Effect of monosodium glutamate on body weight and the histological structure of the zona fasciculata of the adrenal cortex in young male albino rats

Nehal F El-Helbawy; Doaa A Radwan; Maysa F Salem; Manal E El-Sawaf

doi:10.4103/tmj.tmj_11_17

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ORIGINAL ARTICLE

Year : 2017 | Volume : 45 | Issue : 2 | Page : 104-113

Effect of monosodium glutamate on body weight and the histological structure of the zona fasciculata of the adrenal cortex in young male albino rats

Nehal F El-Helbawy, Doaa A Radwan MD, PhD , Maysa F Salem, Manal E El-Sawaf
Department of Anatomy and Embryology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission	11-Feb-2017
Date of Acceptance	28-Jun-2017
Date of Web Publication	13-Oct-2017

Correspondence Address:
Doaa A Radwan
Lecturer in Department of Anatomy and Embryology, Faculty of Medicine, Tanta University, 5 Hassan Kamel Street, Tanta
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/tmj.tmj_11_17

Abstract

Introduction
Monosodium glutamate (MSG) is commonly used as a flavor enhancer in both home and restaurant cooking.
Aim
The aim of this work was to clarify the possible effect of MSG administration on the body weight and the histological structure of the zona fasciculata (ZF) of the adrenal cortex in young male albino rats and to assess whether or not this effect was reversible.
Materials and methods
A total of 40 male albino rats aged 4 weeks were weighed at the onset of the study and then divided into two equal groups. The control rats (group I) were held without medication either for 2 weeks (group Ia) or for 4 weeks (group Ib) and then killed. The experimental rats (group II) received a daily oral dose of MSG (4 mg/g body weight) dissolved in 3 ml of distilled water for 2 weeks and then 10 of them were killed 2 h after the last dose (group IIa); the other 10 rats were held without medication for another 2 weeks and then killed (group IIb). All rats were weighed before scarification. Sections of the adrenal glands were examined with light and electron microscopes. Thickness of the ZF was measured in all subgroups. Data of the final body weight and ZF thickness were analyzed as mean±SD and P values were calculated.
Results
Statistically, the final body weight of rats increased in both groups IIa and IIb and also the thickness of the ZF increased in group IIa as compared with their controls. Histologically, group IIa showed loss of the cord-like architecture of ZF cells. They appeared distended with highly vacuolated cytoplasm that was ultrastructurally full of lipid droplets. Group IIb revealed partial improvement and retained normal thickness and structure of the ZF.
Conclusion
MSG causes histopathological changes in the ZF of the adrenal cortex, increasing its thickness and its secretion, and this could explain the increased final body weight. These changes are reversible but may need a long time to regain their normal values.

Keywords: monosodium glutamate, obesity, zona fasciculata

How to cite this article:
El-Helbawy NF, Radwan DA, Salem MF, El-Sawaf ME. Effect of monosodium glutamate on body weight and the histological structure of the zona fasciculata of the adrenal cortex in young male albino rats. Tanta Med J 2017;45:104-13

How to cite this URL:
El-Helbawy NF, Radwan DA, Salem MF, El-Sawaf ME. Effect of monosodium glutamate on body weight and the histological structure of the zona fasciculata of the adrenal cortex in young male albino rats. Tanta Med J [serial online] 2017 [cited 2023 May 31];45:104-13. Available from: http://www.tdj.eg.net/text.asp?2017/45/2/104/216686

Introduction

Food additives are substances added to the basic foodstuffs to introduce a special color or taste and attract consumers, especially children. Monosodium glutamate (MSG) is a food additive that is marketed in many countries with trade names such as Ajinomoto, Chinese salt, and E621. It is commonly used in many food products such as chips, noodles, and soups [1],[2].

Hermanussen et al. [3] reported that MSG provides its flavoring function through stimulation of the orosensory receptors by improving the palatability of meals. In an aqueous solution, MSG dissociates releasing free glutamate, which binds to taste receptors on taste cells in the oral cavity and activates taste nerves to elicit the unique umami taste, which differs from the four classic tastes of sweet, sour, salty, and bitter. This influences the appetite positively and thus increases the incidence of obesity [4],[5],[6].

