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.

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.

An early-life diet containing large phospholipid-coated lipid globules programmes later-life postabsorptive lipid trafficking in high-fat diet- but not in low-fat diet-fed mice.

Feeding mice in early life a diet containing an experimental infant milk formula (Nuturis®; eIMF), with a lipid structure similar to human milk, transiently lowered body weight (BW) and fat mass gain upon Western-style diet later in life, when compared with mice fed diets based on control IMF (cIMF). We tested the hypothesis that early-life eIMF feeding alters the absorption or the postabsorptive trafficking of dietary lipids in later life. Male C57BL/6JOlaHsd mice were fed eIMF/cIMF from postnatal day 16-42, followed by low- (LFD, American Institute of Nutrition (AIN)-93 G, 7 wt% fat) or high-fat diet (HFD, D12451, 24 wt% fat) until day 63-70. Lipid absorption rate and tissue concentrations were determined after intragastric administration of stable isotope (2H or 13C) labelled lipids in separate groups. Lipid enrichments in plasma and tissues were analysed using GC-MS. The rate of triolein absorption was similar between eIMF and cIMF fed LFD: 3·2 (sd 1·8) and 3·9 (sd 2·1) and HFD: 2·6 (sd 1·7) and 3·8 (sd 3·0) % dose/ml per h. Postabsorptive lipid trafficking, that is, concentrations of absorbed lipids in tissues, was similar in the eIMF and cIMF groups after LFD. Tissue levels of absorbed TAG after HFD feeding were lower in heart (-42 %) and liver (-46 %), and higher in muscle (+81 %, all P < 0·05) in eIMF-fed mice. In conclusion, early-life IMF diet affected postabsorptive trafficking of absorbed lipids after HFD, but not LFD. Changes in postabsorptive lipid trafficking could underlie the observed lower BW and body fat accumulation in later life upon a persistent long-term obesogenic challenge.

Effects of an early life diet containing large phospholipid-coated lipid globules on hepatic lipid metabolism in mice.

We recently reported that feeding mice in their early life a diet containing a lipid structure more similar to human milk (eIMF, Nuturis) results in lower body weights and fat mass gain upon high fat feeding in later life, compared to control (cIMF). To understand the underlying mechanisms, we now explored parameters possibly involved in this long-term effect. Male C57BL/6JOlaHsd mice, fed rodent diets containing eIMF or cIMF from postnatal (PN) day 16-42, were sacrificed at PN42. Hepatic proteins were measured using targeted proteomics. Lipids were assessed by LC-MS/MS (acylcarnitines) and GC-FID (fatty-acyl chain profiles). Early life growth and body composition, cytokines, and parameters of bile acid metabolism were similar between the groups. Hepatic concentrations of multiple proteins involved in ß-oxidation (+ 17%) the TCA cycle (+ 15%) and mitochondrial antioxidative proteins (+ 28%) were significantly higher in eIMF versus cIMF-fed mice (p < 0.05). Hepatic L-carnitine levels, required for fatty acid uptake into the mitochondria, were higher (+ 33%, p < 0.01) in eIMF-fed mice. The present study indicates that eIMF-fed mice have higher hepatic levels of proteins involved in fatty acid metabolism and oxidation. We speculate that eIMF feeding programs the metabolic handling of dietary lipids.

Spontaneous liver disease in wild-type C57BL/6JOlaHsd mice fed semisynthetic diet.

