Inhibition Of Cholesterol Synthesis Research Articles

P68.18 Relationship Between Cholesterol Synthesis in Cancer Cells and Anticancer Effect of Statins

Statins are blood cholesterol-lowering drugs that exert their effects through inhibiting cholesterol synthesis via the mevalonate pathway. In addition to cholesterol, this pathway produces intermediate products such as farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and squalene. It is reported that statins have an antiproliferative effect through depleting FPP and GGPP, suggesting that they could be useful for cancer treatment. Since FPP is located at a branch point between sterol and non-sterol production, squalene synthase (FDFT1) that converts FPP to squalene is thought to have an important role to control the flow of the mevalonate pathway; however, the effects of statins on this branch point remain unclear.

Intermittent Fasting Inhibits High-Fat Diet-Induced Atherosclerosis by Ameliorating Hypercholesterolemia and Reducing Monocyte Chemoattraction.

Atherosclerosis is a major pathology for cardiovascular diseases (CVDs). Clinically, the intermittent fasting (IF) has been observed to reduce the risk of CVDs. However, the effect of IF on the development of atherosclerosis has not been fully elucidated. Herein, we determined the protection of IF against high-fat diet–induced atherosclerosis in pro-atherogenic low-density lipoprotein receptor deficient (LDLR-/-) mice and the potentially involved mechanisms. The LDLR-/- mice were scheduled intermittent fasting cycles of 3-day HFD feeding ad libitum and 1 day fasting, while the mice in the control group were continuously fed HFD. The treatment was lasted for 7 weeks (∼12 cycles) or 14 weeks (∼24 cycles). Associated with the reduced total HFD intake, IF substantially reduced lesions in the en face aorta and aortic root sinus. It also increased plaque stability by increasing the smooth muscle cell (SMC)/collagen content and fibrotic cap thickness while reducing macrophage accumulation and necrotic core areas. Mechanistically, IF reduced serum total and LDL cholesterol levels by inhibiting cholesterol synthesis in the liver. Meanwhile, HFD-induced hepatic lipid accumulation was attenuated by IF. Interestingly, circulating Ly6Chigh monocytes but not T cells and serum c-c motif chemokine ligand 2 levels were significantly reduced by IF. Functionally, adhesion of monocytes to the aortic endothelium was decreased by IF via inhibiting VCAM-1 and ICAM-1 expression. Taken together, our study indicates that IF reduces atherosclerosis in LDLR-/- mice by reducing monocyte chemoattraction/adhesion and ameliorating hypercholesterolemia and suggests its potential application for atherosclerosis treatment.

Defining the Cholesterol Lowering Mechanism of Bergamot (Citrus bergamia) Extract in HepG2 and Caco-2 Cells.

Bergamot, a Mediterranean citrus fruit native to southern Italy, has been reported to have cholesterol-lowering properties; however, the mechanism of action is not well understood. Due to structural similarities with 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitors, it has been proposed that the phenolic compounds in bergamot may also inhibit HMGCR. Statins are widely used for their cholesterol-lowering properties; however, they are not universally well tolerated, suggesting there is a need to identify novel cholesterol-lowering strategies. In the present study, we investigated bergamot fruit extract (BFE) and its principal components (neoeriocitrin, naringin, neohesperidin, melitidin, and brutieridin) for their ability to regulate cholesterol levels in HepG2 and Caco-2 cells. BFE at increasing concentrations decreased the levels of total and free cholesterol in HepG2 cells. BFE and its constituents did not directly inhibit HMGCR activity. However, BFE and neohesperidin decreased HMGCR levels in HepG2 cells, suggesting that neohesperidin and BFE may downregulate HMGCR expression. An increase in AMP-kinase phosphorylation was observed in BFE and neohesperidin-treated cells. In Caco-2 cells, brutieridin exhibited a significant reduction in cholesterol uptake and decreased the level of Niemann-Pick C1 Like 1, an important cholesterol transporter. Taken together, our data suggest that the cholesterol-lowering activity of bergamot is distinct from statins. We hypothesize that BFE and its principal constituents lower cholesterol by inhibiting cholesterol synthesis and absorption.

Low dose red yeast rice with monacolin K lowers LDL cholesterol and blood pressure in Japanese with mild dyslipidemia: A multicenter, randomized trial.