MSG is the sodium salt of glutamic acid composed of white odorless crystals readily soluble in water. It is considered quite stable as it does not change in the appearance or quality during prolonged storage at room temperature. It does not decompose during food processing or cooking, but in acidic conditions and high temperatures it is partially dehydrated and converted into pyrrolidone-2-carboxylate [7],[8].

Freeman [9] reported that overconsumption of MSG in restaurants resulted in complex of symptoms termed ‘Chinese restaurant syndrome’, including numbness, weakness, flushing, sweating, dizziness, and headaches. In addition, many studies proved the damaging effect of excessive MSG use on multiple body organs, such as neurotoxic effects on the cerebellum [10], steatohepatitis, and preneoplastic changes in the liver [11]. Moreover, damage of pancreatic structure including both exocrine and endocrine cells [12] and varying degrees of cytoarchitectural distortion of the kidney [8] were reported with prolonged consumption of MSG.

Hawkins [13] reported that the blood–brain barrier is very effective in blocking the passive transport of glutamate into the central nervous system even when the levels of glutamate are elevated in the plasma. Some areas − for example, the hypothalamus − do not have an impermeable blood–brain barrier. Thus, free glutamic acid from food sources can enter into the hypothalamus and injure its neurons.

Seo et al. [14] reported that the responsiveness of the hypothalamic–pituitary–adrenal axis could be impaired by overstimulation of the hypothalamus and simultaneous accumulation of excessive glutamate due to chronic MSG administration. In rats, glutamate injection into the third ventricle elevated plasma adrenocorticotrophic hormone (ACTH) levels with a gradual increase in body weight. Therefore, this experimental study was designed to clarify the possible effect of MSG administration on both the body weight and the histological structure of the zona fasciculate (ZF) of the adrenal cortex in young male albino rats and assessed whether or not this effect was reversible.

Materials and methods

This experimental study was conducted on 40 young male albino rats aged 4 weeks and weighed 80–100 g. They were collected immediately after weaning and housed in clean, properly ventilated separate cages under similar environmental condition and fed on the same laboratory diet in the animal house of the Anatomy Department, Tanta University. Experiment was carried out according to guidelines of Committee for Research and Ethical Issues of Tanta University. The rats were divided into two groups as follows:

Group I (the control group): It consisted of 20 rats. They were kept without any medication and equally subdivided into two subgroups:
1. Group Ia: It consisted of 10 rats. Rats were killed after 2 weeks from the onset of this study.
2. Group Ib: It consisted of 10 rats. Rats were killed after 4 weeks from the onset of this study.
Group II (the experimental group): It consisted of 20 rats. Each rat received 4 mg of MSG per gram body weight dissolved in 3 ml of distilled water and given daily through orogastric tube for 2 weeks [15]. This group was equally subdivided into two subgroups:
1. Group IIa: It consisted of 10 rats. Rats were killed 2 h after the administration of the last dose of MSG (after 2 weeks from the onset of the study).
2. Group IIb: It consisted of 10 rats. Rats were held without any medication for another 2 weeks after the administration of the last dose of MSG and then killed (after 4 weeks from the onset of the study).

MSG was purchased from Sigma Chemical Product Co, Quesna, Monofeya, Egypt. It was in the form of white crystals.

Rats of all subgroups were weighed at the beginning of the experiment and just before scarification. At the appropriate time, the control and experimental rats were killed with a suitable dose of ether. A median xiphipubic laparotomy was performed, with deviation of the intestinal loops, opening of the retroperitoneal space, and careful dissection of the structures. Each kidney was exposed with the adrenal gland present on its upper pole. It was removed carefully using a blunt probe. Before removal, the bilateral adrenal glands were isolated from the surrounding visceral fat tissue [16],[17].

Histological examination

The specimens of the adrenal glands of different subgroups were divided into two halves.

One half was fixed in 10% formol saline for 24 h. Thereafter, the tissues were dehydrated in ascending grades of ethanol, cleared in xylene, and then embedded in paraffin blocks. Sections of 5 µm thickness were prepared using the microtome and stained with hematoxyline and eosin (H&E) stain to study the general histological features of the adrenal gland [18].

