Alkali-soluble polysaccharides from mushroom fruiting bodies improve insulin resistance

Abstract

Insulin resistance is the main cause of type 2 diabetes. Polysaccharide is one of the main active components in mushrooms. Some mushroom polysaccharides have been reported to have beneficial effects against type 2 diabetes. However, the structural features and mechanisms of polysaccharides with hypoglycemic activity have not been elucidated. In this paper, six alkali-soluble total polysaccharides, six neutral polysaccharides and six acidic polysaccharides were extracted and purified from mushrooms fruiting bodies of different species. The effects of these polysaccharides on improving insulin resistance were compared using high fatty acids and glucose-induced hepatocytes. Among them, the neutral polysaccharide AAMP-N of Armillaria mellea fruiting body showed the most significant activity, indicating that mannogalactoglucan is the main active domain. AAMP-N enhanced insulin sensitivity in vitro. Also AAMP-N lowered blood glucose, and modulated lipid metabolism in db/db mice. In addition, AAMP-N protected damaged pancreatic islets in mice. Our results demonstrated the role of natural polysaccharides from mushrooms in the improvement of insulin sensitivity and lipid metabolism, and provided basis for the development of mushroom polysaccharides as hypoglycemic healthy food.

1. Introduction

Diabetes is a metabolic disorder characterized by hyperglycemia, mainly including type 1 diabetes and type 2 diabetes [1]. Type 1 diabetes is caused by pancreatic β-cell dysfunction, leading to impaired post-prandial insulin secretion [2]. Type 2 diabetes is caused by insulin resistance and insufficient insulin secretion, which contributes to several life-threatening complications, including nephropathy and retinopathy [3].

Mushrooms are a popular healthy food worldwide, mainly containing proteins, lipids, minerals, and polysaccharides [4]. Most health-promoting properties have been attributed to polysaccharides [5]. The main structural types of mushroom polysaccharides include homoglucans (β-1,3-glucan), heteroglucans (galactomannoglucan), heterogalactans (mannogalactan) and heteromannans [6]. They have been reported to lower blood glucose in a variety of manners, including inhibiting activity of α-glucosidase [7]; increasing glucose transporter 4 to promote glucose intake [8]; and reducing the release of inflammatory factors to improve insulin resistance [9].

In our present study, we extracted polysaccharides from six kinds of mushrooms, evaluated their activities on high fatty acid and glucose-induced insulin resistance in hepatocytes, and identified the structural feature of the mushroom polysaccharide and its mechanism of amelioration of insulin resistance in mice.

2. Materials and methods

2.1. Materials

Fruiting bodies of Amillariella mellea, Gomphidius rutilus, Agrocybecy lindracea, Hypsizygus marmoreus, Pleurotus eryngii and Pleurotus ostreatus were collected from Changbai mountain in Jilin Province, China and identified using rDNA-ITS sequencing analysis. DEAE-cellulose was purchased from Amersham Pharmacia Biotech. All other chemicals were of analytical grade and commercially available or produced in China.

2.2. Polysaccharides extraction and fractionation

2.2.1. Extraction of the alkali-soluble polysaccharides

The polysaccharides were extracted according to our previous literature [1]. Briefly, Fruiting bodies were defatted with 95% ethanol (w/v, 1:10), and the residues were extracted with distilled water (w/v, 1:20) three times at 100 °C for 4 h. After filtration, the resulting residues were dried and extracted using 0.5 M NaOH with trace amounts of NaBH₄ at 80 °C (w/v, 1:20). Extracts were neutralized with glacial acetic acid, concentrated under vacuum at 60 °C, and 95% ethanol was added at a final concentration of 75% to precipitate the crude polysaccharides. After centrifugation (4000 rpm, 15 min), the precipitate was collected and re-dissolved in water, then dialyzed and lyophilized to give alkali-soluble polysaccharide. Six alkali-soluble polysaccharides were obtained from Amillariella mellea (named AAMP), Gomphidius rutilus (named AGRP), Agrocybecy lindracea (named AACP), Hypsizygus marmoreus (named AHMP), Pleurotus eryngii (named APEP) and Pleurotus ostreatus (named APOP), respectively.