Red yeast rice contains monacolin K, an inhibitor of cholesterol synthesis, and gamma-aminobutyric acid, a neurotransmitter. The daily dose of red yeast rice and monacolin K in previous studies was relatively high; therefore, there were safety concerns. We aimed to examine the effects of low daily dose red yeast rice on arteriosclerosis in patients with mild dyslipidemia. Eighteen patients without known cardiovascular disease and unsatisfactory low-density lipoprotein cholesterol (3.96±0.19 mmol/L) controlled only by diet therapy were randomly allocated to receive low dose red yeast rice (200 mg/day) containing 2 mg monacolin K or diet therapy alone for 8 weeks. The primary outcome was the absolute change in low-density lipoprotein cholesterol. Secondary outcomes included total cholesterol, apolipoprotein B, and blood pressure. Low-density lipoprotein cholesterol decreased significantly in the red yeast rice group than in the diet therapy group (median [interquartile range]: control -0.20 [-0.62, 1.19] mmol/L vs. red yeast rice -0.96 [-1.05, -0.34] mmol/L, p=0.030). The red yeast rice group also exhibited significant decreases in total cholesterol, apolipoprotein B, and blood pressure. No severe treatment-related adverse effects on muscles, liver, or renal function were observed. We found that patients in the red yeast rice group exhibited significant reductions in lowdensity lipoprotein cholesterol, total cholesterol, apolipoprotein B, and blood pressure without any recognised adverse effect. This suggests that low daily dose red yeast rice could reduce cardiovascular risk in patients with dyslipidemia.

Salsalate reduces atherosclerosis through AMPKβ1 in mice

ObjectiveSalsalate is a prodrug of salicylate that lowers blood glucose in people with type 2 diabetes. AMP-activated protein kinase (AMPK) is an αβγ heterotrimer which inhibits macrophage inflammation and the synthesis of fatty acids and cholesterol in the liver through phosphorylation of acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), respectively. Salicylate binds to and activates AMPKβ1-containing heterotrimers that are highly expressed in both macrophages and liver, but the potential importance of AMPK and ability of salsalate to reduce atherosclerosis have not been evaluated. MethodsApoE−/− and LDLr−/− mice with or without (−/−) germline or bone marrow AMPKβ1, respectively, were treated with salsalate, and atherosclerotic plaque size was evaluated in serial sections of the aortic root. Studies examining the effects of salicylate on markers of inflammation, fatty acid and cholesterol synthesis and proliferation were conducted in bone marrow–derived macrophages (BMDMs) from wild-type mice or mice lacking AMPKβ1 or the key AMPK-inhibitory phosphorylation sites on ACC (ACC knock-in (KI)-ACC KI) or HMGCR (HMGCR-KI). ResultsSalsalate reduced atherosclerotic plaques in the aortic roots of ApoE−/− mice, but not ApoE−/− AMPKβ1−/− mice. Similarly, salsalate reduced atherosclerosis in LDLr−/− mice receiving wild-type but not AMPKβ1−/− bone marrow. Reductions in atherosclerosis by salsalate were associated with reduced macrophage proliferation, reduced plaque lipid content and reduced serum cholesterol. In BMDMs, this suppression of proliferation by salicylate required phosphorylation of HMGCR and the suppression of cholesterol synthesis. ConclusionsThese data indicate that salsalate suppresses macrophage proliferation and atherosclerosis through an AMPKβ1-dependent pathway, which may involve HMGCR phosphorylation and cholesterol synthesis. Since rapidly-proliferating macrophages are a hallmark of atherosclerosis, these data indicate further evaluation of salsalate as a potential therapeutic agent for treating atherosclerotic cardiovascular disease.

Characterization of Citrus Pectin Oligosaccharides and Their Microbial Metabolites as Modulators of Immunometabolism on Macrophages.

We characterized the structure of prepared citrus pectin oligosaccharides (POS) and investigated the immunometabolism-modulating effects of POS and their microbial metabolites on human macrophages. Both POS and metabolites activated immune responses and exhibited anti-inflammatory properties in the presence of lipopolysaccharide (LPS) via regulating expressions of inflammatory cytokines and nuclear factor-kappa B. Cholesterol efflux was also facilitated via increased gene expressions of the liver X receptor-α-adenosine triphosphate-binding cassette transporter (ABC) A1/ABCG1 pathway and suppressed cholesterol synthesis via suppressing expressions of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Microbial degradation prevented POS from attenuating palmitoyl-3-cysteine-serine-lysine-4-induced inflammation and promoting M2 polarization, but it is capable of inhibiting cholesterol uptake-related genes CD36 and SR-A. These findings indicate that immunometabolism-modulating effects of POS are not solely microbiota-dependent effects. Both POS and their microbial metabolites are potential immunometabolism modulators via different mechanisms.

