Enzyme In Cholesterol Synthesis Research Articles

6826 Mevalonate Treatment Worsened Hypocholesterolemia During Prolonged Sepsis Without Affecting Adrenal And Muscle Function

Abstract Disclosure: L. De Bruyn: None. S. Derde: None. F. Van Beek: None. S. Vander Perre: None. I. Derese: None. L. Pauwels: None. G. Van den Berghe: None. L. Langouche: None. Introduction: Sepsis is hallmarked by an immediate and sustained reduction in total-, HDL- and LDL-cholesterol (-C), associated with illness severity and a potential contributor to the development of adrenal failure and muscle weakness. The underlying mechanisms that might be involved in sepsis-induced hypocholesterolemia are multifactorial and partly understood. We hypothesized that treatment with the cholesterol precursor mevalonate might improve cholesterol availability, thereby improving adrenal gland and skeletal muscle function. Our hypothesis was tested in a validated and clinically relevant mouse model of prolonged sepsis. Methods: In a catheterized mouse model of cecal ligation and puncture-induced, fluid resuscitated and antibiotics treated polymicrobial abdominal sepsis (5 d), septic mice received continuous infusion with either mevalonate (78 mg/d) or placebo solution, whereas healthy mice served as controls (N=50). Plasma total-C (HDL- + LDL-C), CORT and total bile acids were measured, in addition to ex vivo muscle force and adrenocortical cholesterol ester content. Gene expression markers of inflammation (Tnf-α, Il-6) were measured in the adrenal gland, as well as hepatic mRNA expression of cholesterol synthesis enzymes (Hmgcs1, Hmgcr, Fdft1). Metabolites of the mevalonate pathway (mevalonic acid, squalene, lanosterol, desmosterol and (7-dehydro) cholesterol) were quantified in the liver with LC-MS. Results: The sepsis-induced reduction in plasma total-C was further decreased in mevalonate-treated septic mice (26±3 mg/dL) as compared with placebo (53±3 mg/dL) and healthy controls (108±3 mg/dL) (p<0.0001). Metabolites of the mevalonate pathway were all increased in the liver after mevalonate treatment (p<0.01), whereas hepatic cholesterol content was decreased as compared with placebo (p<0.05). Also, gene expression markers of cholesterol synthesis enzymes were further decreased in the liver of mevalonate-treated septic mice as compared with placebo (p<0.05). In the context of the unexpected further reduction in plasma total-C, no additional effect on plasma CORT, total bile acids, and sepsis-induced loss of muscle force and depletion in adrenocortical cholesterol ester content was observed. In addition, the sepsis-induced rise in markers of adrenal inflammation (Tnf-α, Il-6) was not further affected by mevalonate treatment. Conclusion: Mevalonate treatment further reduced the sepsis-induced hypocholesterolemia in prolonged septic mice, potentially due to increased feedback inhibition on hepatic cholesterol biosynthesis, but without further affecting adrenal and muscle function. Other therapies such as substitution with bovine serum HDL-C are currently investigated to improve altered cholesterol availability during sepsis. Presentation: 6/2/2024

Evaluation of plasma phytosterols in sitosterolemia, their kindreds and hyperlipidemia subjects

BackgroundPatients suffering from sitosterolemia with ABCG5/8 mutation typically present with early-onset or rapidly progressive atherosclerosis. Their kindreds with partial genetic deficiencies of ABCG5/8 are often considered healthy. However, discerning sitosterolemia from its familial kindreds and hyperlipidemia subjects has remained challenging. MethodsHere we retrospectively recruited seven families including 8 individuals diagnosed with sitosterolemia subjects, and 14 kindreds carrying single gene mutations. Additionally, 17 individuals with hyperlipidemia and 130 healthy controls served as positive and negative controls, respectively. A total of 6 phytosterols combined with cholesterol absorption indices (including sitosterol, campesterol, stigmasterol, and cholestanol) and cholesterol synthesis markers (desmosterol and 7-dehydrocholesterol), was compared across the aforementioned four groups. ResultsAs expected, the sitosterolemia subjects with double mutations demonstrated significantly elevated levels of sitosterol and other cholesterol absorption indices. Meanwhile, sitosterolemia kindreds with single gene mutation showed a similar pattern of activated cholesterol-absorption ability to the hyperlipidemia group, but not as high as the double mutation group. Notably, the cholesterol-synthesis enzyme 7-dehydrocholesterol reductase displayed an increase in the hyperlipidemia group but a decrease in the sitosterolemia kindred group, suggesting a potential discriminative role of 7-dehydrocholesterol in distinguishing between these two groups. The combination of phytosterols was more valuable than clinical lipid index for sitosterolemia diagnosis. ConclusionOur study revealed mild disruptions of cholesterol absorption capacities in sitosterolemia kindreds with single mutations. Furthermore, the combination of 6 phytosterols proved effective in distinguishing between sitosterolemia, its single mutation carriers, and hyperlipidemia patients.

