Adipose Tissue In Cancer Cachexia Research Articles

Cancer cell-derived exosomal miR-425-3p induces white adipocyte atrophy

ABSTRACT White adipose tissue wasting plays a critical role in the development and progression of cancer cachexia. However, the mechanism behind the loss of adipose tissue remains ill-defined. In this study, we found that cancer cell-derived exosomes highly expressed miR-425-3p. Administration of cancer cell-derived exosomes significantly inhibited proliferation and differentiation of human preadipocytes-viscereal (HPA-v) cells. In mature adipocytes, cancer cell-derived exosomes activated cAMP/PKA signalling and lipophagy, leading to adipocyte lipolysis and browning of white adipocytes. These exosomes-induced alterations were almost abolished by endocytosis inhibitor cytochalasin D (CytoD) and antagomiR-425-3p, or reproduced by miR-425-3p mimics. In addition, bioinformatics analysis and luciferase reporter assay revealed that miR-425-3p directly targeted proliferation-related genes such as GATA2, IGFBP4, MMP15, differentiation-related gene CEBPA, and phosphodiesterase 4B gene (PDE4B). Depletion of PDE4B enhanced cAMP/PKA signalling and lipophagy, but had no effects on HPA-v proliferation and differentiation. Taken together, these results suggested that cancer cell-derived exosomal miR-425-3p inhibited preadipocyte proliferation and differentiation, increased adipocyte lipolysis, and promoted browning of white adipocytes, all of which might contribute to adipocyte atrophy and ultimately the loss of adipose tissue in cancer cachexia. Abbreviations: ADPN: adiponectin; aP2: adipocyte protein 2 or fatty acid binding protein 4 (FABP4); BCA: bicinchoninic acid assay; BFA: bafilomycin A1; BMI: body mass index; C/EBP: CCAAT/enhancer binding protein; CEBPA: CCAAT/enhancer-binding protein-alpha; C-Exo: cancer cell-derived exosomes; CNTL: control; CREB: cAMP-response element binding protein; CytoD: cytochalasin D; ECL: chemiluminescence; GATA2: GATA Binding Protein 2; HFD: high fat diet; HSL: hormone-sensitive lipase; IGFBP4: insulin like growth factor binding protein 4; IRS-1: insulin receptor substrate-1; ISO: isoproterenol hydrochloride; KD: knockdown; KO: knock out; LC3: microtubule-associated protein 1A/1B-light chain 3; LMF: lipid mobilizing factor; LPL: lipoprotein lipase; MMP15: matrix metallopeptidase 15; Mir-Inh-C-Exo: cancer cell-derived exosomes with miR-425-3p inhibition; mTOR: mammalian target of rapamycin; Mut: mutant; N-Exo: normal cell-derived exosomes; NSCLC: non-small cell lung cancer; PBS, phosphate buffered saline; PGC-1: peroxisome proliferator-activated receptor-gamma coactivator-1; PDEs: phosphodiesterases; PKI: PKA inhibitor; PKA: cAMP-dependent protein kinase; PLIN1: Perilipin 1; PTHRP: parathyroid hormone-related protein; PVDF: polyvinylidene difluoride; shRNA: short hairpin RNA; UCP1: uncoupling protein 1; WT: wild type.

Lipases and lipid droplet-associated protein expression in subcutaneous white adipose tissue of cachectic patients with cancer

BackgroundCancer cachexia is a multifactorial metabolic syndrome characterized by marked loss of adipose tissue and skeletal muscle. Fat loss from adipose tissue in cancer cachexia is partly the result of increased lipolysis. Despite the growing amount of studies focused on elucidating the mechanisms through which lipolysis-related proteins regulate the lipolytic process, there are scarce data concerning that profile in the adipose tissue of cancer cachectic patients. Considering its fundamental importance, it was our main purpose to characterize the expression of the lipolysis-related proteins in the white adipose tissue of cachectic cancer patients.MethodsPatients from the University Hospital were divided into three groups: control, cancer cachexia (CC), and weight-stable cancer patients (WSC). To gain greater insight into adipose tissue wasting during cancer cachexia progression, we have also analyzed an experimental model of cachexia (Walker 256 carcinosarcoma). Animals were divided into: control, intermediate cachexia (IC) and terminal cachexia (TC). Subcutaneous white adipose tissue of patients and epidydimal white adipose tissue of animals were investigated regarding molecular aspects by determining the protein content and gene expression of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), comparative gene identification-58 (CGI-58), perilipin 1, leptin, adiponectin, visfatin, and tumour necrosis factor alpha (TNF-alpha).ResultsWe found augmented lipolysis in CC associated with increased HSL expression, as well as upregulation of ATGL expression and reduction in perilipin 1 content. In IC, there was an imbalance in the secretion of pro- and anti-inflammatory factors. The alterations at the end-stage of cachexia were even more profound, and there was a reduction in the expression of almost all proteins analyzed in the animals.ConclusionsOur findings show that cachexia induces important morphological, molecular, and humoral alterations in the white adipose tissue, which are specific to the stage of the syndrome.

