Adipose Triglyceride Lipase Contributes to Cancer-Associated Cachexia

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Science  08 Jul 2011:
Vol. 333, Issue 6039, pp. 233-238
DOI: 10.1126/science.1198973

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Cachexia is a multifactorial wasting syndrome most common in patients with cancer that is characterized by the uncontrolled loss of adipose and muscle mass. We show that the inhibition of lipolysis through genetic ablation of adipose triglyceride lipase (Atgl) or hormone-sensitive lipase (Hsl) ameliorates certain features of cancer-associated cachexia (CAC). In wild-type C57BL/6 mice, the injection of Lewis lung carcinoma or B16 melanoma cells causes tumor growth, loss of white adipose tissue (WAT), and a marked reduction of gastrocnemius muscle. In contrast, Atgl-deficient mice with tumors resisted increased WAT lipolysis, myocyte apoptosis, and proteasomal muscle degradation and maintained normal adipose and gastrocnemius muscle mass. Hsl-deficient mice with tumors were also protected although to a lesser degree. Thus, functional lipolysis is essential in the pathogenesis of CAC. Pharmacological inhibition of metabolic lipases may help prevent cachexia.

Cachexia (kakos hexis, Greek for “bad condition”) is a devastating syndrome that frequently occurs in patients suffering from chronic infection, such as tuberculosis or AIDS, and other diseases, including chronic obstructive pulmonary disease, chronic kidney disease, and chronic heart failure. Most commonly, however, cachexia is observed in cancer. The highest frequency of cancer-associated cachexia (CAC) occurs in pancreatic and gastric cancer (13). CAC is an important adverse prognostic factor and the immediate cause of death in an estimated 15% of all cancer patients (35). Wasting results from depletion of both adipose tissue and skeletal muscle (6, 7). In contrast to starvation, the nonmuscle protein compartment of the body is relatively unaffected in CAC patients (7), implying a tumor-associated metabolic condition that specifically targets adipose tissue and muscle. Thus, anorexia is unlikely to be solely responsible for the loss of skeletal muscle in patients with CAC. Indeed, nutritional supplementation has largely failed to reverse the wasting process (8).

The pathogenesis of CAC is multifactorial (9). Central mechanisms regulate appetite, food intake, and energy consumption. Contributing peripheral mechanisms control lipid and carbohydrate metabolism in various tissues. Severe lipid loss in CAC is driven by changes in lipid catabolism (1015) and, possibly, lipogenesis (16). The concept that increased triacylglycerol (TG) degradation may contribute decisively to CAC is strongly underscored by increased plasma levels of fatty acids (FAs) and glycerol; increased lipolytic rates upon epinephrine stimulation; and increased expression of lipid-mobilizing factors, such as zinc-α2 glycoprotein-1 (AZGP1), tumor necrosis factor α (TNF-α), and interleukins (IL)-1 and -6 (17, 18). The breakdown of fat requires lipolysis of TG stored in cellular lipid droplets and is mediated by adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) (19). This led to our hypothesis that disruption of fat catabolism may prevent the initiation and/or progression of CAC. In vertebrates, lipolysis is most active in adipose tissue, with ATGL predominantly responsible for the initial step of TG hydrolysis (formation of diacylglycerol) and HSL for the hydrolysis of diacylglycerol. We investigated whether one or both of these lipases are essential for CAC.

To assess the role of lipases in CAC, we used two different cachexia models in mice lacking either Atgl or Hsl. Cachexia was induced in wild-type C57BL/6 (WT) mice, Atgl-deficient (Atgl−/−) mice (20), and Hsl-deficient (Hsl−/−) mice (21) by subcutaneous injection of Lewis lung carcinoma (LLC) cells (22) or B16 melanoma cells (23). Tumor growth was observed in 100% of treated animals. Tumor weights tended to be lower in nonfasted (Fig. 1, A to C) and overnight (o/n)-fasted (fig. S1, A and B) (24) Atgl−/− and Hsl−/− mice than in WT mice. However, none of the differences reached statistical significance.

