Patent Application
20150190369
The present disclosure relates generally to quercetin-3-glucoside. More particularly, the present disclosure relates to quercetin-3-glucoside and its use in reducing plasma cholesterol in a patient.
The body derives its lipids from food and endogenous biosynthesis. Lipids circulate in the body in association with apolipoproteins (apo), forming lipoprotein particles of different densities, depending on their relative content in cholesterol, phospholipids, and triglycerides. Low-density lipoprotein (LDL) is the major cholesterol transporter in humans. The plasma level of LDL-cholesterol (LDL-C) is primarily modulated by the liver. This organ synthesizes cholesterol and packages it into very-LDL (VLDL) particles, which it secretes into the bloodstream. Through its LDL receptor (LDLR), the liver takes up cholesterol from the bloodstream and excretes it into the intestine in bile acids [1]. Excess plasma cholesterol is a risk factor for atherosclerosis and related cardiovascular diseases.
Hepatic clearance of plasma LDL-C is down regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), the ninth member of the family of proprotein convertases. These subtilases are involved in the post-translational activation or inactivation of secretory proteins by limited endoproteolysis. Human PCSK9 is biosynthesized in the endoplasmic reticulum (ER) as a 692-amino acid preproPCSK9, which, after co-translational removal of a 30-amino acid signal peptide, becomes proPCSK931-692.
This proPCSK931-692 zymogen cleaves itself between Gln152 and Ser152, generating the PCSK931-152 prosegment and the PCSK9153-692 mature enzyme. The prosegment and the mature enzyme remain attached in a non-covalent, enzymatically inactive complex, which is secreted into the extracellular milieu. The endoproteolytic processing of its zymogen is required for PCSK9 secretion [2]. This has been recently corroborated in humans by the identification of a Gln152His mutation that prevents the cleavage site, causing PCSK9 intracellular retention [3].
Besides endoproteolysis, other post-translational modifications of PCSK9 may include N-glycosylation at Asn533, sulfation at Tyr38, and phosphorylation at Ser47 and Ser688 [2,4,5].
The PCSK9/prosegment complex binds to LDLR at the cell surface and, after co-endocytosis, prevents the receptor from returning to the cell surface, rerouting it into lysosomes where it is degraded [6]. The complex is dissociated by a furin-mediated cleavage between Arg218 and Gln219 in the mature enzyme, producing the ΔNT-PCSK9219-692 devoid of LDLR-degradation activity [4,7]. Thus, hepatic LDLR/PCSK9 expression or activity ratio strongly influences the circulating levels of cholesterol. In humans, hypercholesterolemia has been associated with loss-of-function mutations in the LDLR gene, as well as gain-of-function mutations in the PCSK9 gene [8,9].
High plasma cholesterol levels (i.e. hypercholesterolemia) is a risk factor for atherosclerosis and related cardiovascular diseases. Today, atherosclerosis and related cardiovascular diseases have become global epidemics [10,11]. Statins are the drugs most commonly used to combat them [12]. However, for all their success, statin inhibitors of cholesterol biosynthesis occasionally cause serious side effects, such as myopathy and hepatotoxicity [13], precluding their therapeutic use in a growing number of hypercholesterolemic patients.
Statins reduce intracellular cholesterol biosynthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR), the rate-limiting enzyme in cholesterol biosynthesis. This inhibition results in compensatory up-regulation of sterol regulatory element-binding protein 2 (SREBP-2), the transcription factor that drives cholesterol biosynthesis. SREBP-2 activates transcription of both the LDLR and the PCSK9 genes in hepatocytes [14]. Furthermore, therapeutic use of statins in humans is associated with increased plasma levels of PCSK9 [15-17].
The coordinated up-regulation of both the LDLR and PCSK9 genes by statins limits the increase of hepatic LDLR, the efficiency at plasma LDL-C clearance and, therefore, the therapeutic efficacy of the drugs. However, targeted reduction of PCSK9 expression or activity has been shown to potentiate the hypocholesterolemic effect of statins [18-20]. Accordingly, it is believed that PCSK9 inhibitors represent a promising novel class of anti-cholesterol drugs [9,21].
In order to reduce the levels of plasma cholesterol, it is desirable to provide a compound to both increase the level of LDLR and reduce the level of functional, secreted PCSK9 in a patient administered such a compound, since such changes would be expected to increase the cellular uptake of LDL from the blood stream and reduce the levels of plasma cholesterol in the patient.
It is an object of the present disclosure to provide the use of quercetin-3-O-β-D-glucoside (Q3G) for increasing the amount of cell surface low-density lipoprotein receptor (LDLR) on a hepatocyte cell and reducing the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the hepatocyte cell, where the Q3G is formulated for administration to the hepatocyte cell, and where the increase in cell surface LDLR and the decrease in secretion of functional PCSK9 is in comparison to the hepatocyte cell not exposed to Q3G.
The Q3G may be formulated for administration to provide a concentration of Q3G at the hepatocyte cell, in the extracellular medium, between about 0.1 μM and about 100 μM.
The Q3G may be formulated for administration to a patient having dyslipidemia where the increased amount of cell surface LDLR on the hepatocyte cell and the reduced amount of functional PCSK9 secreted by the hepatocyte cell is for treating metabolic syndrome, or a hypercholesterolemia related-disease or disorder.
The hypercholesterolemia related-disease or disorder may be an obesity-related disease, atherosclerosis, coronary artery disease, stroke, or type 2 diabetes.
The Q3G may be formulated for oral administration.
