Molecular actions of hypocholesterolaemic compounds from edible mushrooms

Abstract

Cholesterol levels are strictly regulated to maintain its homeostasis; therefore, if it is not absorbed with the diet, the cholesterol biosynthetic pathway is enhanced and vice versa. Nowadays, the commonly prescribed therapeutic treatments for hypocholesterolemic patients are targeted toward the reduction of both cholesterol intestinal absorption and/or its endogenous biosynthesis. But, when hypercholesterolemia is still moderate the consumption of food products with cholesterol-lowering capacities is more desirable than using drugs. Marketed foods supplemented with hypocholesterolemic compounds are only inhibiting mechanisms for cholesterol absorption (i.e. phytosterols and cereal β-glucans). However, certain fungal extracts obtained from edible mushrooms might be able to modulate cholesterol levels by both strategies, pharmaceutical drugs and functional foods. In vitro and in vivo studies indicated that fungal sterols down-regulated genes involved in cholesterol homeostasis (such as Srebf2 and Nr1h4 (FXR)) and other specific mushroom extracts (β-glucans and other water-soluble compounds) also stimulated transcriptional profiles similar to simvastatin or ezetimibe (two hypocholesterolemic drugs). These and other observations suggested that the hypocholesterolemic effect of mushroom extracts could be due to transcriptional and post-transcriptional modulations besides other indirect effects.

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1. Introduction

Although cardiovascular disease (CVD) incidence has been decreasing over the past decades due to medical advances and advice, they are still the second leading cause of premature death in the Western world after cancer.¹ Already for many years, health authorities have been reinforcing efforts to inform people about what CVDs are, their main symptoms, their health consequences and the way to decrease the risk of suffering them.

CVD risks are influenced by genetic factors such as specific tendencies to obesity, hypertension, etc., and gender or age; however, many risk factors are also modulated by lifestyle habits such as smoking, sedentary/involved in sports or in-/adequate diets² and this is the reason that CVDs are considered as multifactorial diseases.

Several biomarkers are used to evaluate the real individual CVD risk, i.e. elevated homocysteine, coronary artery calcium-phosphate calcification or elevated fibrinogen blood level but, the most useful marker for CVD risk is the LDL (low-density lipoprotein)-cholesterol concentration in serum, one of the major transport cholesterol complexes. LDL-cholesterol constitutes 60–70% of total serum cholesterol and a blood concentration higher than 200 mg dL⁻¹ is considered, nowadays, as a risky level.^3,4

Due to the increasing interest of modern society for natural products as agents able to decrease the consumption of pharmaceutical drugs, new food products with high levels of hypocholesterolemic compounds are being explored. Nowadays, the cholesterol-lowering effect of compounds such as plant sterols (phytosterols, phytostanols and derivatives);^5,6 dietary fibers such as β-glucans or chitosans;⁷ peptides such as those derived from soy proteins⁸ or bovine milk β-lactoglobulins⁹ have been studied and therefore, several functional food products supplemented with these compounds are available at the supermarkets.^10–14

Some edible mushroom species are able to lower cholesterol levels in serum since they are a good source of fungal sterols (with high structural similarity to phytosterols), dietary fibers such as β-glucans and chitins (fungal polysaccharides), β-lactones, specific proteo/glucan complexes or compounds such as eritadenine.¹⁰ Recent studies pointed out that ergosterol-enriched extracts and other fungal compounds were also able to modulate cholesterol-related gene expression in animal intestine and liver.¹⁵

Therefore, in this work, the molecular events occurring during cholesterol synthesis and absorption are reviewed analyzing the influence of some hypocholesterolemic compounds obtained from edible mushrooms.

2. Cholesterol metabolism

Cholesterol, together with phospholipids, modulates membrane fluidity influencing transport through them, permeability, configuration of membrane proteins or enzyme activities. Furthermore, cholesterol is involved in many metabolic pathways since it is a precursor of a wide range of biological molecules such as bile acids (i.e. cholic acid), steroid hormones (i.e. testosterone) and lipophilic vitamins (i.e. vitamin D₃).⁷ This sterol is synthesized mainly in the liver, besides other organs such as adrenal glands, intestine or ovaries. But, it can also be incorporated from the diet after the digestion process. Thus, well-balanced mechanisms of cholesterol synthesis, bile acid catabolism, cholesterol intake and excretion through faeces will maintain healthy and stable cholesterol values in the serum (homeostasis). Until a few years ago, the liver was considered the main control center of cholesterol homeostasis; however, more recent studies indicated the intestine as a tissue highly involved in the regulation of plasma cholesterol levels and homeostasis.^13,16,17

2.1. Molecular events occurring during cholesterol digestion

Although food digestion in humans starts in the mouth with mechanical chewing and starch degradation by salivary enzymes (mastication), fat remains undigested until it reaches the stomach. Gastric digestion is mainly oriented toward protein degradation; however, some lipid-degraded enzymes are also active at this step. Afterwards, the main fat digestion take place in the duodenal lumen where all lipid compounds from the gastric bolus (including free or esterified diet cholesterol) are mixed with the bile and pancreatic juices.

Cholesterol molecules from diet, bile, intestinal secretions and desquamated cells together with the bile secreted compounds such as phospholipids (lecithin) and bile acids (salts of taurocholic and deoxycholic acids, etc.) form small emulsified droplets. Then, pancreatic lipase, phospholipase A₂ and cholesterol esterase transform the emulsified particles into a series of colloidal structures including vesicles, micelles or dietary mixed micelles (DMM).¹⁰

2.2. Molecular events occurring during cholesterol absorption

Most of the micellated lipid-like compounds are incorporated into the organism through the second part of the small intestine (jejunum), except for bile acids that can be absorbed at the jejunum and ileum levels by an apical sodium-dependent bile acid transporter (ASBT).^18,19 The SR-B1 scavenger receptor (encoded by the SCARB1 gene in humans), mainly located at both apical and basolateral membranes^17,20 of adrenal glands, hepatocytes and enterocytes (Fig. 1), is involved in the regulation of endocrine metabolism, vitamin absorption or bile secretion. It also plays a role in cholesterol transport through membranes as a receptor of HDL (high-density lipoprotein)-cholesterol but not in the small intestine absorption context.^20,21 SR-B1 transport allows a passive bi-directional cholesterol efflux depending on concentration gradients²² pointing to SR-B1 as an important modulator of reverse cholesterol transport (RCT) (described later).²⁰ However, although SR-B1 contributes to enterocytic cholesterol absorption, recent studies demonstrated that Niemann-Pick C1-like protein (NPC1L1) is the main sterol transporter from the intestinal lumen to the enterocyte cytoplasm, being imperative for non-esterified cholesterol absorption.²³

Fig. 1 Cholesterol and fat digestion and absorption pathway. PL: phospholipids, FA: fatty acids, BA: bile acids, TAG: triacylglycerols, CL: cholesterol, LPA: lysophosphatidic acid, MAG: monoacylglycerols, LPA: lysophosphatidic acid, CE: cholesterol esters, ABCG5: ATP-binding cassette subfamily G member 5, ABCG8: ATP-binding cassette subfamily G member 8, ABCA1: ATP-binding cassette subfamily A member 1, ACAT2: acetyl-CoA acetyltransferase 2, FABPpm: plasma membrane fatty acid-binding protein, NPC1L1: Niemann-Pick C1-like protein, SRB1: scavenger receptor class B member 1, AGPAT: 1-acylglycerol-3-phosphate O-acyltransferase, LPAT: lysophosphatidate acyltransferase, MGAT2: monoacylglycerol acyltransferase-2, DGAT1: diacylglycerol O-acyltransferase 1, MTTP: microsomal triglyceride transfer protein large subunit, APOB48: apolipoprotein B-48, APOA1: apolipoprotein A-1, ER: endoplasmatic reticulum, GB: Golgi body, PCM: pre-chylomicron; CM: chylomicron.

