Gum Arabic Modifies Glucocorticoid Metabolic Enzymes Gene Associated with Decreased Plasma Corticosterone Levels in Mice

various ABSTRACT Glucocorticoids (GCs) are a class of stress hormones that play a large number of biological actions in the body. The levels of GCs regulated by the intracellular enzyme 11 β -hydroxysteroid dehydrogenases (11 β -HSD 1 & 2) and are associated with the pathogenesis of metabolic syndrome. Here we aimed to investigate effect of Gum Arabic (GA) on GCs plasma corticosterone concentrations and its metabolic enzymes gene expression in mice. In the present study, 40 female CD-1 mice of 90 days old were randomly divided into two groups (20 of each group). Control group and GA group offered GA in the form of drink (10% w/v) for 15 weeks. GA in drinking water significantly (P<0.05) decreased food intake, body weight associated with reduction of visceral adipose tissue. GA supplementation significantly (P<0.01) decreased blood glucose, total cholesterol, and very low-density lipoprotein (VLDL) whereas increased high-density lipoprotein (HDL) concentrations compared to the control. However, supplementation of GA did not change triglycerides or very LDL. Interestingly, GA significantly (P<0.05) decreased plasma corticosterone associated with downregulation of hepatic 11 β -HSD1 and glucocorticoid receptor (GR) mRNA expression compared to the control. Conversely, the treatment of GA increased hepatic 11 β -HSD2 mRNA expression. In addition, the treatment of GA significantly (P<0.05) decreased muscular 11 β -HSD1 compared to the control. No changes were observed in muscular GR and mineralocorticoid receptor. In conclusion, GA may enhance metabolic disorders through modification of hepatic 11 β -HSDs mRNA expression which may ameliorate metabolic disorder complications. Further studies required to elucidate the molecular mechanisms of GA on GCs.

tissues in the body [5], regulating various physiological processes [6] including stress response [7], immune response [8], energy metabolism [9], cell proliferation [10], skeletal muscle growth [11], and reproduction [12]. GCs exert their action through the glucocorticoid receptor (GR) [13], which is a transcription factor that belongs to the superfamily of nuclear hormone receptors [14]. GR acts via different mechanisms [15,16], one of the major mechanisms is transcriptional regulation of its primary target genes through genomic glucocorticoid response elements thereby directly binding to DNA or tethering onto other DNA-binding transcription factors [17]. Their secretion is mainly regulated by the hypothalamic-pituitary-adrenal axis [18].
Deregulation of HSD2 enzyme activity has been associated with a number of metabolic diseases [38]. In addition, inhibition of 11βHSD type 2 has been obviously shown to induce a congenital or acquired syndrome of mineralocorticoid exposure [27], thus contribute to essential hypertension [30].

Animals and Experimental Design
Forty female albino laboratory mice (age, 90 days) old were obtained from Sudanese National Research Center and housed in 8 plastic cages (each containing 5 mice) in a room kept at 25 C with a 12-h light and dark cycle. The animals were allowed to access freely to a commercial pelleted diet for the adaptation and drinking water throughout the experiment at least for one weekday. After 7 days of adaptation, the animals were divided into two groups of 20 mice in each group. Control group and GA group. GA group was provided drinking water containing GA whereas; the control group was given tape water. These mice received 0.5% of GA aqueous solution as drinking water for 7 days to adapt GA, and then 10% solution for a further 15 weeks consecutively. The control group was remained on the same drinking water as in the acclimatization. Body weight and food consumption were recorded throughout the period of the experiment. On day 105, the mice were killed. Liver and visceral adipose tissues were dissected and weighed. The tissue and blood samples were collected and stored at-80°C.

Blood Lipid Profile Biochemistry
Plasma lipids biochemistry including total cholesterol, triglycerides, HDL, VLDL, and LDL were determined using commercially assay kits according to the manufacturers' instructions (Nanjing Jiancheng Bioengineering Company, Nanjing, China).

Blood Glucose Measurement
Plasma samples were obtained from blood through centrifugation (2,400 rpm, 20 min, and 3.5°C) and then stored at -20 °C until samples analyzed. Blood glucose concentrations were measured using assay kits according to the manufacturers' instructions. (Nanjing Jiancheng Bioengineering Company, Nanjing, China).

Plasma Corticosterone Measurement
After decapitation of mice, blood samples were obtained from the ruptured cervical blood vessels in heparinized tubes for corticosterone (CORT). The plasma samples were prepared after centrifugation (2,400 rpm, 20 min, 3.5°C) in a refrigerated device and frozen at -20°C until the measurement of the hormone.
Plasma CORT levels were measured using radioimmunoassay method (RIA), using the CORT commercial kit according to the manufacturers' instructions (Biochem Immuno System). All plasma samples were dosed in the same assay, to avoid inter-assay errors.
The lower detection limit for CORT was 0.064 ng/mL, with a 4% intra-assay error.

