The effects of a high-fat diet on the liver of pregnant albino rats and their developing offspring

ABSTRACT Studies have demonstrated that the consumption of a high-fat diet (HFD) is linked to a higher occurrence of obesity and metabolic disorders. Maternal dietary habits can have long-lasting effects on the health of offspring. This study aimed to evaluate the impact of a HFD on the liver of pregnant rats and their offspring. 30 pregnant rats were used in this study. They were divided into two equal groups; control and HFD group. HFD group were fed on 15% lard from 6th week before conception till the end of weaning period. The mother rats and their offspring were sacrificed; blood samples were collected for estimation of biochemical parameters. The liver was removed for histological, immunohistochemical, and ultrastructural investigations. HFD mothers displayed elevated serum levels of LDL, triglycerides and total cholesterol and elevated blood glucose compared to the control. Also, remarkable histopathological and ultrastructural signs were recorded in the liver tissue of HFD treated mothers and their offspring. Additionally, the liver tissue displayed strong expression for caspase 3 and obvious reduction for PCNA. These results emphasize the importance of a balanced diet during pregnancy to prevent long-term liver complications in both mothers and their offspring.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is a prevalent health concern primarily triggered by the excessive intake of high-fat fast food, resulting in obesity.In the United States NAFLD has become increasingly prevalent, affecting approximately 95 million individuals [1], and its global incidence has reached 25% of the world's population [2].In nonalcoholic patients, the disease can develop into cirrhosis and hepatocellular carcinoma.
Studies conducted on mice fed a HFD have shown that their livers developed conditions such as steatosis (accumulation of fat), cell injury, inflammation in the portal and lobular areas and fibrosis [3,4].
Cholesterol is a fundamental constituent of a diet rich in fats.The liver naturally produces cholesterol to support the body's normal cellular functions.As a general aspect, HFD was shown to elevate the levels of cholesterol in the bloodstream resulting in a condition known as hypercholesterolemia, which significantly increases the development of liver steatosis [5].Furthermore, hypercholesterolemia significantly contributes to the risk of developing atherosclerosis [6].It had been reported that maternal hypercholesterolemia is associated with a substantial increase in the formation of fatty streaks in the arteries of human fetuses and accelerates the progression of atherosclerosis during childhood.Additionally, hypercholesterolemia induced by HFD during gestation is enough to enhance the development of hepatic lesions in the fetuses [7] and to increased postnatal atherogenesis in response to hypercholesterolemia [8].
HFD supplementation, has a significant role in the emergence of NAFLDs that are characterized by an overload of fatty acids and their oxidation within the mitochondria, peroxisomes and microsomes of the liver cells.This is associated with the liberation of excess reactive oxygen species (ROS) [9,10].
The consumption of HFD by the mother is implicated in promoting adverse effects on some developing organs of their incomes, increasing their vulnerability to disease later in life.These adverse effects depend upon the time and duration of exposure to HFD [11,12].
Accordingly, this work aimed to evaluate the adverse effects of HFD on the liver of female rats during gestation and lactation periods and consequence complications on the liver of their fetuses.

Experimental work
A total of 40 sexually mature female and male albino rats weighing approximately 100-110 g were used.The rats were sourced from the animal house unit at the University of Jazan, Saudi Arabia, and were maintained in group cages with unrestricted access to food and water.A one-week period was provided for the rats to acclimate to their environment.Throughout the study, the rats were housed in a well-ventilated room with a light-dark cycle of approximately 12 h each.
The experimental animals were categorized into two sets, comprising 20 individuals each, 5 males just for breeding and 15 females: a control group and a HFD group.The regular-pellet diet utilized in our experiment consisted of 2% fat, 65% carbohydrate, 18% protein, 5% vitamin and mineral mix and 10% fiber.The females of HFD were fed a normal-pellet diet supplemented with 15% soft animal fat (lard) for 6 weeks before becoming pregnant (the females only fed on HFD).Following this period, both the control and HFD groups were allowed to breed.Mating was conducted by placing one male with three females overnight in plastic cages, and the presence of sperm in a vaginal smear confirmed conception the next morning, determining the first day of pregnancy.Throughout pregnancy and until the 21st day postpartum, the control group received the normal-pellet diet mentioned earlier, while the HFD group continued to receive the HFD.
At the 21st day postpartum, the mother rats and their offspring from both experimental groups were sacrificed.Maternal blood samples were collected and serum was separated and stored for analysis of glucose and lipid profiles.Furthermore, liver samples from both the mothers and offspring were promptly collected and prepared for examination under both light and transmission electron microscopy.