NPC1L1 is involved in this transmembrane sterol efflux due to a sterol-sensing domain (SSD) and it is co-localized at the cellular and intracellular vesicular membranes. The distribution of non-esterified cholesterol determines the main location of NPC1L1 proteins. At low intracellular concentrations, NPC1L1 will be mostly exposed at the brush-border enterocyte membrane and it will be translocate inside the cell at high levels of non-esterified cholesterol.²⁴ In humans, NPC1L1 genes are not only expressed in the enterocytes of the small intestine but also in the liver where they are expressed in large amounts. Human hepatocytic NPC1L1 protein is located at the canalicular membrane facilitating the uptake of newly secreted biliary cholesterol and therefore, showing a role similar to intestinal NPC1L1.²⁰ The transcriptional regulation of NPC1L1 has not yet been elucidated but, it seems to be influenced by sterol regulatory element-binding protein or SREBP2 (encoded by SREBF2 gene in humans) that are sensors activating different answers depending on intracellular cholesterol concentrations. At low cholesterol levels, SCAP (integral membrane protein) goes along with SREBP2 from the endoplasmic reticulum (ER) to the Golgi body (GB) for subsequent processing and activation. In contrast, at high or enough cholesterol levels the SCAP-SREBP2 complex is retained by INSIG proteins (Insulin induced gene 1 protein located in the ER membrane) to avoid SREBP2 maturation impairing its transcription.²⁵ Moreover, other reports indicated PPARδ (peroxisome-proliferator-activated receptor δ) as another NPC1L1 modulator since down-regulation of the cholesterol transporter has been induced by PPARδ activation in mice.¹¹

Intracellular non-esterified cholesterol concentrations could also be modulated by ATP-binding cassette (ABC) transporters such as ABCG5/ABCG8 and ABCA1, located respectively at apical and basolateral enterocyte sides. ABCG5 and ABCG8 proteins, expressed in the liver and small intestine, are involved in the reverse cholesterol transport (RCT) of sterols, from the intracellular environment to the lumen. Independent expression of both genes is necessary for the proper function of this heterodimer.²⁶ Over-expression of the ABCG5/8 heterodimer increases non-esterified cholesterol excretion to the lumen reducing its internal concentration and consequently inducing activation of the cholesterol synthesis rate.²⁷ In the small intestine, ABCG5/8 gene expression seems to be regulated by an LXR-dependent member of the nuclear receptor family named RXR (retinoid X receptor)^26–28 while in the liver, the heterodimer is directly modulated by LXR.³⁰ Apparently, the latter receptor, along with PPARδ, is also a modulator of ABCA1 expression.^10,26 ABCA1 is a transport protein directly involved in the excretion of exceeding non-esterified cholesterol into HDL. LXR agonist administration or high cholesterol concentrations in the cytosol stimulate a direct effect on the transcriptional modulation of these transport proteins although its specific regulatory mechanisms remain still unclear. A protein–protein interaction with another transcription factor affecting the transcription rate of the ABC proteins has been hypothesized.²⁹

Due to confusions noticed in several publications, it is worth defining the specific role of two widely mentioned enzymes involved in the transferring of acyl groups within the cholesterol metabolism. Acetyl-Coenzyme A transferase (ACAT) and sterol O-acyltransferase (SOAT) are two enzymes belonging to the acyltransferase family; however, they do not catalyze the same reaction (Table 1). ACAT isoforms (ACAT1/ACAT2) are responsible for the reversible formation of acetoacetyl-CoA from two molecules of acetyl-CoA. ACAT1 and ACAT2 genes are respectively located in chromosome 11 (11q22.3) and chromosome 6 (6q25.3).^30,31 However, SOAT isoforms (SOAT1/SOAT2) catalyze the formation of fatty acid-cholesterol esters from cholesterol and acyl-CoA molecules and are encoded by two genes located at loci 1q25.2 and 12q13.13.^32,33 Then, since in some of the mentioned works the text wrongly referred to ACAT when it should indicate SOAT, personal advice (^AvS mark) was given pointing attention where required.

Table 1 Chemical reaction, location within the metabolic pathway and within the cell of ACAT and SOAT, enzymes involved in cholesterol metabolism. M: mitochondrion; ER: endoplasmic reticulum; Cy: cytoplasm; PM: plasmatic membrane; ExC: extracellular compartment; +: relative gene abundance

	Reaction	ACAT/SOAT are involved in			mRNA subcellular location
		Cholesterol synthesis		Cholesterol absorption
		Pre-HMGCR	Post-HMGCR	Small intestine (ER membrane)	M	ER	Cy	PM	ExC
ACAT [EC 2.3.1.9]		Yes	No	No	+++	+	++	ND	ND
SOAT [EC 2.3.1.26]		No	Yes	Yes	ND	+++	ND	++	+

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Once non-esterified cholesterol reaches the cytoplasm it becomes a substrate of ACAT2^(AvS), an integral membrane protein mainly expressed in the small intestine and liver. ACAT2^(AvS) decreases cytoplasmic amounts of non-esterified cholesterol promoting its esterification and integration into the ER pre-chylomicrons modulating the cholesterol transmembrane absorption rate from the intestinal lumen.³⁴ It also plays an important role in maintaining the dynamic equilibrium (homeostasis) between free-cholesterol and esterified-cholesterol.³⁵ More than 50% of sterol esterification within the enterocytes is carried out by ACAT2^(AvS) with a higher affinity for cholesterol esterification rather than for other non-cholesterol sterols.