2.2.2. Analytical chromatography on DEAE-cellulose

Alkali-soluble polysaccharide (10 mg) was dissolved in distilled water (2 ml). After centrifugation (10,000 rpm, 5 min), the supernatant was loaded on a DEAE-Cellulose (Cl⁻) column (1.5 × 14 cm) pre-equilibrated with distilled water. The column was first eluted with distilled water at 1.0 ml/min to yield a neutral polysaccharide fraction, and then with a linear gradient from 0.0 to 0.5 M NaCl to obtain an acidic polysaccharide fraction (Fig. S1). The eluate was collected at 4 ml per tube and assayed for total sugar and uronic acid contents. According to the analysis results, the concentration of eluate was determined as 0.4 M NaCl solution.

2.2.3. Preparation of neutral and acidic polysaccharide fractions by DEAE-cellulose

Alkali-soluble polysaccharide (10 g) was dissolved in distilled water (1000 ml). After centrifugation (4500 rpm, 15 min), the supernatant was separated by using a DEAE-cellulose column (7.5 × 30 cm, Cl⁻). The column was first eluted with dH₂O, the eluate was collected, concentrated, and lyophilized to give the neutral fraction, then eluted with 0.4 M NaCl to yield the acidic polysaccharide fraction.

2.3. Chemical detection methods

Total carbohydrate content was determined by using the phenol sulfuric acid method with glucose as the standard [10]. Protein content was determined using the Bradford assay with bovine serum albumin as the standard [11]. Uronic acid content was determined by using the m-hydroxydiphenyl method with glucuronic acid as standard [12].

Monosaccharide composition was determined by using high performance liquid chromatography (HPLC) as described by Zhang et al. [13]. Briefly, polysaccharide samples (2 mg) were initially hydrolyzed with 1 ml anhydrous methanol containing 2 M HCl at 80 °C for16 h and then with 1 ml 2 M trifluoro acetic acid (TFA) at 120 °C for 1 h. After derivatization with 1-phenyl-3-methyl-5-pyrazo-lone (PMP), the derivatives were analyzed by HPLC.

2.4. Cell culture

Mouse hepatoma Hepa1–6 cell line was purchased from ATCC, and cultured in Dulbecco’s modified Eagle’s medium (DMEM, 25 mM glucose, Gibco) equilibrated with 5% CO₂ at 37 °C.

2.5. High glucose and fatty acid-induced insulin resistance in vitro

According to our previous study [14], Hepa1–6 cells were cultured in low glucose (5.5 mM) DMEM supplemented with 10% FBS and 1% anti-biotics in 96 well plate for 24 h. Cell culture medium was changed to FG conditioned medium (25 mM glucose and 0.5 mM palmitic acid and oleic acid) with or without polysaccharides for 24 h. Subsequently the cells were treated with 1 nM insulin for 12 h in serum-free low glucose DMEM. Cells were collected for glucose uptake were measured with an assay kit (Applygen, China) using a microplate reader (Infinite 50, Tecan, Switzerland).

2.6. Western blots

Cells were prepared in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, proteinase inhibitor cocktail (Roche Applied Sciences) and phosphatase inhibitor cocktail (Thermo Scientific). Western blots were performed as described in the previous publication [14] using the following primary antibodies: anti-p-insulin receptor (Cell Signaling), anti-p-AKT (Cell Signaling), anti-insulin receptor (Cell Signaling), anti-AKT (Cell Signaling), or anti-actin (Abcam) antibodies.

2.7. Experimental mice

All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Northeast Normal University. Male C57BL/6J and male db/db mice were obtained from Model Animal Research Center of Nanjing University (Nanjing, China). The animals at 5 weeks of age were used. The animals were housed in a room maintained at 23 ± 2 °C with relative air humidity of 45% to 55% on a 12-hour light/12-hour dark cycle. Mice were provided a standard laboratory chow and water.

2.8. Glucose tolerance test (GTT) and insulin tolerance test (ITT)

For GTT, mice were fasted for 6 h followed by an intraperitoneal injection of 2 g/kg body weight glucose. Blood glucose levels were monitored before and 15, 30, 60 and 120 min after injection using blood collected from the tail using the One Touch Ultra Easy Glucometer (Johnson, New Brunswick, NJ, USA). For ITT, mice were fasted for 6 h, followed by injection with insulin (1.5 U/kg body weight). Blood from tail was collected before and 15, 30, 60 and 120 min after injection and glucose levels were determined as described above.