Temporal changes in mouse hippocampus transcriptome after pilocarpine-induced seizures.

Status epilepticus (SE) is a seizure lasting more than 5 min that can have lethal consequences or lead to various neurological disorders, including epilepsy. Using a pilocarpine-induced SE model in mice we investigated temporal changes in the hippocampal transcriptome. We performed mRNA-seq and microRNA-seq analyses at various times after drug treatment. At 1 h after the start of seizures, hippocampal cells upregulated transcription of immediate early genes and genes involved in the IGF-1, ERK/MAPK and RNA-PolII/transcription pathways. At 8 h, we observed changes in the expression of genes associated with oxidative stress, overall transcription downregulation, particularly for genes related to mitochondrial structure and function, initiation of a stress response through regulation of ribosome and translation/EIF2 signaling, and upregulation of an inflammatory response. During the middle of the latent period, 36 h, we identified upregulation of membrane components, cholesterol synthesis enzymes, channels, and extracellular matrix (ECM), as well as an increased inflammatory response. At the end of the latent period, 120 h, most changes in expression were in genes involved in ion transport, membrane channels, and synapses. Notably, we also elucidated the involvement of novel pathways, such as cholesterol biosynthesis pathways, iron/BMP/ferroptosis pathways, and circadian rhythms signaling in SE and epileptogenesis. These temporal changes in metabolic reactions indicate an immediate response to injury followed by recovery and regeneration. CREB was identified as the main upstream regulator. Overall, our data provide new insights into molecular functions and cellular processes involved at different stages of seizures and offer potential avenues for effective therapeutic strategies.

In vitro human colonic fermentation of coffee arabinogalactan and melanoidin-rich fractions

Coffee beverage is a source of dietary fiber composed by arabinogalactans, which can also be associated to proteins and phenolic compounds, originating melanoidins. Human colonic in vitro fermentations of coffee fractions, one rich in melanoidins (Mel) and the other in its parental polysaccharide arabinogalactans (AG), were performed in order to evaluate the metabolites produced by microbiota, namely short-chain fatty acids (SCFA), phenolic compounds, and bile acids. After 48 h of fermentation, a higher fermentability of the carbohydrate fraction of AG (62 %) than that of Mel (27 %) was observed, resulting in a SCFA content of 63 mM and 22 mM, respectively. Supplementation with AG and Mel fractions decreased the acetate:propionate ratio from 4.7 (in the absence of coffee fractions) to 2.5 and 3.5, respectively, suggesting a potential inhibition of HMG-CoA reductase, a rate-limiting enzyme for cholesterol synthesis. The fermentation of coffee fractions yielded dihydroferulic and dihydrocaffeic acids, known to have antioxidant properties. In the presence of Mel, it was observed a decrease (from 0.25 to 0.16 mg/mL) in the production of secondary bile acids, whose high content is associated to the development of several diseases, such as colorectal cancer, neurodegenerative and cardiovascular.

In-Vitro Study of the Binding of Atorvastatin with Adenine using Multi-Spectroscopic Approaches.

Atorvastatin-an oral lipid regulating drug is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), which is the rate determining enzyme for cholesterol synthesis. Adenine is a purine nucleobase that is found in deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) to generate genetic information. The binding mechanism of atorvastatin and adenine was studied for the first time utilizing various techniques, including UV-visible spectrophotometry, spectrofluorimetry, synchronous fluorescence spectroscopy (SF), Fourier transform infrared (FTIR), fluorescence resonance energy transfer (FRET), and metal ion complexation. The fluorescence spectra of the complex indicated that atorvastatin is bound to adenine via hydrophobic interaction through a spontaneous binding process, and the fluorescence quenching mechanism was found to be static quenching with a binding constant of 1.4893 × 104 Lmol-1 at 298 K. Various temperature settings were used to investigate thermodynamic characteristics, such as binding forces, binding constants, and the number of binding sites. The interaction parameters, including the standard enthalpy change (ΔHο) and standard entropy change (ΔSο) were calculated using Van't Hoff's equation to be 42.82 kJmol-1 and 208.9 Jmol-1K-1, respectively. The findings demonstrated that the adenine- atorvastatin binding was endothermic. Furthermore, the results of the experiments revealed that some metal ions (K+, Ca+2, Co+2, Cu+2, and Al+3) facilitate the binding interaction between atorvastatin and adenine. Slight changes are observed in the FTIR spectra of adenine, indicating the binding interaction between adenine and atorvastatin.