Fig. 1

Ablation of Atgl protects mice from cancer-associated weight loss and cancer-associated loss of adipose tissue. (A to C) Tumor weights 14 days (d) and 21 days after injecting LLC and 16 days after injecting B16 melanoma (B16) cells were slightly lower in lipase-deficient mice compared with WT. (D to F) WT mice significantly lost weight with tumor progression after injection of LLC and B16 tumor cells, whereas Atgl−/− animals did not develop cachexia. Hsl−/− mice also lost weight but less than WT mice did. (G) Normalized gonadal and epididymal WAT was reduced by about 55% in nonfasted tumor-bearing WT mice 21 days after LLC tumor implantation, whereas lipase-deficient mice were protected from WAT loss. (H to J) No or minimal gonadal and epididymal WAT was detected upon dissection of nonfasted WT B16 tumor-bearing mice after 16 days and fasted WT LLC tumor-bearing mice after 21 days compared to saline injected control mice. Atgl−/− tumor-bearing mice retained gonadal and epididymal WAT, whereas partial loss of gonadal and epididymal WAT was observed in Hsl−/− tumor-bearing mice. WAT was dissected, and its mass normalized to tibia length as described in (24). Black bars indicate control (saline-injected) animals; white bars, tumor-bearing animals. To allow direct comparison, values were determined after removal of the respective tumor. ***P < 0.001, **P < 0.01, *P < 0.05, n = 5 to 7 except for (A) to (E) and (G) LLC, Hsl−/−, n = 3 and (D) LLC, 14 days, normal, n = 2. (J) Scale bars represent a length of 1 mm.

Next we analyzed body weight, plasma metabolite concentrations, and fat mass in mice of all genotypes with or without tumors. Because all of these parameters strongly depend on the feeding status of the mice and to account for any nutritional bias, they were determined in nonfasted and o/n-fasted animals. Total body weight (after subtraction of tumor weight) differed drastically in lipase-deficient mouse models compared with WT mice in response to LLC and B16 (Fig. 1, D to F). Whereas non–tumor-bearing WT mice on normal chow diet gained weight over a period of 3 weeks, WT mice with LLC started to lose weight 14 days after tumor injection, resulting in an average weight loss of 1.8 g after 21 days. In contrast, body weight in Atgl−/− mice with LLC was identical to Atgl−/− without tumors at all times. Hsl−/− mice exhibited an intermediate phenotype. Compared to non–tumor-bearing Hsl−/− mice, body weight was reduced. However, the loss was less extreme than in WT mice carrying the tumor. Similar results were obtained in B16-treated mice (Fig. 1 F). Compared with WT mice without tumors, B16-treated mice weighed 3.3 g less 16 days after tumor injection. In contrast, B16-treated Atgl−/− mice maintained weights similar to those of untreated Atgl−/ mice. Hsl−/− animals were less protected than Atgl−/− mice and lost on average 2.7 g of body weight. The differences were even more pronounced in o/n-fasted mice, a condition when lipolysis is physiologically induced (fig. S2). Whereas WT mice with LLC weighed 2.1 g (after 14 days) and 5.5 g (after 21 days) less than WT mice without tumors, Atgl−/− mice were totally resistant to weight loss and Hsl−/− animals reached intermediate values. Differences in weight loss in response to the tumors were not explained by variable food intake (fig. S3, A and B), because it was similar in all animals during the initial phase of the experiment and decreased uniformly in all tumor-carrying mice during the final 2 to 4 days. Thus, in the mouse, protection from CAC-associated weight loss can be entirely conferred by the lack of ATGL and partially by the absence of HSL.

