Liver under attack: impacts of high-fat diet on murine model

O fígado sob ataque: impactos da dieta rica em gordura em modelo murino

Abstract

At present, non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide, with obesity recognized as a global epidemic and type 2 diabetes a worldwide disease. In this study, 10 C57BL/6 mice were divided into two groups: the control group (SC) and the high-fat diet (HF) group. Both groups were fed their respective diets for 8 weeks. The animals were analyzed for body weight, glucose/insulin resistance, hepatic steatosis, and fibrosis to diagnose NAFLD. Results showed that the HF group animals had significantly higher body weight (P<0.0001), glucose resistance (P=0.0002), insulin resistance (P=0.0009), and blood glucose levels (P<0.05) compared to the SC group. The HF group exhibited increased hepatic steatosis (P<0.0001) and fibrosis (P<0.0001) compared to the SC group. These findings led to the conclusion that the animals in the HF group had grade and stage 2 NAFLD. Furthermore, the HF group animals were classified as obese, indicating a higher risk for developing insulin resistance and, subsequently, type 2 diabetes mellitus (T2DM). Understanding the risk factors and complications associated with NAFLD, obesity, and T2DM is crucial for preventing and treating metabolic alterations linked to a high-fat diet.

Keywords:
hepatic alterations; high-fat diet; metabolic diseases

Resumo

Atualmente, a doença hepática gordurosa não alcoólica (DHGNA) é a doença hepática mais prevalente em todo o mundo, com a obesidade reconhecida como uma epidemia global e o diabetes tipo 2, uma doença mundial. Neste estudo, 10 camundongos C57BL/6 foram divididos em dois grupos: o grupo controle (SC) e o grupo dieta rica em gordura (HF). Ambos os grupos foram alimentados com suas respectivas dietas por 8 semanas. Os animais foram analisados quanto ao peso corporal, resistência a glicose, esteatose hepática e fibrose para diagnosticar a DHGNA. Os resultados mostraram que os animais do grupo HF tiveram peso corporal significativamente maior (P<0,0001), resistência à glicose (P=0,0002), resistência à insulina (P=0,0009) e níveis de glicose no sangue (P<0,05) em comparação com o grupo SC. Além disso, o grupo HF apresentou aumento da esteatose hepática (P<0,0001) e fibrose (P<0,0001) em comparação com o grupo SC. Esses achados levaram à conclusão de que os animais do grupo HF apresentavam DHGNA de grau e estágio 2. Além disso, os animais do grupo HF foram classificados como obesos, indicando um maior risco de desenvolvimento de resistência à insulina e, posteriormente, diabetes mellitus tipo 2 (TDM2). Diante dos achados em modelo murino foi destaque a necessidade de compreender os fatores de risco e as complicações associadas à DHGNA, obesidade e TDM2. Sendo crucial para a prevenção e tratamento das alterações metabólicas associadas a dieta rica em lipídios.

Palavras-chave:
alterações hepáticas; dieta hiperlipídica; doenças metabólicas

1. Introduction

Non-alcoholic fatty liver disease (NAFLD) has become a prevalent cause of chronic liver disease, attributed to the rising incidence of obesity (Sarwar et al., 2018). Obesity arises from an imbalance between caloric intake, basal metabolism, and energy expenditure (Wang and Liao, 2012). NAFLD is a group of conditions characterized by the accumulation of fat in liver tissue, which may lead to hepatitis, fibrosis, cirrhosis, and, in some instances, hepatocellular carcinoma (Sayiner et al., 2016; Benedict and Zhang, 2017; Lahmi et al., 2023).

Consuming a high-fat diet leads to the expansion of adipose tissue, where triacylglycerol (TAG) is stored. These TAG molecules can be broken down to release glycerol and free fatty acids (FFA). The FFA produced from lipolysis are transported to the liver, where they are used to produce TAG, leading to its accumulation and contributing to the development of NAFLD (Milić et al., 2014; Seo et al., 2018; McPherson et al., 2022). Furthermore, NAFLD has been associated with metabolic disorders such as type 2 diabetes and arterial hypertension (Sarwar et al., 2018).

This study aimed to investigate whether C57BL/6 mice fed a high-fat diet would develop NAFLD and obesity. Additionally, it study sought to evaluate the effects of the high-fat diet on factors such as blood glucose levels and glucose/insulin resistance, which may be linked to the development of NAFLD and obesity.