Glycosaminoglycan from Ostrea rivularis attenuates hyperlipidemia and regulates gut microbiota in high-cholesterol diet-fed zebrafish.

Hyperlipidemia an immense group of acquired or genetic metabolic disorders that is characterized by an excess of lipids in the bloodstream. Altogether, they have a high prevalence worldwide and constitute a major threat to human health. Glycosaminoglycans (GAG) are natural biomolecules that have hypolipidemic activity. The purpose of this study was to investigate the potential hypolipidemic effect of glycosaminoglycans extracted from Ostrea rivularis (OGAG) on hyperlipidemic zebrafish, as well as the possible underlying mechanism of such effect. Dietary supplementation with OGAG during 4 weeks significantly reduced the serum and hepatic lipid levels and the hepatosomatic index in hyperlipidemic zebrafish. In addition, histopathological showed that OGAG supplementation decreases the volume and number of lipid droplets in hepatocytes. Transcriptome and real‐time quantitative polymerase chain reaction analysis revealed that the gene expression levels of PPARγ, SCD, HMGRA, ACAT2, HMGCS, and HMGCR were significantly downregulated by OGAG treatment in hepatocytes, whereas those of CD36, FABP2, FABP6, ABCG5, and CYP7A1 were significantly upregulated. This suggests that the hypolipidemic effect of OGAG relies on increasing the ketogenic metabolism of fatty acids, inhibiting cholesterol synthesis, and enhancing the transformation of cholesterol to bile acid. Furthermore, OGAG treatment improved gut microbiota imbalance by reducing the Firmicutes‐to‐Bacteroidetes ratio, increasing the relative abundance of beneficial bacteria (Bacteroidetes, Verrucomicrobia, Acidobacteria, and Sphingomonas), and reducing the relative abundance of harmful bacteria (Proteobacteria, Cohaesibacter, Vibrio, and Terrisporobacter). These findings highlight the potential benefit of implementing OGAG as a dietary supplement to prevent and treat hyperlipidemia.

A chemokine regulatory loop induces cholesterol synthesis in lung-colonizing triple-negative breast cancer cells to fuel metastatic growth

Triple-negative breast cancer (TNBC) has a high propensity for organ-specific metastasis. However, the underlying mechanisms are not well understood. Here we show that the primary TNBC tumor-derived C-X-C motif chemokines 1/2/8 (CXCL1/2/8) stimulate lung-resident fibroblasts to produce the C-C motif chemokines 2/7 (CCL2/7), which, in turn, activate cholesterol synthesis in lung-colonizing TNBC cells and induce angiogenesis at lung metastatic sites. Inhibiting cholesterol synthesis in lung-colonizing breast tumor cells by pulmonary administration of simvastatin-carrying HER3-targeting nanoparticles reduces angiogenesis and growth of lung metastases in a syngeneic TNBC mouse model. Our findings reveal a novel, chemokine-regulated mechanism for the cholesterol synthesis pathway and a critical role of metastatic site-specific cholesterol synthesis in the pulmonary tropism of TNBC metastasis. The study has implications for the unresolved epidemiological observation that use of cholesterol-lowering drugs has no effect on breast cancer incidence but can unexpectedly reduce breast cancer mortality, suggesting interventions of cholesterol synthesis in lung metastases as an effective treatment to improve survival in individuals with TNBC.

