Hepatocyte-specific deletion of adipose triglyceride lipase (adipose triglyceride lipase/patatin-like phospholipase domain containing 2) ameliorates dietary induced steatohepatitis in mice.

BACKGROUND AND AIMS: Increased fatty acid (FA) flux from adipose tissue to the liver contributes to the development of NAFLD. Because free FAs are key lipotoxic triggers accelerating disease progression, inhibiting adipose triglyceride lipase (ATGL)/patatin-like phospholipase domain containing 2 (PNPLA2), the main enzyme driving lipolysis, may attenuate steatohepatitis. APPROACH AND RESULTS: Hepatocyte-specific ATGL knockout (ATGL LKO) mice were challenged with methionine-choline-deficient (MCD) or high-fat high-carbohydrate (HFHC) diet. Serum biochemistry, hepatic lipid content and liver histology were assessed. Mechanistically, hepatic gene and protein expression of lipid metabolism, inflammation, fibrosis, apoptosis, and endoplasmic reticulum (ER) stress markers were investigated. DNA binding activity for peroxisome proliferator-activated receptor (PPAR) α and PPARδ was measured. After short hairpin RNA-mediated ATGL knockdown, HepG2 cells were treated with lipopolysaccharide (LPS) or oleic acid:palmitic acid 2:1 (OP21) to explore the direct role of ATGL in inflammation in vitro. On MCD and HFHC challenge, ATGL LKO mice showed reduced PPARα and increased PPARδ DNA binding activity when compared with challenged wild-type (WT) mice. Despite histologically and biochemically pronounced hepatic steatosis, dietary-challenged ATGL LKO mice showed lower hepatic inflammation, reflected by the reduced number of Galectin3/MAC-2 and myeloperoxidase-positive cells and low mRNA expression levels of inflammatory markers (such as IL-1ß and F4/80) when compared with WT mice. In line with this, protein levels of the ER stress markers protein kinase R-like endoplasmic reticulum kinase and inositol-requiring enzyme 1α were reduced in ATGL LKO mice fed with MCD diet. Accordingly, pretreatment of LPS-treated HepG2 cells with the PPARδ agonist GW0742 suppressed mRNA expression of inflammatory markers. Additionally, ATGL knockdown in HepG2 cells attenuated LPS/OP21-induced expression of proinflammatory cytokines and chemokines such as chemokine (C-X-C motif) ligand 5, chemokine (C-C motif) ligand (Ccl) 2, and Ccl5. CONCLUSIONS: Low hepatic lipolysis and increased PPARδ activity in ATGL/PNPLA2 deficiency may counteract hepatic inflammation and ER stress despite increased steatosis. Therefore, lowering hepatocyte lipolysis through ATGL inhibition represents a promising therapeutic strategy for the treatment of steatohepatitis.

A Mixed-Lipid Emulsion Containing Fish Oil for the Parenteral Nutrition of Preterm Infants: No Impact on Visual Neuronal Conduction.

Fish oil is rich in omega-3 fatty acids and essential for neuronal myelination and maturation. The aim of this study was to investigate whether the use of a mixed-lipid emulsion composed of soybean oil, medium-chain triglycerides, olive oil, and fish oil (SMOF-LE) compared to a pure soybean oil-based lipid emulsion (S-LE) for parenteral nutrition had an impact on neuronal conduction in preterm infants. This study is a retrospective matched cohort study comparing preterm infants <1000 g who received SMOF-LE in comparison to S-LE for parenteral nutrition. Visual evoked potentials (VEPs) were assessed longitudinally from birth until discharge. The latencies of the evoked peaks N2 and P2 were analyzed. The analysis included 76 infants (SMOF-LE: n = 41 and S-LE: n = 35) with 344 VEP measurements (SMOF-LE: n= 191 and S-LE n = 153). Values of N2 and P2 were not significantly different between the SMOF-LE and S-LE groups. A possible better treatment effect in the SMOF-LE group was seen as a trend toward a shorter latency, indicating faster neural conduction at around term-equivalent age. Prospective trials and follow-up studies are necessary in order to evaluate the potential positive effect of SMOF-LE on neuronal conduction and visual pathway maturation.

Absence of Adiponutrin (PNPLA3) and Monoacylglycerol Lipase Synergistically Increases Weight Gain and Aggravates Steatohepatitis in Mice.

