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.

In vivo and ex vivo regulation of visfatin production by leptin in human and murine adipose tissue: role of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways

Visfatin is an adipogenic adipokine with increased levels in obesity, properties common to leptin. Thus, leptin may modulate visfatin production in adipose tissue (AT). Therefore, we investigated the effects of leptin on visfatin levels in 3T3-L1 adipocytes and human/murine AT, with or without a leptin antagonist. The potential signaling pathways and mechanisms regulating visfatin production in AT was also studied. Real-time RT-PCR and Western blotting were used to assess the relative mRNA and protein expression of visfatin. ELISA was performed to measure visfatin levels in conditioned media of AT explants, and small interfering RNA technology was used to reduce leptin receptor expression. Leptin significantly (P<0.01) increased visfatin levels in human and murine AT with a maximal response at leptin 10(-9) M, returning to baseline at leptin 10(-7) M. Importantly, ip leptin administration to C57BL/6 ob/ob mice further supported leptin-induced visfatin protein production in omental AT (P<0.05). Additionally, soluble leptin receptor levels rose with concentration dependency to a maximal response at leptin 10(-7) M (P<0.01). The use of a leptin antagonist negated the induction of visfatin and soluble leptin receptor by leptin. Furthermore, leptin-induced visfatin production was significantly decreased in the presence of MAPK and phosphatidylinositol 3-kinase inhibitors. Also, when the leptin eceptor gene was knocked down using small interfering RNA, eptin-induced visfatin expression was significantly decreased. Thus, leptin increases visfatin production in AT in vivo and ex vivo via pathways involving MAPK and phosphatidylinositol 3-kinase signaling. The pleiotropic effects of leptin may be partially mediated by visfatin.

Effect of eicosapentaenoic acid (EPA) on expression of a lipid mobilizing factor in adipose tissue in cancer cachexia

Adipose tissue of mice bearing a cachexia-inducing murine tumour (MAC16) shows increased expression of zinc-α2-glycoprotein (ZAG), a lipolytic factor thought to be responsible for the increased lipolysis. The anti-cachectic agent eicosapentaenoic acid (EPA) (0.5 g/kg) attenuated the loss of body weight in mice bearing the MAC16 tumour, and this was accompanied by downregulation of ZAG expression in both white and brown adipose tissue, as determined by Western blotting. Glucocorticoids may be responsible for the increased ZAG expression in adipose tissue. Dexamethasone (1.68 μM) stimulated lipolysis in 3T3-L1 adipocytes, and this effect was attenuated by EPA (50 μM). In addition the lipolytic action of dexamethasone was attenuated by anti-ZAG antibody, suggesting that the induction of lipolysis was mediated through an increase in ZAG expression. This was confirmed by Western blotting, which showed that dexamethasone (1.68 μM) induced a two-fold increase in ZAG expression in both cells and media, and that this was attenuated by EPA (50 μM). These results suggest that EPA may preserve adipose tissue in cachectic mice by downregulation of ZAG expression through interference with glucocorticoid signalling. © 2005 Elsevier Ltd. All rights reserved.

Effect of cancer cachexia on triacylglycerol/fatty acid substrate cycling in white adipose tissue

The effect of cancer cachexia on the TAG/FA substrate cycle in white adipose tissue was determined in vivo using the MAC16 murine model of cachexia. When compared with non-tumor-bearing animals, the rate of TAG-glycerol production was found to be increased almost threefold in animals bearing the MAC13 tumor, which does not induce cachexia, but was not further elevated in animals bearing the MAC16 tumor. In both cases TAG-glycerol production and de novo synthesis of TAG-FA were also increased above non-tumor-bearing animals. In animals bearing the MAC16 tumor, the TAG-FA rates were significantly higher than in animals bearing the MAC13 tumor. This suggests that the presence of the tumor alone is sufficient to cause an increase in cycling rate, and in the absence of an elevated energy intake (MAC16) this may contribute to the depletion of adipose tissue.

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.