The internal ER triglyceride re-assemblage is carried out by several enzymes such as lysophosphatidate acyltransferase (AGPAT), phosphatidate phosphatase (LPAP), 2-acylglycerol O-acyltransferase 2 (MGAT2) and diacylglycerol O-acyltransferase 1 (DGAT1). Then, esterified-cholesterol together with the triglycerides is packed into pre-chylomicrons by the microsomal triglyceride transfer protein (MTTP) and apolipoprotein B48 (an isoform derived from the APOB gene characteristic of enterocytes). MTTP and APOB48 proteins constitute an active heterodimeric complex linked by ionic interactions with a particular feedback assembly and secretion system i.e. the larger the APOB48 subunit is, the lower binding capacity with MTTP is noticed and less APOB48 is secreted. Therefore, the APOB48-MTTP binding process plays an important role in lipoprotein biogenesis.^36–38

The combined regulatory effect of NPC1L1, ABCA1, ABCG5/8 and ACAT2^(AvS) activities play a critical role in modulating the amount of esterified cholesterol that will be integrated in the pre-chylomicrons with the assistance of apolipoporotein B48 (APOB48), microsomal triglyceride transfer protein (MTTP) and diacylglycerol-O-acyltransferase (DGAT1/2).³⁹

Once prechylomicrom structure is assembled, it is further transformed into chylomicrons in the GB and excreted by exocytosis into the lymph system through the enterocyte basolateral membrane. On the other hand, the non-esterified cholesterol remaining in the cytoplasm could bind to the APOA1 protein for further transport to the lymphatic vessels leading to nascent HDL lipoproteins. Thus, HDL as well as chylomicrons are released free into the bloodstream and transported to the liver and peripheral organs such as adrenal glands.¹¹

2.3. Molecular events occurring during cholesterol synthesis

Total blood cholesterol levels are not only dependent on exogenous cholesterol absorption but also on endogenous cholesterol synthesis. Several tissues are involved in de novo cholesterol biosynthesis i.e. enterocytes, adrenal glands, ovaries or testicles but, mostly it is generated by hepatic cells. In fact, one of the liver's main roles is the production of the bile salts from cholesterol as constitutive compounds of biliary fluids needed for the digestion processes while the cholesterol synthesized in adrenal glands or intestine is used respectively as a hormone precursor and a cholesterolemia modulator.¹¹ Cholesterol biosynthesis is carried out by a combination of mevalonate and steroid biosynthetic pathways⁴⁰ (Fig. 2).

Fig. 2 Cholesterol biosynthetic pathway from glycolysis product, acetyl coenzyme A and acetoacetyl coenzyme A. Ac-CoA: acetyl coenzyme A; AcAc-CoA: acetoacetyl coenzyme A; CoA: coenzyme A; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA; A: cholesta-7,24-dien-3β-ol; B: cholesta-8-en-3β-ol; C: 7-dehydro-desmosterol; D: lathosterol; E: desmosterol; F: 7-dehydrocholesterol; ACAT2: acetyl-CoA acetyltransferase 2; HMGCS: hydroxymethylglutaryl-CoA synthase; HMGCR: 3-hydroxy-3-methylglutaryl coenzyme A reductase; SQS: farnesyl-diphosphate farnesyltransferase or squalene synthetase; SQLE: squalene monooxygenase; DHCR24: delta24-sterol reductase; DHCR7: 7-dehydrocholesterol reductase; SOAT: sterol O-acyltransferase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is considered the key enzyme of cholesterol synthesis although the activities of many enzymes involved in the biosynthetic pathway, such as ACAT2, hydroxymethylglutaryl-CoA synthase (HMGCS), delta24-sterol reductase (DHCR24), farnesyl-diphosphate farnesyltransferase (FDFT1/SQS) or 7-dehydrocholesterol reductase (DHCR7), are also susceptible to modulation. Recently, Gill et al. (2011) suggested that squalene monoxygenase (SQLE) might be the second critical modulatory point despite its lower specificity within cholesterol metabolism compared to HMGCR (after in vitro experiments).⁴¹

However, other enzymes such as SQS and SQLE are also gaining attention as a potential stop-point of cholesterol biosynthesis because SQS is involved in the transformation of farnesyl pyrophosphate into squalene, being the first specific reaction at the branching point between sterol and non-sterol biosynthesis. The SQS transcriptional product and protein are modulated by cholesterol since low levels of this sterol activate the SQS promoter by sterol regulatory element binding proteins (mostly SREBP2). In contrast, SQS mRNA concentration decreases in response to cholesterol excess. SQLE is a monoxygenase (also named squalene epoxidase) that catalyzes the next step after squalene transformation by SQS, a crucial oxygenation process yielding squalene 2,3-epoxide (Fig. 2). Modulation of SQLE activity seems also cholesterol-dependent and apparently regulation is mediated by proteosome activity.⁴¹

HMGCR includes seven domains inserted in the ER (endoplasmic reticulum) membrane with an active carboxyl chain located at the cytosol. HMGCR is ubiquitously expressed i.e. in immune (white blood cells), nervous, muscle, small intestine or reproductive (ovary cells) human tissues.⁴² HMGCR transcription and degradation depends on a sterol/non-sterol feedback regulation but is not directly controlled by the cholesterol molecule. Under cholesterol depleted conditions, the SREBP-SCAP complex is formed in the ER membrane (without any INSIG interaction); then, it is transported to GB where SREBP is activated by proteolytic events facilitating its translocation into the nucleus to activate HMGCR transcription (Fig. 3a). In contrast, under cholesterol exceeding conditions, high amount of oxysterols are synthesized by the mitochondrial sterol 27-hydroxylase (CYP27A) and several mechanisms reducing HMGCR transcriptional rates are activated.⁴³ HMGCR regulatory mechanisms could be classified as INSIG-dependent (modulating at transcriptional and post-transcriptional levels), or INSIG-independent.

Fig. 3 Molecular regulation pathway of HMGCR transcription under low (a) and high (b) intracellular cholesterol levels. INSIG: insulin induced gene 1 protein; SREBP: sterol regulatory element-binding protein; SCAP: SREBP cleavage-activating protein; COP-II: coat complex protein II; SPH2: sphingosine kinase II; TMUB1: transmembrane and ubiquitin like domain containing 1; gp78: membrane-bound ubiquitin E3 ligase; Ubc2: ubiquitin-conjugating enzyme E2 2; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; SRE: sterol regulatory element; RXR: retinoic X receptor; LXR: liver X receptor; ABCA1: ATP-binding cassette subfamily A member 1; ABCG5/8: ATP-binding cassette subfamily G member 5/8; SOAT: sterol O-acyltransferase; UBQ: ubiquitin; AMPK: AMP-activated protein kinase; PP2A: phosphatase 2A.

INSIG-dependent HMGCR regulation mechanisms (Fig. 3b):

- INSIG can disrupt SREBP activation by binding to SCAP when high sterol concentrations are noticed in the cytosol. Once the INSIG-SCAP complex is formed, SCAP is structurally altered impairing SREBP recognition and stopping the assembly of the SREBP-SCAP complex for its further transport from the ER (endoplasmic reticulum) to the GB (Golgi body). In consequence, SREBP is not translocated to the nucleus and HMGCR transcription is inactivated.^17,44

- Under similar conditions, INSIG can also bind to the N-terminal region of HMGCR and conjugate it with ubiquitin. Ubiquitation is carried out by gp78 (membrane-bound ubiquitin E3 ligase) assisted by Ubc7 (an E2 ubiquitin conjugating enzyme) providing active ubiquitins and a few other enzymes. Then, ubiquitinated HMGCR is released into the cytosol and subsequently proteosome-degraded with the participation of p97/VCP (ATPase associated to the membrane).