2.9. H&E staining

The liver was fixed in 4% paraformaldehyde (Sangon Biotech, A500684–0500), and then embedded in paraffin, and sectioned at 5 μm. H&E staining were performed following standard protocols. Photos were taken using a light microscope.

2.10. Measurement of biochemical indicators

Enzymatic methods were used to evaluate the levels of non-esterified fatty acid (NEFA), TG, ALT and AST in serum. The kits were from Jiancheng Bioengineering Institute (Nanjing, China). Serum insulin levels were assayed with a Mouse Insulin ELISA Kit (IBL).

2.11. Immunohistochemistry

The pancreas was removed from a centrifuge tube with 4% paraformaldehyde and then a small part of the complete pancreas was cut. The pancreas tissues were soaked in 15% sucrose and dehydrated overnight. Then tissues were transferred into 30% sucrose and dehydration overnight. The dehydrated tissue was taken out and embedded in OCT Compound (SAKURA, 4583) and frozen in −80 °C. The tissues were subsequently sliced into 8 μm sections using freezing microtome (LEICA CM1850UN).

Sections were fixed with 4% paraformaldehyde and permeabilized for 20 min. After twice washes with PBS for 5 min, permeabilization with 0.5% Triton X-100/SDS/PBS for 20 min at room temperature, followed by three washed in PBS. Slides were blocked in 5%FBS/PBS/1% BSA for 3 h at room temperature and incubated in the indicated antibodies at 4 °C overnight. After twice washed with PBST for 5 min and washed with PBS for 5 min, dark incubated slides with FITC-labeled secondary antibody (Bioss, bs-0358G) for 2 h at room temperature. Slides were then washed twice with PBST for 5 min and washed with PBS for 5 min, followed were incubated with Hoechest 33342 for 10 min at room temperature. After three washes with PBS, the slides were covered with fluorescence decay resistant medium (BOSTER, AR1109). All slides were quantified by fluorescence microscopy.

2.12. Statistical analysis

The results were expressed as the means ± s.e.m. Statistical analysis of the data was performed using student t-test and one-way ANOVA (SPSS Statistics 17.0). Differences were considered significant when p < 0.05.

3. Results

3.1. Properties of fruiting body polysaccharides from mushrooms

Six alkali-soluble polysaccharides were extracted from the fruiting bodies of Amillariella mellea (AAMP), Gomphidius rutilus (AGRP), Agrocybecy lindracea (AACP), Hypsizygus marmoreus (AHMP), Pleurotus eryngii (APEP) and Pleurotus ostreatus (APOP), by using 0.5 M NaOH solution extraction, ethanol precipitation, dialysis and lyophilization. Monosaccharide composition analysis indicated that all polysaccharides were mainly composed of glucose (Glc), followed by galactose (Gal) and mannose (Man) (Table 1). Although these polysaccharides were rich in Glc, the results from I₂-KI assays indicated that they did not contain starch-type glucans.

Table 1.

Chemical characterization of the polysaccharides extracted from six species of edible mushrooms.

Mushrooms polysaccharides	Amillariella mellea	Gomphidiusr utilus	Agrocybecy lindracea	Hypsizygus marmoreus	Pleurotus eryngii	Pleurotus ostreatus
	AAMP	AGRP	AACP	AHMP	APEP	APOP
Yield (%)	12.8b	16.6b	15.0b	1.41a	1.03a	1.67a
Total sugar (%)	68.4	59.4	72.2	56.9	78.6	55.6
Protein (%)	14.3	1.5	14.9	4.9	8.6	8.9
Uronic acid (%)	3.7	0.3	8.6	1.8	1.2	1.6
Ash (%)	16.1	4.9	11.9	11.4	4.2	10.9
Starch (%)	-	-	-	-	-	-
Sugar composition (%)
Glucose (Glc)	58.6	70.8	70.4	69.2	76.8	77.8
Galactose (Gal)	19.8	13.2	22.6	16.9	5.7	7.6
Mannose (Man)	18.1	6.7	-	7.8	10.4	8.7
Xylose (Xyl)	-	6.1	-	-	2.1	-
Glucuronic acid (GlcA)	3.3	-	3.3	4.3	3.2	2.8
Fucose (Fuc)	1.5	3.1	4.0	1.8	-	1.5

^aRelation to the wet weights of mushroom materials.