The second half was fixed in 2% phosphate-buffered gluteraldehyde, washed three times in PBS and fixed in 1% phosphate-buffered osmium tetroxide, and thereafter dehydrated in ascending grades of ethanol and embedded in epoxy resin. Ultrathin sections (40–50 nm in thickness) were obtained with a Leica ultratome (Ultracut UCT; Leica Microsystems GmbH, Vienna, Austria) and stained with uranyl acetate and lead citrate [19],[20]. Sections were examined and photographed with JEOL-100 JEM (Jeol, Tokyo, Japan) in the Electron Microscopic Unit of the Faculty of Medicine, Tanta University.

Morphometric study

The mean thickness of the ZF of the adrenal cortex was measured using ImageJ software (https://en.wikipedia.org/wiki/National_Institutes_of_Health) [21]. For different subgroups, 10 values were obtained from each slide at 200 magnifications ([Figure 1]).

Figure 1 A photomicrograph showing the measurement of the mean thickness of the zona fasciculata using Image J software. For each subgroup, 10 values were obtained from the slide at 200 magnification. ZF, zona fasciculate; ZG, zona glomerulosa; ZR, zona reticularis

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Statistical analysis

The numerical results of the final body weight of the rats at the time of scarification as well as the mean thickness of the ZF in all subgroups were expressed as the mean±SD. For multiple comparisons, the statistical significance was assessed using one-way analysis of variance followed by the Scheffe test to compare pairs of groups. The difference was considered significant when P value was 0.05 or less and highly significant if P value was 0.001 or less. If P value was more than 0.05, the difference was considered nonsignificant [22].

Results

Results of statistical analysis in different subgroups

Statistical analysis of the final body weight at time of scarification in different subgroups

initial body weights were approximated among the study subgroups (80–100 g). There was marked increase in the mean final body weight in group IIa (198.1 g), which was highly significant (P<0.001) than that in group Ia (117.6 g). In group IIb, after withdrawal of MSG for 2 weeks, the mean value of the final body weight (184.1 g) was still significantly higher (P<0.05) than that of its control group Ib (164.8 g). The current study revealed an increase in body weights in both MSG-treated subgroups compared with controls. However, the range of increase was less in group IIb compared with that in group IIa ([Table 1] and Histogram 1).

Table 1 Quantitative measurements of the final body weight of the rats at the time of scarification and their statistical comparison in different subgroups

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Statistical analysis of the thickness of the zona fasciculata of the adrenal cortex in different subgroups

The mean thickness of the ZF of the adrenal cortex in the control rats of group Ia was 45.663 μm. In rats of group IIa administrated MSG for 2 weeks, there was a highly significant increase (P<0.001) in mean thickness of the ZF (71.141 μm) compared with the control group Ia with the same age. This increase in the mean thickness of the ZF after MSG administration was decreased again after withdrawal of the drug for 2 weeks in group IIb to be 56.429 μm, which was nearly equal to the mean thickness of the ZF in its control group Ib with the same age (52.877 μm); there were no significant differences between groups Ib and IIb (P>0.05) ([Table 2] and Histogram 2).

Table 2 Quantitative measurements of the mean thickness of the zona fasciculata of the adrenal cortex and their statistical comparison in different subgroups

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Light microscopic study

In the present study, light microscopic examination of H&E-stained sections of the suprarenal gland of control rats killed either at 6 weeks of age (group Ia) or at 8 weeks of age (group Ib) revealed that the adrenal gland was formed of an outer cortex and an inner medulla and surrounded by a connective tissue capsule. Beneath the capsule, the cortex was divided into three zones; zona glomerulosa, ZF, and zona reticularis ([Figure 2]a and [Figure 2]c). The cells of ZF appeared large and polyhedral in shape containing large rounded nuclei. Their cytoplasm was acidophilic containing tiny vacuoles. Many blood sinusoids lined with the flat endothelial cells appeared separating the cellular cords ([Figure 2]b and [Figure 2]d).