Consistent with our previous findings (20, 21), plasma glucose, FA, and TG levels were reduced in o/n-fasted Atgl−/− versus WT mice (figs. S4 to S6). In o/n-fasted Hsl−/− mice, FA and TG levels were also reduced (although less than in Atgl−/− mice), whereas plasma glucose concentrations were unchanged compared with those of WT mice (figs. S4 to S6). The presence of LLC for 14 days did not affect plasma glucose levels in o/n-fasted mice of any genotype (fig. S4A). After 21 days, plasma glucose levels were decreased in tumor-bearing, o/n-fasted WT (–43.2%) and Hsl−/− mice (–27.3%) but remained unchanged in Atgl−/− mice when compared to mice of the same genotype without tumors (fig. S4B). These differences in glucose levels in LLC-treated WT and Hsl−/− mice were not observed in nonfasted animals (fig. S4C). Similarly, plasma glucose and FA levels of nonfasted B16-treated mice matched those in untreated animals (fig. S4D). Presence of LLC caused an increase in FA levels in WT (fasted: +24.0% at 14 days and +9.9% at 21 days; nonfasted: +54.5% at 21 days) and Hsl−/− mice (fasted: +23.4% at 14 days and +25.0% at 21 days) (fig. S5). In contrast, FA levels in tumor-bearing Atgl−/− mice remained unchanged compared with those of Atgl−/− mice without tumors independent of the feeding status. Increased FA levels were also observed in nonfasted B16-tumor-bearing WT mice (+27%) (fig. S5D). Serum TG levels were not significantly different in o/n-fasted animals with or without tumors (fig. S6).

To assess the contribution of adipose tissue loss to the tumor-induced weight loss, we determined white adipose tissue (WAT) mass by visual inspection, weighing of surgically removed adipose depots (gonadal and epididymal adipose tissue), and in vivo nuclear magnetic resonance (NMR) WAT quantitation. In nonfasted LLC-bearing WT mice, WAT weight decreased by 55% after 21 days of tumor growth (Fig. 1G), which corresponded to a loss of 1.7 g of WAT in NMR analysis (fig. S7A). Nonfasted B16-bearing WT mice lost 85% of WAT weight after 16 days of tumor growth (Fig. 1H) (–2.0 g of adipose tissue mass in NMR analysis, fig. S7B). In contrast, none of the tumors affected WAT mass of Atgl−/− mice. In fact, weight of body fat depots and total body fat was increased in Atgl−/− mice independent of the presence or absence of tumors when compared to non–tumor-bearing WT mice (Fig. 1, G and H, and fig. S7). This confirms our earlier observation that Atgl deficiency causes obesity in mice kept on a normal chow diet (20) and shows that WAT mass is independent of the presence of the tumor. In nonfasted Hsl−/− mice, LLC did not affect the weight of WAT depots. However, NMR analyses revealed a total WAT reduction by 0.7 g in (Fig. 1G and fig. S7A). B16 in Hsl−/− mice caused a 32% reduction of adipose tissue weight and a 0.9 g WAT loss in NMR analysis (Fig. 1H and fig. S7B). In o/n-fasted animals, the differences were even more striking (Fig. 1, I and J, and fig. S7C). After 21 days, LLC tumors caused the loss of more than 95% of WAT weight (2 g in NMR analysis) in WT mice, whereas adipose mass was again completely retained in LLC-treated Atgl−/− mice. Hsl−/− mice exhibited an intermediate loss of 37% of WAT weight. Results for LLC-bearing animals were substantiated by magnetic resonance imaging analysis. Taken together, these results show that, independently of feeding status and tumor type, ATGL deficiency completely and HSL deficiency partially protects mice from the loss of WAT.

