Functional identity of receptors for proteolysis-inducing factor on human and murine skeletal muscle.

BACKGROUND: Cachexia in both mice and humans is associated with tumour production of a sulphated glycoprotein called proteolysis-inducing factor (PIF). In mice PIF binds with high affinity to a surface receptor in skeletal muscle, but little is known about the human receptor. This study compares the human PIF receptor with the murine. METHODS: Human PIF was isolated from the G361 melanoma and murine PIF from the MAC16 colon adenocarcinoma. The human PIF receptor was isolated from human skeletal muscle myotubes. Protein synthesis and degradation induced by human and murine PIF was studied in human and murine skeletal muscle myotubes. RESULTS: Both the human and murine PIF receptors showed the same immunoreactivity and Mr 40 000. Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody. Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody. CONCLUSIONS: These results suggest that the murine and human PIF receptors are identical.

Attenuation of muscle atrophy by an N-terminal peptide of the receptor for proteolysis-inducing factor (PIF).

BACKGROUND: Atrophy of skeletal muscle in cancer cachexia has been attributed to a tumour-produced highly glycosylated peptide called proteolysis-inducing factor (PIF). The action of PIF is mediated through a high-affinity membrane receptor in muscle. This study investigates the ability of peptides derived from the 20 N-terminal amino acids of the receptor to neutralise PIF action both in vitro and in vivo. METHODS: Proteolysis-inducing factor was purified from the MAC16 tumour using an initial pronase digestion, followed by binding on DEAE cellulose, and the pronase was inactivated by heating to 80°C, before purification of the PIF using affinity chromatography. In vitro studies were carried out using C(2)C(12) murine myotubes, while in vivo studies employed mice bearing the cachexia-inducing MAC16 tumour. RESULTS: The process resulted in almost a 23,000-fold purification of PIF, but with a recovery of only 0.004%. Both the D- and L-forms of the 20mer peptide attenuated PIF-induced protein degradation in vitro through the ubiquitin-proteosome proteolytic pathway and increased expression of myosin. In vivo studies showed that neither the D- nor the L-peptides significantly attenuated weight loss, although the D-peptide did show a tendency to increase lean body mass. CONCLUSION: These results suggest that the peptides may be too hydrophilic to be used as therapeutic agents, but confirm the importance of the receptor in the action of the PIF on muscle protein degradation.

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-α(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-α(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.

The role of zinc in the anti-tumour and anti-cachectic activity of D-myo-inositol 1,2,6-triphosphate.

BACKGROUND: D-myo-inositol-1,2,6-triphosphate (alpha-trinositol, AT) is a polyanionic molecule capable of chelating divalent metal ions with anti-tumour and anti-cachectic activity in a murine model. METHODS: To investigate the role of zinc in this process, mice bearing cachexia-inducing MAC16 tumour were treated with AT, with or without concomitant administration of ZnSO(4). RESULTS: At a dose of 40 mg kg(-1), AT effectively attenuated both weight loss and growth of the MAC16 tumour, and both effects were attenuated by co-administration of Zn(2+). The concentration of zinc in gastrocnemius muscle increased with increasing weight loss, whereas administration of AT decreased the levels of zinc in plasma, skeletal muscle and tumour, which were restored back to control values after administration of ZnSO(4). CONCLUSION: These results suggest that zinc is important in both tumour growth and cachexia in this animal model.

Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II.

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and D-alpha-tocopherol (10 microM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin-proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-kappaB (NF-kappaB), through inhibition of phosphorylation of the NF-kappaB inhibitor protein (I-kappaB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 microM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 microM), a specific inhibitor of cytosolic phospholipase A2, U-73122 (5 microM) and D609 (200 microM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 microM) and NSC23766 (10 microM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 microM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 microM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with D-alpha-tocopherol (1 mg kg(-1)) attenuated protein degradation and increased protein synthesis in skeletal muscle.

Induction of protein degradation in skeletal muscle by a phorbol ester involves upregulation of the ubiquitin-proteasome proteolytic pathway.

