Inhibition Of Lipid Synthesis Research Articles

Deterioration of muscle quality caused by ammonia exposure in rainbow trout (Oncorhynchusmykiss)

In order to explore deterioration of muscle quality caused by ammonia exposure in rainbow trout (Oncorhynchus mykiss), and then preliminarily investigating the regulatory mechanism of muscle quality in O. mykiss after ammonia exposure based on the lipid metabolism, different concentrations of ammonia stress experiment were conducted, and the physicochemical properties of muscle, antioxidant system, products of energy and lipid metabolism were investigated at total ammonia concentration of 0 (control group), 10 mg/L (T1), 20 mg/L (T2), 30 mg/L (T3), 45 mg/L (T4) and 60 mg/L (T5). In comparison with the control group, the fillets after ammonia stress exhibited higher a* value, expressible moisture, essential amino acids (EAA), the bitter amino acid and mono-saturated fatty (MUFA), less the umami and sweet amino acids and polyunsaturated fatty acid (PUFA), worse flavor and taste. The lower ammonia exposure (lower than 30 mg/L) did not change the content of EPA and DHA while significant decreases were observed after higher ammonia exposure (higher than 30 mg/L). Meanwhile, the lower ammonia exposure activated the antioxidant system and higher ammonia exposure inactivated antioxidant enzymes to defend against reactive oxygen species (ROS). Moreover, the expression of d9D, FAS, LPL, PPARa, PPARy and SREBP were inhibited and the expression of CPT1 was promoted after ammonia stress, inhibiting lipid synthesis and promoting lipid oxidation. The results are important in establishing the relationship among muscle quality, lipid metabolism and the environmental adaptability of rainbow trout (O. mykiss).

UCP2-induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis.

Cellular lipid metabolism has been linked to immune responses; however, the precise mechanisms by which de novo fatty acid synthesis can regulate inflammatory responses remain unclear. The NLRP3 inflammasome serves as a platform for caspase-1–dependent maturation and secretion of proinflammatory cytokines. Here, we demonstrated that the mitochondrial uncoupling protein-2 (UCP2) regulates NLRP3-mediated caspase-1 activation through the stimulation of lipid synthesis in macrophages. UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis. Moreover, UCP2 expression was increased in human sepsis. Consistently, UCP2-deficient mice displayed impaired lipid synthesis and decreased production of IL-1β and IL-18 in response to LPS challenge. In macrophages, UCP2 deficiency suppressed NLRP3-mediated caspase-1 activation and NLRP3 expression associated with inhibition of lipid synthesis. In UCP2-deficient macrophages, inhibition of lipid synthesis resulted from the downregulation of fatty acid synthase (FASN), a key regulator of fatty acid synthesis. FASN inhibition by shRNA and treatment with the chemical inhibitors C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation and inhibited NLRP3 and pro–IL-1β gene expression in macrophages. In conclusion, our results suggest that UCP2 regulates the NLRP3 inflammasome by inducing the lipid synthesis pathway in macrophages. These results identify UCP2 as a potential therapeutic target in inflammatory diseases such as sepsis.

Improved Topical Drug Delivery: Role of Permeation Enhancers and Advanced Approaches.

The delivery of drugs via transdermal routes is an attractive approach due to ease of administration, bypassing of the first-pass metabolism, and the large skin surface area. However, a major drawback is an inability to surmount the skin's stratum corneum (SC) layer. Therefore, techniques reversibly modifying the stratum corneum have been a classical approach. Surmounting the significant barrier properties of the skin in a well-organised, momentary, and harmless approach is still challenging. Chemical permeation enhancers (CPEs) with higher activity are associated with certain side effects restricting their advancement in transdermal drug delivery. Furthermore, complexity in the interaction of CPEs with the skin has led to difficulty in elucidating the mechanism of action. Nevertheless, CPEs-aided transdermal drug delivery will accomplish its full potential due to advancements in analytical techniques, synthetic chemistry, and combinatorial studies. This review focused on techniques such as drug-vehicle interaction, vesicles and their analogues, and novel CPEs such as lipid synthesis inhibitors (LSIs), cell-penetrating peptides (CPPs), and ionic liquids (ILs). In addition, different types of microneedles, including 3D-printed microneedles, have been focused on in this review.

Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

The roles of lamin A/C in adipocyte differentiation and skeletal muscle lipid metabolism are associated with familial partial lipodystrophy of Dunnigan (FPLD). We confirmed that LMNA knockdown (KD) in mouse adipose-derived mesenchymal stem cells (AD-MSCs) prevented adipocyte maturation. Importantly, in in vitro experiments, we discovered a significant increase in phosphorylated lamin A/C levels at serine 22 or 392 sites (pLamin A/C-S22/392) accompanying increased lipid synthesis in a liver cell line (7701 cells) and two hepatocellular carcinoma (HCC) cell lines (HepG2 and MHCC97-H cells). Moreover, HCC cells did not survive after LMNA knockout (KO) or even KD. Evidently, the functions of lamin A/C differ between the liver and adipose tissue. To date, the mechanism of hepatocyte lipid metabolism mediated by nuclear lamin A/C remains unclear. Our in-depth study aimed to identify the molecular connection between lamin A/C and pLamin A/C, hepatic lipid metabolism and liver cancer. Gain- and loss-of-function experiments were performed to investigate functional changes and the related molecular pathways in 7701 cells. Adenosine 5' monophosphate-activated protein kinase α (AMPKα) was activated when abnormalities in functional lamin A/C were observed following lamin A/C depletion or farnesyltransferase inhibitor (FTI) treatment. Active AMPKα directly phosphorylated acetyl-CoA-carboxylase 1 (ACC1) and subsequently inhibited lipid synthesis but induced glycolysis in both HCC cells and normal cells. According to the mass spectrometry analysis, lamin A/C potentially regulated AMPKα activation through its chaperone proteins, ATPase or ADP/ATP transporter 2. Lonafarnib (an FTI) combined with low-glucose conditions significantly decreased the proliferation of the two HCC cell lines more efficiently than lonafarnib alone by inhibiting glycolysis or the maturation of prelamin A.

Steroid hormone 20-hydroxyecdysone disturbs fat body lipid metabolism and negatively regulates gluconeogenesis in Hyphantria cunea larvae.

The steroid hormone 20-hydroxyecdysone (20E) has been described to regulate fat body lipid metabolism in insects, but its accurate regulatory mechanism, especially the crosstalk between 20E-induced lipid metabolism and gluconeogenesis remains largely unclear. Here, we specially investigated the effect of 20E on lipid metabolism and gluconeogenesis in the fat body of Hyphantria cunea larvae, a notorious pest in forestry. Lipidomics analysis showed that a total of 1907 lipid species were identified in the fat body of H. cunea larvae assigned to 6 groups and 48 lipid classes. The differentially abundant lipids analysis showed a significant difference between 20E-treated and control samples, indicating that 20E caused a remarkable alteration of lipidomics profiles in the fat body of H. cunea larvae. Further studies demonstrated that 20E accelerated fatty acid β-oxidation, inhibited lipid synthesis, and promoted lipolysis. Meanwhile, the activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase were dramatically suppressed by 20E in the fat body of H. cunealarvae. As well, the transcriptions of genes encoding these 4 rate-limiting gluconeogenic enzymes were significantly downregulated in the fat body of H. cunealarvae after treatment with 20E. Taken together, our results revealed that 20E disturbed fat body lipid homeostasis, accelerated fatty acid β-oxidation and promoted lipolysis, but negatively regulated gluconeogenesis in H. cunea larvae. The findings might provide a new insight into hormonal regulation of glucose and lipid metabolism in insect fat body.

Comparative evaluation on the effects of dietary docosahexaenoic acid on growth performance, fatty acid profile and lipid metabolism in two sizes of abalone Haliotis discus hannai Ino