Tumor-associated loss of adipose tissue in animal models has been mostly attributed to an increase in WAT lipolysis (1016). Consistent with these reports, the release of FAs and glycerol from WAT explants was increased in WT mice with LLC (38% and 31%, respectively) and B16 (39% and 21%, respectively) compared with mice without tumors (Fig. 2, A to D). This increase in lipolysis did not occur in LLC- or B16-tumor–bearing Atgl−/− mice. WAT lipolysis was also attenuated in tumor-bearing Hsl−/− mice. Whereas FA and glycerol release from WAT was similar in Hsl−/− mice with or without LLC, B16 melanoma formation caused 28% and 19% increases, respectively. To investigate whether changes in TG hydrolase activities underlie the observed differences in lipolytic rates, we measured ATGL and HSL enzyme activities in WAT in response to tumor growth (fig. S8). LLC in o/n-fasted WT mice caused a twofold increase in total lipase activity because of increased ATGL and HSL activity. No tumor-induced increase in WAT lipase activity was observed in Atgl−/− mice, whereas in Hsl−/− mice total TG lipase activity increased by 2.1-fold because of increased ATGL activity. Thus, LLC and B16 cause an induction of WAT TG hydrolase activity, leading to an increased release of FAs and glycerol from WAT. This induction of lipolysis is not observed in the absence of ATGL and significantly reduced in the absence of HSL. Thus, lipase deficiency blocks tumor-induced WAT loss.

Fig. 2

Loss of WAT in tumor-bearing animals is mainly attributable to lipolysis. Both LLC and B16 tumors significantly increased FAs (A and C) and glycerol (B and D) release from WAT explants in nonfasted tumor-bearing WT mice. LLC-bearing lipase-deficient mice did not exhibit increased FA or glycerol release from WAT explants, whereas increased FA and glycerol release was observed from WAT explants of B16-tumor-bearing Hsl−/− mice. WAT explants from B16-tumor-bearing Atgl−/− mice did not show increased FA or glycerol release. (E to J) Lipolytic agonists (TNF-α, IL-6, and AZGP1) were increased in both LLC- and B16-tumor–bearing mice from all genotypes. Black bars indicate control (saline-injected) animals; white bars, tumor-bearing animals. ***P < 0.001, **P < 0.01, *P < 0.05, n = 7 except for (A) and (B), n = 3 to 5.

Several reports have shown that the induction of lipolysis during CAC is mediated by inflammatory cytokines (17), AZGP1 (also designated lipid mobilizing factor, LMF) (18), or cytotoxin-induced death executor proteins (CIDE), such as CIDE-A (25). To investigate whether these lipolytic agonists are increased in cachexia models lacking specific lipases, we assessed their plasma levels. In response to LLC, TNF-α and IL-6 levels were increased (between 2- to 3.2-fold) in all genotypes (Fig. 2, E and F). Similarly, quantitative Western blot analysis revealed that plasma AZGP1 levels were higher in LLC-bearing animals of all genotypes (Fig. 2G). In response to B16, the induction of cytokine release was even more pronounced (Fig. 2, H and I). Plasma levels of TNF-α and IL-6 increased about five- to ninefold and 18- to 21-fold, respectively. Plasma AZGP1 levels were also consistently higher in B16-treated mice of all genotypes compared with untreated mice (Fig. 2J). This suggests that in WT mice the increased concentration of inflammatory and lipolytic agonists induce lipolysis via ATGL and HSL leading to the uncontrolled loss of WAT and cachexia. In the absence of lipases, particularly in the absence of ATGL, this process is disrupted and WAT is retained.

