Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia resulting from a defect in insulin secretion or action or both 1. Insulin-dependent diabetes mellitus (IDDM) is frequently induced in experimental animals by streptozotocin (STZ), which has toxic effects on islet β cells 2,3.
Long-standing hyperglycemia has side effects on other tissues, especially the liver, which is the cardinal organ of the body with the function of glucose homeostasis 4. It is one of the tissues that bear the load of chronic hyperglycemia as glucose is freely permeable into its cells 5. Liver dysfunction has been observed in diabetic patients, especially those with uncontrolled blood sugar levels 6.
The epidermal growth factor receptor (EGFR) family is one of the tyrosine kinase receptor families that regulate liver pathophysiology. This receptor is part of a complex signaling system that includes multiple ligands, mainly transforming growth factor-α and epidermal growth factor (EGF). Both transforming growth factor-α and EGF bind and activate the same receptor, EGFR. Ligand binding results in the activation of a distinct intracellular signaling cascade that mediates cell proliferation, migration, differentiation, and evasion from apoptosis 7.
Together with hepatocyte growth factor, EGFR ligands represent the only complete mitogens in adult hepatocytes in serum-free medium and are the most important growth factors involved in the proliferative response during liver regeneration 8. In addition to its regulatory role in proliferation, EGFR is a major survival pathway in the liver 9 and its signaling contributes toward the proliferation, survival, and differentiation of liver stem/progenitor cells 10.
Aim of the work
This work was carried out in order to demonstrate the histological and EGFR immunohistochemical changes that occur in the liver of STZ-induced diabetic rats.
Materials and methods
Twenty healthy male albino rats weighing 180–220 g were used in this study. They were housed under standard laboratory conditions (light period: 7:00 a.m.–7:00 p.m., temperature: 22–25°C) and provided food and tap water ad libitum.
Animals were divided into two groups (10 rats each).
- Group I served as a control group and was further subdivided into two subgroups: Ia and Ib (five rats each). They were administered a single dose of intraperitoneal saline equivalent to STZ dose.
- Group II served as a diabetic group and was further subdivided into two subgroups: IIa and IIb (five rats each). Diabetes was induced in this group by a single intraperitoneal injection of STZ, 40 mg/kg of body weight, freshly dissolved in 5 mmol/l citrate buffer, pH 4.5 11.
Twenty-four hours after the STZ injection, development of diabetes in group II rats was confirmed by measuring blood glucose levels in blood samples obtained from a tail vein using an Ames One Touch Glucometer (LifeScan; Johnson and Johnson, New Brunswick, New Jersey, USA). Rats with blood glucose levels of 250 mg/dl or more were considered diabetic. Five rats from each subgroup were starved overnight for 12 h and then sacrificed 2 weeks (Ia and IIa) and 4 weeks (Ib and IIb) after the STZ injection. Blood samples from all animals were collected by cardiac puncture using heparinized syringes and then centrifuged to separate plasma used for the detection of serum glucose and insulin concentrations. Serum glucose was determined using the hexokinase method and insulin was determined using an enzyme-linked immunosorbent assay.
Specimens from the liver of all animals were fixed in Bouin’s solution, dehydrated, cleared, and embedded in paraffin. Serial paraffin sections of 5 μm thickness were obtained and stained with
- H&E stain 12.
- Periodic Acid-Schiff’s (PAS) reaction 13.
- Masson’s trichrome stain 14.
- Immunohistochemical stain 15: using anti-EGFR.
The immunocytochemical reaction was performed using the Avidin Biotin peroxidase Complex (ABC) technique. Specific mouse monoclonal antibody (anti-EGFR; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) was used at a dilution of 1:50.
Endogenous peroxidase activity was inhibited by 3% H2O2 in distilled water for 30 min. Sections were then washed in tap water for 30 min and in distilled water for 10 min. Nonspecific binding of antibodies was blocked by incubation with normal goat serum (X 0907; Dako, Carpinteria, California, USA) with PBS, diluted 1:4. Sections were incubated with monoclonal mouse antisera against EGFR (EGFR-10: sc-373746; Santa Cruz Biotechnology Inc.) diluted 1:50 and then left at room temperature for 1 h. The sections were then washed in PBS 3×3 min and incubated with biotinylated anti-mouse IgG (LSAB 2 Kit; Dako), followed by washing in PBS 3×3 min and then incubation with avidin–biotin–peroxidase complex solution (LSAB 2 Kit; Dako). The antibody bound to sections was visualized by treating with 0.05% (w/v) 3,3′-diaminobenzidine tetrachloride (Sigma Chemicals Co., St. Louis, MO, USA) in 10 mmol Tris-buffered saline. Then, the sections were stained by Meyer’s hematoxylin as a counter stain.
