Focal liver lesions (FLLs) are abnormal lumps or masses that form in the liver. They are usually found accidentally when doctors are imaging other organs. With the increased use of imaging technologies, many people are finding liver lesions, even if they do not have symptoms. Although discovering a liver lesion can be concerning, the majority of focal liver lesions are benign and do not require treatment. It is important to evaluate these lesions to rule out more serious conditions, like liver cancer.
Understanding Focal Liver Lesions (FLLs)
Focal liver lesions (FLLs) refer to abnormal areas of tissue within the liver, often discovered incidentally during imaging tests such as ultrasounds, CT scans, or MRIs. Most FLLs are found during imaging tests done for unrelated reasons, such as investigating abdominal pain or other conditions. Incidental liver findings, like benign lesions, usually do not cause symptoms and are not dangerous. In many cases, imaging tests alone can determine whether a liver lesion is benign or malignant. Specialized imaging techniques like contrast-enhanced ultrasounds or MRIs with contrast can provide detailed insights without needing invasive procedures. Once a benign liver lesion is confirmed, regular monitoring may not be required. However, your doctor might suggest follow-up imaging if there is uncertainty about the lesion or if you have risk factors for liver cancer. With accurate imaging and proper monitoring, your healthcare team can guide you through the next steps.
The Link Between Diet and Liver Health
Metabolic disorders (obesity, hypertriacylglycerolaemia, hypercholesterolaemia, hyperinsulinaemia, insulin resistance and hypertension) are a risk factor for CHD and atherosclerosis. They are also likely to result in fatty liver and cholesterol gallstones. Hypercholesterolaemia is considered a major risk factor for atherosclerosis and was found to cause hepatic damage. Excess of dietary cholesterol reportedly contributes to inflammatory diseases such as atherosclerosis.
Investigating the Effects of a High-Cholesterol Diet on the Liver
We investigated the effects of a high-cholesterol (HC) diet administered long term (25 or 55 weeks) on metabolic disorders including hepatic damage in mice. The mice were fed the HC diet (15 % milk fat, 1.5 % cholesterol and 0.1 % cholic acid, w/w) for 25 or 55 weeks. Body and adipose tissue weights were similar to those of mice fed a control diet. Consumption of the HC diet long term resulted in hypercholesterolaemia, hepatic steatosis and gallstones. In addition, focal nodular hyperplasia (FNH) and mild fibrosis of the liver developed in all mice fed the HC diet for 55 weeks. Plasma levels of monocyte chemoattractant protein (MCP)-1 were elevated, and the level of hepatic platelet-derived growth factor (PDGF)-B protein was increased in mice fed the HC diet compared with those fed the control diet. Thus, it seems likely that the liver fibrosis and FNH caused by the long-term consumption of a HC diet may be partly due to an elevation of plasma MCP-1 and hepatic PDGF expression.
Materials and Methods Used in the Study
The Triglyceride E-Test, Triglyceride-Test, Total Cholesterol E-Test and NEFA C-Test were obtained from Wako Pure Chemical (Osaka, Japan). (Yokohama, Japan), respectively. (Minneapolis, MN, USA). 3-Hydroxy-3-methyl [3-14C]glutaryl-coenzyme A and [1-14C]acetyl-coenzyme A were obtained from Amersham Biosciences UK Limited (Little Chalfont, Bucks, UK). Other chemicals were of reagent grade.
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Animal and Diet Details
Male C57BL/6J mice, aged 4 weeks, were obtained from Japan SLC (Shizuoka, Japan). They were housed in a room with a 12 h light-dark cycle and controlled temperature and humidity. After this period of adaptation, they were allowed free access to water and the experimental diets listed in Table 1 for 25 or 55 weeks. A low-fat and low-sucrose diet was used as the control diet in the present study. Animal experiments were performed according to the ethical guidelines of the Animal Experimentation, Ehime University and Japanese Pharmacological Society, and guide for the care and use of laboratory animals of the National Institutes of Health. The animal experiments were approved by the Ethics Committee on Animal Experimentation, Ehime University (approval no.
Plasma Analysis
At 25 or 55 weeks, after fasting overnight, blood was obtained from each mouse by venous puncture under anaesthesia with Nembutal® (Dainippon Pharmaceutical Co. Blood samples were chilled in test-tubes containing heparin and centrifuged to give plasma. The plasma was frozen at − 20°C before analysis. TAG and total cholesterol concentrations in the plasma were determined using the TAG E-Test and Total Cholesterol E-Test kits. The concentrations of MCP-1, TNF-α, leptin and adiponectin in the plasma were measured using the respective ELISA kits.
Oral Glucose Tolerance Test
The oral glucose tolerance test was carried out at weeks 25 and 55. After at least 4 h of food deprivation, glucose (100 mg/mouse) was administered orally to the mice. Blood samples were taken from the tail 0, 10, 20, 30 and 60 min after glucose administration. The blood glucose concentrations were measured using GLUCOCARD® (GT-1640; Arkray Co., Kyoto, Japan).
