asymmetricdimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase. ADMA is synthetized by protein methyltransferases (PRMTs), metabolized via dimethylargininedimethylaminohydrolase (DDAH) and transported across cell membrane by cationic amino-acid transporters (CATs). Aims: This study investigated whether, in a rat model of non-alcoholic fatty liver disease (NAFLD) changes in serum levels of ADMA occur and the mechanisms involved. Material and Methods: Male Wistar rats underwent to NAFLD induced by 4 weeks of feeding with an MCD diet (methionine/choline-deficient diet). Blood samples and hepatic biopsies were collected after 1, 2, 3, and 4 weeks. Serum hepatic enzymes (AST, ALT and Alkaline Phosfatase), total and direct bilirubin and ADMA were evaluated. Hepatic biopsies were used for in situ NAD(P)H autofluorescence detection and for mRNA expression of PRMT-1, DDAH-1 and ADMA transporters (CAT-1, CAT-2A and CAT-2B) by RT-PCR. Tissue DDAH activity and content of lipid peroxides, glutathione and ATP were also quantified. Results: NAFLD injury was confirmed by altered serum levels of hepatic enzymes. A marked increase in total and direct bilirubin was detected. A time-dependent decrease in serum ADMA levels and mRNA expression of CAT-2A and CAT-2B was obtained. On the contrary, an increase in mRNA expression of DDAH-1, PRMT-1 and CAT-1 was found. The hepatic DDAH activity decreased with a concomitant increase in oxidative stress, as demonstrated by high lipid peroxide levels and low GSH content. A decrease in ATP levels and in the NAD(P)Hbound/free ratio reflecting the mitochondria alterations was also detected in MCD rats. Conclusions: These results indicate that while an increase in DDAH mRNA was found, the oxidative stress observed can contribute to the reduction of DDAH activity. This enzyme is a cysteine hydrolase that may be inhibited by increased reactive oxygen species associated to mitochondria dysfunction. The observed decrease in serum ADMA found using an animal model of NAFLD may be due to the increase in the ADMA transporter CAT-1.These data confirm and support the crucial role of the liver in the control of ADMA levels by taking up large amounts of ADMA from the systemic circulation. (Supported by Fondazione Cariplo, grant n° 2011-0439).
Changes in serum levels of asymmetric-dymethylarginine (ADMA) in a rat model of NAFLD: role of cationic transporters
Bottiroli G;Croce AC;
2015
Abstract
asymmetricdimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase. ADMA is synthetized by protein methyltransferases (PRMTs), metabolized via dimethylargininedimethylaminohydrolase (DDAH) and transported across cell membrane by cationic amino-acid transporters (CATs). Aims: This study investigated whether, in a rat model of non-alcoholic fatty liver disease (NAFLD) changes in serum levels of ADMA occur and the mechanisms involved. Material and Methods: Male Wistar rats underwent to NAFLD induced by 4 weeks of feeding with an MCD diet (methionine/choline-deficient diet). Blood samples and hepatic biopsies were collected after 1, 2, 3, and 4 weeks. Serum hepatic enzymes (AST, ALT and Alkaline Phosfatase), total and direct bilirubin and ADMA were evaluated. Hepatic biopsies were used for in situ NAD(P)H autofluorescence detection and for mRNA expression of PRMT-1, DDAH-1 and ADMA transporters (CAT-1, CAT-2A and CAT-2B) by RT-PCR. Tissue DDAH activity and content of lipid peroxides, glutathione and ATP were also quantified. Results: NAFLD injury was confirmed by altered serum levels of hepatic enzymes. A marked increase in total and direct bilirubin was detected. A time-dependent decrease in serum ADMA levels and mRNA expression of CAT-2A and CAT-2B was obtained. On the contrary, an increase in mRNA expression of DDAH-1, PRMT-1 and CAT-1 was found. The hepatic DDAH activity decreased with a concomitant increase in oxidative stress, as demonstrated by high lipid peroxide levels and low GSH content. A decrease in ATP levels and in the NAD(P)Hbound/free ratio reflecting the mitochondria alterations was also detected in MCD rats. Conclusions: These results indicate that while an increase in DDAH mRNA was found, the oxidative stress observed can contribute to the reduction of DDAH activity. This enzyme is a cysteine hydrolase that may be inhibited by increased reactive oxygen species associated to mitochondria dysfunction. The observed decrease in serum ADMA found using an animal model of NAFLD may be due to the increase in the ADMA transporter CAT-1.These data confirm and support the crucial role of the liver in the control of ADMA levels by taking up large amounts of ADMA from the systemic circulation. (Supported by Fondazione Cariplo, grant n° 2011-0439).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