Altered lipid metabolic pathways including hydrolysis of triglycerides are key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Whether adiponutrin (patatin-like phospholipase domain containing protein-3-PNPLA3) and monoacylglycerol lipase (MGL) synergistically contribute to disease progression remains unclear. We generated double knockout (DKO) mice lacking both Mgl and Pnpla3; DKO mice were compared to Mgl-/- after a challenge by high-fat diet (HFD) for 12 weeks to induce steatosis. Serum biochemistry, liver transaminases as well as histology were analyzed. Fatty acid (FA) profiling was assessed in liver and adipose tissue by gas chromatography. Markers of inflammation and lipid metabolism were analyzed. Bone marrow derived macrophages (BMDMs) were isolated and treated with oleic acid. Combined deficiency of Mgl and Pnpla3 resulted in weight gain on a chow diet; when challenged by HFD, DKO mice showed increased hepatic FA synthesis and diminished beta-oxidation compared to Mgl-/-.DKO mice exhibited more pronounced hepatic steatosis with inflammation and recruitment of immune cells to the liver associated with accumulation of saturated FAs. Primary BMDMs isolated from the DKO mice showed increased inflammatory activities, which could be reversed by oleic acid supplementation. Pnpla3 deficiency aggravates the effects of Mgl deletion on steatosis and inflammation in the liver under HFD challenge.

Monoacylglycerol lipase reprograms lipid precursors signaling in liver disease.

Dietary oversupply of triglycerides represent the hallmark of obesity and connected complications in the liver such as non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, which eventually progress to cirrhosis and hepatocellular carcinoma. Monoacylglycerol lipase is the last enzymatic step in the hydrolysis of triglycerides, generating glycerol and fatty acids (FAs), which are signaling precursors in physiology and disease. Notably, monoacylglycerol lipase (MGL) also hydrolyzes 2-arachidonoylglycerol, which is a potent ligand within the endocannabinoid system, into arachidonic acid - a precursor for prostaglandin synthesis; thus representing a pivotal substrates provider in multiple organs for several intersecting biological pathways ranging from FA metabolism to inflammation, pain and appetite. MGL inhibition has been shown protective in limiting several liver diseases as FAs may drive hepatocyte injury, fibrogenesis and de- activate immune cells, however the complexity of MGL network system still needs further and deeper understanding. The present review will focus on MGL function and FA partitioning in the horizons of liver disease.

PNPLA3 I148M Variant Impairs Liver X Receptor Signaling and Cholesterol Homeostasis in Human Hepatic Stellate Cells.

The patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148M variant predisposes to hepatic steatosis and progression to advanced liver injury with development of fibrosis, cirrhosis, and cancer. Hepatic stellate cells (HSCs) drive the wound healing response to chronic injury, and lack of liver X receptor (LXR) signaling exacerbates liver fibrogenesis by impairing HSC cholesterol homeostasis. However, the contribution of the I148M variant to this process is still unknown. We analyzed LXR expression and transcriptional activity in primary human HSCs and overexpressing LX-2 cells according to PNPLA3 genotype (wild type [WT] versus I148M). Here we demonstrate that LXRα protein increased whereas LXR target gene expression decreased during in vitro activation of primary human HSCs. Notably, LXRα levels and signaling were reduced in primary I148M HSCs compared to WT, as displayed by decreased expression of LXR target genes. Moreover, reduced expression of cholesterol efflux and enzymes generating oxysterols was associated with higher total and free cholesterol accumulation whereas endogenous cholesterol synthesis and uptake were diminished in I148M HSCs. Luciferase assays on LXR response element confirmed decreased LXR transcriptional activity in I148M HSCs; in contrast the synthetic LXR agonist T0901317 replenished LXR functionality, supported by adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1) induction, and reduced collagen1α1 and chemokine (C-C motif) ligand 5 expression. Conversely, the peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone had only partial effects on the LXR target gene ABCA1, and neither diminished expression of proinflammatory cytokines nor increased de novo lipogenic genes in I148M HSCs. Conclusion: As a consequence of reduced PPARγ activity, HSCs carrying I148M PNPLA3 show impaired LXR signaling, leading to cholesterol accumulation. The use of a specific LXR agonist shows beneficial effects for diminishing sustained HSC activation and development of liver fibrogenesis.

Inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver.