However, the presence of high levels of sterols is not mandatory for the ubiquitination process but it stimulates HMGCR degradation by enhancing INSIG-HMCR bindings.⁴⁴

INSIG-independent HMGCR regulation mechanisms (Fig. 3b):

- HMGCR activity could be also modulated in situations of cellular stress (low ATP levels) by AMP kinase (AMP-activated protein kinase). In this case, HMGCR is inactivated by a serine phosphorylation due to AMP kinase activity. It is a reversible reaction and HMGCR can also be activated by protein phosphatase 2A (PP2A).^44,45

- Non-sterol isoprenoids might also modulate HMGCR translation by a mechanism still unclear, according to Burg et al. (2011).⁴⁴

2.4. Molecular events occurring during cholesterol excretion

For several decades, classical reverse cholesterol transport (RCT) was considered the main mechanism to eliminate cholesterol; however, recent studies suggest another possible pathway called transintestinal cholesterol excretion (TICE).¹⁷

RCT is a derivative branch of the hepatobiliary pathway. Lipoproteins such as HDL or LDL make available cholesterol for hepatic absorption as esterified or non-esterified molecules. Esterified cholesterol is transformed by the hepatic cholesteryl ester hydrolase (NCEH1) into the non-esterified form after hydrolysis of the ester linkage. Thus, the generated forms are directly excreted through the ABCG8/5 heterodimer protein or transformed into bile salts.⁴⁶ CYP7A1 (cholesterol 7-alpha-monooxygenase) is the enzyme responsible for cholesterol transformation into primary bile salts (cholic and chenodeoxycholic acid) within the neutral bile acid pathway in the liver. Synthesized bile salts are secreted to the bile canaliculus by the bile salt export pump (BSEP) or the multidrug resistance-associated protein 2 (MRP2) and become part of bile fluids. Recent in vivo studies have suggested the involvement in the RCT of other cholesterol transporters such as NPC1L1^20,47 although its role in biliary cholesterol excretion has not yet been elucidated. But, it might involve adjustments in cholesterol balance to avoid excessive loss of the metabolite through the intestinal track.

TICE has been suggested as an alternative cholesterol excretion mechanism where the sterol is directly eliminated from blood through the intestinal mucosa and excreted via the feces.¹⁷ The hypothesis was drawn after unexpected results obtained by several authors that showed an unwarranted balance between cholesterol inputs and outputs in mouse models. They showed a higher amount of fecal cholesterol than the sum of dietary intake and biliary secretion⁴⁸ or an unaltered cholesterol excretion rate in knockout NPC1L1 mice with a decreasing of 90% biliary excretion.⁴⁹

These results questioned the complete RCT classical concept⁵⁰ as well as the contribution of biliary or non-biliary cholesterol to its RCT excretion.⁵¹ Although the involvement of several membrane transports in TICE have been studied (i.e. SR-BI,⁵² NPC1L1,⁵³ APOA1,⁵⁴ LDLR or ABCG5/8⁵⁵), it is still unknown whether TICE is carried out through basolateral or apical transporters or whether HDLs are involved;⁵⁶ therefore, the mechanism is not yet elucidated. There is also scientific controversy about the importance of TICE; some authors suggest that it could be only a compensatory cholesterol excretion mechanism in case of biliary cholesterol depletion but, other authors indicated it as the main mechanism in cholesterol excretion. Van der Velde et al. (2007, 2010) estimated the contribution of TICE as 70 and 30% of total cholesterol were excreted in mice and humans, respectively.^48,56

2.5. Molecular events occurring during cholesterol transport

HDL, IDL (intermediate-density lipoprotein), LDL and VLDL (very low-density lipoprotein) are the connecting structures responsible for transporting cholesterol molecules through the bloodstream from one tissue to another until they are detected by cellular membrane receptors such as SR-B1 for HDL or LDLR (LDL-receptor) for both structures.

The non-esterified cholesterol eliminated by ABCA1 through the basolateral membrane of enterocytes is bound to APOA1 generating nascent-HDL and the esterified cholesterol is similarly assembled with APOB48 in ER synthetized pre-chylomicrons further transformed into chylomicrons in GA and excreted by exocytosis to the intracellular space. Therefore, both structures become cholesterol transporters and they distribute it via the bloodstream to the rest of the organism.

Nascent HDL is transformed into mature HDL by the accumulation of non-esterified cholesterol molecules secreted by hepatocyte and enterocyte ABCA1 protein. Once mature HDLs are formed, some cholesterol molecules are esterified by the action of lecithin–cholesterol acyltransferase (LCAT) along with their blood transport. Esterified and non-esterified cholesterol are detected by SR-B1 located in the basolateral membrane of cells from the liver, small intestine or gland adrenals allowing the incorporation of esterified cholesterol inside the cell. Intracellular esterified cholesterol is transformed into non-esterified cholesterol (by SOAT action) and HDL is turned into LDL. The non-esterified cholesterol can be used for bile salt synthesis and their further intestinal secretion by BSEP or to be directly excreted via ABCA1 (basolateral membrane) and ABCG5/ABCG8 (apical membrane) activity. In turn, secreted non-esterified cholesterol again could be attached to nascent HDL to create mature HDL and continue with cholesterol transport.

LDL molecules are recognized by the liver, intestine and adrenal gland LDLRs as esterified cholesterol suppliers. LDL particles can be generated not only by VLDL transformation (by SR-B1 activity) but also by the addition of esterified cholesterol to APOB100 (apolipoprotein isoform characteristic of hepatocytes) leading to very low density lipoproteins (VLDL). These structures are secreted to the bloodstream by hepatocytes. VLDL lipids are used by the muscle and peripheral tissues as they pass through the bloodstream generating IDL and LDL by lipoprotein lipase (LPL) activity. Moreover, the esterified cholesterol of chylomicron structures is also recognized by LDLR providing to the hepatocyte those cholesterol molecules assembled in enterocytic ER after the digestion process.

The complex cholesterol biosynthesis and absorption engineering and the multifactorial regulation system make the control of cholesterol metabolism a difficult challenge for the scientific community, particularly because some of the involved compounds are also intermediates of other metabolic pathways i.e LXR modulates ABCG5/8 activity but also DIO1, a selenoprotein involved in thyroid hormone metabolism. However, these factors also make it a flexible system that could be modulated from different critical points.

3. Fungal molecules modulating cholesterol metabolism

Many food compounds have been described as modulators of cholesterol homeostasis interacting with specific control points and inhibiting or altering cholesterol metabolism; even some in vivo studies suggest synergistic hypocholesterolaemic activities between several inhibitors.^57–59 Those compounds showed different targets depending on whether they modulate cholesterol absorption, synthesis, excretion or transport.