A 93-day feeding trial was conducted to comparatively evaluate the effects of graded levels of dietary docosahexaenoic acid (DHA) (0.06%, 0.46%, 0.99%, 1.57%, 1.95% and 2.25%) on growth performance, fatty acid profile and lipid metabolism in two different sizes of abalone Haliotis discus hannai (initial body weight: 2.83 ± 0.03 g and 26.81 ± 0.17 g, respectively). Results showed that 0.99%–1.95% of dietary DHA contents significantly increased the weight gain rate (WGR) in large size of abalone (P < 0.05), while the WGR in small size of abalone was significantly increased by the 0.46%–0.99% of dietary DHA contents (P < 0.05). There was no significant difference in the survival of abalone among all the six groups (P > 0.05). Based on the WGR, the optimal dietary DHA content for large and small size abalone were estimated to be 1.32% and 0.88%, respectively. Dietary DHA supplementation significantly increased the activities of trypsin and lipase in the intestine, the content of ∑n-3 polyunsaturated fatty acids (PUFA) and ∑n-3/n-6 PUFA ratio in digestive gland in both sizes of abalone (P < 0.05). The total glyceride concentration in the cell-free hemolymph in two sizes of abalone was decreased. In large size of abalone, 1.95%–2.25% of dietary DHA reduced the lipid content in digestive gland. Lipogenic genes in the digestive gland, including sterol regulatory element binding protein-1 (srebp-1) and diacylglycerol acyltransferase, were up-regulated in the 0.46%–1.57% DHA group (P > 0.05). The mRNA expression levels of hormone sensitive lipase (hsl) and carnitine palmitoyl transferase-1 (cpt-1) of lipolytic genes in the digestive gland were significantly increased in the 0.46% and 0.99% DHA groups, respectively (P < 0.05). In the small size of abalone, the lipid content in digestive gland and soft body decreased significantly in the 1.95% and 2.25% DHA groups, respectively (P < 0.05). Lipogenic genes in the digestive gland, including srebp-1 and acetyl-CoA carboxylase, were significantly down-regulated in the 0.99%–1.95% DHA group (P < 0.05). Lipolytic genes in the digestive gland, including hsl and cpt-1, were not significantly different from the control group (P > 0.05). In conclusion, based on the WGR, the dietary DHA requirements for large and small size of abalone were estimated to be 1.32% and 0.88%, respectively. Dietary DHA reduced lipid deposition in both sizes of abalone by promoting lipid decomposition and inhibiting lipid synthesis.

Induction of autophagy via the PI3K/Akt/mTOR signaling pathway by Pueraria flavonoids improves non-alcoholic fatty liver disease in obese mice

Non-alcoholic fatty liver disease (NAFLD) is the most common among lipid metabolism disorders. Autophagy plays an important role in lipid metabolism in NAFLD. Pueraria flavonoids, the main active ingredients of Pueraria lobata, exert antioxidant and anti-inflammatory effects. Herein, we report the potential lipid-lowering and anti-inflammatory effects of Pueraria flavonoids on NAFLD induced by a high-fat diet. In vivo and in vitro experiments showed that Pueraria flavonoids reduced intracellular lipid deposition by inhibiting lipid synthesis and the release of pro-inflammatory cytokines. We analyzed the autophagy flux by mRFP-GFP-LC3 plasmid transfection to assess the role of autophagy in intracellular scavenging. After treating mice fed on high fat and HepG2 cells with Pueraria flavonoids, the number of autophagosomes increased significantly, along with the level of autophagy. The autophagy loss after siRNA transfection aggravated lipid deposition and the release of inflammatory cytokines. Mechanistically, Pueraria flavonoids trigger autophagy through PI3K/Akt/mTOR signaling pathway to reduce lipid deposition and inflammation. In summary, our results showed that Pueraria flavonoids stimulated autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway, thereby reducing intracellular lipid accumulation and inflammation levels and alleviating NAFLD.

Isosilybin regulates lipogenesis and fatty acid oxidation via the AMPK/SREBP-1c/PPARα pathway

It is well known that the excessive accumulation of lipid in hepatocytes is one of the important causes of non-alcoholic fatty liver disease (NAFLD). The purpose of this study was to explore the effects of isosilybin on lipid metabolism in free fatty acids (FFAs) or TO901317-induced HepG2 cells. Cells were treated with FFAs (oleic acid: palmitic acid, 2:1) or TO901317 to induce steatosis in vitro. Intracellular triglyceride (TG) content was quantified using commercial assay kits. The mRNA and protein expression of genes involved in fatty acid uptake, synthesis and oxidation were analyzed by RT-qPCR and western blotting. Selected biological pathways regulated by isosilybin treatment were determined by GO and KEGG analysis. The results showed that isosilybin significantly reduced TG levels in FFAs- and TO901317-induced HepG2 cells. Further studies showed that isosilybin treatment decreased the mRNA and protein expression of lipid synthesis genes Srebp-1c, Pnpla3, Acc and Fas, as well as the mRNA expression of fatty acid uptake gene CD36, whereas increased the mRNA levels of lipid oxidation genes Pparα, Acox1 and Cpt1α, as well as the mRNA expression of lipid export gene Mttp, in FFAs-induced HepG2 cells. Moreover, TO901317 was employed to induce endogenous lipid synthesis and steatosis, and the expression of Srebp-1c and its target genes in TO901317-induced hepatocytes was basically similar to that in FFAs-induced hepatocytes following isosilybin treatment. We also observed the increased level of phosphorylated AMP kinase (AMPK) after isosilybin treatment, while this effect was reversed after further treatment with AMPK inhibitor, compound C. The results of GO and KEGG analysis indicated that the pathways of fatty acid and TG metabolism were regulated by isosilybin. Interestingly, we found that treatment with the diastereoisomer A of isosilybin increased TG level, while exposure to the diastereoisomer B of isosilybin decreased TG level in FFAs-induced HepG2 cells. The above results suggest that isosilybin can inhibit lipid synthesis and activate lipid oxidation through AMPK signaling pathway, thereby improving steatosis of hepatocytes, and isosilybin B is the basis of its active substance.