In cancer patients, CAC not only emaciates adipose tissue but also consumes skeletal muscle and cardiac muscle (9, 22, 26, 27). Similarly, we observed that LLC and B16 melanoma in mice lowers skeletal muscle mass and heart weight. Surgically removed gastrocnemius muscle of WT mice injected with LLC (21 days) and B-16 (16 days) weighed 36% and 25% less than that of WT mice, respectively (Fig. 3, A and D), suggesting that muscle loss was less pronounced than the loss of adipose mass. Remarkably, Atgl−/− mice suffered no significant loss of gastrocnemius muscle weight in response to the tumors (Fig. 3, A and D). Similarly as in WT mice, LLC and B16 in Hsl−/− mice diminished gastrocnemius muscle weight (Fig. 3, A and D), albeit to a lesser degree (–27% and –18%, respectively). Wasting of gastrocnemius muscle was also reflected by a reduction of total muscle protein in WT (–36%) and Hsl−/− mice (–22%) in response to B16, whereas Atgl−/− mice with B16 melanoma were resistant to the loss of muscle protein (fig. S9). The weight of soleus muscle also decreased in LLC-injected WT mice after 21 days (33%, not statistically significant) and B16-injected WT mice (–27%) (fig. S10). Both LLC and B16 caused a moderate decrease in heart weight (–7.4% and –9.9%, respectively) and total cardiac protein content (–20.1% and –24.9%, respectively) in WT mice but not in Atgl−/− or Hsl−/− mice (fig. S11). Consistent with our previous observations (20), Atgl−/− mice exhibited a twofold increased TG content in gastrocnemius muscle and a 12- to 15-fold increased TG content in cardiac muscle (fig. S12) compared with WT animals.

Fig. 3

Atgl−/− mice were protected from tumor-associated skeletal muscle loss. (A and D) Muscle mass of gastrocnemius (normalized to tibia length) of surgically prepared gastrocnemius illustrate skeletal muscle loss in tumor-bearing WT and Hsl−/− mice but not in Atgl−/− mice. (B and E) Proteasome activity in gastrocnemius muscle homogenates is significantly increased in tumor-bearing WT and Hsl−/− mice but not in Atgl−/− mice. (C and F) Increased caspase 3 and 7 activity in gastrocnemius muscle homogenates demonstrates activation of apoptosis in tumor-bearing WT and Hsl−/− mice. Caspase 3 and 7 activity in gastrocnemius muscle of Atgl−/− mice was not affected by tumor growth. Black bars indicate control (saline-injected) animals, white bars, tumor-bearing animals. ***P < 0.001, **P < 0.01, *P < 0.05, n = 7.

Muscle atrophy may originate from a decrease in protein synthesis (28) or an increase in protein degradation (9, 22). Studies in a number of experimental models of cachexia suggest that both processes occur simultaneously (9). Animal models of cancer cachexia as well as studies in cancer patients provided evidence that the ubiquitin-proteasome pathway mainly degrades myofibrillar proteins, particularly at later stages of cachexia when patients lost more than 10% of their body weight (29). Additionally, apoptotic cell death characterized by increased activity of caspases contributes to the loss of gastrocnemius muscle in mice bearing the cachexia-inducing MAC16 tumor (30). Consistent with these observations, we detected a marked increase in proteasome activity (Fig. 3, B and E) and an increase in caspase 3 and 7 activity (Fig. 3, C and F) in gastrocnemius muscle of WT mice and Hsl−/− mice 21 days after LLC injection and 16 days after B16 injection. In contrast, no significant change in proteasome or caspase 3 and 7 activity was observed in gastrocnemius muscle of Atgl−/− mice in response to both tumors (Fig. 3 B, C, E, and F). Changes in the weight of gastrocnemius muscle, caspase 3 and 7 activity, and proteasome activity were not observed in WT and Hsl−/− mice, 2 weeks after LLC injection (fig. S13), suggesting that loss of WAT precedes the loss of muscle mass, which is consistent with earlier observations (10).

Previous work showed that cachexia is associated with increased FA oxidation in gastrocnemius muscle (9). Our data confirm these findings, showing 1.8- to 2.5-fold increased mRNA expression levels of genes involved in the regulation of cellular FA uptake (CD36 and fatty acid transport protein 1, Fatp-1), FA transport into mitochondria (carnitin palmitoyltransferase-1β, Cpt-1β), and mitochondrial function (peroxisome proliferator-activated receptor-γ coactivator-1α, Pgc-1α) in gastrocnemius muscle samples of WT mice with LLC (fig. S14). mRNA levels in muscle samples of Atgl−/− mice were not affected in response to LLC. In Hsl−/− mice, mRNA levels increased in the presence of the tumor, although to a lesser extent than in WT mice. This suggests that the catabolic state in CAC mobilizes FA from adipose tissue, leading to an energy substrate switch from glucose to FA use in skeletal muscle.