OBJECTIVE: Sustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury. DESIGN: C57BL/6J mice and mice with global invalidation of MAGL (MAGL -/- ), or myeloid-specific deletion of either MAGL (MAGLMye-/-), ATG5 (ATGMye-/-) or CB2 (CB2Mye-/-), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells. RESULTS: MAGL -/- or MAGLMye-/- mice exposed to CCl4 or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6Clow macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGLMye-/- BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGLMye-/- mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2Mye-/- mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5Mye-/- BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited. CONCLUSION: MAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.

PNPLA3 expression and its impact on the liver: current perspectives.

A single-nucleotide polymorphism occurring in the sequence of the human patatin-like phospholipase domain-containing 3 gene (PNPLA3), known as I148M variant, is one of the best characterized and deeply investigated variants in several clinical scenarios, because of its tight correlation with increased risk for developing hepatic steatosis and more aggressive part of the disease spectrum, such as nonalcoholic steatohepatitis, advanced fibrosis and cirrhosis. Further, the I148M variant is positively associated with alcoholic liver diseases, chronic hepatitis C-related cirrhosis and hepatocellular carcinoma. The native gene encodes for a protein that has not yet a fully defined role in liver lipid metabolism and, according to recent observations, seems to be divergently regulated among distinct liver cells type, such as hepatic stellate cells. Therefore, the aim of this review is to collect the latest data regarding PNPLA3 expression in human liver and to analyze the impact of its genetic variant in human hepatic pathologies. Moreover, a description of the current biochemical and metabolic data pertaining to PNPLA3 function in both animal models and in vitro studies is summarized to allow a better understanding of the relevant pathophysiological role of this enzyme in the progression of hepatic diseases.

AQP3 is regulated by PPARγ and JNK in hepatic stellate cells carrying PNPLA3 I148M.

Aquaglyceroporins (AQPs) allow the movement of glycerol that is required for triglyceride formation in hepatic stellate cells (HSC), as key cellular source of fibrogenesis in the liver. The genetic polymorphism I148M of the patatin-like phospholipase domain-containing 3 (PNPLA3) is associated with hepatic steatosis and its progression to steatohepatitis (NASH), fibrosis and cancer. We aimed to explore the role of AQP3 for HSC activation and unveil its potential interactions with PNPLA3. HSC were isolated from human liver, experiments were performed in primary HSC and human HSC line LX2. AQP3 was the only aquaglyceroporin present in HSC and its expression decreased during activation. The PPARγ agonist, rosiglitazone, recovered AQP3 expression also in PNPLA3 I148M carrying HSC. When PNPLA3 was silenced, AQP3 expression increased. In liver sections from patients with NASH, the decreased amount of AQP3 was proportional to the severity of fibrosis and presence of the PNPLA3 I148M variant. In PNPLA3 I148M cells, the blockade of JNK pathway upregulated AQP3 in synergism with PPARγ. In conclusion, we demonstrated profound reduction of AQP3 in HSC carrying the PNPLA3 I148M variant in parallel to decreased PPARγ activation, which could be rescued by rosiglitazone and blockade of JNK.

The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells.

The genetic polymorphism I148M of patatin-like phospholipase domain-containing 3 (PNPLA3) is robustly associated with hepatic steatosis and its progression to steatohepatitis, fibrosis, and cancer. Hepatic stellate cells (HSCs) are key players in the development of liver fibrosis, but the role of PNPLA3 and its variant I148M in this process is poorly understood. Here we analyzed the expression of PNPLA3 during human HSC activation and thereby explored how a PNPLA3 variant impacts hepatic fibrogenesis. We show that expression of PNPLA3 gene and protein increases during the early phases of activation and remains elevated in fully activated HSCs (P < 0.01). Knockdown of PNPLA3 significantly decreases the profibrogenic protein alpha-smooth muscle actin (P < 0.05). Primary human I148M HSCs displayed significantly higher expression and release of proinflammatory cytokines, such as chemokine (C-C motif) ligand 5 (P < 0.01) and granulocyte-macrophage colony-stimulating factor (P < 0.001), thus contributing to migration of immune cells (P < 0.05). Primary I148M HSCs showed reduced retinol (P < 0.001) but higher lipid droplet content (P < 0.001). In line with this, LX-2 cells stably overexpressing I148M showed augmented proliferation and migration, lower retinol, and abolished retinoid X receptor/retinoid A receptor transcriptional activities but more lipid droplets. Knockdown of I148M PNPLA3 (P < 0.001) also reduces chemokine (C-C motif) ligand 5 and collagen1α1 expression (P < 0.05). Notably, I148M cells display reduced peroxisome proliferator-activated receptor gamma transcriptional activity, and this effect was attributed to increased c-Jun N-terminal kinase, thereby inhibiting peroxisome proliferator-activated receptor gamma through serine 84 phosphorylation and promoting activator protein 1 transcription. Conversely, the c-Jun N-terminal kinase inhibitor SP600125 and the peroxisome proliferator-activated receptor gamma agonist rosiglitazone decreased activator protein 1 promoter activity. CONCLUSIONS: These data indicate that PNPLA3 is required for HSC activation and that its genetic variant I148M potentiates the profibrogenic features of HSCs, providing a molecular mechanism for the higher risk of progression and severity of liver diseases conferred to patients carrying the I148M variant. (Hepatology 2017;65:1875-1890).