Abstract PR016: Investigating vulnerabilities associated with chromosome arm aneuploidy in cancer

Abstract Aneuploidy – loss or gain of whole chromosomes or chromosome arms, is rare and poorly tolerated in normal cells but occurs in ∼90% of solid tumors; however, the mechanisms through which specific aneuploidies affect cancer development are unclear. Additionally, generating mammalian models of specific chromosome arm alterations is technically difficult, limiting further study. Cancers have tumor, cell, and tissue type-specific patterns of chromosome arm copy-number alterations that influence tumor evolution and sensitivity to anti-cancer therapies. Squamous cell carcinomas (SCCs) affecting lung, head and neck, esophageal, and cervical tissues have been shown to feature conserved early losses and gains of arms 3p and 3q, respectively, and are associated with relatively few oncogenic drivers and treatment options. These aneuploidies are particularly well established in lung SCC and have been shown to promote tumorigenesis, metastasis, and poor prognosis. To study cancer vulnerabilities associated with chromosome 3 arm aneuploidies, we used CRISPR-Cas9 to delete a 3p copy in human immortalized lung epithelial cell lines—the lung SCC cell-of-origin. A subset of clones duplicated a wild type chromosome 3 copy, transitioning to 3q gain and providing us with independent models for chromosome 3 wild type, 3p loss, and 3q gain. Next, we performed a CRISPRi screen and found that 3q gain cells are more sensitive to knockdown of sterol regulatory element-binding factor 1 (SREBF1)—a master regulator of cholesterol and fatty acid biosynthesis, its co-factor SREBP cleavage activating protein (SCAP), and downstream target HMG-CoA reductase (HMGCR)—the rate-limiting enzyme for cholesterol synthesis. 3q gain cells also showed up-regulated fatty acid metabolites, ceramides, triglycerides, and phosphatidylinositols. After performing a chemical screen, 3q gain cells showed reduced proliferation, increased apoptosis, and DNA damage in response to statins (HMGCR inhibitors). Statins have previously been shown to induce tumor-specific apoptosis and reduced cancer risk, although these studies have associated with variable efficacy and underlying mechanisms. We hypothesize that 3q gain is a biomarker of statin response and sensitivity in squamous cancers. We aim to study the mechanism through which aneuploidy alters lipid metabolism and contributes to cancer. Investigating aneuploidy-induced vulnerabilities will help us understand how cancers exploit aneuploidies to promote tumorigenesis and to elucidate novel and specific therapeutic targets for cancers with shared copy-number profiles. Citation Format: Nadja Zhakula-Kostadinova, Sejal Jain, Laura Byron, Matthew L. Meyerson, Alison M. Taylor. Investigating vulnerabilities associated with chromosome arm aneuploidy in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR016.

High Salt Diet Regulates Intrarenal Sex Steroid Biosynthesis Pathway in a Sex-Specific Manner

