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Murine hepatic carboxylesterase 2c (Ces2c) and the presumed human ortholog carboxylesterase 2 (CES2) have been implicated in the development of nonalcoholic fatty liver disease (NAFLD) in mice and obese humans. These studies demonstrated that Ces2c hydrolyzes triglycerides (TGs) in hepatocytes. Interestingly, Ces2c/CES2 is most abundantly expressed in the intestine, indicating a role of Ces2c/CES2 in intestinal TG metabolism. Here we show that Ces2c is an important enzyme in intestinal lipid metabolism in mice. Intestine-specific Ces2c overexpression (Ces2c(int)) provoked increased fatty acid oxidation (FAO) in the small intestine accompanied by enhanced chylomicron clearance from the circulation. As a consequence, high-fat diet-fed Ces2c(int) mice were resistant to excessive diet-induced weight gain and adipose tissue expansion. Notably, intestinal Ces2c overexpression increased hepatic insulin sensitivity and protected mice from NAFLD development. Although lipid absorption was not affected in Ces2c(int) mice, fecal energy content was significantly increased. Mechanistically, we demonstrate that Ces2c is a potent neutral lipase, which efficiently hydrolyzes TGs and diglycerides (DGs) in the small intestine, thereby generating fatty acids (FAs) for FAO and monoglycerides (MGs) and DGs for potential re-esterification. Consequently, the increased availability of MGs and DGs for re-esterification and primordial apolipoprotcin B(48 )particle lipidation may increase chylomicron size, ultimately mediating more efficient chylomicron clearance from the circulation. Conclusion: This study suggests a critical role for Ces2c in intestinal lipid metabolism and highlights the importance of intestinal lipolysis to protect mice from the development of hepatic insulin resistance, NAFLD, and excessive diet-induced weight gain during metabolic stress.

During inflammation, activated leukocytes release cytotoxic mediators that compromise blood–brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO−/− compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.

Objective Medium-chain fatty acids (MCFAs) play an increasing role in human nutrition. In the liver, one fraction is used for synthesis of MCFA-containing triacylglycerol (MCFA-TG), and the rest is used for oxidative energy production or ketogenesis. We investigated which enzymes catalyse the synthesis of MCFA-TG and how inhibition of MCFA-TG synthesis or fatty acid (FA) oxidation influences the metabolic fate of the MCFAs. Methods FA metabolism was followed by time-resolved tracing of alkyne-labelled FAs in freshly isolated mouse hepatocytes. Quantitative data were obtained by mass spectrometry of several hundred labelled lipid species. Wild-type hepatocytes and cells from diacylglycerol acyltransferase (DGAT)1−/− mice were treated with inhibitors against DGAT1, DGAT2, or FA β-oxidation. Results Inhibition or deletion of DGAT1 resulted in a reduction of MCFA-TG synthesis by 70%, while long-chain (LC)FA-TG synthesis was reduced by 20%. In contrast, DGAT2 inhibition increased MCFA-TG formation by 50%, while LCFA-TG synthesis was reduced by 5–25%. Inhibition of β-oxidation by the specific inhibitor teglicar strongly increased MCFA-TG synthesis. In contrast, the widely used β-oxidation inhibitor etomoxir blocked MCFA-TG synthesis, phenocopying DGAT1 inhibition. Conclusions DGAT1 is the major enzyme for hepatic MCFA-TG synthesis. Its loss can only partially be compensated by DGAT2. Specific inhibition of β-oxidation leads to a compensatory increase in MCFA-TG synthesis, whereas etomoxir blocks both β-oxidation and MCFA-TG synthesis, indicating a strong off-target effect on DGAT1., Graphical abstract Medium-chain fatty acids (red) are oxidized or incorporated into triglycerides by DGAT1 in the liver. The popular experimental drug etomoxir inhibits the oxidation and, unexpectedly, blocks triglyceride formation by DGAT1.Image 1

Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal -/- ) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s). We studied metabolic adaptations in Lal -/- mice. Despite loss of adipose tissue, Lal -/- mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [3H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal -/- mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal -/- mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal -/- mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal -/- mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal -/- mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels. Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Lal -/- mice. We conclude that decreased plasma VLDL production enhances glucose uptake into skeletal muscle to compensate for the lack of energy supply.