In another aspect, there is provided the use of quercetin-3-O-β-D-glucoside (Q3G) for reducing the amount of cell surface low-density lipoprotein receptor (LDLR) on a pancreatic beta cell and increasing the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the pancreatic beta cell, where the Q3G is formulated for administration to the pancreatic beta cell, and where the decrease in cell surface LDLR and the increase in secretion of functional PCSK9 is in comparison to the pancreatic beta cell not exposed to Q3G.
The Q3G may be formulated for administration to provide a concentration of Q3G at the pancreatic beta cell, in the extracellular medium, between about 4 μM and about 100 μM.
The Q3G may be formulated for administration to a patient having dyslipidemia where the decreased amount of cell surface LDLR on the pancreatic beta cell and the increased amount of functional PCSK9 secreted by the pancreatic beta cell is for reducing cytotoxic effects associated with cholesterol uptake by the pancreatic beta cell.
The hypercholesterolemia related-disease or disorder may be an obesity-related disease, atherosclerosis, coronary artery disease, stroke, or type 2 diabetes.
The Q3G may be formulated for oral administration.
In yet another aspect, there is provided the use of quercetin-3-O-β-D-glucoside (Q3G) in combination with a statin for increasing the amount of cell surface low-density lipoprotein receptor (LDLR) on a hepatocyte cell and reducing the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the hepatocyte cell, where the Q3G and the statin are formulated for administration to the hepatocyte cell, where the increase in cell surface LDLR is in comparison to the hepatocyte cell not exposed to either the Q3G or the statin, and where the decrease in secretion of functional PCSK9 is in comparison to the hepatocyte cell exposed to the statin but not exposed to Q3G.
The Q3G may be formulated for administration to provide a concentration of Q3G at the hepatocyte cell, in the extracellular medium, between about 0.1 μM and about 100 μM.
The statin may be simvastatin.
The Q3G and the statin may be formulated for administration to a patient having dyslipidemia where the increased amount of cell surface LDLR on the hepatocyte cell and the reduced amount of functional PCSK9 secreted by the hepatocyte cell is for treating metabolic syndrome, or a hypercholesterolemia related-disease or disorder.
The hypercholesterolemia related-disease or disorder may be an obesity-related disease, atherosclerosis, coronary artery disease, stroke, or type 2 diabetes.
The Q3G may be formulated for oral administration.
In still another aspect, there is provided a composition comprising quercetin-3-O-β-D-glucoside (Q3G) and a statin, the composition for increasing the amount of cell surface low-density lipoprotein receptor (LDLR) on a hepatocyte cell and reducing the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the hepatocyte cell, where the increase in cell surface LDLR is in comparison to the hepatocyte cell not exposed to either the Q3G or the statin, and where the decrease in secretion of functional PCSK9 is in comparison to the hepatocyte cell exposed to the statin but not exposed to Q3G.
The statin may be simvastatin.
In yet another aspect, there is provided a composition comprising quercetin-3-O-β-D-glucoside (Q3G) and a statin, the composition for: increasing the amount of cell surface low-density lipoprotein receptor (LDLR) on a hepatocyte cell and reducing the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the hepatocyte cell, where the increase in cell surface LDLR is in comparison to the hepatocyte cell not exposed to either the Q3G or the statin, and where the decrease in secretion of functional PCSK9 is in comparison to the hepatocyte cell exposed to the statin but not exposed to Q3G; and reducing the amount of cell surface low-density lipoprotein receptor (LDLR) on a pancreatic beta cell and increasing the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the pancreatic beta cell, where the decrease in cell surface LDLR is in comparison to the pancreatic beta cell not exposed to either the Q3G or the statin, and where the increase in secretion of functional PCSK9 is in comparison to the pancreatic cell exposed to the statin but not exposed to Q3G.
In a further aspect, there is provided a method of increasing the amount of cell surface low-density lipoprotein receptor (LDLR) on a hepatocyte cell and reducing the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the hepatocyte cell, the method including: treating the hepatocyte cell with an effective concentration of quercetin-3-O-β-D-glucoside (Q3G) the increase in cell surface LDLR and the decrease in secretion of functional PCSK9 being in comparison to the hepatocyte cell prior to treatment with the Q3G.
The effective concentration of Q3G at the hepatocyte cell, in the extracellular medium, may be between about 0.1 μM and about 100 μM.
In a still further aspect, there is provided a method of not substantially changing, or of decreasing the amount of cell surface low-density lipoprotein receptor (LDLR) on a pancreatic beta cell, and increasing the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the pancreatic beta cell, the method including: treating the pancreatic beta cell with an effective concentration of quercetin-3-O-β-D-glucoside (Q3G) the increase in cell surface LDLR and the decrease or lack of substantial change in secretion of functional PCSK9 being in comparison to the pancreatic beta cell prior to treatment with the Q3G.
The effective concentration of Q3G at the pancreatic beta cell, in the extracellular medium, may be between about 4 μM and about 100 μM.
In another aspect, there is provided a method of increasing the amount of cell surface low-density lipoprotein receptor (LDLR) on a hepatocyte cell and reducing the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the hepatocyte cell, the method including: treating the hepatocyte cell with an effective amount of quercetin-3-O-β-D-glucoside (Q3G) and a statin, the increase in cell surface LDLR being in comparison to the hepatocyte cell not exposed to either the Q3G or the statin, and the decrease in secretion of functional PCSK9 being in comparison to the hepatocyte cell exposed to the statin but not exposed to Q3G.
In a still further aspect, there is provided a method of reducing plasma cholesterol levels in a patient in need thereof, the method including: administering to the patient a therapeutically effective amount of quercetin-3-O-β-D-glucoside (Q3G) to increase the amount of cell surface low-density lipoprotein receptor (LDLR) on a hepatocyte cell and to reduce the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the hepatocyte cell, thereby increasing rate of cellular uptake of exogenous LDL from the plasma of the patient and reducing the plasma cholesterol levels in the patient, the increase in cell surface LDLR and the decrease in secretion of functional PCSK9 being in comparison to the hepatocyte cell prior to exposure to the Q3G.
