The investigation of the effects of monosodium glutamate on healthy rats and rats with STZ‐induced diabetes

Monosodium glutamate (MSG, E621) is a flavor‐enhancing food additive used widely in the food preparation industry and consumed regularly. It is considered that long‐term consumption of MSG causes metabolic syndrome and obesity. Diabetes mellitus (DM) is a chronic metabolic disease characterized by high blood sugar, polyuria, polydipsia, and polyphagia, in which insulin secreted from pancreatic β cells is inadequate for maintaining blood glucose homeostasis. Rats were application 65 mg/kg streptozotocin (STZ) solution intraperitoneally and a diabetes model was created. For this purpose, freshly prepared STZ was injected into the peritoneum. Tumor necrosis factor‐α, interleukin (IL)‐10, IL‐6, and IL‐1β levels in STZ, MSG, and STZ + MSG groups were found to be significantly increased in inflammation parameters measured on the 28th day of administration when compared to the Control Group (p < 0.001). Also, although malondialdehyde (MDA) levels increased significantly in the STZ + MSG group when compared to the control group (p < 0.001), glutathione (GSH), and superoxide dismutase (SOD) levels were significantly decreased in the STZ, MSG, and STZ + MSG groups when compared to the control group (p < 0.001). Also, although glucose levels increased significantly in STZ and STZ + MSG at the end of the 28th day (p < 0.01), insulin levels decreased in STZ, MSG, and STZ + MSG groups when compared to the control groups (p < 0.01). As a result, it was found that STZ and MSG application significantly increased cytokine production, increased MDA, which is an oxidant parameter in pancreatic tissue, and decreased antioxidants (GSH and SOD) when compared to the control groups. It was also found that MSG disrupted the normal histological structure in pancreatic cells, and the damage was much more in both exocrine and endocrine pancreatic areas in the STZ + MSG group when compared to the STZ and MSG groups. It was considered that with the increased use of MSG, the susceptibility to DM might increase along with tissue damage significantly in diabetic groups, therefore, MSG must be used in a limited and controlled manner.


| INTRODUCTION
Monosodium glutamate (MSG), also (E-621), is a food additive known as a flavor enhancer, which is used widely in the world in addition to table salt. [1]MSG is a derivative of glutamate, which is not an essential amino acid. [2]This molecule was described nearly 100 years ago by Kikunae Ikeda as the fifth taste in addition to sweet, sour, salty, and bitter.MSG, which has a unique taste-umami, was first used widely in Asia and then in western countries. [3,4][6] MSG is often added to sauces, soups, and spice mixes.
The European Food Safety Authority determined the allowable daily amount of glutamic acid as 30 mg/kg body weight in 2017 and clarified the amounts that could cause symptoms when used depending on the daily intake.In this respect, it was reported that MSG causes symptoms at a daily level of >42.9 mg/kg, headache when consumed at 85.8 mg/kg, insulin increase at >143 mg/kg, and blood pressure increase of 150 mg/kg. [7]Because of these healthcare issues such as acute headache, flushing, and sweating, also called Chinese restaurant syndrome, its amount must be calculated by considering both healthy people and sensitive individuals. [8]It was reported that long-term high-dose MSG use might cause metabolic syndrome and obesity. [9]It was also reported in animal studies that excessive MSG causes oxidative stress, cardiac arrhythmia, neurotoxic, hepatotoxic, neoplastic, [10] kidney tissue toxicity, fibrosis, diabetes, behavioral disorders, and muscle wasting. [2,11]abetes mellitus (DM) is a chronic metabolic disease that is characterized by high blood sugar, polyuria, polydipsia, and polyphagia, in which insulin secreted from pancreatic β cells is inadequate to maintain blood glucose homeostasis. [12]Carbohydrates, fats, and proteins cannot be adequately utilized because of insulin deficiency or inadequate effect in DM. [13] DM is divided into two groups in etiopathogenic terms.Type 1 diabetes is an autoimmune disease destroying insulin-producing pancreatic beta cells.Type 2 diabetes is caused by insulin resistance and insufficiency of beta cells. [12,14]tokines are pharmacologically active, low molecularweight polypeptide molecules with characteristics, such as autocrine, paracrine, and junkstacrin.Cytokines are also grouped as proinflammatory and anti-inflammatory cytokines.Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 are proinflammatory cytokines and IL-10 are cytokines with antiinflammatory characteristics.Their amounts increase in the blood because of inflammations. [15]It was reported that chronic low inflammation and immune system activation, which have therapeutic potential and have important roles in many physiological reactions, are associated with the pathogenesis of DM because cytokines are produced by a wide variety of cells in the body. [16,17]DM, proinflammatory cytokines, such as IL-1β and TNF-α, which cause an increase in TNF-α and IL-1β, as well as the proliferation of monocyte chemotactic protein, destroying pancreatic endocrine islets through islet infiltrating immune cells. [18]1][22][23][24][25] Free radicals that form in diabetes cause glucose oxidation, nonenzymatic glycation of proteins, and oxidative degradation of glycated proteins causing increased lipid peroxidation, insulin resistance, damage to cellular organelles and enzymes, with the abnormal increase of free radicals and the simultaneous decrease of antioxidant defense systems.These effects, which are caused by oxidative stress, cause the development of DM complications. [26] his review with the title "Updating the Food Safety of Monosodium Glutamate," Henry-Unaeze reported that inadequate studies were conducted by conducting double-blind studies with sensitive people using MSG or placebo in foods. [27]MSG is a flavorenhancing chemical widely used in the food industry, especially in processed foods as a flavor enhancer.With the increasing industrialization, people's interest in ready-to-eat foods has increased.For this reason, the daily consumption rate MSG has increased regularly.The purpose of this study was to determine how the use of MSG affects the course of the disease in patients with DM.In the present study, the effects of MSG use on pancreatic tissue were investigated in type 1 diabetes-induced rats.