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) to generate fatty acids (FA) and cholesterol. LAL deficiency (LAL-D) in both humans and mice leads to hepatomegaly, hypercholesterolemia, and shortened life span. Despite its essential role in lysosomal neutral lipid catabolism, the cell type-specific contribution of LAL to disease progression is still elusive. To investigate the role of LAL in the liver in more detail and to exclude the contribution of LAL in macrophages, we generated hepatocyte-specific LAL-deficient mice (Liv-Lipa−/−) and fed them either chow or high fat/high cholesterol diets (HF/HCD). Comparable to systemic LAL-D, Liv-Lipa−/− mice were resistant to diet-induced obesity independent of food intake, movement, and energy expenditure. Reduced body weight gain was mainly due to reduced white adipose tissue depots. Furthermore, Liv-Lipa−/− mice exhibited improved glucose clearance during glucose and insulin tolerance tests compared to control mice. Analysis of hepatic lipid content revealed a massive reduction of TG, whereas CE concentrations were markedly increased, leading to CE crystal formation in the livers of Liv-Lipa−/− mice. Elevated plasma transaminase activities, increased pro-inflammatory cytokines and chemokines as well as hepatic macrophage infiltration indicated liver inflammation. Our data provide evidence that hepatocyte-specific LAL deficiency is sufficient to alter whole-body lipid and energy homeostasis in mice. We conclude that hepatic LAL plays a pivotal role by preventing liver damage and maintaining lipid and energy homeostasis, especially during high lipid availability.

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (−/−) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)−/− mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO mice is likely not a consequence of potentiated CB1R signaling but rather dependent on the activation of alternative signaling pathways. We conclude that MGL deficiency causes complex metabolic changes including cholesterol metabolism and regulation of gut transit independent of the endocannabinoid system.

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1(-/-)) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1(-/-) and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia.

Graphical abstract, The β-lactam cholesterol absorption inhibitor ezetimibe is so far the only representative of this class of compounds on the market today. The goal of this work was to synthesize new amide ezetimibe analogs from trans-3-amino-(3R,4R)-β-lactam and to test their cytotoxicity and activity as cholesterol absorption inhibitors. We synthesized six new amide ezetimibe analogs. All new compounds exhibited low toxicity in MDCKIIwt, hNPC1L1/MDCKII and HepG2 cell lines and showed significant inhibition of cholesterol uptake in hNPC1L1/MDCKII cells. In addition, we determined the activity of the three compounds to inhibit cholesterol absorption in vivo. Our results demonstrate that these compounds considerably reduce cholesterol concentrations in liver and small intestine of mice. Thus, our newly synthesized amide ezetimibe analogs are cholesterol absorption inhibitors in vitro and in vivo.

During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.