The spirotetramat inhibits growth and reproduction of silkworm by interfering with the fatty acid metabolism

Spirotetramat is a novel insecticide and acaricide that can effectively control many species of piercing-sucking pests by inhibiting lipid synthesis. The silkworm is an economically important insect and a model organism for genetics and biochemical research. However, the toxic effect on their development and reproduction remain unclear. In this study, we demonstrated the negative effects of spirotetramat on the development, vitality, silk protein synthesis, and fecundity of silkworm. We also compared expression changes of silkworm genes using digital gene expression (DGE). A total of 1567 differentially expressed genes (DEGs) were detected, of which 874 genes were downregulated and 693 genes were upregulated. Gene Ontology (GO) enrichment analysis showed that the DEGs were enriched in the oxidation-reduction process, oxidoreductase activity, and fatty-acyl-CoA reductase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were mainly enriched in fatty acid metabolism and lysosome pathways. We detected the relative expression of silkworm genes related to fatty acid synthesis and decomposition pathways and the degradation pathway of juvenile hormone by quantitative real-time PCR. The expression levels of Acetyl CoA carboxylase (ACC), fatty acyl-CoA reductase (FACR), Enoyl-CoA hydratase (ECH), very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase (LCHAD), juvenile hormone epoxide hydrolase (JHEH), and phytanoyl-CoA dioxygenase (PCD) genes were downregulated. These data demonstrate the effects of spirotetramat on silkworm and its effects on genes involved in fatty acid metabolism.

Activity Evaluation of Alcohol Extract of Ginseng-Clove in Inhibiting Lipid Synthesis of Mortierella alpina

Activity Evaluation of Alcohol Extract of Ginseng-Clove in Inhibiting Lipid Synthesis of Mortierella alpina

Glucagon-like peptide-1 receptor mediates the improvement in glycolipid metabolism disorder via AKT and AMPK signalling pathways in L02 cells with insulin resistance

Glucagon-like peptide 1 (GLP-1) has previously been found to regulate glycolipid metabolism in the liver by promoting insulin secretion. However, the presence of GLP-1 receptors (GLP-1R) in the human liver has not been determined. In this research, GLP-1R was found to be expressed in L02 cells and decreased in insulin resistant (IR) cells. Compared with LG treatment group, GLP-1R knockdown significantly decreased glucose utilization (from 143.13% to 118.41%.) and intracellular glycogen content (from 122.77% to 105.72%). Furthermore, the transcription levels of PEPCK and G6Pase increased, and the phosphorylation levels of AKT, FOXO1 and GSK3β decreased in siGLP-1R group. Similarly, the contents of triglyceride and total cholesterol in siGLP-1R group increased by 43.23% and 24.19%, respectively. PCR and WB results showed that knockdown of GLP-1R increased the levels of ACC, FAS, and SREBP-1c, and decreased the levels of CPT-1 via AMPK pathway. GLP-1R knockdown changed the LG-induced promotion of glucose utilization and inhibition of lipid synthesis. These findings demonstrated that GLP-1R is the molecular basis for GLP-1 to improve glycolipid metabolism in L02 cells.

