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Cited by 129Liver Disease Diagnosis and TreatmentMetabolism and Genetic DisordersMitochondrial Function and Pathology

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Oxidation of Hepatic Carnitine Palmitoyl Transferase-I (CPT-I) Impairs Fatty Acid Beta-Oxidation in Rats Fed a Methionine-Choline Deficient Diet

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Serviddio, Gaetano, Giudetti, Anna M., Bellanti, Francesco, Priore, Paola, Rollo, Tiziana, Tamborra, Rosanna, Siculella, Luisa, Vendemiale, Gianluigi, Altomare, Emanuele, Gnoni, Gabriele V.·2011·PLoS ONE
DOI 10.1371/journal.pone.0024084PMC PMC3164715
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Abstract

There is growing evidence that mitochondrial dysfunction, and more specifically fatty acid β-oxidation impairment, is involved in the pathophysiology of non-alcoholic steatohepatitis (NASH). The goal of the present study was to achieve more understanding on the modification/s of carnitinepalmitoyltransferase-I (CPT-I), the rate-limiting enzyme of the mitochondrial fatty acid β-oxidation, during steatohepatitis. A high fat/methionine-choline deficient (MCD) diet, administered for 4 weeks, was used to induce NASH in rats.We demonstrated that CPT-I activity decreased, to the same extent, both in isolated liver mitochondria and in digitonin-permeabilized hepatocytes from MCD-diet fed rats.At the same time, the rate of total fatty acid oxidation to CO(2) and ketone bodies, measured in isolated hepatocytes, was significantly lowered in treated animals when compared to controls. Finally, an increase in CPT-I mRNA abundance and protein content, together with a high level of CPT-I protein oxidation was observed in treated rats. A posttranslational modification of rat CPT-I during steatohepatitis has been here discussed.