The Effect of Three-Month Atorvastatin and α-Calcidol Administration on Some Bone Tissue Morphometric Parameters

Background.Osteoporosis is the fourth most common disease after cardiovascular diseases, cancer, and diabetes. All these diseases have common pathogenetic mechanisms associated with impaired cholesterol metabolism. In recent decades, the use of the key enzyme of cholesterol synthesis inhibitors — statins, which can stimulate osteogenesis, has become widespread. However, statins affect the production of the vitamin D active form by reducing the production of testosterone and thus reducing the activity of 1α-hydroxylase. The combined use of statins and α-calcidol (α-C) for the osteoporosis prevention seems promising. The aim of the study was to evaluate the effect of long-term atorvastatin (ATV) and α-C administration on morphometric growth parameters and bone vascularization in the experiment.Materials and Methods. The experiment was conducted for three months on 120 laboratory male rats, which were injected daily intragastrically with ATV and α-C. After 90 days of the experiment, the animals were decapitated under ether anesthesia. For the study, the right femur and jawbone were taken from the animals. The rat bone sections were impregnated with silver, decalcified, and the histosections were stained according to van Gieson. The distribution of the studied features was evaluated according to the Shapiro-Wilk test. The differences were considered statistically significant at p0.05.Results.It was found that ATV, both separately and together with α-C, increased the size of the newly formed bone in the endoostal and periosteal zones of the femur by 64.8; 40.4 and 15.8; 29.1%, respectively. The combined use of ATV and α-C had a positive effect on the growth of blood vessels in the femur (+23.4%). ATV increased the size of the newly formed bone from the periodontal and vestibular surfaces of the lower jaw by 18.3 and 29.5%, respectively. α-C potentiated the effect of ATV on the size of the newly formed bone tissue in the periodontal and vestibular growth zones of the mandibular bone by 10.1 and 15.0%, respectively. As for the number of vessels in the jawbone tissue, thanks to ATV, it increased by 17.2%, α-C had no effect.Conclusion. ATV increases the thickness of the newly formed bone layer in the growth areas of the femur and jawbone and increases the number of vessels in the jawbone. α-calcidol increases the number of vessels in the bone tissue of the femur and potentiates the effect of ATV on the jawbone growth zones. The combined use of ATV and α-C shows that they positively complement each other.

Greater than expected reduction in low-density lipoprotein-cholesterol (LDL-C) with bempedoic acid in a patient with heterozygous familial hypercholesterolemia (HeFH)

Bempedoic acid is an adenosine triphosphate-citrate lyase (ACL) inhibitor that reduces levels of low-density lipoprotein-cholesterol (LDL-C) in the plasma by inhibition of cholesterol synthesis in hepatic cells, which leads to up-regulation of hepatic LDL receptors. Bempedoic acid is approved as an adjunct to diet and maximally tolerated statin therapy for the treatment of adults with heterozygous familial hypercholesterolemia (HeFH) who require additional lowering of LDL-C. In this case study, we describe a patient with HeFH who had a prior excellent response to statin but unable to take the same, and a less than expected response to PCSK9i, in whom initiation of bempedoic acid led to a substantial reduction of LDL-C. Our findings suggest that patients who are quite responsive to statins may also be quite responsive to bempedoic acid, a medication that works in the same biochemical pathway as HMG-CoA reductase inhibitors. Additionally, this medication may be particularly effective at lowering LDL-C among individuals not on background statin therapy.

Cholesterol synthesis inhibition or depletion in axon regeneration.

Cholesterol is biosynthesized by all animal cells. Beyond its metabolic role in steroidogenesis, it is enriched in the plasma membrane where it has key structural and regulatory functions. Cholesterol is thus presumably important for post-injury axon regrowth, and this notion is supported by studies showing that impairment of local cholesterol reutilization impeded regeneration. However, several studies have also shown that statins, inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, are enhancers of axon regeneration, presumably acting through an attenuation of the mevalonate isoprenoid pathway and consequent reduction in protein prenylation. Several recent reports have now shown that cholesterol depletion, as well as inhibition of cholesterol synthesis per se, enhances axon regeneration. Here, I discussed these findings and propose some possible underlying mechanisms. The latter would include possible disruptions to axon growth inhibitor signaling by lipid raft-localized receptors, as well as other yet unclear neuronal survival signaling process enhanced by cholesterol lowering or depletion.