Hypertension is more prevalent in men than age-matched pre-menopausal women. Sodium (Na+) homeostasis plays a major role in the control of blood pressure. Renal medullary estrogenic signaling promotes Na+ excretion in female, but not male Sprague Dawley (SD) rats implicating estrogen as a potential female-specific natriuretic factor. Recently we showed that the components of estrogen biosynthesis pathway are expressed in rat kidney suggesting the kidneys can produce estrogen locally to invoke this natriuretic response. It is unknown how dietary Na+ impacts biosynthesis of sex hormones. We hypothesize that a high salt (HS) diet enhances intrarenal estrogen biosynthesis pathway, and this enhancement is more pronounced in females compared to males. To test this hypothesis, we harvested the renal cortex from 14 week old male and female SD rats maintained on either a normal salt (NS 0.3% NaCl) or HS (4% NaCl) diet for 5 days. Given that cholesterol is the main precursor of steroid hormones, we measured the mRNA expression of HMG-CoA reductase (HMGCR), the key enzyme for cholesterol synthesis; and the steroidogenic acute regulatory protein (StAR), which transports cholesterol into the mitochondria for steroid commitment. We also assessed the mRNA expression of steroid 5α reductase (SRD5A), which converts testosterone to the non-aromatizable androgen, dihydrotestosterone. Further, we assessed aromatase, the key enzyme in estrogen biosynthesis, via immunohistochemistry. We found that renal HMGCR was more abundant in female compared to male rats maintained on a NS diet (P=0.0467). Increasing dietary salt intake upregulated renal HMGCR expression in male (P=0.0020), but not female rats (NS male; 100.0 ± 26.8 vs. NS female; 235.3 ± 23.2 vs. HS male 294.6 ± 42.0 vs HS female 314.6 ± 21.0 relative expression % NS male n=6-7/group). On a NS diet, renal StAR was more abundant in female compared to male rats (NS male; 100.0 ± 25.3 vs. NS female; 217.2 ± 36.5 relative expression % NS male P=0.0255 n=5-7/group). Increasing dietary salt did not alter expression of StAR in either sex. Similarly, renal SRD5A expression was higher in female compared to male (P=0.0141) rats. In addition, a HS diet increased the expression of SRD5A in female rats (0.0093) and tended to increase SRD5A expression in male (P=0.0849) rats (NS male; 100.0 ± 11.6 vs. NS female; 297.8 ± 37.7 vs. HS male 243.0 ± 39.2 vs HS female 498.8 ± 44.8 relative expression % NS male n=6-7/group). We also revealed that aromatase was expressed at a comparable level in male and female renal cortex, regardless of the diet (NS male; 2.4 ± 0.3 vs. NS female; 2.6 ± 0.3 vs. HS male 2.8 ± 0.3 vs HS female 1.9 ± 0.1 Average intensity x area mM2 n=6-9/group). Together, this data suggests that (i) the female kidney may be more primed for steroid biosynthesis and (ii) there is a sex-specific upregulation of the intrarenal sex steroid biosynthesis pathway in response to increasing dietary salt intake. Additional work is required to investigate the potential role of the kidney in androgen and estrogen production. A better understanding of this steroidogenic pathway could reveal novel sex-specific therapeutics for hypertension. T32DK007569-34 Carl Gottschalk Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lanosterol synthase deficiency promotes tumor progression by orchestrating PDL1-dependent tumor immunosuppressive microenvironment.

Lipid metabolic reprogramming is closely related to tumor progression with the mechanism not fully elucidated. Here, we report the immune-regulated role of lanosterol synthase (LSS), an essential enzyme in cholesterol synthesis. Database analysis and clinical sample experiments suggest that LSS was lowly expressed in colon and breast cancer tissues, which indicates poor prognosis. The biological activity of tumor cell lines and tumor progression in NOD scid gamma (NSG) mice were not affected after LSS knockdown, whereas LSS deficiency obviously aggravated tumor burden in fully immunized mice. Flow cytometry analysis showed that LSS knockdown significantly promoted the formation of tumor immunosuppressive microenvironment, characterized by the increase in M2 macrophages and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), as well as the decrease in anti-tumoral T lymphocytes. With the inhibition of myeloid infiltration or loss function of T lymphocytes, the propulsive effect of LSS knockdown on tumor progression disappeared. Mechanistically, LSS knockdown increased programmed death ligand 1 (PDL1) protein stability by 2,3-oxidosqualene (OS) binding to PDL1 protein. Anti-PDL1 therapy abolished LSS deficiency-induced immunosuppressive microenvironment and cancer progression. In conclusion, our results show that LSS deficiency promotes tumor progression by establishing an OS-PDL1 axis-dependent immunosuppressive microenvironment, indicative of LSS or OS as a potential hallmark of response to immune checkpoint blockade.

The constitutively active form of a key cholesterol synthesis enzyme is lipid droplet-localized and upregulated in endometrial cancer tissues

Cholesterol is essential for both normal cell viability and cancer cell proliferation. Aberrant activity of squalene monooxygenase (SM, also known as squalene epoxidase or SQLE), the rate-limiting enzyme of the committed cholesterol synthesis pathway, is accordingly implicated in a growing list of cancers. We previously reported that hypoxia triggers the truncation of SM to a constitutively active form, thus preserving sterol synthesis during oxygen shortfalls. Here, we show SM truncation is upregulated and correlates with the magnitude of hypoxia in endometrial cancer tissues, supporting the in vivo relevance of our earlier work. To further investigate the pathophysiological consequences of SM truncation, we examine using complementary immunofluorescence and cell fractionation approaches its lipid droplet-localized pool and find that it exclusively comprises the truncated enzyme. This partitioning is facilitated by the loss of an endoplasmic reticulum-embedded region at the SM N-terminus, whereas the catalytic domain containing membrane-associated C-terminal helices is spared. Moreover, we determined multiple amphipathic helices contribute to the lipid droplet localization of trunSM. Taken together, our results expand on the striking differences between the two forms of SM and suggest upregulated truncation may contribute to SM-related oncogenesis.