Administration of the Q3G may increase the amount of functional PCSK9 secreted by a pancreatic beta cell and decrease the amount of cell surface LDLR on the pancreatic beta cell, the decrease or lack of substantial change in cell surface LDLR and the increase in secretion of functional PCSK9 being in comparison to the pancreatic beta cell prior to exposure to the Q3G.
The reduction of plasma cholesterol may result in the treatment or prevention of metabolic syndrome, or a hypercholesterolemia related-disease or disorder.
The hypercholesterolemia related-disease or disorder may be an obesity-related disease, atherosclerosis, coronary artery disease, stroke, or type 2 diabetes.
The Q3G may be orally administered to the patient.
In still a further aspect, there is provided a method of reducing plasma cholesterol levels in a patient in need thereof, the method including: administering to the patient a therapeutically effective amount of quercetin-3-O-β-D-glucoside (Q3G) and a therapeutically effective amount of a statin; where treatment of the patient with the Q3G and the statin increases the amount of cell surface low-density lipoprotein receptor (LDLR) on a hepatocyte cell when compared to the hepatocyte cell not exposed to either the Q3G or the statin, and reduces the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the hepatocyte cell in comparison to the hepatocyte cell exposed to the statin but not exposed to Q3G, the increased amount of hepatocyte cell surface LDLR and reduced amount of functional PCSK9 secreted by the hepatocyte cell resulting in an increased rate of cellular uptake of exogenous LDL from the plasma of the patient and a reduced level of plasma cholesterol in the patient.
The treatment of the patient with the Q3G may increase the amount of functional PCSK9 secreted by a pancreatic beta cell and decrease or not substantially change the amount of cell surface LDLR on the pancreatic beta cell, the decrease or lack of substantial change in cell surface LDLR and the increase in secretion of functional PCSK9 being in comparison to the pancreatic beta cell prior to exposure to the Q3G.
The reduction of plasma cholesterol may result in the treatment or prevention of metabolic syndrome, or a hypercholesterolemia related-disease or disorder.
The hypercholesterolemia related-disease or disorder may be an obesity-related disease, atherosclerosis, coronary artery disease, stroke, or type 2 diabetes.
The Q3G may be orally administered to the patient.
Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.
Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.
Generally, the present disclosure provides a compound that both increases the amount of cell-surface LDL-receptor on a hepatocyte cell and reduces the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the hepatocyte cell. The compound is quercetin-3-O-β-D-glucoside (Q3G). For example, Q3G reduces the amount of PCSK9 secreted by the hepatocyte cell, increasing the half-life of cell-surface LDL-receptor on the hepatocyte cell, and stimulating cholesterol clearance from the blood.
The Q3G also decreases the amount, or does not substantially change the amount, of cell-surface LDL-receptor on a pancreatic beta cell, and increases the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the pancreatic beta cell. In one example, Q3G increases the amount of PCSK9 secreted by the pancreatic beta cell, reducing the half-life of cell-surface LDL-receptor on the pancreatic beta cell, and protecting the beta cell from lipotoxic effects of excessive LDL-cholesterol uptake mediated by the LDL-receptor.
In the context of the present disclosure, it would be understood that “not substantially changing the amount of cell-surface LDL-receptor on a pancreatic beta cell” would correspond to an increase or a decrease of no more than 50% in comparison to the amount of cell-surface LDL-receptor on the pancreatic beta cell which has not been exposed to Q3G. For example, Example 7 and
Q3G may be considered a PCSK9 antagonist in hepatocyte cells, and a PCSK9 agonist in pancreatic beta cells.
An increase in the amount of cell-surface LDL-receptor and reduction in the amount of functional, secreted PCSK9 in hepatocyte cells may reduce plasma cholesterol levels in a patient treated with the compound due to accelerated cellular uptake of exogenous LDL. Increasing the amount of functional, secreted PCSK9 cells, while at the same time reducing or not substantially changing the amount of cell-surface LDL-receptor on pancreatic beta cells may reduce the likelihood of insulin insufficiency, impaired glucose-stimulated insulin secretion, or both.
Reduction in plasma cholesterol levels may be beneficial in treating metabolic syndrome, or hypercholesterolemia related-diseases or disorders. Examples of diseases or disorders which may be treated through a reduction in plasma cholesterol levels include: obesity-related diseases, atherosclerosis, coronary artery disease, stroke, and type 2 diabetes.
Other diseases or disorders which may be treated through a reduction in plasma cholesterol include: Alzheimer's disease, cancer and infectious diseases such as malaria and human immunodeficiency virus (HIV), since cholesterol and cholesterol-rich lipid rafts have been implicated in these diseases. It is believed that reduction of the level of circulating cholesterol may interfere with the pathophysiology of these diseases or disorders. Generally, any disease requiring high cholesterol for its progression may be targeted for treatment with a compound that both increases the amount of LDL-receptor on hepatocyte cells and reduces the amount of functional, secreted PCSK9 secreted by the hepatocyte cells.
The amount of cell-surface LDL-receptor in the liver, 70-85% by mass of which is made up of hepatocyte cells, may be indirectly measured by measuring clearance of plasma LDL levels since liver LDL-receptors are responsible for about 90% of the clearance of plasma LDL. Plasma LDL may be measured by standard techniques. Secreted PCSK9 may be determined using an ELISA assay, such as in commercially available assays from MBL International or R&D Systems.
