INTRODUCTION

Various studies have indicated an association between modification in dietary macronutrient composition and liver apoptosis, but the exact mechanism in the development of apoptosis under these circumstances is not entirely understood ^1-4.

Apoptosis is an innately programmed process responsible for the death of unwanted cells. It occurs mainly during embryogenesis or in tissues with the capacity for regeneration such as the liver ⁵. Senescent and mutagenic cells that have completed their mission are eliminated by apoptosis, and new cells are formed when necessary to replace them ⁶. Dysregulation occurs when the regenerative capacity is lower than the apoptosis rate, and the physiological pathway becomes pathological.

Apoptosis leads to the development of numerous diseases such as cancer, autoimmune diseases, viral or bacterial infections, alcoholic liver disease, viral hepatitis, cholestatic liver disease, hepatocellular carcinoma, Wilson's disease, and nonalcoholic steatohepatitis ⁵.

There are two forms of triggered apoptosis. The first is mediated by agents that penetrate the cell directly, and the second occurs with the participation of mediators like tumor necrosis factor (TNF) ¹. Heat shock, stress, free radicals, ultraviolet radiation, drugs, synthetic peptides, toxins, and numerous enzymes are examples of agents in the first process ⁶. Zhang et al. demonstrated for the first time that apoptosis was induced by TNF-α in an in vitro steatotic hepatocyte model ¹.

Cells are complex genetic machines that once activated directly or indirectly enter into an apoptotic cycle. The cycle begins with the activation of a caspase -a cysteine protease that mediates intracellular proteolytic cascade and cleaves the cellular substrate and activates transcription factors ⁷. Once active, the transcription factors engender the expression of specific genes that determine the production of pro-apoptotic and anti-apoptotic factors ⁵.

In liver, excess fatty acid mobilization or lipid transport leads to apoptosis. Palmitate, a saturated fatty acid, has well-studied lipotoxic effects. The inability to convert fatty acid to triglycerides raises the risk of hepatocyte apoptosis and can probably be avoided by the use of an antioxidant. Hepatocytes are injured because the liver receives more fatty acids than can be oxidized or transported by VLDL ^8-11. Hepatocyte damage begins with modification in mitochondrial morphology from fission to fusion, which decreases cellular adenosine triphosphate (ATP) levels and increases reactive oxygen species (ROS) ¹².

Oxidative stress results from an imbalance between substances that promote oxidation and others with antioxidant effects; the consequence is a cellular lesion when pro-oxidants predominate ¹³. ROS are pro-oxidant substances that are responsible for intracellular lipid peroxidation that produces malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE). It alters the protein, lipid, polysaccharide, and DNA synthesis ^14,15.

The objective of this study is to discuss if and how modifications in a metabolic pathway due to a high-protein, high-fat, and low-carbohydrate diet can increase liver damage.

METHODS

RESULTS

No statistically significant differences were found between the groups for capillary glucose, insulin, glucagon, IL-6, or TNF-α. The β-hydroxybutyrate and MDA were expressed at the highest levels (p = 0.001 and p = 0.009, respectively) and triglycerides at the lowest levels (p = 0.005), in the experimental group. The percentage of nonviable cells in the experimental diet group was 2.18 times that of the control group (p = 0.001) (Table I).

Table I Results of analytical methods and flow cytometry 

The adipose tissue of rats fed on the experimental diet weighted less than the adipose tissue of rats that received the control diet. Mean EDG weight was 6.926 ± 1.118 g and mean CDG weight was 12.10 ± 1.849 g (p = 0.0339).

The results show a positive and significant correlation between the percentage of nonviable cells and MDA nmoL/mL levels (95% confidence interval, p = 0.0217) and a statistically significant negative correlation with triglycerides (TGA) mg/dL (p = 0.006) (Fig. 1).

Figure 1 Pearson's correlation coefficient between: MDA and inviable cells with r = 0,5517, R2 = 0,3044 and p = 0,0217 and TGA and inviable cells with r = - 0,5956, R = 0,3548 and p = 0,0056. 

DISCUSSION

Although the macronutrients composition of the experimental diet is not adequate for rats, it is carbohydrate content that regulates gluconeogenesis ¹⁸. Pichon et al. considered carbohydrate levels the most important factor in lipid metabolism ¹⁹ (Fig. 2).

Figure 2 The metabolic pathway responsible for hepatic damage associated with low-carbohydrate diet, high protein and high fat diet. 

