1. Bioactive Lipid Species and Metabolic Pathways in Progression and Resolution of Nonalcoholic Steatohepatitis 
Giovanni Musso, Maurizio Cassader, Elena Paschetta, Roberto Gambino 
Gastroenterology 
Volume 155, Issue 2, Pages e8 (August 2018)
DOI: /j.gastro
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2. Gastroenterology 2018 155, 282-302. e8DOI: (10. 1053/j. gastro. 2018
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3. Figure 1
Mechanisms of SFA and LPC lipotoxicity in the hepatocyte. In the hepatocyte, SFAs exert their lipotoxicity through various mechanisms. First, they can bind and activate plasma membrane receptors, including death receptor TRAIL-R2 and damage-associated molecular pattern receptors, such as TLR4. The TRAIL-2 signaling pathway triggers caspase 8 proteolytic autoactivation, which activates caspases 3, 6, and 7, eventually executing cell apoptosis directly or indirectly through MOMP and Cyt c release into cytosol. MOMP is executed by engagement of proapoptotic proteins of the Bcl-2 family, including Bim, Bid, Bak, and Bax, with the latter 2 oligomerizing to form pores on the mitochondrial outer membrane. Caspase 8 cleaves Bid, a member of the BH3-domain-only subgroup of the BCL-2 family, into its truncated form, which translocates to the mitochondria outer membrane and induces intramembranous Bak oligomerization into a pore for Cyt c efflux. SFAs also can induce MOMP through other mechanisms, including enhanced expression of the proapoptotic Bcl-2 family protein Bim through upregulation of the nuclear transcription factor FOXO3A, and activation of the proapoptotic protein PUMA by activation of JNK and ER stress. In addition, SFA can trigger the lysosomal pathway of apoptosis through induction of Bax-mediated lysosomal permeabilization with cathepsin B release. TLR4 pathway activation triggers NF-κB–mediated synthesis of proinflammatory cytokines TNF-α, IL-6, pro–IL-1, and pro–IL-18 (subsequently activated by caspase 1) and activate stress kinases, including JNK. SFAs also can enter the cell and induce ceramide-mediated NLRP3 inflammasome activation, ER stress, and JNK activation. ER stress upregulates CHOP-mediated PUMA expression and induces the release of EVs, which promote progression of nonalcoholic steatohepatitis by cell-to-cell communication. The cargoes of EVs mediate their lipotoxicity: C-X-C motif ligand 10 and ceramide-enriched EVs induce monocyte and macrophage chemotaxis to the liver, TRAIL-enriched EVs contribute to macrophage activation, and miR-128-3p-laden EVs promote hepatic stellate cell activation and fibrogenesis. JNK, a member of the mitogen-activated protein kinase family, is a major mediator of hepatic lipotoxicity: in hepatocytes JNK inactivates IRS-1, inducing insulin resistance, and interacts with the outer membrane mitochondrial protein SH3BP5 (Sab) to impair mitochondrial respiration and enhance ROS generation. Furthermore, JNK suppresses PPAR-α–mediated FGF-21 expression and mitochondrial and peroxisome β-oxidation and activates the proapoptotic proteins PUMA and Bim. LPC is a glycerophospholipid generated intracellularly by PLA2, which hydrolyzes PC into LPC and arachidonic acid, the latter being a precursor of proinflammatory eicosanoids. Most lipotoxic mechanisms of LPC, including JNK and ER stress activation and EV generation, overlap with those of SFA. An additional mechanism of lipotoxicity is the depletion of membrane PC, which disrupts hepatocyte membrane functional integrity, resulting in release of lipotoxic and proinflammatory lipids and hepatocyte apoptosis. ASC, apoptosis-associated speck-like protein containing a CARD; Bak, Bcl-2–associated K protein; Bax, Bcl-2–associated X protein; Bim, Bcl-2 protein family member; Cer, ceramide; CHOP, CAAT/enhancer binding homologous protein; Cyt c, cytochrome c; ETC, electron transport chain; EV, extracellular vesicle; FGF-21, fibroblast growth factor-21; FOXO3A, forkhead box-containing protein, class O, member 3a; IRS-1, insulin receptor substrate-1; LPA, lysophosphatidic acid; MCP, monocyte chemoattractant protein; NLRP3, nucleotide-binding and oligomerization domain-like receptor pyrin domain-containing protein 3; PUMA, p53-upregulated modulator of apoptosis; TLR4, Toll-like receptor-4.
