Pancreatic cancer associated diabetogenic peptide induces nitric oxide synthesis in rat hepatocytes

Background: In this study we verified whether MIA PaCa 2 conditioned medium (CM) and its low molecular weight (10,000 Da) fraction (LMWCM) alter hepatocytes glucose metabolism by inducing nitric oxide (NO) production and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) mRNA synthesis, since this enzyme catalyzes a critical reaction in the pathway of gluconeogenesis and can be inhibited by NO-mediated nytrosilation. Methods: Five different experiments were performed. Isolated and perfused rat hepatocytes were incubated for two hours with: 1. non conditioned medium (NCM); 2. CM and 3. LMWCM. In the hepatocytes supernatants we measured glucose, lactate, nitrate and nitrite levels (colorimetric assay), which are final products of NO in vivo. Total RNA was extracted from lysed hepatocytes; after reverse transcription into cDNA, a quantitative PCR was performed to measure the number of GAPDH mRNA copies (BioSource, USA). Results: Glucose declined similarly in the different experimental conditions. Lactate significantly decreased over time in CM (F 4.7, p 0.05) and in LMWCM (F 4.1, p 0.05), but not in NCM (F 1.6, p:ns). Nitrate production was significantly higher in LMWCM with respect to NCM after 30 (t 2.3, p 0.05), 60 (t 3.7, p 0.01), 90 (t 2.9, p 0.05) and 120 (t 5.0, p 0.01) minutes of incubation. A similar pattern was observed in CM, although the variations were not statistically significant. The number of GAPDH mRNA copies, although reduced in LMWCM (40,000) with respect to NCM (63,000) after 120 min of incubation, did not statistically differ in the two experimental conditions. Conclusion: The low molecular weight pancreatic cancer associated diabetogenic peptide induces the production of NO in isolated and perfused rat hepatocytes. NO does not seem to significantly alter the synthesis of GAPDH mRNA, a glycolytic enzyme possibly not involved in causing pancreatic cancer associated diabetes mellitus.