As some plants have been shown to display anti-cholesterolemic properties [22], these plants were analyzed to determine if they contained compounds that increased the amount of cell-surface LDL-receptor, reduced the amount of functional, secreted PCSK9, or both increased the amount of cell-surface LDL-receptor and reduced the amount of functional, secreted PCSK9. Specifically, Moringa oleifera, Lam (M. oleifera), a perennial plant of the tropics, whose leaves have been shown to exhibit anti-dyslipidemic properties in experimental animals and in humans [23-27] was analyzed.
It was observed that exposure of Huh7 human hepatocytes in culture to an aqueous extract of M. oleifera leaves significantly reduced the amounts of PCSK9 secreted in the culture medium, in a concentration dependent manner, as illustrated in
The bioflavonoid quercetin was identified as a candidate compound for the observed anti-PCSK9 activity of the plant. Quercetin is found in amounts as high as 1 mg/g of Moringa oleifera leaf powder [28], predominantly as quercetin-3-O-β-D-glucoside (Q3G) [29,30] (
It was further observed that exposure of MIN6 β-cells (a mouse insulinoma cell line) in culture to Q3G stimulates PCSK9 expression and secretion, without affecting glucose-stimulated insulin secretion (GSIS).
Based on the results discussed herein, it has now been established that quercetin-3-O-β-D-glucoside: increases the amount of cell-surface LDLR and inhibits PCSK9 secretion in hepatocytes. It has also been established that the Q3G stimulates PCSK9 secretion while at the same time reduces or does not substantially change the cell-surface level of LDL-receptor in pancreatic beta cells.
Accordingly, the present disclosure provides a method of increasing the amount of cell-surface LDL-receptor on hepatocyte cells and reducing the amount of functional, secreted PCSK9 secreted by the hepatocyte cells. For example, Q3G reduces the amount of PCSK9 secreted by the hepatocyte cell, increasing the half-life of cell-surface LDL-receptor on the hepatocyte cell, and stimulating cholesterol clearance from the blood.
The present disclosure also provides a method of not substantially changing or decreasing the amount of cell-surface LDL-receptor on a pancreatic beta cell while at the same time increasing the amount of functional proprotein convertase subtilisin/kexin type 9 (PCSK9) secreted by the pancreatic beta cell. For example, Q3G increases the amount of PCSK9 secreted by the pancreatic beta cell, reducing the half-life of cell-surface LDL-receptor on the pancreatic beta cell, and protecting the beta cell from lipotoxic effects of excessive LDL-cholesterol uptake mediated by LDL-receptor.
Such an increase in the amount of cell-surface LDL-receptor on the hepatocytes and reduction in the amount of functional, secreted PCSK9 secreted by the hepatocytes is expected to reduce plasma cholesterol levels in a patient treated with quercetin-3-O-β-D-glucoside due to accelerated cellular uptake of exogenous LDL. Reduction in plasma cholesterol levels are expected to be beneficial in treating metabolic syndrome, or hypercholesterolemia related-diseases or disorders. Examples of diseases or disorders which are expected to be treated through a reduction in plasma cholesterol levels include: atherosclerosis, coronary artery disease, stroke, and type 2 diabetes.
The treatment with Q3G may be especially beneficial to the cardiovascular system when the treatment results in: hepatocytes with increased amounts of cell-surface LDL-receptor; and pancreatic beta cells with increased amount of secreted PCSK9 and with substantially unchanged or reduced amounts of LDL-receptor. Increasing the amount of secreted PCSK9 in pancreatic beta cells, while reducing or leaving the amount of LDL-receptor substantially unchanged, protects the pancreatic beta cells from lipotoxicity resulting from excessive LDL-cholesterol uptake mediated by the LDL-receptor, and therefore helps maintain glucose homeostasis.
Q3G may be administered orally, for example in an oral dose between 150 mg and 1 g. It is believed that oral administration of Q3G will result in an increase in the amount of cell-surface LDL-receptor on hepatocyte cells and a reduction in the amount of functional, secreted PCSK9 secreted by the hepatocyte cells since i) Moringa leaf powder taken orally can effectively reduce cholesterol in animal; ii) Q3G is the predominant form of quercetin in Moringa leaf powder; and iii) Q3G can be taken up by the intestine and its derivatives (sulfated, methylated or glucuronylated) are found in the blood. Q3G may also be administered parenterally (intravenously). Q3Q has been administered intravenously to treat hypertension, as discussed by M. Russo et al. in Biochemical Pharmacology 83 (2012) 6-15.
The results discussed herein indicate that in vitro exposure of Huh7 hepatocytes with Q3G (i) stimulates proSREBP-2 proteolytic activation, (ii) increases the levels of LDLR mRNA and protein, (iii) increases the cell surface density of LDLR, (iv) reduces the cellular levels of PCSK9 mRNA, (v) reduces PCSK9 accumulation in the culture medium and (vi) accelerates cellular uptake of exogenous LDL.
Although the examples disclosed herein were performed at low micromolar concentrations (i.e. concentrations between 2 μM and 50 μM), it is expected that Q3G may be administered at an in vivo concentration of about 0.1 μM to about 100 μM and still result in, in hepatocytes: (i) stimulation of proSREBP-2 proteolytic activation, (ii) increased levels of LDLR mRNA and/or protein, (iii) increased cell surface density of LDLR, (iv) reduced levels of PCSK9 mRNA, (v) reduced PCSK9 accumulation in the culture medium, (vi) accelerated cellular uptake of exogenous LDL, or (vii) any combination thereof. In certain examples, a therapeutically effective dose is a dose administered such that the recipient's plasma level of Q3G is in the range of 0.5 to 5 μM. This may be achieved, for example, through the oral administration of about 2 mg of Q3G/kg of body weight. See, for example, K. Murota et al. Achives of Biochemistry and Biophysics 501 (2010) 91-97.
