CAVII is a physiologically relevant isoform of the enzyme carbonic anhydrase with high CO2 hydration activity. In humans and rodents, CAVII is a cytosolic isoenzyme mainly localized in brain tissues where it contributes in generating neuronal excitation. Recently, S-glutathionylation of two cysteine residues from the hCAVII was reported and the effect of native and tetramutated hCAVII in a cell culture was analyzed by stressing cells with an oxidant agent. Results suggested that hCAVII could function as an oxygen radical scavenger for protecting cells from oxidative damage. Here, we aimed to investigate the CAVII protective role toward oxidative insult in vivo, using obese Zucker rats (OZRs) as an animal model. The genetically obese (fa/fa) Zucker rats, due to a recessive mutation of the leptin 9 Proceedings of the 61st Congress of the Italian Embryological Group (GEI) and the 36th Congress of the Italian Society of Histochemistry receptor gene, exhibit hyperphagia and develop hallmark features of metabolic syndrome. The study was performed on the liver of OZRs and their lean counterparts (LZRs) at different ages (12, 16, and 20 weeks from birth; 6 animals each group). Serum values of glucose, insulin, total cholesterol and triglycerides were measured as parameters of dismetabolism. Thiobarbituric acid reactive substances (TBARS) were evaluated in serum and liver samples as a marker of oxidative stress. A specific polyclonal anti-CAVII serum (from S. Parkilla, Finland) was applied for either Western blot analysis and immunohistochemical investigation, this latter performed on 5 µm thick sections from paraffin embedded liver samples. The TBARS levels in OZRs where higher than in the LZRs, confirming the oxidative stress in the obese rats. At all stages examined, CAVII proved to maintain its expression in the liver of obese rats. Indeed, in the immunoblots a band with a MW consistent with that known for CAVII could be identified in either OZR and LZR lysates. An additional higher MW band, detected in all samples, requires to be further characterized. Differences in the immunohistochemical patterns, however, suggested a distinct CAVII distribution in the liver of OZRs, compared with LZRs. Unlike a weak, diffuse staining found in LZR liver parenchyma, a marked cytoplasmic staining was localized in some OZR hepatocytes zonally distributed in the hepatic lobules, in close association with cells showing morphological features of steatosis. Studies are in progress to clarify a possible relation of such preliminary findings to the proposed role of CAVII in oxidative stress.

THE OBESE ZUCKER RATS: AN IN VIVO MODEL FOR STUDYING THE LIVER CARBONIC ANHYDRASE CAVII BIOLOGICAL ROLE

GABRIELLI, Maria Gabriella;TOMASSONI, Daniele;CATALINI, LAURA;TAYEBATI, Seyed Khosrow;AMENTA, Francesco
2015

Abstract

CAVII is a physiologically relevant isoform of the enzyme carbonic anhydrase with high CO2 hydration activity. In humans and rodents, CAVII is a cytosolic isoenzyme mainly localized in brain tissues where it contributes in generating neuronal excitation. Recently, S-glutathionylation of two cysteine residues from the hCAVII was reported and the effect of native and tetramutated hCAVII in a cell culture was analyzed by stressing cells with an oxidant agent. Results suggested that hCAVII could function as an oxygen radical scavenger for protecting cells from oxidative damage. Here, we aimed to investigate the CAVII protective role toward oxidative insult in vivo, using obese Zucker rats (OZRs) as an animal model. The genetically obese (fa/fa) Zucker rats, due to a recessive mutation of the leptin 9 Proceedings of the 61st Congress of the Italian Embryological Group (GEI) and the 36th Congress of the Italian Society of Histochemistry receptor gene, exhibit hyperphagia and develop hallmark features of metabolic syndrome. The study was performed on the liver of OZRs and their lean counterparts (LZRs) at different ages (12, 16, and 20 weeks from birth; 6 animals each group). Serum values of glucose, insulin, total cholesterol and triglycerides were measured as parameters of dismetabolism. Thiobarbituric acid reactive substances (TBARS) were evaluated in serum and liver samples as a marker of oxidative stress. A specific polyclonal anti-CAVII serum (from S. Parkilla, Finland) was applied for either Western blot analysis and immunohistochemical investigation, this latter performed on 5 µm thick sections from paraffin embedded liver samples. The TBARS levels in OZRs where higher than in the LZRs, confirming the oxidative stress in the obese rats. At all stages examined, CAVII proved to maintain its expression in the liver of obese rats. Indeed, in the immunoblots a band with a MW consistent with that known for CAVII could be identified in either OZR and LZR lysates. An additional higher MW band, detected in all samples, requires to be further characterized. Differences in the immunohistochemical patterns, however, suggested a distinct CAVII distribution in the liver of OZRs, compared with LZRs. Unlike a weak, diffuse staining found in LZR liver parenchyma, a marked cytoplasmic staining was localized in some OZR hepatocytes zonally distributed in the hepatic lobules, in close association with cells showing morphological features of steatosis. Studies are in progress to clarify a possible relation of such preliminary findings to the proposed role of CAVII in oxidative stress.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11581/388197
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