Gastroenterology  , e8DOI: ( /j.gastro )
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4. Figure 2
Mechanisms of SFA and LPC lipotoxicity in non-parenchymal liver cells. Hepatic non-parenchymal cells also are involved in SFA and LPC lipotoxicity: SFAs activate TLR4 on HSCs, Kupffer cells, and macrophages. In Kupffer cells and macrophages, TLR4 pathway activation triggers NOX-2–mediated ROS generation, resulting in NF-κB signaling pathway activation directly or by JNK activation. NF-κB induces the transcription of proinflammatory and pro-fibrotic cytokines, including IL-1, IL-6, TNF-α, TGF-β, and TIMP-1, and chemokines such as MCP-1 to induce proinflammatory M1 polarization, chemotaxis, and HSC activation. In HSCs, TLR4 downregulates, through the adaptor molecule MyD88, the membrane receptor Bambi, a pseudo-receptor for TGF-β1 with negative regulatory function. The removal of this inhibitor sensitizes HSCs to activation by TGF-β1 and secretion of chemokine MCP-1 that recruits circulating macrophages to the liver. Kupffer cells secrete TGF-β and further activate HSCs in a paracrine manner. LPC is generated through the hydrolysis of hepatocyte plasma membrane phosphatidylcholine by phospholipase A2 (Figure 1) and released extracellularly, where it is converted by extracellular enzyme autotaxin to the potent pro-fibrogenic lipid LPA, which activates HSCs. Hepatocyte-released extracellular EVs released from hepatocytes (Figure 1) carry different cargoes and activate Kupffer cells, macrophages, and HSCs. Cer, ceramide; CXCL10, C-X-C motif ligand 10; LPA, lysophosphatidic acid; LPA-R, lysophosphatidic acid receptor; MCP, monocyte chemoattractant protein; NOX-2, nicotinamide adenine dinucleotide phosphate oxidase-2; TIMP, tissue inhibitor of metalloproteinase; TLR4, Toll-like receptor-4.
Gastroenterology  , e8DOI: ( /j.gastro )
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5. Figure 3
Metabolic circuits of CER, Sph, and S1P synthesis and catabolism (“sphingolipid rheostat”) and intracellular targets of sphingolipid lipotoxicity in the hepatocyte. CER can be synthesized de novo from serine and palmitate by the sequential action of 3 ER resident enzymes—serine palmitoyltransferase, ceramide synthase, and dihydro-ceramide desaturase (DES)—or generated from hydrolysis of plasma membrane sphingomyelin into ceramide and phosphocholine by the enzyme sphingomyelinase. Once generated from ceramide deacylation, sphingosine can be phosphorylated by sphingosine kinases to form S1P. S1P is degraded by reversible dephosphorylation to sphingosine by phosphatases. In the hepatocyte, CER inhibits Akt phosphorylation and activation, thereby impairing Akt-mediated insulin signaling and promoting insulin resistance, an effect that is antagonized by S1P. The mitochondria are another major cellular target of CER, which impairs fatty acid β-oxidation through inactivation of ETC complexes II and IV and promotes ROS production and triglyceride accumulation. Furthermore, ceramide triggers Bax-dependent mitochondrial membrane permeabilization and Cyt c release, leading to apoptosis. Further mechanisms of hepatic lipotoxicity of CER overload in NASH include ER stress activation, which leads to ER stress-mediated apoptosis. CER synergizes with S1P to activate SREBP-1c and SREBP-2 to enhance de novo lipogenesis and cholesterol synthesis and with NF-κB and NLRP3 inflammasome to induce proinflammatory cytokine and chemokine secretion. Further mechanisms of lipotoxicity include impairment of autophagy and upregulation of hepatic hepcidin, which leads to hepatic iron overload. In addition, ASMAse activation might promote liver injury independently of CER accumulation by disrupting methionine and phosphatidylcholine metabolism, which promotes lysosomal membrane permeabilization. CER, ceramide; CoA, coenzyme A; Cyt c, cytochrome c; ETC, electron transport chain; MCP, monocyte chemoattractant protein; NLRP3, nucleotide-binding and oligomerization domain-like receptor pyrin domain-containing protein 3; SM, sphingomyelinase; Sph, sphingosine.