2. Material and Methods

All procedures were conducted in compliance with the animal experimentation guidelines set forth by the National Institutes of Health (NIH No. 85-23, revised in 1996), and all experimental protocols were approved by the Ethics Committee for Animal Use at the University of the State of Rio de Janeiro (Protocol Number: CEUA/009/2012).

Ten male C57BL/6 mice (3 months old) were randomly allocated into two groups: the control group (SC), which received a standard chow diet containing 15 kJ/g of total energy, and the high-fat group (HF), which received a diet containing 21 kJ/g of total energy. The mice were kept in a controlled environment with standardized temperature conditions and were provided with unlimited access to water and their respective diets for a duration of 8 weeks. The diets were formulated in accordance with the recommendations of the American Institute of Nutrition for the maintenance phase (AIN 93M) (Reeves et al., 1993).

2.1. Body weight

The mice's body weight was measured weekly using a digital scale with a precision of 0.01 g (BL-3200BH). To maintain consistency, the measurements were taken at the same time of day and on the same day of the week. The body weight data was presented in a graph to illustrate the mean ± standard error of the mean (SEM) for comparative purposes (Figure 1).

Figure 1
Body mass. Weeks 1-8 correspond to the period of obesity induction. The values are presented as the mean ± SEM. Significant differences (P < 0.05) were determined using a t-test. Control group (SC), High-fat (HF).

2.2. Food and energy intake

The daily food intake of the mice was determined by subtracting the amount of food not ingested from the total amount of food provided, to determine the appropriate energy intake for the rodents-based recommendations (Reeves et al., 1993). The energy intake was calculated by multiplying the amount of food consumed (g) by the energy content of each diet (Kj).

2.3. Carbohydrate metabolism

The oral glucose tolerance test (OGTT) was conducted at the conclusion of the experiment, following a six-hour fast. Blood samples were obtained from a small incision at the tip of the tail to determine plasma glucose concentration. A glucose solution (1.0 g/kg) was then administered by orogastric gavage, and plasma glucose concentration was measured before glucose administration and at 15, 30, 60, and 120-minute intervals using a glucometer (Accu-chek, Roche Diagnostics, Germany).

The intraperitoneal insulin tolerance test (IPITT) was carried out in the final week of the experiment following a four-hour fast. After measuring fasting plasma glucose concentration, insulin (Humalog Lispro, Lilly) was administered intraperitoneally at a dose of 0.5 IU/kg. Blood samples were collected from the tail at 15, 30, 60, and 120-minute intervals following insulin administration, and blood glucose concentrations were measured using a glucometer.

The area under the curve (AUC) was calculated in arbitrary units (a.u.) for both analyses, considering the total time from zero to 120 minutes. The trapezoidal tool of the GraphPad Prism program (version 6.02 for Windows, GraphPad Software, La Jolla, CA, USA) was utilized to evaluate glucose tolerance and insulin resistance.

2.4. Euthanasia

At the end of week 8, the mice were fasted for 6 hours and injected with 0.03 mL sodium heparin via intraperitoneal injection. Blood was collected by cardiac puncture and plasma was separated by centrifugation at room temperature (120g for 15 minutes). The plasma was immediately aliquoted, frozen in liquid nitrogen, and stored in a freezer at -80 °C for further analysis. The liver was removed from the abdominal cavity and fixed in 10% aqueous formaldehyde for 72 hours.

2.5. Plasma insulin and glucagon levels

The levels of plasma insulin and glucagon were measured by employing a mouse-specific metabolic panel (Milliplex MMHMAG-44K) and analyzed in duplicate using the Luminex xMAP equipment (Millipore, Billerica, MA, USA). The insulin sensitivity index (Si) was calculated using data from the IPITT, as (basal blood glucose – blood glucose 15 min)/15.

2.6. Liver analysis

The liver tissue was fixed in formaldehyde and embedded in paraffin. Thin slices of 4μm were cut from paraffin blocks using a manual microtome and processed in the usual manner. Hematoxylin and eosin (H&E) and Masson’s trichrome were used to stain the slides, which were examined under an optical microscope (Olympus® BX51) connected to image analysis software (ProgRes® Capture Pro 2.5).

