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. 2014 Mar 31;2(1):6.
doi: 10.1186/2049-3002-2-6.

Leucine supplementation differentially enhances pancreatic cancer growth in lean and overweight mice

Kristyn A Liu #  1 , Laura M Lashinger #  1 , Audrey J Rasmussen #  1 , Stephen D Hursting  1   2
Affiliations

Leucine supplementation differentially enhances pancreatic cancer growth in lean and overweight mice

Kristyn A Liu et al. Cancer Metab. .

Abstract

Background: The risk of pancreatic cancer, the 4th deadliest cancer for both men and women in the United States, is increased by obesity. Calorie restriction (CR) is a well-known dietary regimen that prevents or reverses obesity and suppresses tumorigenesis in a variety of animal models, at least in part via inhibition of mammalian target of rapamycin (mTOR) signaling. Branched-chain amino acids (BCAA), especially leucine, activate mTOR and enhance growth and proliferation of myocytes and epithelial cells, which is why leucine is a popular supplement among athletes. Leucine is also increasingly being used as a treatment for pancreatic cancer cachexia, but the effects of leucine supplementation on pancreatic tumor growth have not been elucidated.

Results: Supplementation with leucine increased pancreatic tumor growth in both lean (104 ± 17 mm3 versus 46 ± 13 mm3; P <0.05) and overweight (367 ± 45 mm3 versus 230 ± 39 mm3; P <0.01) mice, but tumor enhancement was associated with different biological outcomes depending on the diet. In the lean mice, leucine increased phosphorylation of mTOR and downstream effector S6 ribosomal protein, but in the overweight mice, leucine reduced glucose clearance and thus increased the amount of circulating glucose available to the tumor.

Conclusions: These findings show that leucine supplementation enhances tumor growth in both lean and overweight mice through diet-dependent effects in a murine model of pancreatic cancer, suggesting caution against the clinical use of leucine supplementation for the purposes of skeletal muscle enhancement in cachectic patients.

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Figures

Figure 1
Figure 1
Effects of calorie restriction (CR) and/or leucine (LEU) supplementation on body composition glucose tolerance, and hormones. (A) Caloric intake and (B) body weight of C57BL/6 male mice on control and CR diets with and without leucine supplementation reported until glucose tolerance test (GTT) and quantitative magnetic resonance imaging (qMRI) performed (21 weeks, n = 22/group; P <0.001 between groups with different letters). (C) qMRI quantification of body fat and (D) lean mass between mice fed control or CR diets with and without leucine supplementation for 21 weeks (n = 10/group; P <0.001 between all groups with different letters). (E) GTT performed after 21 weeks on diet (n = 10/group; P <0.05 between control and CR, P <0.001 between all other groups with different letters). (F) Fasting glucose levels after 21 weeks on diet (prior to tumor injection; control, n = 14; all other groups, n = 15) (P <0.001 between all groups with different letters). (G-J) Serum hormone analyses after 21 weeks on diet (prior to tumor injection) of (G) insulin (P <0.05 between control and CR + LEU), (H) IGF-1 (P <0.001 between groups with different letters), (I) leptin (P <0.001 between control and both CR groups; P <0.01 between control + LEU and both CR groups), and (J) adiponectin (P <0.001 between CR and both control groups; P <0.05 between all other groups with different letters) (control groups, n = 9; CR groups, n = 10). All data are presented as the mean with error bars indicating the SD (A,B) or SEM (C-J). Differences are considered significant if P <0.05. Abbreviations: CON, control diet; CR,calorie restriction diet; LEU, leucine-supplemented diet.
Figure 2
Figure 2
Effects of leucine supplementation on Panc02 tumor growth and apoptosis. (A) Differences in tumor volume between mice on control and calorie restriction (CR) diets with and without leucine (LEU) supplementation 4 weeks after tumor cell injections (control, control + LEU, and CR, n = 14/group; and CR + LEU, n = 13) (P <0.01 between control and both leucine-supplemented groups; P <0.001 between all other groups with different letters). (B) Comparison of immunohistochemical analyses performed on tumor sections for Ki-67 (n = 5/group; P <0.001 between all groups with different letters) and cleaved-caspase 3 (CC3) (n = 5/group; P <0.01 between the two leucine-supplemented groups; P <0.05 between all other groups with different letters). Scale bars represent 200 μm. Tumor volume is presented as mean ± SD, and Ki-67 and CC3 data are presented as mean ± SEM. Differences are considered significant if P <0.05. Abbreviations: CON, control diet; CR,calorie restriction diet; LEU, leucine-supplemented diet.
Figure 3
Figure 3
Effects of calorie restriction (CR) and/or leucine (LEU) supplementation on energy responsive signals in Panc02 tumors. (A) Comparison of immunohistochemical analyses on tumor sections for phospho-mTOR (P <0.05 between groups with different letters), phospho-S6 (P <0.001 between control and CR; P <0.01 between control + LEU and CR; P <0.05 between CR and CR + LEU), cyclin D1 (P <0.05 between groups with different letters), phospho-ACC (P <0.05 between the two leucine-supplemented groups; P <0.001 between all other groups with different letters) (n = 5/group). Scale bars represent 200 μm. All data are presented as the mean ± SEM. Differences are considered significant if P <0.05. Abbreviations: CON, control diet; CR,calorie restriction diet; LEU, leucine-supplemented diet.
Figure 4
Figure 4
Effects of leucine supplementation on viability of Panc02 tumor cells. (A-B) Comparison of relative viability of cells grown in media with either (A) 10% fetal bovine serum (FBS) or (B) 1% FBS as assessed by MTT assays after 48 hours of 0.3 mM leucine supplementation (* = P <0.05, ** = P <0.01). All data are presented as the mean ± SEM. Differences are considered significant if P <0.05.
Figure 5
Figure 5
Effects of leucine supplementation on energy responsive signals of Panc02 tumor cells. (A-C) Western blot analysis of phosphorylated AMPK, ACC, mTOR, p70S6K, and S6 after 20 minutes of 0.3 mM leucine administration after pretreatment with respective media for 3 hours. Data shown are representative blots from three biological replicates, and images for each protein are from the same blot. (B-C) Relative phosphorylation of p-AMPK, p-ACC, p-mTOR, p-p70S6K, and p-S6 in cells grown in media with either (A) 10% FBS or (B) 1% FBS with or without leucine supplementation (* = P <0.05). All data are presented as the mean ± SEM. Differences are considered significant if P <0.05.
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