Black yeast beta glucan for insulin Resistance Prevention through IL-33, ST2 and Leptin Level: An In vivo Study of an Obesity model using Sprague dawley Rats

Ifana Anugraheni¹, Sri Andarini², Dian Handayani³, Titin Andri Wihastuti⁴*

¹Medical Sciences Doctoral Program, Faculty of Medicine, University of Brawijaya Malang.

²Departement of Public Health, Faculty of Medicine, University of Brawijaya Malang.

³School of Nutrition, Faculty of Medicine, University of Brawijaya Malang.

⁴School of Nursing, Faculty of Medicine, University of Brawijaya.

*Corresponding Author E-mail: titinwihastuti@gmail.com

ABSTRACT:

Obesity is defined as a condition of excessive fat accumulation in adipose tissue. Adipose hypertrophy stimulates the production of pro-inflammatory cytokines and recruitment and polarization of M1 macrophages. To maintain homeostasis, there is an increase IL-33 expression and a decrease ST2s in obesity. IL-33 expression correlates with leptin expression in adipose tissue. Black yeast beta glucan decreases the pro-inflammatory modulator and increases anti-inflammatory factors, as well as decreases adipose macrophage tissue infiltration levels. This study aims to prove the effect of black yeast beta glucans on IL-33, ST2 and leptin levels. This research design is true experimental with post test only group design. The experimental animals used were male Sprague Dawley strain rats. Rats were divided into 5 (five) groups (normal feed, high fat feed and 3 high fat feed groups with black yeast beta glucan doses of 0.135grams, 0.270grams and 0.540grams). Elisa method is used to analyze IL-33, ST2 and Leptin levels. Statistical analysis used two way ANOVA and continued with the least significant difference test (LSD). There was an increase in IL-33 and leptin levels, and a decrease in ST2 levels. There was no difference in the effect of black yeast beta glucans on IL-33, ST2 and leptin levels. However, with further statistical tests, there is a difference in the effect of black yeast beta glucan on IL-33 and Leptin levels at a dose of 0.270grams. The conclusion of this study is black yeast beta glucan can increase IL-33 and leptin levels at a dose of 0.270grams.

KEYWORDS: Black yeast, beta glucans, insulin resistance, obesity.

INTRODUCTION:

Obesity has reached epidemic proportions globally, with more than 1 billion adults overweight - at least 300 million of them clinically obese - and are a major contributor to the global burden of chronic illness and disability. In 2018, the proportion of overweight in adults ≥18 years in Indonesia is 13.6%, while obesity is 21.8%. In people with obesity occurs adipose cell hypertrophy. The existence of large adipose cells can recruit new cells that can trigger adipocyte hyperplasia^1–4.

Excess energy causes expansion of adipocytes and infiltration of inflammatory cells into adipose tissue. This event is associated with the generation of Reactive Oxygen Species (ROS) and nuclear factor κB (NFκB) transactivation. Reactive oxygen species (ROS) is a component of oxygen-derived molecules that, in the case of excessive activity, is considered to be harmful oxidant. Some of these radical forms are free^5,6. It includes molecular oxygen, since ground-state oxygen has two unpaired electrons and cause oxidative stress.^7–9. ROS is a key modulator that activates adipose tissue inflammation resulting in pro-inflammatory cytokine production and recruitment and polarization of M1 macrophages.

Physiologically, ROS acts as a regulator in various defense mechanisms, differentiation, proliferation and migration of body cells. Interleukin (IL) - 33 is a member of the newly identified IL-1 cytokine family that induces Th2 cytokine production in adipose tissue and macrophage polarization against alternative macrophages (M2). Interleukin (IL) - 33 acts through the ST2 receptor (Suppression of Tumorigenicity) and is involved in the pathogenesis of inflammatory disorders. Based on the results of research that has been done, the expression of IL-33 and ST2 increases significantly in omental and subcutaneous adipose tissue of people with severe obesity and in diet-induced obese rats. IL-33 expression is highly correlated with leptin expression in human adipose tissue^10–14. Leptin is an adipose tissue hormone that helps in regulating energy intake and energy expenditure, including appetite and metabolism ^15,16. It is a hormone that made by fat cells which regulates the amount of fat stored in the body.^17,18

Beta glucans are the most common polysaccharides found in bacterial and fungal cell walls, in the form of soluble or temporary water soluble. Beta glucans which are secreted by yeast are important sources of beta glucans for potential medical use due to the fact that they can be recovered and purified more easily. Yeast beta glucans reduce the pro-inflammatory modulator TNF-α, IL-6, IL-1β, CCL2, and SAA3, and increase the anti-inflammatory factors in Azgp1 (2.53±0.02-fold changes) at the protein and/or mRNA. Yeast beta glucans also reduced the level of infiltration of adipose macrophage tissue by 82.5±8.3%, especially the newly recruited adipose macrophage tissue. In addition, yeast beta glucans also increase Th2 GATA3 regulator cells (7.72±0.04 fold) and decrease IL-10 and IL-1ra immunosuppressors, suggesting that yeast beta glucans cause inflammation inhibition through stimulation of the immune response.^19–22