Liver steatosis and fibrosis were evaluated using STEPanizer© 1.0 software with a point system, with a total of 36 points (Pt). The area was calculated using Delesse’s Principle, with A = Pp/Pt = Vv (%), where A is the Area, Pp is the number of points that hit the structure, and Vv is the volume density. Results were presented as mean ± standard deviation of Vv (%). The classification for NAFLD was based on the degrees of steatosis (Sanyal, 2002; Hashimoto et al., 2015; Nassir et al., 2015), with 1st degree being mild (0-33%), 2nd degree being moderate (33-66%), and 3rd degree being severe (>66%). For fibrosis classification, the staging proposed by Sanyal (2002) and Hashimoto et al. (2015) was used. Stage 1 is perisinusoidal/pericellular fibrosis in a focal or extensive form; Stage 2 is perisinusoidal/pericellular fibrosis with periportal fibrosis, focal or extensive; Stage 3 is perisinusoidal/pericellular fibrosis with bridge periportal fibrosis, focal or extensive, and Stage 4 is cirrhosis. Additionally, we assessed steatosis for the presence of micro or macrovesicular steatosis.

2.7. Statistical analysis

The data were assessed for normality, Shapiro Wilk test - and expressed as mean ± standard error of the mean (SEM). Student's t-test was used to compare the groups, with a significance level of P<0.05 (GraphPad Software, version 6.03 for Windows, La Jolla, CA, USA).

3. Results

3.1. Body weight

At the beginning of the study, there were no significant differences between the groups. However, at the end of the experiment (week 8), the HF group showed a body weight that was 23% higher than the SC group (P<0.0001) (Figure 1).

3.2. Carbohydrate metabolism

The area under the curve (AUC) for the oral glucose tolerance test (OGTT) was 72% higher in the HF group than the SC group (P=0.0002), with an AUC of 20597 ± 1333 and 12734 ± 359.3, respectively (Figure 2A). The insulin tolerance test showed that the HF group had an AUC 62% higher (P=0.0009) than the SC group, with values of 14733 ± 1099 and 9095 ± 1156, respectively (Figure 2B). The fasting blood glucose levels of the HF group were 43% higher than the SC group (P<0.0001), with values of 150.3 ± 8.2 and 104.9 ± 6.4 mg/dL, respectively.

Figure 2
Area under the curve for the oral glucose tolerance test (OGTT) (A) and Intraperitoneal insulin tolerance test (IPITT) (B). The values are presented as the mean ± SEM. Significant differences (P < 0.05) were determined using a t-test. Control group (SC), High-fat (HF).

3.3. Insulin and glucagon levels

The insulin level in the HF group was significantly higher than the SC group (P<0.05), with levels greater than 1 x 10³ pg/mL, while in the SC group, it was less than 1.0 X 10³ pg/mL (Figure 3A). The glucagon levels in the HF group were 125% higher than the SC group (P<0.0001), with levels greater than 40 pg/mL in the HF group, while in the SC group, it was smaller than 20 pg/mL (Figure 3B).

Figure 3
Plasma insulin (A) and glucagon (B) levels. The values are presented as the mean ± SEM. Significant differences (P < 0.05) were determined using a t-test. Control group (SC), High-fat (HF).

3.4. Hepatic steatosis

The steatosis in the SC group was characterized by Vv=A=4.65 ± 1.80%, with macrovesicular steatosis as the predominant form. In contrast, the steatosis in the HF group was characterized by Vv=33.02 ± 1.77%, with the presence of macro and microvesicular steatosis, and the difference between the groups was statistically significant (P<0.0001) (Figure 4). The steatosis in the HF group was 7.10 times higher than in the SC group.

Figure 4
Hepatic steatosis (A). Photomicrography of the liver stained with H&E. SC group (B) and HF group (C). The values are presented as the mean ± SEM. Significant differences (P < 0.05) were determined using a t-test. Control group (SC), High-fat (HF). Bar = 40 μm.

3.5. Hepatic fibrosis

The hepatic fibrosis in the SC group was characterized by Vv=0.73 ± 0.23%, while in the HF group, it was characterized by Vv=2.72 ± 0.43%, and the difference was statistically significant (P<0.0001) (Figure 5). The area of fibrosis in the HF group was 2.93 times bigger than in the SC group.

Figure 5
Hepatic fibrosis (A). Photomicrography of the liver stained with Masson’s trichrome of SC (B) and HF (C). The values are presented as the mean ± SEM. Significant differences (P < 0.05) were determined using a t-test. Control group (SC), High-fat (HF). Bar = 40 μm.