Although muscle atrophy is common to a number of disease states there is incomplete knowledge of the cellular mechanisms involved. In this study murine myotubes were treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to evaluate the role of protein kinase C (PKC) as an upstream intermediate in protein degradation. TPA showed a parabolic dose-response curve for the induction of total protein degradation, with an optimal effect at a concentration of 25 nM, and an optimal incubation time of 3 h. Protein degradation was attenuated by co-incubation with the proteasome inhibitor lactacystin (5 microM), suggesting that it was mediated through the ubiquitin-proteasome proteolytic pathway. TPA induced an increased expression and activity of the ubiquitin-proteasome pathway, as evidenced by an increased functional activity, and increased expression of the 20S proteasome alpha-subunits, the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme E2(14k), also with a maximal effect at a concentration of 25 nM and with a 3 h incubation time. There was also a reciprocal decrease in the cellular content of the myofibrillar protein myosin. TPA induced activation of PKC maximally at a concentration of 25 nM and this effect was attenuated by the PKC inhibitor calphostin C (300 nM), as was also total protein degradation. These results suggest that stimulation of PKC in muscle cells initiates protein degradation through the ubiquitin-proteasome pathway. TPA also induced degradation of the inhibitory protein, I-kappaBalpha, and increased nuclear accumulation of nuclear factor-kappaB (NF-kappaB) at the same time and concentrations as those inducing proteasome expression. In addition inhibition of NF-kappaB activation by resveratrol (30 microM) attenuated protein degradation induced by TPA. These results suggest that the induction of proteasome expression by TPA may involve the transcription factor NF-kappaB.

Biology of cachexia.

About half of all cancer patients show a syndrome of cachexia, characterized by loss of adipose tissue and skeletal muscle mass. Such patients have a decreased survival time, compared with the survival time among patients without weight loss, and loss of total body protein leads to substantial impairment of respiratory muscle function. These changes cannot be fully explained by the accompanying anorexia, and nutritional supplementation alone is unable to reverse the wasting process. Despite a falling caloric intake, patients with cachexia frequently show an elevated resting energy expenditure as a result of increases in Cori cycle (i.e., catalytic conversion of lactic acid to glucose) activity, glucose and triglyceride-fatty acid cycling, and gluconeogenesis. A number of cytokines, including tumor necrosis factor-apha, interleukins 1 and 6, interferon gamma, and leukemia-inhibitory factor, have been proposed as mediators of the cachectic process. However, the results of a number of clinical and laboratory studies suggest that the action of the cytokines alone is unable to explain the complex mechanism of wasting in cancer cachexia. In addition, cachexia has been observed in some xenograft models even without a cytokine involvement, suggesting that other factors may be involved. These probably include catabolic factors, which act directly on skeletal muscle and adipose tissue and the presence of which has been associated with the clinical development of cachexia. A polyunsaturated fatty acid, eicosapentaenoic acid, attenuates the action of such catabolic factors and has been shown to stabilize the process of wasting and resting energy expenditure in patients with pancreatic cancer. Such a pharmacologic approach may provide new insights into the treatment of cachexia.

Lipolytic factors associated with murine and human cancer cachexia.

We have identified a lipolytic factor in extracts of a cachexia-inducing murine carcinoma (MAC16) that shows characteristics of an acidic peptide and appears to be composed of three fractions of apparent molecular weights corresponding to 3 kd, 1.5 kd, and 0.7 kd, as determined by exclusion chromatography. Material with identical chromatographic and molecular weight characteristics was also present in the serum of patients with clinical cancer cachexia but absent from normal serum, even under conditions of starvation. The MAC16 lipid factor, when injected into animals bearing the non-cachexia-inducing tumor MAC13, was capable of inducing weight loss without a significant reduction in food intake. Similar lipolytic material, although in lower concentration, was also found in the MAC13 tumor extracts. These findings suggest that cachexia may arise from the enhanced expression of a lipolytic factor associated with tumor cells.

Characterization of a transplantable adenocarcinoma of the mouse colon producing cachexia in recipient animals.