The intake of dietary fibers (DF) from cereals etc., increases cholesterol and bile acid excretion rates lowering their bioavailability because they might act as bile acid scavengers. This mechanism impairs bile acid re-absorption stimulating the hepatic synthesis of new bile acids from cholesterol reducing its blood concentration^10,60,61 although for particular β-glucans, modulation of cholesterol-related gene expressions have been also noticed.⁶²

Plant sterols (phytosterols and phytostanols) are considered direct cholesterol competitors for their incorporation into DMMs because of their structural similarity and due to the limited capacity of DMMs to solubilize lipophilic and water-insoluble molecules.^63–65 Some reports also suggested that phytosterols might modulate SOAT activities although it still remains partially unclear. Cholesterol enterocytic esterification by ACAT^(AvS) was decreased by sterol competition although the enzyme showed lower esterification efficiency for plant sterol than cholesterol. Other authors suggested that plant sterols influenced MTTP and APOB48 lipoprotein in vitro models.⁶⁵

Compounds such as novel amino β-lactam derivates⁶⁶ or curcuminoid polyphenols were described as potential NPC1L1 inhibitiors by showing high binding affinity or acting by indirect influence on SREBP1.³⁷ ABCG5/8 postranscriptional regulations were also described for spironolactones or polyphenols from Aronia melanocarpa.^67,68

Other compounds from natural sources have been reported as ACAT^(AvS) inhibitors reducing cholesterol esterification rates such as alkamides from Piper nigrum,⁶⁹ shikonin derivatives from Lithospermum erythrorhizon,⁷⁰ an isoprenyl flavonoid identified as grabol from licorice roots,⁷¹ ursolic acid (indirect inhibition via (PPAR)-α activation)⁷² or flucoxanthin from marine plants.⁷³

3.1. Hypocholesterolemic compounds of edible mushrooms

A few specific bioactive compounds are already indicated as responsible for the hypocholesterolaemic properties of mushrooms. The most extensively investigated are fungal sterols (ergosterol and its derivatives such as ergost-22-ene-1,3-diol, ergosta-5,7-dien-3b-ol, (22E)-ergosta-1,4,6,22-tetraen-3-one, etc.) and β-glucans together with other dietary fibres.

Ergosterol (ergosta-5,7,22-trien-3β-ol) is considered the major fungal sterol (from 53% up to 80% of the fungal sterols, w/w) followed by ergosterol derivatives such as ergosta-5,8,22-trien-3-ol, ergosta-7,22-dien-3-ol, ergosta-5,7-dien-3-ol and ergosta-7-en-3-ol (fungisterol).⁷⁴ However, their precise concentration in the different mushroom strains was dependent on environmental conditions (developmental stage, cultivation conditions, etc.) or species. For instance, Chantharellus cibarius and Craterellus cornucopioides showed almost exclusively ergosterol, while other species showed more ergosta-5,7-dienol than fungisterol (Lyophyllum shimeji or Pleurotus ostreatus). Flammulina velutipes contained high amounts of ergosta 5,8,22-trien-3β-ol.¹⁰

Apparently, the hypocholesterolaemic activities of fungal sterols were mainly due to their structural similarity with cholesterol as noticed for plant sterols. Therefore, these compounds such as phytosterols and phytostanols were able to hinder cholesterol incorporation into DMMs. Ergosterol-enriched fractions obtained using green technologies, such as supercritical fluid extraction (SFE), were more effective than β-sitosterol in displacing cholesterol from DMMs during an in vitro digestion model.⁵⁹ Moreover, fungal sterol-enriched extracts also showed HMGCR inhibitory activities in vitro⁷⁵ and ergosterol was also considered as a competitive inhibitor of C24-reductase due to its double bond at C-22 in the side chain of its structure (as also noticed for stigmasterol and brasicasterol). C24-Reductase is another enzyme involved in the cholesterol biosynthetic pathway down-stream from HMGCR.⁷⁶

Fungal β-glucans showed different molecular structures from cereal polysaccharides. Their branching profiles are (1→3) and (1→3)(1→6) conferring on them many biological activities such as antitumoral, antioxidant, immunomodulatory, etc. apart from hypocholesterolaemic properties.^10,77,78 Some reports suggested that those bioactive properties are due to their different tridimensional configuration depending on their monomer composition, branching degree or conformation while other authors considered their water solubility (that is also partially dependent on their glycosidic linkages) a more relevant factor, considering those water-insoluble as responsible for the cholesterol lowering activities by impairing cholesterol absorption. Moreover, other publications indicated β-glucan degradation products (generated i.e. after a digestion process) as hypocholesterolemic molecules more bioactive than their larger precursors.^10,79 However, until now the precise structural requirements for the observed hypocholesterolemic action of fungal β-glucans remain unclear awaiting further studies.

β-Glucans from a large number of mushroom species have been studied such as lentinan from shiitake mushrooms (Lentinula edodes), schizophyllan from Schizophyllum commune, grifolan from Grifola frondosa, and many others from Agaricus blazei, Ganoderma lucidum (a medicinal mushroom consumed as a nutraceutical or dietary supplement), and P. ostreatus as well as (1→3) β-glucans and glucuronoxylomannans from Auricularia polytricha and Tremella fuciformis.^80,81 All showed different conformations, and solubilities and therefore biological properties.

Chitin, a special type of water-insoluble β-(1→4)-glucan including N-acetylglucosamine monomers, is also considered a hypocholesterolaemic polysaccharide while its derivative, named chitosan, was more studied because of its antitumor and immunomodulation activities.^10,77 Several polysaccharide fractions (containing high percentages of fungal β-, α-glucans and chitooligosacharides among others) from L. edodes, P. ostreatus and A. bisporus extracted by using pressurized technologies (PSE) showed bile acid binding capacities in vitro similar to those shown by cereal fibers⁸² indicating that this might impair cholesterol absorption as previously suggested for plant β-glucans.

According to suggestions by Gunde-Cimerman et al. (1993, 1995), lovastatin an inhibitor of HMGCR (3-hydroxy-3-methylglutaryl-Coenzime A reductase) was naturally present in some Pleurotus spp. fruiting bodies such as P. ostreatus or P. eryngii^83,84 and in other species such as A. bisporus or B. edulis. However, recent studies did not detect any statin in mushroom species showing interesting HMGCR inhibitory activity pointing to some water soluble polysaccharides and proteoglucans as responsible for the noticed in vitro inhibition.^10,75

HMGCR inhibition was also mediated by an AMP kinase via phosphorylation and green and black tea polyphenols induced a direct increase of HMGCR phosphorylation possibly via AMP kinase phosphorylation. Their precise mechanism of action is still unclear but, it seems to involve the activation of regulatory factors such as PPAR.^85,86

Only a few compounds are still nowadays indicated as potential inhibitors of SQS such as resveratrol from wine⁸⁷ as well as zaragozic acids isolated from the liquid broth of certain ascomycetes.⁸⁸

24(S),25-Epoxycholesterol was also pointed out as an inhibitor of DHCR24 activity. This enzyme catalyzes the transformation of desmosterol into cholesterol. The inhibitor did not modify DHCR24 protein levels, but increased desmosterol accumulation decreasing cholesterol levels in in vitro studies due to its structural similarity with desmosterol.⁸⁹

3.2 Molecular events modulated by mushroom extracts

The presence of certain natural molecules in mushrooms does not only modify cholesterol absorption or the metabolic pathway of consumers but can also modulate the expression of some genes related to cholesterol homeostasis. Recent studies carried out on particular edible mushrooms studied their influence as an attempt to further identify the most interesting fungal compounds to treat moderate hypercholesterolemia.

