The mode of action of a lipid mobilising factor in cancer cachexia

Cachexia is characterised by a progressive weight loss due to depletion of both skeletal muscle and adipose tissue. The loss of adipose tissue is due to the production of a tumour-derived lipid mobilising factor (LMF), which has been shown to directly induce lipolysis in isolated epididymal murine white adipocytes. The administration of LMF to a non-tumour bearing mice produced a rapid weight loss, with a specific reduction in carcass lipid with also some redistribution of lipid with the accumulation of lipid in the liver. There was also up-regulation of uncoupling protein-1 and -2 mRNA and protein expression in brown adipose tissue, suggesting that an adaptive process occurs due to increased energy mobilisation. There was also up-regulation of UCP-2 in the livers of LMF treated mice, suggesting a protective mechanism to the build up of lipid in the livers, which would produce free radical by-products. LMF was also shown to stimulate cyclic AMP production in CHO-K1 cells transfected with human -3 adrenergic receptors and inhibited by the -β3 antagonist SR59230A. LMF binding was also inhibited by SR59230A in isolated receptors. This suggests that LMF mediates its effects through a β3 adrenergic receptor. There were also changes in glucose and fatty acid uptake in LMF treated mice, which suggests metabolic changes are occurring. The study suggests that a tumour derived lipolytic factor acts through the 3 adrenoceptor producing effects on lipid mobilisation, energy expenditure and glucose metabolism.

Mechanism of action of a tumour derived lipid mobilising factor

Cancer cachexia comprises unintentional and debilitating weight loss associated with certain tumour types. Fat loss in cachexia is mediated by a 43kDa Lipid Mobilising Factor (LMF) sharing homology with endogenous Zinc-α2-Glycoprotein (ZAG). LMF and ZAG induced significant lipolysis in isolated epidydimal adipose tissue. This is attenuated by co-incubation with 10μM of antagonist SR59230A and partially attenuated by 25μM PD098059 (indicating β3-AR and MAPK involvement respectively). LMF/ZAG induced in vitro lipid depletion in differentiated 3T3-L1 adipocytes that seen to comprise a significant increase in lipolysis (p<0.01), with only a modest decrease in lipid synthesis (p=0.09). ZAG significantly increased in vitro protein synthesis (p<0.01) in C2C12 myotubes (without an effect on protein degradation). This increase was activated at transcription and attenuated by co-incubation with 10μM SR59230A. Proteolytic digestion of ZAG and LMF followed by sephadex G50 chromatography yielded active fragments of 6-15kDa, indication the entire molecule was not required for bioactivity. Cachexigenic MAC16 cells demonstrated significant in vitro ZAG expression over non-cachexigenic MAC13 cells (p<0.001). WAT and BAT excised from MAC16 mice of varying weight loss demonstrated increased ZAG expression compared to controls. Dosing of NMRI mice with s/c ZAG failed to reproduce this up-regulation, thus another cachectic factor is responsible. 0.58nM LMF conferred significant protection against hydrogen peroxide, paraquat and bleomycin-induced oxidative stress in the non-cachexigenic MAC13 cell line. This protection was attenuated by 10μM SR59230A indicating a β3-AR mediated effect. In addition, 0.58nM LMF significantly up regulated UCP2 expression (p<0.001), (a mitochondrial protein implicated in the detoxification of ROS) implying this to be the mechanism by which survival was achieved. In vitro, LMF caused significant up-regulation of UCP1 in BAT and UCP2 and 3 in C2C12 myotubes. This increase in uncoupling protein expression further potentiates the negative energy balance and wasting observed in cachexia.

Second messenger pathways in the action of cachectic factors

Cachexia in cancer is characterised by progressive depletion of both adipose tissue stores and skeletal muscle mass. Two catabolic factors produced by cachexia-inducing tumours have the potential for inducing these changes in body composition: (i) proteolysis-inducing factor (PIF) which acts on skeletal muscle to induce both protein degradation and inhibit protein synthesis, (ii) lipid-mobilising factor (LMF), which has been shown to directly induce lipolysis in isolated epididymal murine white adipocytes. Administration of lipid-mobilising factor (LMF) to mice produced a specific reduction in carcass lipid with a tendency to increase non-fat carcass mass. Treatment of murine myoblasts, myotubes and tumour cells with tumour-produced LMF, caused concentration dependent stimulation of protein synthesis, within a 24hr period. It produced an increase in intracellular cyclic AMP levels, which was linearly related to the increase in protein synthesis. The observed effect was attenuated by pretreating cells with the adenylate cyclase inhibitor, MDL12330A and was additive with stimulation produced by forskolin. Both propranolol and a specific 3 adrenergic antagonist SR59230A, significantly reduced the stimulation of protein synthesis induced by LMF. LMF also affected protein degradation in vitro, as demonstrated by a reduction in proteasome activity, a key component of the ubiquitin-dependent proteolytic pathway. These effects were opposite to those produced by PIF which caused both a decrease in the rate of protein synthesis and an elevation on protein breakdown when incubated in vitro.Incubation of LMF with a fat cell line produced alterations in the levels of guanine-nucleotide binding proteins (G proteins). This was also evident in adipocyte plasma membranes isolated from mice bearing the tumour model of cachexia, MAC16 adenocarcinoma and from patients with cancer cachexia. Progression through the cachectic state induced an upregulation of stimulatory G proteins paralleled with a downregulation of inhibitory G proteins. These changes would contribute to the increased lipid mobilisation seen in cancer cachexia.