MAC16 is a chemically induced, transplantable adenocarcinoma of the colon passaged in inbred NMRI mice. At small tumor burdens (less than 1% of the host weight), weight loss was observed without a reduction in food intake. As the tumor mass increased, weight loss also increased and reached 33% of host body weight in females and 20% in males when compared with the weight of age-matched controls. The reduction in host body weight was directly proportional to the tumor size and was reversible when the tumor was excised. There was a preferential loss of body fat in tumor-bearing animals with an increase in the plasma level of free fatty acids, although there was a minimal elevation of ketone bodies. Tumor growth was accompanied by progressive hypoglycemia and a reduction in the plasma insulin levels. The decrease in plasma insulin may have contributed to the catabolic effects of progressive tumor growth.

Nitrogen excretion in cancer cachexia and its modification by a high fat diet in mice.

Animals transplanted with the MAC16 colon adenocarcinoma showed a loss of body weight as the tumor weight increased, without a reduction in food intake. Both adipose tissue and muscle mass decreased in tumor-bearing animals, although loss of body fat exceeded that of muscle mass for given tumor weight. Urinary nitrogen excretion was significantly elevated when the weight loss did not exceed 3 to 4 g, but above this weight loss there was a conservation of nitrogen and the excretion level fell to or below that found in non-tumor-bearing animals. The presence of a tumor alone was not sufficient to account for the elevated nitrogen excretion, since animals bearing a related colon adenocarcinoma (MAC13) that did not induce weight loss had a nitrogen excretion pattern similar to that of non-tumor-bearing controls. Feeding an isocaloric isonitrogenous diet in which 80% of the calories were supplied as medium chain triglycerides, which significantly elevated plasma levels of ketone bodies, reduced both tumor weight and host weight loss and restored both the nitrogen balance and urea excretion to that of non-tumor-bearing animals. The plasma levels of amino acids, which were reduced in the cachectic state, were also restored to control values in animals fed the medium chain triglyceride diet. These results suggest that excessive nitrogen catabolism in the cachectic state can be prevented by suitable dietary modification.

Inhibition of weight loss by omega-3 fatty acids in an experimental cachexia model.

The effect of substitution of the carbohydrate component of the diet by calories derived from fish oil on host body weight loss and tumor growth rate has been studied in an experimental colon adenocarcinoma (MAC16). This tumor produces extensive host weight loss and reductions in both total body fat and muscle dry weight, without a reduction in food intake. Diets containing fish oil significantly reduced host body weight loss, with almost complete protection occurring when the fish oil comprised 50% of the calories, without an alteration of total calorie consumption or nitrogen intake. There was also a significant reduction in tumor growth rate, although the reduction in host weight loss was greater than might be expected from a smaller tumor burden. The reduction of host body weight loss was associated with an increase in total body fat and muscle mass. The effect appears specific to the type of fat since comparable results were not obtained with a gamma-linolenic acid-enriched diet. When compared with cyclophosphamide and 5-fluorouracil the fish oil diet exerted a similar antitumor effect at the maximum dose. Whereas the antitumor effect of the former agents was achieved with considerable host toxicity, the latter produced no toxicity and almost completely abolished the cachectic effect of the tumor. These results suggest that fish oil is a nontoxic, highly effective anticachectic agent with the added advantage of antitumor activity.

Production of lipolytic and proteolytic factors by a murine tumor-producing cachexia in the host.

Animals given transplants of the MAC16 colon adenocarcinoma show a progressive decrease in carcass weight as the tumor size increases without a reduction in either fluid or caloric intake when compared with non-tumor-bearing controls. There is a decrease in both carcass fat and muscle mass which is directly proportional to the weight of the tumor. In male animals weight loss occurs when the tumor mass comprises more than 0.3% of the body weight and reaches 30% when the tumor represents 3% of the body weight. There is evidence for the production by the tumor of both lipolytic and proteolytic factors, which may be responsible for the cachexia, since two related mouse adenocarcinomas, which do not produce weight loss, have little lipolytic or proteolytic activity. The lipolytic factor is nondialyzable and is destroyed by both heat and acid. Both insulin and 3-hydroxybutyrate suppress the lipolytic activity of the tumor extract. The MAC16 tumor also contains a serine protease, the activity of which is also completely abolished by insulin and 3-hydroxybutyrate. Animals bearing the MAC16 tumor have an elevated plasma lipolytic and proteolytic activity when compared with non-tumor-bearing controls, suggesting a peripheral effect of the tumor products. The catabolic factors elaborated by the MAC16 adenocarcinoma may be responsible for the loss of both the fat and nonfat carcass mass, but they do respond to normal metabolic controls.