Potential of Squid Ink Compounds as Insilicate FabH Protein In Silico

Tuberculosis (TB) is a disease with the second number of cases in Indonesia. The therapy process is carried out by utilizing one of the waste materials, namely squid ink bags, which are predicted to have active compounds. In the TB cycle, the enzyme FabH is catalyzed as a mediator of lipid synthesis. Inhibition of FabH became the main target through the role of squid ink compounds. The squid ink has a potential as bactery inhibitor. The purpose of this study was to analyze the potential of squid ink as an alternative to TB therapy by inhibiting lipid synthesis (FabH). In silico approach was used in this research. Bioactive compound 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (CID: 119405), cinnamic acid (CID: 444539), betaine (CID: 247) were downloaded from PubChem, and FabH protein (PDB ID 2QO1) was downloaded from the protein data bank database. (GDP). Docking used HeX 8.0.0.0, visualization and analysis using Discovery studio ver 21.1.1. Results from the analysis, we found compounds 5,6-dihydroxyindole-2-carboxylic acid (DHICA), cinnamic acid, betaine contained in squid ink molecularly have potential as an alternative TB therapy by inhibiting FabH, the interaction lead to treatment the TB. In vitro and in vivo studies are needed to analyze further before human studies.

Phospholipid synthesis inside phospholipid membrane vesicles

Construction of living artificial cells from genes and molecules can expand our understanding of life system and establish a new aspect of bioengineering. However, growth and division of cell membrane that are basis of cell proliferation are still difficult to reconstruct because a high-yielding phospholipid synthesis system has not been established. Here, we developed a cell-free phospholipid synthesis system that combines fatty acid synthesis and cell-free gene expression system synthesizing acyltransferases. The synthesized fatty acids were sequentially converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of intermediates inhibiting lipid synthesis, sub-millimolar phospholipids could be synthesized within a single reaction mixture. We also performed phospholipid synthesis inside phospholipid membrane vesicles, which encapsulated all the components, and showed the phospholipids localized onto the mother membrane. Our approach would be a platform for the construction of self-reproducing artificial cells since the membrane can grow sustainably.

Mulberry Leaf Extract Improves Metabolic Syndrome by Alleviating Lipid Accumulation In Vitro and In Vivo.

Metabolic syndrome (MS) is a metabolic disease with multiple complications. Mulberry leaf extract (MLE) is rich in flavonoids and has great potential in alleviating glucose and lipid metabolism disorders. This study evaluated the effect and mechanism of MLE on the alleviation of MS. The components of the MLE were analyzed, and then the regulation of lipid metabolism by MLE in vitro and in vivo was determined. In a hepatocyte model of oleic acid-induced lipid accumulation, it was found that MLE alleviated lipid accumulation and decreased the expression of genes involved in lipogenesis. Furthermore, MLE improved obesity, insulin resistance, plasma lipid profile, and liver function in MS mice after a 15-week intervention. MLE decreased the expression of SREBP1, ACC, and FAS through the AMPK signaling pathway to inhibit lipid synthesis and increase the level of CPT1A to promote lipid decomposition to achieve its hypolipidemic effect. Meanwhile, MLE was also shown to affect the composition of the gut microbiota and the production of short-chain fatty acids, which contributed to the alleviation of lipid accumulation. Our results suggest that MLE can improve MS by improving lipid metabolism through multiple mechanisms and can be developed into dietary supplements for the improvement of MS.

Preventive and Therapeutic Effects of Krill Oil on Obesity and Obesity-Induced Metabolic Syndromes in High-Fat Diet-Fed Mice.

Obesity increases the risks of metabolic syndromes including nonalcoholic fatty liver disease (NAFLD), diabetic dyslipidemia, and chronic kidney disease. Dietary krill oil (KO) has shown antioxidant and anti-inflammatory properties, thereby being a therapeutic potential for obesity-induced metabolic syndromes. Thus, the effects of KO on lipid metabolic alteration were examined in a high-fat diet (HFD)-fed mice model. The HFD model (n = 10 per group) received an oral gavage with distilled water as a control, metformin at 250 mg/kg, and KO at 400, 200, and 100 mg/kg for 12 weeks. The HFD-induced weight gain and fat deposition were significantly reduced in the KO treatments compared with the control. Blood levels were lower in parameters for NAFLD (e.g., alanine aminotransferase, and triglyceride), type 2 diabetes (e.g., glucose and insulin), and renal dysfunction (e.g., blood urea nitrogen and creatinine) by the KO treatments. The KO inhibited lipid synthesis through the modification of gene expressions in the liver and adipose tissues and adipokine-mediated pathways. Furthermore, KO showed hepatic antioxidant activities and glucose lowering effects. Histopathological analyses revealed that the KO ameliorated the hepatic steatosis, pancreatic endocrine/exocrine alteration, adipose tissue hypertrophy, and renal steatosis. These analyses suggest that KO may be promising for inhibiting obesity and metabolic syndromes.

