ABSTRACT
Diabetic rats were daily fed with a high-cholesterol diet containing 1% or 3% freeze-dried whole submerged G. lucidum culture or its mycelia for 5 weeks. Body weight, adipose tissue weight and plasma triglyceride levels were reduced, while high-density lipoprotein-cholesterol levels were elevated in rats fed with G. lucidum powder supplement diets. Notably, G. lucidum supplements downregulated the activities of hepatic acetyl-CoA carboxylase, fatty acid synthase and lipoprotein lipase, but upregulated the activity of hormone-sensitive lipase in the perirenal adipose tissues. Moreover, G. lucidum supplements increased the faecal triglyceride excretion. Therefore, daily supplementation of submerged G. lucidum culture, especially mycelia, can ameliorate dyslipidemia and reduce visceral fat accumulation in diabetic rats fed with a high-fat diet, which is closely related to the modulation of lipid synthesis, metabolism, and excretion.
Introduction
Type 2 diabetes is a chronic metabolic disease with hyperglycaemia and insulin resistance. It is mainly caused by obesity and accounts for 90% of diabetes cases. (Wu et al. 2014). The symptoms of type 2 diabetes usually develop slowly. As a result, complications such as cardiovascular disease have often been associated with the diagnosis of diabetes (Grundy et al. 1999). Increasing evidence suggests that lipid metabolism and glucose metabolism are equally important in the development of diabetes. Visceral fat is thought to be the culprit in the development of insulin resistance, which plays an important role in the pathogenesis of dyslipidemia in type 2 diabetes (Patel and Abate 2013). Dyslipidemia is a well-recognised and modifiable risk factor and should be detected early to prevent cardiovascular diseases (Nelson 2013). Type 2 diabetes is associated with abnormal plasma lipids and lipoproteins, including decreased high-density lipoprotein cholesterol (HDL-C), predominant low-density lipoprotein cholesterol (LDL-C), and elevated triglyceride (TG) levels. These dyslipidemia characteristics increase the risk of cardiovascular disease (Nelson 2013). Increased liver secretion of very low-density lipoprotein (VLDL) and impaired clearance, leading to its conversion to LDL, are crucial in the pathophysiology of dyslipidemia (Krauss 2004). Although proper diet, exercise, and weight management may improve diabetic dyslipidemia and body fat accumulation, pharmacological treatment is still needed (Krauss 2004).
Ganoderma lucidum is a well-known medicinal mushroom that has been used as a health supplement (Galor et al. 2011). G. lucidum is widely consumed because it has various medicinal properties including antitumor, immune regulation, liver protection, antioxidant, anti-ageing, hypoglycaemic and hypolipidemic effects (Galor et al. 2011). In recent decades, G. lucidum has attracted great interest from scientists due to its potential as an alternative adjuvant therapy for diabetes. Both carbohydrate and lipid metabolism are targets of G. lucidum for improving the outcome of diabetes. For example, 26-oxysterols isolated from G. lucidum fruiting bodies showed an inhibitory effect on cholesterol synthesis (Hajjaj et al. 2005). Lanostane triterpenes harvested from G. lucidum fruiting bodies suppressed 3T3-L1 adipocytes differentiation (Lee et al. 2010). Genetic resources of Mexican G. lucidum showed cholesterol-lowering properties in mice fed a high-cholesterol diet (Meneses et al. 2016). The extracts of G. lucidum fruiting bodies, spores, and a proteoglycan PTP1B inhibitor isolated from G. lucidum also showed hypolipidemic activities in diabetic rodents (Wang et al. 2012, 2015; Bach et al. 2018). Moreover, water extracts of mycelia of G. lucidum have been reported to reduce the weight of the body, liver, and adipose tissue of mice fed a high-fat diet (Chang et al. 2015). At the same time, the results of clinical studies showed that G. lucidum could improve the occurrence of diabetic dyslipidemia (Chu et al. 2012). Therefore, it has been considered as a potential therapeutic candidate for lipogenic diseases.
