Purpose: In this study we verified whether MIA PaCa2 conditioned medium (CM) and its low molecular weight (!10,000 D) fraction (LMWCM) alter hepatocyte glucose metabolism by (1) inducing nitric oxide (NO) production and (2) inhibiting glyceraldehyde-3- phospate dehydrogenase (GAPDH) mRNA synthesis. Methods: Five different experiments were performed. Isolated and perfused rat hepatocytes were incubated for 2 h with: (1) non conditioned medium (NCM); (2) CM and (3) LMWCM. In the hepatocyte 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, n.s.). 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) min of incubation. A similar pattern was observed in CM, although the variations were not statistically significant. The number of GAPDH mRNA copies was reduced in LMWCM (40,000) with respect to NCM (63,000) after 120 min of incubation. Conclusions: The low molecular weight pancreatic cancer- associated diabetogenic peptide induces the production of NO in isolated and perfused rat hepatocytes. NO might alter hepatocyte glucose metabolism by inhibiting the synthesis of the glycolytic key enzyme GAPDH.

Pancreatic cancer associated diabetogeni peptide induces nitric oxide synthesis in rat hepathocytes

GRECO, ELIANA;BASSO, DANIELA;VALERIO, ANNA CANDIDA;FOGAR, PAOLA;ZAMBON, CARLO-FEDERICO;PEDRAZZOLI, SERGIO;PLEBANI, MARIO
2001

Abstract

Purpose: In this study we verified whether MIA PaCa2 conditioned medium (CM) and its low molecular weight (!10,000 D) fraction (LMWCM) alter hepatocyte glucose metabolism by (1) inducing nitric oxide (NO) production and (2) inhibiting glyceraldehyde-3- phospate dehydrogenase (GAPDH) mRNA synthesis. Methods: Five different experiments were performed. Isolated and perfused rat hepatocytes were incubated for 2 h with: (1) non conditioned medium (NCM); (2) CM and (3) LMWCM. In the hepatocyte 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, n.s.). 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) min of incubation. A similar pattern was observed in CM, although the variations were not statistically significant. The number of GAPDH mRNA copies was reduced in LMWCM (40,000) with respect to NCM (63,000) after 120 min of incubation. Conclusions: The low molecular weight pancreatic cancer- associated diabetogenic peptide induces the production of NO in isolated and perfused rat hepatocytes. NO might alter hepatocyte glucose metabolism by inhibiting the synthesis of the glycolytic key enzyme GAPDH.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/2431834
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