Abstract 2862: BMP4 mediated suppression of cholesterol synthesis and breast cancer metastasis

Abstract Breast cancer affects one in seven women and kills more than 600,000 women each year. Compared to the 98.9% survival rate of patients with localized breast cancer, the five-year survival rate of patients with distant metastasis is only 28.1%. Using cDNA arrays and patient datasets, we found that a protein called bone morphogenetic protein 4 (BMP4) is downregulated in highly metastatic breast cancer cells and in high-grade breast tumors. BMP4 is a ligand in the TFGβ/BMP cytokine family and has the capacity to suppress TGFβ signaling through induction of SMAD6 and SMAD7. In several different preclinical metastasis models, we have shown that restored BMP4 expression in breast cancer cells with metastatic capacity does not alter primary tumor growth but results in significantly reduced metastasis to lung, liver and spine (p< 0.01), following primary tumor resection. MDA-MB-231HM cells with or without enforced BMP4 expression were recovered from primary mammary tumors and subjected to transcriptomic profiling by RNAseq. This analysis revealed that BMP4 suppressed multiple genes involved in the cholesterol biosynthesis pathway (p=2.56×10-7) and down-regulated cholesterol levels in the tumors (p=0.042). In a patient cohort of over 2000 predominantly luminal breast cancers, we found that stage, as measured by size and lymph node status, was not altered by statin use, however statin users had less high grade tumors and more luminal A tumors. In terms of recurrence, we found a marked risk reduction in patients with luminal B tumors (p=0.014) and particularly in Her2+ luminal B tumors (p=0.045). Lung, liver and brain metastases were most reduced in statin users. Current experiments involve testing a statin therapy, with the knowledge that statins are not effective in mice, but should inhibit cholesterol synthesis in human tumors in xenograft models. In addition, we are testing an inhibitor of the nuclear receptor RORγ that has been reported to activate cholesterol synthesis through SREBP2, a master regulator of cholesterol synthesis. Citation Format: Robin L. Anderson, Lap Hing Chi, Allan D. Burrows, Suraya Roslan, Andrew Redfern. BMP4 mediated suppression of cholesterol synthesis and breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2862.

Upregulation of Alternatively Glycosylated Smoothened (SMO) Is Involved in Doxorubicin (DXR) Resistance in Diffuse Large B-Cell Lymphoma

▪Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Chemoresistance remains the most urgent clinical challenge in the management of DLBCL patients. The salvage therapy options for patients with relapsed disease are poor (responses rates less than 20%) and survival is usually short. Therefore, the development of less toxic yet effective therapeutic approaches for DLBCL patients will address a significant immediate medical need. Smoothened (SMO), a Frizzled-class G-protein-coupled-receptor (GPCR), is activated by cholesterol and regulated indirectly by Hedgehog (Hh) ligands through the release of PTCH1-mediated suppression. SMO contains an N-terminal extracellular segment with the cysteine-rich domain (CRD), a heptahelical transmembrane domain (TMD), and a C-terminal intracellular segment. Although SMO localizes in cilia to function as Hh signal transducer, our data support that SMO is also a constituent of lipid rafts, and plays a fundamental role in lipid raft organization in DLBCL. SMO in doxorubicin (DXR) resistant cell lines shows increased glycosylation of its extracellular segments and drug resistance is sensitive to modulation of cellular cholesterol levels, both features associated with protein localization to lipid raft and lipid raft composition, a poorly understood aspect in drug resistance. To improve our current therapeutic approaches for lymphoma patients we need to increase our understanding of the cellular mechanisms that contribute to tumor progression and drug resistance.We established a DXR-resistant cell line (SUDHL4) through long-term exposure to escalating doses of DXR (DXR-resistant cell line: virtually no cell death up to a DXR concentration of 120 nM; parental cell line DXR-IC50: 40nM). We observed that DXR-resistant SUDHL4 cells were also resistant to escalating doses of vincristine in comparison with the parental (sensitive) cells. Studies on the response of DXR resistant lymphoma cells to DXR confirmed high levels of DNA damage in resistant cells (measured by phosphorylation of histone H2AX) confirming that DXR resistance was not due to increase drug efflux. In addition, in this experimental model, both parental and DXR-resistant lymphoma cells have mutated TP53 (missense mutation p.V157A) showing that TP53 deficiency was not a factor in inducing DXR resistance.Several lines of evidence support a direct role of SMO in the DXR-resistance observed in DLBCL cells. First, DXR-resistant lymphoma cells in comparison with the parental cells showed a strong increase in SMO mRNA, but not its canonical downstream target of signaling, the transcription factor GLI1. Concordantly, SMO protein levels were substantially elevated in DXR-resistant cells (as detected by immunofluorescence and western blot). Moreover, upregulation of the expression of SMO was detected by immunohistochemistry in 4 of 5 relapsed DLBCL tumors in comparison with the matched tumors at the time of the initial diagnosis and before chemotherapy treatment. Second, we found that SMO is localized in the lipid rafts and mediates the assembly of a TRAF6 E3 ligase-dependent machinery that enhances AKT signaling. Additionally, we observed that SMO is highly glycosylated in DXR-resistant SUDHL4 cells but not in the parental cell line; previously, it has been found that SMO has 4 potential N-glycosilation sites that are dispensable for canonical Hh signaling but needed to induce a non-canonical SMO signal through Gα1. Finally, depletion of SMO using shRNA and a lentiviral-tetracycline inducible system and pharmacologically inhibition of SMO using the classic SMO inhibitor (cyclopamine-KAAD) or a cholesterol synthesis inhibitor (lovastatin) re-established sensitivity to DXR in DXR-resistant cells. Interestingly, lovastatin did not modify DXR sensitivity in the parental cell line. In summary, this study suggests a novel function of SMO in the development of DXR-resistance in DLBCL and opens new opportunities to regain or enhance DXR sensitivity through targeting this novel mechanism. DisclosuresNo relevant conflicts of interest to declare.