^bRelation to the dry weights of mushroom materials.

AAMP, AGRP, AACP, AHMP, APEP and APOP were all fractionated by anion-exchange chromatography (Fig. 1). Based on the chromatography procedure, we obtained one neutral fraction eluted with distilled water, and one acidic fraction eluted with 0.4 M NaCl from each polysaccharide. Thus, we obtained a total of six neutral polysaccharide fractions (AAMP-N, AGRP-N, AACP-N, AHMP-N, APEP-N and APOP-N), and six acidic polysaccharide fractions (AAMP-A, AGRP-A, AACP-A, AHMP-A, APEP-A and APOP-A). Monosaccharide composition results showed that all the neutral polysaccharides contained Glc and Gal as the major monosaccharides, followed by Man or Fuc. Therefore, these neutral fractions are likely to be heterogalactans or heteroglucans. Acidic polysaccharide fractions were all mainly composed of Glc, together with small amounts of Gal, Man (or Fuc) and GlcA, indicating that they are glucans (Table 2).

Fig. 1.

Extraction and fractionation scheme for mushroom polysaccharides. According to the procedure, six neutral polysaccharides and six acidic polysaccharides were prepared.

Table 2.

Yield and monosaccharide composition of sub-fractions isolated from six species of mushroom polysaccharides.

Mushrooms	Fractions	Yield (%)a	Monosaccharide composition (mol%)
			Glc	Gal	Man	GlcA	Fuc	Xyl
Amillariella	AAMP-N	35.0	49.8	25.2	18.2	-	-	-
mellea	AAMP-A	47.5	81.6	6.0	3.3	3.5	3.1	-
Gomphidius	AGRP-N	9.8	53.0	29.4	9.8	-	4.4	3.4
rutilus	AGRP-A	17.5	69.3	14.2	10.9	0.6	2.1	2.8
Agrocybecy	AACP-N	24.6	15.6	67.6	1.9	-	14.9	-
lindracea	AACP-A	62.5	77.6	10.2	2.6	3.6	5.9	-
Hypsizygus	AHMP-N	12.0	34.2	40.7	12.3	-	12.8	-
marmoreus	AHMP-A	51.2	76.7	12.0	4.2	3.5	3.6	-
Pleurotus	APEP-N	14.4	50.2	11.0	27.5	-	1.3	8.0
eryngii	APEP-A	53.4	86.9	5.2	5.6	2.2	-	-
Pleurotus	APOP-N	15.0	60.6	19.4	13.8	-	2.1	-
ostreatus	APOP-A	53.4	92.0	4.5	3.5	-	-	-

^aRelation to the crude polysaccharides.

3.2. The effects of fruiting body polysaccharides from mushrooms on insulin-stimulated glucose uptake

Next, we examined the improvement effects of six alkali-soluble polysaccharides on insulin resistance induced by high glucose and fatty acids (FG). As shown in Fig. 2a, glucose uptake in Hepa1–6 cells was significantly reduced by FG treatment compared with control, indicating FG-induced insulin resistance. Metformin (20 μM), an anti-diabetic drug, significantly attenuated FG-induced insulin resistance by 60%. AAMP, AGRP and AACP enhanced glucose uptake compared to FG, even at a low concentration (0.01 mg/ml); whereas AHMP, APEP and APOP had little effects. These results indicated that AAMP, AGRP and AACP may improve insulin sensitivity.

Fig. 2.

Mushrooms polysaccharides improved insulin resistance. Hepa1–6 cells were induced with FG-conditioned medium. (a) Effects of six alkali-soluble total polysaccharides; (b) Effects of sub-fractions of AAMP; (c) Effects of sub-fractions of AGRP; (d) Effects of sub-fractions of AACP; (e) Effects of sub-fractions of AHMP; (f) Effects of sub-fractions of APEP; (g) Effects of sub-fractions of APOP. Results represent mean ± s.e.m. (n = 8 incubations in each group) *p < 0.05; **p < 0.01; ***p < 0.001 as compared to FG.

