Diabetes mellitus (DM) is a metabolic disorder caused by a lack of insulin and/or pancreatic dysfunction characterized by hyperglycemia. DM is a common, morbid and costly disease, affecting more than 1 in 10 adults in both United States and China [1]. DM is the leading cause of new blindness, amputation and end-stage renal diseases and it also contributes to a host of other conditions. Although there is no published data for the medical cost of diabetes in China in recent years, the medical costs of managing DM and its complications exceeded $115 billion in the United States alone in 2011 and the indirect costs added another $58 billion [1]. Evidence suggests that DM complications can be markedly attenuated with appropriate control of blood pressure and hyperglycemia and with successful treatment of hyperlipidemia. Thus, there is a great interest in novel approaches to indirect DM management. However, despite the increasing number of drugs available for DM treatment, significant improvements in the control of DM have not been observed [2].

Previous studies have confirmed that the treatment with natural antioxidants can reduce diabetic complications [3] and the continuous efforts to discover new antioxidants as useful drug candidates to combat diabetic complications are on-going. Ganoderma lucidum (Leyss; Fr) Karst. (Ganodermataceae) is a well-known Chinese traditional medicine which has been clinically used in China, Japan and Korea for more than 2000 years. Mushrooms of the genus Ganoderma have been shown to be a rich source of biologically active metabolites [4], containing many bioactive components, including triterpenoids, polysaccharides, nucleotides, sterols, steroids, peptides and other bioactive ingredients [5]. G. lucidum spores contain high levels of ganoderic acids, ergosterol peroxide and pentadecanoate [6]. Many are active against current major chronic diseases. For example, ganoderic acids, one group of triterpenoids existing in the fruiting body of G. lucidum showed anti-androgenic, anti-5 α-reductase, anti-inflammatory and anti-tumor and a range of other biological activities [7]–[9].

Although the fruiting body of G. lucidum has been used as a traditional herbal medicine since ancient times, the spores were utilized only in the late 20th century [10]. The spores contain many bioactive substances, including lanostane type triterpenes [11] and polysaccharides [12] similar to those in the fruiting body [13]. Other characteristics of the bioactive compounds existing in the spores are those they are also rich in fatty acids, in particular long-chain C-19 fatty acids. Previous study demonstrated that these fatty acids could inhibit tumor cell proliferation and induce apoptosis in the HL-60, promyelocytic leukemic cell line [14]. Meanwhile, other research also showed the potential anti-hyperglycemic effect in diabetic rats using polysaccharides extracted from G. lucidum fruiting body [15]. However, to the date, few detailed studies described the effects of G. lucidum spores on blood glucose and lipid compositions in streptozotocin (STZ) induced diabetic rats and neither of the investigation of G. lucidum spores intervention on the gene expression of glucose and lipid metabolisms has been reported in above diabetic model. To the best of our knowledge, there are few reports in the literature evaluating the feasibility of using G. lucidum spores as a potential anti-diabetic agent and the descriptions of the molecular mechanism(s) involved in these processes are also very rare. Moreover, the co-existing of the multi-active compounds in G. lucidum spores might provide a stronger synergistic or positively effect on improving the diabetic status than the consumption of the single active compound. Therefore, in this study, STZ-induced-diabetic rats are used to investigate the changes in the expression levels of genes involved in lipid and glucose metabolisms after G. lucidum spores treatment.

Although previous research has shown that the fruiting bodies of G. lucidum could reduce blood glucose and plasma cholesterol levels [17]–[19], few studies have investigated the molecular mechanisms associated with these activities. Our study combines the data of biochemical analysis and gene expression and indicates that GLSP intervention could result in a partial recovery of body weight, reduction of blood glucose level and improvement of lipid compositions and even attenuation of oxidative stress in the STZ-induced diabetic rats. These improvements might be highly associated with the changes in the expression levels of the related genes.

The current study would suggest that GLSP intervention could manipulate hyperglycemic and hyperlipidemic status with a significant reduction of blood glucose level and the improvement of blood lipid compositions (TG, TC, HDL-c) in type 2 diabetic rats (P < 0.01). The GLSP dosage used in this study was 1 g per day, which was about 3 % of the total diet. Although this dosage did not achieve a completely functional recovery of the DM rats to a normal status, it could be used as a reference dosage for improving the symptoms of DM. In contrast, Winther et al.[20] reported that G. lucidum as monotherapy lowered C-peptide significantly (P < 0.001), leaving HDL-c parameter unchanged.