Scap structures highlight key role for rotation of intertwined luminal loops in cholesterol sensing

The cholesterol-sensing protein Scap induces cholesterol synthesis by transporting membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi apparatus for proteolytic activation. Transport requires interaction between Scap's two ER luminal loops (L1 and L7), which flank an intramembrane sterol-sensing domain (SSD). Cholesterol inhibits Scap transport by binding to L1, which triggers Scap's binding to Insig, an ER retention protein. Here we used cryoelectron microscopy (cryo-EM) to elucidate two structures of full-length chicken Scap: (1) a wild-type free of Insigs and (2) mutant Scap bound to chicken Insig without cholesterol. Strikingly, L1 and L7 intertwine tightly to form a globular domain that acts as a luminal platform connecting the SSD to the rest of Scap. In the presence of Insig, this platform undergoes a large rotation accompanied by rearrangement of Scap's transmembrane helices. We postulate that this conformational change halts Scap transport of SREBPs and inhibits cholesterol synthesis.

Bempedoic Acid: A New Avenue for the Treatment of Dyslipidemia.

Uncontrolled dyslipidemia, specifically elevation of low-density lipoprotein cholesterol, is a major risk factor for developing cardiovascular disease. Currently, statin therapy remains as first-line treatment for reducing both serum cholesterol levels and cardiovascular risk. However, certain patients are unable to achieve desired serum cholesterol levels despite maximally tolerated statin therapy. As a result, several nonstatin therapy avenues have been evaluated for their potential benefits in reducing cholesterol and cardiovascular risk. Bempedoic acid is one such nonstatin therapy option, which has been explored over the past few years to potentially assist patients in further reducing serum cholesterol. Bempedoic acid is a novel prodrug that inhibits cholesterol synthesis upstream of statins by inhibiting adenosine triphosphate-citrate lyase. Bempedoic acid has been studied as a single, once daily 180 mg dose. Administered as monotherapy or in combination with statin or ezetimibe, bempedoic acid significantly reduces low-density lipoprotein cholesterol. Furthermore, bempedoic acid was generally well tolerated by patients and rates of adverse events were similar to placebo with few exceptions. Despite proven reductions in cholesterol and favorable safety profile, bempedoic acid will likely remain a third- or fourth-line agent for the treatment of dyslipidemia behind other nonstatin therapies until the improvement of cardiovascular outcomes is demonstrated in future clinical trials.

α-globulin-rich rice cultivar, low glutelin content-1 (LGC-1), decreases serum cholesterol concentration in exogenously hypercholesterolemic rats.