Negative control sections were placed under the same conditions after omitting the primary antibodies. Positive controls were included within the tissue sections and a positive reaction was observed in the skin.
Immunostained sections from all groups were examined for assessment of the intensity of staining with anti-EGFR antibodies. The intensity of staining was graded as follows: 0, negative; +, weak; ++, moderate; and +++, strong immunoreaction.
- Binucleated hepatocyte count: Binucleated hepatocytes were counted in five high-power fields at a magnification (×1000) in the pericentral region in H&E-stained sections from each animal in all groups.
- The area% of EGFR immunoreaction: The area% of EGFR immunoreaction in the liver was estimated using a computerized image analyzer system (Leica Q 500 MC Program; Leica, Cambridge, UK). Five sections from each animal from all groups were examined.
Statistical analysis was carried out using the statistical package for social science (SPSS) program (SPSS version 10 Inc., Chicago, Illinois, USA). The data were expressed as mean and SD. Student’s t-test was used to compare the two animal groups. The difference between the two groups was significant when the P value was <0.05.
Gross examination of the liver showed normal color of the liver in the control and group IIa rats. However, yellowish brown discoloration was observed in the livers of rats in group IIb.
The liver appeared normal and the hepatic lobule was formed of a central vein with cords of hepatocytes radiating from the central vein toward the periphery of the lobule. The cords of hepatocytes were separated by blood sinusoids. Hepatocytes appeared normal with an acidophilic vacuolated granular cytoplasm and large vesicular nuclei (Fig. 1). The portal tracts appeared normal and contained branches of the hepatic artery, portal vein, and bile ducts (Fig. 2). PAS-stained sections showed PAS-positive granules in the cytoplasm of hepatocytes (Fig. 3). Masson’s trichrome-stained sections showed a minimal amount of collagenous fibers around the central vein (Fig. 4). Immunostained sections showed strong positive (+++) EGFR immunoreactivity in the hepatocytes in the pericentral and periportal regions and in the hepatocytes under the liver capsule. Moderate positive (++) EGFR immunoreactivity was observed in the hepatocytes elsewhere in the liver (Figs 5 and 6). Strong positive (+++) EGFR immunoreactivity was observed in the bile duct epithelium; however, this immunoreaction was negative (0) to weakly positive (+) in other structures of the portal tracts (Fig. 7).
In the rats in group IIa, the central vein and blood sinusoids appeared congested (Fig. 8). Hepatocytes with vacuolated cytoplasm and shrunken nuclei and others with pyknotic nuclei were observed (Fig. 9). Most of the hepatocytes in the periportal region showed accumulation of lipid droplets in their cytoplasm (Fig. 10). The portal tract showed some lymphocytic infiltration and congestion of portal vein radicle (Fig. 11). In PAS-stained sections, accumulation of PAS-positive granules was observed in the hepatocyte cytoplasm, especially at the periphery of the hepatic lobules (Fig. 12). Immunostained sections showed a marked decrease in EGFR immunoreactivity. This immunreaction appeared moderate (++) in the hepatocytes in the pericentral region and under the liver capsule and appeared weak (+) in the hepatocytes in the periportal region. However, this immunreaction was negative to weakly positive in the hepatocytes elsewhere in the liver (Figs 13 and 14). Structures of portal tract showed a weakly positive (+) or a negative EGFR immunoreaction (Fig. 15).
In the rats in group IIb, marked dilatation of the central vein and congestion of blood sinusoids were observed (Fig. 16). Some hepatocytes showed an acidophilic granular cytoplasm and smaller condensed nuclei. Large swollen hepatocytes with vacuolated cytoplasm and deeply stained shrunken nuclei were also observed (Fig. 17). In the periportal region, most of the hepatocytes showed a pale cloudy nongranular cytoplasm with either normal or shrunken nuclei. Some hepatocytes with pyknotic nuclei were also observed in this region (Fig. 18). Fatty change with the accumulation of lipid droplets was also observed in some hepatocytes in the periportal region (Fig. 19). In the pericentral region, large binucleated hepatocytes were observed (Fig. 20). The portal tracts showed marked congestion of the portal vein radicles and the bile ducts appeared smaller and lined with low cubical epithelium (Fig. 21). In PAS-stained sections, marked accumulation of PAS-positive granules was observed in the hepatocyte cytoplasm (Fig. 22). Masson’s trichrome-stained sections showed increased collagenous fibers around the central vein (Fig. 23). Moderate to strong EGFR immunoreactivity was restricted to hepatocytes in a narrow zone around the central vein and under the liver capsule (Figs 24 and 25). The structures in the portal tracts showed negative EGFR immunoreactivity (Fig. 26).