Inhibition of preadipocyte differentiation and adipogenesis by zinc-α2-glycoprotein treatment in 3T3-L1 cells.

Zinc-α2-glycoprotein (ZAG) is associated with the loss of adipose tissue in cancer cachexia, and has recently been proposed to be a candidate factor in the regulation of bodyweight. The aim of the study was to investigate the effects of ZAG on the proliferation and differentiation of 3T3-L1 preadipocytes. 3-(4,5-Dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) spectrophotometry, Oil Red O staining, intracellular triglyceride assays, real-time quantitative reverse transcription polymerase chain reaction and transient transfection methods were used to explore the action of ZAG. Ectopic ZAG expression significantly stimulates 3T3-L1 cells proliferation in a dose- and time-dependent manner. The maximum influence of ZAG on proliferation was 1.43-fold higher than what was observed in control cells. This effect was observed 144h after transfection with 0.16μg of murine ZAG (mZAG) plasmid (P<0.001). The intracellular lipids content in mZAG over-expressing cells were decreased as much as 37% when compared with the control cells after differentiation (P<0.05, P<0.01). The messenger ribonucleic acid levels of peroxisome proliferators-activated receptor-γ (PPARγ), CCAAT enhancer-binding protein-α (C/EBPα) and the critical lipogenic gene, fatty acid synthase (FAS), are also downregulated by up to 50% in fully differentiated ZAG-treated adipocytes. ZAG suppresses FAS messenger ribonucleic acid expression by reducing FAS promoter activity. Zinc-α2-glycoprotein stimulates the proliferation and inhibits the differentiation of 3T3-L1 murine preadipocytes. The inhibitory action of ZAG on cell differentiation might be a result of the attenuation of the expression of PPARγ, C/EBPα and the lipogenic-specific enzyme FAS by reducing FAS promoter activity.

A role of active brown adipose tissue in cancer cachexia?

Until a few years ago, adult humans were not thought to have brown adipose tissue (BAT). Now, this is a rapidly evolving field of research with perspectives in metabolic syndromes such as obesity and new therapies targeting its bio-energetic pathways. White, brown and socalled brite adipose fat seem to be able to trans-differentiate into each other, emphasizing the dynamic nature of fat tissue for metabolism. Human and animal data in cancer cachexia to date provide some evidence for BAT activation, but its quantitative impact on energy expenditure and weight loss is controversial. Prospective clinical studies can address the potential role of BAT in cancer cachexia using 18F-fluorodeoxyglucose positron emission tomography-computed tomography scanning, with careful consideration of co-factors such as diet, exposure to the cold, physical activity and body mass index, that all seem to act on BAT recruitment and activity.

A role of active brown adipose tissue in cancer cachexia?

Until a few years ago, adult humans were not thought to have brown adipose tissue (BAT). Now, this is a rapidly evolving field of research with perspectives in metabolic syndromes such as obesity and new therapies targeting its bio-energetic pathways. White, brown and so-called brite adipose fat seem to be able to trans-differentiate into each other, emphasizing the dynamic nature of fat tissue for metabolism. Human and animal data in cancer cachexia to date provide some evidence for BAT activation, but its quantitative impact on energy expenditure and weight loss is controversial. Prospective clinical studies can address the potential role of BAT in cancer cachexia using 18F-fluoro- deoxyglucose positron emission tomography-computed tomography scanning, with careful consideration of co-factors such as diet, exposure to the cold, physical activity and body mass index, that all seem to act on BAT recruitment and activity.

Adipose tissue pathways involved in weight loss of cancer cachexia

Background:The regulatory gene pathways that accompany loss of adipose tissue in cancer cachexia are unknown and were explored using pangenomic transcriptome profiling.Methods:Global gene expression profiles of abdominal subcutaneous adipose tissue were studied in gastrointestinal cancer patients with (n=13) or without (n=14) cachexia.Results:Cachexia was accompanied by preferential loss of adipose tissue and decreased fat cell volume, but not number. Adipose tissue pathways regulating energy turnover were upregulated, whereas genes in pathways related to cell and tissue structure (cellular adhesion, extracellular matrix and actin cytoskeleton) were downregulated in cachectic patients. Transcriptional response elements for hepatic nuclear factor-4 (HNF4) were overrepresented in the promoters of extracellular matrix and adhesion molecule genes, and adipose HNF4 mRNA was downregulated in cachexia.Conclusions:Cancer cachexia is characterised by preferential loss of adipose tissue; muscle mass is less affected. Loss of adipose tissue is secondary to a decrease in adipocyte lipid content and associates with changes in the expression of genes that regulate energy turnover, cytoskeleton and extracellular matrix, which suggest high tissue remodelling. Changes in gene expression in cachexia are reciprocal to those observed in obesity, suggesting that regulation of fat mass at least partly corresponds to two sides of the same coin.