Rice α-globulin has been reported to have serum cholesterol-lowering activity in rats. However, it is still unclear whether α-globulin exerts this effect when taken as one of the dietary components. In the present study, we investigated the effect of two cultivars of rice, low glutelin content (LGC)-1 and LGC-Jun, on reducing serum cholesterol in exogenously hypercholesterolemic (ExHC) rats. LGC-1 is enriched in α-globulin (10.6mg g-1 rice flour, which is an approximately 1.5 times higher α-globulin content than in Koshihikari a predominant rice cultivar in Japan), whereas LGC-Jun is a globulin-negative cultivar. ExHC rats, the model strain of diet-induced hypercholesterolemia, were fed 50% LGC-1 or LGC-Jun and 0.5% cholesterol-containing diets for 2 weeks, followed by measurement of cholesterol metabolism parameters in serum and tissues. Serum cholesterol and non-high-density lipoprotein cholesterol levels were significantly lower in the LGC-1 group compared to the LGC-Jun group. Cholesterol intestinal absorption markers, hepatic and serum levels of campesterol and β-sitosterol, and lymphatic cholesterol transport were not different between the two groups. Levels of 7α-hydroxycholesterol, an intermediate of bile acid synthesis, showed a downward trend in the livers of rats that were fed LGC-1 (P=0.098). There was a significant decrease in the hepatic mRNA expression of Cyp7a1 (a synthetic enzyme for 7α-hydroxycholesterol) in the LGC-1 group compared to the LGC-Jun group. Dietary LGC-1 significantly decreased serum cholesterol levels in ExHC rats. The possible mechanism for the cholesterol-lowering activity of LGC-1 is partial inhibition of bile acid and cholesterol synthesis in the liver. © 2021 Society of Chemical Industry.

Discovery of analogues of non-β oxidizable long-chain dicarboxylic fatty acids as dual inhibitors of fatty acids and cholesterol synthesis: Efficacy of lead compound in hyperlipidemic hamsters reveals novel mechanism

Background and aimsCholesterol and triglycerides are risk factors for developing cardiovascular disease. Therefore, appropriate cells and assays are required to discover and develop dual cholesterol and fatty acid inhibitors. A predictive hyperlipidemic animal model is needed to evaluate mechanism of action of lead molecule for therapeutic indications. Methods and resultsPrimary hepatocytes from rat, hamster, rabbit, and humans were compared for suitability to screen compounds by de novo lipogenesis (DNL) using14C-acetate. Hyperlipidemic hamsters were used to evaluate efficacy and mode of action.In rat hepatocytes DNL assay, both the central moiety and carbon chain length influenced the potency of lipogenesis inhibition. In hyperlipidemic hamsters, ETC-1002 decreased plasma cholesterol and triglycerides by 41% and 49% at the 30 mg/kg dose. Concomitant decreases in non-esterified fatty acids (−34%) and increases in ketone bodies (20%) were associated with induction of hepatic CPT1-α. Reductions in proatherogenic VLDL-C and LDL-C (−71% and −64%) occurred partly through down-regulation of DGAT2 and up-regulation of LPL and PDK4. Activation of PLIN1 and PDK4 dampened adipogenesis and showed inverse correlation with adipose mass. Hepatic concentrations of cholesteryl ester and TG decreased by 67% and 64%, respectively. Body weight decreased with concomitant decreases in epididymal fat. Plasma and liver concentrations of ETC-1002 agreed with the observed dose–response efficacy. ConclusionsTaken together, ETC-1002 reduced proatherogenic lipoproteins, hepatic lipids and adipose tissues in hyperlipidemic hamsters via induction of LPL, CPT1-α, PDK4, and PLIN1, and downregulation of DGAT2. These characteristics may be useful in the treatment of fatty livers that causes non-alcoholic steatohepatitis.

Statin-boosted cellular uptake and endosomal escape of penetratin due to reduced membrane dipole potential.

Cell penetrating peptides are promising tools for delivery of cargo into cells, but factors limiting or facilitating their cellular uptake are largely unknown. We set out to study the effect of the biophysical properties of the cell membrane on the uptake of penetratin, a cell penetrating peptide. Using labelling with pH-insensitive and pH-sensitive dyes, the kinetics of cellular uptake and endo-lysosomal escape of penetratin were studied by flow cytometry. We report that escape of penetratin from acidic endo-lysosomal compartments is retarded compared with its total cellular uptake. The membrane dipole potential, known to alter transmembrane transport of charged molecules, is shown to be negatively correlated with the concentration of penetratin in the cytoplasmic compartment. Treatment of cells with therapeutically relevant concentrations of atorvastatin, an inhibitor of HMG-CoA reductase and cholesterol synthesis, significantly increased endosomal escape of penetratin in two different cell types. This effect of atorvastatin correlated with its ability to decrease the membrane dipole potential. These results highlight the importance of the dipole potential in regulating cellular uptake of cell penetrating peptides and suggest a clinically relevant way of boosting this process.