Ganoderma fungus is composed of fruiting body (the basidiocarp), mycelium and spores (Galor et al. 2011). Compared with the cultivation of fruiting bodies and spores, the submerged G. lucidum culture has the advantages of lower production and time cost, less space requirements, easy-to-control culture conditions, and higher yield, purity and regeneration ability. (Galor et al. 2011). However, the components and their relative proportions in G. lucidum from basidiocarp cultivation are different from those in submerged culture (Galor et al. 2011).
As the effects of submerged G. lucidum culture on blood lipid and visceral fat accumulation are unknown, we used a type 2 diabetic rat model to deepen the understanding of the ameliorating effects of submerged G. lucidum cultures and their mycelia on hyperlipidaemia and visceral fat accumulation. Streptozotocin (STZ)-induced diabetic rats were daily fed with a high-cholesterol diet containing freeze-dried whole submerged G. lucidum cultures or mycelia for 5 weeks. Body weight and adipose tissue weight as well as TG, total cholesterol (TC), HDL-C, LDL-C, and VLDL-C levels were measured. Since dyslipidemia and visceral fat accumulation are involved in lipid synthesis, metabolism and excretion, the activities of key enzymes and faecal excretion of TG and TC were studied to clarify the underlying mechanism behind the beneficial effects of submerged G. lucidum cultures.
Materials and methods
Culture, chemicals and reagents
G. lucidum BCRC 36123 was obtained from Bioresources Collection and Research Centre (Hsinchu, Taiwan). All chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) unless otherwise stated. AIN-93 vitamin mixture and AIN-93 mineral mixture were purchased from MP Biomedicals LLC (Santa Ana, CA, USA). The rat insulin enzyme-linked immunosorbent assay kit was purchased from Randox Laboratories, Ltd. (Crumlin, UK), and the glucose detection kit was purchased from Audit Diagnostics (Cork, Ireland).
Submerged culture of G. lucidum and sample preparation
Pieces of mycelium pad (0.5 x 0.5 cm2) of G. lucidum BCRC 36123 grown on the Gano medium (2.4 g glucose, 0.6 g yeast extract in 100 mL deionised water) agar plate at 30°C for 7 days were put into 1 mL frozen preserving medium (10 mL glycerol, 2 g dextran, 2 g trehalose dehydrate in 90 mL deionised water) for storage at −80°C. The frozen pieces of G. lucidum mycelium pad inoculated on Gano medium agar plates were incubated at 30°C for 7 days and two mycelial pieces (1 x 1 cm2) from the culture plate were added into a flask containing 250 mL Gano medium and incubated at 30°C, 110 rpm for 7 days. Based on the method described by Chang et al. (2006), the extracellular polysaccharides and mycelia in cultures were separated and measured. Whole submerged cultures and collected mycelia were freeze-dried and stored at −20°C for further experiments.
Animals and experimental design
Male Sprague Dawley rats (8 weeks old) were purchased from BioLASCO Taiwan Co., Ltd (Taipei, Taiwan). The rats were housed in stainless steel cages with diet and water provided ad libitum in a temperature- and humidity-controlled animal room. All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Taiwan Ocean University, and the rats were handled and euthanised in accordance with its guidelines.
On arrival, rats were fed with normal diet for 1 week and then randomly divided into six groups (n = 8): normal control (NC), diabetic control (DC), and four experimental groups including diabetic rats fed with 1% or 3% freeze-dried whole submerged culture of G. lucidum powder supplement diet (1G or 3G), and diabetic rats fed with 1% or 3% freeze-dried submerged culture of G. lucidum mycelia powder supplement diets (1M or 3M). The rats for diabetic control and experimental groups were subcutaneously injected with nicotinamide (230 mg/kg B.W.) and STZ (65 mg/kg B.W.) for the induction of type 2 diabetes (Masiello et al. 1998). One week later, oral glucose tolerance test was performed to confirm the successful induction of diabetes (Figure S1), and the rats were then daily fed with a high-cholesterol diet containing 1G, 3G, 1M, or 3M for 5 weeks. The composition of employed diets is shown in Table 1. Body weight and adipose tissue weights were measured, and samples of blood, faeces, liver, and perirenal and epididymal adipose tissues were harvested after sacrifice for further analysis