Effects of Diet, Gender, and Age on Liver Metabolism Upon Feeding a High Fat Diet

Background/Objective: Currently, 30% of the USA population has non-alcoholic fatty liver disease (NAFLD), a condition that is increasing in parallel with the global obesity epidemic and life expectancy and is a risk factor for type 2 diabetes and cardiovascular disease. Aberrant transcriptional control of genes is a hallmark of metabolic disease. Thus, it is of interest to characterize how diet, age, and sex impacts various metabolic and signaling pathways upon progression to NAFLD.
 Methods: A cohort of 7-week or 52-week-old male and female mice (n=8) were assigned a high-fat diet (HFD) or a low-fat diet (LFD) feeding regimen for 10 weeks, after which serum chemistries were obtained. Subsequently, gene expression in hepatic tissue samples was analyzed using RNA-seq and Western Blot.
 Results: In both males and females, HDL, LDL, and total cholesterol were significantly increased with HFD in both age groups. In females, there was no significant difference in fasting blood glucose and insulin levels between any groups, while in males, both markers were significantly increased with HFD. RNA-seq data showed significant changes in gene expression correlating with diet, gender, and age. KEGG pathway analysis showed clustering of genes in fatty acid oxidation and synthesis pathways in mice fed a HFD, without affecting cholesterol synthesis enzymes. In aged mice, genes involved in extracellular matrix-receptor interaction were among the most affected. In males and females, young and aged, Western Blot analysis showed decreased expression of lipogenic enzymes ATP-citrate lyase and fatty acid synthase with HFD, but no changes of enzymes of cholesterol synthesis, confirming the RNA-seq data. No significant alterations were observed in insulin signaling.
 Conclusion and Potential Impact: A HFD leads to a more severe phenotype in males than females, including increased fasting and non-fating glucose as well as insulin, and age exacerbates these abnormalities. Cholesterol is increased in both genders and is also exacerbated by age.

Exploiting in silico structural analysis to introduce emerging genotype-phenotype correlations in DHCR24-related sterol biosynthesis disorder: a case study.

Desmosterolosis is a rare sterol biosynthesis disorder characterized by multiple congenital anomalies, failure to thrive, severe developmental delay, progressive epileptic encephalopathy, and elevated levels of desmosterol caused by biallelic mutations of DHCR24 encoding 3-β-hydroxysterol Δ-24-reductase. DHCR24 is regarded as the key enzyme of cholesterol synthesis in the metabolism of brain cholesterol as it catalyzes the reduction of the Δ-24 double bond of sterol intermediates during cholesterol biosynthesis. To date, 15 DHCR24 variants, detected in 2 related and 14 unrelated patients, have been associated with the desmosterolosis disorder. Here, we describe a proband harboring the never-described DHCR24 homozygous missense variant NM_014762.4:c.506T>C, NP_055577.1:p.M169T, whose functional validation was confirmed through biochemical assay. By using molecular dynamics simulation techniques, we investigated the impact of this variant on the protein stability and interaction network with the flavin adenine dinucleotide cofactor, thereby providing a preliminary assessment of its mechanistic role in comparison to all known pathogenic variants, the wild-type protein, and a known benign DHCR24 variant. This report expands the clinical and molecular spectra of the DHCR24-related disorder, reports on a novel DHCR24 deleterious variant associated with desmosterolosis, and gives new insights into genotype-phenotype correlations.

Mechanisms of 3-Hydroxyl 3-Methylglutaryl CoA Reductase in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide and has a high incidence in the elderly. Unfortunately, there is no effective therapy for AD owing to its complicated pathogenesis. However, the development of lipid-lowering anti-inflammatory drugs has heralded a new era in the treatment of Alzheimer's disease. Several studies in recent years have shown that lipid metabolic dysregulation and neuroinflammation are associated with the pathogenesis of AD. 3-Hydroxyl 3-methylglutaryl CoA reductase (HMGCR) is a rate-limiting enzyme in cholesterol synthesis that plays a key role in cholesterol metabolism. HMGCR inhibitors, known as statins, have changed from being solely lipid-lowering agents to neuroprotective compounds because of their effects on lipid levels and inflammation. In this review, we first summarize the main regulatory mechanism of HMGCR affecting cholesterol biosynthesis. We also discuss the pathogenesis of AD induced by HMGCR, including disordered lipid metabolism, oxidative stress, inflammation, microglial proliferation, and amyloid-β (Aβ) deposition. Subsequently, we explain the possibility of HMGCR as a potential target for AD treatment. Statins-based AD treatment is an ascent field and currently quite controversial; therefore, we also elaborate on the current application prospects and limitations of statins in AD treatment.