Effect of a tumour-derived lipid-mobilising factor on glucose and lipid metabolism in vivo

Treatment of ex-breeder male NMRI mice with lipid mobilising factor isolated from the urine of cachectic cancer patients, caused a significant increase in glucose oxidation to CO2, compared with control mice receiving phosphate buffered saline. Glucose utilisation by various tissues was determined by the 2-deoxyglucose tracer technique and shown to be elevated in brain, heart, brown adipose tissue and gastrocnemius muscle. The tissue glucose metabolic rate was increased almost three-fold in brain, accounting for the ability of lipid mobilising factor to decrease blood glucose levels. Lipid mobilising factor also increased overall lipid oxidation, as determined by the production of 14CO2 from [14C carboxy] triolein, being 67% greater than phosphate buffered saline controls over a 24 h period. There was a significant increase in [14C] lipid accumulation in plasma, liver and white and brown adipose tissue after administration of lipid mobilising factor. These results suggest that changes in carbohydrate metabolism and loss of adipose tissue, together with an increased whole body fatty acid oxidation in cachectic cancer patients, may arise from tumour production of lipid mobilising factor. © 2002 Cancer Research UK.

Maternal periconceptional and gestational low protein diet affects mouse offspring growth, cardiovascular and adipose phenotype at 1 year of age

Human and animal studies have revealed a strong association between periconceptional environmental factors, such as poor maternal diet, and an increased propensity for cardiovascular and metabolic disease in adult offspring. Previously, we reported cardiovascular and physiological effects of maternal low protein diet (LPD) fed during discrete periods of periconceptional development on 6-month-old mouse offspring. Here, we extend the analysis in 1 year aging offspring, evaluating mechanisms regulating growth and adiposity. Isocaloric LPD (9% casein) or normal protein diet (18% casein; NPD) was fed to female MF-1 mice either exclusively during oocyte maturation (for 3.5 days prior to mating; Egg-LPD, Egg-NPD, respectively), throughout gestation (LPD, NPD) or exclusively during preimplantation development (for 3.5 days post mating; Emb-LPD). LPD and Emb-LPD female offspring were significantly lighter and heavier than NPD females respectively for up to 52 weeks. Egg-LPD, LPD and Emb-LPD offspring displayed significantly elevated systolic blood pressure at 52 weeks compared to respective controls (Egg-NPD, NPD). LPD females had significantly reduced inguinal and retroperitoneal fat pad: body weight ratios compared to NPD females. Expression of the insulin receptor (Insr) and insulin-like growth factor I receptor (Igf1r) in retroperitoneal fat was significantly elevated in Emb-LPD females (P&0.05), whilst Emb-LPD males displayed significantly decreased expression of the mitochondrial uncoupling protein 1 (Ucp1) gene compared to NPD offspring. LPD females displayed significantly increased expression of Ucp1 in interscapular brown adipose tissue when compared to NPD offspring. Our results demonstrate that aging offspring body weight, cardiovascular and adiposity homeostasis can be programmed by maternal periconceptional nutrition. These adverse outcomes further exemplify the criticality of dietary behaviour around the time of conception on long-term offspring health. © 2011 Watkins et al.

Resistin down-regulates insulin receptor expression, and modulates cell viability in rodent pancreatic beta-cells

The adipokine resistin is known to induce insulin resistance in rodent tissues. Increases in adipose tissue mass are known to have a negative effect on pancreatic beta-cell function, although the mechanisms are poorly understood. This study investigated the effects of resistin on insulin secretion, insulin receptor expression and cell viability in pancreatic beta-cells. BTC-6 or BRIN-BD11 cells were treated for 24h with resistin, and insulin receptor expression, insulin secretion and cell viability were measured. Incubation with 40ng/ml resistin caused significant decreases in insulin receptor mRNA and protein expression, but did not affect insulin secretion. At low concentrations, resistin caused significant increases in cell viability. These data implicate resistin as a factor that may regulate beta-cell function/viability, and suggests a potential mechanism by which increased adiposity causes beta-cell dysfunction.