As shown in Table 3, the level of oxidative stress in diabetic rats was improved with the reduction of MDA and the increase of SOD and GSH-Px following GLSP intervention. Meanwhile, The GLSP intervention also led to a reduction of ROS level compared to the model control, although the difference was not significant. Considering that MDA is produced from ROS, the higher level of MDA may promote polyunsaturated fatty acid peroxidation. Previous studies have also shown a strong relationship between MDA levels and different pathological stages of diabetes, because MDA concentration increased considerably in diabetes mellitus [21]. Thus, the controlling of MDA concentration would be helpful in maintaining a suitable level of oxidative stress. Antioxidant enzymes, including SOD and GSH-Px are vital defenses against ROS and they are important in inhibiting oxygen radical formation and usually act as biomarkers for indicating ROS production. This study found that the activities of antioxidant enzymes SOD and GSH-Px were significantly decreased in the model control group compared to the normal controls, indicating a lower antioxidant defense caused by diabetes. However, the administration of GLSP significantly enhanced the activity of SOD and GSH-Px (Table 3), suggesting that the antioxidant compounds present in GLSP might enhance plasma antioxidant capacity in diabetic rats. One plausible mechanism for interpreting the anti-hyperglycemic function of GLSP might be through its scavenging ability to protect pancreatic cells from oxygen-radical damage, supporting an increased secretion of insulin. Previous research has also demonstrated that a high dosage of antioxidant compounds had a favorable effect on glucose homeostasis in obese subjects with the improvements in their Homeostasis Model Assessment (HOMA) index, thus exerting a positive effect on insulin sensitivity [22].

Previous reports have shown the anti-hyperglycemic activity of GLSP [17], [18] and this function was further highlighted in this study (Table 1). A significant elevation of insulin levels in rats was also demonstrated after the administration of GLSP (unpublished data), which was also accompanied by a decreased blood glucose level. The current study is consistent with previously reported work [23]. Thus, the current results could further support the evidence from previous studies on the anti-hyperglycemic effect of GLSP through decreased glucose level in animals accompanied with increased insulin levels and improvements in pancreatic cell function [24]–[27]. However, the mechanisms associated with this benefit are not completely clear. Therefore, in light of our current results, it might be hypothesized that the benefits derived from GLSP intervention in the diabetic rats could be partially associated with its roles in promoting glycogen synthesis although gluconeogenesis was not significantly affected (PECPK and G6PC1 in Fig. 1). Another plausible reason may be associated with insulin regulation. The expression level of insulin induced genes, in particular Insig-1, was enhanced significantly in GLSP intervention group compared to model control group. It has been shown that Insig-1 and Insig-2 play an important role in glucose homeostasis [28] and they also have a key function in the regulation of intracellular cholesterol and fat metabolism [29]. Consistently, previous reports have confirmed that a lower glucose concentration could promote the expression levels of Insig-1 and Insig-2 genes [30] and Insig-1 inhibited lipid accumulation and free fatty acid (FFA) synthesis in a time-dependent manner [31]. In addition, the hepatic over-expression of Insig-1 (or Insig-2) would also inhibit the activation of SREBP-1 c in the rat liver [32]–[34], a key factor in the control of hepatic glucose metabolism and the manipulation of glucose homeostasis associated with insulin. Although there was no significant change in the expression level of SREBP-1 with the increased expression level of Insig genes in our study, post transcriptional regulation might play some roles as well.

In this study, the significant improvement of blood lipid compositions confirmed that GLSP could be used as an efficient therapy for treating hyperlipidemia caused by diabetes and these improvements in blood cholesterol and triglyceride levels might be related to the reduced blood glucose level in the diabetic rats [35], [36]. Nevertheless, other study also suggested that G. lucidum consumption could result in a great suppression of elevated total cholesterol levels, which might be associated with the inhibition of the hepatic phosphoenolpyruvate carboxykinase gene expression [17]. The HMG CoA reductase activity associated with lipoprotein metabolism may also contribute to the hypolipidemic effects following GLSP treatment, which is similar to the effects of other plant medicines [37], [38]. Since Insig genes are also associated with lipid metabolism, acyl-CoA oxidase plays an important role in plasma lipid compositions. Fatty acid degradation in most organisms occurs primarily via β-oxidation. Both mitochondrial and peroxisomal β-oxidation catalyze the shortening of acyl-CoA esters chains, which requires the participation of β-oxidation enzymes, including acyl-CoA dehydrogenase and acyl-CoA oxidase (Acox) [39]. In our study, the Acox1 gene was up-regulated by over 5-folds in the diabetic rats following GLSP treatment, which might provide some clear evidences that GLSP could reduce TG level mainly through promoting lipid β-oxidation (Fig. 2). Thus, based on above results in this study, a relationship between GLSP intervention and the expression of genes involved in glucose and lipid metabolisms in the diabetic rats is summarized in Fig. 3. In brief, GLSP in the diet of type 2 diabetic rats could reduce the blood glucose level possibly through the increasing of the gene expression level of glycogen synthesis (GS2 and GYG1) and glucose homeostasis (Insig-1 and Insig-2). In particular, plasma lipid compositions (TG and TC) decreased concomitantly with increased gene expression levels of Acox1 and Insig-1/2.

In summary, this study has shown that G. lucidum spores could be used as an ingredient for attenuating diabetic mellitus through potential anti-hyperglycemic and anti-hyperlipidemic activities. More importantly, the relationships between GLSP intervention and the changes in the gene expression levels of the related glucose and lipid metabolic pathways might highlight a potential model for interpreting the action of GLSP treatment.