Beneficial effects of 1,3–1,6 β-glucans produced by Aureobasidium pullulans on non-esterified fatty acid levels in diabetic KKAy mice and their potential implications in metabolic dysregulation

Abstract

Objectives

In this study, we used an obese and diabetic mouse model to compare two strains of Aureobasidium pullulans (AFO-202 and N-163) produced beta-glucans (β-glucans), which alleviate lipotoxicity.

Methods

Four groups of KK-Ay mice were used, with six subjects in each group. Group 1: sacrificed on day 0 for baseline values; Group 2: control (drinking water); Group 3: AFO-202 beta glucan—200 mg/kg/day; Group 4: N-163 beta glucan—300 mg/kg/day for 28 consecutive days.

Results

Group 4 (N-163) had the lowest non-esterified fatty acids (NEFA) levels and marginally decreased triglyceride levels compared to the other groups. There were no significant differences in blood glucose, hemoglobin A1c (HbA1c), triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol levels. N-163 β-glucans decreased NEFA levels after 28 days.

Conclusion

These results, although modest, warrant further in-depth research into lipotoxicity and associated inflammatory cascades in both healthy and diseased subjects for the prevention and management of metabolic dysregulation and associated diseases such as non-alcoholic fatty liver disease (NAFLD).

Introduction

Fatty acids are a major component of lipids and an important source of fat fuel for the body. Non-esterified fatty acids (NEFA), also called free fatty acids (FFA) are the circulating form of fatty acids in the plasma [1]. FFA is an important link between obesity, insulin resistance, inflammation, and the development of diabetes, hypertension, dyslipidaemia, coagulation disorders, and cardiac diseases [2]. Lipotoxicity due to the destructive effects of excess fat accumulation impairs the function of several metabolic pathways [3]. In particular, chronic excessive level of plasma FFA leads to insulin resistance and influences the synthesis of hepatic triglycerides. Hepatic fatty acid metabolism, which is closely linked to inflammation, leads to hepatic steatosis (non-alcoholic steatohepatitis (NASH)), which progresses to fatty liver disease, cirrhosis, and cancer [3]. Therefore, modulating and normalising NEFA is a key target to alleviate the effects of the entire cascade of lipotoxicity [4] and the dysregulation of lipid metabolism described above. Both statin and non-statin pharmacological agents have been studied for their effects on NEFA with limited outcomes [5].

Beta-glucans (β-glucans) are polysaccharides with many beneficial therapeutic effects, including ameliorating glucose metabolic disorders such as diabetes and dyslipidaemia [6], and enhancing immunity to fight viral infections and cancer [7]. The effects of 1,3–1,6 β-glucan derived from the AFO-202 strain of the black yeast Aureobasidium pullulans (Fig. 1) in individuals with type II diabetes [8] and dyslipidaemia have been reported [9]. N-163 is another strain of A. pullulans which produces a novel variant of 1,3–1,6 β-glucan (Fig. 2). In vitro studies of N-163 β-glucan have shown the positive influence of N-163 β-glucan on decreasing inflammatory cytokines [10]. In this study, we evaluated the effects of β-glucans derived from AFO-202 and N-163 A. pullulans in KKAy mice, an obese diabetic mouse model. KKAy mice are used as animal models for research on obesity and metabolic disorders, and were originally developed by crossing diabetic KK mice with yellow obese mice (A^y mice) [11].

Fig. 1.

Structure of β-glucan derived from the AFO-202 strain with the chemical formula (C₆H₁₀O₅)_n

Fig. 2.

Structure of β-glucan derived from N-163 with the chemical formula (C₆H₁₀O₅)_n

Materials and methods

AFO-202 and N-163 beta-glucans

A. pullulans is a harmless, naturally occurring black yeast that was originally isolated from the soil. Common cultures based on potato dextrose agar (PDA) and potato dextrose broth (PDB) were used for the initial culture in the lab, which were later scaled up for large cultures using each specific medium so that the AFO-202 and N-163 strains would produce β-glucan as an exopolysaccharide with a chemical structure of (C₆H₁₀O₅)_n. For the AFO-202 culture, rice bran was used as a nitrogen source along with a natural medium consisting primarily of rice bran, ascorbic acid, and glucose. The N-163 strain was cultured with nitrate instead of rice bran and a synthetic medium consisting of thiamine nitrate, yeast extract, and glucose. The storage and incubation temperatures for both the strains were approximately 25 °C. The incubation period was five days for the N-163 strain and six days for the AFO-202 strain. The product, in gel form, was then heat sterilised to yield a consumable product. The pH of both the products was 5.0 ± 1.0. The produce of the N-163 strain was more viscous and formed more threads than the produce from the AFO-202 strain. This difference may be due to the difference in the structural formula of β-glucans (Figs. 1, 2).

Animal study

Protocol approval was obtained from the Ethics Committee of Toya Laboratory, HOKUDO Co., Japan (ref. no. HKD47047). The study was conducted in accordance with the HOKUDO Animal Experiment Regulations following the Act on Welfare and Management of Animals (Ministry of the Environment, Japan, Act No. 105 of October 1, 1973) standards relating to the care and management of laboratory animals and relief of pain (Notice No. 88 of the Ministry of the Environment, Japan, April 28, 2006) and the guidelines for proper conduct of animal experiments (Science Council of Japan, June 1, 2006). All animal experiments were performed at Toya Laboratory, HOKUDO Co., Hokkaido, Japan.

Healthy 6-week-old male KKAy/TaJcL mice (Nippon Clare Co., Japan) were purchased for this study. The animals were acclimatised for three weeks from their date of arrival. Healthy animals with no abnormalities during the acclimatisation period were divided into four groups (six males per group) using a weight-stratified randomisation method, such that the average weight of each group was as uniform as possible. The animals were kept in a rearing environment at a room temperature of 23 ± 2 °C (acceptable limit range: 20–26 °C), relative humidity of 55 ± 10% (acceptable limit range: 30–70%), and 12 h of light and dark (light hours: 7:00 a.m. to 7:00 p.m.). The mice were housed in micro barrier cages made of polysulfone (external dimensions: W x D x H: 196 mm × 306 mm × 166 mm) with bedding chips (Dohoh Rika Sangyo Co., Ltd.). One mouse was placed in each cage. The cages and bedding were changed at least once per week. The animals were fed solid feed (CE-2, Feed One Co., Ltd.) manufactured within the past year. The solution used for all the groups was groundwater that had been sterilized by adding sodium hypochlorite to achieve a residual chlorine level of 0.3-–0.4 mg/L using a facility water sterilizer (MJ25SR, Kawamoto Manufacturing Co., Ltd.). The water bottles were changed at least twice a week.

The groups and doses of the test substances were as follows:

• Group 1 (n = 6): Euthanized on day 0 for baseline values

• Group 2 (n = 6): Control (solvent—drinking water)

• Group 3 (n = 6): AFO-202 β-glucan: 200 mg/kg/day; 20 mg/ml concentration in solution

• Group 4 (n = 6): N-163 β-glucan: 300 mg/kg/day; 30 mg/ml concentration in solution.

The dose of each test substance was determined to be the same as the expected daily intake dose for humans. In other words, the estimated daily human intake of each test substance was 10 g in the gel form of AFO-202 β-glucan (5 mg of active ingredient of β -glucan per gram) and 15 g in the gel form of N-163 β-glucan (6 mg of active ingredient of β-glucan per gram).

Because the test substance was a food material, oral administration via gavage was selected, which is commonly used for oral administration in rodents. The drug was forcibly administered orally into the stomach of the animals, using a gastric tube (KN-348, oral administration needle, Natsume Corporation) and a disposable syringe (Terumo Corporation), once daily for 28 consecutive days (between 08:00 a.m. and 15:00 p.m.).

The animals in group 1 were weighed using an electronic balance (FX-1200I, A&D Co., Ltd.) on the day before the start of treatment (day 0). All the animals underwent laparotomy under isoflurane anaesthesia (isoflurane, Fujifilm Wako Pure Chemical Co., Ltd.), and blood was collected from the aorta. The blood was divided into two portions, one of which was heparinised and the whole blood was frozen for HbA1c measurement. The other portion was centrifuged to obtain serum, which was then frozen and stored to measure blood glucose, triglyceride, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and free fatty acid levels. Centrifugation was performed at 3500 rpm at 4 °C for 15 min.

The general condition of all the subjects in groups 2 and 3 was observed at least once a day from the day of administration (day 1) to the day of autopsy. Body weight was measured before dosing (between 08:00 a.m. and 13:00 p.m.) on days 1, 7, 14, 21, and 28 (the last dosing day) for all the subjects in groups 2, 3, and 4. On the day of autopsy, the body weight of each mouse was measured before fasting. Body weight was measured using an electronic balance (FX-1200I, A&D Co. Ltd.). The animals were euthanised using isoflurane anaesthesia (Fujifilm Wako Pure Chemical Co., Ltd., Japan).

The average daily food intake was calculated from the food intake over 3 days. To measure the acclimation period, the amount of food fed on day 18 and the amount of food remaining on day 21 were measured for all the cases. For the acclimation period, feed intake was measured on days 3, 10, 17, and 24, and residual feed intake was measured on days 6, 13, 20, and 27. From these data, the mean daily food intake was calculated at doses 3–6, 10–13, 17–20, and 24–27.

Blood glucose levels were measured using a blood glucose monitoring device (Freestyle Freedom Lite, Nipro Corporation) using whole blood obtained from the tail vein on the day before the start of dosing ( day 0). After 14 days of administration, the blood glucose levels were again measured using whole blood obtained from the tail vein in the same way, before afternoon (13:00–15:00 p.m.).

Blood glucose, triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol, and NEFA tests were conducted using refrigerated blood and frozen serum samples before the start of dosing (day 0), and the frozen blood and frozen serum samples on the day following the last day of dosing (day 29). The tests were outsourced to Nagahama Life Science Laboratory, Japan.

We calculated the mean body weight and standard deviation for each group on days 1, 7, 14, 21, and 28, the average daily food intake during the acclimation period, average daily food intake on days 3–6, 10–13, 17–20, and 24–27, blood glucose levels over time, HbA1c on the day after the last dose, blood glucose in serum, and triglycerides. The total means and standard deviations were calculated for each group for total cholesterol, LDL cholesterol, HDL cholesterol, and free fatty acids. Furthermore, Bartlett’s test was used to test for equality of variance using Excel statistics (Social Information Service Co., Ltd.). Equal variances were analysed using one-way analysis of variance (ANOVA), and unequal variances were analysed using the Kruskal–Wallis test. When a significant difference was found in one-way ANOVA, Dunnett's multiple comparison test was used to compare the means with the control group. When significant differences were found using the Kruskal–Wallis test, the means were compared with the control group using Dunnett's nonparametric multiple comparison test. The significance level was set at P < 0.05.

Results

The mean weight of the animals in group 1 was 41.47 g. Their average daily food intake was 6.71 g/day during the acclimation period (18–21 days). Their blood glucose was 730 mg/dL, HbA1c was 6.89%, triglycerides were 663 mg/dL, total cholesterol was 164 mg/dL, LDL cholesterol was 7 mg/dL, HDL cholesterol was 96 mg/dL, and free fatty acids were 1113 μEq/L (Table 1).

Table 1.

Body weight changes in male KKAy mice that were administered test solutions orally for 28 days

Group	Category	Dose (mg/kg/day)	Total animal number		Day of administration
					0a)	1	7	14	21	28
1	–	–	6	Mean SD	41.47 3.05
2	Solvent (Water for injection)	0	6	Mean SD		41.29 3.00	41.50 2.97	42.29 3.32	43.24 3.46	43.89 3.84
3	β-glucan nyan-wan glucan	200	6	Mean SD		41.21 2.24	41.27 2.76	41.90 2.35	43.25 3.06	43.91 3.16
4	β-glucan N-163	300	6	Mean SD		41.78 1.83	42.00 2.35	42.46 2.50	43.69 2.73	43.94 2.62

There were no significant differences in body weight or food intake between groups 2, 3, and 4. None of the animals showed dose-related abnormalities.

After 28 days, the average blood glucose levels were 592 mg/dL in group 2, 615 mg/dL in group 3, and 599 mg/dL in group 4. The differences were not statistically significant. At the end of the treatment period, the HbA1c levels of each test substance and the control ranged between 7.85% and 8.35%. There was no significant difference in blood glucose, HbA1C, total, or LDL, or HDL cholesterol between the groups (Table 2) on days 14 and 28 of treatment.

Table 2.

Blood chemical findings in male KKAy mice that were administered test solutions orally for 28 days

Group	Category	Dose (mg/kg/day)	Total animal number		Glucose (mg/dL)	HbA1c (%)	Triglyceride (mg/dL)	Total-cholesterol (mg/dL)	LDL-Cholesterol (mg/dL)	HDL-Cholesterol (mg/dL)	NEFA (μEq/L)
1a)	–	–	6	Mean SD	730 63	6.89 0.37	663 121	164 19	7 1	96 9	1,113 103
2	Solvent (Water for injection)	0	6	Mean SD	592 86	8.21 0.67	521 183	179 26	9 3	111 11	1,888 830
3	β-glucan nyan-wan glucan	200	6	Mean SD	615 95	8.17 0.70	499 142	181 24	10 2	114 13	1,886 456
4	β-glucan N-163	300	6	Mean SD	599 96	8.26 0.41	491 114	179 17	10 2	110 11	1,691 536

NEFA levels were lower in group 4 (N-163) (mean ± SD, 1691 ± 536 mg/dL) than in groups 3 (AFO-202) (1886 ± 456 mg/dL) and 2 (control) (1888 ± 830 mg/dL) (Fig. 3). Similarly, triglyceride levels were lower in group 4 (N-163) (491 ± 114 mg/dL), than in group 3 (AFO-202, 499 ± 142 mg/dL) and group 2 (control, 521 ± 183 mg/dL) (Fig. 4).

Fig. 3.

NEFA levels in male KKAy mice that were administered solution orally for 28 days, showed a better reduction in group 3 (N-163 β-glucan) compared to groups 2 (AFO-202 β-glucan) and 1 (control)

Fig. 4.

Triglyceride levels in male KKAy mice that were administered solution orally for 28 days, showed a marginal reduction in group 3 (N-163 β-glucan) compared to groups 2 (AFO-202 β-glucan) and 1 (control)

Discussion

Dyslipidaemia is an emerging epidemic worldwide, with nearly 39% of the global population having elevated cholesterol levels. According to the Global Burden of Diseases, Injuries, and Risk Factors study, dyslipidaemia is the cause of 26.9% of morbidity and 28% of mortality cases [12]. The liver plays a major role in glucose and lipid metabolism. In particular, NEFAs have been indicated as a major cause of the progression of non-alcoholic liver injury because the liver is responsible for taking up serum FFA and manufacturing, storing, and transporting lipid metabolites. The circulating NEFA pool contributes to the majority of FFAs that flow to the liver; hence, elevated NEFA contributes to fatty liver disease progression [13]. In addition, several reports have pointed out the key role of chronic inflammation in the pathogenesis of obesity-related metabolic dysfunction and non-alcoholic fatty liver disease (NAFLD) [14]. This inflammatory pathway, caused by a high-fat diet and elevated lipid levels, leads to insulin resistance and the progression of metabolic syndrome and NAFLD [15]. Adipose tissue is a potential source of elevated circulating inflammatory cytokines, such as tumour necrosis factor (TNF)-α and interleukin (IL)-6, especially because the increased numbers of macrophages that accumulate in adipose tissue in obese individuals contributes to the secretion of these cytokines [16]. NEFA induces macrophages to secrete inflammatory cytokines [16]. Thus, the entire cascade of metabolic syndrome, diabetes, obesity, and inflammatory state contributing to cancer, can be attributed to lipotoxicity; therefore, individuals may benefit from strategies that target lipids, especially those that normalise NEFA levels.

β-Glucans, especially those derived from A. pullulans, are potent immunomodulators that have already been shown to decrease IL-6 and TNF-α levels and positively regulate the Akt/PI3K and peroxisome proliferator-activated receptor γ (PPARγ) signalling pathways, which are principal regulators of adipogenesis and glucose metabolism [17]. These β-glucans are capable of modulating the immune response by suppressing inflammatory cytokines [18] without over-activation. β-glucans can elicit a unique immune response, binding directly with immune cells such as macrophages, and, importantly, downregulating the abnormal macrophages while activating normal macrophages [19–21]. Therefore, β-glucans are considered a potential strategy to counter the ill effects of dyslipidaemia and reduce elevated NEFA levels.

In the current study, N-163 β-glucan showed a beneficial reduction of NEFA and triglyceride levels, compared to the AFO-202 β-glucan and control. At the end of the treatment period, the blood glucose levels in groups 2 and 3 were within 588–615 mg/dL. In literature (KKAy/Ta Jcl Mouse Data Collection, Nihon Clare Co., Ltd., 1994), the blood glucose level, when not fasting, in 10-week-old male KKAy mice was reported to be 333 ± 33 mg/dL. This suggests that the present experiment reproduced a hyperglycaemic state similar to or higher than that reported in earlier studies, throughout the treatment period, thus reproducing the metabolic syndrome that occurs in humans. The significant decrease in NEFA but not in blood glucose, HbA1c or LDL and HDL cholesterol, we presume could be due to the acute effect of N-163 beta glucan on vascular/inflammation related parameters in metabolic syndrome rather than the glucose related parameters [22]. This finding was further corroborated in another study in a stelic animal model (STAM), in which AFO-202 strain produced β-glucan had profound effects on glucotoxicity, while N-163 produced β-glucan efficiently reduced inflammation and liver fibrosis; together, they were able to mitigate hepatic steatosis and reduce the NAFLD score [23].

One major limitation of the study is that the dosage was based on human consumption levels of β-glucans, and earlier reports [8, 9] have indicated the normalisation of lipid levels at least two months after the treatment. Because the present study lasted only 28 days, a dose-escalation study will be more useful in the context of studying the effects of β-glucans in a shorter time duration. Further evaluation of these two β-glucans regarding balancing the parameters relevant to metabolic syndrome, glucotoxicity, and lipotoxicity, and ensuring control of the inflammatory cascade in both healthy and diseased patients may shed more light on their efficacy and probable mechanisms.

In conclusion, repeated oral administration of β-glucans derived from the AFO-202 and N-163 strains of A. pullulans, for 28 days, resulted in lower triglyceride and NEFA levels in male KKAy mice than before the start of treatment. In particular, the NEFA levels in N-163-fed mice were lower than those in the AFO-202 group; therefore, further research on anti-inflammatory efficacy is essential, which may help with designing effective therapeutic strategies for preventing and managing metabolic dysregulation-induced fibrotic diseases such as NAFLD.

Abbreviations

NEFA

Non-esterified fatty acids

FFA

Free fatty acids

NASH

Non-alcoholic steatohepatitis

PDA

Potato dextrose agar

PDB

Potato dextrose broth

LDL

Low-density lipoprotein

HDL

High-density lipoprotein

ANOVA

Analysis of variance

NAFLD

Non-alcoholic fatty liver disease

STAM

Stelic animal model

TNF

Tumour necrosis factor

IL

Interleukin

PPARγ

Peroxisome proliferator-activated receptor γ

Author contribution statement

N.I and S.A. contributed to conception and design of the study. Y.I and M.N helped with technical assistance. R.S helped in literature search. S.A, M.R and S.P. drafted the manuscript. G.K, V.D and S.V performed critical revision of the manuscript. All the authors read, and approved the submitted version.

Data availability

All data generated or analysed during this study are included in this manuscript.

Declarations

Ethics approval

The protocol approval was obtained from the ethics committee of Toya Laboratory, HOKUDO Co., Japan (Ref no: HKD47047). The study was conducted in accordance with the HOKUDO Animal Experiment Regulations following the Act on Welfare and Management of Animals (Ministry of the Environment, Japan, Act No. 105 of October 1, 1973), standards relating to the care and management of laboratory animals and relief of pain (Notice No.88 of the Ministry of the Environment, Japan, April 28, 2006) and the guidelines for proper conduct of animal experiments (Science Council of Japan, June 1, 2006). All animal experiments took place at Toya Laboratory, HOKUDO Co., Hokkaido, Japan.

Potential conflict of interests

Author Samuel Abraham is a shareholder in GN Corporation, Japan which holds shares of Sophy Inc., Japan., the manufacturers of novel beta glucans using different strains of Aureobasidium pullulans; a board member in both the companies and also an applicant to several patents of relevance to these beta glucans.