| Creating a diabetes model
The rats were housed in an environment with 24 ± 1°C, 45 ± 5% humidity, and a 12-h light/dark cycle.Animals were fed standard rodent chow and water ad libitum.The rats were administered intraperitoneally with 65 mg/kg streptozotocin (STZ) (BioVision) solution and a diabetes model was created. [28]For this purpose, the solution of freshly prepared STZ in citrate buffer with a pH of 4.5 was injected into the peritoneum of the rats that were fasted overnight.A solution that contained 10% dextrose was given to the animals as drinking water for one time only after the STZ application.The blood samples that were taken from the tail vein were measured with the On-Call Plus Blood Glucose Monitor after 72 h, and the rats with glucose levels above 200 mg/dL were considered diabetic, and study groups were formed. [29]total of 30 male rats (Sprague-Dawley) aged 3 months and weighing 200-300 g were used in the study.The rats were divided into five groups with six animals in each group.Group I (n = 6) was used as the healthy control.Group II (n = 6) animals were injected with sterile 0.9% NaCl once a day (intraperitoneal [ip]) for 1 day.
Group III animals (n = 6) were given 30 mg/kg/day MSG (gavage) for 28 days.Diabetes was induced in Group IV by giving 65 mg/kg/day of streptozotazine (ip) (n = 6) for 1 day.Animals in Group V (n = 6) were given 65 mg/kg/day of streptozotazine (ip) for 1 day and

| Malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) analysis in the pancreatic tissues
The pancreatic tissue samples were taken, washed with phosphate buffer solution (PBS), and dissected with Qiagen Tissue Lyser II at 30 Hz for 3 min by adding liquid nitrogen.Then, 0.1 g tissue samples were homogenized at 30 Hz for 30 s by adding homogenate buffers, and these tissues were prepared for analysis.SOD enzyme activity was measured in tissue samples according to Sun et al. [30] MDA levels were determined according to Ohkawa et al. [31] and GSH levels were measured according to the method of Sedlak and Lindsay. [32]

| Histopathological analysis
The pancreatic tissues that were taken for histopathological evaluations were fixed in 10% neutral buffered formaldehyde solution at room temperature for 48 h.Then, the tissues, which were washed under running water, were passed through graded alcohol and xylol series and embedded in paraffin blocks and 5-μm-thick sections were cut from paraffin blocks with a Leica RM2125RT microtome (Leica Microsystems).Crossman's modified triple staining technique was used for all sections for histopathological evaluations.Photographs of the sections that were examined under a camera-attached light microscope (Nikon Eclipse i50) were taken.