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Figure 1 Histological analysis of liver specimens stained with haematoxylin and eosin (H&E) from controls (A) and rats fed the MCD diet (B). Liver of rats fed the MCD diet shows steatohepatitis characterized by panacinar steatosis predominantly as macrovescicular fat, together with lobular inflammation (H&E ×100).
Figure 1 Histological analysis of liver specimens stained with haematoxylin and eosin (H&E) from controls (A) and rats fed the MCD diet (B). Liver of rats fed the MCD diet shows steatohepatitis characterized by panacinar steatosis predominantly as macrovescicular fat, together with lobular inflammation (H&E ×100).CC BY · PubMed Central
Figure 2 Carnitine palmitoyl transferase-I (CPT-I) activity and sensitivity to malonyl-CoA in isolated mitochondria. Activity of CPT-I (A) and its sensitivity to malonyl-CoA (B) was measured in isolated liver mitochondria from both control (CTRL) and methionine-choline deficient (MCD) diet fed rats. CPT-I activity is reported as nmoles [14C]carnitine incorporated into acylcarnitine/min/mg protein. CPT-I sensitivity to malonyl-CoA was expressed as percentage of inhibition to 10 µM malonyl-CoA. Data are expressed as means ± SD of five experiments for each group. Statistical differences were assessed using unpaired t-test assuming variance homogeneity. *Significantly different from the control.
Figure 2 Carnitine palmitoyl transferase-I (CPT-I) activity and sensitivity to malonyl-CoA in isolated mitochondria. Activity of CPT-I (A) and its sensitivity to malonyl-CoA (B) was measured in isolated liver mitochondria from both control (CTRL) and methionine-choline deficient (MCD) diet fed rats. CPT-I activity is reported as nmoles [14C]carnitine incorporated into acylcarnitine/min/mg protein. CPT-I sensitivity to malonyl-CoA was expressed as percentage of inhibition to 10 µM malonyl-CoA. Data are expressed as means ± SD of five experiments for each group. Statistical differences were assessed using unpaired t-test assuming variance homogeneity. *Significantly different from the control.CC BY · PubMed Central
Figure 3 Effects of the methionine-choline deficient (MCD) diet on 3-hydroxy-acyl-CoA dehydrogenase (3-HAD) activity. Activity of 3-HAD of control (CTRL) and methionine-choline deficient (MCD) diet fed rats was measured spectrophotometrically as described in “Methods”. Data are expressed as means ± SD of five experiments for each group. Statistical differences were assessed using unpaired t-test assuming variance homogeneity. *Significantly different from the control.
Figure 3 Effects of the methionine-choline deficient (MCD) diet on 3-hydroxy-acyl-CoA dehydrogenase (3-HAD) activity. Activity of 3-HAD of control (CTRL) and methionine-choline deficient (MCD) diet fed rats was measured spectrophotometrically as described in “Methods”. Data are expressed as means ± SD of five experiments for each group. Statistical differences were assessed using unpaired t-test assuming variance homogeneity. *Significantly different from the control.CC BY · PubMed Central
Figure 4 Activity and protein level of acetyl-CoA carboxylase (ACC). ACC activity (A) and protein level (B) in control (CTRL) and methionine-choline deficient (MCD) diet fed rats were measured as reported in “Methods”. ACC activity is expressed as nmoles [14C]NaHCO3 incorporated into malonyl-CoA/min/mg protein. α-Tubulin was used for signal normalization. Data are expressed as means ± SD of five experiments for each group. Statistical differences were assessed using unpaired t-test assuming variance homogeneity. *Significantly different from the control.
Figure 4 Activity and protein level of acetyl-CoA carboxylase (ACC). ACC activity (A) and protein level (B) in control (CTRL) and methionine-choline deficient (MCD) diet fed rats were measured as reported in “Methods”. ACC activity is expressed as nmoles [14C]NaHCO3 incorporated into malonyl-CoA/min/mg protein. α-Tubulin was used for signal normalization. Data are expressed as means ± SD of five experiments for each group. Statistical differences were assessed using unpaired t-test assuming variance homogeneity. *Significantly different from the control.CC BY · PubMed Central
Figure 5 Palmitate oxidation by rat hepatocytes. Hepatocytes were incubated with [1-14C]palmitate. Total fatty acid oxidation was obtained as the sum of CO2 and total acid-soluble products (ASP). Results, expressed as [1-14C]palmitate into product/h/106 cells, correspond to means ± SD of three different experiments. *Significantly different from the control.
Figure 5 Palmitate oxidation by rat hepatocytes. Hepatocytes were incubated with [1-14C]palmitate. Total fatty acid oxidation was obtained as the sum of CO2 and total acid-soluble products (ASP). Results, expressed as [1-14C]palmitate into product/h/106 cells, correspond to means ± SD of three different experiments. *Significantly different from the control.CC BY · PubMed Central
Figure 6 mRNA and protein levels of rat liver CPT-I. The histogram (A) represents CPT-I mRNA abundance determined using RT-quantitative PCR and expressed as relative amount (18s rRNA as a reference) in liver from control (CTRL) and methionine-choline deficient (MCD) diet fed rats (A). Data are shown as mean values ± SD for the CPT-I gene relative to an arbitrary value of 1, which was assigned to the expression level in control animals. Western blot analysis of CPT-I in liver from CTRL and MCD rats (B) was performed as reported in “Methods”. Porin was used for signal normalization. The content of CPT-I was quantified by densitometric analysis. Data are mean values ± SD of five experiments for each group. *Significantly different from the control.
Figure 6 mRNA and protein levels of rat liver CPT-I. The histogram (A) represents CPT-I mRNA abundance determined using RT-quantitative PCR and expressed as relative amount (18s rRNA as a reference) in liver from control (CTRL) and methionine-choline deficient (MCD) diet fed rats (A). Data are shown as mean values ± SD for the CPT-I gene relative to an arbitrary value of 1, which was assigned to the expression level in control animals. Western blot analysis of CPT-I in liver from CTRL and MCD rats (B) was performed as reported in “Methods”. Porin was used for signal normalization. The content of CPT-I was quantified by densitometric analysis. Data are mean values ± SD of five experiments for each group. *Significantly different from the control.CC BY · PubMed Central
Figure 7 HNE-protein adducts in liver mitochondria. (A) Levels of HNE-protein adducts in liver mitochondria from methionine-choline deficient (MCD) diet and control rats (CTRL) were measured by fluorimetric analysis and evaluated in terms of arbitrary fluorescent units (A.F.U.) at 355 nm excitation and 460 nm emission. Data are expressed as means ± SD of five experiments for each group. (B) Western blot analysis was performed to reveal HNE-CPT-I adducts from CTRL and MCD rats. Signals were quantified by densitometric analysis and expressed as % of the total CPT-I protein content measured in mitochondria from CTRL and MCD rats. Statistical differences were assessed using unpaired t-test assuming variance homogeneity. *Significantly different from the control.
Figure 7 HNE-protein adducts in liver mitochondria. (A) Levels of HNE-protein adducts in liver mitochondria from methionine-choline deficient (MCD) diet and control rats (CTRL) were measured by fluorimetric analysis and evaluated in terms of arbitrary fluorescent units (A.F.U.) at 355 nm excitation and 460 nm emission. Data are expressed as means ± SD of five experiments for each group. (B) Western blot analysis was performed to reveal HNE-CPT-I adducts from CTRL and MCD rats. Signals were quantified by densitometric analysis and expressed as % of the total CPT-I protein content measured in mitochondria from CTRL and MCD rats. Statistical differences were assessed using unpaired t-test assuming variance homogeneity. *Significantly different from the control.CC BY · PubMed Central