Gastroenterology  , e8DOI: ( /j.gastro )
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6. Figure 4
Molecular targets of CER and S1P in cells other than hepatocytes. In HSCs, CER and S1P activate HSCs to increase ECM deposition and secretion of pro-angiogenetic cytokines angiopoietin and VEGF. The secretion of pro-angiogenetic cytokines has been shown to be specifically induced by S1P upon binding to its receptors S1PR1 and S1PR3. In the hypothalamus, CER decreases sympathetic tone and sympathetic-induced energy expenditure. In adipocytes, CER impairs mitochondrial ETC activity and β-oxidation and triggers ER stress, leading to activation of the proinflammatory transcription factor NF-κB. S1PR1 and S1PR3 activation by S1P synergizes with ER stress to activate NF-κB. NF-κB activation upregulates secretion of TNF-α and IL-6 and inhibits secretion of adiponectin and IL-10, promoting inflammation and insulin resistance. Furthermore, in adipocytes and macrophages, CER activates NLRP3 inflammasome to induce proinflammatory cytokine IL-1 and IL-18 secretion. In endothelial cells, S1PR1 and SIPR3 activation by S1P enhances integrity and barrier function. CER, ceramide; NLRP3, nucleotide-binding and oligomerization domain-like receptor pyrin domain-containing protein 3; VEGF, vascular endothelial growth factor.
Gastroenterology  , e8DOI: ( /j.gastro )
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7. Figure 5
Scheme of short-, long- and n-6 and n-3 PUFA biosynthesis leading to membrane phospholipid synthesis. The metabolism of PUFA is a complex process involving several enzymes of desaturation, elongation, and β-oxidation. The pathway of n-6 and n-3 PUFA metabolism to more unsaturated long-chain members of each family is illustrated. The long-chain saturated fatty acids and unsaturated fatty acids of the n-3, n-6, n-7, and n-9 series can be synthesized from myristic acid (C14:0) and palmitic acid (C16:0). Long-chain fatty acids of the n-6 and n-3 series also can be synthesized from precursors obtained from dietary precursors to elongation (ELOVL) and desaturation steps as indicated in these pathways. ACC, acetyl-CoA carboxylase; Cer, ceramide; ELOVL, elongase of very-long-chain fatty acid; FADS, fatty acid desaturase; FASN, fatty acid synthase; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelinase.
Gastroenterology  , e8DOI: ( /j.gastro )
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8. Supplementary Figure 1
Chemical structures of main lipotoxic and anti-inflammatory and pro-resolving lipids. (A) Intermediates (acetyl-CoA and malonyl-CoA) in fatty acid synthesis, saturated fatty acids, and monounsaturated fatty acids. (B) Diacylglycerol and triacylglycerol. A diacylglycerol or diglyceride is a glyceride consisting of 2 fatty acid chains (green area) covalently bonded to a glycerol molecule (blue area) through ester linkages. A triacylglycerol or triglyceride is a glyceride consisting of 3 fatty acid chains (green area) covalently bonded to a glycerol molecule (blue area) through ester linkages. (C) The ω-3 and ω-6 polyunsaturated fatty acids. The ω-3 polyunsaturated fatty acids have a carbon–carbon double bond located 3 carbons from the methyl end of the chain. The ω-6 polyunsaturated fatty acids have a carbon–carbon double bond located 6 carbons from the methyl end of the chain. (D) Lysophosphatidylcholine consists of a fatty acid chain (green area) and a phosphocholine (yellow area) covalently bonded to a glycerol molecule (blue area) through ester linkages. (E) Sphingolipids, sphingosine, sphingosine-1-phosphate, and ceramide. Ceramide consists of a fatty acid chain (green area) covalently bonded to a sphingosine molecule (dark red area) through amino linkage. (F) Leukotrienes and prostaglandins. Leukotrienes are a family of eicosanoid inflammatory mediators produced in leukocytes by the oxidation of arachidonic acid and the essential fatty acid eicosapentaenoic acid by the enzyme arachidonate 5-lipoxygenase. Prostaglandins are composed of unsaturated fatty acids that contain a cyclopentane (5-carbon) ring and are derived from the 20-carbon, straight-chain, polyunsaturated fatty acid precursor arachidonic acid by the enzyme cyclo-oxygenase through endoperoxides. (G) Resolvins are members of polyunsaturated fatty acid–derived metabolites termed specialized pro-resolving mediators. Resolvin Ds are metabolites of the 22-carbon polyunsaturated fatty acid docosahexaenoic acid. Resolvin Es are metabolites of the 20-carbon polyunsaturated fatty acid eicosapentaenoic acid. (H) Maresins are 12-lipoxygenase-derived specialized pro-resolving mediators synthesized from the omega-3 fatty acid docosahexaenoic acid.