Figure 2 Photomicrographs of sections in the adrenal glands of control rats from group Ia (a, b) and group Ib (c, d) showing the adrenal gland formed of outer cortex and inner medulla (M) and surrounded by connective tissue capsule (C). The cortex consists of the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR) (a, c, H&E ×200). The cells of ZF are arranged in cords (*) and appear large and polyhedral (arrows) containing large rounded nuclei and tiny cytoplasmic vacuoles (V). Blood sinusoids (S) lined with endothelial cells (E) separate the cellular cords (b, d, ×1000)

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In group IIa (rats were administered MSG for 2 weeks and were killed at 6 weeks of age), sections stained with H&E showed apparent increase in the thickness of the ZF and the medulla was not seen ([Figure 3]a) compared with that found in its control group Ia ([Figure 2]a). There was disorganization of the cells in the ZF with loss of the cord-like architecture. The cells appeared swollen and distended with highly vacuolated cytoplasm. Some cells showed karyolitic nuclei, whereas others lost their nuclei. Some cells partially lost their cellular membranes and a cytoplasmic syncetium was formed between them. The blood sinusoids appeared collapsed in-between the cells ([Figure 3]b).

Figure 3 (a, b) Adrenal glands from group IIa showing increased thickness of zona fasciculata ZF (↔) and the medulla is not seen (a, H&E, ×200). Its cells are disorganized and distended with highly vacuolated cytoplasm (V) and collapsed sinusoids (S) in-between. They show karyolitic (→) or lost (L) nuclei. A cytoplasmic syncetium (CS) is seen between the cells (b, ×1000). (c and d) The adrenal glands from group IIb showing normal thickness of ZF (↔) (c, ×200). Its cells appear in cords (*) with apparent sinusoids (S) in-between. They show rounded (N) or shrunken (→) nuclei and cytoplasmic vacuolations (►) (3, ×1000)

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Sections stained with H&E. of the withdrawal group IIb (rats were administered MSG for 2 weeks and were held without any medication for another 2 weeks and killed at 8 weeks of age) showing apparently normal thickness of the ZF ([Figure 3]c) compared with that of its control group Ib ([Figure 2]c). Higher magnifications showed apparently normal sized cells of the ZF with reappearance of the cord-like arrangement of cells. Some cells had normal, large, rounded nuclei. However, some cells had small shrunken nuclei and cytoplasmic vacuolations. The sinusoids were well-apparent lined with flat endothelial cells ([Figure 3]d).

Transmission electron microscopic study

Ultrastructural examination of the adrenal gland of control rats killed either at 6 weeks of age (group Ia) or at 8 weeks of age (group Ib) revealed that the cells of the ZF contained normal euchromatic nuclei and many mitochondria as well as lipid droplets of various densities were seen in the cytoplasm ([Figure 4]a and [Figure 4]c). Higher magnifications showed the characteristic rounded mitochondria with closely packed vesiculated cristae as well as the well-developed smooth endoplasmic reticulum were present ([Figure 4]b and [Figure 4]d).

Figure 4 Transmission electron micrographs of an ultrathin section in the adrenal gland of a control male albino rat either from group Ia (a, b) or group Ib (c, d) showing a zona fasciculata cell with a normal euchromatic nucleus (N) and many mitochondria (M) and lipid droplets (L) of various densities are seen in the cytoplasm (a, c, ×3000). Higher magnifications show the characteristic rounded mitochondria (M) with closely packed vesiculated cristae. In addition, a well-developed smooth endoplasmic reticulum (sER) is present (b, d, ×5000)

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In group IIa (rats were administered MSG for 2 weeks and killed at 6 weeks of age), ultrastructural examination of the ZF cells revealed shrunken nuclei surrounded by a perinuclear electron lucent zone in some cells ([Figure 5]a and [Figure 5]b). The cytoplasm showed numerous lipid droplets of variable size; some of them were full of secretory material and others exhibited marked depletion of secretory material. Furthermore, confluent lipid droplets could be noticed ([Figure 5]c). Higher magnifications of the cytoplasm showed dilated smooth endoplasmic reticulum and swollen mitochondria with disrupted cristae and vesicles inside ([Figure 5]d).