Mushrooms such as Pleurotus ostreatus, Grifola frondosa and Hypsizigus marmoreus were able to differently modulate the gene expression patterns of livers of mice fed with each mushroom (10–14%) for 4 weeks. Triglyceride levels in the liver and plasma decreased in the mice fed with P. ostreatus compared with those in the control group. Moreover, liver cholesterol decreased while plasma total cholesterol increased probably due to HDL values that were also increased. Cholesterol in the liver was lower in the group fed with G. grondosa than in the control group but no changes were found in the H. marmoreus-fed group. DNA microarray analysis of the livers revealed that CTP1A and FABP families were upregulated in the P. ostreatus-fed group, which were considered to promote lipid transport and β-oxidation. In the G. frondosa-fed group, not only the gene involved in signal transduction of innate immunity via TLR3 and interferon but also virus resistance genes (such as MX1, RSAD2 and OAS1) were upregulated.⁹⁰

Administration of Agaricus brasiliensis (also known as Agaricus blazei) to hypercholesterolemic albino Fischer rats during 6 weeks lowered cholesterol levels in serum and induced significant changes in the expression of cholesterol-related genes. HMGCR mRNA expression was not influenced but LDLR upregulation was noticed together with the upregulation of CYP7A1, the rate-limiting enzyme for bile acid synthesis, and mRNA levels of the ABCG5/8 carriers. These increases were accompanied by a significant increase in the content of cholesterol excreted in the faeces and by a concomitant increase in NR1H3 (LXR) mRNA levels. However, in this case PPAR-α was not significantly upregulated compared with levels noticed in the hypercholesterolemic control group.⁹¹

In a few reports specific extracts, and not the whole fruiting body, were studied. For instance, ethanol (95%) extracts obtained from P. ostreatus were administered to hypercholesterolemic male Wistar rats for 14 days and a reduction of triglycerides and HDL cholesterol was noticed in the plasma. Moreover, the extract was able to upregulate the genes that were downregulated after hypercholesterolemia induction such as those related to fatty acid oxidation such as acyl CoA oxidase (ACO) and synthetase (ACS) together with carnityl palmityl transferase-1 (CPT-1) and PPAR-α. They also downregulated genes related to fatty acid biosynthesis and cholesterol metabolism (FAS fatty acid synthase, SREBF1, APOC3) to similar expression than during normocholesterolemia.⁹²

Hericium erinaceus hot water and ethanol extracts were administered, together with a high-fat diet, to C57BL76J mice for 4 weeks. Incorporation of the extracts in the diet resulted in a significant decrease in body weight, fat and serum and hepatic trigliceride levels compared to control. The ethanol extract acted as an agonist of PPAR-α since it was able to up-regulate mRNAs usually modulated by PPAR-α (ACAT, ApoA1, LPL or SREBP1) in spite of the fact that the PPAR-α expression itself did not change.⁹³

An extract obtained from the cauliflower mushroom (Sparassis crispa) significantly enhanced hepatic cholesterol catabolism when administered to male Sprague-Dawley rats fed a cholesterol-rich diet for 4 weeks because they were able to up-regulate CYP7A1 mRNA expression concomitant with HMGCR downregulation after. Additionally, the extract supplementation resulted in cholesterol and bile acid fecal excretion.⁹⁴

3.3 Molecular events modulated by fungal sterols

Since plant and fungal sterols share structural similarities, similar modulation of cholesterol related genes might also be expected. However, in the few studies carried out using fungal sterols different effects were noticed.

Ergosterol was able to regulate sterol regulatory element binding protein (SREBP) cleavage in yeast as response to cellular oxygen levels⁹⁵ suggesting that it might also modulate these elements in mammalians. Studies carried out using cell cultures revealed that DMMs isolated from the in vitro digestion of ergosterol and extracts containing ergosterol mixed with cholesterol did not influence the transcriptional levels of SREBF1 differently than when only cholesterol was administered. Moreover, only a slight inhibition was noticed for SREBF2 mRNA of Caco2 cells when treated with an ergosterol-containing extract but not with ergosterol itself. However, other cholesterol related genes were also overexpressed such as LDLR (when an ergosterol-enriched extract from Agaricus bisporus was applied).¹⁵ When the lower compartment of the Caco2 cells supplemented with the sterol-containing digested extracts were added to HepG2 cells, higher modulation of genes related to lipid metabolism was noticed than those more directly related to cholesterol homeostasis such as DGAT2 upregulation.

When similar extracts were given for 4 weeks to C57BL/6JRj mice previously fed for 4 weeks with hypercholesterolemic diets, inhibition of SREBF2 and NR1H4 (gene enconding FXR) were noticed in the jejunum up to similar levels than hypocholesterolemic drugs such as ezetimibe and simvastatin.¹⁵ This downregulation was due to their ergosterol content because supplementation with purified ergosterol induced similar modulation. FXR is the farnesoid X receptor, a nuclear receptor also involved in the regulation of cholesterol homeostasis.⁹⁶ FXR is involved in the activation or inhibition of bile acid synthesis and transport acting as a sensor of their concentration.⁹⁷ The extract induced the downregulation of DGAT2 in the liver as observed in cell cultures but upregulation of FDFT1 might be to compensate for the reduction of the ratio TC/HDL recorded. Reduction of the triglyceride levels was also noticed in the liver, perhaps because of the observed modulation of DGAT2 gene transcription. Moreover, this extract induced the overexpression of DIO1 mRNA in the jejunum, the tissue where cholesterol absorption is higher.⁹⁸ Type I iodothyronine deiodinase is a selenoprotein encoded by the DIO1 gene and plays a major role in normal thyroid function, but it also indirectly influences cholesterol homeostasis by regulating LDLR gene expression. Increased DIO1 activity in the liver correlated with up-regulation of LDLR mRNA and lowering of TC, TG and LDL levels in serum.¹⁵ However, no significant effect was noticed by Gil-Ramirez et al., 2015⁹⁸ on the latter gene; perhaps the supplemented concentration was sufficient to stimulate DIO1 but it did not revert on LDLR readjustments. In contrast, ergosterol administered as a single compound induced the downregulation of DIO1 mRNA; therefore the effect of the ergosterol enriched extract could be due to some other sterol or compound present in the extract.⁹⁸

However, when ergosterol containing extracts were incorporated into lard and the mixture was supplemented together with the diet to induce hypercholesterolemia, insignificant effects were noticed on any SREBP, other cholesterol regulatory nuclear receptor or genes involved in cholesterol metabolism except for ABCG5 and 8 mRNAs expression in cecum that were up-regulated. Perhaps the addition of the extract as an ingredient inside a lipid matrix prevented their proper bioavailability (because it was also unable to lower cholesterol levels in serum, HDLc or LDLc) or perhaps the beneficial properties of ergosterol were not sufficient to overcome the detrimental effect of lard and the hypercholesterolemic diet when administered simultaneously.⁹⁹

