Abstract IA27: Exercise, energy balance, and cancer
Authors: Hakimi Parvin , Feng Zhaoyang , Kalhan Satish , Berger Nathan A. 
Published: May 2016

PhosphoEnol Pyruvate Carboxy Kinase (PEPCK) catalyzes the GTP dependent conversion of oxaloacetate to CO2 and phospo enol pyruvate, which is subsequently converted to glyceraldehyde-3-phosphate, which then serves as precursor for both gluconeogenesis and/or glyceroneogenesis. Using the mouse gene for the cytoplasmic form of PEPCK-Cmus linked to the skeletal muscle actin promoter, PEPCK-Cmus transgenic mice (TG) were engineered on an SJL/B6 background. The PEPCK-Cmus transgenic mice bear 4 allelic copies of PEPCK; they show a nine-fold overexpression of PEPCK in skeletal muscle associated with an increase in muscle glyceroneogenesis and a 4 fold greater concentration of triglycerides per gram muscle compared with controls. The PEPCK-Cmus TG mice have a remarkable behavioral phenotype of significantly increased and spontaneous exercise, evident from 10 days of age and continuing throughout their extended lifespan. In their home cages, the TG mice show more spontaneous activity, including rearing and running such that they exhibit more than 6 times the physical activity of control mice. Running on a 25° inclined treadmill, the PEPCK-Cmus mice run 25 times further and for longer time periods compared to controls. After 10 minutes treadmill running, the wild type controls show marked increase in Respiratory Exchange Rate (RER) and lactate production, while PEPCK-Cmus TG mice maintain relatively stable RER and lower lactate levels, the latter indicating lipid oxidation to support exercise in TG mice compared to glucose oxidation in controls. TG mice are able to run at faster speeds than controls, 50M/m vs 20M/m up until 18 months of age and continue to run at 30 M/m till at least 24 months of age, a time when controls no longer run.

In order to support the increased energy demand associated with the enhanced exercise phenotype, the PEPCK-Cmus TG mice eat 60% more than controls, but weigh 30% less than controls (35 vs 20 grams body weight). MRI shows that PEPCK TG mice maintain a lean body habitus with minimal subcutaneous and visceral fat compared to controls. TG mice appear to live longer and show extended fertility. Histology of the soleus and gastrocnemius muscles from the TG mice compared to controls show that the TG mice have greater intra myocellular lipid (IMCL), increased mitochondrial enzymes including succinate dehydrogenase and NADH dehydrogenase. The increase in the number of mitochondrial enzymes is associated with an increase in mitochondria which was confirmed by eletronmicroscopy and by increased mitochondrial DNA. Compared to control mice, the TG mice showed significantly lower circulating triglycerides and free fatty acid levels but no significant difference in glucose levels.

In more recent studies, we have shown that TG mice have lower levels of the hormone insulin, lower levels of the adipocytokine leptin. In ongoing studies we are comparing age dependent, longitudinal changes in inflammatory cytokines TNFα, IL-6, IL-1β and the adipocytokine, adiponectin. PEPCK TG mice have extended lifespans compared to non TG Mice. These observations of increased life span in association with increased physical activity (PA) shown by the PEPCK-Cmus TG mice is in agreement with the recent demonstration in humans of a 40% lower mortality rate in humans engaging in highest PA level, for example, elite athletes who complete the Tour de France.

As a result of overexpression in skeletal muscle of the single enzyme, PEPCK, the TG mice synthesize more muscle triglycerides, accumulate more IMCL, exercise more, consume more food, weigh less, have minimal visceral or subcutaneous fat, and have lower circulating FFA and triglycerides but no difference in serum glucose. As expected with a reduced body fat mass, we have shown they have low leptin levels. These characteristics are those of a well trained endurance athlete. And like endurance athletes, we have shown the PEPCK-Cmus TG mice show increased IMCL but lower levels of insulin than controls and no signs of insulin resistance. In fact, we have now shown that the PEPCK-Cmus TG mice are hypersensitive to externally administered insulin. This increase in IMCL with no signs of insulin resistance is the classic “athletes paradox.” The low insulin levels despite increased need for glucose consumption by muscle is in agreement with the proposal that increased glucose transport in exercising muscle is significantly achieved by non insulin dependent transport systems ie independent of Glut4 and also that alternate energy sources are used for fuel, ie substrates for oxidation.

Since the PEPCK-TG mice spontaneously demonstrate the characteristics of successful aerobic training with an “elite athlete phenotype,” they provide a robust model to study the impact of exercise on cancer and the possibility that exercise leads to extended lifespan, even in the presence of malignant disease. To examine this possibility, we are studying the effects of PEPCK overexpression in skeletal muscle on progression of colon tumors in APCMin/+ mice with a hereditary mutation in the APCmin gene such that mice with this genotype develop and die from progression of intestinal neoplasia. Tumor bearing APCMin/+ PEPCK-TG mice maintain their high physical activity and enhanced exercise phenotype. Compared to controls, the TG mice with the enhanced exercise phenotype showed the slowest increase in tumor number and size. We found that the overall survival of the PEPCK TG tumor bearing mice was significantly extended. Thus, overexpression of PEPCK in mouse muscle and enhanced exercise was associated with a decreased rate of tumor growth and increased life span.

In ongoing experiments, we are continuing to examine serum samples obtained from these mice to analyze circulating inflammatory and growth promoting cytokines and hormones and potentially tumor growth retarding myokines for differences between the transgenic and control mice.

Citation Format: Parvin Hakimi, Zhaoyang Feng, Satish Kalhan, Nathan A. Berger. Exercise, energy balance, and cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr IA27.