Liver Lipid Contents and Cytokine Levels, and Cholesterol Content in Gallstones
TAG and total cholesterol levels in liver were measured by the methods of Fletcher and Zak et al. The TGF-β1 and IL-1β levels in the supernatant fraction were measured using the respective ELISA kits. Gallstones were collected from gallbladder and extracted with an acetone-ethanol (1:1, v/v) mixture, and then cholesterol contents in gallstones were measured by the methods of Zak et al.
Histological Examination
Liver was fixed in 10 % (v/v) buffered formalin, embedded in paraffin, and sectioned. The liver segments were stained with haematoxylin and eosin and with the Azan stain. The main artery was embedded in Tissue-Tek® (OTC compound; Sakura Finetech Co.
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Enzyme Activity
3-Hydroxymethylglutaryl-CoA Reductase
The liver microsomal fraction was separated by the method of Daniele et al. Briefly, the liver was cut and homogenised immediately on ice in 10 mm-HEPES buffer (pH 7·2) containing 0·25 m-sucrose and protease inhibitor, using a Teflon glass homogeniser. The homogenate was centrifuged at 9000 g for 20 min at 4°C. Then the supernatant fraction was further centrifuged at 105 000 g for 60 min at 4°C. The microsomal pellet was suspended in 0·1 m-phosphate buffer (pH 7·4), and added to a reaction mixture containing 0·128 mm-3-hydroxymethylglutaryl (HMG)-CoA ([14C]HMG-CoA, 144 MBq/mmol), 1 mm-NADPH, 10 mm-dithiothreitol and 10 mm-EDTA in 0·12 m-phosphate buffer (pH 7·4). The mixture was incubated at 37°C for 15 min, added to 2 m-HCl, and further incubated for 15 min to allow lactonisation of the mevalonic acid produced. This reaction mixture was spotted on a silica gel 60 F254 TLC plate (Merck, Germany), and developed with benzene-acetone (1:1, v/v). The radioactive spot corresponding to mevalonate lactone was measured using a BAS 1000 image analyser (Fuji Film, Tokyo, Japan). The HMG-CoA reductase activity was expressed as nmol mevalonic acid per mg protein.
Fatty Acid Synthase
Liver was homogenised on ice in 10 mm-HEPES buffer (pH 7·2) containing 0·25 m-sucrose and protease inhibitor, and centrifuged at 12 000 g for 10 min at 4°C. The activity was determined by measuring the incorporation of [14C]acetyl-CoA into fatty acids. The supernatant fraction was added to a reaction mixture containing 0·062 mm-[14C]acetyl-CoA (148 MBq/mmol), 0·195 mm-malonyl-CoA, 0·5 mm-NADPH, 0·45 mm-dithiothreitol, 1 mm-EDTA and 0·93 % bovine serum albumin in 100 mm-HEPES buffer (pH 6·8), and then incubated at 37°C for 10 min. The reaction was stopped by the addition of chloroform-methanol (2:1, v/v), and mixed with a vortex. After centrifugation, the supernatant fraction was removed by aspiration, and the residue was washed with water in acidic conditions twice. The residual radioactivity was quantified with a liquid scintillation counter. The fatty acid synthase (FAS) activity was expressed per mg protein.
Immunoblotting
Liver was homogenised in a solubilising buffer (50 mm-HEPES buffer (pH 7·4) containing 5 mm-EDTA, 150 mm-NaCl, 1 % TritonX-100, 0·5 % sodium deoxycholate and protease inhibitor) and then centrifuged at 12 000 g for 15 min at 4°C. The amount of protein in the supernatant fraction was measured using a protein assay reagent (Bio-Rad, Hercules, CA, USA). After being denatured at 99°C, the protein was resolved by 12 % SDS-PAGE. The gel was transferred to a polyvinylidene fluoride membrane, and the membrane was blocked with 5 % skimmed milk. Immunoreactivity was visualised with alkaline phosphatase-conjugated goat anti-rabbit IgG (ICN Pharmaceuticals, Cleveland, OH, USA) and BCIP/NBT solution (Sigma, St Louis, MO, USA). The blots were measured using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). The ratio is the intensity of the PDGF blots normalised to the blot of glyceraldehyde-3-phosphate dehydrogenase.
Statistical Analysis
All values are expressed as the mean values with their standard errors. The Dunnett test was used to compare the two groups. Differences were considered significant at P < 0·05.