The mean blood glucose concentration showed a significant increase in subgroup IIa (397.8±15.12 mg/dl) compared with subgroup Ia (101.7±2.73 mg/dl) and in subgroup IIb (382.1±38.2 mg/dl) compared with subgroup Ib (98.7±5.86 mg/dl) (Table 1). The mean blood insulin was significantly decreased in subgroup IIa (11.4±0.47 mU/l) compared with subgroup Ia (57.5±3.02 mU/l) and in subgroup IIb (10.6±1.16 mU/l) compared with subgroup Ib (57.4±2.08 mU/l) (Table 2).
The number of binucleated hepatocytes per high power field (HPF) in the pericentral region showed a nonsignificant increase in group IIa (3.8±1.04/HPF) compared with group Ia (3.28±1.17/HPF) and a significant increase in group IIb (5.64±1.41/HPF) compared with group IIa (3.8±1.04/HPF) (Table 3).
The area% of EGFR immunoreactivity decreased significantly in group IIa (39.52±3.29) and group IIb (31.24±3.14) compared with groups Ia (88.92±2.47) and Ib (88.48±3.13), respectively (Table 3).
DM is one of the most common endocrine diseases characterized by a state of hyperglycemia and caused by a defect in the insulin secretion, insulin action, or even both 16. IDDM is frequently induced in experimental animals by STZ, which has toxic effects on islet β cells 3.
Long-standing hyperglycemia has side effects on different tissues, especially the liver, which bears the load of chronic hyperglycemia as glucose is freely permeable into its cells 5. Liver dysfunction has been reported in diabetic patients, especially in those with uncontrolled blood sugar levels 6.
Liver pathophysiology is regulated by several growth factors and cytokines 7. EGF, a polypeptide mitogen, and its tyrosine kinase receptor (EGFR) have been proposed to play vital roles in liver regeneration and transformation 17,18.
This work has been carried out in order to demonstrate the histological and EGFR immunohistochemical changes that occur in the liver of STZ-induced diabetic rats.
In the present work, an injection of STZ resulted in increased plasma glucose and reduced insulin levels. STZ was found to induce pancreatic β cell damage that resulted in insulin deficiency, which in turn led to increased blood glucose 19,20. This chronic hyperglycemia in DM leads to disorders of carbohydrate, fat, and protein metabolism and induces complications in different organs including the liver 16.
This work showed marked congestion and dilatation of the central vein, portal vessels, and blood sinusoids in diabetic rats. These findings are in agreement with previous studies that demonstrated enlargement in sinusoids 21 and dilatation and congestion of portal vessels and central veins 22,23 in the liver of diabetic rats. However, narrowing and reduction in the volume of sinusoids have been reported in the liver of diabetic rats 24.
The present study found degenerative changes in hepatocytes of diabetic rats such as vacuolated cytoplasm and shrunken nuclei (hydropic degeneration) and others with pyknotic nuclei. Similarly, previous workers reported periportal necrosis, degeneration, and nuclear shrinkage of hepatocytes 21,25. Mild centrilobular degeneration was observed 2 weeks after induction of diabetes by STZ 23. Focal necrosis of hepatocytes was also reported in the liver of alloxan-induced diabetic rats 26.
Large swollen hepatocytes with vacuolated cytoplasm and deeply stained shrunken nuclei were observed in the rats in group IIb. Previous workers 27,28 found hepatocytes with swollen cytoplasmic hydropic and microvesicular vacuoles in the livers of STZ-induced diabetic neonatal rats 12 weeks after induction of diabetes. However, reduction in the volume of hepatocytes and their nuclei was reported 4 weeks after an injection of STZ 24.
In the rats in group IIb, some hepatocytes showed granular degeneration with an acidophilic granular cytoplasm and smaller condensed nuclei. Similar observations have been reported previously 27. The presence of degenerated hepatocytes is possibly associated with the production of free radicals in the liver of diabetic rats 29. It was reported that free radicals play an important role in the initiation and progression of liver injury and induce apoptosis, necrosis, and regeneration in hepatocytes and endothelial cells 30,31. Moreover, oxidative stress may cause oxidative damage of cellular membranes and changes in subcellular organelles, resulting in several complications in diabetic disease 32,33. These degenerative changes are mainly because of insulin deficiency and not STZ toxicity as these changes could be prevented by the administration of insulin 34.
Fatty change with the accumulation of lipid droplets in the cytoplasm of hepatocytes was observed in groups IIa and IIb. Previous workers have reported deposition of lipid droplets 23 and global microvesicular steatosis 22 in the hepatocytes of diabetic rats. Periportal fatty infiltration was also described in the livers of alloxan-induced diabetic rats 26. This fatty change may explain the yellowish brown discoloration of the livers from rats in group IIb. Fatty liver and hyperlipidemia were also reported in IDDM of STZ-treated rats and shrews 3,23. This fatty change could be because of the increased influx of fatty acids into the liver as a result of hypoinsulinemia and the low capacity of excretion of lipoprotein secretion from the liver caused by a deficiency in apolipoprotein B synthesis 3. In addition, hyperlipidemia could be another factor for fatty liver formation as reported previously in diabetic monkeys and rats 3,35.
