Reduced Liver Lipid Peroxidation in Subcellular Fractions Is Associated with a Hypometabolic State in Rats with Portacaval Anastomosis

Abstract

A surgical connection between portal and inferior cava veins was performed to generate an experimental model of high circulating ammonium and hepatic hypofunctioning. After 13 weeks of portacaval anastomosis (PCA), hyperammonemia and shrinkage in the liver were observed. Low glycemic levels accompanied by elevated levels of serum alanine aminotransferase were recorded. However, the activity of serum aspartate aminotransferase was reduced, without change in circulating urea. Histological and ultrastructural observations revealed ongoing vascularization and alterations in the hepatocyte nucleus (reduced diameter with indentations), fewer mitochondria, and numerous ribosomes in the endoplasmic reticulum. High activity of hepatic caspase-3 suggested apoptosis. PCA promoted a marked reduction in lipid peroxidation determined by TBARs in liver homogenate but specially in the mitochondrial and microsomal fractions. The reduced lipoperoxidative activity was also detected in assays supplemented with Fe²⁺. Only discreet changes were observed in conjugated dienes. Fluorescent probes showed significant attenuation in mitochondrial membrane potential, reactive oxygen species (ROS), and calcium content. Rats with PCA also showed reduced food intake and decreased energy expenditure through indirect calorimetry by measuring oxygen consumption with an open-flow respirometric system. We conclude that experimental PCA promotes an angiogenic state in the liver to confront the altered blood flow by reducing the prooxidant reactions associated with lower metabolic rate, along with significant reduction of mitochondrial content, but without a clear hepatic dysfunction.

Figure 1

Histological and ultrastructural characterization of hepatocytes. (a) Histological characterization in H&E stained section of the liver. (A) Liver from sham operated rat. CV: central vein; PV: portal vein. (B) Experimental animal with PCA showing formation of new blood vessels. (b) Ultrastructural characteristics of hepatic cells. (A–D) Normal hepatocyte of sham-operated rats. (F–H) Hepatocyte cells of PCA-operated rats. N: nucleus; M: mitochondrial; ER: endoplasmic reticulum. Images are representative of 6 independent experimental observations.

Figure 2

Prooxidant reactions in the serum and liver homogenate. (a) Conjugated dienes (not measured in serum). (b) Basal TBARs. (c) TBARs supplemented with FeSO₄ 50 μM. Data obtained from sham and PCA rats after 13 weeks of surgical procedures. Values represent mean ± SEM of 12-15 independent experimental observations; P < 0.05.

Figure 3

Prooxidant reactions in mitochondrial and microsomal fractions of the liver. (a) Conjugated dienes. (b) Basal TBARs. (c) TBARs supplemented with FeSO₄ 50 μM. Subcellular fractions were obtained by differential centrifugation (see Methods). Data obtained from sham and PCA rats after 13 weeks of surgical procedures. Values represent mean ± SEM of 12-15 independent experimental observations; P < 0.05.

Figure 4

Prooxidant reactions in the plasma membrane and cytosolic microsomal fractions of the liver. (a) Conjugated dienes. (b) Basal TBARs. c) TBARs supplemented with FeSO₄ 50 μM. Subcellular fractions were obtained by differential centrifugation (see Methods). Data obtained from sham and PCA rats after 13 weeks of surgical procedures. Values represent mean ± SEM of 12-15 independent experimental observations; P < 0.05.

Figure 5

Mitochondrial parameters by fluorescent dyes. (a) MitoTracker for assessment of mitochondrial membrane potential ΔΨ_m. (b) Rhod-2 to measure mitochondrial calcium. (c) MitoSOX for mitochondrial reactive oxygen species (ROS) detection. Histograms at the right side show the quantification of the fluorescent signals, expressed as average ± SEM. Statistical analysis was done by Student's t-test with P < 0.05; ^∗ means a significant difference. Graphics are representative of 12-15 independent experimental observations.

Figure 6

Food intake, respiratory quotient, and energy expenditure in rats with PCA. Data were collected over a period of 1-2 days using metabolic cages (PANLAB, Spain). Darker section depicts period with light off. (a) Cumulative food intake, expressed in g, during 24 h. (b) Respiratory quotient (ratio of CO₂ produced by the O₂ consumed) recorded for 2 days. (c) Energy expenditure recorded for 2 days. Data from panels (b) and (c) were recorded every 12 min and the results shown in the graphics correspond to values calculated with the average of every hour. To make simpler the display of the data, only the mean of 6 independent experimental observations are represented; SEM values (corresponding from 8 to 13% of the mean) were omitted. PCA rats showed significant reduction in food intake and respiratory quotient during the dark period and along the 24 h recording in energy expenditure.