To test whether the activity of metabolic lipases in WAT also associates with CAC in humans, we determined ATGL- and HSL-mediated TG hydrolase activities of visceral WAT from autopsy samples of 27 patients. Twelve of these individuals had been diagnosed with various forms of malignancies (two adenocarcinomas of the lung, two adenocarcinomas of the colon, two ductal adenocarcinomas of the breast, two adenocarcinomas of the prostate, one hepatocellular carcinoma, one clear cell carcinoma of the kidney, one squamous cell carcinoma of the esophagus, and one malignant germ cell tumor). Six out of the 12 patients were designated as cachectic according to the definition of Evans et al. (31). Total lipase, ATGL, and HSL activities were significantly higher in visceral WAT of cancer patients compared with individuals without cancer and significantly higher in cancer patients with cachexia compared with cancer patients without cachexia (Fig. 4, A to C). Lipase activities in cancer patients without cachexia were similar to those of noncancer patients. A significant inverse correlation was found between total lipase, ATGL, and HSL activities in WAT of cancer patients and their body mass index (BMI) (Fig. 4, D to F). In contrast, lipolytic activities in WAT of noncancer patients showed no correlation with their BMI (fig. S15). Thus, our study provides compelling evidence that the previously observed increase in FA and glycerol release from WAT of patients with CAC (9, 10) is due to up-regulation of ATGL and HSL activities and that increased lipase activities strongly correlate with cachexia.

Fig. 4

CAC patients show increased TG hydrolase activity compared with noncachectic patients. Total lipase activity (A), specifically inhibited (HSL, 76-0079) lipase activity (mainly ATGL activity) (B), and HSL activity (determined by subtraction of HSL-inhibited lipase activity from total lipase activity) (C) in visceral WAT of cancer patients compared with those of noncancer patients. Lipase activities in WAT of cachectic cancer patients are significantly higher than in noncachectic cancer patients. Ranges indicate mean ± standard deviation. (D to F) Total, HSL-inhibited, and HSL lipase activities show negative correlation with BMI of cancer patients. FFA, free fatty acid.

In summary, our data are consistent with the view that lipolysis plays an instrumental role in the pathogenesis of CAC. The increased catabolism of adipose lipid stores leads to the complete loss of WAT followed by a reduction in muscle mass. The absence of ATGL and, to a lesser degree, HSL reduces FA mobilization, retains WAT and muscle mass, and prevents CAC. Whether the protection of adipose and muscle loss in lipase-deficient mice is a consequence of defective tissue autonomous lipolysis or due to endocrine signaling from the tumor or WAT remains to be elucidated. However, pharmacological inhibition of lipases may represent a powerful strategy to avoid the devastating condition of cachexia in response to cancer or other chronic diseases.

Supporting Online Material

Materials and Methods

Figs. S1 to S15


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: We thank E. Zechner and C. Schober-Trummler for reviewing the manuscript. The research was supported by the doctoral program Molecular Medicine of the Medical University of Graz (S.D.); GOLD–Genomics of Lipid-Associated Disorders as part of the Austrian Genome Project GEN-AU funded by Forschungsförderungsgesellschaft and Bundesministerium für Wissenschaft und Forschung (Ru.Ze.); SFB LIPOTOX grant no. F30 (Ru.Ze., G.H.), the Wittgenstein Award 2007 grant no. Z136 funded by the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung (Ru.Ze.). S.K.D., Ro.Zi., G.H., and Ru.Ze. hold a patent related to the modulation of ATGL for prevention and treatment of cachexia.
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