Adenosine Triphosphate Citrate Lyase and Fatty Acid Synthesis Inhibition

Adenosine triphosphate citrate lyase (ACLY) is a key regulatory enzyme of glucose metabolism, cholesterol and fatty acid synthesis, and the inflammatory cascade. Bempedoic acid, an ACLY inhibitor, significantly reduces atherogenic lipid markers, including low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol, and apolipoprotein B. Additional effects of ACLY inhibition include antitumor growth; reduction of triglycerides and proinflammatory molecules such as high-sensitivity C-reactive protein; less insulin resistance; reduction of hepatic lipogenesis; and weight loss. While numerous ACLY inhibitors have been identified, most of the clinical data have focused on bempedoic acid. The Cholesterol Lowering via Bempedoic Acid, an ACL-Inhibiting Regimen (CLEAR) program was a series of phase 3 clinical trials that evaluated its effects on lipid parameters and safety, leading to US Food and Drug Administration approval in 2020. CLEAR Outcomes was a phase 3, double-blind, randomized, placebo-controlled trial in individuals with a history of statin intolerance, serum LDL-C level of 100 mg/dL or higher, and a history of, or at high risk for, cardiovascular disease. Bempedoic acid modestly reduced the primary 4-way cardiovascular composite end point as well as the individual components of myocardial infarction and coronary revascularization but did not reduce stroke, cardiovascular death, or all-cause mortality. Rates of gout and cholelithiasis were higher with bempedoic acid, and small increases in serum creatinine, uric acid, and hepatic-enzyme levels were also observed. ACLY inhibition with bempedoic acid has been established as a safe and effective therapy in high-risk patients who require further LDL-C lowering, particularly for those with a history of statin intolerance. The recently published CLEAR Outcomes trial revealed modest reductions in cardiovascular events with bempedoic acid, proportional to its LDL-C lowering, in high-risk individuals with statin intolerance and LDL-C levels of 100 mg/dL or higher. The additional effects of ACLY inhibition have prompted a more thorough search for novel ACLY inhibitors for conditions such as cancer, hypertriglyceridemia, chronic inflammation, type 2 diabetes, fatty liver disease, obesity, and metabolic syndrome. Similarly, therapies that reduce fatty acid synthesis are being explored for their use in cardiometabolic conditions.

Predictive Modeling of HMG-CoA Reductase Inhibitory Activity and Design of New HMG-CoA Reductase Inhibitors.

As blood cholesterol increases, it accumulates in the intima of blood vessels, elevating the risk of atherosclerosis and coronary artery disease. Drugs that inhibit enzymes essential for cholesterol synthesis are effective in improving blood cholesterol levels. Statins are used to treat hypercholesterolemia as they inhibit 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), the rate-limiting enzyme in cholesterol synthesis. Statins are known to exert their effects by translocating to the liver, where they are taken up by the organic anion transporting polypeptide 1B1 (OATP1B1). Therefore, we hypothesized that a compound with high HMGR inhibitory activity and high affinity for OATP1B1 would be an excellent new therapeutic agent for hypercholesterolemia with increased liver selectivity and fewer side effects. In this study, we developed two models for predicting HMGR inhibitory activity and OATP1B1 affinity to propose the chemical structure of a new therapeutic agent for hypercholesterolemia with both high inhibitory activity and high liver selectivity. HMGR inhibitory activity and OATP1B1 affinity prediction models were constructed with high prediction accuracy for the test data: r2 = 0.772 and 0.768, respectively. New chemical structures were then input into these models to search for candidate compounds. We found compounds with higher HMGR inhibitory activity and OATP1B1 affinity than rosuvastatin, the most recently developed statin drug, and compounds that did not have a common structure of statins with high HMGR inhibitory activity.

High fat diet induces brain injury and neuronal apoptosis via down-regulating 3-β hydroxycholesterol 24 reductase (DHCR24).