| Immunohistochemical analysis
All of the sections were taken on adhesive (poly-L-lysin) slides for the immunoperoxidase examination and a deparaffinized-dehydrated procedure by passing through a xylol and alcohol series was performed.Then, the sections were washed in distilled water for 5 min.After that, they were washed with PBS (pH 7.2) for 5 min.kept in 3% H 2 O 2 for 10 min., and endogenous peroxidase inactivated.
Then, the tissues were boiled in 1% antigen retrieval (citrate buffer (pH +6.0) ab93678) solution and allowed to cool at room temperature.After washing for 5-10 min in PBS, the sections were incubated for 5 min with a protein block (LOT: PHL659660; Thermo Fisher Scientific) that is compatible with all primary and secondary antibodies to prevent nonspecific background staining.After the excess of the block solution remaining on the tissue sections at the end of the incubation was poured, anti-insulin primary antibody (Abcam rabbit polyclonal antibody, 1:100, ab63820) was dripped without washing.In accordance with the primary antibody, it was incubated at room temperature for 1 h or at +4°C for 1 night.After incubation, the sections were washed with PBS again, two times for 5 min, and incubated with a biotinized secondary antibody (LOT: PHL659660; Thermo Fisher Scientific) for 10-30 min at room temperature.The sections washed again with PBS and streptavidinperoxidase for 10-30 min.Then, 3-3′ diaminobenzidine chromogen (LOT: HDX47396; Thermo Fisher Scientific) was dripped onto the sections and then washed with distilled water.Between 1 and 2 min of Mayer's hematoxylin was performed for background staining.
Then, they were washed with tap water.Alcohol and xylol series were conducted, covered with a coverslip and examined under a light microscope (Nikon Eclipse i50) and photographed.The intensity of anti-insulin immunoreactivity in the endocrine sections of the pancreas was scored semiquantitatively with values between 0 and 3 for each section; 0: no immunoreactivity, 1: weak immunoreactivity, 2: moderate immunoreactivity, and 3: severe immunoreactivity. [33]

| Statistical analysis
The data that were obtained in the study were compared by using the one-way analysis of variance and Tukey post hoc tests in the SPSS 20.00 statistical program.

| Effect of MSG on TNF-α, IL-1β, IL-6, IL-10, and insulin levels in the serum samples
After the application of STZ, blood glucose levels of the STZ group increased when compared to the control group for 30 days.Also, although there were increased blood glucose levels in the MSG applied groups when compared to the control group (p < 0.01), this increase was found to be higher in the STZ + MSG group (p < 0.001).
Although serum insulin levels decreased in the MSG group when compared to the control group, this was not at a statistically significant level (p > 0.05), and this decrease was found to be significant in the STZ + MSG group when compared to the MSG administered group (p < 0.05) (Figure 1).
It was found that the serum TNF-α levels were increased in groups with STZ when compared to healthy control groups (p < 0.001).It was found that serum IL-1β levels were higher in the groups that were treated with STZ and MSG when compared to the URCAR GELEN ET AL. healthy control groups (p < 0.001).Also, the IL-1β level was found to be higher in the STZ + MSG group than in the MSG group (p < 0.001).
IL-6 levels increased in blood serum samples in STZ groups when compared to healthy control groups (p < 0.001).However, IL-6 levels were found to be decreased in the STZ + MSG and MSG groups when compared to the STZ-only group (p < 0.001).
When IL-10 levels in serum samples were compared, it was found that STZ (p < 0.001), STZ + MSG (p < 0.01), and MSG (p < 0.05) groups had increased levels when compared to healthy control groups (Figure 2).

| Effect of MSG on MDA, GSH, and SOD levels in the pancreatic tissues
It was found that MDA levels increased in the study groups when compared to the healthy control groups, and this increase was more in the STZ + MSG group (p < 0.001).It was found that GSH levels decreased in pancreatic tissue samples in the application groups when compared to the healthy control groups (p < 0.001).The increase in the GSH level was not significant in MSG, STZ + MSG groups when compared to the STZ groups (p > 0.05).SOD activity that was measured in pancreatic tissue samples was decreased in the treatment groups when compared to the healthy control groups (p < 0.001).It was also found that SOD activity was decreased in the STZ + MSG group when compared to the MSG group (p < 0.05) (Figure 3).