Real-time PCR and Gene Expression
About 100 mg of liver and muscle were ground in liquid N2, and a portion of about 50 to 100 mg were used for extraction of RNA using TRIzol total RNA kit (Invitrogen, Biotechnology Co, Ltd, Carlsbad, CA, USA) according to the manufacturer's instruction. Two approaches were taken to ensure that all the total RNA preparations are free of genomic DNA contamination. Firstly, total RNAs were treated with 10 U DNase I (Rnase Free, D2215, Takara, Japan) for 30 min at 37°C, and purified according to the manufacturer's protocol.

Statistical Analysis
Descriptive statistics analysis was performed to check the normality and homogeneity of variances before using parametric analyses. Body weight, food intake, organs weight, blood lipids profile, CORT, as well as the relative quantitative data of gene expression were analyzed by one-way ANOVA using SPSS 21.0 for Windows, followed by a least-significant difference (LSD) test for individual comparisons. A P-value ≤0.05 was considered significant.

Weight, and Liver Weight
In the present study, the supplementation of GA significantly   (Table 1). No changes were observed both in triglycerides or VLDL concentrations regarding GA administration.

Plasma Corticosterone and Blood Glucose Concentrations
In the current study, the treatment of GA significantly (P<0.05) decreased plasma CORT concentrations when compared to the control group (Figure 2A). In addition, the administration of GA significantly (P<0.05) decreased blood glucose compared to the control group (Table 1).

Hepatic and Muscle GR and MR mRNA Expression
In the present study, the treatment of GA significantly (P<0.05) decreased hepatic GR ( Figure 3C) mRNA expression when compared to the control group. However, no significant differences observed in hepatic MR ( Figure 3C) mRNA expression compared to the control group. Likewise, no significant differences were observed both in muscular GR and MR mRNA expression regarding to the treatment of GA ( Figure 3D).

Discussion
There are several reports confirming the association between The reduction effect of GA on food intake and body weight may be due to the fact that several studies confirmed that the dietary fiber have bulk properties [56] and viscosity [57] therefore, it promoting satiety [58], satiation [59,60] and lowering body weight [61].
In addition, intake of dietary fiber associated with increases in satiety and decreased blood glucose levels [61]. Moreover, it was confirmed that supplementation of GA inhibited glucose absorption in intestine through interference of membrane abundance of sodium-glucose transporter 1 in mice [62]. Abdominal obesity is considered the most common signs of metabolic syndrome [63].
As a result, metabolic syndrome is a fatal consequence of visceral obesity [64]. Here we reported that supplementation of GA decreased visceral adipose tissue (VAT) accumulation associated with reduction in blood cholesterol, and VLDL. However, the treatment of GA increased HDL concentrations. Our findings are consistent with previous reports that dietary fibre decreased adiposity [65], lowered blood cholesterol [66] and blood VLDL levels [67]. A variety of mechanisms have been suggested to elucidate the hypercholesterolemic effect of GA [68,69]. Some reports suggested that the viscosity of fermentable dietary fibers in GA contribute significantly to lipid lowering action [65,70]. In addition, mechanism it was found that administration of GA increased fecal bile acid excretion together with decreased medication in lipid digestion and absorption [71].
Glucocorticoids (GCs) play a vital role in a wide array of physiological processes in the body. The 11β-hydroxysteroid dehydrogenases (11β-HSDs) enzyme catalyze interconversion of intracellular GC in liver [72]. Deregulations of both 11β-HSD1 and 11β-HSD2 have been associated with several types of metabolic disorders [73]. In the present study, the supplementation of and blood lipid profile, thus may attenuate atherosclerosis [74].
Downregulation of muscular 11β-HSD1 within the muscle may also protect against the unfavorable effects of local inflammation [75]. However, the mechanism through which GA downregulates 11β-HSD1 mRNA is unclear the.
Therefore, additional studies are essential to disclose such effects. 11β-HSD2 plays a crucial role in the prevention of inappropriate activation of the mineralocorticoid receptor (MR) from improper activation via GCs by inactivating GCs in mineralocorticoid target tissues [76]. In the present study, the treatment of GA upregulated hepatic 11β-HSD2 mRNA expression.
Chronic exposure elevated levels of GCs cause metabolic disorders such as insulin resistance [78] elevation of fasting glucose [79] and development of type 2 diabetes mellitus [80]. Here we reported for the first time that the administration of GA decreased GR mRNA expression in the liver but not muscle. Downregulation of hepatic GR mRNA could therefore decrease the development of metabolic risk factors [81] such as obesity [82], cardiovascular diseases [83], and diabetes [84].

Conclusion
We concluded that the supplementation of GA reduced food intake, body weight, VAT, plasma lipids profile and plasma CORT concentration which were associated with modification of hepatic