The results showed no statistically significant differences in glucose, insulin, or glucagon levels between the groups. The glucose levels are preserved by gluconeogenesis ²⁰.

Low insulin and high glucagon may not occurs when gluconeogenesis is associated with high protein amounts that increases the secretion of insulin and glucagon ^21-23. Our results are similar to the datas observed by Blouet et al. ²³. Bielohuby et al. found low fasting glucose and low insulin levels associated with low carbohydrate and protein, and high fat diet ²⁴. Westman et al. reported lower glucose, insulin, and C-peptide in humans receiving high fat versis high carbohydrate diet ²⁰.

Corroborating previous studies, the adipose tissue weight was lower in the experimental diet than in the control ^{19,21,23,25,26}. Low-carbohydrate decreased fat reserves and high-protein increased energy expenditure as protein needs more energy for digestion and metabolism ^26,27. In adipose tissue, the lipolysis of triglyceride occurs with the supply of glycerol that will be converted to glucose, and fatty acids ²⁸. In the liver, the fatty acids enter in the mitochondria, but are not stored as triglycerides, in the cytoplasm, because lipogenesis is inhibited ^10,21,29. Botion et al. showed that rats fed with high-protein and carbohydrate free diet had a reduction of fat acids synthesis and low triglycerides in liver and plasma ³⁰. Monteiro et al. demonstrated that hepatic steatosis was not found in rat livers that received high-fat, high-protein and low-carbohydrate diet. The diet composition was the same as the present work ³¹.

The plasma triglyceride levels in the present work are lower in the experimental group than the controls (Fig. 2). Kennedy et al. reported decreased plasma and hepatic triglycerides associated with a low-carbohydrate diet in rats fed on a ketogenic diet ³². Westman et al. conducted a thematic review with humans and found similar results ²⁰.

In the mitochondria, fatty acids from the adipose tissue and the high-fat diet are broken down to produce energy through -oxidation. When the concentration of acetyl-CoA is elevated, it is converted to ketoacids that can be used as fuel for the brain, muscle, and heart ³³. The pyruvate formed by an excess supply of amino acids from a high-protein diet enters the hepatocytes by a carrier-mediated transport. In the mitochondria, the pyruvate is converted to oxalacetate that may take two metabolic pathways -one to glucose be formed (the main one) or another to acetyl-CoA. When the offer of amino acids is elevated the second pathway becomes the mainly one ³⁴. These data confirm this: the β-hydroxybutyrate level was 2.7 times higher in the experimental diet group than controls. This result supports the conclusion that gluconeogenesis is the main metabolic process and that low-carbohydrate diet is a ketogenic diet.

Kim et al. reported that when β-oxidation is abnormal (uncontrolled), levels of intermediate products increase and lead to inadequate energy production with a decrease in the NAD/NADH ratio and ATP. Consequently, lipid peroxidation occurs. This biochemical cascade leads to cellular membrane damage via the formation of reactive oxygen species (ROS), causing mitochondrial damage, oxidative stress and apoptosis ³⁵. The elevated levels of β-hydroxybutyrate are another cause of lipid peroxidation and its consequences ³⁶.

In our research the plasma MDA levels were higher in experimental diet versus the AIN-93M diet demonstrating the presence of lipid peroxidation (Fig. 2). The current study showed a positive correlation between the percentage of nonviable liver cells (apoptosis + non-apoptotic death) and MDA levels and a negative correlation with TGA levels (Fig. 1). This result suggests plasma MDA and triglycerides as probable indicators of hepatocyte damage.

Malondialdehyde (MDA) may reach distant tissues and extend cellular damage causing an increase in pro-inflammatory cytokines such as IL-6 and TNF-α ¹⁴. Here, plasma IL-6 and TNF-α levels did not differ significantly between the two groups. Oarada et al. found similar results when they fed rats on a low-carbohydrate, high-protein diet ³⁷. Ellenbroek et al. showed that rats receiving a low-carbohydrate diet for 12 weeks had increased plasma IL-6. The diet in this study consisted of low protein and high fat (93.1%) ³⁸.

In conclusion the metabolic pathway leads to modifications on plasma MDA and triglycerides doing them indicators of liver damage in rats fed with an experimental Atkins-type diet containing 59.46% of protein, 31.77% of fat and 8.77% of carbohydrate.