4. Discussion

The present study investigated the effects of a high-fat (HF) diet on metabolic parameters and hepatic steatosis in a rodent model. The results showed that at the end of the study period, the HF group had a significantly higher body weight compared to the standard control (SC) group. In addition, the HF group had impaired carbohydrate metabolism, as evidenced by higher area under the curve values for oral glucose tolerance test and insulin tolerance test, and elevated fasting blood glucose levels. The HF group also had significantly higher levels of insulin and glucagon compared to the SC group. Furthermore, hepatic steatosis was markedly higher in the HF group compared to the SC group, characterized by the presence of both macro and microvesicular steatosis. These findings suggest that the HF diet may have detrimental effects on metabolic health and contribute to the development of hepatic steatosis.

Our study revealed some compelling findings regarding the impact of high-fat diets on animal body weight. The HF group animals displayed a remarkable 23% increase in body weight compared to the SC group, a result that aligns with previous research in C57BL/6 mice over a 12-week period Lang et al. (2019). Our data showed that the HF group animals weighed approximately 29g after 8 weeks, while the SC group animals weighed around 24g. These results suggest that body weight can increase substantially over several weeks, leading to obesity. Interestingly, C57BL/6 mice have been proven to be an excellent model for studying obesity, as they develop hyperinsulinemia and hyperglycemia when fed ad libitum with high-fat diets. While previous studies suggested that obesity would take 16 weeks to manifest, our study revealed that the mice developed obesity after only 8 weeks, while (Lang et al., 2019) reported obesity development after 12 weeks.

Regarding the glucose levels, our study results are consistent with those of (Lang et al., 2019) who reported that C57BL/6 mice fed with HF diets exhibited significantly higher glucose and insulin levels after 12 weeks of observation. However, our study found significant alterations in insulin and blood glucose levels in just 8 weeks, indicating that obesity can have a rapid impact on glucose metabolism. These findings are particularly concerning since impaired glucose tolerance and insulin resistance are the most important co-morbid conditions in type 2 diabetes mellitus. The evidence suggests that obesity increases the likelihood of developing insulin resistance, potentially leading to the development of type 2 diabetes in a shorter time frame (Lang et al., 2019).

The hepatocytes in the HF group were visibly enlarged, which occurred due to the presence of macro and microvesicles of fat inside the cells, leading to an increase in liver size. These findings are consistent with (Vansaun et al., 2009), whose experiment with murine models fed HF diets showed liver enlargement due to the presence of fat vesicles.

In our own study, we also found significant steatosis in the HF group after just 8 weeks, with a 609% increase compared to the SC group. This aligns with (Souza Marinho et al., 2019), who reported a 468% increase in hepatic steatosis after 8 weeks of HF diets in mice. Remarkably, our results were comparable to (Catta-Preta et al., 2011), whose mice displayed 600% higher hepatic steatosis after 16 weeks of HF diet, with a similar area of 33.5% steatosis in the liver, close to our findings of 33%. These results highlight the dangers of consuming high-fat diets, which can lead to the accumulation of fat in liver cells and ultimately contribute to liver disease.

Steatosis, also known as fatty liver disease, can be classified into two types: macrovesicular steatosis, where the fat is stored in one large vesicle, and microvesicular steatosis, where small deposits of fat are stored in little vesicles (Catta-Preta et al., 2011). Our analysis of hepatic steatosis showed that the SC group had macrovesicular steatosis, while the HF group had both macro and microvesicular steatosis, with microvesicular steatosis being more prevalent after only 8 weeks of evaluation. This is concerning as microvesicular steatosis has a worse prognosis than macrovesicular steatosis (Catta-Preta et al., 2011).

The evidence base for the investigation and management of NAFLD is substantial and growing; therefore, evidence-based recommendations have been developed for the management and care of patients (McPherson et al., 2022). In our study, we found that the animals showed a moderate degree of NAFLD (Grade 2) after only 8 weeks of HF diet, with 33.02% of hepatic steatosis attested, according to the graduation proposed by Sanyal (2002) and Nassir et al. (2015).