Figure 5 Transmission electron micrographs of ultrathin sections in adrenal cortex of a rat from group IIa showing a cell of zona fasciculata containing shrunken nucleus (N) surrounded by a perinuclear electron lucent zone (→) in some cells (a, b, ×3000). The cytoplasm shows numerous lipid droplets of variable size, some of them are full (L1) and others revealing marked depletion of secretory material (L2). Furthermore, confluent lipid droplets (star) can be seen (c, ×3000). The cytoplasm shows dilated smooth endoplasmic reticulum (sER) and swollen mitochondria (M) with disrupted cristae (d, ×5000)

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Ultrastructural examination of the cells of ZF of group IIb (rats were administered MSG for 2 weeks and were held without any medication for another 2 weeks and then killed at 8 weeks of age) showed partial restoration of the normal architecture of ZF cells. The cells contained apparently small euchromatic nucleus with indented nuclear membrane, and small perinuclear electron lucent zone could be seen in some cells ([Figure 6]a and [Figure 6]b). The cytoplasm contained normal amount of lipid droplets; however, some cytoplasmic vacuolation can be noticed ([Figure 6]a and [Figure 6]c). Higher magnification showed numerous apparently normal rounded mitochondria with closely packed vesiculated cristae. The smooth endoplasmic reticulum appeared slightly dilated ([Figure 6]d).

Figure 6 Transmission electron micrographs of ultrathin sections in the adrenal cortex of a rat from group IIb showing a cell of zona fasciculata containing apparently small euchromatic nucleus (N) with indented nuclear membrane (►) or small perinuclear electron lucent zone (→) (a, b, ×3000). The cytoplasm contains normal amount of lipid droplets (L); however, some cytoplasmic vacuolation (V) can be noticed (a, c, ×3000). Slightly dilated smooth endoplasmic reticulum (sER) and numerous apparently normal rounded mitochondria (M) with closely packed vesiculated cristae appear in the cytoplasm (d, ×5000)

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Discussion

Obesity is a serious metabolic disorder whose prevalence has increased in epidemic proportions around the world. It has long been recognized as an independent risk factor for various comorbidities such as type 2 diabetes mellitus, dyslipidemias, hypertension, stroke, and cardiovascular disease [23].

In the present study, the effects of MSG evaluated after a period of 14 days’ treatment in young rats showed a statistically highly significant increase (P<0.001) in the mean body weight of rats compared with controls. Obesity without hyperphagia and stunted growth after MSG treatment was also a common outcome of previous studies [3],[24].

Magarinos et al. [25] and Macho et al. [26] attributed the MSG-induced obesity to salt and water retention as a result of increased plasma cortisol level in MSG-treated rats, which resulted in Cushing’s obesity. Hermanussen et al. [3] added that MSG-induced obesity may also be due to elevated levels of glucagon and insulin levels, leading to a feeling of hunger and therefore overeating.

Furthermore, Hawkins [13] and Banks [27] reported that the hypothalamus does not have an impermeable blood–brain barrier. Thus, free glutamic acid from food sources can get into the hypothalamus, injuring and frequently killing its neurons. They demonstrated that MSG treatment destroys growth hormone-releasing hormone neurons within the hypothalamic arcuate nucleus in neonatal mice, and thus decreases serum growth hormone concentration and retards linear growth.

In addition, Cekic et al. [28] and Matyskova et al. [29] found that obesity induced after administration of MSG in newborn mice was of rapid onset due to lesions in hypothalamic arcuate nucleus and impaired leptin and insulin signaling in this region. Leptin is an appetite-suppressing hormone that regulates energy and controls appetite and body weight. In MSG-treated rats much adipose tissue was located mainly within the greater omentum and behind the peritoneum [30].

In contrast to our results, Miskowiak and Partyka [31] stated that there was no difference in body weight between control and MSG-treated rats in the course of the experiment. Furthermore, Merrett [32] found that the animals that ingested MSG were reported to have significantly less weight gain and reduced abdominal fat mass compared with animals that ingested only water. Moreover, lower plasma leptin levels were observed. However, Abd El-Aziz et al. [33] showed that prolonged administration of MSG causes an initial increase in weight gain followed by terminal suppression, despite increased food consumption. This could be explained by the inefficient food digestion and absorption due to gastric mucosal.

In this work, statistical analysis of the recovery group (group IIb) that stopped MSG treatment for 14 days before scarification revealed a highly significant increase as compared with their control group (P<0.001) in the mean body weight of rat. However, the range of weight gain in this group was less than that seen in group IIa denoting partial regression of weight gain on stoppage of MSG administration.

