RESUMO
BACKGROUND: The mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-sensing pathway and a key regulator of amino acid and glucose metabolism. Dysregulation of the mTOR pathways is implicated in the pathogenesis of metabolic syndrome, obesity, type 2 diabetes, and pancreatic cancer. OBJECTIVES: We investigated the impact of inhibition of mTORC1/mTORC2 and synergism with metformin on pancreatic tumor growth and metabolomics. METHODS: Cell lines derived from pancreatic tumors of the KPC (KrasG12D/+; p53R172H/+; Pdx1-Cre) transgenic mice model were implanted into the pancreas of C57BL/6 albino mice (n = 10/group). Two weeks later, the mice were injected intraperitoneally with daily doses of 1) Torin 2 (mTORC1/mTORC2 inhibitor) at a high concentration (TH), 2) Torin 2 at a low concentration (TL), 3) metformin at a low concentration (ML), 4) a combination of Torin 2 and metformin at low concentrations (TLML), or 5) DMSO vehicle (control) for 12 d. Tissues and blood samples were collected for targeted xenometabolomics analysis, drug concentration, and cell signaling. RESULTS: Metabolomic analysis of the control and treated plasma samples showed differential metabolite profiles. Phenylalanine was significantly elevated in the TLML group compared with the control (+426%, P = 0.0004), whereas uracil was significantly lower (-38%, P = 0.009). The combination treatment reduced tumor growth in the orthotopic mouse model. TLML significantly decreased pancreatic tumor volume (498 ± 104 mm3; 37%; P < 0.0004) compared with control (1326 ± 134 mm3; 100%), ML (853 ± 67 mm3; 64%), TL (745 ± 167 mm3; 54%), and TH (665 ± 182 mm3; 50%) (ANOVA and post hoc tests). TLML significantly decreased tumor weights (0.66 ± 0.08 g; 52%) compared with the control (1.28 ± 0.19 g; 100%) (P < 0.002). CONCLUSIONS: The combination of mTOR dual inhibition by Torin 2 and metformin is associated with an altered metabolomic profile and a significant reduction in pancreatic tumor burden compared with single-agent therapy, and it is better tolerated.
RESUMO
Pancreatic Cancer (PC) is a devastating lethal disease. Therefore, there is an urgent need to develop new intervention strategies. The mammalian Target of Rapamycin (mTOR) is a conserved kinase and master regulator of metabolism and cell growth. mTOR is dysregulated in chronic diseases including diabetes and pancreatic cancer. Recent reports indicate that 50% of Pancreatic Ductal Adenocarcinoma (PDAC) patients are diabetic at the time of diagnosis. Furthermore, the anti-diabetic drug, metformin, which indirectly inhibits mTOR, has emerged as a potential therapeutic target for PC. The objective of this study is to determine the targeted-metabolomics profile in PDAC cell line (HPAF-II) with mTOR inhibition and the interaction between mTOR ATP-competitive inhibitor (Torin 2) and metformin as potential combined therapy in PC. HPAF-II cell lines were cultured in the presence of either Torin 2, metformin, both, or control vehicle. We utilized targeted LC/MS/MS to characterize the alterations in glycolytic and tricarboxylic acid cycle metabolomics, and employed Western Blot analysis for cell signaling activation by phosphorylation. Comparisons between groups were analyzed using one-way Analysis of Variance followed by secondary post-hoc analysis. After 1 h incubation with metformin, AMP concentration was significantly increased compared to other groups (p<0.03). After 24 h, Torin-2 significantly decreased glycolysis intermediates (fructose 1,6-bisphosphate (FBP), and 2-phosphoglycerate/3-phosphoglycerate), TCA intermediate metabolites (citrate/isocitrate, and malate), as well as Nicotinamide Adenine Dinucleotide (NAD+) and Flavin Adenine Dinucleotide (FAD), and ATP levels. When HPAF-II cells were incubated with both Torin-2 and metformin, there was a significant reduction in NAD+ and FAD, suggesting decreased levels of the energy equivalents that are available to the electron transport chain. Targeted metabolomics data indicate that mTOR complexes inhibition by Torin 2 reduced glycolytic intermediates and TCA metabolites in HPAF- II and may synergize with metformin to decrease the electron acceptors NAD+ and FAD which may lead to reduced energy production.
RESUMO
Significant weight loss following Roux-en-Y gastric bypass surgery (RYGB) in obese humans correlates with enhanced secretion of anorexigenic gut hormones glucagon-like peptide-1 (GLP-1) and peptide YY(3-36) (PYY(3-36)). Our aim here was to identify a dosing strategy for intraperitoneal (IP) infusion of GLP-1 homologue exendin-4 alone and with PYY(3-36) that produces a sustained reduction in daily food intake and body weight in diet-induced obese (DIO) rats. We tested 12 exendin-4 strategies over 10 weeks. Exendin-4 infused during the first and last 3 h of the dark period at 15-20 pmol/h (0.15 nmol/kg/day) produced a sustained 24 ± 1% reduction in daily food intake for 17 days, and decreased body weight by 7%. In a separate group of DIO rats, none of seven dosing strategies combining exendin-4 and PYY(3-36) produced a similar reduction in daily food intake for >10 days. The subsequent decline in efficacies of exendin-4 alone and with PYY(3-36) on food intake and body weight in each experiment suggested possible receptor downregulation and tolerance to treatments. However, when treatments were discontinued for 1 day following losses in efficacies, daily food intake significantly increased. Together, these results demonstrate that (i) intermittent IP infusion of a low dose of exendin-4 can produce a relatively prolonged reduction in daily food intake and body weight in DIO rats, (ii) co-infusion of exendin-4 and PYY(3-36) does not further prolong this response, and (iii) activation of an orexigenic mechanism gradually occurs to counteract the inhibitory effects of exendin-4 alone and with PYY(3-36) on food intake and body weight.