A significant increase in the number of large binucleated hepatocytes was observed in the pericentral region of group IIb. This increase may be a sign of hepatocyte regeneration. Similarly, previous workers 36 found an increase in large cytomegalic hepatocytes in diabetic rats. It was suggested that this increase may be because of early hyperplasia and decreased apoptosis in the liver of rats with STZ-induced diabetes 37.
Increased collagenous fibers around the central vein were observed in rats in group IIb. Previous researchers 23,38 also reported an increase in the fibrous content of diabetic rat liver. However, some workers 27 did not find any increase in the collagenous material in the diabetic rat liver.
The lymphocytic infiltration observed in the portal tracts of diabetic rats in this study is in agreement with the findings of other researchers 25,27. In rats in group IIb, bile ducts appeared small and lined with low cubical epithelium. Similarly, destruction of some bile ducts was reported in diabetic rat liver 23.
In this work, the glycogen content of the hepatocytes was increased in diabetic rats. Excess glycogen accumulation in the liver is observed in 80% of diabetic patients 39. In patients with chronic diabetes, glycogen accumulation is observed and it is postulated that long-standing insulin deficiency may facilitate glycogen synthase activity. This facilitated enzyme activity and enhanced gluconeogenesis may cause glycogen accumulation in diabetes 40. However, some workers 23,38 reported a decrease in hepatocyte glycogen in diabetic rats. Studies carried out on animals with recently induced diabetes showed that glycogen synthesis in the liver is impaired because of defective activation of glycogen synthase 40. The discrepancy between the present work and the previous studies may be because of differences in rat strain, duration of the experiment, or differences in blood insulin and glucose levels.
This work found positive EGFR immunoreactivity in hepatocytes and bile duct epithelium of the control group. Immunohistochemical analysis indicated strong cytoplasmic staining for EGFRs in normal hepatocytes 41. It was documented that hepatocytes express the tyrosine kinase EGFR and quickly respond to exogenous EGF in-vivo42 and in primary culture 43. It was also reported that both quiescent and proliferative hepatocytes maintain significant levels of the transmembrane EGFR; however, its functional role in liver homeostasis has remained controversial 44. Moreover, cellular distribution of EGFR gene expression in the human liver showed a cell-specific pattern restricted to hepatocytes 42.
In the present work, immunohistochemical staining of control liver sections showed strong EGFR immunoreactivity in the hepatocytes around the central veins, around the portal tracts, and under the liver capsule. Moderate immunoreaction was observed in hepatocytes elsewhere in the liver. However, it was reported that the EGFR shows acinar heterogeneity with a portal-to-central concentration gradient 41. Moreover, it was found that, in the individual hepatic lobules, mRNA and EGFR expression was nonuniformly distributed, showing a peripheral bias 42.
The present study found a significant decrease in the area% of EGFR immunoreactivity in rat liver 2 and 4 weeks after induction of diabetes. Similarly, previous workers 45 reported a decrease in the binding of EGF to liver membranes by 43–52% within 3 h after the injection of alloxan. They added that the maximal decrease in EGF binding was 70% and persisted for 20 days. Moreover, a reduction in the number of hepatocyte surface EGFRs was reported in alloxan-induced diabetic rats 45 and in hepatocytes freshly isolated from STZ-diabetic rats 46. This decrease in the number of EGFRs is mainly because of inhibition of EGFR synthesis, with almost no changes in their affinity 45,46.
In this work, fatty and degenerative changes appear to be localized in the hepatocytes around the portal tract, which is the main site of decreased EGFR immunoreactivity. This colocalization suggests that these fatty and degenerative changes may be the cause of decreased expression of EGFR on the surface of hepatocytes. However, in the rats in group IIb, strong EGFR immunoreactivity was restricted to hepatocytes in a narrow zone around the central vein. This strong immunoreaction may provide an explanation for the signs of hepatocyte regeneration such as increased large binucleated hepatocytes observed in this region.
DM is associated with marked hepatic congestion, degenerative and fatty changes in the hepatocytes, and decreased EGFR immunoreactivity in the liver.
As DM is associated with a reduction in the hepatic EGFR that affects the function and regenerative capacity of the liver, better care should be provided to diabetics with hepatic diseases and precautions must be taken to avoid liver injurious agents in diabetic patients.
Conflicts of interest
There is no conflict of interest to declare.
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