Cholesterol-lowering properties of Ganoderma lucidum in vitro, ex vivo, and in hamsters and minipigs

Ex vivo hepatic HMG-CoA reductase and fractional cholesterol synthesis rate in hamsters

The observed inhibition of ex-vivo HMG-CoA reductase activity in hamsters treated with Gl has similarly been observed with Gl in rats [51], and with pure lanosterol analogs [44,52]. Our lack of effect with lovastatin (4.3 μmol/kg body wt) contrasts results with the related statin, simvastatin, where 10, 30, and 60 μmol/kg body wt/d increased ex-vivo hepatic HMG-CoA reductase activity 2-, 17-, and 50-fold, respectively [53]. Lovastatin could have different effects on HMG-CoA reductase and other enzymes than simvastatin, and was not however examined in the above study.

Lanosterol analogs such as those found in Gl are known to inhibit translation of HMG-CoA reductase mRNA, and may also accelerate protein degradation [44,52]. Gl may also affect cholesterol biosynthesis at latter biosynthetic steps such as the conversion of lanosterol [51], which could in turn, indirectly inhibit HMG-CoA reductase activity, as reported for statins in minipigs [53]. Indeed, it was reported that repression of the lanosterol 14 α-demethylase step can result in accumulation of 3 β-hydroxy-lanost-8-en-32-al, a known translational downregulator of HMG-CoA reductase [54].

If Gl had direct physical effects on HMG-CoA reductase activity, this implies that even after the 16 h fast employed in hamsters, Gl components were still bound to the enzyme during the assay procedure [55]. After the 16 h fast, lovastatin could have been removed from the enzyme accounting for the lack of observed effects of lovastatin on ex-vivo HMG-CoA reductase activity. Due to removal of the drug, other statins have even been found to increase ex-vivo HMG-CoA reductase activity [56]. Hepatic ex-vivo HMG-CoA reductase activity and whole body cholesterol FSR are entirely different types of measurements. It is not clear why Gl and lovastatin did not influence cholesterol FSR in hamsters. In principle, the low saturated fat-cholesterol condition employed via use of a chow diet, should have led to a high endogenous rate of cholesterol synthesis, one that could be inhibited by Gl and lovastatin. It is conceivable that the Gl and lovastatin became decomposed in the dietary mixture. To test this hypothesis, we re-extracted Gl and lovastatin from stored diets after culmination of the experiments, and found no differences in bioactive components analyzed, compared to the original starting materials (before addition to the diets; data not shown).

Cholesterol and triacylglycerol in hamsters and minipigs

Hamsters were fed a low-cholesterol chow-based diet with no added exogenous cholesterol or saturated fat. Under these conditions, there was not sufficient cholesterol to redistribute cholesterol from the HDL to LDL pool [29]. This is why in hamsters, 5% Gl and lovastatin reduced D18 TC and HDL, but not LDL [57,58].

Using the same types of diet, lovastatin was similarly found to preferentially reduce HDL in hamsters; and only when dietary saturated fat was added, were both LDL and HDL reduced [57].

Another factor contributing to the lack of strong effects in hamsters, and the total lack of effect in minipigs may be that the dose of lovastatin was insufficient. In hamsters, the employed dose of 2 mg lovastatin/100 g diet is ca. 4.3 μmol lovastatin/kg body wt. Himber et al. [57] treated hamsters with 25 μmol lovastatin/kg body wt, which lowered HDL; or 50 μmol, which lowered LDL and HDL [57]. Morand et al. [53] found that 20–200 μmol simvastatin/kg body wt was sufficient to reduce LDL. Ma et al. [59] reduced lipoproteins in hamsters with 100 mg lovastatin/100 g diet. In minipigs, we utilized a dose of 80 mg lovastatin/minipig/d, which may also have been on the low side. A dose of 24–42 mg was sufficient to lower lipoproteins in Hyde Park minipigs [60]. Nevertheless, our particular species, strain, and location of minipigs may have responded less aggressively to lovastatin (M. Huff, Personal Communication, December 2000). In Göttingen minipigs, a dose of 80 mg simvastatin lowered LDL, whereas 240 mg lowered LDL and HDL [53]; simvastatin is likely more effective in minipigs than lovastatin at a similar dietary weight percent [61,62].

The reduction in TAG with Gl was likely due to lower D1 TAG values in the Gl groups relative to control. TAG reductions in hamster models typically occur under conditions of higher saturated fat intake [6,63]. In the only other peer-reviewed study examining cholesterol lowering properties of Gl in a small animal model, 5 dietary wt% dried Reishi mushroom powder was found to decrease TC in SHR rats; effects on VLDL, LDL and HDL were not studied [2]. In minipigs, with the high fat-cholesterol feeding conditions employed, a Gl-induced inhibition of cholesterol synthesis should result in less availability of hepatic cholesterol for lipoprotein synthesis. In turn, this has the potential effect of reducing plasma VLDL cholesterol secretion, reducing LDL direct secretion; and possibly reducing VLDL-LDL conversion [64,65]. In the present work, we did not observe differences in TAG or VLDL in pigs fed either Gl or lovastatin, however this effect could have been missed since the VLDL pool represented only a small lipoprotein pool and/or there was efficient VLDL-LDL conversion. The reductions in both LDL and HDL with Gl is consistent with that seen with higher statin doses [53].

Fecal bile acids and neutral sterols in hamsters and minipigs

In hamsters, Gl increased fecal total bile acids and chenodeoxycholate, whereas both doses, increased coprostanol 3-one; the 5% dose decreased cholestanol for unclear reasons. An increase in fecal chenodeoxycholate likely indicates production or recycling of chenodeoxycholate was enhanced.

Plasma levels of cholestanol are positively associated with cholesterol absorption [66]; whereas decreased fecal cholestanol may indicate plasma cholestanol was increased and cholesterol absorption was enhanced. In minipigs, Gl tended to increase fecal cholestanol, the opposite pattern to that of hamsters fed 5% Gl. Coprostanol and coprostanol 3-one are the bacterial products of cholesterol, which are increased when fecal cholesterol is increased, or when gut flora are altered [67]. Since fecal cholesterol and coprostanol levels were not changed by either dose of Gl, it is not obvious why coprostanol 3-one accumulated.

Bile salts are now known to possess many different functions acting as detergents, activators of protein kinase C and phosphatidylinositol-3 kinase; and being important gene regulators [68,69]. Chenodeoxycholate, deoxycholate, and their glycine and taurine conjugates can lead to farnesoid X receptor/retinoid X receptor (FXR/RXR)-induced activation of intestinal bile acid binding protein transcription (I-BABP), and suppression of CYP7α RNA and protein levels (FXR prevents liver X receptor (LXRα)-induced transactivation of CYP7α). CYP7α regulates the committed step in classical bile acid synthesis. Overall, an increased fecal level of chenodeoxycholate would mean less chenodeoxycholate is available to activate FXR. Less activation of FXR would lead to less bile acid recycling and less inhibition of bile acid synthesis, more hepatic cholesterol converted to bile acids, and a lowering of plasma cholesterol.

Overall, it is likely that fibrous and/or lipophilic sterol-like molecules in Gl altered the absorption and recycling of bile acids and neutral sterols, leading to altered fecal accumulation. Monitoring plasma levels of neutral sterols and bile acids, and quantifying conjugated and de-conjugated bile acids, should help to clarify the potential importance of the observed trends.