In view of the present disclosure, it is expected that in vivo exposure of hepatocytes to Q3G would similarly: (a) increases the cell surface density of LDL-receptor on the hepatocytes and (b) reduce the level of functional, secreted PCSK9 secreted by the hepatocytes. This increased cell surface density of LDLR and reduced levels of functional, secreted PCSK9 would similarly be expected to accelerate cellular uptake of plasma LDL and lead to a reduction in plasma cholesterol levels, though the reduction in plasma cholesterol levels is due, in vivo, to hepatocytes and the impact of extrahepatic tissues in plasma cholesterol levels is overshadowed by the impact of the hepatocytes. Such a reduction in plasma cholesterol levels is expected to be beneficial in treating metabolic syndrome, or hypercholesterolemia related-diseases or disorders. Examples of diseases or disorders which may be treated through a reduction in plasma cholesterol levels include: obesity-related diseases, atherosclerosis, coronary artery disease, stroke, and type 2 diabetes.
Without wishing to be bound by theory, the in vitro accelerated uptake of exogenous LDL is believed to at least partially be due to a higher density of LDLR at the cell surface of the hepatocytes, following stimulated expression of its gene by SREBP-2. However, the 2× increase of LDLR mRNA could not, alone, account for the 4× increase in the LDLR level. It is also believed that the protein half-life was also increased, since the level of secreted PCSK9 decreased. Indeed, although an intracellular LDLR-degrading activity has been suggested for PCSK9 [39], the remarkable hypocholesterolemic efficacy of parenteral therapy using anti-PCSK9 antibodies [40,41] is evidence that the primary mechanism of action of PCSK9 involves its prior secretion and its subsequent binding to the LDL receptor at the cell surface. The attenuation of LDLR increase when Huh7 cells were exposed to Q3G above 2-digit micromolar concentrations may be due to feedback repression of the LDLR gene following the intracellular accumulation of cholesterol caused by the flavonoid.
Without wishing to be bound by theory, the reduction of cellular levels of PCSK9 mRNA in hepatocytes following treatment with Q3G is believed to result from invalidation of co-activators of the PCSK9 gene promoter, induction of repressors of this promoter, increased instability of the transcript, or a combination thereof. Berberine (BBR) which, like Q3G, is a plant-derived hypocholesterolemic compound, reduces PCSK9 gene transcription by inducing decreased expression of hepatocyte nuclear factor la (HNF-1α). This factor cooperates with SREBP-2 to activate the PCSK9 promoter. In its absence, the promoter activity is reduced [42]. Unlike BBR, Q3G does not change the level of HNF-1α (
The data discussed herein indicate that chronic exposure of Huh7 cells to Q3G reduces PCSK9 accumulation in the culture medium by delaying its transit through the secretory pathway. The delay appears not to be caused by impaired proteolytic processing of its precursor. Quercetin is known to bind, covalently in some cases, to selected cellular proteins [43-45]. Without wishing to be bound by theory, the spectroscopy data discussed herein suggest that Q3G can bind to recombinant human PCSK9 in vitro, as illustrated in
PCSK9 has been recently shown to interact with Apo B, protecting it from autophagic degradation [46]. Quercetin aglycone, at 5-30 μM, has been shown to inhibit Apo B secretion by intestinal Caco-2 cells. The inhibition was selective since there was no difference between treated and untreated cells in the overall amount of secreted proteins after a 2-h metabolic pulse-labeled with radioactive amino acids. In this case, inhibition of Apo B secretion appeared to be caused by reduced packaging of triacylglyceride to the protein [47]. Interference with normal intermolecular interactions is one of possible mechanisms of Q3G-induced delay of PCSK9 secretion.
Inhibition of PCSK9 secretion or an increase in LDLR level in Huh7 cells exposed to quercetin aglycone was not observed at the concentrations of Q3G discussed herein. Another recent study has reported LDLR up-regulation in HepG2 hepatocytes with 75 μM of the non-glycosylated form of quercetin [48]. Without wishing to be bound by theory, it is believed that the greater effectiveness of the glycosylated form of quercetin may be due to its ability to enter into cells more efficiently, to interact more strongly with functional proteins at the cell surface or within the cell, or a combination thereof.
In pigs and dogs fed a meal supplemented with either quercetin aglycone, Q3G, or quercetin-3-O-glucorhamnoside (rutin), quercetin bioavailability was significantly greater with Q3G as a supplement than with the other two forms of quercetin [49,50]. Intestinal Na-dependent glucose transporter 1 (SGLT1) appears to mediate this preferential uptake [51]. Yet quercetin aglycone has been shown to penetrate, passively or actively, inside a variety of other cell types [52], including HepG2 hepatocytes, where it elicited significant changes in gene expression [53].
Statins induce expression of LDLR and PCSK9. However, unlike Q3G, statins do not reduce PCSK9 secretion. Administration of Q3G to a patient may be used to reduce the level of functional, secreted PCSK9 secreted by hepatocyte cells which is stimulated by the administration of an inhibitor of HMGCoA reductase, for example a statin such as simvastatin, to the patient. Example 4 discusses the treatment of hepatocytes with Q3G and/or simvastatin. The results suggest that simvastatin and Q3G stimulated LDLR expression through similar mechanism; but that Q3G possesses, in addition, distinct anti-PCSK9 production/secretion properties. It is expected that Q3G could similarly be used to reduce the stimulated level of functional, secreted PCSK9 in a patient administered a statin other than simvastatin. The level of secreted, plasma PCSK9 in a patient may be measured using commercially available ELISA kits.