The body weight (g) of pregnant mothers
In the experimental groups, body weight gain was recorded once a week for 3 months.The final body weight was recorded at 21st day post-partum.

The body weight (g) of offspring
The body weight of offspring of the experimental groups was also recorded twice a week for 3 weeks after birth.

Absolute and relative liver weight (g) of mothers and their offspring
At the end of the experiment (21st day postpartum) the net weight of the liver for both mothers and their offspring was recorded for each rat.After weighing the livers of every mother and their offspring, the relative liver weight of the mothers and their offspring was calculated by comparing the liver weight to each animal's individual body weight.

Maternal blood glucose levels and serum lipid profile
At the end of the experiment (at 21st day postpartum), the mothers and their offspring were weighed, anesthetized using a chloroform solution, and subsequently euthanized.Cardiac puncture was performed on each rat to collect blood samples, which were subsequently placed into individual heparinized tubes.The collected blood samples were processed to determine the plasma levels of glucose (GL), low-density lipoproteins (LDL), total cholesterol (TC) and triglycerides (TG) for both the control and HFD mothers groups.

Histological and transmission electron microscope investigations
According to [13] and [14], investigations were conducted on the livers of mothers and their offspring in the control and HFD groups using light and a transmission electron microscope (Joel 100CXl, Musashino 3-chome; Akishima, Tokyo, Japan).

Immunohistochemical labeling of PCNA and caspase-3
Formalin-fixed liver tissue sections, 5 μm in thickness, were placed on glass slides coated with polylysine and kept at room temperature.The tissue sections were subsequently exposed to the PCNA antibody (Thermo Fisher Scientific, Fremont, CA, USA, Cat.No. 31-033) and the caspase-3 antibody obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, NB100-56116).In order to enhance background staining, the tissue sections were treated with hematoxylin as a counterstain.Subsequently, the specimens were examined and photographed.The generated images were processed and analyzed using Video Test morphology software.The software facilitated the measurement and recording of the percentage area for the analyzed images.

Flow cytometric analysis of caspase 3
Apoptosis analysis was conducted using a fluorescence-activated cell sorting (FACS) flow cytometer (Becton Dickinson, Sunnyvale, CA) that was equipped with a 15-mW air-cooled 488 nm argonion laser.To assess caspase 3 activity, the hepatocyte suspension was subjected to treatment with 1 μg of Fc antibody, followed by staining with FL1-H and phycoerythrin (PE) for cell surface staining.The cells, with a concentration of 1 × 106 cells, were subsequently stained with FL1-H -labeled Caspase 3 and propidium iodide for subsequent analysis.

Statistical analysis
The statistical analysis included calculating the mean values and standard error of the means (SEM) for all data.The data were presented in the format of mean ± standard error of the mean.Data analysis was performed using SPSS data analysis software, specifically version 17.The analysis involved conducting a one-way ANOVA followed by Scheffe and Duncan tests.A significance level of P < 0.05 was used to determine the statistical significance.

Body weight gain of pregnant mothers
Body weight gain of the experimental groups was measured on a weekly basis for a duration of 12 weeks, before, during and after pregnancy.In comparison to the control group, the HFD group had more weight gain (Table 1).

Body weight of offspring
The body weight of offspring of the experimental groups was recorded twice a week for 3 weeks after delivery.Offspringof HFD group also recorded higher weight gain as compared to that of control group (Table 2).

Absolute and relative liver weight of mother rats and their offspring
The absolute and relative weights of liver for both mothers and their offspring were recorded for each rat.The absolute and relative liver weights of HFD mothers and their offspring were significantly higher in HFD group compared with control group (Table 3).

Maternal blood glucose levels and serum lipid profile
Compared to the control, treated mother rats ingesting HFD exhibited significant increases in blood glucose levels, as well as serum triglycerides (TG), total cholesterol (TC) and low-density lipoprotein (LDL).(Table 4)