Each total polysaccharide is composed of neutral polysaccharides and acidic polysaccharides. Therefore, we analyzed which component contributes to the observed effect. The results in Fig. 2b–d showed that the neutral fraction had better activity than the acidic fraction. AAMP-N and AGRP-N significantly increased glucose uptake, even at a low concentration of 0.01 mg/ml (Fig. 2b and c). However, for the three inactive total polysaccharides, their neutral fraction and acidic fraction had little effects (Fig. 2e–g). Combined with the analysis of monosaccharide composition results of AAMP-N and AGRP-N, they appear to have the similar domain as mannogalactoglucan, implying that mannogalactoglucan may play an important role in insulin sensitization. Considering the yield of AAMP-N and AGRP-N (Table 2), we selected AAMP-N to further investigate its hypoglycemic roles in vivo.

3.3. AAMP-N increases insulin sensitivity

Since polysaccharides from Amillariella mellea, especially the neutral fraction AAMP-N, significantly increased insulin-stimulated glucose absorption, we investigated the antagonizing effect of AAMP-N on FG-induced insulin resistance in Hepa1–6 cells. The results are presented in Fig. 3, showing that 0.1 mg/ml of AAMP, 0.1 mg/ml of AAMP-N or 20 μM of MET treatment enhanced insulin-stimulated phosphorylation of insulin receptor (IR) and its downstream kinase AKT, to a similar level as AAMP. In contrast, AAMP-A showed little effect. Thus, we proposed that AAMP-N mediates the elevation of insulin sensitivity.

Fig. 3.

AAMP-N enhanced insulin signaling. Phosphorylation of IR, Akt and AMPK with or without 10 min of 50 nM insulin incubation in Hepa1–6 cells was detected by western blot (left) and quantified (right). Prior to insulin stimulation, cells were sensitized overnight in serum-free experiments. Results represent mean ± s.e.m.

3.4. The oral hypoglycemic effect of AAMP-N in db/db mice

Given AAMP-N ameliorated insulin resistance induced by FG in hepatocytes, we decided to study the metabolic effects of AAMP-N in vivo. We found that in the db/db mice, which are defective in leptin receptor [15], AAMP significantly lowered blood glucose after 3-week treatment at a dose of 50 mg/kg/d (Fig. S2a). At the same dose, neutral polysaccharide AAMP-N had better activity than the acidic polysaccharide AAMP-A. Moreover, the hypoglycemic effect of either polysaccharide at a dose of 50 mg/kg/d was better than 25 mg/kg/d (Fig. S2b). We continued the gavage treatment of AAMP-N at 50 mg/kg/d for 7 weeks, and found that the mouse blood glucose was significantly reduced with AAMP-N treatment after 4 weeks of administration, and remained unchanged from 5 weeks to 7 weeks (Fig. S2c). These results demonstrated the oral hyperglycemic effects of AAMP-N.

In C57BL/6J mice, oral gavage of polysaccharides at a dose of 50 mg/kg/d for 4 weeks had no effect on body weight (Fig. S3a), blood glucose (Fig. S3b), liver weight (Fig. S3c), or adipose tissue mass (Fig. S3d), indicating that the polysaccharides have no evident side effects on normal mice.

The daily administration of AAMP and AAMP-N at a dose of 50 mg/kg/d for 4 weeks in db/db mice caused a significant reduction in the blood glucose level compared to the control (Fig. 4a). The blood glucose reduction ratios were 24% and 30% caused by AAMP and AAMP-N, respectively (Fig. 4b), whereas AAMP-A showed little effects on blood glucose. Polysaccharides had no effects on body weight gain in mice during administration (Fig. 4c). In glucose tolerance tests (GTT), we found that treatment of AAMP and AAMP-N reversed impaired glucose tolerance in db/db mice during the 2 h period of the test (Fig. 4d). They also responded more dramatically to insulin injection, suggesting that AAMP and AAMP-N increase insulin sensitivity (Fig. 4e). Moreover, AAMP-N treatment resulted in a significant decline in serum insulin (Fig. 4f). Taken together, AAMP-N reduces blood glucose and increases insulin sensitivity.

Fig. 4.

AAMP-N treatment protected diabetic mice form severe insulin resistance. Diabetic mice were treated with 50 mg/kg/d of metformin and polysaccharides for 4 weeks (n = 10). (a) Blood glucose. (b) Blood glucose reduction. (c) Body weight. (d) Glucose tolerance test. (e) Insulin tolerance test. (f) Serum insulin level. Results represent mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.001.