| Effect of MSG on histopathological and immunohistochemical results in the pancreatic tissues
In the light microscopic examination of pancreatic tissues stained with Crossman's modified triple staining technique, it was determined that Langerhans islets had very smooth borders and a rich capillary network in the sections belonging to the healthy control and buffer control groups, and the exocrine pancreatic acini and Langerhans islet cells were found to be in normal histological structure (Figure 4 A,B).
When the pancreatic sections of the STZ group were compared with the healthy control group, the deterioration in the structure of the islets of Langerhans and degenerative and necrotic changes in the islet cells were remarkable.It was determined that in the islets of Langerhans, with a significant loss of cells and disruption of the cellular order, shrinkage occurred, and the borders of the islets became irregular.Cytoplasmic vacuolization and pycnotic nuclei were observed in necrotic cells.Hydropic degeneration was found predominantly in the cytoplasm of degenerative and necrotic cells, while cells with pycnotic nuclei had dark eosinophilic cytoplasms (Figure 4C).In the pancreatic sections of the MSG group, it was observed that the normal histological structure was highly deteriorated, the islets of Langerhans developed with more irregular borders compared to the control group, and hyperplasia and hypertrophy occurred in the size of the islets.Vacuolization, pycnotic nuclei, necrotic areas between cells, congestion, and dilatation in blood vessels were observed in the cytoplasm of islet cells (Figure 4D).
In the STZ + MSG group, it was determined that the damage was much more in both the exocrine and endocrine pancreas areas compared to the STZ and MSG groups.In the exocrine part, very intense lymphocyte cell infiltrations were striking, together with degenerative areas that caused cellular losses.In the islets of Langerhans, which form the endocrine part, intense cell losses, a reduction in islet sizes, and an increase in degeneration areas were observed (Figure 4E).Anti-insulin antibody was used for immunohistochemical staining.After staining of pancreatic tissues, it was determined that the groups with the highest immunopositivity were the healthy control and buffer control groups.In the immunohistochemical staining of the pancreatic tissues of the healthy control group, a very strong anti-insulin immunoreactivity was detected in the cytoplasmic areas of Langerhans islet cells.In the immunohistochemical staining of pancreatic tissues of rats in the STZ group, it was determined that there was anti-insulin immunoreactivity in a small number of cells in the islets of Langerhans compared to the healthy control group.Pancreatic sections of the MSG group had less anti-insulin immunoreactivity compared to the control group.On the other hand, in the sections of the STZ + MSG group, islets of Langerhans were observed in very small sizes compared to all groups, and at the same time, anti-insulin immunoreactivity was found to be negligible.Staining and scoring results for immunoreactivity are shown in Figure 4F-J and Table 1.

