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/EBPa), CCAAT/enhancer binding protein beta, peroxisome proliferatoractivated receptor gamma, and sterol regulatory element binding protein-1c (SREBP-1c) in adipose tissue, which was accompanied by reduced protein content of C/EBPa 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.

The potential for inhibitors of nuclear factor-kB (NF-kB) activation to act as inhibitors of muscle protein degradation in cancer cachexia has been evaluated both in vitro and in vivo. Activation of NF-kB is important in the induction of proteasome expression and protein degradation by the tumour factor, proteolysis-inducing factor (PIF), since the cell permeable NF-kB inhibitor SN50 (18 mM) attenuated the expression of 20S proteasome a-subunits, two subunits of the 19S regulator MSS1 and p42, and the ubiquitinconjugating enzyme, E2 14k , as well as the decrease in myosin expression in murine myotubes. To assess the potential therapeutic benefit of NF-kB inhibitors on muscle atrophy in cancer cachexia, two potential inhibitors were employed; curcumin (50 mM) and resveratrol (30 mM). Both agents completely attenuated total protein degradation in murine myotubes at all concentrations of PIF, and attenuated the PIF-induced increase in expression of the ubiquitin -proteasome proteolytic pathway, as determined by the 'chymotrypsin-like' enzyme activity, proteasome subunits and E2 14k . However, curcumin (150 and 300 mg kg À1 ) was ineffective in preventing weight loss and muscle protein degradation in mice bearing the MAC16 tumour, whereas resveratrol (1 mg kg À1 ) significantly attenuated weight loss and protein degradation in skeletal muscle, and produced a significant reduction in NF-kB DNAbinding activity. The inactivity of curcumin was probably due to a low bioavailability. These results suggest that agents which inhibit nuclear translocation of NF-kB may prove useful for the treatment of muscle wasting in cancer cachexia.

No abstract

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose -response curve and with a maximal effect between 0.05 and 0.1 mM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose -response curve, and with a maximal effect between 1 and 2.5 mM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 mM) and MG132 (10 mM), suggesting that the effect was mediated through upregulation of the ubiquitin -proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome 'chymotrypsin-like' enzyme activity as well as an increase in protein expression of 20S proteasome a-subunits, the 19S subunits MSS1 and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin -proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitort) (30 mg kg À1 ) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model.

Loss of adipose tissue in cancer cachexia in mice bearing the MAC16 tumour arises from an increased lipid mobilisation through increased expression of zinc-a 2 -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 À1 ) attenuated both the loss of body weight and ZAG expression in WAT. Dexamethasone (66 mg kg À1 ) 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 mM) to stimulate lipolysis and increase ZAG expression, and both were attenuated by RU38486 (10 mM), anti-ZAG antibody (1 mgml À1 ), and the b3-adrenoreceptor (b3-AR) antagonist SR59230A (10 mM). Zinc-a 2 -glycoprotein also increased its own expression and this was attenuated by SR59230A, suggesting that it was mediated through the b3-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.

No abstract

Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipocyte plasma membranes, by a tumour-produced lipid mobilizing factor, was attenuated by low concentrations (10 77 -10 75 M) of the specific b3-adrenoceptor antagonist SR59230A. Lipid mobilizing factor (250 nM) produced comparable increases in intracellular cyclic AMP in CHOK1 cells transfected with the human b3-adrenoceptor to that obtained with isoprenaline (1 nM). In both cases cyclic AMP production was attenuated by SR59230A confirming that the effect is mediated through a b3-adrenoceptor. A non-linear regression analysis of binding of lipid mobilizing factor to the b3-adrenoceptor showed a high affinity binding site with a Kd value 78+45 nM and a B max value (282+1 fmole mg protein 71 ) comparable with that of other b3-adrenoceptor agonists. These results suggest that lipid mobilizing factor induces lipolysis through binding to a b3-adrenoceptor.

Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E2 14k , has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E2 14k were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E2 14k mRNA and protein levels in gastrocnemius muscle with increases of 6 -8-fold for C2 and two-fold for E2 14k between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25 -27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased two-fold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18 -20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E2 14k mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss.