Beneficial effects of the AFO-202 and N-163 strains of Aureobasidium pullulans produced 1,3-1,6 beta glucans on non-esterified fatty acid levels in obese diabetic KKAy mice: A comparative study

TLDR: In this paper, the authors compared two strains (AFO-202 and N-163) that produce beta glucans to differentiate their efficacy in alleviating lipotoxicity, and they found that N163 produced by A. pullulans decreased NEFA in a diabetic mice model in 28 days.

Abstract: Background Obesity, metabolic syndrome, associated lipotoxicity and its cascade of events contribute to the majority of the burden related to non-communicable diseases globally. Preventive lifestyle changes aside, several beneficial effects have been reported in type II diabetes mellitus and dyslipidaemia patients with biological response modifier glucans (BRMG) produced as an exopolysaccharide by Aureobasidium pullulans. In this study, we compared two strains (AFO-202 and N-163) that produce beta glucans to differentiate their efficacy in alleviating lipotoxicity. Methods This study was performed in obese diabetic mice model of KK-Ay mice, in four groups 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. The animals in groups 2–4 had the test solutions forcibly administered orally into the stomach using a gastric tube once daily for 28 consecutive days. Biochemical analyses were conducted of blood glucose, triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol and non-esterified fatty acids (NEFA). Results Group 4 (N-163) had the lowest NEFA levels, as compared to the other groups, and marginally decreased triglyceride levels. The groups had no significant differences in blood glucose, HbA1c, triglycerides, or LDL and HDL cholesterol. Conclusion N-163 produced by A. pullulans decreased NEFA in a diabetic mice model in 28 days. These results, although modest, warrant further in-depth research into lipotoxicity and associated inflammatory cascades in both healthy and disease affected subjects to develop novel strategies for prevention and management.

Figures

Figure 2: Structure of strain beta glucan derived from N-163 with the chemical formula C6H10O5)n

Figure 1: Structure of beta glucan derived from the AFO-202 strain with the chemical formula (C6H10O5)n

Full text

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Title: Beneficial effects of novel strains of Aureobasidium pullulans produced 1,3-1,6
β
-
glucans on non-esterified fatty acid levels in diabetic KKAy mice
Running title: Beta-glucans in regulating free fatty acids
Authors:
Nobunao Ikewaki
1,2
(nikewaki@phoenix.ac.jp)
Takashi Onaka
3
(onaka-t@sophy-inc.co.jp)
Yasunori Ikeue
3
(ikeue-y@sophy-inc.co.jp)
Mitsuru Nagataki
3
(nagataki-m@sophy-inc.co.jp)
Gene Kurosawa
4,5
(gene@fujita-hu.ac.jp)
Vidyasagar Devaprasad Dedeepiya
6
(dedeepiya_76@yahoo.co.in)
Mathaiyan Rajmohan
7
(mrm@nichimail.jp)
Suryaprakash Vaddi
8
(suryaprakashuro@gmail.com)
Rajappa Senthilkumar
7
(rsk@nichimail.jp)
Senthilkumar Preethy
7
(drspp@nichimail.jp)
Samuel JK Abraham
6,9,10
(drsam@nichimail.jp)
Affiliations:
1. Dept. of Medical Life Science, Kyushu University of Health and Welfare, Japan
2. Institute of Immunology, Junsei Educational Institute, Nobeoka, Miyazaki, Japan
3. Sophy Inc., Kochi, Japan
4. Department of Academic Research Support Promotion Facility, Center for Research
Promotion and Support, Fujita Health University, Aichi, Japan.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 3, 2021. ; https://doi.org/10.1101/2021.07.22.453362doi: bioRxiv preprint

2
5. MabGenesis KK, Nagoya, Japan
6. Mary-Yoshio Translational Hexagon (MYTH), Nichi-In Centre for Regenerative
Medicine (NCRM), Chennai, India.
7. Fujio-Eiji Academic Terrain (FEAT), Nichi-In Centre for Regenerative Medicine
(NCRM), Chennai, India.
8. Department of Urology, Yashoda Hospitals, Hyderabad, India
9. Centre for Advancing Clinical Research (CACR), University of Yamanashi - School
of Medicine, Chuo, Japan.
10. Antony- Xavier Interdisciplinary Scholastics (AXIS), GN Corporation Co. Ltd., Kofu,
Japan
Corresponding Author Information:
Dr. Samuel JK Abraham,
University of Yamanashi - School of Medicine, Chuo, Japan,
Correspondence Address: 3-8, Wakamatsu, Kofu, Yamanashi 400-0866, Japan.
Email id- drsam@nichimail.jp
; Alternate email id: drspp@nichimail.jp
Phone: +81-55-235-7527
Funding:
No external funding was received for the study
Ethics Approval:
The protocol approval was obtained by the ethics committee of Toya Laboratory, HOKUDO
Co (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
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 3, 2021. ; https://doi.org/10.1101/2021.07.22.453362doi: bioRxiv preprint

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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).
Potential Conflict of Interests:
1. Author Samuel Abraham is a shareholder in GN Corporation, Japan which in turn is a
shareholder in the manufacturing company of the Beta Glucans described in the study.
2. Author Takashi Onaka is a shareholder and Yasunori Ikeue is member of the board in
the manufacturing company of the Beta Glucans described in the study.
Acknowledgements:
The authors thank
a. Mr. Yoshio Morozumi, Ms. Yoshiko Amikura of GN Corporation, Japan for their
liaison assistance with the conduct of the study.
b. Loyola-ICAM College of Engineering and Technology (LICET) for their support
to our research work.
Author Contribution Statement:
N.I and S.A. contributed to conception and design of the study. T.O, 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.
Availability of data and material
All data generated or analysed during this study are included in this manuscript
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 3, 2021. ; https://doi.org/10.1101/2021.07.22.453362doi: bioRxiv preprint

4
Abstract:
Obesity, metabolic syndrome, associated lipotoxicity and its cascade of events contribute to
the majority of the burden related to non-communicable diseases globally. Preventive
lifestyle changes aside, several beneficial effects have been reported in type II diabetes
mellitus and dyslipidaemia patients with biological response modifier glucans (BRMG)
produced as an exopolysaccharide by Aureobasidium pullulans. In this study, we compared
two strains (AFO-202 and N-163) that produce beta glucans in alleviating lipotoxicity. This
study was performed in obese diabetic mice model of KK-Ay mice, in four groups 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. The animals in groups 2–4 had the test solutions administered by
gavage once daily for 28 consecutive days. Biochemical analyses were conducted of blood
glucose, triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol and non-esterified
fatty acids (NEFA). Group 4 (N-163) had the lowest NEFA levels, as compared to the other
groups, and marginally decreased triglyceride levels. The groups had no significant
differences in blood glucose, HbA1c, triglycerides, or LDL and HDL cholesterol. N-163
produced by A. pullulans decreased NEFA in a diabetic mice model in 28 days. These results,
although modest, warrant further in-depth research into lipotoxicity and associated
inflammatory cascades in both healthy and disease affected subjects to develop novel
strategies for prevention and management.
Keywords: Beta glucans; Non-esterified fatty acids (NEFA); Lipotoxicity; Free fatty acids
(FFA); Aureobasidium pullulans; AFO-202; N-163
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 3, 2021. ; https://doi.org/10.1101/2021.07.22.453362doi: bioRxiv preprint

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Introduction:
Fatty acids are a major component of lipids and are an important source of fat fuel for the
body. The non-esterified fatty acids (NEFA) of free fatty acids (FFA) are the circulating form
of fatty acids in the plasma [1]. FFA are an important link between obesity, insulin resistance
and inflammation and the development of diabetes, hypertension, dyslipidemia, coagulative
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 levels of plasma FFA, leading to insulin resistance, influences the synthesis of
hepatic triglycerides. The hepatic fatty acid metabolism, which is closely linked to
inflammation, leads to hepatic steatosis (non-alcoholic steatohepatitis [NASH]), progressing
to fatty liver disease, cirrhosis and cancer [3]. Therefore, modulating and normalizing NEFA
can considered as 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
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 3, 2021. ; https://doi.org/10.1101/2021.07.22.453362doi: bioRxiv preprint

Frequently asked questions

Q1. What have the authors contributed in "Title: beneficial effects of novel strains of aureobasidium pullulans produced 1,3-1,6 β- glucans on non-esterified fatty acid levels in diabetic kkay mice running title: beta-glucans in regulating free fatty acids authors:" ?