3.5. AAMP-N regulates lipid metabolism in db/db mice

Abnormal deposition of fat results in insulin resistance. Therefore, we examined whether AAMP-N regulates lipid metabolism in mice. AAMP-N reduced ALT and AST levels in db/db mice (Fig. 5a and b), showing a protective effect of AAMP-N on liver. Besides that, TG and NEFA were significantly higher in diabetic mice, whereas AAMP-N treatment effectively reduced their levels (Fig. 5c and d). Consistent with these findings, diabetic mice treated with AAMP-N showed less accumulation of hepatic lipid droplets than untreated diabetic mice (Fig. 5e).

Fig. 5.

AAMP-N treatment suppressed lipid accumulation. (a) Serum ALT level. (b) Serum AST level. (c) Serum triglyceride level. (d) Serum non-esterified fatty acid level. (e) Representative images of liver sections from normal mice and diabetic mice. Scale bar: 100 μm. Results represent mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.001.

3.6. AAMP-N restored enlarged pancreatic islets

Although both AAMP and AAMP-N regulated lipid accumulation, the ability of AAMP-N in lowering blood glucose is better than that of AAMP (Fig. 4b). Therefore, we investigated whether AAMP-N affected pancreatic islets as another mechanism. Compared with normal mice, the islets of db/db mice underwent compensatory expansion and were significantly enlarged, likely due to peripheral insulin resistance (Fig. 6). In contrast, islets from AAMP-N-treated db/db diabetic mice were significantly smaller and denser in structure, suggesting that AAMP-N restored the size of islets and protected pancreatic islets from compensatory enlargement.

Fig. 6.

AAMP-N protected pancreatic islet integrity in diabetic mice. Representative images of immunofluorescence staining for insulin (green) and nucleus (blue) in the pancreatic islet of normal mice and diabetic mice, or treated with polysaccharides (n = 10). Scale bars: 100 μm.

4. Discussions

Mushrooms polysaccharides have hypoglycemic activity, but due to the different extraction and purification methods, the structures and activities of the active polysaccharides obtained vary widely [16]. To screen the most active polysaccharide component from mushrooms, we prepared total polysaccharides from six species fruiting bodies of mushrooms based on a unified purification method, and obtained neutral fractions and acidic fractions using DEAE-cellulose column chromatography. According to the monosaccharide composition, the neutral polysaccharides mainly contained Glc, Gal and Man, and acidic polysaccharide fraction mainly contained Glc. It was worth noting that the structural features of AAMP-N and AGRP-N were similar, presumably containing mannogalactoglucan domain.

In previous studies, we established insulin resistance cell model in which FG-treatment of hepatocytes results in reduced glucose uptake, mimicking the pathogenesis of obesity and type 2 diabetes [14]. Through this model, we found that neutral polysaccharides had better promotion in glucose uptake than acidic polysaccharides, especially AAMP-N and AGRP-N exerted the best activities. Thus, we speculated that mannogalactoglucan is a key active domain of mushroom polysaccharide contributed to ameliorate insulin resistance.

We used db/db mice to further elucidate the oral hypoglycemic effect of Amillariella mellea polysaccharides. From the results of blood glucose, GTT and ITT analysis, AAMP-N and AAMP showed pronounced hypoglycemic effect. Both of them could regulate lipid metabolism and improve insulin resistance to lower blood glucose. It was worth noting the results of AAMP were not consistent with in vivo and in vitro. We speculated that AAMP-N and AAMP might be degraded into some common active ingredients after oral administration. In addition, AAMP-N could restore damaged islets. However, the mechanism that AAMP-N protected islets from damage remained to be further investigated.

Accumulating literatures reported that polysaccharides extracted from nearly 20 mushroom species have hypoglycemic effects, most of them are from mycelia or fermentation broth [5]. Our study is first time to investigate the effect of mannogalactoglucan extracted from fruiting bodies of mushrooms on improving insulin resistance.

5. Conclusion

Insulin resistance is the main cause of diabetes. Mushrooms are a popular healthy food worldwide that have been used in the prevention and treatment of diseases. AAMP-N, the neutral polysaccharide of Armillaria mellea fruiting body, is identified as mannogalactoglucan. AAMP-N increases insulin sensitivity, lowers blood glucose, modulates lipid metabolism and protects damaged pancreatic islets in db/db mice. Combining these results, AAMP-N could be used as functional foods or drugs against diabetes.