Adiponectin regulates aquaglyceroporin expression in hepatic stellate cells altering their functional state.

BACKGROUND AND AIM: Obesity is a major risk factor for liver fibrosis and tightly associated with low levels of adiponectin. Adiponectin has antifibrogenic activity protecting from liver fibrosis, which is mainly driven by activated hepatic stellate cells (HSC). Aquaporins are transmembrane proteins that allow the movement of water and, in case of aquaglyceroporins (AQPs), of glycerol that is needed in quiescent HSC for lipogenesis. Expression of various AQPs in liver is altered by obesity; however, the mechanisms through which obesity influences HSCs activation and AQPs expression remain unclear. This study aimed to identify obesity-associated factors that are related to HSC AQPs expression activation and lipid storage. METHODS: Correlations between serum adipokine levels and hepatic AQPs gene expression were analyzed from a cohort of obese patients. AQP and fibrotic gene expression was determined in a HSC line (LX2) and in a hepatocyte cell line (HepG2) after stimulation with adiponectin using quantitative real-time polymerase chain reaction. RESULTS: We found that serum adiponectin significantly correlated with liver AQP3, AQP7, AQP9 gene expressions. In vitro, adiponectin induced upregulation of AQP3 gene and AQP3 protein expression in human HSCs, but not in hepatocytes, while AQP7, AQP9 remained undetectable. Accordingly, HSC stimulated with adiponectin increased glycerol uptake, lipogenic gene expression, and lipid storage while downregulating activation/fibrosis markers. CONCLUSIONS: These findings demonstrate that adiponectin is a potent inhibitor of HSC activation and induces AQPs expression. Thus, low serum levels of adiponectin could be a mechanism how obesity affects the functional state of HSC, thereby contributing to obesity-associated liver fibrosis.

Osteopontin is a key player for local adipose tissue macrophage proliferation in obesity.

OBJECTIVE: Recent findings point towards an important role of local macrophage proliferation also in obesity-induced adipose tissue inflammation that underlies insulin resistance and type 2 diabetes. Osteopontin (OPN) is an inflammatory cytokine highly upregulated in adipose tissue (AT) of obese and has repeatedly been shown to be functionally involved in adipose-tissue inflammation and metabolic sequelae. In the present work, we aimed at unveiling both the role of OPN in human monocyte and macrophage proliferation as well as the impact of OPN deficiency on local macrophage proliferation in a mouse model for diet-induced obesity. METHODS: The impact of recombinant OPN on viability, apoptosis, and proliferation was analyzed in human peripheral blood monocytes and derived macrophages. Wild type (WT) and OPN knockout mice (SPP1KO) were compared with respect to in vivo adipose tissue macrophage and in vitro bone marrow-derived macrophage (BMDM) proliferation. RESULTS: OPN not only enhanced survival and decreased apoptosis of human monocytes but also induced proliferation similar to macrophage colony stimulating factor (M-CSF). Even in fully differentiated monocyte-derived macrophages, OPN induced a proliferative response. Moreover, proliferation of adipose tissue macrophages in obese mice was detectable in WT but virtually absent in SPP1KO. In BMDM, OPN also induced proliferation while OPN as well as M-CSF-induced proliferation was similar in WT and SPP1KO. CONCLUSIONS: These data confirm that monocytes and macrophages not only are responsive to OPN and migrate to sites of inflammation but also they survive and proliferate more in the presence of OPN, a mechanism also strongly confirmed in vivo. Therefore, secreted OPN appears to be an essential player in AT inflammation, not only by driving monocyte chemotaxis and macrophage differentiation but also by facilitating local proliferation of macrophages.

Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage.

Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.