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.

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.

Cancer cachexia

Causative factors: Nutritional supplementation or pharmacological manipulation of appetite are unable to control the muscle atrophy seen in cancer cachexia. This suggests that tumour and/or host factors might be responsible for the depression in protein synthesis and the increase in protein degradation. An increased expression of the ubiquitin-proteasome proteolytic pathway is responsible for the increased degradation of myofibrillar proteins in skeletal muscle, and this may be due to tumour factors, such as proteolysis-inducing factor (PIF), or host factors such as tumour necrosis factor-α (TNF-α). In humans loss of adipose tissue is due to an increase in lipolysis rather than a decrease in synthesis, and this may be due to tumour factors such as lipid-mobilising factor (LMF) or TNF-α, both of which can increase cyclic AMP in adipocytes, leading to activation of hormone-sensitive lipase (HSL). Levels of mRNA for HSL are elevated twofold in adipose tissue of cancer patients, while there are no changes in lipoprotein lipase (LPL), involved in extraction of fatty acids from plasma lipoproteins for storage. Treatment for cachexia: This has concentrated on increasing food intake, although that alone is unable to reverse the metabolic changes. Agents interfering with TNF-α have not been very successful to date, although more research is required in that area. The only agent tested clinically that is able to interfere with the action of PIF is eicosapentaenoic acid (EPA). EPA attenuates protein degradation in skeletal muscle by preventing the increased expression of the ubiquitin-proteasome pathway, but has no effect on protein synthesis. When used alone EPA prevents further wasting in cachectic patients, and, when it is combined with an energy- and protein-dense nutritional supplement, weight gain is seen, which is totally lean body mass. These results suggest that mechanistic studies into the causes of cancer cachexia will allow appropriate therapeutic intervention.

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.

Cancer cachexia

Purpose of review: Although cachexia has a major effect on both the morbidity and mortality of cancer patients, information on the mechanisms responsible for this condition is limited. This review summarizes recent data in this area. Recent findings: Cachexia is defined as loss of muscle, with or without fat, frequently associated with anorexia, inflammation and insulin resistance. Loss of adipose mass is due to an increased lipolysis through an increased expression of hormone-sensitive lipase. Adipose tissue does not contribute to the inflammatory response. There is an increased phosphorylation of both protein kinase R (PKR) and eukaryotic initiation factor 2 on the α-subunit in skeletal muscle of cachectic cancer patients, which would lead to muscle atrophy through a depression in protein synthesis and an increase in degradation. Mice lacking the ubiquitin ligase MuRF1 are less susceptible to muscle wasting under amino acid deprivation. Expression of MuRF1 and atrogin-1 is increased by oxidative stress, whereas nitric oxide may protect against muscle atrophy. Levels of interleukin (IL)-6 correlate with cachexia and death due to an increase in tumour burden. Ghrelin analogues and melanocortin receptor antagonists increase food intake and may have a role in the treatment of cachexia. Summary: These findings provide impetus for the development of new therapeutic agents. © 2010 Wolters Kluwer Health

White Adipose Tissue Re-growth after Partial Lipectomy in High Fat Diet Induced Obese Wistar Rats

The effects of partial removal of epididymal (EPI) and retroperitoneal (RET) adipose tissues (partial lipectomy) on the triacylglycerol deposition of high fat diet induced obese rats were analyzed, aiming to challenge the hypothesized body fat regulatory system. Male 28-day-old wistar rats received a diet enriched with peanuts, milk chocolate and sweet biscuits during the experimental period. At the 90th day of life, rats were submitted to either lipectomy (L) or sham surgery. After 7 or 30 days, RET, EPI, liver, brown adipose tissue (BAT), blood and carcass were obtained and analyzed. Seven days following surgery, liver lipogenesis rate and EPI relative weight were increased in L. After 30 days, L, RET and EPI presented increased lipogenesis, lipolysis and percentage of small area adipocytes. L rats also presented increased liver malic enzyme activity, BAT lipogenesis, and triacylglycerol and corticosterone serum levels. The partial removal of visceral fat pads affected the metabolism of high fat diet obese rats, which leads to excised tissue re-growth and possibly compensatory growth of non-excised depots at a later time.

