Diabetes mellitus, or simply diabetes is the metabolic disorder with a chronic high blood glucose level [1]. This could yet provoke serious clinical complications, such as retinopathy, neuropathy, and nephropathy, subsequently leading to blindness, renal failure, amputation, coma, and death [2][3]. Nearly 50 million US population might have diabetes (including undiagnosed) in 2025 and its incidence keeps rising every year [4]. Hence, it is urgently needed to control or stop steadily growing such an incidence.

Diabetes is classified into two types: Type 1 (insulin-dependent diabetes) and Type 2 (non-insulin-dependent diabetes) [5]. Type 1 diabetes is primarily caused by insulin deficiency, representing <10% of all cases of diabetes, while Type 2 diabetes with a >90% incidence rate [6] involves multiple factors, such as defects in insulin secretion, insulin resistance (at muscle and adipose tissue), and elevated hepatic glucose production [7][8]. Regarding treatment, Type 1 diabetes, due directly to insulin deficiency, is relatively more manageable through insulin injection; however, Type 2 diabetes is not primarily linked to insulin deficiency but rather linked to peripheral insulin resistance, i.e. insulin insensitivity at peripheral sites [8]. Due to this insulin resistance, current oral therapy using sulfonylurea derivatives [7], stimulating insulin secretion from pancreas, was often unsatisfactory. Medications such as troglitazone and metformin were also developed to enhance peripheral insulin sensitivity [9][10]. Despite some improvements in glycemic control, they have been reported to have potential adverse effects. Because of these drawbacks, it makes even harder to adequately and effectively treat Type 2 diabetes. Hence, more effective treatment must rely mainly on how to overcome insulin resistance, but it should be also safer unlike drugs currently being used.

We were particularly interested in a natural extract called, SX-fraction (SXF), obtained from maitake mushroom (Grifolafrondosa). SXF is a water-soluble glycoprotein with a molecular weight of ~20,000 Da, and a number of scientific/medical studies have been performed, including its hypoglycemic and anti-diabetic activity on diabetic mice [11]-[13]. Hence, the safety of SXF is acceptable based on these animal studies and it unlikely has side effects because of its natural nature. Now, it is worth mentioning that maitake mushroom is not just another mushroom but has promising potentials. While it is an edible, tasty mushroom often used in cooking, it has provided us with certain health benefits as well. Prior to SXF, another distinct extract called, Maitake D-fraction (PDF), was initially extracted from this mushroom. A number of studies conducted on PDF in the past 40 years then revealed its significant medicinal and physiological properties, including anticancer, immunostimulatory, anti-viral activities etc. [14]-[16]. In addition, the safety of PDF was also granted by the facts that the US Food and Drug Administration (FDA) exempted it from a Phase I Toxicology study and approved it for the Investigational New Drug (IND) application as well [17]. Thus, this further confirms the safety of maitake mushroom extracts (PDF and SXF), which can be taken safely by patients as well as normal subjects.

Accordingly, we investigated if original maitake mushroom or a different dose of SXF given would indeed have anti-diabetic activity on diabetic KK-A^y mice, which are widely used as an excellent model for the studies related to Type 2 diabetes [18]. Several diabetic and lipid parameters, such as the levels of serum glucose (GLC), insulin (INS), triglycerides (TGL), total cholesterol (CHL), and high-density lipoprotein (HDL), were recorded for 8 weeks. Any positive or negative effects on these parameters were rationally interpreted and discussed herein.

To find a safer and more effective option to treat diabetes, specifically Type 2 diabetes, we investigated if natural products, instead of ordinary medications, might have anti-diabetic activity to improve diabetic conditions. The natural bioactive extract, SX-fraction (SXF) obtained from maitake mushroom, was chosen to study its potential anti-diabetic activity in diabetic KK-A^y mice. We examined the effects of original/raw maitake mushroom powder, SXF (with the high or low dose), or Actos^® (ACT) used as a reference drug, on several diabetic parameters in mice. Those included body weight, GLC, 1,5-AG, INS, TGL, CHL, and HDL, which were monitored and evaluated during the 8-week experimental period.

All mice in the 5 groups (Sham, Orig-M, SXF (H)/(L), and ACT) gradually gained weight and grew normally during a course of study. Hence, those different materials given to mice had little effects on their body weight or may not affect their overall metabolism.

We found that the GLC levels significantly declined with Orig-M or SXF (L)/(H), demonstrating the hypoglycemic effect (i.e., the lowering of the GLC level). In fact, such a GLC decrease with SXF (L)/(H) was yet lower or better than that with Actos^® (ACT) medication used as a reference. This is rather interesting and quite significant.

The 1,5-AG level was also examined because it was often used for assessing the short-term glycemic control [21][23]. Actually, this 1,5-AG test has been commercially available since 1991 in Japan, and it is structurally similar to glucose but specifically the 1-deoxy form of glucose [24]. Because of such a structural similarity (to glucose), it has a physiological significance that the absorption of 1,5-AG is competitively inhibited by glucose in the kidneys (specifically renal tubules) [24]. Normally, nearly all of 1,5-AG filtered by the kidney will be reabsorbed, maintaining its consistent serum levels. However, when the serum glucose levels are high (in diabetic patients), the reabsorption of 1,5-AG is prevented by glucose, resulting in the excretion of 1,5-AG in the urine and thus decreasing its serum levels [23]. Hence, as soon as a rise in serum glucose occurs, 1,5-AG will fall immediately. This is why 1,5-AG is a useful marker for assessing the short-term glycemic control. Nevertheless, no significant 1,5-AG changes in the experimental groups were observed in this study.