Mother rats
Histologically the control liver was composed of normally oriented plates, one cell-layer thick forming a lobular structure radially oriented from the central vein.The hepatocytes appeared polyhedral with vesicular round nuclei and eosinophilic cytoplasm.Finely distributed hepatic sinusoids were distributed in between the hepatic cords and enclosed by finely arranged Kupffer cells (Figure 1a).
At ultrastructure level, the hepatocyte nuclei possessed peripheral margination of heterochromatin along the nuclear envelope and the bulk of the nuclei is composed of euchromatin.Abundant mitochondria and rough endoplasmic reticulum were distributed within the   cytoplasm of hepatocyte.Microvilli were abundant on the hepatocytes adjacent to the blood vessel; canaliculi were also observed (Figure 2).High fat treated mother rats demonstrated histopathological signs in hepatic tissues.These were characterized by abundant lipid deposits within cytoplasm of the hepatocytes and mild fibrotic change.There were dense perivascular leukocytic infiltrations and congested blood vessels.Discrete foci of grouping leukocytes and dilated blood sinusoids were also visualized among hepatic tissue (Figures.1(b-d)).At ultrastructural level, high fat treated mothers exhibited accumulation of cytoplasmic lipid droplet, atrophied mitochondria and vesiculated rough endoplasmic reticulum.Numerous hepatocytes appeared with clumped chromatin materials manifesting apoptosis or even with karyolitic chromatin material.Damaged canaliculi and congested blood vessels were also observed.Degenerating Kupffer cells and sparsely infiltrating leukocytes were detected (Figure 3).

Offspring
In rat offspring of control mothers, the histological structure of haptic tissue was composed of organized hepatic plates, one or two cell layers    thick with characteristic polygonal shape with centrally located nuclei.The central veins were ensheathed by endothelial cells (Figure 4a).At ultrastructural level, the hepatocytes had a nucleus with a thin peripheral heterochromatin and inner core of euchromatin material.The cytoplasm contained abundant rough endoplasmic reticulum and mitochondria with differentiated inner cristae (Figure 5).
In offspring maternally treated with HFD, massive degeneration of hepatocytes associated with perivascular leukocytic cell infiltration was observed.Hepatocytes' nuclei were either pyknotic or karyolysed.Vacuolar degeneration was prominent.The blood vessels appeared congested and many of the megakaryocytes distributed throughout blood sinusoids are dilated (Figure 4b-d).
At ultrastructural level, there was a marked increase in hepatocytes with pyknotic nuclei.The cytoplasm possessed abundant accumulation of lipid globules causing displacement of mitochondria.A significant number of mitochondria exhibited signs of atrophy and a lack of differentiation in their inner compartment.Leukocytes with abnormal nuclear envelope were distributed within blood sinusoids (Figure 6).

Immunohistochemical reaction of PCNA and caspase 3
The hepatic tissue of control mother rats and their offspring possessed increased PCNA immunohistochemical reaction reflecting normal nuclear proliferation.However, liver of HFD mother rats and their offspring possessed a significant reduction of the immunohistochemical reaction reflecting degeneration of the hepatocytes (Figure 7).
Furthermore, in the HFD mother rats and their offspring, there was a noticeable increase in the intensity of the dark brown immunohistochemical reaction of caspase 3 in the cytoplasm of hepatocytes, in comparison to the control animals (Figure 8).

Flow cytometry results of caspase 3
HFD mother rats and their offspring revealed an increase in the mean percentage value of caspase 3 positively expressed liver cell compared to control (Figure 9).