New insights into adipose tissue atrophy in cancer cachexia

Profound loss of adipose and other tissues is a hallmark of cancer cachexia, a debilitating condition associated with increased morbidity and mortality. Fat loss cannot be attributable to reduced appetite alone as it precedes the onset of anorexia and is much more severe in experimental models of cachexia than in food restriction. Morphological examination has shown marked remodelling of adipose tissue in cancer cachexia. It is characterised by the tissue containing shrunken adipocytes with a major reduction in cell size and increased fibrosis in the tissue matrix. The ultrastructure of 'slimmed' adipocytes has revealed severe delipidation and modifications in cell membrane conformation. Although the molecular mechanisms remain to be established, evidence suggests that altered adipocyte metabolism may lead to adipose atrophy in cancer cachexia. Increased lipolysis appears to be a key factor underlying fat loss, while inhibition of adipocyte development and lipid deposition may also contribute. Both tumour and host-derived factors are implicated in adipose atrophy. Zinc-alpha2-glycoprotein (ZAG), which is overexpressed by certain malignant tumours, has been identified as a novel adipokine. ZAG transcripts and protein expression in adipose tissue are up regulated in cancer cachexia but reduced with adipose tissue expansion in obesity. Studies in vitro demonstrate that recombinant ZAG stimulates lipolysis. ZAG may therefore act locally, as well as systemically, to promote lipid mobilisation in cancer cachexia. Further elucidation of ZAG function in adipose tissue may lead to novel targets for preventing adipose atrophy in malignancy.

Regulation of inflammation in the adipose tissue in cancer cachexia: effect of exercise

The paraneoplastic syndrome of cachexia is considered a degenerative chronic inflammatory disease, being deeply related to the increase of pro-inflammatory factors, especially tumour necrosis factor alpha (TNF-alpha). It is known that the adipose tissue is affected by cachexia and contributing with the secretion of pro-inflammatory factors which reach the adjacent tissues and the circulation. The effect of pro-inflammatory factors is balanced by the effect of anti-inflammatory factors, such as interleukin 10 (IL-10). The IL-10/TNF-alpha ratio has been recently postulated as a marker for the assessment of the degree of inflammation, which correlates with disease-associated morbidity and mortality. In order to counteract inflammation in chronic disease, our group has currently adopted chronic endurance exercise in models of cancer cachexia and chronic heart failure. Since it is clear that white adipose tissue is strongly implicated in the secretion of both pro- and anti-inflammatory factors in disease, we chose to address its contribution to cachexia-related inflammation and the effect of endurance training on the capacity of cytokine expression and secretion by this tissue. Our results show an enhancement of IL-10 adipose tissue content, and increased IL-10/TNF-alpha ratio induced by endurance training. The mechanisms are discussed.

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- α 2-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.

The role of glucocorticoids in the induction of zinc-α2-glycoprotein expression in adipose tissue in cancer cachexia

Loss of adipose tissue in cancer cachexia in mice bearing the MAC16 tumour arises from an increased lipid mobilisation through increased expression of zinc-α2-glycoprotein (ZAG) in white (WAT) and brown (BAT) adipose tissue. Glucocorticoids have been suggested to increase ZAG expression, and this study examines their role in cachexia and the mechanisms involved. In mice bearing the MAC16 tumour, serum cortisol concentrations increased in parallel with weight loss, and the glucocorticoid receptor antagonist RU38486 (25 mg kg−1) attenuated both the loss of body weight and ZAG expression in WAT. Dexamethasone (66 μg kg−1) administration to normal mice produced a six-fold increase in ZAG expression in both WAT and BAT, which was also attenuated by RU38486. In vitro studies using 3T3-L1 adipocytes showed dexamethasone (1.68 μM) to stimulate lipolysis and increase ZAG expression, and both were attenuated by RU38486 (10 μM), anti-ZAG antibody (1 μgml−1), and the β3-adrenoreceptor (β3-AR) antagonist SR59230A (10 μM). Zinc-α2-glycoprotein also increased its own expression and this was attenuated by SR59230A, suggesting that it was mediated through the β3-AR. This suggests that glucocorticoids stimulate lipolysis through an increase in ZAG expression, and that they are responsible for the increase in ZAG expression seen in adipose tissue of cachectic mice.

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

The plasma protein zinc-α 2-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 C 2C 12 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.

Induction of lipolysis in vitro and loss of body fat in vivo by zinc-α 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-α 2-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 β3-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.

Modulation of adipocyte G-protein expression in cancer cachexia by a lipid-mobilizing factor (LMF).