When hepatic steatosis is not related to alcohol ingestion, it is referred to non-alcoholic hepatic steatosis (NASH). NASH occurs when the liver tissue becomes inflamed, leading to the expansion of the hepatocytes, a phenomenon known as “ballooning” that causes degeneration, which could lead to a change in NAFLD and raise the risks of progression to fibrosis and cirrhosis (Nassir et al., 2015). Hashimoto et al., (2015) define NASH as the presence of hepatic steatosis and inflammation, with or without fibrosis.

Hepatocyte damage can stimulate an inflammatory response in the liver. When hepatocytes become injured, they release inflammatory mediators that attract white blood cells to the site of the injury. These white blood cells produce pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α, which further exacerbate the inflammation. T lymphocytes are also recruited to the site of injury, leading to an amplified inflammatory response (Pellicoro et al., 2014; Prystupa et al., 2015). The liver in the fatty liver group revealed many abnormalities, including lymphocyte aggregation among hepatocytes, hazy swelling and vacuolization of the cytoplasm, mononuclear inflammatory cell infiltration with congestion and bleeding, hydropic changes, and Kupffer cell hyperplasia (Lahmi et al., 2023). The healthy control group's liver sections showed no histological alterations during the trial, and the hepatocytes had typical radial patterns around the hepatic cords.

In the present study, inflammation was widespread in the livers of the HF group animals, accompanied by both micro and macrovesicular steatosis types. Fibrosis was also present. Based on these findings, it can be concluded that the animals in the HF group likely developed non-alcoholic hepatic steatosis (NASH), as described by Nassir et al. (2015). NASH can lead to degeneration, hepatic lesions, and subsequent necrosis of the hepatocytes.

According to Jiao et al. (2009) hepatic fibrosis is a response to damage in the liver. This chronic lesion leads to the deposition of extracellular matrix and is typically characterized by the presence of myofibroblasts. In the HF group, hepatic fibrosis was observed mainly in the proximity of large vessels, where microvesicular hepatic steatosis degenerated. Myofibroblasts, stimulated by Transforming Growth Factor β (TGF- β), were responsible for the extracellular matrix deposition in the cicatricial process. The continuous lesions, associated with the inflammatory process, are believed to have led to the death of hepatocytes, triggering the repair process. The myofibroblasts of the liver, stimulated by TGF- β, began the repair process near large vessels, leading to periportal fibrosis. Overall, these findings suggest that BMC transplantation may be a promising therapeutic strategy for liver fibrosis.

The HF group mice in this study were found to have NAFLD at Stage 2 according to the grading system proposed by Sanyal (2002) and Hashimoto et al. (2015). This indicates that within just 8 weeks of observation, the mice in the HF group developed Stage 2 NAFLD. The combination of steatosis and fibrosis indicates that the mice in the HF group had NAFLD of both degree and stage 2.

Velázquez et al. (2019) found that administering HF diets for 80 weeks mimicked the pathophysiological behavior of NAFLD. However, based on the classifications proposed by Sanyal (2002), Hashimoto et al. (2015) and Nassir et al. (2015), we can confirm the presence of NAFLD after just 8 weeks of HF diet administration. It is well-established that steatosis and fibrosis are both consequences of NAFLD (Sayiner et al., 2016; Benedict and Zhang, 2017).

Nowadays, NAFLD is one of the most significant causes of chronic liver disease worldwide. It is a disease that presents with liver abnormalities unrelated to alcohol ingestion and can include hepatic steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (Nassir et al., 2015). Additionally, NAFLD is linked with other disorders such as type 2 diabetes (Sarwar et al., 2018). The presence of NAFLD in the mice in this experiment was associated with higher blood glucose levels and glucose and insulin resistance, indicating a potential risk for developing a progression of NAFLD and insulin resistance that could lead to type 2 diabetes.

The findings of this study provide evidence that the administration of high-fat diets in mice can lead to the development of non-alcoholic fatty liver disease (NAFLD), which is a significant and growing health concern worldwide. The presence of NAFLD in the mice was associated with abnormalities in glucose metabolism and insulin resistance, which may increase the risk of developing type 2 diabetes. The development of hepatic fibrosis in response to the hepatic lesions observed in the HF group highlights the importance of early intervention and treatment to prevent the progression of NAFLD to more severe liver diseases.

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Publication Dates

• Publication in this collection
  31 Jan 2025
• Date of issue
  2024

History

• Received
  06 Mar 2024
• Accepted
  06 Sept 2024