Materials
Huh7 human liver cells and the rabbit anti-human PCSK9 antibody for immunoblotting were obtained from Dr. Nabil G Seidah. The rabbit anti-human PCSK9 antibody for immunoprecipitation was produced in house. The following antibodies were from commercial sources: anti-LDLR (RD Systems), anti-β-actin and simvastatin (Sigma), anti-SREBP-2 (Santa Cruz), Horseradish peroxidase (HRP)-conjugated antirabbit or mouse immunoglobulins (Ig) (GE HealthCare) or anti-goat Ig (Santa Cruz). The chemiluminescence revelation kit was from PerkinElmer; the PCSK9 ELISA kit from Circulex or RD Systems; the RNeasy extraction kit from Qiagen. Superscript II RNase H-Reverse Transcriptase, bodipy-LDL, non-conjugated LDL; lipoprotein-depleted serum (LPDS), and Alexa Fluor 488™ were from Invitrogen. The FastStart TaqMan ProbeMaster-Rox master mix, primer pairs, and Universal Probe Library (UPL) fluorescent probes and Protease Inhibitor Cocktail (PIC) were from Roche, and Amplify fluor solution from Amersham Biosciences. Q3G was obtained from Sigma; goat anti-mouse LDLR from Cederlane; anti-β-actin monoclonal primary anti-body and horseradish (HRP)-conjugated donkey anti-goat IgG from Santa Cruz; HRP-conjugated sheep anti-mouse IgG from GE HealthCare; ELISA kit for mouse PCSK9 and mouse insulin from R & D Systems, and Crystal Chem, respectively; the protease inhibitor cocktail (PIC), the FastStart TaqMan ProbeMaster-Rox master mix, primer pairs and fluorescent probes from Roche; the RNA extraction kit from Qiagen, Super-script II RNase H-Reverse Transcriptase from Invitro-gen, the Western Lightning Chemiluminescence Reagent Plus a chemiluminescence-based revelation kit from Perkin-Elmer.
Cell Culture and Lysis
At passage, Huh7 cells were routinely seeded at sub-confluence (˜106 cells/10-cm dish) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) or LPDS (for experiments) and 50 μg/ml gentamycin. They were incubated overnight at 37° C., in a humidified 5% CO2-95% air atmosphere. Cells were treated or not with Q3G at defined concentrations and for defined lengths of time. Media were collected and centrifuged at 200 g for 5 min to sediment suspended cells; supernatants were collected and supplemented with 0.33 volumes of a 3×-concentrated PIC. Cell monolayers were rinsed with ice-cold phosphate-buffered saline (PBS); they were overlaid with 0.5 of the RIPA lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate and 0.1% SDS) supplemented with 1× PIC. After 20 min in an ice bath, the lysates were centrifuged at 14,000 g and 4° C. for 20 min, and supernatants were collected. Conditioned media and lysates were stored at −20° C. until analysis.
Mouse insulinoma MIN6 cells were cultured in a 5% CO2-95% air atmosphere at 37° C. in DMEM medium containing 10% heat-inactivated fetal bovine serum, 1 mM Na-pyruvate, 2 mM L-glutamine, 25 mM D-glucose, and 28 μM β-mercaptoethanol. Q3G at a specific final concentration was supplemented to the culture medium and incubation was conducted for a selected length of time. Media were collected, spun at 600 g to sediment suspended cells, supplemented with 0.5 volumes of 3×RIPA-PIC (1×: 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate and 0.1% SDS and PIC). Cells were lysed in 1×RIPA-PIC for immuno-blotting, or in RNA extraction buffer for qRT-PCR.
Metabolic Labeling
For metabolic labeling, Huh7 cells were seeded in a 12-well plate 8×105 cells/well in 1.5 ml/well of complete medium and incubated overnight. After a rinse with Dulbecco's PBS (PBS-D), cell monolayers were overlaid with 1.5 ml of DMEM/10% LPDS without or with 5 μM Q3G, and were incubated for 24 h. Fresh serum-free medium (SFM, 1.5 ml) was substituted, and cells were allowed to incubate for 30 min to reduce endogenous Met and Cys. The medium was removed and replaced with fresh SFM (0.75 ml/well) containing 300 μCi/ml35S-Met/Cys, and cells were incubated at 37° C. for 20 min to label de novo biosynthesized proteins (pulse-labeling). The radioactive medium was replaced with DMEM/0.5% LPDS containing 10 mM non-radioactive Met/Cys and cells were incubated at 37° C. for 0, 15, 30, 60, 90 and 120 min (chase). Conditioned media and cell lysates were processed as described above.
Flow Cytometry
Cells were seeded at 4×104 cell/10-cm dish in 3 ml of completed medium and incubated overnight. LPDS medium containing or not 5 μM Q3G was substituted and incubation resumed for 24 h. Subsequent steps were conducted with ice-cold solutions and at 4° C. Cell monolayers were rinsed with PBS, overlaid with PBS containing rabbit anti-LDLR antibody for 1 h, then with PBS containing Alexa Fluor-488-conjugated goat anti-rabbit Ig antibody for another 1 h. After a PBS rinse, the cells were overlaid with Versene, suspended in DMEM and analyzed in Benson-Dickenson XL flow cytometer at 492 nm and 520 nm excitation and emission wavelengths, respectively. Cell autofluorescence and non-specific fluorescence were assessed using cells not treated with the secondary and the primary antibody, respectively.