Mechanism of inhibition of a tumor lipid-mobilizing factor by eicosapentaenoic acid.

Stimulation of lipolysis or adenylate cyclase activity by either isoprenaline or a tumor-derived lipid-mobilizing factor was effectively attenuated in isolated white adipocytes or in adipocyte plasma membranes pretreated with eicosapentaenoic acid (EPA) dissolved in ethanol or from mice dosed p.o. with EPA (1.25 g/kg). A similar effect was observed with docosahexanoic acid (DHA) that may be partly due to retroconversion to EPA. Stimulation of adenylate cyclase activity by forskolin, which acts directly on the enzyme without the involvement of a receptor, was also decreased in membranes of mice treated with EPA, suggesting a direct interaction between EPA and adenylate cyclase. Pertussis toxin eliminated the inhibition of lipolysis and the stimulation of adenylate cyclase by isoprenaline and lipid-mobilizing factor in the presence of EPA, but not DHA. This suggests that the attenuation of hormonal stimulation of adenylate cyclase by EPA was due, at least in part, to an inhibitory guanine nucleotide-binding protein-mediated inhibition of adenylate cyclase activity. The effect of DHA may be due to a direct inhibition of the cyclase catalytic component. The ability of EPA to preserve fat stores during the process of cachexia seems to arise from the attenuation of the stimulation of adenylate cyclase.

Anticachectic and antitumor effect of eicosapentaenoic acid and its effect on protein turnover.

The effect of the polyunsaturated fatty acids eicosapentaenoic acid (EPA) and gamma-linolenic acid (GLA) on host body weight loss and tumor growth has been investigated in mice bearing a cachexia-inducing colon adenocarcinoma, the MAC16. EPA effectively inhibited both host weight loss and tumor growth rate in a dose-related manner with optimal effects being observed at a dose level of 1.25 to 2.5 g/kg. At these concentrations host body weight was effectively maintained, and there was a delay in the progression of growth of the tumor, such that overall survival was approximately doubled in EPA-treated animals, using the criteria dictated by the United Kingdom Coordinating Committee for the welfare of animals with neoplasms. Even when tumor growth resumed, weight loss did not occur. Animals bearing the MAC16 tumor showed a decreased protein synthesis and an increased degradation in skeletal muscle. Treatment with EPA significantly reduced protein degradation without an effect on protein synthesis. The effect of GLA on both host body weight loss and tumor growth was much less pronounced than that of EPA, with an effect only being seen at a dose of 5 g/kg, at which some toxicity was observed. In vitro studies showed that while EPA was effective in inhibiting tumor-induced lipolysis, GLA was ineffective in this respect. However, prostaglandin E1, which is formed from GLA in vivo, showed partial reversal of tumor-induced lipolysis and probably accounted for the anticachectic effect of GLA. These results suggest that EPA as the pure fatty acid should be considered for clinical investigation as both an anticachectic and antitumor agent, since prior work has shown that the other major component of fish oil docosahexaenoic acid is without pharmacological activity in this system.

Effect of a cancer cachectic factor on protein synthesis/degradation in murine C2C12 myoblasts: modulation by eicosapentaenoic acid.

The effect of a proteolysis inducing factor (PIF) on protein synthesis and degradation and the modulation of this effect by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA), have been examined using a surrogate model system, C2C12 myoblasts in vitro. After 90 min of incubation, PIF produced a significant inhibition of protein synthesis in a dose-dependent manner, with maximal inhibition at a concentration of 4 nM. The effect was attenuated both by treatment with a monoclonal antibody to PIF and by treatment with insulin at physiological concentrations (1 nM) and below (0.1 nM), but not by EPA (50 microM). The inhibitory effect on protein synthesis was transitory and was not seen after prolonged incubation with PIF. An increased rate of protein degradation was observed in C2C12 myoblasts after addition of PIF, which was also maximal at a concentration of PIF of 4 nM. Higher concentrations of PIF did not produce an increase in protein degradation. Unlike the effect on protein synthesis, the enhanced protein degradation was completely abolished by pretreatment with 50 microM EPA, suggesting that the two effects are mediated by different mechanisms. PIF produced an increased release of [3H]arachidonic acid from prelabeled myoblasts with a dose-response curve parallel to that of protein degradation and with a maximum at 4 nM PIF. Release of [3H] arachidonic acid was completely blocked in cells pretreated with 50 microM EPA, suggesting that the effect was related to protein degradation. The [3H]arachidonic acid was rapidly metabolized to prostaglandins E2 and F2alpha and to 5-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs). Production of all eicosanoids was attenuated in cells pretreated with EPA. Of all of the metabolites, only 15-HETE produced a significant increase in protein degradation in C2C12 myoblasts with a maximal effect at 30 nM and with a bell-shaped dose-response curve similar to that produced by PIF. These results suggest that PIF enhances protein degradation as a result of an increased production of 15-HETE.