Adipose atrophy in cancer cachexia:morphologic and molecular analysis of adipose tissue in tumour-bearing mice

Extensive loss of adipose tissue is a hallmark of cancer cachexia but the cellular and molecular basis remains unclear. This study has examined morphologic and molecular characteristics of white adipose tissue in mice bearing a cachexia-inducing tumour, MAC16. Adipose tissue from tumour-bearing mice contained shrunken adipocytes that were heterogeneous in size. Increased fibrosis was evident by strong collagen-fibril staining in the tissue matrix. Ultrastructure of 'slimmed' adipocytes revealed severe delipidation and modifications in cell membrane conformation. There were major reductions in mRNA levels of adipogenic transcription factors including CCAAT/enhancer binding protein alpha (C/EBPα), CCAAT/enhancer binding protein beta, peroxisome proliferator-activated receptor gamma, and sterol regulatory element binding protein-1c (SREBP-1c) in adipose tissue, which was accompanied by reduced protein content of C/EBPα and SREBP-1. mRNA levels of SREBP-1c targets, fatty acid synthase, acetyl CoA carboxylase, stearoyl CoA desaturase 1 and glycerol-3-phosphate acyl transferase, also fell as did glucose transporter-4 and leptin. In contrast, mRNA levels of peroxisome proliferators-activated receptor gamma coactivator-1alpha and uncoupling protein-2 were increased in white fat of tumour-bearing mice. These results suggest that the tumour-induced impairment in the formation and lipid storing capacity of adipose tissue occurs in mice with cancer cachexia. © 2006 Cancer Research UK.

Ageing, adipose tissue, fatty acids and inflammation

A common feature of ageing is the alteration in tissue distribution and composition, with a shift in fat away from lower body and subcutaneous depots to visceral and ectopic sites. Redistribution of adipose tissue towards an ectopic site can have dramatic effects on metabolic function. In skeletal muscle, increased ectopic adiposity is linked to insulin resistance through lipid mediators such as ceramide or DAG, inhibiting the insulin receptor signalling pathway. Additionally, the risk of developing cardiovascular disease is increased with elevated visceral adipose distribution. In ageing, adipose tissue becomes dysfunctional, with the pathway of differentiation of preadipocytes to mature adipocytes becoming impaired; this results in dysfunctional adipocytes less able to store fat and subsequent fat redistribution to ectopic sites. Low grade systemic inflammation is commonly observed in ageing, and may drive the adipose tissue dysfunction, as proinflammatory cytokines are capable of inhibiting adipocyte differentiation. Beyond increased ectopic adiposity, the effect of impaired adipose tissue function is an elevation in systemic free fatty acids (FFA), a common feature of many metabolic disorders. Saturated fatty acids can be regarded as the most detrimental of FFA, being capable of inducing insulin resistance and inflammation through lipid mediators such as ceramide, which can increase risk of developing atherosclerosis. Elevated FFA, in particular saturated fatty acids, maybe a driving factor for both the increased insulin resistance, cardiovascular disease risk and inflammation in older adults.

Numerical simulation of mass transfer during the osmotic dehydration of biological tissues

In this paper a mathematical model based on mass transfer in plant tissues is developed. The model takes into account the diffusion and convection of each constituent within the tissue. The driving force for the convection is assumed to be the gradient of hydrostatic pressure. The mass balance equation for the transport of each constituent is established separately for intracellular and extracellular volumes but taking into account the mass exchange across the cell membrane between the intracellular and extracellular volumes. The mass transfer results in not only the change of intracellular and extracellular volumes but also the shrinkage of whole tissue. The model allows us to quantitatively simulate the time evolution of intracellular and extracellular volumes, which was observed in histological sections under the microscope. © 2005 Elsevier B.V. All rights reserved.