The transcription of some ABC transporters (ABCG5/8 etc.) was induced by the LXR factor in enterocytes (but not in hepatocytes)⁶⁵ although, there is a controversy about its influence on other transporters (ABCA1). Oxysterols such as 22(R)-hydroxycholesterol, 24(S),25-epoxycholesterol or 27-hydroxycholesterol, etc., are considered as endogenous natural LXR agonists; however, plant sterol derivatives showed higher LXR agonist activity. Brasicasterols from unicellular algae and Brassica sp. (i.e. rapeseed) induced large variations in the gene expression of ABC transporters due to their ability to act as the LXR factor in mice. Moreover, sitostanol (in mice), sitosterol (in Caco2 cells) and a few 4-desmethylsterol derivatives were also able of inducing ABCA1 up-regulation using their LXR agonist activity.^5,11,13,65 However, no direct effects of phytosterols or fungal sterols were noticed on the transcriptional levels of the transporters because, on the one hand, in ABCA1 and ABCG5/8-deficient mice decreasing of cholesterol intestinal absorption was also noticed after phytosterol administration indicating that ABC transporters were not their direct targets. On the other hand, no significant overexpression or repression was noticed in hypercholesterolemic C57BL/6JRj mice treated with ergosterol-containing extracts.¹⁵

Other reports indicated that phytosterol/stanols modulated HMGCR expression via ACAT^(AvS) inhibition. Apparently, lower ACAT^(AvS) activity led to higher free cholesterol amounts, inhibiting the cholesterol biosynthetic pathway and HMGCR expression beside others. It also reduced chylomicron assembling, and promoted the back efflux of non-esterified sterols to the lumen.^11,65,100 However, their influence on SOAT seemed to be by chemical inhibition more than by molecular modulation. Direct down-regulation of HMGCR mRNA (together with inhibition of the enzyme) was only noticed in organic extracts obtained from the Reishi mushroom (Ganoderma lucidum) containing oxygenated lanosterol derivatives. They also inhibited cholesterol synthesis in T9A4 hepatocytes and reduced total cholesterol in hamsters.¹⁰¹

Several in vitro studies pointed out that SREBP2, NPC1L1 and SR-B1 gene expression was modulated by plant sterols such as stigmasterol and β-sitosterol toward the reduction of cholesterol absorption. Surprisingly, HepG2 cells treated with these sterols showed simultaneous down-regulation of NPC1L1 and SR-B1 when an opposite effect on these two molecules could be expected. This modulation was not noticed when fungal sterols were applied to hepatic cells¹⁵ or when administered to mice together with a hypercholesterolemic diet.⁹⁹ Studies using homozygous and heterozygous knockout mice (NPC1L1^−/− and NPC1L1^+/−) showed a lower cholesterol absorption in homozygous animals; however, heterozygous mice showed higher HMGCR mRNA levels in gut and liver tissues than wild-type animals without changes in ABC transporter expression rates. Authors explained their results by compensatory effects: difficulties for cholesterol absorption were compensated by a stimulation of endogenous cholesterol synthesis to maintain physiological plasma levels.^102,103 Moreover, when the knockout mice were treated with plant sterols or ezetimibe, the wild-type and heterozygous animals showed a reduction in cholesterol and TG levels. Absorption of campesterol and β-sitosterol was reduced in NPC1L1^+/− and almost absent in NPC1L1^−/− mice indicating that certain modulation of NPC1L1 took place.^102,103 However, no modulation of NPC1L1 expression was noticed in C57BL/6 mice fed a fungal sterol extract together with a hypercholesterolemic diet.⁹⁹

Moreover, β-sitosterol addition to Caco2 cultures induced the down-regulation of HMGCR expression although this effect was not noticed in mice. Only sitosterolemic individuals showed reduction in HMGCR activity in the ileum; so apparently only large amounts of plant sterols or long-term 2% (w/w) plant sterol administration can induce such an effect.^11,104

Other reports indicated that phytosterols might modulate the expression of other closely related genes such as those encoding the hepatic cholesterelogenic farnesyl phosphate synthase (FFPS), liver CYP7A1 (cytochrome P450 family 7 subfamily A polypeptide 1) or the annexin 2-caveolin 1 (ANXA2-CAV1) protein complex.¹¹ Therefore, fungal sterols might be involved in the modulation of CYP7A1 noticed during the administration of the complete mushroom fruiting bodies to hypercholesterolemic mice.⁹¹

3.4 Molecular events modulated by fungal polysaccharides

At present, not many studies have been carried out concerning the molecular effect of dietary fibers independently of their plant or fungal origin. However, already in 1996, reports by Cheung suggested that fungal polysaccharide extracts could modulate cholesterol related enzyme activity (HMGCR) by several mechanisms and not only via direct inhibition. Later on, several studies were carried out to investigate the molecular role of fungal polysaccharides on cholesterol metabolism.⁷⁸

Fukushima et al. (2000)¹⁰⁵ examined the effects of a fiber extract obtained from Agaricus bisporus on LDLR mRNA expression after feeding rats with cholesterol-free diets during 4 weeks. Despite a higher relative liver weight in the control group (cellulose supplementation) compared with those animals fed with the mushroom fiber extract as supplement, serum total cholesterol, VLDL and LDL concentrations in the control group were significantly greater than in animals fed the A. bisporus fiber. Added to this, once LDLR mRNA hepatic levels were analyzed, results showed that mushroom LDLR up-regulation was significantly higher than that induced by cellulose fiber supplement.¹⁰⁵ One year later, the same group reported that maitake (Grifola frondosa) and enokitake (Flammulina velutipes) fiber extracts lowered total serum cholesterol levels by two mechanisms, by scavenging cholesterol molecules inducing their fecal excretion and by enhancement of LDLR mRNA expression in rat liver.¹⁰⁶ However, no up-regulation was significantly noticed when rats were fed shiitake (Lentinula edodes) fiber extracts.

Dietary fibre fractions from P. ostreatus, L. edodes and A. bisporus fruiting bodies obtained as described by Jeurink et al. (2008)¹⁰⁷ including a low α-glucan amount and high concentrations of chitin derivatives and other β-glucans⁸² were also added to Caco2 cells to study their effect on the most interesting cholesterol-related genes. The transcriptomic profile was studied after 1 and 24 h application and results indicated that the fiber extracts obtained from P. ostreatus were able to modulate the transcription of more genes related to cholesterol metabolism than the other two mushrooms studied at longer incubation times. Up-regulation of FDFT1 and NPC1L1 was noticed together with slight modulation of a few other mRNAs; therefore, in vivo experiments were carried out using P. ostreatus fiber extract using two different experimental settings. In the first one, C57BL/6J mice were firstly administered hypercholesterolemic diets and then, supplemented with the fiber extract (as palliative treatment) and in the second experiment, mice were simultaneously fed with hypercholesterolemic diet plus fiber extract (preventive treatment). Results indicated that their molecular responses were completely dependent on the supplementation setting. In the palliative setting, administration of the fiber extract reduced hepatic triglyceride levels and it might be because of the DGAT1 downregulation also recorded. In the preventive setting, the fiber extract modulated cholesterol-related gene expression similar to simvastatin and ezetimibe in the liver (i.e. by inhibiting FDFT1, NR1H3 (LXR) and NR1H4 (FXR) mRNA expression) although no changes in plasma and liver biochemical data were recorded.¹⁰⁸ Later on, when a similar extract was applied in a higher concentration and mixed with lard, a hypocholesterolemic effect was noticed (as reduction of TC and LDLc levels) but no relevant modulation of cholesterol-related gene transcription was noticed despite the overexpression of NPC1L1 in mice liver and jejunum.⁹⁹