Mechanism of attenuation of skeletal muscle protein catabolism in cancer cachexia by eicosapentaenoic acid.

Cancer cachexia is characterized by selective depletion of skeletal muscle protein reserves. Soleus muscles from mice bearing a cachexia-inducing tumor (MAC16) showed an increased protein degradation in vitro, as measured by tyrosine release, when compared with muscles from nontumor-bearing animals. After incubation under conditions that modify different proteolytic systems, lysosomal, calcium-dependent, and ATP-dependent proteolysis were found to contribute to the elevated protein catabolism. Treatment of mice bearing the MAC16 tumor with the polyunsaturated fatty acid, eicosapentaenoic acid (EPA), attenuated loss of body weight and significantly suppressed protein catabolism in soleus muscles through an inhibition of an ATP-dependent proteolytic pathway. The ATP-ubiquitin-dependent proteolytic pathway is considered to play a major role in muscle catabolism in cachexia, and functional proteasome activity, as determined by "chymotrypsin-like" enzyme activity, was significantly elevated in gastrocnemius muscle of mice bearing the MAC16 tumor as weight loss progressed. When animals bearing the MAC16 tumor were treated with EPA, functional proteasome activity was completely suppressed, together with attenuation of the expression of 20S proteasome alpha-subunits and the p42 regulator, whereas there was no effect on the expression of the ubiquitin-conjugating enzyme (E2(14k)). These results suggest that EPA induces an attenuation of the up-regulation of proteasome expression in cachectic mice, and this was correlated with an increase in myosin expression, confirming retention of contractile proteins. EPA also inhibited growth of the MAC16 tumor in a dose-dependent manner, and this correlated with suppression of the expression of the 20S proteasome alpha-subunits in tumor cells, suggesting that this may be the mechanism of tumor growth inhibition. Thus EPA antagonizes loss of skeletal muscle proteins in cancer cachexia by down-regulation of proteasome expression, and this may also be the mechanism for inhibition of tumor growth.

Increased gene expression of brown fat uncoupling protein (UCP)1 and skeletal muscle UCP2 and UCP3 in MAC16-induced cancer cachexia.

Weight loss in cancer cachexia is attributable to decreased food intake and/or enhanced energy expenditure. We investigated the roles of the uncoupling proteins (UCPs) UCPI, -2, and -3 in a murine model of cachexia, the MAC16 adenocarcinoma. Weight fell to 24% below that of non-tumor-bearing controls (P < 0.01) 18 days after MAC16 inoculation, with significant reductions in fat-pad mass (-67%; P < 0.01) and muscle mass (-20%; P < 0.01). Food intake was 26-60% lower (P < 0.01) than in controls on days 17-18. Non-tumor-bearing mice, pair-fed to match MAC16-induced hypophagia, showed less weight loss (10% below controls, P < 0.01; 16% above MAC-16, P < 0.01) and smaller decreases in fat-pad mass (21% below controls, P < 0.01). Core temperature in MAC16 mice was significantly lower (-2.4 degrees C, P < 0.01) than in controls, and pair-feeding had no effect. MAC16 mice showed significantly higher UCP1 mRNA levels in brown adipose tissue (BAT) than in controls (+63%, P < 0.01), and pair-feeding had no effect. UCP2 and -3 expression in BAT did not differ significantly between groups. By contrast, UCP2 mRNA levels in skeletal muscle were comparably increased in both MAC16 and pair-fed groups (respectively, 183 and 163% above controls; both, P < 0.05), with no significant difference between these two groups. Similarly, UCP3 mRNA was significantly higher than controls in both MAC16 (+163%, P < 0.05) and pair-fed (+253%, P < 0.01) groups, with no significant difference between the two experimental groups. Overexpression of UCP1 in BAT in MAC16-bearing mice may be an adaptive response to hypothermia, which is apparently induced by tumor products; increased thermogenesis in BAT could increase total energy expenditure and, thus, contribute to tissue wasting. Increased UCP2 and -3 expression in muscle are both attributable to reduced food intake and may be involved in lipid utilization during lipolysis in MAC16-induced cachexia.