Study on the Potential Mechanism of Tonifying Kidney and Removing Dampness Formula in the Treatment of Postmenopausal Dyslipidemia Based on Network Pharmacology, Molecular Docking and Experimental Evidence.

BackgroundManagement of menopausal dyslipidemia is the main measure to reduce the incidence of cardiovascular disease in postmenopausal women. Tonifying Kidney and Removing Dampness Formula (TKRDF) is a traditional Chinese medicine (TCM) formula that ameliorates dyslipidemia in postmenopausal women. This study applied network pharmacology, molecular docking, and in vitro and in vitro experiments to investigate the underlying mechanism of TKRDF against postmenopausal dyslipidemia.MethodsNetwork pharmacology research was first conducted, and the active compounds and targets of TKRDF, as well as the targets of postmenopausal dyslipidemia, were extracted from public databases. Protein–protein interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to identify the potential targets and signaling pathways of TKRDF in postmenopausal dyslipidemia. Molecular docking was then performed to evaluate the combination of active compounds with principal targets. Finally, an ovariectomized rat model was used for the in vivo experiment and alpha mouse liver 12 (AML12) cells treated with palmitic acid were used for the in vitro experiments to provide further evidence for the research.ResultsBased on network pharmacology analysis, we obtained 78 active compounds from TKRDF that acted on 222 targets of postmenopausal dyslipidemia. The analysis results indicated that IL6, TNF, VEGFA, AKT1, MAPK3, MAPK1, PPARG and PIK3CA, etc., were the potentially key targets, and the PI3K/AKT signaling pathway was the possibly crucial pathway for TKRDF to treat postmenopausal dyslipidemia. Molecular docking suggested that the active compounds have good binding activity with the core targets. The in vivo and in vitro experiments demonstrated that TKRDF ameliorates postmenopausal dyslipidemia by regulating hormone levels, inhibiting inflammation, promoting angiogenesis and inhibiting lipid synthesis, which appear to be related to TKRDF’s regulation of the ERK1/2 and PI3K/AKT signaling pathways.ConclusionThis study clarified the active ingredients, potential targets, and molecular mechanisms of TKRDF for treating postmenopausal dyslipidemia. It also provided a feasible method to uncover the scientific basis and therapeutic mechanism for prescribing TCM in the treatment of diseases.

Theabrownin and Poria cocos Polysaccharide Improve Lipid Metabolism via Modulation of Bile Acid and Fatty Acid Metabolism.

Nonalcoholic fatty liver disease (NAFLD) is prevalent worldwide, while no pharmaceutical treatment has been approved. Natural herbs are promising for their amelioration effect on lipid metabolism. Theabrownin (TB) and Poria cocos polysaccharide (PCP) have been reported to have effect on hyperlipidemia and diabetes. Here, we compared the effect of individual TB or PCP and the combination of TB and PCP (TB + PCP) on NAFLD phenotypes and the alteration of metabolism in the mice with high-fat diet. The results showed that TB, PCP, and TB + PCP reduced serum and hepatic lipid levels, among which TB + PCP was the most effective. Serum metabolomic profile and liver mRNA analyses revealed that the treatments altered metabolic pathways involved in fatty acid metabolism, bile acid metabolism, and tricarboxylic acid cycle, which was also most significant in the TB + PCP group. This study demonstrated that TB, PCP, especially the combination of TB and PCP could be potential therapeutic formula for NAFLD that promoted lipid utilization and inhibited lipid synthesis and absorption.

Abstract 2308: Inhibition of fatty acid synthesis and concurrent lipid deprivation decreases neuroendocrine cancer cell proliferation and colony formation