Adipocytes isolated from cachectic mice bearing the MAC 16 tumour showed over a 3-fold increase in lipolytic response to both low concentrations of isoprenaline and a tumour-derived lipid mobilizing factor (LMF). This was reflected by an enhanced stimulation of adenylate cyclase in plasma membrane fractions of adipocytes in the presence of both factors. There was no up-regulation of adenylate cyclase in response to forskolin, suggesting that the effect arose from a change in receptor number or G-protein expression. Immunoblotting of adipocyte membranes from mice bearing the MAC16 tumour showed an increased expression of Gαs up to 10% weight loss and a reciprocal decrease in Gα. There was also an increased expression of Gαs and a decrease in Gα in adipose tissue from a patient with cancer-associated weight loss compared with a non-cachectic cancer patient. The changes in G-protein expression were also seen in adipose tissue of normal mice administered pure LMF as well as in 3T3L1 adipocytes in vitro. The changes in G-protein expression induced by LMF were attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). This suggests that this tumour-derived lipolytic factor acts to sensitize adipose tissue to lipolytic stimuli, and that this effect is attenuated by EPA, which is known to preserve adipose tissue in cancer cachexia. © 2001 Cancer Research Campaign

Inhibition of lipolysis and muscle protein degradation by EPA in cancer cachexia.

Depletion of muscle and adipose tissue in cancer cachexia appears to arise not only from decreased food intake but also from the production of catabolic factors by certain tumours. Experiments with the cachexia-inducing MAC16 tumour in mice showed that when part of the carbohydrate calories were replaced by fish oil, host body weight loss was inhibited. The effect occurred without an alteration of either the total calorie consumption or nitrogen intake. Instead, one of the polyunsaturated fatty acids (PUFA) in fish oil, eicosapentaenoic acid (EPA), was found directly to inhibit tumour-induced lipolysis. The effect was structurally specific, as two related PUFA, docosahexaenoic acid (DHA) and gamma-linolenic acid (GLA), were without effect. The antilipolytic effect of EPA arose from an inhibition of the elevation of cyclic AMP in adipocytes in response to the lipid mobilizing factor. The increased protein degradation in the skeletal muscle of cachectic animals was also inhibited by EPA. This effect was due to the inhibition of the rise in muscle prostaglandin E2 in response to a tumour-produced proteolytic factor by EPA. Thus, reversal of cachexia by EPA in this mouse model results from its capacity to interfere with tumour-produced catabolic factors. Similar factors have been detected in human cancer cachexia.

Inhibition of lipolysis and muscle protein degradation by epa in cancer cachexia

Depletion of muscle and adipose tissue in cancer cachexia appears to arise not only fromdecreased food intake but also from the production of catabolic factors by certain tumours. Experiments with the cachexia-inducing MAC 16 tumour in mice showed that when part of the carbohydrate calories were replaced by fish oil, host body weight loss was inhibited. The effect occurred without an alteration of either the total calorie consumption or nitrogen intake. Instead, one of the polyunsaturated fatty acids (PUFA) in fish oil, eicosapentaenoic acid (EPA), was found directly to inhibit tumour-induced lipolysis. The effect was structurally specific, as two related PUFA, docosahexaenoic acid (DHA) and γ-linolenic acid (GLA), were without effect. The antilipolytic effect of EPA arose from an inhibition of the elevation of cyclic AMP in adipocytes in response to the lipid mobilizing factor The increased protein degradation in the skeletal muscle of cachectic animals was also inhibited by EPA. This effect was due to the inhibition of the rise in muscle prostaglandin E 2 in response to a tumour-produced proteolytic factor by EPA. Thus, reversal of cachexia by EPA in this mouse model results from its capacity to interfere with tumour-produced catabolic factors. Similar factors have been detected in human cancer cachexia.

Diminished visceral adipose tissue in cancer cachexia.

To estimate the relationship between the visceral adipose tissue (AT) area and cancer cachexia, 13 cachectic patients (7 males, 6 females; age 65.2 +/- 11.0 years; body mass index 20.8 +/- 4.1 kg/m2) were examined by computed tomography (CT) scanning. Cachectic cancer patients who had a 10% decrease of body weight and died within 6 months because of gastrointestinal carcinoma had a significantly smaller visceral AT area than control subjects (mean +/- sd: 43.9 +/- 42.2 cm2 vs. 93.4 +/- 56.0 cm2, P < 0.05, P = 0.014). Otherwise, there were no significant differences between the visceral AT areas of cachectic cancer patients and those of cancer patients with resectable tumors treated by curative operation (mean +/- sd: 68.8 +/- 57.7 cm2) (NS, P = 0.206). There was, however, a tendency for cachectic cancer patients to have a smaller visceral AT area than those with resectable tumors. This result suggests that the visceral AT area is not preserved in the cachectic state associated with cancer.