Lipid Metabolism of Monosodium Glutamate Obese Rats after Partial Removal of Adipose Tissue

We analyzed the effects of partial fat pad removal on retroperitoneal and epididymal fat depots and carcass metabolism of control (C) and MSG-obese (M) rats. Three-month-old C and M male Wistar rats were submitted to either partial surgical excision of epididymal and retroperitoneal fat tissue (lipectomy, L) or sham surgery (S) and studied after 7 or 30 days. Retroperitoneal and epididymal tissue re-growth after lipectomy was not observed, as indicated by the low pads weight of the L groups. The lipolysis rate was stimulated in LC7 and LM7, probably due to surgical stress and low insulin levels. In LM7, but not in LC7, in vivo lipogenesis rate increased in retroperitoneal and epididymal fat tissue, as did the diet-derived lipid accumulation in epididymal fat tissue. Although these local increases were no longer present in LM30, this group showed a large increase in the percentage of small area adipocytes in both pads as well as increased carcass lipogenesis rate. The present data showed that the partial removal of fat depots affected the metabolism of control and MSG-obese rats differently. In the obese animals only, it stimulated both local and carcass lipogenesis rate as well as adipocyte differentiation, i.e. responses likely to favor excised tissue re-growth and/or compensatory growth of non-excised depots.

Nouveaux indices de suppression de la lipolyse par l'insuline déterminés lors de l'hyperglycémie provoquée par voie orale : comparaisons avec le clamp euglycémique-hyperinsulinémique et les paramètres métaboliques chez les femmes

Résumé : Une dysrégulation de la lipolyse des tissus adipeux peut conduire à une surexposition des tissus non-adipeux aux acides gras non-estérifiés (AGNE), qui peut mener à un certain degré de lipotoxicité dans ces tissus. La lipotoxicité constitue, par ailleurs, l’une des causes majeures du développement de la résistance à l’insuline et du diabète de type 2. En plus de ses fonctions glucorégulatrices, l’insuline a pour fonction d’inhiber la lipolyse et donc de diminuer les niveaux d’AGNE en circulation, prévenant ainsi la lipotoxicité. Il n’y a pas d’étalon d’or pour mesurer la sensibilité de la lipolyse à l’insuline. Le clamp euglycémique hyperinsulinémique constitue la méthode étalon d’or pour évaluer la sensibilité du glucose à l’insuline mais il est aussi utilisé pour mesurer la suppression de la lipolyse par l’insuline. Par contre, cette méthode est couteuse et laborieuse, et ne peut pas s’appliquer à de grandes populations. Il existe aussi des indices pour estimer la fonction antilipolytique de l’insuline dérivés de l’hyperglycémie provoquée par voie orale (HGPO), un test moins dispendieux et plus simple à effectuer à grande échelle. Cette étude vise donc à : 1) Étudier la relation entre les indices de suppressibilité des AGNE par l’insuline dérivés du clamp et ceux dérivés de l’HGPO; et 2) Déterminer laquelle de ces mesures corrèle le mieux avec les facteurs connus comme étant reliés à la dysfonction adipeuse : paramètres anthropométriques et indices de dysfonction métabolique. Les résultats montrent que dans le groupe de sujets étudiés (n=29 femmes, 15 témoins saines et 14 femmes avec résistance à l’insuline car atteintes du syndrome des ovaires polykystiques), certains indices de sensibilité à l’insuline pour la lipolyse dérivés de l’HGPO corrèlent bien avec ceux dérivés du clamp euglycémique hyperinsulinémique. Parmi ces indices, celui qui corrèle le mieux avec les indices du clamp et les paramètres anthropométriques et de dysfonction adipeuse est le T50[indice inférieur AGNE] (temps nécessaire pour diminuer de 50% le taux de base – à jeun – des AGNE). Nos résultats suggèrent donc que l’HGPO, facile à réaliser, peut être utilisée pour évaluer la sensibilité de la lipolyse à l’insuline. Nous pensons que la lipo-résistance à l’insuline peut être facilement quantifiée en clinique humaine.

