Restoring adiponectin via rosiglitazone ameliorates tissue wasting in mice with lung cancer

Abstract

Aim: To investigate systemic regulators of the cancer-associated cachexia syndrome (CACS) in a pre-clinical model for lung cancer with the goal to identify therapeutic targets for tissue wasting.

Methods: Using the Kras/Lkb1 (KL) mouse model, we found that CACS is associated with white adipose tissue (WAT) dysfunction that directly affects skeletal muscle homeostasis. WAT transcriptomes showed evidence of reduced adipogenesis, and, in agreement, we found low levels of circulating adiponectin. To preserve adipogenesis and restore adiponectin levels, we treated mice with the PPAR-γ agonist, rosiglitazone.

Results: Rosiglitazone treatment increased serum adiponectin levels, delayed weight loss, and preserved skeletal muscle and adipose tissue mass, as compared to vehicle-treated mice. The preservation of muscle mass with rosiglitazone was associated with increases in AMPK and AKT activity. Similarly, activation of the adiponectin receptors in muscle cells increased AMPK activity, anabolic signaling, and protein synthesis.

Conclusion: Our data suggest that PPAR-γ agonists may be a useful adjuvant therapy to preserve tissue mass in lung cancer.

Keywords: AMPK; adiponectin; cachexia; lung cancer; muscle wasting.

Figure 1.

Cancer cachexia is associated with a decrease in serum adiponectin and a concomitant decline in AMPK/CaMKII signaling in skeletal muscle. A. Principal component analysis (PCA) of white adipose tissue (WAT) RNA-Seq samples showing distinct clustering between CACS (n=10) and NCACS (n=9) in the KL model. B. Gene Set Enrichment Analysis (GSEA) of samples in “A” showing the top 18 pathways differentially expressed in CACS using the Hallmark dataset. C. Serum levels of Adiponectin in wild type (WT, male=10, female=3) and tumor-bearing mice classified either as non-cachectic (NCACS, male=16, female=5) or cachectic (CACS, male=9, female=5). D. GSEA comparing CAMKK signaling pathway (GO:0061762) in the muscles of 3 different models of cancer cachexia. In GSE165856 and GSE107470, the gastrocnemius of CACS (n=10) KL mice are compared with those of NCACS (n=10). In, GSE123310 quadriceps of CACS (n=4) mice with Pancreatic ductal adenocarcinoma are compared to controls (n=3), and in GSE138464 the gastrocnemius of mice implanted with the C26 colon cancer (n=4) cell line are compared to controls (n= 6). Data information: Error bars stand for SD. p < 0.05, NES = Normalized Enrichment Score. The p-value in panel C was calculated using a two-way ANOVA followed by Sidak’s multiple comparison test.

Figure 2.

Serum analysis of cachectic mice treated with or without rosiglitazone. A-C. Rosiglitazone administration (Rosi) through ad libitum food intake (incorporated into regular chow, 100mg/kg) successfully restores total adiponectin, the physiologically active heavy molecular weight (HMW) adiponectin and the ratio of HMW to total adiponectin in cachectic mice (n=16–17 per group). D. No change in circulating insulin levels between Control and Rosi mice (n=17 per group). E. No change in blood glucose levels between Control and Rosi mice (n=17 per group). F. Rosiglitazone administration significantly reduced serum triglyceride levels in cachectic mice (n=17 per group). Data information: Error bars stand for SD. The p-value was calculated by an unpaired t-test. * denotes a p-value of <0.05, ‡ denotes a p-value of <0.001.

Figure 3.

Restoring adiponectin through rosiglitazone results in preserved skeletal muscle and adipose tissue mass of cachectic mice. A. Both groups significantly reduced body weight over the 15-week intervention period (p<0.0001). There was a significant effect for the rosiglitazone intervention (p<0.0001) and a significant interaction effect between time and the rosiglitazone intervention (p<0.05) to affect body weight (n=17 per group). B. Food intake significantly declined over time (p<0.0001) but there was no effect of rosiglitazone (p=0.86) or an interaction between rosiglitazone and time (p=0.77). Food intake was calculated by dividing the total intake per cage by the number of animals per cage (n=4 cages per group). C-E. Skeletal muscle, white adipose tissue (WAT), and brown adipose tissue (BAT) were significantly preserved in the Rosi compared to the Control condition (n=17 per group). F. Rosiglitazone administration did not significantly affect lung weight in cachectic mice (n=17 per group). Data information: Error bars stand for SD. The p-value was calculated by a two-way ANOVA (body weight, food intake), and an unpaired t-test (tissue weights). * denotes a p-value of <0.05, ‡ denotes a p-value of <0.001.

Figure 4.

Restoring circulating adiponectin results in increased AMPK activity and improved maintenance of Akt signaling in skeletal muscle of cachectic mice. A-C. Western blot analysis of CAMKII, AMPK, and p38 signaling in gastrocnemius muscle of cachectic mice (n=12 per group). The bar graphs represent the ratio of phosphorylated to total protein levels. D-E. Western blot analysis of Akt activity and downstream signaling in gastrocnemius muscle of cachectic mice (n=12 per group). The bar graphs represent the ratio of phosphorylated to total protein levels. G. Whole membrane strips corresponding to the quantified western blot data from “A-F”. Data information: Error bars stand for SD. The p-value was calculated by an unpaired t-test. * denotes a p-value of <0.05, ‡ denotes a p-value of <0.001.

Figure 5.

Adiponectin activates AMPK and acutely improves muscle protein synthesis in the presence of IL-6, while chronic effects appear insulin-dependent. A-D. Western blot analysis of AMPK activity, anabolic signaling, and protein synthesis in C2C12 cells after an acute dose-response experiment (n=4 per group). Cells were treated with increasing doses of adiponectin (10, 25, 50μM) together with IL-6 (10ng/mL) for 3 hours. The control condition was untreated, the IL-6 condition received only IL-6 (10ng/mL), and the Drug Ctrl group received only adiponectin (25μM). “A” shows representative pictures of the graphs from “B-D”. Phosphorylated levels of AMPK (B), S6K1 (C), and puromycin (D) were normalized to total protein content per lane. E-H. Western blot analysis of AMPK activity, anabolic signaling, and protein synthesis in C2C12 cells after chronic exposure to adiponectin or rosiglitazone (n=3 per group). Cells were treated with adiponectin (25μM) or rosiglitazone (10μM) together with IL-6 (10ng/mL) for 24 hours. Thirty minutes before collection, half of the cells were treated with 100nM insulin. The Ctrl group was untreated, the IL-6 group only received IL-6 (10ng/mL). “E” shows representative pictures of the graphs from “F-H”. Phosphorylated levels of AMPK (F), S6K1 (G), and puromycin (H) were normalized to total protein content per lane. Data information: Error bars stand for SD. The p-value was calculated by a one-way ANOVA and Dunnett’s multiple comparison test (acute experiment, Figure 5A–D), and a two-way ANOVA and Sidak’s multiple comparison test (chronic experiment, Figure 5E–H). * denotes a p-value of <0.05.