LDL Uptake Assay
Huh7 cells were seeded in 96-well black-bottom plates at 4×104 cells/well in 0.1 ml complete medium and allowed to attach by overnight incubation at 37° C. They were rinsed with PBS-D, overlaid with 0.1 ml of DMEM/10% LPDS and incubated at 37° C. for 24 h. After a PBS-D rinse, they were overlaid with 0.1 ml DMEM/0.5% LPDS containing or not 5 μM Q3G and incubated 37° C. for 24 h. To assay for LDL uptake ability, cells were rinsed, first with pre-warmed (37° C.) PBS-D, then with pre-warmed DMEM/0.5% LPDS. They were overlaid with 75 μl of the latter medium containing 20 mg/ml bodipy-LDL, and then incubated at 37° C. for 15 min or 30 min to allow LDLR-mediated endocytosis of the fluorescent lipoprotein. The process was stopped by substituting ice cold DMEM/0.5% LPDS. After 3 rinses with 0.2 ml of ice-cold PBS-D, the cells were fixed with 0.1 ml of isopropanol for 20 min, in the dark and with gentle shaking. Intracellular fluorescence was measured in a SpectraMax Gemini XS fluorescence plate reader (Molecular Devices) at the excitation and emission wavelengths of 485 and 535 nm, respectively. Non-specific fluorescence was measured by incubating cells in medium containing bodipy-LDL (20 μg/ml) and a 12.5× excess of non-fluorescent LDL (250 μg/ml).
RT-qPCR
Total RNA was extracted using the Qiagen RNeasy extraction kit. It was reverse transcribed into cDNA using random hexameric primers and the Superscript II RNase H-Reverse Transcriptase. The levels of specific cDNAs were quantified by PCR-based fluorogenic Taqman assays [34], using FastStart TaqMan ProbeMaster-Rox master mix, primer pairs and the appropriate fluorescent UPL probes as shown in Table 1, in a Mx3005P thermocycler (Stratagene, LaJolla, Calif.). The probes were designed using an online algorithm at the Roche Universal Probe Library Assay Design Center.
Standard curves were established using varying amounts of purified and quantified cDNA amplicons of each mRNA. The level of mRNA for the TATA-binding protein (TBP) was used for normalization.
qRT-PCR
For the mouse studies, the levels of specific mRNAs were quantified in a PCR-based fluorogenic assay using the Taqman technology (Holland et al., 1991). Briefly, total RNA was extracted using the RNeasy extraction kit and reverse-transcribed into cDNA using random hexameric primers and the Superscript II RNase H-Reverse Transcriptase. The cDNA was used as a template to produce PCR amplicons using FastStart TaqMan ProbeMaster-Rox master mix, primer pairs and the appropriate fluorescent probes) in the Stratagene Mx3005P thermocycler. Standard curves were established using varying amounts of pre-quantified amplicons of each transcript. The level of mRNA for the TATA-box binding protein (TBP) was used for normalization.
ELISA
The assays for PCSK9 and insulin were conducted as prescribed by kit manufacturers, using a Thermo Scientific plate reader. For example, PCSK9 levels in conditioned media were measured using the human PCSK9 ELISA kit from Circulex, as specified the manufacturer. The assay was a sandwich immunoassay using two antibodies (A and B) recognizing different PCSK9 epitopes. Briefly, aliquot of diluted media were overlaid on wells coated with anti-PCSK9 antibody A. After 1-h incubation, the wells were washed, overlaid with a solution of HRP-conjugated anti-PCSK9 antibody B, and incubated for 1 h. They were washed again, and overlaid with a solution of tetra-methylbenzidine as a chromogenic substrate for HRP. After 15 min, the reaction was stopped with ammonium sulfate and the absorbance of the reaction mixtures measured by spectrophotometry at 450 nm. All the steps were performed at room temperature. Standards consisted of recombinant human PCSK9.
Immunoblotting
Cell lysates were fractionated by SDS-PAGE and electrophoretically transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with a goat antihuman LDLR, rabbit anti-PCSK9, or rabbit anti-SREBP-2 polyclonal antibody at 1:1000, 1:1500, and 1:200 dilutions, respectively, and then with a HRP-conjugated heterospecific secondary antibody against the primary Igs at a 1:2000 dilution. It was probed for HRP reaction using the Western Lightning Chemiluminescence Reagent Plus a chemiluminescence-based revelation kit. The signal was captured on X-ray film and immunoreactive bands analyzed by densitometry on a Syngene's ChemiGenius2XE Bio Imaging System (Cambridge, Mass.) within the dynamic range of the instrument. The membrane was stripped and reprobed with the anti-β-actin monoclonal primary antibody at 1:20,000 dilution and HRP-conjugated rabbit anti-mouse IgG secondary antibody at a 1:5000 dilution. The densitometric values of β-actin bands were using for normalization of experimental samples.
Immunoprecipitation
Radioactive conditioned media or cell lysates (0.1 ml) were supplemented with 2 l of normal rabbit serum and 15 μl of a 50% (w/v) suspension of Protein A-agarose. After a 1-h incubation at 4° C. with rotational mixing, the samples were centrifuged at 3,000 g for 5 min at 4° C. Supernatants were supplemented 2 μl of rabbit anti-PCSK9 [35], and incubated as above. The resin with bound immune complexes was then sedimented by centrifugation as above, rinsed three times with RIPA buffer, twice with a buffer containing 1 M NaCl, 10 mM Tris-HCl and 1 mM EDTA, pH 8, and twice with PBS containing 1 mM EDTA. Pellets were suspended in 25 μl of 1× Laemmli buffer each, boiled for 5 min, and sedimented as above. Supernatant was subjected to electrophoresis through polyacrylamide gels (8 or 12%). Gels were fixed for 30 min in a 50% methanol-10% acetic acid solution, treated for 30 min with Amplify fluor solution, dried under vacuum and exposed to phosphorimaging screen overnight. Specific radioactive protein bands were visualized and quantified on a Typhoon Phosphorimager (Molecular Dynamics).