Adipocyte size fluctuation, mechano-active lipid droplets and caveolae

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Effects of feeding carnitine and ractopamine on rainbow trout (Oncorhynchus mykiss, Walbaum 1972)

L-carnitine is required for the transfer of long-chain fatty acids from the cytosol to the mitochondrial matrix for 13-oxidation of them and ractopamine, beta adrenergic agonists, have potential stimulating lipolysis and altering rates of protein degradation and synthesis. Present study was carried out to improve lipid body oxidation and protein-sparing action of fish through addition of L-carnitine and ractopamine to diet of rainbow trout, Oncorhynchus mykiss, Walbaum 1972. An eight-week feeding trial was carried out to evaluate the effects of supplementation of tree levels of L-carnitine tartrate (0, 1 and 2 g/kg) and two levels of ractopamine hydrochloride (0 and 10 ppm) on growth performance, fillet muscle fatty acid compositions and blood biochemical parameters in 288 juvenile rainbow trout (130 g) at 3X2 factorial experimental design. Ractopamine and 1 g/kg carnitine improved the specific growth rate, feed conversion ratio, protein efficiency ratio and weight gain at the end of experiment. The protein and lipid contents of fillet muscle were affected by the inclusion of 10 mg/kg ractopamine in the diet, increasing crude protein and reducing crude fat (P<0.05) of fish fillet muscle. The highest protein and lowest fat contents of fish fillet were observed in diet that contains 2 g/kg carnitine plus ractopamine. Ractopamine and carnitine increased levels of albumin, total protein and globulin in fish blood serum, but carnitine increased triacylglycerol and cholesterol. Fatty acids compositions of fish fillet were also affected by ractopamine and carnitine. All fatty acids except for eicosapentaenoic acid and docosahexaenoic acid, were increased by dietary supplementation of ractopamine. Total saturated fatty acids were not affected by carnitine. Supplementation (P>0.05). However, total n-3 poly unsaturated fatty acids were reduced by carnitine supplementation. A significant interaction was observed between ractopamine and carnitine supplementation regarding the saturated (P<0.01) and n-3 poly unsaturated fatty acid (P<0.001) of fish fillet. This study shows that supplementation of 1 g/kg carnitine and 10 ppm ractopamine could improve performance of juvenile rainbow trout and their combination in diet results in protein increment, fat reduction and change in profile of fatty acids in fillet muscle.

A influência do exercício físico de moderada intensidade e da suplementação com ácido lipóico sobre a função cardíaca, a bioquímica sanguínea e a angiogênese em miocárdio de Rattus norvegicus

Atualmente o exercício físico vem sendo utilizado com intuito de redução de massa corporal, em especial de massa gorda. Entretanto cresce o número de indivíduos que, associado ao exercício físico, utiliza substâncias com característica lipolítica, como é o caso do ácido lipóico. Tanto o exercício físico, quanto a utilização de suplementação com ácido lipóico são responsáveis por remodelagem vascular (devido à interferência no processo de angiogênese) e modificação de fatores de risco cardiovascular (como hipertensão e lipídeos sanguíneos elevados). Desta forma, o presente estudo buscou analisar a influência do exercício físico de moderada intensidade e da suplementação com ácido lipóico sobre a frequência cardíaca, pressão arterial, bioquímica sanguínea e angiogênese no músculo cardíaco e no músculo esquelético de ratos Wistar. Foram utilizados 80 ratos Wistar, divididos em quatro grupos: controle, ácido lipóico (LA), exercício (E) e associação (ELA). Os animais foram submetidos a um programa de adaptação e treinamento de natação (9 e 17 semanas) com um aumento progressivo no tempo natação (até 1h/dia) e intensidade de carga (até 5% do peso corporal). Os animais receberam ácido lipóico 5 vezes por semana (da 10ª à 17ª semana), 60 mg / Kg / dia. O exercício crônico de intensidade moderada promoveu bradicardia, mas sua associação com a suplementação de ácido lipóico interrompeu este benefício. A suplementação com LA mostrou-se eficaz em melhorar o perfil lipídico, mas associado ao exercício não apresentou redução. A angiogênese foi aumentada no coração e gastrocnêmio dos animais exercitados, a largura da fibra de E, LA e ELA foi reduzida no coração, enquanto no gastrocnêmio apresentaram um aumento na largura das fibras apenas por LA e ELA. A espessura do ventrículo esquerdo diminuiu no grupo E, enquanto que a área da câmara do ventrículo esquerdo, e os níveis de VEGF circulantes, não mostraram nenhuma diferença significativa. Foi observada uma interação negativa entre o exercício físico e a suplementação com ácido lipóico (supressão da bradicardia do exercício de um lado, e a perda da adaptação do perfil lipídico induzido por suplementação de AL por outro lado). Este estudo mostra pela primeira vez, a interação entre o exercício crônico de intensidade moderada e a suplementação com ácido lipóico sobre a remodelação cardíaca e angiogênese, confirmando os benefícios da prática física em melhorar o fornecimento de sangue do músculo, que não foi afetado pelo consumo de ácido lipóico. O ácido lipóico em animais não treinados não foi capaz de estimular a 9 angiogênese cardíaca e ao contrário mostram uma tendência para a redução dos novos vasos.