Induction of muscle protein degradation and weight loss by a tumor product.

Splenocytes from mice bearing a cachexia-inducing tumor (MAC16) have been fused with mouse myeloma cells to produce hybridomas, which have been cloned to produce antibody reactive to a material which copurified with a lipid-mobilizing factor isolated from the same tumor. The monoclonal antibody has been used to investigate factors potentially involved in the development of cachexia. The major protein detectable by immunoprecipitation of a partially purified lipid-mobilizing factor was M(r) 69,000, whereas Western blotting showed two bands of M(r) 69,000 and M(r) 24,000. Although the monoclonal antibody did not neutralize lipid-mobilizing activity in an in vitro assay, it did neutralize a serum factor capable of protein degradation in isolated gastrocnemius muscle. Affinity purification of MAC16 tumor homogenates using the monoclonal antibody yielded two immunoreactive bands of M(r) 69,000 and M(r) 24,000, which were further fractionated on a hydrophobic column (C8). This material was capable of inducing tyrosine release from isolated gastrocnemius muscle, and the effect could be blocked with the monoclonal antibody. The two immunoreactive bands from the hydrophobic column were capable of inducing weight loss in mice, whereas nonimmunoreactive fractions had no effect on body weight. The M(r) 24,000 species had a unique amino acid sequence, whereas the M(r) 69,000 species gave the same sequence as the M(r) 24,000 material, together with that for albumin. The M(r) 24,000 species contained carbohydrate, and lectin blotting showed a strong reaction with wheat germ and Erythrina crystagalli agglutinins. This suggests that the material is a glycoprotein or proteoglycan that shows strong binding affinity for albumin, possibly through the carbohydrate residues.

Purification and characterization of a lipid-mobilizing factor associated with cachexia-inducing tumors in mice and humans.

A scheme is described for the purification of a lipid-mobilizing factor from a cachexia-inducing murine tumor (MAC16) using a combination of ion exchange (Mono Q), exclusion (Superose), and hydrophobic (C8) chromatography. This process yields an active material with an apparent molecular weight of 24,000 with an overall purification of 3,500 from the tumor homogenate and representing 0.005% of the total protein present. The material tends to aggregate to high molecular mass, is acidic (pI < 4), and displays heterogeneity of charge as evidenced by a broad elution profile on ion exchange and exclusion chromatography and multiple peaks on hydrophobic columns. The purified material was heat and alkali (pH 10.4) labile and activity could be completely inhibited by sulfatase, suggesting that the negative charge could arise from sulfate residues. There was no evidence that the material possessed triglyceride lipase activity. Animals transplanted with the MAC16 tumor and with a delayed weight loss contained in their serum antibodies that recognized a M(r) 24,000 band on Western blots. This material copurified with the lipid-mobilizing factor. Such antibodies were not present in the serum of mice transplanted with the MAC13 tumor, which does not induce cachexia, suggesting that the antibodies were directed to the induction of cachexia rather than the tumor itself. Urine from patients with cancer cachexia also contained a lipid-mobilizing factor which adhered to DEAE-cellulose and gave an apparent M(r) of 24,000 by exclusion chromatography. Western blotting using serum from MAC16 tumor-bearing animals showed the presence of a band of M(r) 24,000 in such fractions, which was not detected using serum from mice bearing the MAC13 tumor. This band was not present in Western blots of urine from normal subjects. The fact that serum from mice bearing the MAC16 tumor can detect the human lipid-mobilizing activity suggests a high degree of structural similarity between the two and raises the possibility that cachexia in humans may be caused by the same species as in the mouse.