GSIS Assay
Cells were seeded and grown to 80% confluence. Prior to GSIS assay, fresh medium containing 3 mM Glucose and 10% FBS medium (low-glucose medium or LGM) without or with Q3G was substituted and incubation resumed for 6 h to adapt the cells to low glucose. Fresh LGM without or with Q3G was substituted and supplemented or not with glucose to the final concentration of 18 mM. After 30 min of incubation, media were collected as above for insulin-specific ELISA.
Huh7 cells, hepatocyte derived cellular carcinoma cells, were incubated for 24 h in medium containing 10% lipoprotein-depleted serum (LPDS) and 0 to 10 μM Q3G. The level of LDLR mRNA was measured by quantitative real-time RT-PCR; that of the LDLR protein by semi-quantitative immunoblotting. Exposure to Q3G increased the intracellular content of LDLR mRNA in a concentration-dependent manner; the increase reached a 2× maximum at 2 μM (P<0.01, relative to no Q3G) (
In contrast, at the highest Q3G concentration tested, PCSK9 mRNA levels decreased by one-third (P<0.05) (
The kinetics of cellular accumulation of LDLR and PCSK9 at 2 μM Q3G was also examined: PCSK9 accumulation in the cells began after a 3-h lag; that of LDLR after 6-h lag (
The increase in LDLR mRNA content could be attributed to increased transcription of its gene. This transcription is known to be up regulated by SREBP-2 [36], a nuclear transcriptional factor generated through two successive cleavages of its ER membrane bound precursor, proSREBP-2, by the Golgi proteases PCSK8/S1 P and S2P [37]. We therefore examined the effect of Q3G on SREBP-2 expression. The results are shown in
Since PCSK9 can be secreted only after endoproteolytic cleavage of its precursor at the carboxyl end of the prodomain, and the formation of a PCSK9/prosegment complex, it was possible that the reduced secretion of PCSK9 by Q3G-treated Huh7 cells resulted from impaired processing of its precursor. We verified this possibility by pulse-chase analysis. Cells were incubated for 24 h in the absence, or in the presence 5 μM Q3G; they were then metabolically pulse-labeled using radioactive amino acids; the newly biosynthesized radioactive proteins were chased for varying periods of time; PCSK9-related proteins in cell lysates and media were analyzed by immunoprecipitation, SDS/PAGE, and semi-quantitative phosphorimaging. The results are shown in
Statins induce expression of LDLR and PCSK9. However, unlike Q3G, they do not reduce PCSK9 secretion. We examined whether, at a 5 μM concentration of Q3G, simvastatin at 0.2 and 1 μM could further up regulate LDLR expression in Huh7 cells; and, inversely, whether the flavonol can reduce statin-stimulated PCSK9 secretion secreted by Huh7 cells. The results are shown in
To be functionally relevant, Q3G-induced LDLR should accumulate at the cell surface of the hepatocyte cells where it could mediate LDL uptake. To verify the surface localization of the receptor, untreated and pretreated intact Huh7 cells were stained at 4° C. for LDLR by indirect immunofluorescence, and analyzed by fluorescence flow cytometry. The results are shown in
An increase of LDLR expression, combined with a reduction of PCSK9 secretion, should significantly improve the ability of Huh7 cells to take up exogenous LDL. To verify this prediction, cells were incubated overnight in medium supplemented with LPDS to promote expression of the LDLR; they were then treated with 5 μM Q3G for 24 h and exposed to fluorescent bodipy-LDL for 15 or 30 min; after washing, accumulated intracellular LDL was measured by fluorescence spectrometry. As shown in
MIN6 β-cells were incubated for 24 h in the presence of different concentrations of Q3G. Total RNA was extracted and analyzed by qRT-PCR for the levels of mRNA for PCSK9, LDLR, ABCA1 and ABCG1.
The results indicated that, for pancreatic beta cells, at concentrations of up to 4 mM, Q3G does not affect PCSK9 and LDLR mRNA levels, but increases by about 50% the levels of ABCA1 and ABCG1 mRNA. At 8 mM and above, it increased the mRNA levels of the PCSK9 and LDL-receptor while reducing those of ABCA1 and ABCG1. At the maximum Q3G concentration used (32 mM), the increase in PCSK9 mRNA was greater (2.5-fold than that of LDLR mRNA (1.8-fold). These results suggest that Q3G at low micromolar could promote cholesterol efflux using pancreatic beta cells by increasing the levels of ABCA1 and ABCG1, but at two-digit concentrations, would oppose cholesterol influx into the pancreatic beta cells by increasing more PCSK9 expression and effectively opposing LDLR-mediated uptake of cholesterol. The increase of PCSK9 in the medium paralleled that of the transcript (
This increased translation is reflected by the higher intracellular content of proPCSK9, presumably located in the endoplasmic reticulum (
At the protein level, the relative amounts of the LDLR, ABCA1 and ABCG1 in Q3G-treated pancreatic beta cells were in concordance with the relative amounts of mRNA, suggesting that the observed regulation of these cholesterol homeostatic proteins is primarily transcriptional.
Since exogenous and endogenous cholesterol levels can affect the responsiveness of β-cells secretory granules to exocytosis (Hao et al., 2007; Tsuchiya et al., 2010), the authors of the present disclosure examined whether Q3G regulation of cholesterol homeostatic proteins affected insulin secretion by MIN6 β-cells upon stimulation with 18 mM glucose.
As shown in
In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the examples. However, it will be apparent to one skilled in the art that these specific details are not required.
The above-described examples are intended to be exemplary only. Alterations, modifications and variations can be effected to the particular examples by those of skill in the art without departing from the scope, which is defined solely by the claims appended hereto.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2013/050507 | 6/28/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61667736 | Jul 2012 | US |