Abstract Introduction. Gastroenteropancreatic neuroendocrine tumors (GEP-NETs), the second most common digestive cancer in terms of prevalence, can be particularly challenging to treat due to resistance to current clinical regimens. Lipid metabolism provides an important energy source for cancer cells; cells increase lipid supply by endogenous synthesis or exogenous uptake. Both processes can become dysregulated in cancer, but little is known about the role of either pathway in neuroendocrine tumors (NETs). The goal of this project was to determine whether inhibition of lipid synthesis with concurrent lipid deprivation affects NET cell proliferation and survival. Methods. (i) Expression and localization of fatty acid synthase (FASN) in NET patient samples (n=39) and human cell lines (BON, QGP-1, and NT-3) were examined with immunohistochemistry and immunoblotting, respectively. (ii) NET neutral lipid stores were determined with immunofluorescent analysis of Bodipy 493/503 staining. (iii) To study the role of lipid synthesis and lipid uptake on colony formation, NET cells were treated with TVB-3664 with or without FA depletion for 10d. Colonies were visualized with sulforhodamine B (SRB) dye staining and quantified with photospectrometry. (iv) To study the effect of lipid synthesis and uptake on proliferation, NET cells were treated with TVB-3664 with or without FA depletion for 72h, 96h, and 120h. Cellular growth was determined with SRB assays. (v) The effect of TVB-3664 on cyclin D1 and β-catenin expression in NET cells was determined with immunoblotting. Results. (i) FASN was detected in the cytoplasm of all 39 NET primary and metastatic tissue samples; scores of 5 or 6 indicated robust expression in 38/39 samples. Among cell lines, FASN expression was highest in BON cells followed by similar levels in QGP-1 and NT-3 cells. (ii) TVB-3664 treatment or lipid deprivation for 5d reduced neutral lipid storage in BON cells; combined TVB-3664 treatment with FA depletion for 5d fully depleted BON lipid stores. (iii) TVB-3664 treatment, but not FA deprivation, reduced NET colony formation by ~25% in BON cells and ~50% in QGP-1 cells. TVB-3664 treatment with FA deprivation reduced colony formation by ~65% in BON cells and ~75% in QGP-1 cells. (iv) TVB-3664 treatment or FA deprivation reduced NET cell proliferation at 72h, 96h, and 120h. (v) TVB-3664 treatment reduced cyclin D1 expression in BON and QGP-1 cells and β-catenin levels in BON cells. Conclusions. Our findings indicate that FASN is robustly expressed in NET patient samples and cell lines. Inhibition of endogenous lipid synthesis with concurrent lipid deprivation reduces colony formation and proliferation of NET cells. Importantly, our findings show that lipid metabolism promotes NET cell growth and that targeting de novo lipogenesis may be a potential treatment strategy for advanced GEP-NETs. Citation Format: Jeremy Johnson, Yekaterina Y. Zaytseva, Jennifer Castle, B. Mark Evers, Piotr Rychahou. Inhibition of fatty acid synthesis and concurrent lipid deprivation decreases neuroendocrine cancer cell proliferation and colony formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2308.

Abstract 3018: Targeting lipid metabolism and diet in advanced prostate cancer

Abstract Lipid metabolism reprogramming provides the energetic needs of prostate cancer cells (PCa) to support growth and cell progress through the cell cycle, sustaining a highly proliferative phenotype by increasing the expression levels of several enzymes involved in fatty acid (FAs) synthesis. Fatty acid synthase (FASN), a key enzyme in de novo fatty acid lipogenesis (DNL), is upregulated in metastatic, castration-resistant prostate cancer (CRPC), and its overexpression is related to carcinogenesis, growth, and metastasis. We previously demonstrated that FASN inhibitors show antitumor activity suppressing PCa cell proliferation and slowing tumor growth, both in vitro and in vivo, via rewiring of lipid cancer metabolism and inducing endoplasmic reticulum stress.We observed that impairment of DNL increases polyunsaturated fatty acids (PUFA) uptake as a compensatory mechanism of survival. Exogenously acquired PUFAs cause the remodeling of the prostate cancer cell lipidome, manifesting an enhanced acyl chain unsaturation across several lipid species. Lipid peroxidation and ROS accumulation are also observed, suggesting cell susceptible to oxidative damage following FASN blockade. Alterations in metabolic pathways cause a higher uptake of acetate and glutamine in PCa cells, as assessed by 14C-nutrient incorporation following FASN inhibition. Mitochondrial dysfunction is induced by reduction in several respiratory parameters, including ATP production and basal respiration. This may be, at least in part, the result of a reduction in phosphatidylglycerol, a cardiolipin precursor, altering mitochondrial membrane composition. Supplementation with the PUFA docosahexaenoic acid (DHA) increases phosphatidylcholine levels and total phospholipid acyl chain unsaturation, which can lead to membrane disorganization in prostate cancer cells. Superoxide anion production is enhanced by DHA treatment, as expected, and increased levels of glucose and palmitate oxidation by mitochondria are observed. Interestingly, AR and AR-V7 protein expression, as well as c-MYC and PSA, is reduced with DHA treatment. We then evaluated how de novo lipid synthesis inhibition combined with manipulation of the exogenous supply of fatty acids could impair tumor cell progression. Due to the obligate cell dependence on PUFA following DNL suppression, we combined FASN and Acetyl-CoA carboxylase (ACC) inhibitors with DHA supplementation and observed a significant cell growth inhibition in CRPC cell lines and prostate cancer organoids.Altogether, our results suggest a novel mechanism to inhibit the growth of castration-resistant prostate cancer, combining lipogenesis inhibition with PUFA-enriched diet to overcome advanced disease. Citation Format: Silvia D. Rodrigues, Caroline Fidalgo Ribeiro, Guilherme H. Tamarindo, Hubert Pakula, Massimo Loda. Targeting lipid metabolism and diet in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3018.