Black yeast beta glucans 1.3-1.6 are included in the dissolved glucan category. Previous research has shown something noteworthy namely the stimulatory effect of this compound on IL-8 production by peripheral mononuclear cells. Black yeast, Aureobasidium pullulan produces extracellular beta glucans (1,3), (1,6) under certain conditions. Beta glucans produced by A. Pullulan are known as dietary fiber and also immune stimulators, and are believed to have beneficial effects on health through their function as dietary fiber and immune stimulation activity. Beta glucans (1 → 3), (1 → 6) from A. pullulan are produced in a water-soluble form as a viscous liquid, and are ready to be used as food additives, without extraction. It also has almost the same effectiveness with the type of glucans produced by other organisms.^19–22

MATERIAL AND METHODS:

Research Design:

The research design used in this study is true experimental with a post test only control group design approach.

Experimental Animal:

The study was conducted for 12 weeks. The experimental animals used were male Sprague Dawley strain rats aged 3 (three) months with a body weight of 175-250grams, agile, soft, clean, dense, shiny, not fall and smooth-haired, open eyes, and reddish-white skin taken from Laboratorium Penelitian dan Pengujian Terpadu Universitas Gadjah Mada. The sample consisted of 30 heads divided into 5groups (normal feed, high fat feed and 3 groups of high fat feed with black yeast beta glucan doses of 0.135grams, 0.270grams and 0.540 grams) with a total sample of 6 tails each group.

Black Yeast Glucan and Beta Glucans:

Composition of ingredients, energy content and nutrients of high fat feed based on AIN-93G standard modified by ad libithum administration (composition: 20.51% KH; 57.57% fat and 21.90% protein) as much as 20grams/ day.

Black yeast beta glucan is obtained from Badan Pengkajian dan Penerapan Teknologi (BPPT), where 1 sachet is taking as much as 15grams of beta glucan gel. Black yeast beta glucans are given per sonde.

Parameter Measurement:

Measurement of IL-33, ST2 and leptin levels using the Elisa kit brand Bioassay Technology Laboratory, with catalog no. E0938Ra for IL-33, E3305Ra for ST2, and E0561Ra for leptin. The measurement procedure is in accordance with the protocol that the manufacturer has recommended.

Statistic Analysis:

Two way anova was used in this study to determine the effect of black yeast beta glucan on IL-33, ST2 and leptin levels in sprague dawley rats. Then a post hoc test is performed to identify differences between groups. Statistical Package Software for Social Sciences (SPSS) version 20 (IBM Corporation, 590 Madison Avenue, New York, USA) was used to obtain data.

IL-33 levels in all treatment groups ranged from 0.824 ng/ml to 4.157ng/ml. N group had the lowest IL-33 level (0.824 - 3.150ng/ml). Whereas the highest IL-33 level in the HF2 group (1,850 - 4,157ng/ml). ANOVA test with a 95% confidence level showed that administration of black yeast beta glucan did not have a significant effect (P = 0.160) to increase IL-33 levels. Post hoc test using the Least Significance Different (LSD) method showed that IL-33 levels between the normal feed group and the high-fat feed + black yeast beta glucan were significantly different.

ST2 levels in all treatment groups ranged from 12.77ng/ ml to 24.58ng/ml. The HF2 group had the lowest ST2 levels (12.77 - 19.58ng/ml). While the highest ST2 levels in the HF3 group (14.42 - 21.42ng/ml). ANOVA test with a 95% confidence level showed that administration of black yeast beta glucan did not have a significant effect (P = 0.509) to increase ST2 levels. Post hoc test with Least Significance Different (LSD) method showed that ST2 levels between groups did not differ significantly.

Leptin levels in all treatment groups ranged from 0.256 ng/ml to 0.493ng/ml. The HF-group had the lowest leptin levels (0.274 - 0.329ng/ml). While the highest levels of leptin in the HF2 group (0.267 - 0.493ng/ml). ANOVA test with a 95% confidence level showed that administration of black yeast beta glucan did not have a significant effect (P = 0.160) to increase leptin levels. Post hoc test using the Least Significance Different (LSD) method showed that the level of leptin between the normal feed group and the high-fat feed + beta-glucan black yeast glucan was significantly different.