Next, an elevation or increase in the INS level (hyperinsulinemia) is commonly seen in the early to mid-stage of Type 2 diabetic patients [25]. This is due to “insulin resistance” [8], a possible, primary cause of Type 2 diabetes, where insulin is overproduced (in the pancreas) to lower/control the elevated glucose levels. It is the compensatory mechanism for insulin resistance, resulting in the higher or increased insulin levels [25][26]. Our study then showed that the INS levels were somewhat fluctuated with those materials given. Particularly, the effects of SXF (H)/(L) were recognizable in the 8^th week—the INS level was significantly lowered with SXF (H), while SXF (L) also distinctively lowered the level, although that was not statistically significant. Therefore, SXF (H)/(L) appear to effectively decrease or control the INS levels, at least in part demonstrating their anti-diabetic properties.

It was also important to address the effects of materials given on the cardiovascular parameters closely associated with diabetes [22]. We found that the levels of TGL, CHL, and HDL in the different groups were not significantly affected throughout the study period. However, it was rather surprised to see that the levels of both TGL and CHL in the materials-given groups were actually higher than that of the Sham group, while the HDL levels were nearly the same as that of the Sham. These findings are rather unexpected because the reduced TGL and CHL but the elevated HDL levels (with these given materials) would indicate the improved diabetic conditions. However, since such fluctuations in the TGL, CHL, and HDL levels are observable but not statistically significant, it remains uncertain whether they are truly the facts that should be considered and accepted. Hence, more studies might be required for further clarification.

After all, this study reveals the key finding that Orig-M and SXF are indeed capable of significantly lowering the GLC and INS levels in KK-A^y mice, demonstrating the anti-diabetic property, although little effects on serum lipid profile were found.

Moreover, it was also the interesting finding that the effects of SXF on GLC and INS appeared to be similar or more effective than Actos^® (ACT) being clinically used today. Actos^® (pioglitazone) belongs to the thiazolidinedione family and has been approved for the treatment of insulin resistance in Type 2 diabetes by the US FDA [20]. It promotes insulin sensitivity, improving the uptake of blood glucose through the peroxisome proliferator-activated receptor-gamma (PPARγ), ligand-activated transcription factors [27]. They play a key role in numerous metabolic processes, particularly glucose and lipid homeostasis. Pioglitazone is yet known to have side effects, such as fluid retention (peripheral oedema), cardiac hypertrophy, hypoglycemia etc. [28]. This thus suggests that SXF could be a safer and more effective alternative option.

Besides this animal study, we have also conducted the small, volunteer-based clinical study [29] to find the hypoglycemic (lowering GLC) effect of SXF, which was demonstrated in this mice study. Briefly, 10 patients took a SXF tablet (500 mg) three times a day for 4 weeks, and their fasting blood glucose (FBG) levels were measured before SXF intake (Day 0), at 2 weeks (Day 14), and after SXF trial (Day 28). These results are shown in Table 2.

All 10 patients showed a 30-63% decrease of FBG from an average FBG of ~205 mg/ml to that of ~116 mg/dl. That was an average of ~42.3% decline in their FBG levels under a SXF regimen in 2 to 4 weeks. The apparent FBG decreases were yet noticed in 2 weeks after the first SXF intake, and those values kept declining to 4 weeks. Moreover, none of participants presented palpable ailments or adverse effects related to SXF during the trial, confirming its safety even in human use. Although these results are encouraging and promising, more studies with more patients are certainly required for the further confirmation of hypoglycemic effect of SXF on Type 2 diabetic patients.

Table 2.Hypoglycemic effects of SXF on type 2 diabetic patients.

*Before and After SXF indicates the FBG values measured at Day 0 and Day 28, respectively. SXF: SX-fraction; FBG: fasting blood glucose; M: male; F: female.

Lastly, the legitimate question would be raised—how does SXF lower the GLC level? How does it work? Actually, we explored the hypoglycemic mechanism of SXF in vitro, using skeletal muscle cells [29]. We hypothesized that SXF might activate the insulin signal transduction (IST) pathway [30], overcoming insulin resistance, which is believed to be the primary cause of Type 2 diabetes. Briefly, the IST pathway involves the sequential events with several key regulators, starting from activation of insulin receptor (IR), insulin receptor substrate 1 (IRS-1), and protein kinase B (Akt) to translocation of glucose transporter type 4 (GLUT4). Once a cascade of signaling events was successfully completed, extracellular glucose will be transported to the (muscle) cell trough GLUT4, resulting in the increased glucose uptake and the decreased extracellular glucose level. It should be yet noted that the IR plays a critical role in the IST pathway because it is involved in a committed step, triggering the pathway [31]. Hence, (re)activation of IR with SXF could be the critical event, accounting for its underlying hypoglycemic mechanism to overcome insulin resistance.

The present study shows that maitake mushroom itself and its extract, SX-fraction (SXF), have anti-diabetic activity in diabetic KK-A^y mice. Particularly, SXF demonstrated its anti-diabetic property, capable of lowering the serum glucose and insulin levels. However, SXF had little effects on other diabetic or lipid parameters in mice. Since the control of serum glucose and insulin levels is especially crucial in Type 2 diabetic patients, SXF may offer alternative anti-diabetic option with safety and efficacy. Further studies are thus warranted.

We thank Mushroom Wisdom, Inc. for providing us with experimental materials and financial support in this study.

Financial support was generously provided by Mushroom Wisdom, Inc.

Sensuke Konno: Conceptualization, Validation, Writing-Original Draft; Daniel Bassily: Investigation, Software; Charles White: Investigation, Data Curation; Sean Fullerton: Project administration, Data Curation; Cun Zhuang: Methodology, Resources, Supervision.

All data in this study are available from the corresponding author upon appropriate request.