These results are in agreement with Nosseir et al. [34], who reported that through stimulation of the orosensory receptors, MSG influenced the appetite positively and induced weight gain. Nosseir et al. [34] also added that there was a significant reduction in the body weight after a recovery period of 6 weeks cessation of MSG indicating that the effect of MSG administration is temporary.

Many authors demonstrated the effects of MSG administration on the adrenal gland, and some of them attributed the increase in weight gain to its effect on its function [14],[26],[28]. Thus, this work had studied the possible effect of MSG administration on the histological structure of the ZF of the adrenal cortex of young male albino rats and assessed whether or not this effect was reversible after cessation of MSG administration.

In the present study, histological examination of the adrenal cortex in rats treated with MSG for 2 weeks revealed obvious changes indicating increased activity of the gland. H&E-stained sections of the adrenal cortex showed an increase in the thickness of the ZF as compared with the control group. There was disorganization of the cells in the ZF with loss of the cord-like architecture. The cells appeared swollen and distended with highly vacuolated cytoplasm with collapsed sinusoids in-between. Some cells showed karyolitic nuclei, whereas others lost their nuclei. Some cells partially lost their cellular membranes and a cytoplasmic syncetium was formed between them.

These results are in agreement with Cekic et al. [28] and Bojanovic et al. [35], who reported that, besides Cushing’s obesity observed in MSG-treated rats, the most important morphological finding was hypertrophy of both adrenal glands compared with controls. They reported that the cortex was widened and composed predominantly of fasciculata cells with large cells and abundant intracytoplasmic lipid droplets showing microvesicular pattern.

Kovacs et al. [36] found that the hypothalamic neurotoxic effect of MSG occurs not only on growth hormone-releasing hormone but also activates ACTH secretion. In addition, Bojanic et al. [37] reported that hyperplasia of the adrenal cortex can be attributed to hypersecretion of ACTH from the basophilic pituitary cells in the rats treated with MSG.

Furthermore, Baratta et al. [38] stated that both corticosterone and leptin plasma levels are significantly increased in adult MSG-treated rats with a permanent hypothalamic nuclei lesions in these animals.

In this study, the ultrastructural examination of the adrenal cortex in rats that received MSG for 2 weeks confirmed the light microscopic results and revealed marked degenerative changes including shrunken irregular nuclei surrounded by a perinuclear electron lucent zone in some cells. The cytoplasm contained many lipid droplets; most of them showed marked depletion of secretory material, whereas others were full of secretory material. Furthermore, confluent lipid droplets could be noticed. The mitochondria were markedly swollen and some of them showed vesicles inside with destruction of their cristae. Marked dilatation of the smooth endoplasmic reticulum leading to cytoplasmic vacuolation was also seen.

These results go hand in hand with Szabo et al. [39] and Mazroa and Asker [40], who explained that the increase in ACTH resulted in a significant increase in smooth endoplasmic reticulum and mitochondria of the ZF cells. Moreover, the increased lipid droplets indicated increased hormone formation, and the secretory depletion indicated increased secretion with consequent increase in plasma cortisol level.

Djordjevic et al. [41] claimed that ACTH depleted the lipid content in the cells of ZF. They also stated that apparent increase in the number of lipid droplets, swollen mitochondria, and dilated smooth endoplasmic reticulum contained enzymes involved in steroid synthesis and may refer to the high activity in the process of steroidogenesis. They added that lipid droplets are the intracellular stores of cholesterol esters, which are the obligate precursors of steroid hormone.

In this study, there was a significant improvement in the histological and ultrastructural structure of the ZF cells of the adrenal cortex after cessation of treatment with MSG for 2 weeks and the normal structure of the adrenal cortex was retained. In agreement with these results, Nosseir et al. [34] reported that the damaging effect caused by MSG treatment was reversible. In contrary to these findings, Aloa et al. [42] found that withdrawalgroups showed more histologically observed degenerative changes compared with the groups in which the treated rats were immediately sacrificed.

Thus, the major findings in the current study were the increased thickness and secretions of the ZF cells, which may explain the significant increase in body weight in MSG-treated rats. These findings were partially improved on cessation of MSG for 2 weeks before scarification.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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Figures

[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

Tables

[Table 1], [Table 2]

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