Mouse models are frequently used to study mechanisms of human diseases. Recently, we observed a spontaneous bimodal variation in liver weight in C57BL/6JOlaHsd mice fed a semisynthetic diet. We now characterized the spontaneous variation in liver weight and its relationship with parameters of hepatic lipid and bile acid (BA) metabolism. In male C57BL/6JOlaHsd mice fed AIN-93G from birth to postnatal day (PN)70, we measured plasma BA, lipids, Very low-density lipoprotein (VLDL)-triglyceride (TG) secretion, and hepatic mRNA expression patterns. Mice were sacrificed at PN21, PN42, PN63 and PN70. Liver weight distribution was bimodal at PN70. Mice could be subdivided into two nonoverlapping groups based on liver weight: 0.6 SD 0.1 g (approximately one-third of mice, small liver; SL), and 1.0 SD 0.1 g (normal liver; NL; p<0.05). Liver histology showed a higher steatosis grade, inflammation score, more mitotic figures and more fibrosis in the SL versus the NL group. Plasma BA concentration was 14-fold higher in SL (p<0.001). VLDL-TG secretion rate was lower in SL mice, both absolutely (-66%, p<0.001) and upon correction for liver weight (-44%, p<0.001). Mice that would later have the SL-phenotype showed lower food efficiency ratios during PN21-28, suggesting the cause of the SL phenotype is present at weaning (PN21). Our data show that approximately one-third of C57BL/6JOlaHsd mice fed semisynthetic diet develop spontaneous liver disease with aberrant histology and parameters of hepatic lipid, bile acid and lipoprotein metabolism. Study designs involving this mouse strain on semisynthetic diets need to take the SL phenotype into account. Plasma lipids may serve as markers for the identification of the SL phenotype.

Programming effects of an early life diet containing large phospholipid-coated lipid globules are transient under continuous exposure to a high-fat diet.

Breast-feeding is associated with a lower risk of developing obesity during childhood and adulthood compared with feeding infant milk formula (IMF). Previous studies have shown that an experimental IMF (eIMF; comprising Nuturis®) programmed mouse pups for a lower body weight and fat mass gain in adulthood when challenged with a high-fat diet (HFD) compared with a control IMF (cIMF). Nuturis has a lipid composition and structure more similar to breast milk. Here, the long-term effects were tested of a similar eIMF, but with an adapted lipid composition and a cIMF, on body weight, glucose homoeostasis, liver and adipose tissue. Nutrient composition was similar for the eIMF and cIMF; the lipid fractions comprised approximately 50 % milk fat. C57BL/6JOlaHsd mice were fed cIMF or eIMF from postnatal day (PN) 16-42 followed by an HFD until PN168. Feeding eIMF v. cIMF in early life resulted in a lower body weight (-9 %) and body fat deposition (-14 %) in adulthood (PN105). The effect appeared transient, as from PN126 onwards, after 12 weeks' HFD, eIMF-fed mice caught up on controls and body and fat weights became comparable between groups. Glucose and energy metabolism were similar between groups. At dissection (PN168), eIMF-fed mice showed larger (+27 %) epididymal fat depots and a lower (-26 %) liver weight without clear morphological aberrations. Our data suggest the size and coating but not the lipid composition of IMF fat globules underlie the programming effect observed. Prolonged exposure to an HFD challenge partly overrules the programming effect of early diet.

Measurement of Intestinal and Peripheral Cholesterol Fluxes by a Dual-Tracer Balance Method.

Long-term elevated plasma cholesterol levels put individuals at risk for developing atherosclerosis. Plasma cholesterol levels are determined by the balance between cholesterol input and output fluxes. Here we describe in detail the methodology to determine the different cholesterol fluxes in mice. The percentage of absorbed cholesterol is calculated from a stable isotope-based double-label method. Cholesterol synthesis is calculated from MIDA after 13 C-acetate enrichment. Cholesterol is removed from the body via the feces. The fecal excretion route is either biliary or non-biliary. The non-biliary route is dominated by trans-intestinal cholesterol efflux, or TICE. Biliary excretion of cholesterol is measured by collecting bile. Non-biliary excretion is calculated by computational modeling. In this article, we describe methods and procedures to measure and calculate dietary intake of cholesterol, fractional cholesterol absorption, fecal neutral sterol output, biliary cholesterol excretion, TICE, cholesterol synthesis, peripheral fluxes, and whole-body cholesterol balance. © 2016 by John Wiley & Sons, Inc.