Discussion
The prevalence of consuming processed foods with high fat content is closely linked to the emergence of obesity and subsequent metabolic syndromes.Previous reports have demonstrated that elevated levels of maternal serum LDL, triglycerides and total cholesterol are indicative of the likelihood of developing type 2 diabetes [15][16][17].Additionally, HFD intake is one of the main causes of NAFLD [18] that results in significant alterations in the molecular events and function of liver which reflect a considerable modulation in the functional markers of liver [19].
In the present study, a highly significant increase in the body weight was recorded in the HFD exposed mother rats and their pups.Such findings go parallel with the study by Qiao et al. [20], who found that consuming a HFD resulted in an increase in visceral and subcutaneous fat.Lima et al. [21] added that there is a direct correlation between the obesity observed in neonatal rats maternally fed on a HFD with an elevation in visceral fat.Karbaschi et al. [22] discussed that mother rats supplemented with a HFD consequently accompanied with over-stress, which influenced alterations in the programming of the neuroendocrine system in their offspring.It has been observed that infants born to women with higher body mass index (BMI) are frequently born with a larger size [23].
Further results indicated that HFD mother rats and their offspring showed significant increases in absolute and relative liver weights.The obtained results agreed with [24] who explained that the increase in liver weight is mainly attributed to fatty infiltration in hepatic cells which promotes liver injury.Hart et al. [25] employed a HFD containing 35.5% fat to investigate the specific impact of maternal HFD on metabolic dysfunctions in the offspring, which is in agreement with our obtained results.
In this study, the exposure to HFD during pregnancy was associated with the emergence of metabolic syndrome-like traits in the offspring, irrespective of maternal obesity.A previous study proposed that the dysfunction in mesenteric adipose tissue contributes to abnormal glucose metabolism in the offspring, suggesting its significant involvement in fetal metabolic reprogramming triggered by increased maternal fat consumption during pregnancy [26].Moreover, a strong association between the prevalence of pediatric NAFLD and dyslipidemia, spanning from fatty liver to steatohepatitis (NASH) were previously reported [27][28][29][30].
In the current work, severe histopathological signs were recorded in the liver of HFD mother rats and their fetuses included dilated blood  sinusoids, congested central veins and excess Kupffer cells.Similar observations were recorded by [31], who found that exposure of female rats to HFD during gestation is implicated in the induction of severe liver cell injury and development of hepatic steatosis.Further investigation by TEM revealed remarkable deleterious changes in the cellular levels of hepatic cells like abnormal canaliculi, atrophied and swollen mitochondria, dilated rough endoplasmic reticulum, damaged microvilli, and accumulated lipid droplets.Previous reports have declared that exposure to HFD during gestation and lactation periods is implicated in induction of oxidative stress on the liver cells even in mothers or their offspring, resulting in liver cell damage [32,33].Additionally, the offspring of HFD mothers rats were accompanied by liver mitochondrial DNA (mtDNA) damage, as reported by [34].Such finding agrees with our obtained results.
Wang et al. [35] reported that the increased levels of caspase-3 and apoptotic hepatocytes observed in HFD-induced NASH were linked to elevated oxidative stress of hepatic cells.This is in line with our obtained results indicated strong positive expression of caspase-3 in the liver cells of HFD mothers and their offspring.Another confirmational test for discrimination of increased level of hepatic cell apoptosis under the influence of HFD was applied in this study using PCNA (proliferative marker).The obtained result revealed remarkable significant decrease for PCNA expression in the liver tissue.This explains that HFD is a promoting factor inducing apoptosis through activation of caspase-3 and inhibition of PCNA.
In conclusion, the author established that maternal consumption of a high-fat diet led to severe biochemical, histopathological and ultrastructural changes in the liver cells of both mother rats and their offspring.This is basically attributed to the accumulation of lipids within hepatocytes.

Figure 7 .
Figure 7. Photomicrographs of formalin fixed histological sections of liver of mother rats and their offspring immunostained with PCNA antibody.a.Control mother.A1.Mother rat fed on HFD. b. control offspring.B1 Offspring maternally fed on a HFD.

Figure 8 .
Figure 8. Photomicrographs of formalin fixed histological sections of liver of mother rats and their offspring immune-histochemical stained with caspase 3 antibody.a.Control mother.A1.Mother rat fed on a HFD.b. control offspring.B1 Offspring maternally fed on a HFD.

Figure 9 .
Figure 9.A Flow cytometry chart illustrating the mean % value of caspase 3 positively expressed liver cells among mothers and their offspring.

Table 1 .
Body weight gain (g) of HFD and control mother rats.All data were presented as mean ± standard error of the mean.SPSS data analysis software (version 17) was used for data processing.The analysis was performed by one-way ANOVA followed by Scheffe and Duncan test, P value < 0.05 is considered as significance, indicated by*.

Table 3 .
Absolute and relative liver weight of mother rats and their offspring of control and HFD groups.
Note: All data were presented as mean ± standard error of the mean.SPSS data analysis software (version 17) was used for data processing.The analysis was performed by one-way ANOVA followed by Scheffe and Duncan test, P value < 0.05 is considered as significance, indicated by*.

Table 2 .
Body weight of offspring (g) of HFD and control groups.All data were presented as mean ± standard error of the mean.SPSS data analysis software (version 17) was used for data processing.The analysis was performed by one-way ANOVA followed by Scheffe and Duncan test, P value < 0.05 is considered as significance, indicated by*.

Table 4 .
The blood glucose, TG, TC, and LDL levels of HFD and control mother rats.
Note: All data were presented as mean ± standard error of the mean.SPSS data analysis software (version 17) was used for data processing.The analysis was performed by one-way ANOVA followed by Scheffe and Duncan test, P value < 0.05 is considered as significance, indicated by*.