Trabecular Meshwork Cholesterol Levels Regulate Actin Polymerization and Tunneling Nanotubes

Trabecular meshwork (TM) tissue is made of highly contractile mechanosensitive cells regulating aqueous humor (AH) outflow facility and intraocular pressure (IOP). Increased actin‐mediated contraction of TM elevates IOP causing glaucoma and irreversible blindness. This study aims at understanding the role of cholesterol in regulating actin dynamics in human TM (HTM) cells. To test this, HTM cells were treated with SiR‐Actin in serum‐free media overnight and F‐actin changes were recorded using live‐cell time‐lapse (5 min interval) confocal imaging before and during exposure to ‐ A) 10mM methyl‐β‐cyclodextrin (MβCD) for 1h for acute removal of cellular cholesterol, B) 100μM cholesterol‐saturated MβCD (MβCD‐CHOL) for 1h to enrich cholesterol, and C) 10mM MβCD for 1h followed by cholesterol supplementation using MβCD‐CHOL for 1h. F‐actin fluorescence intensity changes were calculated and unpaired t‐test was used for statistical analyses and results were significant if p&lt;0.05 with a sample size of n= 4‐57. HTM cells plated on coverslips were treated as A – C as mentioned above, cholesterol and lipid synthesis inhibitors for 24h ‐ D) 100μM atorvastatin and E) 20μM fatostatin, respectively, F) actin polymerization disruptor ‐ 10μM cytochalasin D (CYTD) for 1h, G) 10μM CYTD for 1h followed by 100μM MβCD‐CHOL for 1h, H) microtubule disruptor ‐ 1μM demecolcine (DEM) for 1h, I) 1μM DEM for 1h followed by 100μM MβCD‐CHOL for 1h. Cells without treatment were used as control. After all these treatments, cells were fixed and immunofluorescence (IF) was performed to check for cholesterol using filipin, F‐actin by phalloidin, focal adhesion (FA) localization based on ‐ vinculin and paxillin distribution. Further F‐actin/G‐actin in vitro assay was performed to quantify the changes in actin polymerization under the conditions A – I compared to the control. The imaging analysis showed cholesterol removal significantly decreased F‐actin fluorescence intensity from 20min sustaining up to 1h and decreased the FA distribution. Cholesterol supplementation led to partial rescue of actin and FA indicating the re‐establishment of actin cytoskeleton and increased tunneling nanotube structures enhancing the cell‐cell communication (Figure 1). Moreover, IF results show that atorvastatin and fatostatin decreased F‐actin and FA distribution. CYTD disrupted F‐actin network leaving remnant filament ends and decreased FA but cholesterol enrichment increased F‐actin branches formed at ~70‐degree angle indicating Arp2/3 activation. DEM treatment increased F‐actin and FA, and cholesterol enrichment induced thick F‐actin filaments. These results were confirmed by quantitative F/G‐actin ratio decrease and increase upon cholesterol removal and enrichment, respectively. Additionally, supplementing cholesterol after removal increased the ratio compared to solely removing cholesterol. Interestingly, F/G‐actin ratio increased under CYTD treatment indicating that CYTD disrupted longer actin filaments but helped polymerization of newly formed actin filaments. Finally, DEM treatment increased F/G‐actin ratio, which was further increased by cholesterol enrichment. This systematic evaluation of cholesterol modulation on TM actin cytoskeleton polymerization identifies the significance of maintaining membrane and cellular cholesterol levels and cellular communication for achieving IOP homeostasis.