Cachexia-associated adipose loss induced by tumor-secreted leukemia inhibitory factor is counterbalanced by decreased leptin
Gurpreet K. Arora, Arun Gupta, Sriram Narayanan, Tong Guo, Puneeth Iyengar, Rodney E. Infante
Gurpreet K. Arora, Arun Gupta, Sriram Narayanan, Tong Guo, Puneeth Iyengar, Rodney E. Infante
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Research Article Metabolism Oncology

Cachexia-associated adipose loss induced by tumor-secreted leukemia inhibitory factor is counterbalanced by decreased leptin

Abstract

Cachexia syndrome consists of adipose and muscle loss, often despite normal food intake. We hypothesized that cachexia-associated adipose wasting is driven in part by tumor humoral factors that induce adipocyte lipolysis. We developed an assay to purify secreted factors from a cachexia-inducing colon cancer line that increases lipolysis in adipocytes and identified leukemia inhibitory factor (LIF) by mass spectrometry. Recombinant LIF induced lipolysis in vitro. Peripheral LIF administered to mice caused >50% loss of adipose tissue and >10% reduction in body weight despite only transient hypophagia due to decreasing leptin. LIF-injected mice lacking leptin (ob/ob) resulted in persistent hypophagia and loss of adipose tissue and body weight. LIF’s peripheral role of initiating lipolysis in adipose loss was confirmed in pair-fed ob/ob mouse studies. Our studies demonstrate that (a) LIF is a tumor-secreted factor that promotes cachexia-like adipose loss when administered peripherally, (b) LIF directly induces adipocyte lipolysis, (c) LIF has the ability to sustain adipose and body weight loss through an equal combination of peripheral and central contributions, and (d) LIF’s central effect is counterbalanced by decreased leptin signaling, providing insight into cachexia’s wasting, despite normophagia.

Authors

Gurpreet K. Arora, Arun Gupta, Sriram Narayanan, Tong Guo, Puneeth Iyengar, Rodney E. Infante

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Figure 1

Biochemical characterization of lipolysis activity from C26c20 cell line medium.

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Biochemical characterization of lipolysis activity from C26c20 cell line...
(A and B) Characterization of cancer cell line medium-induced adipocyte lipolysis. Medium was collected, processed, and protein quantified from C26c20 or MC-38 cells as described in Methods. Differentiated adipocytes in a 12-well format were treated with 1.5 ml of medium E with the indicated amount of C26c20 or MC-38 medium (A) or 150 ng of recombinant IL-6, 150 ng of recombinant TNFα, or either 1.8 mg or 3.1 mg C26c20 medium in the absence or presence of 4.5 μg of the indicated antibody (B). After incubation for 20 hours at 37˚C, medium was collected and glycerol concentration was measured using the adipocyte lipolysis assay described in Methods. Data are shown as mean ± SEM (A) or dot plots with bars representing mean ± SEM (B) of 3 or 4 (A and B, respectively) experiments and represents the absolute increase of medium glycerol concentration over background (A) or as the relative change in medium glycerol concentration compared with conditions containing the indicated protein without antibody (B) (IL-6, 54 and 19 μM; TNFα, 25 and 36 μM; C26c20 medium, 37 and 20 μM). (C) Leukemia inhibitory factor (LIF) expression in medium of cancer cells. Medium (15 ml) from C26c20 and MC-38 was concentrated to a final volume of 150 μl using a 10 kDa MW cut-off Amicon Ultra centrifugal filter, and protein was quantified using a bicinchoninic acid kit. Protein (20 μg) was subjected to IB analysis with anti-LIF and Ponceau S stain described in Methods. (D) Immunodepletion of LIF from partially purified C26c20 medium. C26c20 medium was partially purified as described in Methods. Approximately 14 μg of the elution fractions containing lipolysis activity in Step 1 of the partial purification of C26c20 medium in 300 μl of buffer A with 0.2% BSA was subjected to immunodepletion described in Methods using 2 μg of the indicated antibody. The supernatant fraction from the immunodepletion was collected, and 30 μl was subjected to IB analysis with the indicated antibody and 20 μl subjected to the adipocyte lipolysis assay described in Methods. Data are shown as dot plots with bars representing mean ± SEM of 4 experiments and is represented as the relative change in medium glycerol concentration compared with conditions containing the indicated protein without antibody (33 and 64 μM). *P < 0.05 and ***P < 0.001 based on Student’s t test.