DISCUSSION:

In this journal we describe research that shows that black yeast beta glucans can prevent insulin resistance through changes in IL-33, ST2 and leptin levels. Based on statistical tests that have been done, the results show that administration of black yeast beta glucans does not affect the results of IL-33, ST2 and Leptin levels after being treated for 12 weeks. However, with continued statistical tests using the smallest significant difference test, the results obtained that beta glucan black yeast at a dose of 0.270 grams affect IL-33 and Leptin levels.

Adiposity is associated with an increased risk of metabolic diseases such as insulin resistance and type 2 diabetes mellitus. Some disorders are related to insulin resistance, including a reduction in NO bioavailability, increased ROS production, and a decrease in intracellular signaling pathways. However, some individuals with increased adiposity still have a healthy metabolic system. It was shown that the IL-33/ST2 axis plays a protective role against obesity and/or insulin resistance and type 2 diabetes mellitus in animal models. In addition, there is evidence to suggest that IL-33 circulation can have a metabolic protective effect in humans, especially in individuals of normal weight and normal blood sugar but not with obesity and/or type 2 diabetes mellitus. However, it is not known whether the metabolic effect IL-33 in adipose tissue differs between individuals with normoglycemia, prediabetes, and type 2 diabetes mellitus^14,23

Severe obesity in humans is associated with a roughly threefold increase in IL-33 expression in omental and subcutaneous adipose tissue compared to the control group. Immunoblotting confirms the presence of IL-33 at the protein level, and its increase in human fat adipose tissue which is very fat. The serum concentration of IL-33 does not change in obesity¹²

It is known that obesity is associated with a mild state of chronic inflammation that leads to ongoing activation of the innate immune system. Recruitment of immune cells, specifically macrophages, is a key feature in inflammation caused by obesity. Although macrophage phenotypes in adipose tissue are highly dependent on the microenvironment, macrophages can be divided into two main pheno types generally: alternative activated macrophages (M2) that are metabolically protective and classically activated macrophages (M1) which are proinflammatory. However, macrophages can have a mixed state that does not fall strictly under the M1 / M2 phenotype, and thus, the phenotype may not adequately capture the inflammatory state of adipose tissue²³

Beta glucan preparations originating from different sources can bind to several PRRs, the main signaling beta glucan (1,3) (1,6) which is purified through the dectin-1 receptor, which is expressed on dendritic cells, macrophages and neutrophils. The innate immune response is mediated through a number of PRRs, especially such as Toll-like receptors (TLR) and C-type lectin receptors (CLR). Dectin-1, CLR, plays an important role in the recognition of fungi by binding to beta-glucan-like polysaccharides on their cell walls. These receptors have been shown to work together with TLR in inducing a balance of innate pro and anti-inflammatory immune responses through Antigen Presenting Cells (APC). TLR ligand activity results in improved Treg function and protection of rats from diabetes. TLR2 and dectin-1 bonds produce IL-33 cytokine expression (along with a decrease in its receptor, ST2) and suppress proinflammatory cytokines. This condition will cause improvements in adipose cells, so adipose cells send impulses to the hypothalamus to increase leptin production. High levels of leptin will signal to the central nervous system that there are enough energy stores in the body, so this situation will provide a response back to reduce food intake and increase energy use.

CONCLUSION:

Obesity is characterized as a chronic state of mild inflammation with infiltration of progressive immune cells into adipose tissue. The administration of black yeast beta glucan bound to dectin-1 and in cooperation with TLR induces innate pro and anti-inflammatory immune responses balances through Antigen Presenting Cells (APC). TLR2 and dectin-1 produce IL-33 cytokine expression (along with a decrease in its receptor, ST2) and suppress proinflammatory cytokines. This condition will cause improvements in adipose cells, so adipose cells send impulses to the hypothalamus to increase leptin production

ACKNOWLEDGEMENT:

We acknowledge the Laboratorium Penelitian dan Pengujian Terpadu Universitas Gadjah Mada for preparing the experimental animals.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 30.01.2020 Modified on 19.03.2020

Research J. Pharm. and Tech. 2020; 13(12):6077-6080.

DOI: 10.5958/0974-360X.2020.01059.8

Variable						p
Variable	N (ng/ml) ± SD	HF- (ng/ml) ± SD	HF1 (ng/ml) ± SD	HF2 (ng/ml) ± SD	HF3 (ng/ml) ± SD	p
IL-33	1,67 ± 0,96	2,50 ± 0,66	1,95 ± 0,5	2,84 ± 0,9	2,3 ± 0,75	0,160
ST2	16,49 ± 1,58	16,68 ± 1,39	18, 36 ± 3,88	16,33 ± 2,28	18,42 ± 2,98	0,509
Leptin	0,306 ± 0,176	0,299 ± 0,024	0,336 ± 0,505	0,390 ± 0,087	0,338 ± 0,065	0,103