Results of the High-Cholesterol Diet Study
The body weights and epididymal adipose tissue weights of mice fed the control diet and HC diet were similar at weeks 25 and 55. The mean food consumption/mouse per d for 55 weeks was not different between mice fed the control diet and the HC diet: 54·5 (sem 0·53) kJ/mouse per d (control diet) and 55·2 (sem 0·99) kJ/mouse per d (HC diet). The fasting plasma glucose levels were not significantly different between the groups at weeks 25 and 55. To confirm glucose tolerance, oral glucose tolerance tests were performed at weeks 25 and 55. There were no differences in plasma glucose levels after the oral administration of glucose between the two groups (data not shown). At week 25, plasma levels of total cholesterol were significantly higher in the mice fed the HC diet. Levels of plasma, NEFA, MCP-1, leptin and adiponectin were not significantly different between the groups at week 25, but plasma TAG and NEFA levels were significantly elevated in the mice fed the HC diet at week 55. MCP-1 levels of mice fed the HC diet long term (55 weeks) were markedly increased compared with those of mice fed the control diet. On the other hand, plasma leptin and adiponectin levels did not differ between the two groups at week 55.
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Hepatic Lipid Accumulation and Gallstone Formation
At week 25, liver hypertrophy was observed, and liver weight was significantly increased in the mice fed the HC diet. The hepatic accumulation of lipids was also markedly enhanced in these mice. In addition, consumption of the HC diet resulted in cholesterol gallstones. Hepatic hypertrophy induced by the consumption of cholesterol may have contributed to the hepatic lipid accumulation.
Hepatic Lipogenesis
FAS activity in the liver was greater in mice fed the control diet than in those on the HC diet at weeks 25 and 55. The activity of HMG-CoA reductase, a key enzyme of cholesterol synthesis, was lower in the mice fed the HC diet at weeks 25 and 55.
Liver Focal Nodular Hyperplasia and Fibrosis, and Macrophage Accumulation
Hepatic hyperplastic pseudotumours were found in the mice fed the HC diet for 55 weeks. Based on the histological analysis, the HC diet caused FNH, and lipid accumulation and fibrosis were observed around the hyperplastic region. The chronic intake of the HC diet for 55 weeks caused the accumulation of macrophages into the vessel walls.
Effects on Hepatic IL-1β, Transforming Growth Factor-β1 and Platelet-Derived Growth Factor Protein Levels
TGF-β1 and IL-1β levels did not differ between the two groups at weeks 25 and 55. However, PDGF-B protein levels were significantly increased in the mice fed the HC at weeks 25 and 55.
Discussion of the Findings
The body and adipose tissue weights, and the food consumption of the mice fed the control diet were similar to those of mice fed the HC diet. Chronic consumption of the HC diet resulted in the hepatic accumulation of cholesterol and crystallisation in the gallbladder. Therefore, these findings suggest that the influence on liver function may be due to dietary cholesterol in the diet composition rather than the difference of energy intake of the two diets. Blood and liver total cholesterol levels were increased by feeding the HC diet, and the increases of blood and hepatic total cholesterol might have been due to dietary cholesterol rather than endogenous cholesterol synthesis in the liver, because these mice showed a reduction of HMG-CoA reductase. In fact, the HMG-CoA reductase in the control diet-treated mice was significantly greater than that in the HC diet-treated mice. HMG-CoA reductase was not significantly different between feeding for 25 and 55 weeks in the control diet-fed mice. On the other hand, HMG-CoA reductase in the HC diet-fed mice for 25 weeks tended to be greater than that in the HC diet-fed mice for 55 weeks. Therefore, it seems likely that the longer feeding HC diet results in the reduction of endogenous cholesterol synthesis by the supply of dietary cholesterol; consequently the differences for increasing rate in plasma total cholesterol may have occurred during week 25 to 55 in the control and the…
Dietary Recommendations for FNH and Liver Health
Given the findings of the study on high-cholesterol diets and their impact on liver health, it is crucial to consider dietary recommendations for individuals with FNH or those seeking to maintain optimal liver function.
Limiting Cholesterol Intake
The study demonstrated that a high-cholesterol diet can lead to hypercholesterolemia, hepatic steatosis, gallstone formation, and even FNH and liver fibrosis in mice. While these findings are from animal studies, they suggest that limiting cholesterol intake may be beneficial for liver health.
Emphasizing Whole-Grain Foods
Intake of whole-grain foods was inversely related to risk, with an OR of 0.3 (95% CI = 0.1-0.7) in consumers versus nonconsumers.
Balanced Diet
A balanced diet rich in fruits, vegetables, and whole grains is crucial for overall health, including liver health. These foods provide essential nutrients, antioxidants, and fiber that support liver function and help prevent liver damage.
Other Risk Factors for FNH
Risk factors for focal nodular hyperplasia (FNH) of the liver are largely unknown, except for a possible role of female hormones. Compared to those who never smoked the multivariate ORs were 1.9 (95% CI = 0.6-6.0) in ex-smokers and 3.5 (95% CI = 1.2-9.7) in current smokers, and the risk increased with number of cigarettes smoked to 8.0 (95% CI = 1.7-37.4) for > or = 20 cigarettes/day.