The molecular modulations noticed for fungal β-glucans seemed to be different from other dietary fibers obtained from plants i.e. high viscosity oat or barley β-glucan extracts demonstrated their ability to down-regulate SREBF2 gene expression in intestinal cells (NCl-H716)⁶² but no with in vivo testing. However, according to Hu, Wang & Xu (2008) corn bran dietary fiber up-regulated the expression of other genes such as FXR in ileal cells or PPAR in liver.¹⁰⁹

Hepatic HMGCR up-regulation was observed after the administration of hydroxylpropylmethycellulose or inulin-oligofructose (1 : 1) to animal models;^110,111 however, barley β-glucan administration induced no changes in HMGCR expression.¹¹² Jones (2008)¹¹² noticed hepatic SREBF2 up-regulation when soluble dietary fiber (guar gum, a galactomannan) was administered to pigs. In consequence, LDLR expression was enhanced and reduced the LDLs from the bloodstream. Fungal fibers instead seemed to modulate LDLR mRNA expression directly.^105,106 Hepatic ABCG5/8 gene expression was also enhanced by guar gum consumption leading a higher cholesterol efflux from the liver to the intestinal lumen.¹¹² A similar enhancement together with ABCA1 mRNA was noticed in jejunum from mice administered P. ostreatus fibers (palliative treatment) but without significant cholesterol reduction.¹⁰⁸

Other studies indicated that the scavenging of bile acids by plant fiber (β-glucans) activated CYP7A1 to convert cholesterol into new bile acids^109,113 provoking a hepatic cholesterol decrease that up-regulated LDLR expression and reduced cholesterol blood levels. Due to these changes, the biosynthetic pathway was also activated via HMGCR up-regulation to compensate for the lack of hepatic cholesterol.^113,114 However, the effect of fungal fibers on CYP7A1 is still, at the present, unknown.

Other studies were carried out using extracts containing water soluble polysaccharides from L. edodes. This fraction contained α- and β-glucans and fucomannogalactans that when digested (in vitro) were applied to Caco2 cell cultures and no significant effect was noticed on the modulation of cholesterol-related gene expression. But, when the lower compartment of the cell monolayer was applied to HepG2, modulation of some mRNAs was noticed after 24 h incubation. However, the modulatory pattern fitted more with a low cholesterol level response since enzymes such as HMGCR, FDFT1 and ACAT1 were overexpressed; perhaps the HMGCR inhibitory activity showed by this extract induced a posttranscriptional cholesterol reduction that activated the biosynthetic pathway after 24 h.¹¹⁵ Later on, when this extract was administered to normocholesterolemic mice for 4 weeks similar overexpression of HMGCR (in liver) and FDFT1 (in jejunum) was noticed. However, when the extract was administered to hypercholesterolemic mice the modulation profile showed similarities with those generated after the administration of ezetimibe, simvastatin or both such as for instance the down-regulation of FDFT1 (in the ileum), SREBF2 or NR1H4 (in the jejunum) or the up-regulation of ABCG8 or ACAT1 in the jejunum.

Other polysaccharides extracted from 3 different strains of Pleurotus tuber-regium, when administered to obese-diabetic rats for 8 weeks also showed a hypolipidemic effect that was associated with up-regulated liver PPAR-alpha mRNA expression and protein levels. Moreover, hyperglycemia was also attenuated by polysaccharides; the elevated serum total cholesterol, triglycerides and low-density lipoprotein (LDL) concentrations were controlled, and parallel restoration of decreased high-density lipoprotein (HDL) levels was noticed after their supplementation.¹¹⁶

3.5 Molecular events modulated by other fungal compounds

An adenosine analogue alkaloid (eritadenine) is another compound from L. edodes able to inhibit S-adenosylhomocysteine hydrolase, a key enzyme of hepatic phospholipid metabolism. This inhibition could be related to the lowering of cholesterol levels in serum noticed in animal studies.^117,118 Eritadenine increases hepatic microsomal phosphatidylethanolamine (PE) concentration and decreases liver microsomal Δ6-desaturase activity, altering the fatty acid and molecular species profile in liver and plasma. When it was administered to rats, suppression of Δ6-desaturase activity was accompanied by a significant reduction in the abundance of mRNA for the enzyme suggesting that dietary eritadenine might suppress the activity of liver microsomal Δ6-desaturase by altering the microsomal phospholipid profile and this effect was mediated by the regulation of enzyme transcription.¹¹⁹ Eritadenine was also involved in the up-regulation of the CYP7A1 expression noticed in the liver of hypercholesterolemic mice fed the standard compound or 5, 10 or 20% L. edodes.¹²⁰

Water extracts obtained by PWE (pressurized water extractions) from normal and selenium-enriched A. bisporus were tested in vitro as HMGCR inhibitors. Selenium supplementation enhanced the inhibitory activity of statins and therefore, the latter extracts might improve their HMGCR inhibitory capacity. However, no significant differences were found; both similarly inhibited the enzyme only if the extracts were not thermally treated. When the extracts were applied to HepG2 cells to study their effect at the molecular level they also induced similar responses in most of the cholesterol-related genes. However, after 1 h application overexpression of LDLR mRNA was noticed in non Se-fortified extracts. After 24 h LDLR mRNA was downregulated together with FDFT1. Similar downregulation of the SQS mRNA was also noticed in Se-fortified extracts indicating that non-thermal water extracts from A. bisporus could inhibit transcription of this enzyme that is also a key enzyme downstream the cholesterol metabolic pathway.¹²¹ Reduced expression of GPX3 (glutathione peroxidase, a selenoprotein largely influenced by the presence of selenium in the media) was shown to increase the cell-mediated oxidation of LDL and selenium supplementation (1 ppm) also induced downregulation of APOB and HMGCR expression during hypercholesterolemia in rat models. However, no significant modulation of GPX3⁹⁸ and APOB¹²¹ expression was noticed either in non- or Se-fortified extracts; only a slight up-regulation of HMGCR after 1 h for non-fortified extracts and after 24 h for Se-fortified ones perhaps to compensate the inhibition of the FDFT1 transcription was noticed.¹²¹

4 Conclusion

Consumption of edible mushrooms or functional foods containing specific fungal extracts should be encouraged among people with low to moderate hypercholesterolemia (before the use of pharmaceutical drugs) to lower their cholesterol levels in serum. As noticed for plants, cereals and other food derivatives, they contain specific compounds that can modulate cholesterol homeostasis via different transcriptional and post-translational mechanisms that are nowadays not completely understood but that they could be different than those described for plants. Thus, more studies are necessary to broaden the knowledge toward the molecular effect of fungal compounds on human health since the results obtained up to now are promising, suggesting that their use as hypocholesterolemic foods might be more effective than those products actually offered in the markets.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research was supported by the national R + D program from the Spanish Ministry of Science and Innovation (projects AGL2010-21537 and AGL2014-56211-R) and the regional program from the Community of Madrid, Spain (S2013/ABI-2728).

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