Hyperlipidemia (HLP) is one of the risk factors for memory impairment and cognitive impairment. However, its pathological molecular mechanism remained unclear. 3β-hydroxysterol Δ24- reductase (DHCR24) is a key enzyme in cholesterol synthesis and has been reported to decrease in the affected areas in the brain of neurodegenerativedisorders. In this study, hyperlipidemic mouse model was established to study the effect of high blood lipid on brain. The data obtained from HPLC analysis demonstrated that the cholesterol level in the brain of mice with hyperlipidemia was significantly elevated compared to the control group. While the pathological damages were observed in both cerebral cortex and hippocampus in the brain of hyperlipidemic mice. Furthermore, the protein level of DHCR24 was downregulated accompanied by elevated ubiquitination level in the hyperlipidemic mice brain. The mouse neuroblastoma cells N2a were exposed to the excess cholesterol loading, the cells underwent apoptosis and the mRNA and protein of DHCR24 in cholesterol-loaded N2a cells were significantly reduced. In addition, the expression level of endoplasmic reticulum stress marker protein (Bip and Chop) was markedly increased in response to the cholesterol loading. More importantly, overexpression of DHCR24 in N2a reversed neuronal apoptosis induced by the cholesterol loading. Conclusively, these findings suggested that hyperlipidemia could cause brain tissue injuries via down-regulating DHCR24, and overexpression of DHCR24 may alleviate hyperlipidemia-induced neuronal cells damage by reversing the endoplasmic reticulum stress-mediated apoptosis.

A positive feedback between cholesterol synthesis and the pentose phosphate pathway rather than glycolysis promotes hepatocellular carcinoma.

Hepatic cholesterol accumulation and hypercholesterolemia are implicated in hepatocellular carcinoma (HCC). However, the therapeutic effects of cholesterol-lowering drugs on HCC are controversial, indicating that the relationship between cholesterol metabolism and HCC is more complex than anticipated. A positive feedback between cholesterol synthesis and the pentose phosphate pathway (PPP) rather than glycolysis was formed in tumors of c-Myc mice. Blocking the PPP prevented cholesterol synthesis and thereby HCC in c-Myc mice, while ablating glycolysis did not affect cholesterol synthesis and failed to prevent c-Myc-induced HCC. Unexpectedly, HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) and G6PD (glucose-6-phosphate dehydrogenase), the rate-limiting enzymes of cholesterol synthesis and the PPP, were identified as direct targets of microRNA-206. By targeting Hmgcr and G6pd, microRNA-206 disrupted the positive feedback and fully prevented HCC in c-Myc mice, while 100% of control mice died of HCC. Disrupting the interaction of microRNA-206 with Hmgcr and G6pd restored cholesterol synthesis, the PPP and HCC growth that was inhibited by miR-206. This study identified a previously undescribed positive feedback loop between cholesterol synthesis and the PPP, which drives HCC, while microRNA-206 prevents HCC by disrupting this loop. Cholesterol synthesis as a process rather than cholesterol itself is the major contributor of HCC.

243-OR: A Novel Platform for Genetic Screening of NAFLD-Associated Genes Identifies UBE2G2 and Other Genes That Alter Hepatic Lipid Accumulation

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver failure that is prevalent in 70% of people with diabetes. Human genetic studies (i.e. GWAS) have identified dozens of potentially causal genes/loci increasing NAFLD susceptibility but translation into actionable drug targets has been impeded by a lack of cellular and molecular mechanistic studies. To address this knowledge gap, we developed a platform for high-throughput genetic perturbation and assessment of fatty acid-triggered lipid accumulation (i.e. hepatic steatosis) in human hepatocytes by combining mechanized cell culture in multi-well plates with high-content microscopy and automated image analysis. We then deployed this platform to screen 106 genes nominated by NAFLD biomarker GWAS, knocking out each gene via CRISPR and scoring the phenotypic changes of the resulting engineered cells in response to oleate treatment. Analysis of over 24,000 images across all gene knockouts revealed 32 genes whose deletion caused significant alterations in cellular lipid droplet (LD) numbers and/or droplet size distribution. Here, we highlight ubiquitin-conjugating enzyme E2 G2 (UBE2G2), whose knockout robustly increases the size of cellular LDs that form in response to oleate stimulation in both the primary screen and subsequent validation. UBE2G2 localizes to the LD surface and is essential for the sterol-induced degradation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Thus we propose that deletion of UBE2G2 may increase cholesterol derivative content in LDs, thereby increasing LD size. In summary, we demonstrate the successful development and use of a platform for genetic screening of hepatocyte steatosis in vitro and identify multiple NAFLD-associated genes of previously unknown function that likely operate during this early stage of NAFLD pathogenesis. We acknowledge input: Seunghan Lee, Lifeng Wang, David Frederick, Alessandro Pocai Disclosure S. Hu: None. S. Khandavilli: None. R.Y. Diao: None. J. Raum: Employee; Janssen Research & Development, LLC. N. Azhar: Employee; Janssen Research & Development, LLC. I. Bakaj: Employee; Janssen Research & Development, LLC. G. Cowley: Employee; Janssen Research & Development, LLC. B. Rady: Employee; Janssen Research & Development, LLC. D.F. Reilly: Employee; Janssen Research & Development, LLC. A.R. Majithia: None. Funding National Institutes of Health (RDK01DK123422)