BACKGROUND: The success of allogeneic stem cell transplantation (SCT) is limited by transplant-related mortality (TRM). Death from pulmonary causes is a major contributor to TRM. Several factors influence TRM from pulmonary causes. We have analyzed the effect of cigarette smoking on pulmonary related TRM. METHODS: From January 2000 through April 2006 240 patients (pts) underwent allogeneic related or unrelated SCT. Diagnosis included ALL in 22 pts, AML in 90 pts, CLL in 11 pts, CML in 11 pts, Lymphoma in 49 pts, MDS in 29 pts, MM in 9 pts, SSA in 4 pts, solid tumors in 5 pts, other in 4 pts; median age of the pts was 50 years (range 18–70); 148 were males and 92 females; 48 pts received a reduced intensity regimen and 192 an ablative regimen; 117 pts received TBI; 123 pts were non-smokers (Group I) and 117 pts smokers. Amongst the smokers 86 pts provided pack/year information. In this group 46 pts (Group II) had a pack/year history < 30 and 40 pts (Group III) of ≥ 30. RESULTS: 70/123 pts in Group I are deceased with 44% surviving. In this group causes of death included disease progression/relapse in 17 pts, infection in 17 pts, organ failure in 13 pts, GVHD in 11 pts, other in 3 pts and pulmonary related TRM in 8 pts (6.5 ± 4%). 29/46 pts in Group II are deceased with 36% surviving. In this group causes of death included diseaseprogression/relapse in 10 pts, infection in 8 pts, organ failure in 1pt, GVHD in 3 pts, other in 2 pts and pulmonary related TRM in 5 pts (10.9 ± 9%). 31/40 pts in Group III are deceased with 22% surviving. In this group causes of death included disease progression/relapse in 11 pts, infection in 6 pts, organ failure in 5 pts, GVHD in 2 pts, other in 2 pts and pulmonary related TRM in 5 pts (12.5 ± 10 %). CONCLUSION: Smoking contributes to transplant-related mortality from pulmonary causes after allogeneic stem cell transplantation in a dose-related manner. In future analysis the interaction between smoking and other variables will be explored. | Pt Group | Surviving Pts (%) | Deceased Pts (%) | Pulmonary TRM | |:----------------- | ----------------- | ---------------- | ------------- | | Group I ( n=123) | 44% | 56% | 6.5% | | Group Ii (n=46) | 36% | 64% | 10.9% | | Group III ( n=40) | 22% | 78% | 12.5% | Smoking and Outcomes

BACKGROUND: Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality after allogeneic stem cell transplantation. Valganciclovir, the valine ester of ganciclovir, has excellent oral bioavailability and has the potential to replace intravenous ganciclovir in may situations, namely CMV prophylaxis or pre-emptive therapy after allogeneic stem cell transplantation. METHODS: From August 2002 to December 2005, 58 patients (pts) who were either CMV positive or had a seropositive donor were enrolled in a prospective trial of intravenous (IV) ganciclovir at 5 mg/Kg twice a day for one week, followed by oral valganciclovir 900 mg once daily for a total of 180 days. IV ganciclovir was started at time of engraftment. Dose adjustments of valganciclovir were made according to renal function; growth factors were allowed in the event of neutropenia. Patients were monitored with weekly antigenemia. Study endpoints included incidence of CMV reactivation and disease during the first 180 days after transplantation. For the study, viremia was defined as a positive CMV blood culture by shell vial or conventional culture. A positive antigenemia assay in pts with severe (grade III–IV) GVHD was defined as 1 positive cell on either of 2 duplicate slides. For pts with no or mild GVHD, a positive antigenemia assay was defined as 2 positive cells/slide. RESULTS: Fifty eight pts were enrolled. The median age was 50 years (range 18–71). Twenty eight pts underwent a sibling transplant and 30 pts unrelated transplant. Twenty pts were transplanted using a reduced intensity regimen (12 received Campath-1H) and 38 pts received a standard ablative regimen with T cell depletion. There were 47 seropositive recipients and 33 seropositive donors. Fourteen pts were not able to proceed after consenting due to screening positive antigenemia (2 pts), financial reasons (2 pts), severe gut GVHD unabling oral intake (4 pts), death (4 pts), acyclovir resistant HSV infection treated with other antiviral agents (2 pts). Forty four pts received valganciclovir. Neutropenia was observed in 7 pts, resolving in all of them. Valganciclovir was discontinued temporarily in these pts. Four of them were receiving concomitant mycophenolate mofetil. No adverse effects were otherwise reported. Nineteen pts failed to complete the study due to diagnosis of severe gut GVHD (8 pts), financial reasons (1 pt), death due to disease progression (5 pts), relapse/progression of underlying disease (5pts). Twenty six pts have completed at least 3 months of therapy and 21 pts completed 180 days. One pt experienced CMV reactivation (not taking the precribed dose). No other pt experienced CMV reactivation or disease while on therapy. CONCLUSION: These early results suggest that valganciclovir has a role in the prevention of CMV after allogeneic stem cell transplantation. Valganciclovir is well tolerated and is an attractive oral alternative for prevention of CMV.