| DISCUSSION
MSG is a flavor-enhancing food additive used widely all over the world.FDA declared that limited MSG use is safe, but increased MSG consumption is associated with various potential side effects such as circulatory, cardiac, muscle neurological, and gastrointestinal disorders. [34]DM is a metabolic syndrome characterized by changes in carbohydrate, protein, and lipid metabolism that result from the destruction of pancreatic β cells or defects in insulin secretion or activity. [13]Various toxic chemicals are used to create a diabetes model in rats.For this purpose, chemicals such as streptozotazine and alloxan are used to form type 1 DM in rats. [35,36]A type 2 DM model was defined in animal studies by Cameron et al. in 1976, [37] and a type 2 diabetes model was created by injecting MSG into mice during the neonatal period. [38]Diabetes was formed in rats with high-fat diet and high-carbohydrate diet applications in nutritional diabetes models. [36,39]In the present study, MSG was administered orally to The levels of malondialdehyde (MDA), glutathione (GSH), and the activity of superoxide dismutase (SOD) in pancreatic tissue samples (*p < 0.05; ***p < 0.001).MSG, monosodium glutamate; ns, not significant; STZ, streptozotocin.
T A B L E 1 Immunopositivity scores of anti-insulin antibody in pancreatic sections.Abbreviations: MSG, monosodium glutamate; STZ, streptozotocin.
| 5 of 8 rats with DM for 28 days and the side effects of MSG were investigated in diabetic rats.Various potential health hazards of MSG were also emphasized in animal experimentation studies.
To determine pancreatic functions, the blood serum of the animals was measured for insulin on Day 28 and glucose levels on Days 7 and 28.It was determined that insulin levels decreased with the increase in glucose levels in the STZ and MSG groups when compared to the healthy control groups. [40]It was also found that there was a greater decrease in insulin levels in the group in which STZ + MSG was administered in combination when compared to the group in which only STZ was administered.In the present study, it was determined that there was anti-insulin immunoreactivity (p < 0.05) in a small number of β cells in the islets of Langerhans in the STZ group as a result of immunohistochemical staining in the pancreatic tissue when compared the control group (Figure 4).
Pancreatic sections of the MSG group had less (p < 0.05) anti-insulin immunoreactivity when compared to the control group and there was an immunoreactivity close to the STZ group (p > 0.05) (Table 1).
Although small islets of Langerhans were observed in the STZ + MSG group when compared to all groups, it was also found that the antiinsulin immunoreactivity was negligible when compared to the other groups (p < 0.05).It was shown that in addition to STZ, the application of MSG can be more destructive in pancreatic β cells.It is already known that only MSG causes histopathological changes in the pancreatic cell structure depending on the dose when given orally. [41]e normal histological structure is impaired in pancreatic sections, and islets of Langerhans with more irregular borders develop when compared to the control group.Hyperplasia and hypertrophy occurred in vacuolization in the cytoplasm of the islet cells, pycnotic nuclei, and necrotic areas between the cells along with congestion and dilatations in the blood vessels (Figure 4).All these changes in the pancreatic tissue occur because lipid peroxidation and oxidative stress occurring in biomembranes cause tissue damage, and therefore, cellular dysfunctions and degeneration occur. [40]ile reactive oxygen species (ROS) are involved in cell signaling and tissue homeostasis under normal physiological conditions, they cause lipid peroxidation and DNA damage under overproduction.
MDA is a lipid peroxidation product.The antioxidant system protects cells from oxidative stress by breaking down ROS.SOD converts superoxide to hydrogen peroxide and is then decomposed by catalase into molecular oxygen and water. [42]Oxidative stress and lipid peroxidation are vital for modern humans. [43]Free radicals make up the lipid peroxidation process in an organism.MDA is among the end products of polyunsaturated fatty acids peroxidation.Increased free radicals cause overproduction of MDA.MDA level is shown as a marker of oxidative stress. [44]It was found that MDA is increased in studies with high-dose MSG. [45]It was found in the present study that oral MSG application at a dose of 30 mg/kg increased the MDA levels in pancreatic tissue insignificantly when compared to the Control Group, but MDA increased in the diabetic group when compared to both the STZ group and the healthy control groups.
Also, orally administered MSG increased MDA in diabetic groups, although the GSH level decreased when compared to the control groups both in the MSG and STZ + MSG groups, [46] and SOD activity decreased in the treatment groups when compared to the healthy control groups. [46,47]tokines are pleiotropic polypeptides regulating inflammatory and immune responses.Low-grade inflammation is closely associated with the activation of the innate immune system, diabetes, and the pathogenesis of microvascular complications. [48]The secretion of proinflammatory cytokines is associated with the loss of pancreatic β-cell viability and cell death. [49]Cytokines such as TNF-α and IL-6 and inflammatory signaling molecules might contribute to insulin resistance.
It was shown that the oral administration of 30 mg/kg MSG for 28 days increases the TNF-α levels by causing inflammation. [50]It was determined that the TNF-α level increased in the STZ + MSG group when compared to the healthy control groups, although the IL-6 levels increased significantly in the STZ + MSG group when compared to the healthy control group and only the MSG group.It was also found that IL-1β, which is a proinflammatory cytokine, increased in serum samples of rats only in the MSG-treated group when compared to the healthy Control Groups, but also in the STZ + MSG group, but the increase in the STZ + MSG group was not significant when compared to the STZtreated group.Also, IL-10, which is an anti-inflammatory cytokine, increased in the MSG group when compared to the healthy control groups, and MSG alone can cause inflammation. [51]It was also determined that the change in IL-10 was not significant in the STZ + MSG group when compared to the STZ group.Since proinflammatory cytokines TNF-α, IL-1β, and IL-6 were increased in the MSG group, IL-10, which is an anti-inflammatory cytokine, was also increased. [52]In the pancreatic tissue sections, it was found that the damage was much more in the STZ + MSG group in both exocrine and endocrine pancreatic areas when compared to the groups that only received STZ and only MSG and that there were very intense lymphocyte cell infiltrations with degenerative areas causing cellular losses in the exocrine part.However, there were intense cell losses in the islets of Langerhans in the endocrine part along with a reduction in islet sizes, and an increase in the degeneration areas.

| CONCLUSION
As a result, it was determined that the maximum dose of 30 mg/kg of MSG, which is a food additive, causes inflammation both orally and increases MDA, which is a free radical product, in diabetic groups, decreases antioxidants, and causes damage to β cells in the pancreatic tissue histopathologically.Therefore, it is necessary to limit the use of MSG in both healthy individuals and people with diabetes or to prefer natural flavor enhancers as an alternative to this food additive.
Different letters in the same column represent statistical difference (p < 0.05).

F I G U R E 4
Crossman triple staining and immunohistochemical staining of anti-insülin in pancreas tissues.(A-E) Crossman triple stain; black arrow: islet cells with pycnotic nuclei and eosinophilic cytoplasm, white arrow: islet cells showing cytoplasmic vacuolization, v: dilated vascular structures, star: lymphocyte cell infiltration, ellipse: islet of Langerhans undergoing intense degeneration, and curved arrow: degenerative areas in the acini (×10).(F-J) Light microscopic images showing insulin immunoreactivity in pancreatic sections obtained from all groups.Insulin positive cells are seen stained dark brown (×10).MSG, monosodium glutamate; STZ, streptozotocin.