A proximity labeling strategy enables proteomic analysis of inter-organelle membrane contacts

Inter-organelle membrane contacts are highly dynamic and act as central hubs for many biological processes, but the protein compositions remain largely unknown due to the lack of efficient tools. Here, we developed BiFCPL to analyze the contact proteome in living cells by a bimolecular fluorescence complementation (BiFC)-based proximity labeling (PL) strategy. BiFCPL was applied to study mitochondria-endoplasmic reticulum contacts (MERCs) and mitochondria-lipid droplet (LD) contacts. We identified 403 highly confident MERC proteins, including many transiently resident proteins and potential tethers. Moreover, we demonstrated that mitochondria-LD contacts are sensitive to nutrient status. A comparative proteomic analysis revealed that 60 proteins are up- or downregulated at contact sites under metabolic challenge. We verified that SQLE, an enzyme for cholesterol synthesis, accumulates at mitochondria-LD contact sites probably to utilize local ATP for cholesterol synthesis. This work provides an efficient method to identify key proteins at inter-organelle membrane contacts in living cells.

SIAH1 ubiquitination-modified HMGCR inhibits lung cancer progression and promotes drug sensitivity through cholesterol synthesis

BackgroundsLung cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths worldwide. Deep understanding of chemoresistance will lead to remarkable progress in lung cancer treatment strategy. Cholesterol accumulation was associated with cisplatin resistance in lung cancer treatment. And we found the degree of cisplatin resistance was correlated with the expression of the cholesterol synthesis HMGCR.MethodsWe analyzed a group of 42 lung cancer patients who received cisplatin treatment after lung resection surgery. The expression of HMGCR and its correlation with cholesterol in lung cancer cell lines were determined by qRT-PCR and ELISA analyses. We focus on the function and mechanism of HMGCR in lung cancer and reveal that knockdown of HMGCR expression inhibits the proliferation, colony formation, and migration of lung cancer cell lines in vitro or in vivo and dramatically enhances the efficacy of cisplatin.ResultsThrough mechanism studies, we illustrate that SIAH1, an E3 ubiquitin-protein ligase, ubiquitination modifies HMGCR and inhibits efflux protein activity via regulating cholesterol synthesis. In vivo experiments showed that SIAH1 overexpression or using HMGCR knockdown retard tumor growth and enhanced the efficacy of cisplatin. In summary, HMGCR affects cholesterol metabolism by regulating key enzymes in cholesterol synthesis, thereby reducing drug sensitivity.ConclusionThis study indicates that lung cancer patients with lower HMGCR levels may lead to a better prognosis and provide a potential treatment by SIAH1 overexpression for lung cancer patients with cisplatin resistance.

Contribution of serum lipids and cholesterol cellular metabolism in lung cancer development and progression

Neoplasms of the lungs are the leading cause of cancer incidence and mortality worldwide. Although immunotherapy has increased the overall survival of patients with lung cancer, there is the need to improve this treatment. At this regard, blood lipid levels are thought to be linked to cancer risk and thus a preventive intervention through regulation of the nutrition of patients with lung cancer is gaining much attention. In this study, we therefore asked about the contribution of serum lipids and cholesterol cellular metabolism in lung cancer development and progression. We measured different serum lipids and analyzed cholesterol synthesis enzymes 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and acetyl-coenzyme A cholesterol acetyltransferase 1 (ACAT1) as well as the cholesterol cellular export protein ATP-binding cassette (ABC) A-1 mRNA by quantitative PCR (qPCR) in the control and tumoral regions of post-surgery lung tissues to analyze the accumulation of cholesterol in cancer cells in a cohort of patients with lung adenocarcinoma (LUAD). We found that triglycerides in serum directly correlated with the body mass index (BMI) in patients with LUAD. By contrast, we found that high-density lipoprotein (HDL) cholesterol inversely correlated with the BMI, C-reactive protein (CRP) and overall survival and total cholesterol inversely correlated with the tumor diameter, serum CRP and overall survival in these LUAD patients. Functionally, the role of cholesterol is indispensable for the growth and development of normal animal cells where it is tightly regulated. Excess of cellular cholesterol regulated by HMGCR is converted to cholesteryl esters by the enzyme ACAT1 and exported extracellularly by the cholesterol transporter ABCA1. Here we found HMGCR and ACAT1 upregulated and ABCA1 downregulated in the lung’s tumoral region of our LUAD cohort, indicating cholesterol dysregulated cellular export in lung tumor cells.