Gastroenterology  , e8DOI: ( /j.gastro )
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9. Supplementary Figure 1
Chemical structures of main lipotoxic and anti-inflammatory and pro-resolving lipids. (A) Intermediates (acetyl-CoA and malonyl-CoA) in fatty acid synthesis, saturated fatty acids, and monounsaturated fatty acids. (B) Diacylglycerol and triacylglycerol. A diacylglycerol or diglyceride is a glyceride consisting of 2 fatty acid chains (green area) covalently bonded to a glycerol molecule (blue area) through ester linkages. A triacylglycerol or triglyceride is a glyceride consisting of 3 fatty acid chains (green area) covalently bonded to a glycerol molecule (blue area) through ester linkages. (C) The ω-3 and ω-6 polyunsaturated fatty acids. The ω-3 polyunsaturated fatty acids have a carbon–carbon double bond located 3 carbons from the methyl end of the chain. The ω-6 polyunsaturated fatty acids have a carbon–carbon double bond located 6 carbons from the methyl end of the chain. (D) Lysophosphatidylcholine consists of a fatty acid chain (green area) and a phosphocholine (yellow area) covalently bonded to a glycerol molecule (blue area) through ester linkages. (E) Sphingolipids, sphingosine, sphingosine-1-phosphate, and ceramide. Ceramide consists of a fatty acid chain (green area) covalently bonded to a sphingosine molecule (dark red area) through amino linkage. (F) Leukotrienes and prostaglandins. Leukotrienes are a family of eicosanoid inflammatory mediators produced in leukocytes by the oxidation of arachidonic acid and the essential fatty acid eicosapentaenoic acid by the enzyme arachidonate 5-lipoxygenase. Prostaglandins are composed of unsaturated fatty acids that contain a cyclopentane (5-carbon) ring and are derived from the 20-carbon, straight-chain, polyunsaturated fatty acid precursor arachidonic acid by the enzyme cyclo-oxygenase through endoperoxides. (G) Resolvins are members of polyunsaturated fatty acid–derived metabolites termed specialized pro-resolving mediators. Resolvin Ds are metabolites of the 22-carbon polyunsaturated fatty acid docosahexaenoic acid. Resolvin Es are metabolites of the 20-carbon polyunsaturated fatty acid eicosapentaenoic acid. (H) Maresins are 12-lipoxygenase-derived specialized pro-resolving mediators synthesized from the omega-3 fatty acid docosahexaenoic acid.
Gastroenterology  , e8DOI: ( /j.gastro )
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10. Supplementary Figure 2
Proinflammatory and pro-resolving bioactive lipids derived from arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. Arachidonic acid is metabolized by cyclo-oxygenases 1 and 2 to prostaglandins and thromboxanes and by 5-lipoxygenase to leukotrienes, which are involved in the initiation of the inflammatory response (pink). Lipoxins also are synthesized from arachidonic acid and 5-, 12-, and 15-lipoxygenase are involved. Eicosapentaenoic acid is metabolized to 3-series prostaglandins and 3-series thromboxanes by cyclo-oxygenase (proinflammatory properties; pink) and to E-series resolvins (anti-inflammatory and pro-resolving actions; green) by 15- and 5-lipoxygenase. Resolvins of the D-series, protectins and maresins, are derived from docosahexaenoic acid through the actions of 5-, 12-, and 15-lipoxygenase. The biosynthesis of D-series resolvins is initiated by 15-lipoxygenase, which transforms docosahexaenoic acid into 17S-hydroperoxy-DHA (17S-HpDHA), which is further transformed by 5-lipoxygenase into 7-hydroperoxy-17S-HDHA, and then hydrolyzed to RvD1, RvD2, or RvD5. Alternatively, lipo-oxygenation at the C-4 position by 5-lipoxygenase forms 4-hydroperoxy-17S-HDHA that is subsequently converted to RvD3, RvD4, and RvD6. Docosahexaenoic acid also can be transformed by 15-lipoxygenase into a dihydroxy-containing docosahexaenoic acid derivative named protectin D1. Furthermore, lipo-oxygenation of docosahexaenoic acid by 12-lipoxygenase originates MaR1 and MaR2. More recently, bioactive molecules derived from DPA, a third ω-3 polyunsaturated fatty acid between eicosapentaenoic acid and docosahexaenoic acid, have been identified and called n-3 DPA resolvins, protectins, and maresins. Lipoxins, resolvins, protectins, and maresins have anti-inflammatory and pro-resolving actions (green). DPA, docosapentaenoic acid; MaR, maresin
Gastroenterology  , e8DOI: ( /j.gastro )
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