3-AR, but instead appeared to require mitogen activated protein kinase. These results confirm the ability of ZAG to directly influence UCP expression, which may play an important role in lipid utilization during cancer cachexia. © 2004 Elsevier Ireland Ltd. All rights reserved.

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.

Bone marrow stromal cells stimulate neurite outgrowth over neural proteoglycans (CSPG), myelin associated glycoprotein and Nogo-A

In animal models, transplantation of bone marrow stromal cells (MSC) into the spinal cord following injury enhances axonal regeneration and promotes functional recovery. How these improvements come about is currently unclear. We have examined the interaction of MSC with neurons, using an established in vitro model of nerve growth, in the presence of substrate-bound extracellular molecules that are thought to inhibit axonal regeneration, i.e., neural proteoglycans (CSPG), myelin associated glycoprotein (MAG) and Nogo-A. Each of these molecules repelled neurite outgrowth from dorsal root ganglia (DRG) in a concentration-dependent manner. However, these nerve-inhibitory effects were much reduced in MSC/DRG co-cultures. Video microscopy demonstrated that MSC acted as "cellular bridges" and also "towed" neurites over the nerve-inhibitory substrates. Whereas conditioned medium from MSC cultures stimulated DRG neurite outgrowth over type I collagen, it did not promote outgrowth over CSPG, MAG or Nogo-A. These findings suggest that MSC transplantation may promote axonal regeneration both by stimulating nerve growth via secreted factors and also by reducing the nerve-inhibitory effects of the extracellular molecules present.

In silico modelling of the interaction of flavonoids with human P-glycoprotein nucleotide-binding domain

A three-dimensional model of human ABCB1 nucleotide-binding domain (NBD) was developed by homology modelling using the high-resolution human TAP1 transporter structure as template. Interactions between NBD and flavonoids were investigated using in silico docking studies. Ring-A of unmodified flavonoid was located within the NBD P-loop with the 5-hydroxyl group involved in hydrogen bonding with Lys1076. Ring-B was stabilised by hydrophobic stacking interactions with Tyr1044. The 3-hydroxyl group and carbonyl oxygen were extensively involved in hydrogen bonding interactions with amino acids within the NBD. Addition of prenyl, benzyl or geranyl moieties to ring-A (position-6) and hydrocarbon substituents (O-n-butyl to O-n-decyl) to ring-B (position-4) resulted in a size-dependent decrease in predicted docking energy which reflected the increased binding affinities reported in vitro.

The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment

The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.

The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein

Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.