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  1. M Nakamuta, 2005, Evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease., Int J Mol Med, 16, 631

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Metadata

Authors
Serviddio, G., Giudetti, A. M., Bellanti, F., Priore, P., Rollo, T., Tamborra, R., Siculella, L., Vendemiale, G., Altomare, E., & Gnoni, G. V.
Affiliations
  • University of Foggia (IT)
  • University of Salento (IT)
Corresponding author
Gnoni, Gabriele V.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-09-01
Volume
6
Issue
9
Pages
e24084
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0024084
PMC
PMC3164715
OpenAlex
W2007759293
Cited by
128
References
36
Citations by year
2026: 5 · 2025: 6 · 2024: 7 · 2023: 7 · 2022: 8 · 2021: 10 · 2020: 9 · 2019: 9 · 2018: 11 · 2017: 10 · 2016: 7 · 2015: 14 · 2014: 9 · 2013: 9 · 2012: 6
FWCI
6.03
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • AIRUS (submittedVersion)
Funders
Ministero dell’Istruzione, dell’Università e della Ricerca
Topics
  • Health Sciences › Medicine › Hepatology › Liver Disease Diagnosis and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Metabolism and Genetic Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
Keywords
carnitine palmitoyltransferase 1, fatty acid beta-oxidation, nonalcoholic steatohepatitis, methionine-choline-deficient diet, mitochondrial dysfunction, hepatocytes, liver mitochondria, post-translational modification, ketone synthesis, high-fat diet
MeSH terms
Acetyl-CoA Carboxylase, Animals, Blood Glucose, Carnitine O-Palmitoyltransferase, Cell Membrane Permeability, Choline, Diet, Fatty Acids, Feeding Behavior, Liver, Male, Malonyl Coenzyme A, Methionine, Mitochondria, Liver, Oxidation-Reduction, RNA, Messenger, 3-Hydroxyacyl CoA Dehydrogenases, Gene Expression Regulation, Enzymologic, Rats, Wistar, Palmitic Acid, Hepatocytes, Mitochondrial Membranes, Rats, Enzyme Assays

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