The development and growth of tissues derived from cranial neural crest and primitive mesoderm is dependent on the ligation status of retinoic acid receptor γ:evidence that retinoic acid receptor γ functions to maintain stem/progenitor cells in the absence of retinoic acid

Retinoic acid (RA) signaling is important to normal development. However, the function of the different RA receptors (RARs)-RARα, RARβ, and RARγ-is as yet unclear. We have used wild-type and transgenic zebrafish to examine the role of RARγ. Treatment of zebrafish embryos with an RARγ-specific agonist reduced somite formation and axial length, which was associated with a loss of hoxb13a expression and less-clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist also disrupted formation of tissues arising from cranial neural crest, including cranial bones and anterior neural ganglia. There was a loss of Sox 9-immunopositive neural crest stem/progenitor cells in the same anterior regions. Pectoral fin outgrowth was blocked by RARγ agonist treatment. However, there was no loss of Tbx-5-immunopositive lateral plate mesodermal stem/progenitor cells and the block was reversed by agonist washout or by cotreatment with an RARγ antagonist. Regeneration of the caudal fin was also blocked by RARγ agonist treatment, which was associated with a loss of canonical Wnt signaling. This regenerative response was restored by agonist washout or cotreatment with the RARγ antagonist. These findings suggest that RARγ plays an essential role in maintaining stem/progenitor cells during embryonic development and tissue regeneration when the receptor is in its nonligated state.

Pioglitazone

Pioglitazone is a thiazolidinedione (TZD) antihyperglycemic agent introduced in 1999 for the treatment of type 2 (non-insulin dependent) diabetes mellitus. Another TZD, rosiglitazone, is also used in the treatment of type 2 diabetes. Troglitazone has been withdrawn from clinical use, and other TZDs, such as ciglitazone, have not proceeded into clinical use. Pioglitazone, like other TZDs, improves insulin action mainly by activation of the nuclear peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Peroxisome proliferator-activated receptor-gamma is most strongly expressed in adipose tissue and weakly expressed in liver and skeletal muscle, and activation of PPAR-gammain these tissues reinforces the effects of insulin. Pioglitazone may exert effects on other tissues that express PPAR-gamma ..... © 2007 Copyright © 2007 Elsevier Inc. All rights reserved.

An in vitro comparison of the incorporation, growth, and chondrogenic potential of human bone marrow versus adipose tissue mesenchymal stem cells in clinically relevant cell scaffolds used for cartilage repair

Aim. To compare the incorporation, growth, and chondrogenic potential of bone marrow (BM) and adipose tissue (AT) mesenchymal stem cells (MSCs) in scaffolds used for cartilage repair. Methods. Human BM and AT MSCs were isolated, culture expanded, and characterised using standard protocols, then seeded into 2 different scaffolds, Chondro-Gide or Alpha Chondro Shield. Cell adhesion, incorporation, and viable cell growth were assessed microscopically and following calcein AM/ethidium homodimer (Live/Dead) staining. Cell-seeded scaffolds were treated with chondrogenic inducers for 28 days. Extracellular matrix deposition and soluble glycosaminoglycan (GAG) release into the culture medium was measured at day 28 by histology/immunohistochemistry and dimethylmethylene blue assay, respectively. Results. A greater number of viable MSCs from either source adhered and incorporated into Chondro-Gide than into Alpha Chondro Shield. In both cell scaffolds, this incorporation represented less than 2% of the cells that were seeded. There was a marked proliferation of BM MSCs, but not AT MSCs, in Chondro-Gide. MSCs from both sources underwent chondrogenic differentiation following induction. However, cartilaginous extracellular matrix deposition was most marked in Chondro- Gide seeded with BM MSCs. Soluble GAG secretion increased in chondrogenic versus control conditions. There was no marked difference in GAG secretion by MSCs from either cell source. Conclusion. Chondro-Gide and Alpha Chondro Shield were permissive to the incorporation and chondrogenic differentiation of human BM and AT MSCs. Chondro-Gide seeded with BM MSCs demonstrated the greatest increase in MSC number and deposition of a cartilaginous tissue.

Alternative exon usage in the single CPT1 gene of Drosophila generates functional diversity in the kinetic properties of the enzyme:differential expression of alternatively spliced variants in drosophila tissues

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A.Weperformed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1mRNAwere found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K 0.5) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.