In this paper, the effects of β glucans derived from AFO-202 and N-163 A. pullulans in KK-Ay mice, as obese diabetic mice models, were evaluated. 

Q2. What future works have the authors mentioned in the paper "Title: beneficial effects of novel strains of aureobasidium pullulans produced 1,3-1,6 β- glucans on non-esterified fatty acid levels in diabetic kkay mice running title: beta-glucans in regulating free fatty acids authors:" ?

In particular, the NEFA levels in N-163-fed mice were lower than those in the AFO-202 group, making further research recommended on antiinflammatory efficacy, which may help with designing effective therapeutic strategies for treating and preventing fibrotic diseases that develop due to dyslipidaemia-related cascade. 

Q3. What is the role of beta glucans in reducing NEFA?

Beta glucans, especially derived from A. pullulans, are potent immunomodulators that have already been shown to decrease IL-6 and TNF-α levels, in addition to positively regulating Akt/PI3K and peroxisome proliferator-activated receptor γ (PPARγ) signalling pathways, which are principal regulators of adipogenesis and glucose metabolism [16]. 

Q4. What are the benefits of beta glucans?

Beta glucans are polysaccharides with many beneficial effects to ameliorate glucose metabolic disorders such as diabetes and dyslipidaemia [6], in addition to enhancing immunities for fighting viral infections and cancer [7]. 

Q5. What is the role of beta glucans in the immune system?

The beta glucans are capable of eliciting a unique immune response, binding directly with immune cells such as macrophages and, importantly, downregulating the abnormal macrophages while activating the normal macrophages [18-20]. 

Q6. What is the main limitation of the study?

One major limitation of the study is that the dosage was based on human consumption levels of beta glucans, and the earlier reports [8,9] have indicated the normalization of lipid levels at least 2 months after the treatment. 

Q7. What is the purpose of the study?

Because the present study lasted only 28 days, a doseescalation study will be more useful in the context of studying the effects of beta glucans in a shorter time duration. 

Q8. What is the role of NEFA in diabetes?

modulating and normalizing NEFA can considered as a key target to alleviate the effects of the entire cascade of lipotoxicity [4] and the dysregulation of lipid metabolism described above. 

Q9. What is the effect of beta glucans on NEFA?

In the current study, N-163 beta glucan showed beneficial reductions of NEFA and triglyceride levels, as compared to the AFO-202 beta glucan and the control. 

Q10. How long did the animals receive the test substance?

The animals were forcibly administered orally into the stomach 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 and 15:00). 

Q11. What is the reason for the NEFA in the liver?

In particular, NEFA has been indicated as a major cause for the progression of non-alcoholic liver injury because the liver is responsible for taking up serum FFA and manufacturing, storing and transporting lipid metabolites. 

Q12. How many mice were acclimatized to the study?

Healthy animals with no abnormalities during the acclimatization period were divided into four groups (six males per group) using a weight-stratified randomization method so that the average weights of each group would be as uniform as possible. 

Q13. What was the purpose of the study?

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). 

Q14. What is the role of beta glucans in the prevention of NEFA?

beta glucans are considered a potential strategy to counter the ill effects of dyslipidaemia and reduce elevated NEFA levels. 

Q15. How often did the animals in the groups 2 and 3 receive isoflurane?

The general condition of all 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. 

Q16. What are the main factors contributing to the burden of diabetes?

All data generated or analysed during this study are included in this manuscriptObesity, metabolic syndrome, associated lipotoxicity and its cascade of events contribute to the majority of the burden related to non-communicable diseases globally. 

Q17. What is the blood glucose level in KK-Ay mice?

In the literature (KK-Ay/Ta Jcl Mouse Data Collection, Nihon Clare Co., Ltd., 1994), the blood glucose level in 10-week-old KK-Ay male mice was reported to be 333 ± 33 mg/dL when not fasting. 

Q18. What was the size of the mice?

The mice were housed in microbarrier cages made of polysulfone (external dimensions: W196 mm × D306 mm × H166 mm) with bedding chips (Dohoh Rika Sangyo Co., Ltd.). 

Q19. How many g of beta-glucan was in each test?

In other words, the estimated daily human intake of each test substance is 10 g in gel form of AFO-202 beta-glucan (5 mg of active ingredient of beta-glucan per gm) and 15 g in gel form of N-163 beta-glucan (6 mg of active ingredient of beta-glucan per gm).