NaHS or Lovastatin Attenuates Cyclosporine A-Induced Hypertension in Rats by Inhibiting Epithelial Sodium Channels.

The use of cyclosporine A (CsA) in transplant recipients is limited due to its side effects of causing severe hypertension. We have previously shown that CsA increases the activity of the epithelial sodium channel (ENaC) in cultured distal nephron cells. However, it remains unknown whether ENaC mediates CsA-induced hypertension and how we could prevent hypertension. Our data show that the open probability of ENaC in principal cells of split-open cortical collecting ducts was significantly increased after treatment of rats with CsA; the increase was attenuated by lovastatin. Moreover, CsA also elevated the levels of intracellular cholesterol (Cho), intracellular reactive oxygen species (ROS) via activation of NADPH oxidase p47phox, serum- and glucocorticoid-induced kinase isoform 1 (Sgk1), and phosphorylated neural precursor cell–expressed developmentally downregulated protein 4–2 (p-Nedd4-2) in the kidney cortex. Lovastatin also abolished CsA-induced elevation of α-, ß-, and γ-ENaC expressions. CsA elevated systolic blood pressure in rats; the elevation was completely reversed by lovastatin (an inhibitor of cholesterol synthesis), NaHS (a donor of H2S which ameliorated CsA-induced elevation of reactive oxygen species), or amiloride (a potent ENaC blocker). These results suggest that CsA elevates blood pressure by increasing ENaC activity via a signaling cascade associated with elevation of intracellular ROS, activation of Sgk1, and inactivation of Nedd4-2 in an intracellular cholesterol-dependent manner. Our data also show that NaHS ameliorates CsA-induced hypertension by inhibition of oxidative stress.

Negative Regulation of Human Hepatic Constitutive Androstane Receptor by Cholesterol Synthesis Inhibition: Role of Sterol Regulatory Element Binding Proteins.

The squalene synthase inhibitor squalestatin 1 (Squal1) is a potent and efficacious inducer of CYP2B expression in primary cultured rat hepatocytes and rat liver. To determine whether Squal1 is also an inducer of human CYP2B, the effects of Squal1 treatment were evaluated in primary cultured human hepatocytes, differentiated HepaRG cells, and humanized mouse livers. Squal1 treatment did not increase CYP2B6 mRNA levels in human hepatocytes or HepaRG cells and only slightly and inconsistently increased CYP2B6 mRNA content in humanized mouse liver. However, treatment with farnesol, which mediates Squal1's effect on rat CYP2B expression, increased CYP2B6 mRNA levels in HepaRG cells expressing the constitutive androstane receptor (CAR), but not in cells with knocked-down CAR. To determine the impact of cholesterol biosynthesis inhibition on CAR activation, the effects of pravastatin (Prava) were determined on CITCO-mediated gene expression in primary cultured human hepatocytes. Prava treatment abolished CITCO-inducible CYP2B6 expression, but had less effect on rifampicin-mediated CYP3A4 induction, and CITCO treatment did not affect Prava-inducible HMG-CoA reductase (HMGCR) expression. Treatment with inhibitors of different steps of cholesterol biosynthesis attenuated CITCO-mediated CYP2B6 induction in HepaRG cells, and Prava treatment increased HMGCR expression and inhibited CYP2B6 induction with comparable potency. Transfection of HepG2 cells with transcriptionally active sterol regulatory element binding proteins (SREBPs) reduced CAR-mediated transactivation, and inducible expression of transcriptionally active SREBP2 attenuated CITCO-inducible CYP2B6 expression in HepaRG cells. These findings suggest that Squal1 does not induce CYP2B6 in human hepatocytes because Squal1's inhibitory effect on cholesterol biosynthesis interferes with CAR activation. SIGNIFICANCE STATEMENT: The cholesterol biosynthesis inhibitor squalestatin 1 induces rat hepatic CYP2B expression indirectly by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). This study demonstrates that squalestatin 1 does not similarly induce CYP2B6 expression in human hepatocytes. Rather, inhibition of cholesterol biosynthesis interferes with CAR activity, likely by activating sterol regulatory element binding proteins. These findings increase our understanding of the endogenous processes that modulate human drug-metabolizing gene expression.